Classifications

• • 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
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering N.A.
  • C12N2310/141—MicroRNAs, miRNAs
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2320/00—Applications; Uses
  • C12N2320/10—Applications; Uses in screening processes
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

• the present invention relates to the field of diagnostics.
• the invention relates to a method for determining whether a patient is insensitive to a treatment with aspirin for use in a platelet inhibition treatment.
• anti-platelet and “platelet inhibiting” shall mean any inhibition of platelet activation and/or platelet aggregation and/or platelet adhesion.
• Platelet activation, aggregation and/or adhesion are believed to play significant roles in the pathogenesis of many vaso-occlusive disorders such as unstable angina, acute myocardial infarction, reocclusion of vessels following balloon angioplasty, transient ischemic attacks and strokes.
• vaso-occlusive disorders such as unstable angina, acute myocardial infarction, reocclusion of vessels following balloon angioplasty, transient ischemic attacks and strokes.
• chemical agonists bind with certain binding sites on circulating platelets, causing the platelets to become activated.
• the types of blood vessel wall damage that can trigger platelet activation include perforation or injury to the vessel wall, progression of atherosclerotic plaque, the performance of some interventional procedure (e.g., angioplasty, atherectomy or stenting), which stretches the vessel wall or causes intimal tearing, or other causes.
• platelets When activated, platelets interact with fibrinogen, fibronectin and other clotting factors causing them to adhere to the affected blood vessel wall and to aggregate with one another and with other blood cells (e.g., leukocytes). This activation, adherence and aggregation of platelets leads to the formation of a thrombus or blood clot.
• Aspirin is the most commonly prescribed platelet inhibitor for secondary prevention after a cardiovascular event, but other thromboxane A2 inhibitors or a selective COX-1 inhibitors could also be used. It is known that long-term treatment with aspirin significantly reduces the risk of myocardial infarction (Ml), stroke and vascular death. However, 10 to 20% of treated patients develop recurrent vascular events. A relatively high incidence of recurrent events is due to insensitivity to aspirin treatment. Indeed, it is known that there are large inter-individual differences in aspirin response and in some individuals platelet function is not decreased by aspirin treatment.
• kits for diagnosing or monitoring insensitivity treatment with a thromboxane A2 inhibitor or a selective COX-1 inhibitor, in particular of aspirin are provided, and to use said miRNA levels in the diagnosis of insensitivity to treatment with a thromboxane A2 inhibitor or a selective COX-1 inhibitor.
• the invention is based on the surprising finding that in healthy individuals, lower expression of miR- 19b-l-5p after aspirin use is associated with the insensitivity of platelets for aspirin.
• the inventors demonstrated that in vivo miRNA expression in isolated platelets, and in particular the expression of miR-19b-l-5p, shows a heterogeneous response to medication use in healthy individuals.
• the inventors showed that lower miR-19b-l-5p expression after aspirin use is associated with platelet insensitivity to indomethacin in vitro.
• Other objects of the present invention are to provide a kit for diagnosing or monitoring insensitivity of a patient for the treatment with thromboxane A2 inhibitor or a selective COX-1 inhibitor based upon the miRNA levels according to the invention, and to use said miRNA levels in the diagnosis of insensitivity to treatment with thromboxane A2 inhibitor or a selective COX-1 inhibitor.
• the invention provides a method for determining whether a patient is insensitive to a treatment with a thromboxane A2 inhibitor or a selective COX-1 inhibitor for use in a platelet inhibition treatment comprising the steps of determining the expression level of one or more miRNA(s) selected from the group consisting of miR-19b-l-5p, miR-1271, and miR-1537-5p, in a sample comprising platelet derived nucleic acids; comparing said expression level with a reference level, and determining the aspirin insensitivity of said subject based on the information obtained in the previous step.
• upregulation of said miRNA is indicative for insensitivity to treatment with a a thromboxane A2 inhibitor or a selective COX-1 inhibitor.
• upregulation of said miRNA is indicative for insensitivity to treatment with said thromboxane A2 inhibitor or said selective COX-1 inhibitor.
• said thromboxane A2 inhibitor or a selective COX-1 inhibitor is selected from the group consisting of aspirin and indomethacin.
• the method according to the invention comprises the step of determining the expression level of said miRNA in a sample before, during and/or after treatment with said thromboxane A2 inhibitor or said selective COX-1 inhibitor.
• the method of the invention comprises the step of determining the expression level of said miRNA in a sample after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13 or 14 days after the first doses of said thromboxane A2 inhibitor or said selective COX-1 inhibitor treatment.
• said one or more miRNA(s) comprises miR-19b-l-5p.
• said sample comprises a platelet sample.
• said expression level of said miRNA is normalized using one or more reference miRNAs.
• said one or more reference miRNAs is selected from the group consisting of consisting of miR-151-3p, miR-28-5p, miR-331-3p, miR-29c, miR-148b-3p and miR-18a.
• the detecting said one or more miRNA(s) is performed by reverse amplification of said miRNA and real time detection of amplified products.
• the invention further provides a kit for diagnosing or monitoring aspirin insensitivity comprising a nucleic acid capable of hybridizing under stringent conditions with miR-19b-l-5p, miR-1271, and miR- 1537-5p.
• said kit further comprises one or more reference miRNAs selected from the group consisting of miR-151-3p, miR-28-5p, miR-331-3p, miR-29c, miR-148b-3p and miR-18a.
• Figure 1 shows the inter-individual heterogeneity of medication-induced changes in miRNA microarray expression.
• Figure 2 shows the correlation between the in vivo changes in miRNA expression after aspirin use and in vitro platelet aggregation after indomethacin incubation. Correlation between the change in A) miR-19b-l-5p expression, B) miR-1537-5p expression and C) miR-1271 expression (log fold change of normalised RT-PCR expression after and before medication use) and the sensitivity of the platelets to aspirin as measured by the reduction in platelet aggregation.
• Figure 3 shows that the percentage of serum TXB2 reduction after two weeks of aspirin therapy varied among individuals.
• the article “a” and “an” as used herein refers to one or to more than one (i.e., at least one) of the grammatical object of the article.
• an element means one element or more than one element.
• Thiboxane inhibitors include compounds that inhibit thromboxane synthase and compounds that inhibit, prevent or otherwise interfere with the binding of thromboxane to its receptor
• Thromboxane synthase inhibitors and thromboxane receptor antagonists can be identified using assays described in Tai, H.-H. Assay of thromboxane A synthase inhibitors. Methods in Enzymology Vol 86, 1982 pp. 110-113 and references contained within Hall, S. E. Thromboxane A2 Receptor Antagonists. Medicinal Research Reviews, 11, 503-579 (1991) and Coleman, R. A., Smith, W. L, Narumiya, S.
• the characteristics of the preferred thromboxane inhibitor should include suppression of thromboxane A2 formation (thromboxane synthase inhibitors) and/or blockade of thromboxane A2 and prostaglandin H2 on platelets and vessel wall (thromboxane receptor antagonists). The effects should block platelet activation and therefore platelet function. Thromboxane synthase inhibitors may also increase the synthesis of antiaggregatory prostaglandins including prostacyclin and prostaglandin D2.
• a second class is the tricyclic inhibitor class, which can be further divided into the sub-classes of tricyclic inhibitors with a central carbocyclic ring (examples include SC-57666, 1, and 2); those with a central monocyclic heterocyclic ring (examples include DuP 697, SC-58125, SC-58635, and 3, 4 and 5; and those with a central bicyclic heterocyclic ring (examples include 6, 7, 8, 9 and 10).
• Compounds 3, 4 and 5 are described in U.S. Patent No. 5,474,995.
• the third identified class can be referred to as those which are structurally modified NSAIDs, and includes 11a and structure 11 as example members.
• ASA aspirin
• ortho-acetylsalicylic acid and the pharmaceutically acceptable formulations thereof.
• aspirin insensitivity or "aspirin resistance” refers to an inability to effectively inhibit the biosynthesis of thromboxane A2 after taking a thromboxane A2 inhibitor or a selective COX-1 inhibitor, most preferably aspirin by standard antiplatelet doses of 75-300 mg/day. It is believed that as a result thereof, aspirin loses its protective effect on cardiovascular and cerebrovascular system. In the majority of patients, aspirin can reduce the risk of cardiovascular and cerebrovascular diseases by 25%. However, for patients with aspirin resistance, treatment of cardiovascular and cerebrovascular diseases with aspirin cannot prevent them from the cardiovascular and cerebrovascular events, but instead, can increase the risk of myocardial infarction and stroke.
• determining the level of a certain mi NAs in a sample means assaying a test sample, e.g. a platelet sample from a patient, in vitro to determine the concentration or amount of the miRNAs in the sample. Any convenient qualitative, semi-quantitative or, preferably, quantitative detection method for determining nucleic acids can be used to determine the concentration or amount of the miRNAs in the sample. A variety of methods for determining nucleic acids are well known to those of skill in the art, e.g. determination by nucleic acid hybridization and/or nucleic acid amplification. Exemplary methods to determine the concentration or amount of the miRNAs in the sample are provided below.
• sample comprising platelet derived nucleic acids refers to any type of biological sample from a patient which comprises platelet derived nucleic acids, preferably RNA, in a detectable amount.
• miRNAs are small non-coding RNAs (17-24 nucleotides) that regulate gene expression by binding to partly complementary sequences in messenger RNA transcripts (mRNAs) thereby preventing the mRNAs from being translated into protein. Due to their function as regulators of gene expression they play a critical role in fundamental biological processes, including hematopoietic differentiation, cell cycle regulation, metabolism, cardiovascular biology, and immune function, and have been suggested to be involved in pathological processes. It has been found that the expression level (or expression pattern) of miRNAs varies over time and between tissues/cells.
• miR-19b-l-5p, miR-1271, miR-1537-5p, miR-1280, miR-1260a, miR-718, miR-484, MiR- 130b-3p, miR-342-3p miR-151-3p, miR-28-5p, miR-331-3p, miR-29c-3p, miR-148b-3p, miR-18a-5p refer to the miRNAs as retrieved in miRBase version 21. Exemplary sequences of the miRNAs are listed in Table 1.
• miR-1271 CUUGGCACCUAGCAAGCACUCA
• miR-151-3p CUAGACUGAAGCUCCUUGAGG
• miR-331-3p GCCCCUGGGCCUAUCCUAGAA
• miR-29c UAGCACCAUUUGAAAUCGGUUA
• miR-1225-3p UGAGCCCCUGUGCCGCCCCCAG 16.
• miR-587 UUUCCAUAGGUGAUGAGUCAC
• miR-718 CUUCCGCCCCGCCGGGCGUCG
• miR-484 UCAGGCUCAGUCCCCUCCCGAU
• miR-342-3p UCUCACACAGAAAUCGCACCCGU
• RNA may be extracted from the sample prior to miRNA processing for detection.
• RNA may be purified using a variety of standard procedures as described, for example, in RNA Methodologies, A laboratory guide for isolation and characterization, 2nd edition; 1998, Robert E. Farrell, Jr., Ed., Academic Press.
• miRNeasyTM kit Qiagen
• MagMAXTM kit Life Technologies
• Pure LinkTM kit Life Technologies
• mirVANATM miRNA Isolation Kit Ambion
• small molecular weight RNAs may be isolated by organic extraction followed by purification on a glass fiber filter.
• Alternative methods for isolating miRNAs include hybridization to magnetic beads.
• the determination of aspirin insensitivity is based on comparing the expression level(s) of the miRNAs in the patient's sample with those obtained using relevant controls, e.g. internal standards, samples of purified platelets from subjects known to be sensitive to aspirin treatment.
• relevant controls e.g. internal standards, samples of purified platelets from subjects known to be sensitive to aspirin treatment.
• the "control" may be test results obtained from the same patient at an earlier time, i.e., the patient may be examined for changes in microRNA levels before and after aspirin treatment.
• control i.e., control
• miRNAs or of miRNA ratios
• the step of determining whether a subject is sensitive or insensitive to aspirin treatment is based on the information obtained by comparison between the expression level with a reference level.
• the expression level(s) of the analysed miRNA(s) when statistically analysed will have a threshold whereby expression levels of the individual miRNAs below or above the threshold are indicative for respectively the presence or absence of aspirin insensitivity.
• Threshold miRNA levels for each of the analysed miRNAs can be determined by any suitable algorithm. Such an algorithm may involve classifying a sample between aspirin insensitive and aspirin sensitive groups.
• samples may be classified on the basis of threshold values, or based upon Mean and/or Median miRNA levels in aspirin insensitive patients versus aspirin sensitive (e.g., a cohort from the general population or a patient cohort with diseases unrelated to aspirin insensitivity).
• Mean and/or Median miRNA levels in aspirin insensitive patients versus aspirin sensitive (e.g., a cohort from the general population or a patient cohort with diseases unrelated to aspirin insensitivity).
• Various classification schemes are known for classifying samples between two or more groups, including Decision Trees, Logistic Regression, Principal Components Analysis, Naive Bayes model, Support Vector Machine model, and Nearest Neighbour model.
• the predictions from multiple models can be combined to generate an overall prediction.
• the miRNA expression level (miRNA signature, level, or miRNA concentration) is generated.
• RNA capture is determined from (in) the biological-sample using any of various methods known in the art for quantifying miRNA levels.
• methods include polymerase-based assays, such as Real-Time PCR (e.g., TaqmanTM), hybridization-based assays, for example using microarrays (e.g. miRNome microRNA Profilers QuantiMir Human PCR array (Biocat)), nucleic acid sequence based amplification (NASBA), flap endonuclease-based assays, as well as direct RNA capture with branched DNA
• polymerase-based assays such as Real-Time PCR (e.g., TaqmanTM)
• hybridization-based assays for example using microarrays (e.g. miRNome microRNA Profilers QuantiMir Human PCR array (Biocat)), nucleic acid sequence based amplification (NASBA), flap endonuclease-based assays, as well as direct RNA capture with branched DNA
• the assay format in addition to determining the miRNA levels will also allow for the control of, inter alia, intrinsic signal intensity variation.
• Such controls may include, for example, controls for background signal intensity and/or sample processing, and/or hybridization efficiency, as well as other desirable controls for quantifying miRNA levels across samples (e.g., collectively referred to as "controls").
• controls Many of the assay formats for amplifying and quantitating miRNA sequences, and thus for generating miRNA levels are commercially available and/or have been described, e.g. in WO 2008/153692, WO 2010/139810, and WO 2011/163214, or references cited therein.
• the specific platelet-based miRNAs that are tested for in the present invention include hsa-miR-19b- l-5p (miRBAse, version 21), hsa-miR-1225-3p (miRBAse, version 21), hsa-miR-1271 (miRBAse, version 21), hsa-miR-1537-5p (miRBAse, version 21), hsa-miR-548e-3p (miRBAse, version 21), and hsa-miR- 587 (miRBAse, version 21).
• the designations provided are standard in the art and are associated with specific sequences that can be found at the microRNA registry (http://www.mirbase.org/).
• hsa Homo sapiens
• hsa-miR-19b-l-5p hsa-miR-1271
• hsa-miR-1537- 5p under the standard nomenclature system.
• miRNAs tested for are indicated as RNA sequences, it will be understood that, when referring to hybridizations or other assays,
• RNA sequences may be reverse transcribed and amplified using the polymerase chain reaction (PCR) in order to facilitate detection. In these cases, it will actually be DNA and not RNA that is directly quantitated. It will also be understood that the complement of the reverse transcribed DNA sequences can be analysed instead of the sequence itself.
• the term "complement” refers to an oligonucleotide that has an exactly complementary sequence, i.e. for each adenine there is a thymine, etc.
• assays may be performed for the miRNAs individually, it is generally preferable to assay several miRNAs or to compare the ratio of two or more of the miRNAs.
• the method of the invention can comprise differentiating between patients with aspirin insensitivity and patients who are sensitive to aspirin treatment, wherein any of the miRNAs of the invention up- regulated (increased concentration) in the biological sample from a patient compared to a normal control.
• the method of the invention can further comprise determining the expression level of hsa-miR-19b-l-5p in the sample from the patient.
• Said sample may be any sample from said patient comprising platelet RNA. Examples include for instance, platelet rich plasma, whole blood,
• said sample is a sample of isolated platelets or enriched for platelets.
• said sample comprises a high concentration of platelets, preferably more than 99% of all cell particles in said sample is a platelet, preferably more than 99.5%, 99.6% or 99.7% as determined by FACS analysis.
• the method of the invention can further comprise determining the level of one or more normalization control(s) in the sample.
• the sample can be spiked with the normalization control(s).
• the normalization control can be a non-endogenous RNA or miRNA, or a miRNA not expressed in the sample.
• the normalization control may be one or more exogenously added RNA(s) or miRNA(s) that are not naturally present in the biological sample, e.g. an RNA or miRNA from another organism, and/or one or more human miRNAs not expressed in the sample-sample undergoing analysis.
• said level of the miRNA of the invention is normalized using one or more reference miRNAs which are stably expressed in platelets.
• said one or more reference miRNAs is selected from the group consisting of miR-151-3p, miR-28-5p, miR-331-3p, miR-29c, miR-148b-3p and miR-18a-5p.
• the normalization may suitably be performed as described herein and as described in European patent application EP15166257.
• the invention further provides a kit for quantifying the amount of a target miRNA in a biological sample comprising an amplification primer set, comprising at least one primer comprising a sequence that is complementary to a portion of said first reference miRNA as defined above.
• said amplification primer set further comprises a sequence that is complementary to a portion of said second reference miRNA as defined above.
• the kit of the invention further comprises a second amplification primer set, wherein at least one primer comprises a sequence that is complementary to a portion of a target miRNA.
• the kit according to the invention further comprises a first probe comprising a sequence that is complementary to a portion of the target miRNA and a second probe comprising a sequence that is complementary to a portion of the reference miRNA, wherein the first and second probes are distinguishably detectable.
• the miRNA level (or miRNA concentration) is preferably determined by an amplification-and/or hybridization-based assay.
• the amplification- and/or hybridization-based assay can be quantitative miRNA real-time polymerase chain reaction (RT-PCR), e.g. TaqMan.
• RT-PCR quantitative miRNA real-time polymerase chain reaction
• the miRNA level may also be determined by preparing cDNA, followed by RT-PCR.
• the miRNA level may be determined with the use of a custom kit or array, e.g., to allow particularly for the profiling of the platelet-based miRNAs of the invention. Accordingly, the present invention further provides a kit (or test) for diagnosing or monitoring insensitivity for aspirin treatment based upon the miRNA levels in the biological samples as described herein.
• the kit for diagnosing or monitoring aspirin insensitivity of the invention may comprise means for determining the concentration (expression level) of miR-19b-l-5p, miR-1225-3p, miR-1271, miR- 1537-5p, miR-548e-3p, and miR-587; in a platelet sample from a subject.
• the means for determining the concentration of miR-19b-l-5p, miR-1225-3p, miR-1271, miR-1537-5p, miR-548e-3p, and miR- 587 can be oligonucleotide probes specific for miR-19b-l-5p, miR-1225-3p, miR-1271, miR-1537-5p, miR-548e-3p, and miR-587; or miRNA-specific primers for reverse transcribing or amplifying each of miR-19b-l-5p, miR-1225-3p, miR-1271, miR-1537-5p, miR-548e-3p, and miR-587.
• the means for determining the concentration of miR-19b-l-5p, miR-1225-3p, miR-1271, miR-1537-5p, miR-548e-3p, and miR-587 may be TaqMan probes specific for each miRNA of the kit.
• oligonucleotide probes specific for miR-19b-l-5p, miR-1225-3p, miR-1271, miR-1537- 5p, miR-548e-3p, and miR-587; or miRNA-specific primers for reverse transcribing or amplifying each of miR-19b-l-5p, miR-1225-3p, miR-1271, miR-1537-5p, miR-548e-3p, and miR-587 to detect their expression levels (concentrations) in accordance with suitable assay formats is well known to those of skill in the art, and appropriate probes and/or primers can be commercially purchased.
• the kit may comprise an enzyme for cDNA preparation (e.g. , reverse transcriptase) and/or PCR amplification (e.g., Taq polymerase), and/or a reagent for detecting and/or quantifying miRNA. Additionally, the kit may further comprise include a reagent for miRNA isolation from samples.
• the kit can also comprise one or more normalization control(s). The normalization control(s) can, for example, be provided as one or more separate reagent(s) for spiking samples or reactions.
• the normalization control(s) is/are selected from non-endogenous RNA or miRNA, or a miRNA not expressed in the sample.
• said kit comprises a specific primer for reverse transcribing or amplifying one or more reference miRNAs is selected from the group consisting of miR-151-3p, miR-28-5p, miR-148b-3p and miR-18a.
• aspirin is the most commonly prescribed platelet inhibitor after a cardiovascular event. Many patients, however, suffer from re-events that are thought to be due to platelet insensitivity to aspirin. The aim of this study was to identify a biomarker which could be used as a suitable marker for aspirin insensitivity.
• Microarray cohort For the miRNA microarray experiments the inventors recruited 15 healthy Caucasian male volunteers. This group was part of a previously reported study (Sondermeijer BM et al. , PLoS One [Internet]. 2011 [cited 2013 Aug 12];6:e25946.), in which healthy controls were matched to patients with coronary artery disease (CAD). The original cohort consisted of 40 healthy controls, of which 24 individuals completed the medication regimen described below. Of these, 15 individuals additionally completed the platelet aggregation assays and were therefore included in the microarray cohort. Healthy volunteers were eligible for participation if they were between the age of 35 and 65 years, did not have a personal or family history of cardiovascular disease (CVD) and did not use any medication.
• CVD cardiovascular disease
• the inventors administered 100 mg of acetyl salicylic acid, once daily, for two weeks. Since this cohort was intended as a control group for a group of subjects with CAD, all subjects were also asked to use statins.
• the inventors administered simvastatin 40 mg, once daily, for 6 weeks, of which the last 2 weeks in combination with the administration of acetyl salicylic acid. Blood samples including isolated platelets were collected at baseline in the absence of aspirin and statins and after six weeks of medication use.
• the PC cohort consisted of the 15 healthy volunteers from the microarray cohort and 10 additional healthy volunteers. Additional participants were selected in a similar manner to the controls in the microarray cohort, using identical inclusion and exclusion criteria. The subjects were also treated with simvastatin 40 mg, once daily, for 6 weeks, of which the last 2 weeks in
• Venous blood samples were drawn without stasis, using an open system with a 19-gauge needle.
• Blood samples for platelet isolation were collected in trisodium citrate (each 5 ml containing 0.5 ml 0.105M trisodium citrate). The first sample was discarded.
• Blood samples for platelet aggregation test were collected in citrate tubes.
• Samples for the serum thromboxane A2 assay were collected in glass serum tubes.
• Platelets were isolated as described previously (Sondermeijer BM et al. , PLoS One [Internet]. 2011 [cited 2013 Aug 12];6:e25946.). In short, immediately after withdrawal the samples were centrifuged (180g, 15 min, room temperature) to obtain platelet-rich plasma (PRP). The upper layer of PRP was transferred to a plastic tube to avoid leukocyte contamination. One part of acid-citrate-dextrose (ACD) buffer (0.085 M trisodium citrate, 0.11 M glucose, 0.071 M citric acid) was added to five parts of PRP and then the PRP was centrifuged (800 g, 20 min, room temperature).
• ACD acid-citrate-dextrose
• the platelet-poor plasma was discarded and the platelet pellet carefully resuspended in Tyrode buffer (136.9 mM NaCI, 2.61 mM KCI, 11.9 mM NaHC03, 5.55 mM Glucose, 2 mM EDTA, pH 6.5).
• the platelet suspension was centrifuged (800 g, 20 min, room temperature). The supernatant was discarded and the platelet pellet was resuspended in 50 ml sterile phosphate buffered saline (PBS) and stored at -80 Q C prior to RNA isolation.
• the isolated platelets were investigated by fluorescence-activated cell sorting (FACS) using monoclonal antibodies against CD45 (BD Biosciences), CD235a (DAKO) and CD61 (BD
• RNA isolation The inventors isolated platelet RNA using the mirVana PARIS kit (Ambion, Inc.), according to the manufacturer's protocol for liquid samples. The protocol was modified such that samples were extracted twice with an equal volume of acid-phenol chloroform.
• RNA 6000 Pico kit Agilent Technologies
• Small RNA kit Agilent Technologies
• RNA including microRNAs 100 ng of total RNA including microRNAs was dried down in a Centrivap concentrator (Labconco) and dissolved in 2 ⁇ RNase-free water.
• Sample labeling with Cy3 was performed as described in the miRNA Microarray System with miRNA Complete Labeling and Hyb Kit manual version 2.2 (Agilent Technologies) with the inclusion of spike-ins and the optional desalting step with spin columns (Micro Bio-Spin 6, Bio-Rad). Labeled samples were hybridized on Human 8x15k miRNA microarrays based on Sanger miRBase release 12.0 containing 866 human and 89 human viral miRNAs (G4470C, Agilent Technologies) at 55°C and 20 rpm for 20 hours.
• the arrays were scanned using the Agilent DNA microarray scanner (G2565CA, Agilent Technologies). Data was extracted with Feature Extraction software (vlO.7.3.1, Agilent Technologies) with the miRNA_107_Sep09 protocol for miRNA microarrays miRNA microarray pre-processing and analysis
• qPCR was performed with RNA of isolated platelets as previously described (Tijsen AJ et al., Ore Res [Internet]. 2010 [cited 2013 Jun 24];106:1035-9). A fixed volume of 8 ⁇ of total RNA was used as input in the reverse transcription reaction. Input RNA was reverse transcribed using the miScript reverse transcription kit (Qiagen) according to the manufacturer's protocol. The real-time qPCR was performed using High Resolution Melting Master (Roche). MgCI 2 was used in final concentration of 2.5mmol/L and 2 ⁇ of 8 times diluted cDNA was used in a total volume of 10 ⁇ . The forward primers had the same sequence as the mature miRNA sequence with all U's changed into T's.
• the reverse sequence was GAATCGAGCACCAGTTACGC (SEQ ID NO:21), which is complementary to the adapter sequence of the RT-primer used to create cDNA.
• qPCR reactions were performed on a LightCycler480 system II (Roche).
• the candidate miRNAs were normalized to the geometric mean of a previously established miRNA normalization panel for platelet samples consisting of miR-151-3p, miR-28-5p, miR-148b-3p and miR-18a. These normalization miRNAs were selected from independent microarray experiments and were further validated on PCR data using the geNorm and Normfinder algorithms.
• the original platelet normalization panel consists of 6 miRNAs selected by both algorithms.
• ADP Adenosine diphosphate
• Serum thromboxane B2 (TXB2) was measured at baseline and after 2 weeks aspirin use to check compliance to the therapy.
• TXB2 was measured in duplicate by an enzyme-linked immunosorbent assay (R&D Systems) according to the manufacturer's instructions. The inventors calculated the TBX2 concentration by performing a logistic four-parameter fit of the standard concentrations versus the ratio of absorbance of a particular sample to that of the maximum binding sample.
• Microarray cohort Clinical characteristics of the 15 healthy volunteers included in the microarray cohort are reported in Table 1. The results of the serum TBX2 assay showed that all subjects complied to aspirin therapy.
• PCR cohort This cohort consisted of the 15 subjects of the microarray cohort and 10 additional healthy volunteers recruited in the same manner as the controls in the microarray cohort (Table 1). Clinical characteristics of these 10 healthy volunteers did not differ from those of the microarray cohort. Detailed characteristics of the PCR cohort are listed in Table 1. Also for these 10 healthy volunteers, compliance to aspirin therapy was good, as shown by the serum TBX2 assay.
• the miRNA microarray experiment was performed on platelet RNA samples obtained at baseline and after 6 weeks medication use for all 15 subjects included in the microarray cohort. Each microarray contained 866 human miRNAs as annotated in miRBase 12.0. In total 468 miRNAs were detected in at least one platelet sample.
• Medication- induced changes in expression of each miRNA on the microarray were correlated with the reduction in platelet aggregation after incubation with indomethacin. Changes in expression of six miRNAs correlated strongly with the extent of platelet aggregation reduction (Table 2)
• the inventors were able to perform RT- qPCR on 3 out of 6 candidate miRNAs (miR-1271, miR- 1537-5p and miR-19b-l-5p).
• the expression levels of the other 3 miRNAs were below the detection limit of their PCR system.
• Diastolic blood pressure mmHg ⁇ SD 84 ⁇ 8 84 ⁇ 7
• Triglycerides mmol/L ⁇ SD 1.0 ⁇ 0.5 1.2 ⁇ 0.7
• Continuous data are expressed as mean ⁇ SD, categorical data as absolute number with
• BMI body mass index
• HDL high density lipoprotein
• LDL low density lipoprotein
• n number
• SD standard deviation
• serum TXB2 levels were analysed in samples taken at baseline and after two weeks of aspirin therapy. At these time points the samples for the miRNA expression analyses were also taken. The percentage of serum TXB2 reduction after two weeks of aspirin therapy varied among individuals, but all participants had at least a 30% reduction, indicating good compliance of all individuals.
• Non-fasting venous blood was drawn in CTAD citrate 5,4 ml tubes (Becton Dickinson, Alphen aan de Rijn, the Netherlands) and centrifuged for 10 minutes at 158 g at 20°C without brake to obtain platelet rich plasma (PRP).
• CTAD citrate 5,4 ml tubes Becton Dickinson, Alphen aan de Rijn, the Netherlands
• PRP platelet rich plasma
• MiR-19b-l-5p specific reverse transcription was performed on lOOng of purified total RNA, using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Gent, Belgium). RT-qPCR reactions were carried out in duplicate, on a LightCycler 480 system II (Roche, Basel,
• Table 3 NO values (expression) of miR-19b-l-5p in PRP samples per sample.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Biomedical Technology (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Analytical Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Plant Pathology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=53054891

Family Applications (1)

Country Status (2)

Families Citing this family (3)

Family Cites Families (27)

• 2016
  • 2016-05-04 EP EP16720828.9A patent/EP3292213A1/de not_active Withdrawn
  • 2016-05-04 WO PCT/EP2016/059991 patent/WO2016177776A1/en active Application Filing

Also Published As

Similar Documents

Legal Events