WO2013160474A2 - Expression de miarn dans les maladies hématologiques - Google Patents

Expression de miarn dans les maladies hématologiques Download PDF

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WO2013160474A2
WO2013160474A2 PCT/EP2013/058811 EP2013058811W WO2013160474A2 WO 2013160474 A2 WO2013160474 A2 WO 2013160474A2 EP 2013058811 W EP2013058811 W EP 2013058811W WO 2013160474 A2 WO2013160474 A2 WO 2013160474A2
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seq
variants
precursors
orthologues
combinations
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Pilar Giraldo Castellano
Marta ANDRÉS CORDURAS
Miguel Pocovi Mieras
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Instituto Aragonés De Ciencias De La Salud
Universidad De Zaragoza
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • 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
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    • 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
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to the field of molecular biology. Certain aspects of the invention include the application of microRNA (miRNA) molecules in diagnostics and/or prognostics of hematological diseases, preferably in myelodysplastic syndromes (MDS).
  • miRNA microRNA
  • MDSs myelodysplastic syndromes
  • PB peripheral blood
  • BM dysplastic-appearing bone marrow
  • the French- American-British (FAB) group proposed a classification system based on morphologic features of dysplasia, the presence of ringed sideroblasts, and the number of blasts and monocytes (Bennett et al., 1982). Their classification consists of 5 subcategories: refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-T) and chronic myelomonocytic leukemia (CMML).
  • RA refractory anemia
  • RARS refractory anemia with ringed sideroblasts
  • RAEB refractory anemia with excess blasts
  • RAEB-T chronic myelomonocytic leukemia
  • CMML chronic myelomonocytic leukemia
  • the World Health Organization comprises eight subtypes based on biological, genetic and morphological features.
  • RA refractory anemia
  • RCMD RA with multilineage dysplasia
  • RCMD-RS RA with multilineage dysplasia and ringed sideroblasts
  • RA with excess blasts- 1 RA with excess blasts-2
  • MDS unclassified and MDS associated with isolated deletion chromosome 5q del(5q)
  • the International Prognostic Scoring System which was proposed originally in 1997, is being used in parallel with increasing frequency because of its focus on risk-adapted therapeutic strategy based on karyotype, percentage of BM blasts, cytopenias, life expectancy and the risk of progression to acute myeloid leukemia (AML) (Greenberg et al., 1997).
  • AML acute myeloid leukemia
  • the basis for the selective outgrowth of MDS clones and the decrease in the normal hematopoietic stem/progenitor cells patients over time are unknown, probably due to imperfect interactions between hematopoietic progenitors and marrow stromal cells (Cazzola and Malcovati, 2005).
  • miRNAs are single stranded RNAs (usually 19-25 nt) processed from long hairpin-shaped transcripts precursors (primary miRNAs and pre-miRNAs) and evolutionarily conserved, which are transcribed from the miRNA gene.
  • miRNAs are known to regulate gene expression at the post-transcriptional level by complementariety-mediated binding to target mRNAs resulting in the repression of translation (Doench and Sharp, 2004) or in the cleavage of the target transcript (Mansfield et al., 2004).
  • Deregulation of miRNAs expression has been shown in cancer, where they are thought to act as oncogenes (oncomirs) or as tumor suppressors (Esquela-Kerscher and Slack, 2006).
  • oncomirs oncogenes
  • Esquela-Kerscher and Slack tumor suppressors
  • miRNAs are also involved in the tumour progression and in the metastatic process (Nicoloso et al., 2009).
  • a proportion of human miRNA genes is reported to be located in regions involved in cancer (Calin et al., 2004) and several examples of an association between disrupted expression of specific miRNAs and cancer have been shown in a variety of tissues (Calin et al., 2002; Michael et al., 2003; Metzler et al., 2004; Takamizawa et al., 2004).
  • miRNA expression profiles are now recognized to be more accurate and informative than the mRNA expression profiles, allowing the classification of poorly differentiated tumors that were not discriminated by mRNA profiles (Lu et al., 2005).
  • hematopoiesis-related miRNAs in the BM and PB of patients with MDS were studied (Pons et al., 2009). Twelve miRNAs were over-expressed in BM, six of which were also over-expressed in PB. Also they identified two miRNAs (miR-181a and miR-222) with progressively higher expression from controls to early-stage MDS to advanced MDS to post-MDS AML.
  • miRNAs have been related to MDS: For example, miR-337-5p, which might take part in leukemic transformation (Hussein et al., 2009), the myeloid cell differentiation is arrested by miR-125b-l in MDS and AML with the t(2;l I)(p21 ;q23) translocation (Bousquet et al., 2008) and there is a significant inverse correlation of miR- 150/MYB (myeloblastosis virus oncogene, a transcription factor) and miR-222/p27 (tumor suppressor) in MDS (Hussein et al., 2010b).
  • miR- 150/MYB myeloblastosis virus oncogene, a transcription factor
  • miR-222/p27 tumor suppressor
  • the present invention overcomes these problems in the art by identifying, in a sample obtained from a subject, preferably a sample of blood or plasma and more preferably a sample of peripheral blood or plasma, a miRNAs fingerprint involved in the diagnosis, prognosis and development of MDS. Moreover, the invention also discloses a fingerprint of miRNAs that allows the identification of the stage or progression disease and it might be used to develop an adequate therapeutic response in different patients.
  • the miRNAs disclosed in the present invention which are involved in the diagnosis/prognosis of MDS, are differentially expressed in subjects with MDS in comparison to healthy controls.
  • the present invention refers to a miRNA selected from the group comprising a sequence selected from SEQ ID Nos: 1-20 (Table 1) or variants, precursors, orthologues or combinations thereof for use in the diagnosis/prognosis of MDS.
  • miRNAs involved in the diagnosis/prognosis of MDS miRNAs Accesion number Sequence hsa-miR-26a-5p MIMAT0000082 SEQ ID NO:l hsa-miR-451 a MIMAT0001631 SEQ ID NO:2 hsa-miR-99b-5p MIMAT0000689 SEQ ID NO:3 hsa-miR-24-3p MIMAT0000080 SEQ ID NO:4 hsa-miR-625-3p MIMAT0004808 SEQ ID NO:5 hsa-miR-19b-3p MIMAT0000074 SEQ ID NO:6 hsa-let-7e-5p MIMAT0000066 SEQ ID NO:7 hsa-miR-140-3p MIMAT0004597 SEQ ID NO: 8 hsa-miR-361-3p MIMAT0004682 SEQ ID NO:9 hsa-miR-378a-3p MIMAT0000732 SEQ ID NO:
  • the expression levels of miRNAs disclosed herein have been analyzed by Real Time PCR in a sample obtained from a subject.
  • the sample can be selected from: saliva, blood, plasma, serum, bone marrow, or urine, more preferably, samples of saliva, blood, plasma, serum or bone marrow, and still preferably, samples of peripheral blood or plama.
  • the ability to identify miRNAs that are differentially expressed, over-expressed or under-expressed, in pathological samples compared to control samples can provide high-resolution and high- sensitivity datasets, which may be used in diagnostics, therapeutics, drug development, pharmacogenetics, biosensor development, and other related areas.
  • An expression profile generated by the methods disclosed herein may be a "fingerprint” or “signature” (terms used interchangeably throughout the present invention) of the state of the sample with respect to a number of miRNAs. While two states may have any particular miRNA similarly expressed, the evaluation of a number of miRNAs simultaneously allows the generation of a gene expression profile that is characteristic of the state of the sample. That is, normal sample obtained by healthy controls may be distinguished from samples obtained from MDS patients. By comparing expression profiles of samples in different disease states, information regarding which miRNAs are associated in each of these states of disease development may be obtained. Then, diagnosis may be performed or confirmed to determine whether a sample has the expression profile of normal or disease tissue. This may provide for molecular diagnosis of related conditions.
  • Hsa-miR-181d SEQ ID NO:27 Sokol L. et al. 2011 ; Pons A. et al. 2009
  • Hsa-miR-206 SEQ ID NO:30 Hussein K. et al. 2010a, Sokol L. et al. 2011 ;
  • Hsa-miR-665 SEQ ID NO:55 Dostalova M.M. et al. 2011
  • miRNA cluster refers to a set of miRNAs which are encoded in polycistronic transcripts.
  • the miRNAs hsa-let-7e-5p (SEQ ID No. 7) and has-miR-99b-5p (SEQ ID No: 3) are included in the same cluster (www.mirbase.org).
  • Table 3 Downregulated miRNAs in MDS known in the art. miRNAs Sequence Reference
  • Hsa-miR-1305 SEQ ID NO:81 Dostalova M.M. et al. 2011
  • Hsa-miR-583 SEQ ID NO: 82 Dostalova M.M. et al. 2011
  • Hsa-miR-661 SEQ ID NO:91 Hussein K. et al. 2010a
  • Hsa-miR-636 SEQ ID NO: 102
  • Hsa-miR-206 SEQ ID NO:30 Hussein K. et al. 2010a, Sokol L. et al.
  • the invention refers to the use of at least one of the miRNA or their nucleotide sequences disclosed in the present invention or variants, precursors, orthologues or combinations thereof for the manufacture of a kit for the diagnosis/prognosis of MDS.
  • the invention refers to a kit which comprises at least one probe that hybridizes with at least one of the miRNA or with their nucleotide sequence, disclosed in the present invention or variants, precursors, orthologues or combinations thereof.
  • the present invention refers to the use of the kit disclosed herein for the in vitro diagnosis/prognosis of MDS.
  • the invention refers to an in vitro method of diagnosis/prognosis of MDS which comprises determining in a sample from a subject, the expression level of at least one of the miRNAs disclosed herein or their nucleotide sequences, or variants, precursors, orthologues or combinations thereof and the comparison of said expression level with respect to the expression values obtained from healthy controls.
  • the miRNAs expression levels are analyzed in a sample of peripheral blood of subjects and the miRNAs expression levels of MDS patients are differentially expressed over the miRNAs expression levels of healthy controls.
  • the present invention also relates to an in vitro method of determining the expression level of a disease-associated miRNA, particularly MDS, comprising contacting a sample with a probe or kit of the invention and measuring the amount of hybridization.
  • the expression level of a disease-associated miRNA is information in a number of ways. For example, a differential expression of a disease-associated miRNA compared to a control may be used as a diagnostic that a patient suffers from the disease. Expression levels of a disease-associated miRNA may also be used to monitor the treatment and disease state of a patient. Furthermore, expression levels of the disease-associated miRNA may allow the screening of drug candidates for altering a particular expression profile or suppressing an expression profile associated with the disease.
  • the present invention also relates to a method of diagnosis comprising detecting a differential expression level of a disease-associated miRNA in a sample.
  • the sample may be derived from a patient. Diagnosis of a disease state in a patient allows prognosis and selection of therapeutic strategy.
  • sample refers to a sample of a body fluid, to a sample of isolated cells or to a sample from a tissue or an organ.
  • Samples of body fluids can be obtained by well-known techniques and include, preferably, samples of saliva, blood, plasma, serum, bone marrow, or urine, more preferably, samples of peripheral blood or plasma.
  • subject refers to an animal, preferably a mammal, most preferably a human being, including both young and old human beings of both sexes which may suffer from or are predisposed to a pathology.
  • the subject according to this aspect of the present invention may suffer from a pathology associated with abnormal expression of miRNAs, preferably MDS.
  • the terms “subject” and “patient” could be used interchangeably throughout the present invention.
  • differential expression or differentially expressed may mean qualitative or quantitative differences in the genes, proteins, peptides or polypeptides, specially, miRNAs, expression patterns within and among cells and/or tissue.
  • a differentially expressed gene, protein, peptides or polypeptides can qualitatively have its expression altered, including an activation-inactivation, or an under-expression-over-expression in, e.g., normal versus diseased subjects.
  • Genes, proteins, peptides or polypeptides, specially, miRNAs may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more states.
  • a qualitatively regulated gene or protein or peptide or polypeptide, specially, miRNA, will exhibit an expression pattern within a state or cell type which may be detectable by standard techniques.
  • the difference in expression may be quantitative, e.g., in that expression is modulated, either up-regulated, resulting in an increased amount of transcript (over-expression), or down-regulated, resulting in a decreased amount of transcript (under-expression).
  • the term "transcripts” as used throughout the present invention refers to a sequence of RNA produced by transcription from a DNA template. In this sense the expression "complementary DNA derived from a transcript” refers to DNA synthesized from a mature RNA template. In another sense, the expression “complementary RNA” (cRNA) refers to a synthetic RNA produced by transcription from a specific DNA single stranded template.
  • variant as used throughout the specification is to be understood to mean an nucleotide sequence of a miRNA or amino acid sequence of a protein or polypeptide, preferably, a miRNA that is altered by one or more nucleotides or amino acids, respectively.
  • the variant may have "conservative” changes, wherein a substituted nucleotide or amino acid has similar structural or chemical properties to the replaced nucleotide or amino acid.
  • a variant may also have "non-conservative” changes or a deletion and/or insertion of one or more nucleotides or amino acids.
  • the term also includes within its scope any insertions/deletions of nucleotides or amino acids for a particular miRNA or protein or polypeptide.
  • a "functional variant” will be understood to mean a variant that retains the functional capacity of a reference nucleotide sequence or a protein or polypeptide, specifically a miRNA.
  • complement may mean that each strand of double-stranded nucleic acids such as, DNA and RNA, is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds.
  • adenine (A) bases complement thymine (T) bases and vice versa
  • guanine (G) bases complement cytosine (C) bases and vice versa.
  • RNA it is the same except that adenine (A) bases complement uracil (U) bases instead of thymine (T) bases. Since there is only one complementary base for each of the bases found in DNA and in RNA, one can reconstruct a complementary strand for any single strand.
  • pri-miRNA refers to a reverse-complement DNA sequence that is transcribed into a single-stranded RNA molecule.
  • the miRNA sequence and its reverse-complement base pair to form a double stranded RNA hairpinloop; this pairing form a primary miRNA structure referred to as pri-miRNA.
  • pre-miRNA refers to the precursors to miRNA generated from pri-miRNA.
  • the sequence of the pre-miRNA may comprise a miRNA and/or a miRNA*.
  • the pre-miRNA may also comprise a miRNA or miRNA* and complements or variants thereof.
  • the sequence of pre-miRNA may comprise from 45-90, more preferably 60- 80 or still more preferably 60-70 nucleotides.
  • the sequence of the pre-miRNA may comprise any of the sequences included at the miRBase registry (http://microrna.sanger.ac.uk/sequences/), or variants thereof.
  • miRNA refers to a sequence of a miRNA, miRNA* or a variants thereof. miRNA and miRNA* are derived from the same precursor and miRNA* is the minor product of miRNA biogenesis.
  • miRNA* sequences may be found in libraries of cloned miRNAs but typically at lower frequency than the miRNAs.
  • the miRNA sequence may comprise from 13-33, more preferably 18-24 or still more preferably 21-23 nucleotides.
  • the sequence of the miRNA may comprise the sequence of a miRNA referred to in Table 1, or variants thereof, or any sequence of miRNAs included at the miRBase registry (http://microrna.sanger.ac.uk/sequences/).
  • sequences of the miRNAs comprise the sequences listed in Table 1, along with, further, any other sequences of miRNAs known in the prior art and which are used for the diagnosis/prognosis of MDS (see Tables 2 and 3) as the have a differential expression in MDS patients compared to control subjects.
  • An expression profile generated by the methods disclosed in the present invention may be a "signature/fingerprint" of the state of the sample with respect to a number of miRNAs. While two states may have any particular miRNA similarly expressed, the evaluation of a number of miRNAs simultaneously allows the generation of a gene expression profile that is characteristic of the state of the cell or patient in general. That is, normal tissue or sample may be distinguished from diseased tissue or sample. By comparing expression profiles of tissue or sample in known different disease states, information regarding which miRNAs are associated in each of these states may be obtained. Then, diagnosis may be performed or confirmed to determine whether a tissue sample has the expression profile of normal or disease tissue. This may provide for molecular diagnosis of related conditions.
  • the skilled artisan can make a diagnosis, a prognosis, or a prediction based on the findings. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis, and molecular profiling of the condition of the cells may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes. In this sense, the term prognosis describes the likely outcome of a disease.
  • kits comprising probes and/or primers that hybridize with at least one miRNA, or variants, precursors, orthologues or combinations thereof disclosed in the present invention together with any or all of the following: assay reagents, buffers and sterile saline or another pharmaceutically acceptable emulsion and suspension base.
  • the kits may include instructional materials containing directions (e.g., protocols) for the practice of the methods of this invention.
  • the present invention also relates to a method of identifying miRNAs that are associated with a disease or a pathological condition, preferably MDS, comprising contacting a sample taken from a subject with a probe or kit of the invention and detecting the amount of hybridization.
  • PCR may be used to amplify nucleic acids in the sample, which may provide higher sensitivity.
  • the present invention also provides orthologues of the above human miRNAs, which may be identified by any method known in the art.
  • a database of miRNAs is found at the miRBase registry (http ://microrna. Sanger, ac.uk/sequences/).
  • Figure 1 Analysis of miRNAs expression (median of LOGIO(RQ)) in plasma-MDS patients versus plasma-healthy controls.
  • Figure 2. Analysis of miRNAs expression (median of LOGIO(RQ)) in low, intermediate and high risk plasma-MDS patients versus plasma-healthy controls.
  • the Present invention relates to at least one of the miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof, for use in the diagnosis and/or prognosis of MDS.
  • the miRNAs are selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations, thereof.
  • the miRNAs are selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 18, 19, 20 or variants, or precursors, or orthologues, or combinations, thereof.
  • the miRNAs are selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.
  • the miRNAs disclosed in the present invention may be combined with the miRNAs disclosed in Tables 2 and/or 3 or variants, or precursors, or orthologues, or combinations, thereof, for use in the diagnosis and/or prognosis of MDS.
  • the miRNAs are selected from those consisting in a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof.
  • the miRNAs are selected from those consisting in a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations, thereof. In another particular embodiment of the invention, the miRNAs are selected from those consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 18, 19, 20 or variants, or precursors, or orthologues, or combinations, thereof.
  • the miRNAs are selected from those consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.
  • Present invention also relates a combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof, for use in the diagnosis and/ or prognosis of MD S .
  • the combination of miRNAs sequences consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations thereof.
  • the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 18, 19, 20 or variants, or precursors, or orthologues, or combinations thereof In another particular embodiment of the invention, the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14 or variants, or precursors, or orthologues, or combinations thereof.
  • the combination of miRNAs sequences disclosed above could be further combined with at least one of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 21-103
  • Another object of the present invention refers to miRNA selected from those comprising a sequence selected from SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof for use in the prognosis of MDS.
  • the miRNA is SEQ ID No. 3, which was significantly associated (p ⁇ 0.05) with progression to AML.
  • Another object of the present invention refers to a combination of miRNA sequences consisting in SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof, for use in the prognosis of MDS.
  • the miRNA is SEQ ID No. 3, which was significantly associated (p ⁇ 0.05) with progression to AML.
  • Present invention also relates to the use of at least one of miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof for the manufacture of a kit for the diagnosis and/or prognosis of MDS.
  • said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.
  • said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11,14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.
  • said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.
  • said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized in that at least one miRNAs selected from SEQ ID Nos: 1-15 or variants, or precursors, or orthologues, or combinations, thereof is further combined with those comprising a sequence selected from SEQ ID Nos: 21-103 (Tables 2 and 3) or variants, or precursors, or orthologues, or combinations, thereof.
  • said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group consisting in a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof.
  • said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group consisting in a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.
  • said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.
  • said use for manufacturing a kit for the diagnosis and/or prognosis of MDS is characterized by miRNAs are selected from the group consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.
  • Another object of the present invention refers to the use of a combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof, for the manufacture of a kit for the diagnosis and/or prognosis of MDS.
  • said use of a combination of miRNAs sequences further comprises the combination with those miRNAs comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations, thereof.
  • said use of a combination of miRNAs sequences is characterized by the combination consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations thereof.
  • said use of a combination of miRNAs sequences is characterized by the combination consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11 , 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations thereof.
  • said use of a combination of miRNAs sequences is characterized by the combination consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14 or variants, or precursors, or orthologues, or combinations thereof.
  • Another object of the present invention refers to the use of at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof for the manufacture of a kit for the prognosis of MDS.
  • the miRNA is SEQ ID No. 3, which was significantly associated (p ⁇ 0.05) with progression to AML.
  • Another object of the present invention refers to the use of a combination of miRNAs sequences consisting in SEQ ID Nos: 3, 7, 8, or 10, or variants, or precursors, or orthologues, or combinations, thereof, for the manufacture of a kit for the prognosis of MDS.
  • Present invention also relates a kit comprising at least one probe that hybridizes with at least one miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13- 20, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that further could be comprises at least one probe that hybridizes with at least one miRNA selected from those disclosed in Tables 2 and/or 3.
  • the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those consisting in a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof.
  • the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one of miRNAs selected from those consisting in a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that comprises at least one probe that hybridizes with at least one miRNA selected from those consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that the probe comprises a sequence that specifically hybridizes with at least one complementary DNA derived from the transcript of the corresponding miRNA selected from those comprising in a sequence selected from SEQ ID Nos: 1-103, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that the probe comprises a sequence that specifically hybridizes with at least one complementary DNA derived from the transcript of the corresponding miRNA selected from those consisting in a sequence selected from SEQ ID Nos: 21-103, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that the probe comprises a sequence that specifically hybridizes with at least one complementary RNA derived from the transcript of a specific DNA derived from the transcript of the corresponding miRNA selected from those comprising a sequence selected from SEQ ID No: 1-103, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit disclosed herein is characterized in that the probe comprises a sequence that specifically hybridizes with at least one complementary RNA derived from the transcript of a specific DNA derived from the transcript of the corresponding miRNA selected from those consisting in a sequence selected from SEQ ID No: 21-103, or variants, or precursors, or orthologues, or combinations, thereof.
  • Present invention also disclosed a kit comprising a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof.
  • the kit further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 21- 103 or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit comprises a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17 or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit comprises a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.
  • the kit comprises a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.
  • kits comprising at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 3, 7, 8 or 10, or variants, or precursors, or orthologues, or combinations thereof.
  • the kit further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos:21- 103 or variants, or precursors, or orthologues, or combinations thereof.
  • Present invention also disclosed a kit comprising a combination of probes that hybridizes with a combination of miRNAs sequences consisting in SEQ ID Nos: 3, 7, 8 or 10, or variants, or precursors, or orthologues, or combinations thereof
  • the kit further comprises at least one probe that hybridizes with at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos:21- 103 or variants, or precursors, or orthologues, or combinations, thereof.
  • Present invention also disclosed the use of the kit disclosed herein for the in vitro diagnosis and/or prognosis of MDS.
  • Present invention also disclosed an in vitro method of diagnosis and/or prognosis of MDS which comprises determining in a sample from a subject, the expression level of at least one miRNA selected from those comprising a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof and the comparison of said expression level with respect to the expression values obtained from healthy controls.
  • the in vitro method further comprises the determination of the expression level of at least one miRNAs selected from those disclosed in Table 2 and/or 3, or variants, or precursors, or orthologues, or combinations, thereof.
  • the in vitro method comprises the determination of the expression level of a group of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.
  • the in vitro method comprises the determination of the expression level of a group of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.
  • the in vitro method comprises the determination of the expression level of a group of miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.
  • the in vitro method comprises the determination of the expression level of a group of miRNAs consisting in a sequence selected from SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations, thereof.
  • the in vitro method comprises the determination of the expression level of a group of miRNAs consisting in a sequence selected from SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations, thereof.
  • the in vitro method comprises the determination of the expression level of a group of miRNAs consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations, thereof.
  • the in vitro method comprises the determination of the expression level of a group of miRNAs consisting in a sequence selected from SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations, thereof.
  • the in vitro method disclosed herein is characterized in that the sample of the subject is selected from: saliva, blood, plasma, serum, bone marrow, or urine, more preferably, samples of saliva, plasma and blood and still preferably, samples of peripheral blood and plasma.
  • an in vitro method of diagnosis and/or prognosis of MDS which comprises determining in a sample from a subject, the expression level of a combination of miRNA sequences consisting in SEQ ID Nos: 1-11, 13-20, or variants, or precursors, or orthologues, or combinations thereof and the comparison of said expression level with respect to the expression values obtained from healthy controls.
  • the in vitro method further comprises the determination of the expression level of at least one miRNAs selected from those comprising a sequence selected from SEQ ID Nos: 21-103 or variants, or precursors, or orthologues, or combinations thereof.
  • the in vitro method comprises the determination of the expression level of the combination of miRNAs sequences consisting in SEQ ID Nos: 1, 5, 6, 8, 9, 13, 15, 17, or variants, or precursors, or orthologues, or combinations thereof.
  • the in vitro method comprises the determination of the expression level of the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 11, 14, 16, 17, 18, 19, 20, or variants, or precursors, or orthologues, or combinations thereof.
  • the in vitro method comprises the determination of the expression level of the combination of miRNAs sequences consisting in SEQ ID Nos: 2, 3, 4, 7, 10, 14, or variants, or precursors, or orthologues, or combinations thereof.
  • the in vitro method according to the present invention is characterized by the miRNAs of subjects with MDS are differentially expressed with respect to the corresponding miRNA expression levels of healthy controls.
  • the miRNAs sequences SEQ ID No. 1, 3-7, 9-11, 13-18 and 20 are over-expressed with respect to the corresponding miRNA expression levels of healthy controls and the miRNAs sequence SEQ ID No. 2 is down-expressed with respect to the corresponding miRNA expression levels of healthy controls.
  • the in vitro method it is characterized in that the expression levels are obtained by Real Time-PCR.
  • the miRNA may be further analyzed by using microarrays, locked nucleic acid in situ hybridization, fluorescence in situ hybridization, and other RNA processing methods known in the art.
  • the RNA may be converted to complementary DNA by subjecting the mature miRNA to reverse transcription PCR.
  • the RNA may also be converted to cDNA by any methods known in the art. Non-limiting examples are readily available.
  • the expression profile may be standardized by background subtraction and normalization using a set of housekeeping genes before comparing the expression profile of the at least one miRNA to those obtained from control subjects. Once the RNA or complementary DNA has been processed, the RNA or DNA is analysed to determine the status of the patient. The method may also be used to determine whether a patient is at risk to develop MDS.
  • EXAMPLE 1 Identification of a miRNA profile/fingerprint in peripheral blood from MDS patients.
  • RNA extraction, reverse transcription and quantitative real-time PCR assay F : female; M: male; RA: Refractory Anemia; RARS: Refractory Anemia with ringed sideroblasts; RCMD: Refractory Cytopenia with multilineage dysplasia; RAEB: Refractory Anemia with excess blast; AML: Acute Myeloid Leukemia; CMML: Chronic myelomonocytic leukemia.
  • PAXgene Blood miRNA kit PreAnalytiX, Qiagen/BD Company, Switzerland
  • a screening to identify a miRNA signature/fingerprint associated with diagnosis/prognosis in MDS was determined by quantitative real-time PCR using MegaplexTM Primers Human Pool AV2.1 and B (P/N 4399966 and 4399968 respectively; Applied Biosystems, USA).
  • the kit contained assays for 754 miRNAs of the 1000 currently listed in the Sanger miRBase database (Griffiths-Jones et al., 2006). PCR reactions were performed using an ABI 7900 Fast (Sequence Detection System software version 2.4) (Applied Biosystems, USA).
  • these miRNAs disclosed in the present invention are biological markers for the diagnosis/prognosis of MDS. Additionally, a differential expression of a disease-associated miRNA, specifically MDS, compared to a control may be used as a diagnostic that a patient suffers from the disease. Expression levels of a disease-associated miRNA may also be used to monitor the treatment and disease state of a patient. Diagnosis of a disease state in a patient allows for prognosis and selection of therapeutic strategy.
  • the sequences of miRNAs known in the prior art can be combined together with the miRNAs of the invention, for a greater reliability in the diagnosis and prognosis of MDS.
  • Example 2 Validation and analysis of the miRNA expression profile/fingerprint in plasma from patients diagnosed with MDS.
  • Example 1 To demonstrate that the identified miRNA signature/fingerprint characteristic of 40 MDS 5 patients analyzed in Example 1 could be extrapolated to a wide MDS population, a total of 242 patients diagnosed with MDS were analyzed. In this sense, 192 plasma samples were derived from the project INBIOMED HEMA-001/2006 and 50 corresponding to patients diagnosed in the Haematology department of Miguel Servet University Hospital (2008-2011). Samples were deposited in the Aragon Biobank. recruited patients were informed of their 0 legal and ethical rights through Informed Consent (IC). Clinical characteristics of patients included in the present study are disclosed in Table 6. The control group included 40 healthy individuals.
  • PAZ-018 80 1 1 3 2 0 2
  • VAL-009 50 1 3 5 4 0 3
  • Gender 1 - Male; 2- Female.
  • FAB classification 1 - RA; 2- RARS; 3- RAEB; 4- RAEB-t; 5- CMML.
  • WHO 2008 Classification 1 - RCUD; 2- RARS; 3- RCMD; 5- RAEB-I; 6- RAEB-II; 10- 5q- syndrome; 1 1 - MDS unclassified; 12- Hypoplastic MDS; 13- CMML.
  • Plasma samples were collected in EDTA tubes (BD Vacutainer®). Total RNA was extracted from cryopreserved plasma samples using miRCURYTM RNA Isolation kit- Biofluids (Exiqon) according to manufacturer ' s instructions. Samples were reverse transcribed and 0 quantified by real-time PCR using miRCURY LNATM Universal RT microRNA PCR (Exiqon).
  • PCR reactions were performed using an ABi 7900HT (Sequence Detection System software version 2.4) (Applied Biosystems, USA). Normalization was performed with has-miR-16 (SEQ ID No. 12) because it had the lowest expression variability in the miRNA expression 5 patient dataset.
  • RQ Relative Quantity
  • the data obtained by the present example show that seventeen miRNAs were differentially expressed between plasma-MDS samples and plasma-healthy controls ( Figure 1). Sixteen miRNAs were over-expressed and only one was under-expressed ( Figure 1). As indicated above, the expression levels of a disease-associated miRNA may also be used to monitor the treatment and disease state of a patient. Diagnosis of a disease state in a patient allows for prognosis and selection of therapeutic strategy. In this sense, differences in miRNA levels in low, intermediate and high risk MDS patients were analysed. A non-parametric study (Kruskal-Wallis) was used. The data obtained show that four of these miRNAs were significantly associated with low, intermediate and high risk groups (p ⁇ 0.01) ( Figure 2).
  • hsa-let-7e-5p SEQ ID No. 7
  • hsa-miR-99b-5p SEQ ID No. 3 miRNAs are located in chromosome 19 and are included in the same cluster
  • the present invention analysed the progression to AML of patients with MDS by a non-parametric study (Mann- Whitney U). Only has-miR-99b-5p (SEQ ID No. 3) was significantly associated (p ⁇ 0.05) with progression to AML.
  • these extracellular miRNAs disclosed in the present invention are biological markers for the diagnosis/prognosis of MDS and progression to AML. Additionally, a differential expression of a disease-associated miRNA, specifically MDS, compared to a control may be used as a diagnostic that a patient suffers from the disease. Expression levels of a disease-associated miRNA may also be used to monitor the treatment and disease state of a patient. Diagnosis of a disease state in a patient allows for prognosis and selection of therapeutic strategy. Moreover, in the present invention, the sequences of miRNAs known in the prior art (Tables 2 and 3) can be combined together with the miRNAs of the invention, for a greater reliability in the diagnosis and prognosis of MDS.
  • MicroRNAs genomics, biogenesis, mechanism, and function. Cell.
  • Hussein K Busche G, Muth M, Gohring G, Kreipe H, Bock O. 2011. Expression of myelopoiesis-associated microRNA in bone marrow cells of atypical chronic myeloid leukaemia and chronic myelomonocytic leukaemia. Ann Hematol. 90: 307-313. Hussein, K., N. von Neuhoff, G. Busche, T. Buhr, H. Kreipe, and O. Bock. 2009. Opposite expression pattern of Src kinase Lyn in acute and chronic haematological malignancies. Ann Hematol. 88:1059-1067.
  • MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat Genet. 36:1079-1083.

Abstract

Les microARN (miARN) sont une classe de petits ARN endogènes qui sont des régulateurs négatifs de l'expression des gènes. Les miARN interviennent dans différents processus biologiques y compris l'hématopoïèse, laissant penser qu'ils sont associés au cancer. La présente invention concerne une signature/empreinte de miARN, exprimés différentiellement chez les sujets atteints de MDS relativement aux témoins sains, pour le diagnostic et le pronostic de MDS. De plus, la signature/empreinte des miARN décrite dans la présente invention est associée à des sous-types spécifiques de MDS, permettant l'identification du stade ou de la progression de la maladie, et elle pourrait être utilisée pour mettre au point une réponse thérapeutique adéquate chez différents patients.
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