WO2019138237A1 - Micro arn en tant que marqueur d'une maladie qui peut être associée au vieillissement - Google Patents

Micro arn en tant que marqueur d'une maladie qui peut être associée au vieillissement Download PDF

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WO2019138237A1
WO2019138237A1 PCT/GB2019/050069 GB2019050069W WO2019138237A1 WO 2019138237 A1 WO2019138237 A1 WO 2019138237A1 GB 2019050069 W GB2019050069 W GB 2019050069W WO 2019138237 A1 WO2019138237 A1 WO 2019138237A1
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mir
concentration
sample
related disorder
subject
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PCT/GB2019/050069
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Peter Barnes
Jonathan Baker
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Imperial Innovations Limited
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to novel biological markers for diseases associated with accelerated aging, such as accelerated lung aging, chronic obstructive pulmonary disease (COPD), ischemic heart disease and renal disease, and in particular to the use of microRNAs as diagnostic and prognostic markers in assays for detecting such disorders.
  • the invention extends to the use of the novel biomarkers as a therapeutic target for treating any diseases associated with accelerated aging, and to methods of determining the efficacy of treating a disorder characterised by accelerated aging with a therapeutic agent, as well as kits for carrying out the assays and methods.
  • the assays are qualitative and/or quantitative, and are adaptable to large-scale screening and clinical trials.
  • oxidative stress drives the ageing process with reactive oxygen species (ROS), inducing cellular senescence 2 .
  • ROS reactive oxygen species
  • Senescent cells enter cell cycle arrest but remain metabolically actives, and secrete multiple inflammatory proteins, known as senescence-associated secretory SASP, which include proinflammatory cytokines, chemokines, growth factors and matrix metalloproteinases (MMPs).
  • MMPs matrix metalloproteinases
  • COPD Chronic obstructive pulmonary disease
  • Oxidative stress induces miRNA expression, leading to the induction of cellular senescence 7 ’ 8 .
  • Oxidative stress caused by cigarette smoke exposure accelerates cellular senescence within the lungs of COPD patients via the induction of DNA double-strand breaks, elevated pi6 and p2i expression and proinflammatory cytokine release 9-12 .
  • sirtuin-i which is an NAD + -dependent class III protein deacetylase 1344 .
  • Sirtuin-i has reduced expression and activity in COPD, which has been attributed to the increased oxidative in the lungs 15 ’ 16 .
  • MicroRNAs are small (18-22 nucleotide) regulatory RNAs, which are key modulators of several physiological processes and have been shown in both COPD and other inflammatory diseases to regulate cellular senescence 17-19 . The inventors therefore set out to investigate miRNAs that regulate cellular senescence via the modulation of sirtuin-i.
  • miR-34a is increased by oxidative stress through the activation of the PI3 kinase-mTOR signalling pathway and inhibits sirtuin- 1 in COPD cells. Inhibiting miR-34a with a specific antagomir increases reduced sirtuin-i expression in these cells, suggesting that this might be a good therapeutic target 19 .
  • this miRNA is reduced in non-small-cell lung cancer 20 and therefore there is a risk that its inhibition could increase the risk of lung cancer, which is already high in COPD patients 21 . Accordingly, miR-34a is not a good target for COPD therapy.
  • the inventors examined the role of miR-570-3p in the regulation of senescence in COPD lungs and cells. They observed that miR-570-3p was elevated in the lungs of COPD patients and induced by oxidative stress-dependent P38 MAP kinase signalling via c-Jun mediated transcription. Luciferase gene constructs confirmed that miR-570-3p binds to the 3’UTR of sirtuin-i.
  • SAECs small airway epithelial cells
  • a method for diagnosing a subject suffering from an accelerated age-related disorder, or a pre- disposition thereto, or for providing a prognosis of the subject’s condition comprising analysing the concentration of one or more type of micro RNA molecule in a bodily sample from a test subject, and comparing this concentration with a reference for the concentration of the one or more type of microRNA molecule in an individual who does not suffer from an accelerated age-related disorder, wherein a difference in the concentration of the one or more type of microRNA molecule in the bodily sample from the test subject compared to the reference suggests that the subject is suffering from an accelerated age-related disorder, or has a pre-disposition thereto, or provides a negative prognosis of the subject’s condition.
  • kits for diagnosing a subject suffering from an accelerated age-related disorder, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition comprising:
  • RNA molecule in a sample from a test subject RNA molecule in a sample from a test subject
  • kit is used to identify a difference in the concentration of the one or more type of microRNA molecule in the sample from the test subject compared to the reference concentration, thereby suggesting that the test subject suffers from an accelerated age-related disorder, or has a pre-disposition thereto, or providing a negative prognosis of the subject’s condition.
  • the method of the first aspect may comprise administering or having administered, to the subject, a therapeutic agent, which prevents, reduces or delays progression of an accelerated age-related disorder.
  • a method of treating an individual suffering from an accelerated age-related disorder comprising the steps of:
  • a method for determining the efficacy of treating a subject suffering from an accelerated age-related disorder with a therapeutic agent comprising analysing the concentration of one or more type of micro RNA molecule in a bodily sample from a test subject and comparing this concentration with a reference for the concentration of the one or more type of micro RNA molecule in an individual who does not suffer from an accelerated age- related disorder, wherein a difference in the concentration of the one or more type of micro RNA molecule in the bodily sample compared to the reference is indicative of the efficacy of treating the test subject with the therapeutic agent.
  • kits for determining the efficacy of treating a subject suffering from an accelerated age-related disorder with a therapeutic agent comprising:-
  • kit is used to identify a difference in the concentration of the one or more type of microRNA molecule in the sample from the test subject compared to the reference concentration, the difference in concentration being indicative of the efficacy of treating the test subject with the therapeutic agent.
  • a method for analysing a sample from a test subject comprising: (i) determining the concentration of one or more type of microRNA molecule in a bodily sample from a test subject; and (ii) comparing the concentration of the one or more more type of microRNA molecule determined in step (i) to at least one control concentration, wherein the concentrations of the one or more more type of microRNA molecule are indicative of whether the subject
  • miRNAs can survive in the peripheral blood.
  • these miRNAs can serve as robust biomarkers for accelerated age-related disorders, such as COPD, and can therefore be used for the detection of oxidative stress-driven conditions resulting from reduced levels of the protective molecule, Sirtuin-i, and disease prognosis.
  • the inventors have shown that using miRNAs as biomarkers for oxidative stress-driven accelerated ageing diseases employs an assay which is simple, reproducible and inexpensive, and with minimal
  • miRNAs can be detected in blood plasma and serum, opening the way to a novel, non-invasive diagnostic test.
  • miRNAs are stable in plasma at room temperature (for at least 24 hours) and when subjected to freeze-thaw cycles. Coupled with this, and the data described in the examples, it is clearly possible to use miRNAs derived from blood samples as a minimally invasive diagnostic tool for patients suffering from accelerated age-related disorders.
  • the method according to the first aspect is useful for enabling a clinician to make decisions with regards to the best course of treatment for a subject who is currently or who may suffer from an accelerated age-related disorder in the future, such as COPD, or other oxidative stress-driven accelerated ageing diseases. It is preferred that the method of the first aspect is useful for enabling a clinician to decide how to treat a subject who is suffering from an accelerated age-related disorder. In addition, the methods described herein are useful for monitoring the efficacy of a putative treatment for an accelerated age-related disorder, for example metformin and rapamycin, when treating COPD.
  • kits according to the invention are useful for providing a prognosis of the subject’s condition, such that the clinician can carry out the treatment according to the fourth aspect.
  • the kits of the invention may be used to monitor the efficacy of a putative treatment for age-related disorders.
  • the methods and the kits are therefore very useful for guiding an accelerated age-related disorder treatment regime for the clinician, and to monitor the efficacy of such a treatment regime.
  • the clinician may use the kits of the invention in conjunction with existing diagnostic tests to improve the accuracy of diagnosis.
  • the methods and kits of the invention may be used to predict the risk of progression in an accelerated age-related disorder.
  • the accelerated age-related disorder is preferably a disorder characterised by oxidative stress.
  • the accelerated age-related disorder is most preferably a disorder characterised by cellular senescence.
  • the accelerated age-related disorder is preferably a disorder characterised by reduced levels of sirtuin-i compared to healthy individuals.
  • the accelerated age-related disorder may be selected from the group consisting of: accelerated lung aging, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cardiovascular disease, ischemic heart disease, cardiac failure, diabetes, Type 2 diabetes, and renal disease.
  • COPD chronic obstructive pulmonary disease
  • IPF idiopathic pulmonary fibrosis
  • the accelerated age-related disorder is COPD or IPF.
  • Micro RNA molecules are non-coding, post-transcriptional regulators that normally bind to complementary sequences in the 3’ untranslated regions (3’ UTRs) of target messenger RNA transcripts (mRNAs), usually resulting in gene silencing.
  • miRNAs are short ribonucleic acid (RNA) molecules, on average only about 22 nucleotides long.
  • the miRNA detected in the methods and kits of the invention may be about 15 to 30 nucleotides long, or about 18 to 25 nucleotides long, or about 21 to 23 nucleotides long.
  • the methods and kits of the invention may comprise detecting and analysing the concentration of any of the known miRNAs, and which can be found on the miRBase website (http:/ /www.mirbase.org/).
  • the miRBase (release 18) currently includes 1587 mature human miRNAs, all of which are processed from longer precursors and differ from each other in nucleotide sequence.
  • the current understanding is that each miRNA is expressed in one or more human tissues and binds to one or more target RNA sequences expressed in particular tissues.
  • the binding of this single miRNA, by itself or in combination with other miRNAs and/or proteins to a particular mRNA leads to down-regulation of gene expression, usually by degradation of the target mRNA or repression of protein translation.
  • the methods and kits may comprise detecting miR-570-3p, and measuring its concentration compared to the reference value.
  • the methods and kits may comprise detecting miR-570-3p, and measuring its concentration compared to the reference value.
  • one or more type of microRNA as a biomarker for diagnosing a subject suffering from an accelerated age-related disorder, or a pre-disposition thereto, or for providing a prognosis of the subject’s condition, wherein the one or more type of microRNA molecule is miR-570-3p (SEQ ID No.i), or the complementary sequence thereof, or a variant or fragment thereof.
  • sequence of miR-570-3p is 22 nucleotides long, and is referred to herein as SEQ ID No.i, as follows:
  • the miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.i, or the complementary sequence thereof, or a variant or fragment thereof.
  • the methods and kits of the invention may comprise determining the concentration of miR-570-3p comprising a nucleotide sequence substantially as set out in any or SEQ ID No.i, or a complementary sequence thereof, or variant or fragment thereof.
  • the pattern of expression, where miR-570-3p is found at significantly higher levels in a bodily sample compared to a test subject (e.g. plasma or serum from peripheral blood) maybe termed the“miRNA signature”.
  • the methods and kits comprise detecting an increase in the concentration of the one or more type of microRNA molecule in the bodily sample from the test subject compared to the reference.
  • the complementary sequence of SEQ ID No l (often termed miRNA*, for example miR- 570-5p) may also be detected.
  • This complementary sequence has also been shown to have activity, and therefore also has utility as a biomarker for accelerated age-related disorders.
  • Variants and fragments of any of the miRNA molecules that maybe detected may include truncations or additions of nucleotides of the miRNA molecule, for example SEQ ID No.i.
  • a truncation may comprise the miRNA molecule having been reduced in size by the removal of at least one nucleotide from the 5’ and/or 3’ end of the miRNA, or by deletion of one of more nucleotides from within the core or centre of the miRNA.
  • the truncation may comprise deletion of at least 2, 3, 4 or 5 nucleotides from the miRNA molecule.
  • An addition may comprise the miRNA molecule having been increased in size by the addition of at least one nucleotide to the 5’ and/or 3’ end of the miRNA, or by the introduction of one of more nucleotides into the core or centre of the miRNA.
  • the addition may comprise addition of at least 2, 3, 4, 5, or up to 10
  • the concentration of the at least one type of miRNA molecule may act as a diagnostic and/or prognostic marker for an accelerated age-related disorder, for example COPD.
  • the inventors investigated the expression levels of miR-570-3p in COPD patients, and were annoyed to observe that it was over-expressed in COPD patients compared to the healthy controls.
  • the miRNA signature comprises an increase in miR-570-3p concentration compared to the control concentration.
  • this miRNA molecule which forms a miRNA signature, represents a useful and robust physiological marker of accelerated age-related disorders, such as COPD, and other oxidative stress-driven accelerated ageing diseases. Accordingly, this biomarker can be robustly used for prognostic and diagnostic purposes.
  • the inventors have established that circulating levels of miRNAs in a test subject is highly suggestive of the whether the subject either has, or is pre-disposed to developing, an accelerated age-related disorder, and is sufficiently sensitive to detect the disorder at an early stage. Furthermore, using the invention, it is possible to monitor whether a subject’s condition relapses following a treatment regime. Accordingly, the methods of the invention, in which the concentration of the miRNA molecule is measured, and compared to a control concentration, provides a very reliable prognostic marker for monitoring conditions, both before and after treatment. Accordingly, assaying for miRNA molecules is a substantial improvement over assaying for other markers, because it is more sensitive and also provides enhanced specificity. In addition, assaying for miRNA molecules also provides far more information to the clinician, and will help stratify the disease.
  • detecting one particular type of miRNA molecule may be of use by itself as a biomarker for accelerated age-related disorder. Further, detecting more than one type of miRNA molecule, may provide a more robust diagnosis or prognosis of the disease.
  • the biomarker may also be used in combination with an assay of another biological marker indicative of an accelerated age-related disorder, for example detection of miR-34a, miR-26a and/or miR-126.
  • assaying for one or more miRNA molecules may be used to complement the use of another marker to provide even more information to the clinician.
  • the subject may be any animal of veterinary interest, for instance, a cat, dog, horse etc. However, it is preferred that the subject is a mammal, such as a human, either male or female.
  • kits of the invention may comprise sample extraction means for obtaining the sample from the test subject.
  • the sample extraction means may comprise a needle or syringe or the like. It will also be appreciated that“fresh” bodily samples may be analysed immediately after they have been taken from a subject. Alternatively, the samples may be frozen and stored. The sample may then be de-frosted and analysed at a later date.
  • the sample may be any bodily sample into which miRNA molecules are secreted, e.g. it maybe lymph or interstitial fluid.
  • the sample maybe a urine sample.
  • the inventors were very surprised to observe that miRNAs survive in the blood. Therefore, it is preferred that the miRNA molecule is measured or assayed in a blood sample.
  • the blood sample may be venous or arterial.
  • the miRNA being detected may be encapsulated in extracellular vesicles, and that the detection step preferably comprises isolating these miRNA-containing vesicles prior to detection.
  • further detection of miRNAs encapsulated in extracellular vesicles maybe an alternate method to detect the biomarkers described herein.
  • a miR-570 signature in extracellular vesicles taken from plasma or serum samples may be an alternate biomarker detection method.
  • the kit may comprise a sample collection container for receiving the extracted sample.
  • Blood samples may be assayed for miRNA molecule levels immediately.
  • the blood may be stored at low temperatures, for example in a fridge or even frozen before the miRNA assay is conducted. Measurement of miRNA may be made on whole blood.
  • the blood maybe further processed before the assay is performed.
  • an anticoagulant such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added.
  • the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting.
  • the blood sample may be centrifuged or filtered to prepare a plasma or serum fraction, which maybe used for analysis.
  • the miRNA is analysed or assayed in a blood plasma or a blood serum sample. It is preferred that miRNA concentration is measured in vitro from a blood serum sample or a plasma sample taken from the subject.
  • the inventors monitored the concentration of miR-570- 3p in numerous patients who suffered from COPD, and compared them to the concentration of the same miRNA in individuals who did not suffer from COPD. They demonstrated that there was a statistically significant increase in the concentration of certain miRNA molecules described herein in the patients suffering from COPD. Thus, the difference in concentration may be an increase compared to the reference. It will be appreciated that the concentration of a miR-570-3p in COPD patients is highly dependent on a number of factors, for example how far the disease has progressed, and the age and gender of the subject.
  • the concentration of miRNAs in individuals who do not suffer from an accelerated age-related disorder may fluctuate to some degree, but that on average over a given period of time, the concentration tends to be substantially constant.
  • the concentration of miRNA in one group of individuals who do not suffer from an accelerated age-related disorder may be different to the concentration of those miRNAs in another group of individuals who do not suffer from the disease.
  • the skilled technician will know how to determine the average concentration of certain miRNAs in individuals who do not suffer from an accelerated age-related disorder, and this is referred to as the‘normal’ or‘control’ or‘reference’ concentration of miRNA.
  • the normal concentration corresponds to the reference values discussed above.
  • the miRNA may be extracted from the bodily sample by a variety of techniques. Briefly, these may comprise addition of a protein denaturant (such as Trizol or guanidine thiocyanate) to the sample, centrifugation to remove protein debris, addition of DNasel to remove DNA, and extraction of RNA using a suitable column. RNA samples may be further concentrated by ethanol/isopropanol precipitation and/or centrifugal concentration.
  • a protein denaturant such as Trizol or guanidine thiocyanate
  • RNA samples may be further concentrated by ethanol/isopropanol precipitation and/or centrifugal concentration.
  • the preferred extraction kit used by the inventors is supplied by Qiagen, but other extraction kits could be used, depending on availability and/ or suitability in subsequent downstream reactions.
  • PCR may be used to amplify the one or more type of miRNA molecule.
  • kits according to the invention comprise means for determining the concentration of one or more type of miRNA molecule in a sample from a test subject.
  • the kit may comprise a container in which the means for determining the concentration of one or more type of miRNA molecule in a sample from a test subject may be contained.
  • the kit may also comprise instructions for use.
  • kits may comprise detection means for determining the concentration of the one or more type of miRNA (preferably, miR-570-3p) in the sample once this has been obtained from the subject.
  • the detection means may comprise one or more primer, for use in a PCR method for amplifying the miRNA.
  • detection of the one or more type of miRNA molecule may be achieved by TaqMan quantitative RT-PCR using primer and probe sets specific for particular human miRNAs, as described on the Applied Biosystems website
  • detection may be achieved using an Exiqon micro RNA detection kit (http://www.exiqon.com/ls).
  • Exiqon micro RNA detection kit http://www.exiqon.com/ls.
  • the primers may comprise at least partial sequence identity with the miRNA molecule being detected, for miR-570-3p.
  • the reference values may be obtained by assaying a statistically significant number of control samples (i.e. samples from subjects who do not suffer from an accelerated age- related disorder).
  • the reference (ii) according to the kits of the invention may be a control sample (for assaying).
  • the kit may comprise a positive control (preferably provided in a container), which corresponds to total RNA extracted from a sample (e.g. the plasma) of a subject having an accelerated age-related disorder (e.g. COPD) where it has been established that the miR-570-3p is present at statistically higher level than normal controls (i.e. a normal healthy subject without an accelerated age-related disorder, such as COPD).
  • the positive control miRNA may comprise a nucleotide sequence substantially as set out in SEQ ID No.i, or the complementary sequence thereof, or a variant, or fragment thereof.
  • the kit may comprise a negative control (preferably provided in a container), which corresponds to total RNA extracted from a sample (e.g. the plasma) of a normal, healthy subject without an accelerated age-related disorder (e.g. COPD).
  • a negative control preferably provided in a container
  • a sample e.g. the plasma
  • an accelerated age-related disorder e.g. COPD
  • the kit may comprise the reference, a positive control and a negative control.
  • the kit will also comprise further controls, as necessary, such as “spike-in” controls to provide a reference for concentration, and further positive controls for the“signature” micro RNA, preferably miR-570-3p.
  • the blood plasma concentration of the signature miRNA i.e. miR-570-3p
  • the concentration of this signature miRNA in a patient with an age-related disorder may be at least 5-, 10, 15- or 20-fold higher .
  • the concentration of miR-570-3p in the test individuals was statistically more than the reference concentration (as calculated using the method described in the Example). This may be referred to herein as the‘increased’
  • Methods described herein may comprise an initial step of selecting the test subject and / or selecting the control subject(s).
  • the skilled technician will appreciate how to measure the concentrations of miR-570-3p in a statistically significant number of control individuals, and the concentration of miR-570-3p in the test subject, and then use these respective figures to determine whether the test subject has a statistically significant increase in that miRNA concentration, and therefore infer whether that subject is suffering from an accelerated age-related disorder. Accordingly, the inventors have realised that the difference in concentrations of miRNAs between the normal (i.e. control) and increased levels, can be used as a physiological marker, suggestive of the presence of an age-related disorder in the test subject.
  • a subject has an increased concentration of miR- 570-3p which is considerably higher than the‘normal’ concentration of that miRNA in the reference, control value, then they would be at a higher risk of having an accelerated age-related disorder, or a condition that was more advanced, than if the concentration of that miRNA was only marginally higher than the‘normal’ concentration.
  • the increase in concentration of miRNA compared to the‘normal’ concentration maybe at least 5-, 10, 15- or 20-fold higher than the‘normal’ or reference concentration.
  • Such changes in miRNA concentration infer that the test subject is suffering from an accelerated age-related disorder. Accordingly, a clinician would be able to make a decision as to the preferred course of treatment required, for example the type and dosage of the therapeutic agent administrated in the treatment methods of the invention.
  • corresponding concentration in the reference is indicative of the efficacy of treating the subject’s accelerated age-related disorder with the therapeutic agent, for example metformin or rapamycin.
  • the difference may be an increase or a decrease in the concentration of the one or more type of microRNA molecules in the bodily sample compared to the reference value. In embodiments where the concentration of the microRNA molecule in the bodily sample is lower than the corresponding
  • concentration in the reference then this would indicate that the therapeutic agent is successfully treating the accelerated age-related disorder in the test subject. Conversely, where the concentration of the microRNA molecule in the bodily sample is higher than the corresponding concentration in the reference, then this would indicate that the therapeutic agent is not successfully treating the accelerated age-related disorder.
  • an inhibitor of miR-570-3p for use in therapy.
  • an inhibitor of miR-570-3p for use in treating, preventing or ameliorating an accelerated age-related disorder.
  • a ninth aspect there is provided a method of treating, preventing or ameliorating an accelerated age-related disorder, the method comprising
  • the inhibitor of miR-570-3p may comprise an oligonucleotide configured to prevent other molecules from binding to the desired site on the miR-570-3p micro RNA.
  • the inhibitor is preferably a nucleic acid molecule, which may be double-stranded, but which is preferably single-stranded.
  • the inhibitor is a nucleic acid molecule that is complementary to miR-570-3p, or a variant or fragment thereof. More preferably, the inhibitor is an RNA molecule that is complementary to miR-570-3p, or a variant or fragment thereof.
  • the inhibitor of miR-570-3p comprises an miR- 570-3P antagomir (also known as anti-miR or blockmir).
  • the nucleic acid sequence which is complementary to miR-570-3p i.e. SEQ ID No:i
  • SEQ ID No:i is 22 nucleotides long, and is referred to here as SEQ ID No: 2, as follows:
  • the inhibitor of miR-570-3p comprises or consists of a nucleotide sequence substantially as set out in SEQ ID No.2, or a variant or fragment thereof.
  • Variants and fragments of the inhibitor may include truncations or additions of nucleotides from/to the nucleotide sequence of SEQ ID No: 2.
  • a truncation may comprise the nucleotide sequence of SEQ ID No:2 having been reduced in size by the removal of at least one nucleotide from the 5’ and/or 3’ end of the sequence of SEQ ID NO:2, or by deletion of one of more nucleotides from within the core or centre of the sequence of SEQ ID No:2.
  • the truncation may comprise deletion of at least 2, 3, 4 or 5 nucleotides from the sequence of SEQ ID No:2.
  • An addition may comprise the sequence of SEQ ID No: 2 having been increased in size by the addition of at least one nucleotide to the 5’ and/or 3’ end of the sequence of SEQ ID No: 2, or by the introduction of one of more nucleotides into the core or centre of the nucleotide sequence of SEQ ID NO: 2.
  • the addition may comprise addition of at least 2, 3, 4, 5, or up to 10 nucleotides to the nucleotide sequence of SEQ ID No: 2.
  • the inhibitor may comprise one or more modification configured to render it more resistant to degradation.
  • a suitable modification may include the addition of a 2’-methoxy group and/or phosphorothioate.
  • the inhibitor of miR-570-3p i.e. which is referred to hereinafter as“agent” or“active agent”
  • the inhibitor of miR-570-3p maybe used in a medicament which maybe used in a monotherapy i.e. the (miR-570-3p inhibitor is administered on its own), or as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing an accelerated age-related disorder, such as metformin and rapamycin.
  • the miR-570-3p inhibitor according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid etc. or any other suitable form that may be administered to a person or animal in need of treatment.
  • the vehicle of medicaments according to the invention should be one which is well- tolerated by the subject to whom it is given.
  • Medicaments comprising the agents according to the invention (i.e. the miR-570- 3p inhibitor) may be used in a number of ways.
  • oral administration may be required, in which case the miR-570-3p inhibitor may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid.
  • Preferred compositions comprising the miR-570-3p inhibitor may be administered by inhalation (e.g. by a nebulizer).
  • Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.
  • the miR-570-3p inhibitor according to the invention may also be incorporated within a slow- or delayed-release device.
  • Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
  • the device may be located at least adjacent the treatment site.
  • Such devices may be particularly advantageous when long-term treatment with the miR- 570-3P inhibitor used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
  • the miR-570-3p inhibitor and compositions according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections maybe intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).
  • the amount of the miR-570-3p inhibitor that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the miR-570-3p inhibitor, and whether it is being used as a monotherapy, or in a combined therapy.
  • the frequency of administration will also be influenced by the half-life of the miR-
  • administered may be determined by those skilled in the art, and will vary with the particular miR-570-3p inhibitor in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease.
  • a daily dose of between o.oiug/kg of body weight and 500mg/kg of body weight of the miR-570-3p inhibitor according to the invention may be used. More preferably, the daily dose is between o.oimg/kg of body weight and 400mg/kg of body weight, and more preferably between o.img/kg and 200mg/kg body weight.
  • the miR-570-3p inhibitor maybe administered before, during the or after the onset of disease.
  • the miR-570-3p inhibitor may be administered immediately after a subject has developed a disease.
  • Daily doses maybe given systemically as a single administration (e.g. a single daily injection).
  • the miR-570-3p inhibitor may require administration twice or more times during a day.
  • miR-570-3p inhibitor maybe administered as two (or more depending upon the severity of the disease being treated) daily doses of between 25mg and 7000 mg (i.e. assuming a body weight of 70 kg).
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
  • a slow release device maybe used to provide optimal doses of miR-570-3p inhibitor according to the invention to a patient without the need to administer repeated doses.
  • Known procedures such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations comprising the miR-570-3p inhibitor according to the invention and precise therapeutic regimes (such as daily doses of the miR-570-3p inhibitor and/or the frequency of administration).
  • a pharmaceutical composition comprising a miR-570-3p inhibitor, and a pharmaceutically acceptable vehicle.
  • the composition can be used in the therapeutic amelioration, prevention or treatment of any accelerated age-related disease.
  • the accelerated age-related disease may be selected from the group consisting of: accelerated lung aging, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cardiovascular disease, ischemic heart disease, cardiac failure, diabetes, Type 2 diabetes, and renal disease.
  • COPD chronic obstructive pulmonary disease
  • IPF idiopathic pulmonary fibrosis
  • cardiovascular disease ischemic heart disease
  • cardiac failure ischemic heart disease
  • Type 2 diabetes Type 2 diabetes
  • renal disease Most preferably, the accelerated age-related disorder is COPD or IPF.
  • the invention also provides, in an eleventh aspect, a process for making the
  • composition according to the tenth aspect, the process comprising contacting a therapeutically effective amount of a miR-570-3p inhibitor and a pharmaceutically acceptable vehicle.
  • A“subject” maybe a vertebrate, mammal, or domestic animal.
  • agents, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or maybe used in other mammals.
  • A“therapeutically effective amount” of a miR-570-3p inhibitor is any amount which, when administered to a subject, is the amount of drug that is needed to treat the target disease, or produce the desired effect, e.g. results in the lowering of the concentration of miR-570-3p in the subject, and increases sirtuin-i levels.
  • the therapeutically effective amount of the miR-570-3p inhibitor may be from about o.oi mg to about 8oo mg, and preferably from about o.oi mg to about 500 mg.
  • A“pharmaceutically acceptable vehicle” as referred to herein, is any known
  • the pharmaceutically acceptable vehicle maybe a solid, and the composition may be in the form of a powder or tablet.
  • a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
  • the vehicle may also be an encapsulating material.
  • the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
  • the miR-570-3p inhibitor may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain up to 99% of the active agents.
  • suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
  • the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
  • Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the miR-570-3p inhibitor may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo- regulators.
  • suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo- regulators.
  • suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g.
  • the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
  • the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
  • the miR-570-3p inhibitor maybe prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • the miR-570-3p inhibitor and pharmaceutical compositions of the invention maybe administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • the miR-570-3p inhibitor according to the invention can also be administered orally either in liquid or solid composition form.
  • compositions such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • nucleic acid or variant, derivative or analogue thereof which comprises substantially the nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
  • the terms“substantially the nucleotide sequence”, “functional variant” and“functional fragment”, can be a sequence that has at least 40% sequence identity with the nucleotide sequences of any one of the sequences referred to herein, for example 40% identity with the nucleotide identified as SEQ ID No:i (i.e. miR-570-3p) or SEQ ID No:2 (i.e. the complementary sequence of SEQ ID No:i).
  • nucleotide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • the nucleotide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
  • the skilled technician will appreciate howto calculate the percentage identity between two nucleotide sequences.
  • an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.
  • the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
  • the pair-score matrix e.g. BLOSUM62, PAM250, Gonnet etc.
  • gap-penalty e.g. functional form and constants.
  • percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
  • calculation of percentage identities between two nucleotide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
  • a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No’s: 1-15, or their complements under stringent conditions.
  • stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ 0.1%
  • a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences described herein.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
  • Figure l shows miRNA-570 directly binds the 3’UTR of sirtuin-i and is elevated in patients with COPD.
  • A. Dual luciferase reporter assays using vectors encoding sirtuin-i target site in the 3'-UTR or control, conducted in BEAS2B cell co-transfected with either a miR-570-3p mimic or mimic control.
  • miRNA and RNA were extracted and changes in gene expression assessed by qRT-PCR normalized to GNB2L1 or RNU48. Differences in the gene expression of B. miRNA-570-3p and C.
  • sirtuin-i Changes in the protein expression of sirtuin-i after over-expression of miR-570-3p mimic.
  • Data are means ⁇ SEM and analyzed by a Mann-Whitney U test, unpaired or paired Student t-test or Kruskal-Wallis test with post-hoc Dunn’s test; * P ⁇ 0.05, **P ⁇ o.oi, ***P ⁇ o.ooi;
  • Figure 2 shows airway epithelial cells from COPD patients show cellular senescence.
  • SAEC Small airway epithelial cells
  • C) Effect of PI3KCX (100 nM) siRNA mediated knock-down on miR-570-3p expression in BEAS2B cells (n 3).
  • D. P38 MAPK inhibitor VX745 (100 nM) effect on miR-570-3p gene expression in BEAS2B cells treated with or without H 2 0 2 (n 6).
  • the band density of each blot is represented as a histogram and is the average of all experiments performed.
  • Data are means ⁇ SEM, analyzed by Kruskal-Wallis test with post hoc Dunn’s test, Mann-Whitney U test, unpaired or paired Student t-test and one- way ANOVA with post-hoc Bonferroni correction; * P ⁇ 0.05, ** P ⁇ o.oi, *** P ⁇ o.ooi;
  • Figure 4 shows inhibition of miRNA-570 expression in airway epithelial cells alters cellular growth and SASP release.
  • E Effect of miRNA-570-3p antagomir or oligonucleotide control, in COPD SAECs stained with propidium
  • MMP-2 and MMP-9 expression measured in supernatants from SAEC COPD cells treated with miRNA-570-3p antagomir or oligonucleotide control.
  • Data are means ⁇ SEM, analyzed by unpaired or paired Student t-test and two-way ANOVA with post-hoc Bonferroni correction; * P ⁇ 0.05, ** P ⁇ o.oi, *** P ⁇ o.ooi;
  • Figure 5 shows changes in miRNA and gene expression in IPF patients.
  • Figure 6 is a plasmid map of the backbone of one embodiment of the reporter plasmid used;
  • Figure 7 is a plasmid map showing the positioning and how the SIRTi 3’ UTR was cloned into the reporter plasmid shown in Figure 6;
  • FIG. 8 shows that miR-570-3p acts independently to miR-34a.
  • BEAS2BS were co- transfected with a control and SIRTi luciferase reporter, along with either a miR-570- 3p mimic +/- a miR-34a mimic or mimic control for 24 hours and relative luciferase activity detected.
  • Data are means ⁇ SEM and analyzed by One way Anova post hoc Bonferroni. * P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001;
  • FIG. 9 shows that miR-570-3p mimics modulate cell cycle checkpoint inhibitors, SASP and antioxidant gene expression.
  • SAECs from 5 non-smokers were treated with miR-570-3p mimics for 48 hours.
  • RNA was extracted and changes in gene expression of pi6, p2i, MMP-2, MMP-9, SOD2 and F0X03a were assessed by qRT-PCR using a TaqMan assays normalized to GNB2L1. Data are means ⁇ SEM and analyzed paired student t-test. * P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001;
  • Figure 10 shows the correlation between disease severity and miR-570-3p expression. Correlation data between miR-570-3p expression in lung homogenate samples and FEV (% predicted) and FEV /FVC (% predicted). Data was analyzed by Spearman correlation test;
  • Figure li shows that PI3K signalling does not regulate miR-570-3p expression.
  • BEAS2B cells were treated with either AS-605240 and IC-87114 (10 mM) or vehicle
  • FIG 12 shows that P38 regulates p2i and SIRTi expression.
  • FIG 13 shows that silencing of c-Jun regulates p2i and SIRTi gene expression.
  • Figure 14 shows that over-expression of a miR-570-3p mimic reduces SIRTi expression.
  • BEAS2B cells were transfected with either a miR-570-3p mimic (15 nM) or control and then left untreated or treated for 48 hours with 100 pM H 2 0 2 .
  • Figure 15 shows that inhibition of miR-570-3p induces anti-oxidant gene expression.
  • SAECs from COPD patients were transfected with either a miR-570-3p antagomir (30 nM) or control for 48 hours and RNA extracted. Changes in the gene expression of F0X03a and SOD2 were examined; and Figure 16 shows that over-expression of miR-570-3p induces cytokine release.
  • SAECs from 3 non-smokers were treated with miR-570-3p mimics for 48 hours and supernatant collected, changes in CXCL-8 and IL-ib were detected by ELISA. Examples
  • Hydrogen peroxide H 2 0 2 was purchased from Sigma (Poole, UK).
  • the p38 MAPK inhibitor VX745 was purchased from Tocris Biosciences (Abingdon, UK) and the PI3K inhibitors; PIK75 hydrochloride (PI3KC1) was purchased from Abeam (Cambridge, UK), GSK2636771 (R ⁇ 3Kb), AS-605240 (Pl3Ky) and IC-87114 (R3 ⁇ 4Kd) were purchased from VWR International Ltd. (Lutterworth, UK).
  • COPD severity was staged according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) document 22 .
  • Lung tissues were obtained from an established tissue bank linked to an established patient registry which has previously been used 2 3.
  • 20 Subjects were matched for age and normal smokers and COPD patients for smoking history (Table 1).
  • mRNA and miRNAs were extracted using the miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
  • BEAS2B cells (ATCC Teddington, UK) were cultured in keratinocyte media (Invitrogen, Paisley, UK) containing human recombinant epithelial growth factor and bovine pituitary extracts.
  • Human primary SAEC were cultured as monolayers in LHC-9 media (Invitrogen) on collagen (1% w/v) coated plates.
  • Cells were extracted from lung tissue from patients undergoing lung resection surgery at the Royal Brompton Hospital (London, UK). The subjects were matched for age and smokers and COPD patients for smoking history (Table 2). All subjects gave informed written consent and the study was approved by the NRES London-Chelsea Research Ethics committee (study number 09/H0801/85). All methods were performed in accordance with the relevant guidelines and regulations.
  • BEAS2B cells were transfected with siRNA (c-Jun #6203 or PI3KCX #6359 (Cell Signaling Technology), Negative control #1 (Ambion Silencer Select siRNA,) ioohM) using Lipofectamine RNAimax for 24 hours prior to stimulation with H 2 0 2 .
  • RNA samples were then reverse transcribed and qPCR performed. See Table 3 for patient demographics.
  • COPD chronic obstructive pulmonary disease
  • FVC forced vital capacity
  • FEV t forced expiratory volume in one second
  • Data are expressed as mean value ⁇ standard deviation.
  • RNA and miRNAs were extracted using the miR easy kit (Qiagen) according to the manufacturer’s instructions. RNAs were then reverse-transcribed using the TaqMan normal RNA and MicroRNA Reverse Transcription Kit (Life Technologies). Both normal and miRNA levels were detected by either TaqMan Assays (SIRTi HS01009006, hTERT HS00972656, p2i HS00355782, pi6 HS00923894, p27 2 HSO1597988, CDK4 HSO0364847, MMP9 HSO0234579, MMP2 HSO1548727, Jun Hs
  • Luc-SIRTi 3'UTR was a gift from Charles
  • Protein extracts were prepared using RIPA buffer (Sigma; 150 mM NaCl, 1.0%
  • IGEPAL® CA-630 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0.
  • protease inhibitor cocktail (Roche, Welwyn Garden City, UK).
  • Protein extracts (40pg) were analyzed by SDS-PAGE (Invitrogen) and detected with Western Blot analysis by chemiluminescence (ECL Plus; GE Healthcare, Hatfield, UK). Protein expression levels were expressed relative to b-actin.
  • SAECs Passage 2-3 SAECs from COPD and non-smokers were plated into 24 well plates and left for 24 hours to adhere. Cells were then fixed and senescence-associated b- galactosidase (SA ⁇ -Gal) activity was determined according to the manufacturer's instructions (Abeam, ab0535i). iCELLigence cellular growth assay
  • iCELLigence System Roche, Mannheim, Germany. Cells were then treated with or with miR-570-3p inhibitors or control inhibitors and left for 48 hours; impedance was measured every hour for 48h.
  • the principle of the iCELLigence system is that cells grown on the surface of electrodes produce an increase in electrode impedance whose magnitude is determined by the number of cells and the degree of cell adhesion.
  • MMP2/9 enzyme activity was measured by gelatin zymography using Novex Zymogram Gelatin Gel (Invitrogen, Carlsbad, CA). 15 pl of supernatant was diluted with 5 m ⁇ amount of Novex Tris-Glycine SDS sample buffer (Invitrogen) and applied to each lane of the zymogram gel. After electrophoresis, the gels were incubated with Novex zymogram renatureing buffer (Invitrogen) for 30 minutes. After the gels were rinsed for 30 min in Novex zymogram developing buffer (Invitrogen), the gels were incubated in the developing buffer for 18 h at 37°C. After the incubation, the gels were stained with Colloidal Blue Staining Kit (Invitrogen).
  • CXCL8, IL-ib, TNF, CCL2 and IL-6 were quantified using commercially-available ELISA kits (R&D Systems Europe Ltd, UK) according to manufacturer’s instructions.
  • miR-570-3p (5’- CGAAAACAGCAAUUACCOUUGC-3’ - SEQ ID No:i), as a potential novel inhibitor of sirtuin-i.
  • M1R-570-3P has previously been described in asthma 25 , and has also been shown to have conflicting roles in regulating cellular growth 26 ’ 2 ?, but has not been studied in the context of senescence, sirtuin-i regulation or COPD.
  • MiR-570 is expressed only in primates so cannot be investigated in mice.
  • miR-570-3p was shown to directly regulate SIRTi, its expression in COPD lung homogenate samples was examined and found to be significantly up-regulated in both mild and severe disease compared to age-matched non-smokers and smokers without COPD (see Fig. lE).
  • the up-regulation of miR-570-3p correlated with decreased lung function as measured by the forced expiratory volume in 1 second (FEVO (% predicted) and the ratio of FEV to forced vital capacity (FVC), which is a measurement of airway obstruction (See Fig. 10).
  • the expression of miR-570-3p was also elevated in sputum (see Fig. lF), peripheral blood mononuclear cells (PBMC) (see Fig. lG) and SAEC (see Fig. lH) from COPD patients compared to controls.
  • PBMC peripheral blood mononuclear cells
  • SAEC see Fig. lH
  • COPD SAECs showed evidence of cellular senescence, with increased staining of senescence-associated b-galactosidase (SA- Gal) compared to age- and passage-matched non-smokers (see Fig. 2A).
  • Flow cytometry analysis of propidium iodide staining suggested COPD SAEC were mainly within Gi arrest (see Fig. 2B), with elevated levels of the cell cycle checkpoint inhibitors (pi6, p2i and P27) (see Fig. 2C-E), and reduced levels of CDK4 and SIRTi (see Fig. 2F, 2G).
  • COPD SAECs also showed elevated levels of MMP-2 and MMP-9 at both the mRNA and protein level (see Fig. 2H-J).
  • Peripheral lung homogenates from COPD patients displayed a similar phenotype with elevated mRNA levels of MMP-9, IL-8 and p2i, and reduced expression of CDK4 and SIRTi (see Fig. 2K-O). These data confirm that the lung displays a cellular senescence phenotype when compared to age-matched non- smokers.
  • oxidative stress is known to induce cellular senescence
  • the inventors then investigated whether increased oxidative stress elevated miR-570-3p.
  • Treatment of a bronchial epithelial cell line (BEAS2B) with increasing levels of hydrogen peroxide as a surrogate of oxidative stress induced a significant increase in miR-570-3p (see Fig. 3A).
  • miR-34a another senescence modulating miRNA, is induced by oxidative stress in a Pl3Ka-dependent manner 19 .
  • PI3KC1 inhibition using the pharmacological inhibitor PIK75, failed to reduce miR-570- 3p expression (see Fig.
  • NF-kB nuclear factor-kB
  • antagomir of miR-570-3p prevented oxidative stress-dependent reduction of sirtuin-i at the mRNA and protein levels (see Fig. 3F, 3G), whereas over- expression of miR-570-3p mimics caused a further reduction of sirtuin-i in both non- treated and H 2 0 2 -treated cells (See Fig. 14).
  • the miR-570-3p antagomir, a mirVanaTM miRNA Inhibitor has-miR-570-3p
  • MH12799 (30 or 60 nM) (Ambion Life Technologies, Foster City, CA) was transfected into cells using Lipofectamine RNAimax (Invitrogen, Paisley) for 48 h.
  • the sequence of the antagomir is SEQ ID No:2, i.e. complementary to SEQ ID No:i.
  • the inventors assessed that the over-expression of the antagomir did in fact reduce miR-570-3p expression in SAEC from COPD patients (see Fig. 3H), whilst also increasing the levels of SIRTi at the mRNA and protein levels (see Fig. 31, 3J).
  • a reduction in the mRNA levels of pi6 and p27 was observed, along with increased expression of CDK4 and a key component of telomerase, hTERT (see Fig. 3K, 3N), as well as F0X03a and SOD2 (See Fig. 15). All of these data suggest that the inhibition of miR-570-3p in COPD SAEC induces sirtuin-i expression, leading to sirtuin-i dependent inhibition of senescence markers and cellular rejuvenation.
  • Cell cycle arrest is a key phenotype of a senescent cell, with the cells remaining metabolically active but ceasing to divide 2 ’ 33 .
  • the inventors observed that SAEC from COPD patients had impaired cellular growth compared to non-smoking, passage- and age-matched controls (see Fig. 4A).
  • the growth of these cells increased, suggesting the prevention of SAECs from going into arrest (see Fig. 4B).
  • SAEC from non-smokers could be driven into Gi arrest by overexpressing a miR-570-3p mimic (see Fig.
  • miRNA-34a and miR-570-3p were examined in primary fibroblasts taken from IPF patients.
  • Preliminary data shown in Figure 5 suggest that both miRNAs were elevated in IPF patients, as well as a reduction in the gene expression of sirtuin-i and sirtuin-6.
  • These data suggest that miRNA-34a and miR-570-3p to be elevated in IPF patients, as well as these sirtuins suggesting a similar pattern of expression to that seen in COPD.
  • miR-570-3p is elevated in COPD patients and plays an important role in driving a senescent phenotype within the lung.
  • miR-570-3p is induced by oxidative stress via P38 MAPK-dependent AP-i mediated transcription, leading to the down-regulation of sirtuin-i in the lungs of COPD.
  • Up-regulation of miR-570-3p induces cycle arrest as well as increased pro- inflammatory cytokine and MMP expression.
  • MicroRNA 217 modulates endothelial cell senescence via silent information regulator 1. Circulation 120, 1524-1532 (2009).
  • Oxidative stress dependent microRNA-34a activation via PI3K alpha reduces the expression of sirtuin-1 and sirtuin-6 in epithelial cells. Scientific reports 6(2016).

Abstract

L'invention concerne de nouveaux marqueurs biologiques pour des maladies associées au vieillissement accéléré, tels que le vieillissement accéléré du poumon, la bronchopneumopathie chronique obstructive (BPCO), une cardiopathie ischémique et une maladie rénale, et en particulier l'utilisation de micro-ARN en tant que marqueurs de diagnostic et de pronostic dans des dosages pour détecter de tels troubles. L'invention s'étend à l'utilisation des nouveaux biomarqueurs en tant que cible thérapeutique pour traiter toute maladie associée à un vieillissement accéléré, et des méthodes de détermination de l'efficacité de traitement d'un trouble caractérisé par un vieillissement accéléré avec un agent thérapeutique, ainsi que des kits pour la réalisation des dosages et des méthodes. Les dosages sont qualitatifs et/ou quantitatifs, et peuvent être adaptés à un criblage à grande échelle et à des essais cliniques.
PCT/GB2019/050069 2018-01-12 2019-01-11 Micro arn en tant que marqueur d'une maladie qui peut être associée au vieillissement WO2019138237A1 (fr)

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Citations (3)

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WO2010129860A2 (fr) * 2009-05-08 2010-11-11 The Ohio State University Research Foundation Profil d'expression et ciblage de micro-arn dans un tissu pulmonaire atteint d'une bronchopneumopathie chronique obstructive (bpco) et leurs méthodes d'utilisation
EP2341145A1 (fr) * 2009-12-30 2011-07-06 febit holding GmbH Empreinte ARNm dans le diagnostic de maladies
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