WO2008068047A1 - Microarn ciblant ets1 - Google Patents

Microarn ciblant ets1 Download PDF

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WO2008068047A1
WO2008068047A1 PCT/EP2007/011074 EP2007011074W WO2008068047A1 WO 2008068047 A1 WO2008068047 A1 WO 2008068047A1 EP 2007011074 W EP2007011074 W EP 2007011074W WO 2008068047 A1 WO2008068047 A1 WO 2008068047A1
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mir
ets
use according
rna
antisense rna
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Cesare Peschle
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Istituto Superiore Di Sanita
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    • 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
    • C12N15/113—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
    • C12N15/1135—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 against oncogenes or tumor suppressor genes
    • 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
    • C12N2310/11—Antisense
    • 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.
    • 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
    • C12N2330/00—Production
    • C12N2330/10—Production naturally occurring

Definitions

  • the present invention relates to the use of micro RNAs in therapy.
  • Micro RNAs are a recently discovered class of small ( ⁇ 22nt) RNAs, which plays an important role in the negative regulation of gene expression by base- pairing to complementary sites on the target mRNAs (1).
  • MiRs first transcribed as long primary transcripts (pri-miRs), are processed in the nucleus by the RNase III enzyme Drosha to generate a 60-120 nucleotide precursor containing a stem-loop structure, known as pre-miR (2). This precursor, exported into the cytoplasm by the nuclear export factor Exportin-5 and the Ran-GTP cofactor, is finally cleaved by the RNase enzyme Dicer to release the mature miR (3).
  • MiRs mostly bind to the 3' untranslated regions (UTR) of their target mRNAs. This process, requiring only partial homology, leads to translational repression. Target mRNAs which are more stringently paired may be cleaved (4, 5).
  • miRs are phylogenetically conserved (6-9). Their expression pattern is often developmentally determined and/or tissue-specific, although some miRs are steadily expressed throughout the whole organism (10).
  • Growing evidence indicates that miRs are involved in basic biological processes, e.g.: cell proliferation and apoptosis (11,12); neural development and haematopoiesis (13); fat metabolism; stress response; and cancer (14-16), via the targeting of key functional mRNAs. Little is known of the functional role of miRs in mammals, and even less on the targets in mammals (13,16).
  • microRNAs are considered key regulators of cell growth and differentiation, little is known on their function in hematopoiesis.
  • Ets-1 a transcription factor up-regulated in megakaryopoiesis, and which transactivates relevant Mk genes.
  • Ets-1 is also implicated in cancer, especially in digestive tract cancers such as colon cancer, where it is thought to suppress tumourigenesis. Numerous cancers list changes in Ets-1 expression as a marker or controlling factor, for instance colon, lung and B cell lymphoma, the latter being particularly preferred.
  • the Ets-1 protein also has an important role in the production of platelets by activating various platelet factors such as PF4 and platelet integrin GPIIb (CD41), which is involved in cell adhesion and platelet aggregation.
  • MK cells are the progenitors to platelets.
  • miR can each inhibit or block translation or activity of Ets-1 mRNA.
  • the present invention provides the use of antisense RNA specific for Ets-1 in therapy, most preferably in the treatment or prophylaxis of cancers or tumours where it is desirable to repress Ets-1 expression.
  • cancers or tumours where it is desirable to repress Ets-1 expression.
  • These may include colon, lung and B cell lymphoma.
  • the antisense RNA is miR 155. It is also preferred that the antisense RNA is miR 221 or miR 222, or combinations of any of miR 155, miR 221 and miR
  • the present invention provides the use of Ets- 1 antagomirs or anti-miRs, inhibitors of antisense RNA specific for Ets-1, in therapy.
  • the miRs and/or anti-miRs of the present invention can be used in methods of blood cell replacement, especially MK cells and platelets, methods of replacing defective platelet production, particularly upon cancer chemotherapy, and in other types of piastropenia, or controlling excessive platelet production. They are also useful in methods for the treatment or prophylaxis of cancer, preferably digestive tract cancers, most preferably colon cancer, and in methods of modulating, preferably suppressing, tumourigenesis.
  • micro RNA capable of interacting with the 3 'untranslated region of Ets-1 protein mRNA is useful in treating Ets-1 -dependent tumours, and inhibitors therefor are useful in treating suppressed megakaryopoiesis in cancer patients or abnormal megakaryopoiesis.
  • Micro RNA and inhibitors therefore are also useful in in vivo and ex vivo expansion of megakaryocytes and platelets.
  • the 3' untranslated region (UTR) of human Ets-1 protein mRNA targeted by miR-155, -221 and -222 are provided as accompanying SEQ ID NOS. 4, 5 and 6 respectively.
  • Antisense RNA may be specific for any part of the 3' UTR of Ets-1 protein mRNA, and it will be appreciated that the 3' UTR may vary slightly from individual to individual.
  • miR need not be 100% faithful to the target, sense sequence. Indeed, where they are 100% faithful, this can lead to cleavage of the target mRNA through the formation of dsRNA. While the formation of dsRNA and cleavage of Ets-1 protein mRNA is included within the scope of the present invention, it is not a requirement that the antisense RNA be 100% faithful to the target sequence, provided that the antisense RNA is capable of binding the target 3' UTR to inhibit or prevent translation.
  • the antisense RNA of the present invention need only exhibit as little as 60% or less homology with the target region of the 3' UTR. More preferably, the antisense RNA exhibits greater homology than 60%, such as between 70 and 95%, and more preferably between 80 and 95%, such as around 90% homology. Homology of up to and including 100%, such as between 95 and 100%, is also provided. Suitable programs for assessing homology are well known in the art and include BLAST, for instance. The above also applies to inhibitors of the miRs (i.e. sense sequences or antagomirs).
  • the antisense RNA of the present invention may be as long as the 3' UTR, or even longer. However, it is generally preferred that the antisense RNA is no longer than 50 bases, and it may be a short as 10 bases, for example. More preferably, the antisense RNA of the present invention is between about 12 bases and 45 bases in length, and is more preferably between about 15 and 35 bases in length.
  • Preferred miRs are miR 155, miR 221 and miR 222. Combinations such as miR 155 and miR 221, miR 155 and miR 222, and miR 221 and miR 222 are also preferred.
  • SEQ. ID NO's 1, 2 and 3 Their mature sequences are shown hereinafter as SEQ. ID NO's 1, 2 and 3, and have a mature length of 23 or 24 bases. Thus, a particularly preferred length is between 20 and 25 bases, and especially 23 or 24.
  • the area of the 3' UTR to be targeted may be any that prevents or inhibits translation of the ORF, when associated with an antisense RNA of the invention.
  • the particularly preferred regions are those targeted by miR 155, miR 221 and miR 222, and targeting either of these regions with antisense RNA substantially reduces translation of Ets-1 protein.
  • Regions of the 3' UTR that it is preferred to target include the central region of the 3' UTR and regions between the central region and the ORF. Such regions which are proximal to the ORF are particularly preferred.
  • Ets-1 mRNA sequences such as the coding region for instance, may also be targeted.
  • the antisense RNA of the present invention is a short interfering RNA or a micro RNA.
  • the present invention further provides mutants and variants of these miRs.
  • a mutant may comprise at least one of a deletion, insertion, inversion or substitution, always provided that the resulting miR is capable of interacting with the 3' UTR to inhibit or prevent translation of the associated coding sequence. Enhanced homology with the 3' UTR is preferred.
  • a variant will generally be a naturally occurring mutant, and will normally comprise one or more substitutions.
  • miR-155 matches Ets-1 3'UTR in two different sites from 2504 to 2526 and from 4570 to 4592 nucleotides ( Figure 3A, top)
  • the miR-222 seed sequence matches with nucleotides 4980-5002 in Ets-1 3'UTR ( Figure 3B, top right) while the seed sequence in miR-221 matches the same sequence with lower complementarity ( Figure 3B, top left).
  • Particularly preferred stretches of the microRNA of the present invention correspond to the so-called “seed” sequences, i.e., short sequences matching specific regions of the 3'UTR of Ets-1 mRNA (SEQ ID NOS. 4, 5 and 6)
  • any sequence encompasses mutants and variants thereof, caused by substitutions, insertions or deletions, having levels of sequence homology (preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%, and most preferably at least 99.5% sequence homology), or corresponding sequences capable to hybridising to the reference sequence under highly stringent conditions (preferably 6x SSC).
  • the antisense RNAs of the present invention may be provided in any suitable form to the target site.
  • the target site may be in vivo, ex vivo, or in vitro, for example, and the only requirement of the antisense RNA is that it interacts with the target 3' UTR sufficiently to be able to inhibit or prevent translation of the Ets-1 ORF.
  • the antisense RNA may be provided directly, or a target cell may be transformed with a vector encoding the antisense RNA directly, or a precursor therefor.
  • Suitable precursors will be those that are processed to provide a mature miR, although it is not necessary that such precursors be transcribed as long primary transcripts, for example.
  • RNA is provided directly, then this may be provided in a stabilised form such as is available from Dharmacon (www.dharmacon.com. Boulder, CO, USA).
  • Dharmacon www.dharmacon.com. Boulder, CO, USA.
  • a large number of microRNAs are known from WO 2005/013901, the patent specification of which alone is over 400 pages. This publication discloses, in particular, the sequences of miR221, miR222. However, no specific function is provided therefor. Similarly, WO 2005/017145 also discloses at least one of the above mentioned miRNAs and provides it with a role in gene expression.
  • microRNAs are known, we are the first to establish that naturally-occurring RNA sequences, in particular miR 155, 221 and 222, or inhibitors thereof, are in fact capable of modulating the expression of Ets-1 protein.
  • the present invention does not extend to these compounds per se. However, the present invention extends to these and all other antisense RNAs provided by the present invention, for use in therapy and other processes.
  • the present invention provides the use of antisense RNA specific for all or part of the 3' untranslated region of Ets-1 protein mRNA in therapy.
  • antisense RNAs of the present invention may be used in the treatment of cancers or tumours, i.e, treatment with anti-miRs or antagomirs in tumors associated with deficient megakaryocyte (Mk) production, such as those treated with chemotherapy or hematopoietic stem cell transplantation.
  • Mk megakaryocyte
  • Solid, non-diffuse tumours may be targeted by direct injection of the tumour with a transforming vector, such as lentivirus, or adenovirus.
  • a transforming vector such as lentivirus, or adenovirus.
  • the virus or vector may be labelled, such as with FITC (fluorescein isothiocyanate), in order to be able to monitor success of transformation.
  • FITC fluorescein isothiocyanate
  • the present invention is used in the modulation of Megakaryopoiesis and/or the prophylaxis or treatment of MK-dependent cancer or tumour cell growth, preferably by Ets-1 down-modulation.
  • systemic administration may be appropriate, and antisense RNA may be administered by injection in a suitable vehicle, for example.
  • RNA to be administered will be readily determined by the skilled physician, but may vary from about 1 ⁇ g/kg up to several hundred micrograms per kilogram.
  • the present invention further provides miR 155, miR 221 and miR 222 inhibitors, and their use in therapy. These are referred to as “sense inhibitors" in that they are complementary, at least in part, to the antisense miRNA of the present invention.
  • inhibitors can be delivered in much the same way as described above for the miR's themselves.
  • levels of homology or sequence hybridisation to a reference sequence are as described above in relation to the antisense RNA.
  • the inhibitors or antagomirs of the present invention are complementary or capable of binding under highly stringent conditions to the antisense RNA. Therefore, in some embodiments, the antagomirs are complementary or capable of binding under highly stringent conditions to SEQ ID NOS 1-3 (miRs 221, 222 and 155 respectively). SEQ ID NOS 17-19 are therefore particularly preferred as these are the complementary sequences to SEQ ID NOS 1-3, respectively.
  • inhibitors comprising stretches comprising the "seed" sequences of Ets-A, which the antisense RNA or the specific miRs recognise.
  • MiR 155, miR 221 and miR 222 are naturally occurring, and high levels of these micro RNAs inhibit megakaryopoiesis, and this effect can be undesirable, such as with cancer patients undergoing chemotherapy, which can repress megakaryopoiesis.
  • the present invention provides the use of an miR155, miR 221 and miR 222 inhibitor in therapy.
  • a sense or antisense polynucleotide according to the present invention in the treatment or prophylaxis of the conditions specified herein.
  • the invention also provides the use of a sense or antisense polynucleotide according to the present invention in the manufacture of a medicament for the treatment or prophylaxis of the conditions specified herein.
  • a second inhibitor and preferably a third inhibitor to the other miR(s) is also provided, in order to enhance Ets-1 protein expression.
  • an inhibitor for miR 155, miR 221 and for miR 222 in any such therapy, although combinations of just two are also preferred such as miR 155 and miR 221, miR 155 and miR 222, and miR 221 and miR 222.
  • Suitable inhibitors for miR 155, miR 221 and miR 222 include antibodies and sense RNA sequences capable of interacting with these miRs. Such sense RNAs may correspond directly to the concomitant portion of the 3' UTR of kit mRNA, but there is no requirement that they do so. Indeed, as miRs frequently do not correspond entirely to the 3' UTR that they target, while the existence of dsRNA often leads to destruction of the target RNA, then it is a preferred embodiment that the inhibitor of miR 155, miR 221 or of miR 222 is entirely homologous for the corresponding length of miR 155, miR 221 or miR 222. The length of the inhibitor need not be as long as miR 155, miR 221 or miR 222, provided that it interacts sufficiently at least to prevent either of these miRs interacting with the 3' UTR or Ets-1 mRNA, when so bound.
  • Conditions treatable by miR 155, miR 221 and miR 222 inhibitors include different types of piastrinopenia, e.g., immunological based or chemotherapy induced.
  • Antagomirs being sense inhibitors of said oncomirs, bind or hybridise to the oncomirs and thereby form dsRNA, stimulating an RNAi reaction, leading to degradation and therefore removal of said antisense miRs. This leads to greater Ets-1 activity (de-repression of Ets-1 caused by removal of Ets-1 inhibition by said antisense miRs.
  • Suitable sequences will include sufficient nucleotides to hybridise to the oncomir and induce an RNAi response. It will be appreciated that exact sequence correspondence is not required, but that there should, preferably, be at a stretch of at least 5 and more preferably 10 consecutive nucleotides that correspond to, or are capable of binging to, an equivalent stretch of the antisense miR. This is discussed further below.
  • a preferred antagomir sequence is an anti-miR sequence.
  • the anti- miR sequences can be chemically modified as to increase in vivo effectiveness, for instance by adding cholesterol moieties in order to facilitate cellular trans-membrane passage and, optionally, 5' methylation to reduce cellular degradation.
  • Anti-miR sequences more suitable for use in vitro can also be readily provided.
  • the antagomirs may be thought of as inhibitors or suppressors of the present antisense miRs.
  • 2 '-O-Methyl antagomir oligonucleotides directed against the antisense miRs are chemically modified antisense oligonucleotides that bind and irreversibly inactivate miRs. These provide valuable tools for selectively suppressing miRs function in vitro and in vivo.
  • preferred antagomirs of the present invention include chemically modified nucleotides, particularly 2 '-O-Methyl oligonucleotides.
  • the antisense miRNA of the present invention may also be used for the restriction or inhibition of potentiation of ex vivo production or expansion of primitive megakaryopoietic cells and for the inhibition of the proliferative, differentiation and maturation effects of Ets-1 in MK cells and in platelets, whether such cells be of a normal or abnormal phenotype.
  • inhibitors or suppressors of the antisense RNA for instance "antagomirs" of the present invention inhibit the miRs themselves and can, therefore, be used to restrict their effects. For instance, if it is thought that too much miR has been administrated or if the effects of the miRs needs to be reduced and Ets-1 de-repressed (relieving the miR repression of Ets-1, leading to increased Ets-1 activity), then antagomirs of the present invention can also be administered.
  • the sense inhibitors or suppressors are particularly useful in the treatment or prophylaxis of cancers or tumours associated with decreased Ets-1 expression or where it is desired to increase Ets-1 expression in order to treat the patient.
  • Preferred methods of delivery of the antisense miRNA or sense inhibitors may be by any gene therapy method known in the art, as will be readily apparent to the skilled person. Such methods include the so-called “gene-gun” method or delivery within viral capsids, particularly adenoviral or lentiviral capsids encapsulating or enclosing said polynucleotides, preferably under the control of a suitable promoter.
  • Preferred means of administration by injection include intravenous, intramuscular, for instance.
  • the polynucleotides of the present invention can be administered by other methods such as transdermally or per orally, provided that they are suitably formulated.
  • MiR expression profiling in unilineage megakaryopoietic (MK) culture of human cord blood (CB) CD34 + hematopoietic progenitor cells (HPCs) indicated that miR-155, - 221 and -222 are abundant in HPCs, but then sharply decline starting from initial MK differentiation. We hypothesized that this decline may promote megakaryopoiesis by favouring expression of a key functional target.
  • miR-155, -221 and -222 target Ets-1, a transcription factor up-regulated in megakaryopoiesis, which transactivates relevant Mk genes. This was confirmed by Luciferase assays, which indicated a direct interaction of each of these miRs with the 3'UTR of Ets-1.
  • miR-155 matches Ets-1 3'UTR in two different sites from 2504 to 2526 and from 4570 to 4592 nucleotides ( Figure 3A, top)
  • the miR-222 seed sequence matches with nucleotides 4980-5002 in Ets-1 3'UTR ( Figure 3B, top right) while the seed sequence in miR-221 matches the same sequence with lower complementarity ( Figure 3B, top left).
  • Ets-1 is upregulated during normal megakaryopoiesis (12, 13) and transactivates relevant Mk genes, including the thrombopoietin receptor (14), the platelet factor 4 (PF4) (15, 16) the glycoprotein GPIX (CD42) (17) and von Willebrand factor (18). Moreover, Ets-1 upregulates the transcription of platelet GPIIb integrin (CD41): this Mk-specific marker (19) heterodimerizes with GPIIIa (CD61) and regulates cell adhesion and platelet aggregation by binding fibrinogen and von Willebrand factor.
  • Mk-specific marker (19) heterodimerizes with GPIIIa (CD61) and regulates cell adhesion and platelet aggregation by binding fibrinogen and von Willebrand factor.
  • one of the advantages of the present invention is that naturally-occurring microRNA sequences, which are antisense to the 3' UTR of the Ets-1 mRNA, or sense sequences which inhibit said antisense microRNAs, can be used to modulate the level of Ets-1 protein expression and, therefore, the transactiavtion of certain genes involved in MK growth, differentiation, proliferation, maturation and also in platelet growth. It will be understood, of course, that the progenitor cell for platelets is the megakaryocyte.
  • the present invention is useful in methods of blood cell replacement, especially MK cells and platelets, for instance after chemotherapy or due to other damage or cancer. It is also useful in methods or replacing defective platelets or controlling excessive platelet production. Such methods may be in vivo and are most preferably ex vivo in MK, and preferably thereafter platelet, expansion.
  • test kit capable of testing the level of expression of the Ets-1 protein such that the physician or patient can determine whether or not levels of the Ets- 1 protein should be increased or decreased by the sense or antisense sequences of the present invention.
  • the present invention also encompasses a polynucleotide sequence, particularly a DNA sequence, which encodes the microRNAs of the present invention, operably linked to a suitable first promoter so that the MicroRNAs can be transcribed in vivo.
  • the present invention also provides a polynucleotide, particularly DNA, providing polynucleotides encoding the sense microRNA inhibitors of the present invention, also operably linked to a suitable second promoter for in vivo expression of said sense microRNA inhibitors.
  • first and second promoters mentioned above can be controlled by a third element, such that the level of expression of the antisense microRNA and the level of expression of the sense microRNA inhibitors can be controlled in a coordinated manner.
  • a feedback mechanism is also included for establishing this level of control.
  • Chimeric molecules are also provided, consisting of a polynucleotide according to the present invention, i.e. the antisense MicroRNAs or the sense microRNA inhibitors, linked to a second element.
  • the second element may be a further polynucleotide sequence or may be a protein sequence, such as part or all of an antibody. Alternatively, the second element may have the function or a marker so that the location of microRNAs can be followed.
  • both the antisense RNA sequences and the sense inhibitors or suppressors are useful in diagnostic methods to evaluate cancer or tumour progression. It is therefore preferred that by assaying for levels of the antisense RNA sequences (especially the miR- 155/221/222), the progress of the cancerous disease state can be evaluated. For instance, an increase over time in the levels of the oncomirs in a sample or biopsy is indicative of tumour progression or growth, whereas a decrease over time in the levels of the oncomirs in a sample or biopsy is indicative of tumour regression or a successful anti-cancer treatment.
  • the present invention provides a method of assessing the progression of a tumour over time, comprising assaying levels of the antisense RNA sequences over time.
  • the proximal promoters of MK-specific genes mostly contain functional GATA and Ets binding sites (6).
  • the Ets family of transcription factors comprises at least 30 members, all sharing an Ets binding domain that recognizes specific purine rich DNA sequences containing a conserved GGA core (7).
  • FIi-I (10, 11) and Ets-1 (11-19) play important regulatory functions.
  • Ets-1 is upregulated during normal megakaryopoiesis (12, 13) and transactivates relevant Mk genes, including the thrombopoietin receptor (14), the platelet factor 4 (PF4) (15, 16) the glycoprotein GPIX (CD42) (17) and von Willebrand factor (18). Moreover, Ets-1 upregulates the transcription of platelet GPIIb integrin (CD41): this Mk-specific marker (19) heterodimerizes with GPIIIa (CD61) and regulates cell adhesion and platelet aggregation by binding fibrinogen and von Willebrand factor.
  • Ets-1 was a potential target of miR-155, -221 and -222.
  • the miR-155 seed sequence is conserved among mammals whereas the miR-221 and -222 complementarity with Ets-1 3'UTR is conserved in mammals and chicken.
  • Ets-1 Both Ets-1 mRNA and protein increased during MK differentiation and were expressed at elevated level through terminal maturation (Figure 2B). Consistent with our hypothesis that Ets-1 may be a direct target of miR-155, -221, and -222, Ets-1 expression is inversely correlated to the levels of these miRs in MK culture ( Figure 2A and 2B). Ets-1 is a direct target for miR-155, -221 and -222.
  • Ets-1 mRNA is a target of miR-155, -221 and -222
  • the miR-222 seed sequence matches with nucleotides 4980-5002 in Ets-1 3'UTR (Figure 3B, top right) while the seed sequence in miR-221 matches the same sequence with lower complementarity (Figure 3B, top left).
  • the Ets-1 3'UTR sequences targeted by miRs 155, 221 and 222 are also shown in SEQ ID NOS 4a, b, 5 and 6, respectively.
  • Reporter constructs containing the mutated miR-155 (pGL3/155-up -downstream 3'UTR mt) or -221/-222 (pGL3/221/222 3'UTR mt) target sites were produced as additional control.
  • the mutations of all of the complementary bases abolished the interaction between miR-155, -221, -222 and Ets-1 3'UTR ( Figure 3A and 3B bottom panels).
  • Enforced expression of miR-155, -221 and -222 in MK cultures down-modulates Ets-1 expression and impairs proliferation, differentiation and maturation.
  • CD34+ HPCs were grown in unilineage MK liquid suspension culture and transfected on day 1 with miR-155, -221, -222 precursors (pre-miRs) or the control pre-miR oligonucleotide at 16OnM. To determine the transfection efficiency cells were also transfected with a FITC-conjugated dsRNA and analyzed by FACS 24 hr post- transfection. 72-85% of cells were FITC positive (not shown). Analysis of apoptosis showed no toxicity of the transfected oligoribonucleotides (not shown).
  • Ets-1 Small interfering RNA against Ets-1 (siEts-1) was also utilized to confirm the role of Ets-1 in megakaryopoiesis and to define the functional relevance of Ets-1 targeting by miR- 155/221/222, as related to MK growth and differentiaton.
  • MK transfected cells Morphological analysis of MK transfected cells indicated a marked reduction of cell with polylobated nuclei. Moreover, at late stages of culture (day 13), scr miR transfected cells showed jagged and light coloured cytoplasms, typical of mature MKs, whereas several cells with immature cytoplasms (azurophil smooth edge) were still observed in cultures treated with miR-155, -222 precursors or siEts-1 ( Figure 5B). Similar results were obtained in miR-221 precursors transfected MK cells (data not shown).
  • the present studies indicate that in MK culture the decline of miR- 155/221/222 promotes megakaryopoiesis at both progenitor and precursor level by favouring Ets-1 expression at post-transcriptional level.
  • miR- 155/221/222 transfection in MK culture inhibits both commitment of HPCs and differentiation-maturation of precursor cells, mainly through Ets-1 mRNA degradation.
  • the miRs action on HPCs is indicated by clonogenic studies, while the effect on MK precursors is documented by liquid phase culture experiments.
  • miRs The miR-induced degradation of Ets-1 mRNA is of interest. It is now well accepted that miRs not only repress translation, but also degrade mRNAs bearing imperfect complementary sequences. The mechanisms of miR-induced mRNA degradation are under scrutiny. MiRs can accelerate mRNA decapping (25) and deadenylation (26), leading to rapid mRNA decay. MiRs also enhance cleavage and degradation of target mRNA containing AU-rich sequences (ARE) in the 3' UTR (27, 28). The presence of ARE motif(s), even in an AU-rich region, does not guarantee a destabilizing function (23).
  • ARE AU-rich sequences
  • Ets-1 3'UTR revealed the presence of several AU-rich sequences: interestingly we also observed that the 3' UTR downstream region per se down-regulates luciferase expression (unpublished data), suggesting that the AU-rich element present in the 3 ' region mediates RNA instability.
  • the mechanism of action of miR 155/221/222 in megakaryopoiesis deserves discussion. As mentioned above, these miRs target Ets-1, which plays a major role in megakaryocytopoiesis. Specifically, it transactivates MK-specific genes (11-19), thereby promoting MK differentiation and maturation (13).
  • Ets-1 expression in HPCs by either RNA interference or miR- 155/221/222 transfection similarly impairs commitment, differentiation and maturation of MK cells.
  • the similarity is also observed at quantitative level. This clearly indicates that in the MK lineage Ets-1 is the major functional target of miR- 155/221/222. However, we cannot exclude that these miRs may also target other mRNAs involved in the control of megakaryopoiesis.
  • miR-221/222 control the translation of the kit receptor (5 and WO 2006/108718 by the present inventors), which is also expressed in MK differentiation (unpublished data). Furthermore, bioinformatics analysis suggests that miR-155, -221 and -222 may target transcription factors with positive function in the control of megakaryopoiesis, e.g. Meis-1, a member of the TALE family of homeobox genes (29, 30).
  • miRs act as fine regulators of post-transcriptional gene expression. This regulation seems to play a pivotal role during developmental and differentiation processes, where transition from one state to another may require gene expression fine tuning, associated or not with on/off regulation (1, 33).
  • miR- 155/221/222 fine tune MK commitment of HPCs, as well as differentiation-maturation of MK precursors.
  • miR-based regulatory mechanisms also operate in other cell types, such the E (5) lineage through pivotal targets, i.e., miR-221/222 through kit receptor.
  • K562 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and antibiotics.
  • FCS fetal calf serum
  • For luciferase assay cells were co-transfected with 0.8 ⁇ g of pGL3-3'UTR plasmid, 50 ng of Renilla and 40 pmol of either a stability-enhanced nontargeting dsRNA control oligonucleotide (Dharmacon) or a stability-enhanced miR- 155 miR-221, miR-222 (Dharmacon), all combined with Lipofectamine 2000 (Invitrogen).
  • Dharmacon stability-enhanced nontargeting dsRNA control oligonucleotide
  • miR- 155 miR-221, miR-222 Dharmacon
  • HPCs purification, MK culture and transfection with miR oligonucleotides HPCs purification, MK culture and transfection with miR oligonucleotides.
  • CB CD34+ HPCs were isolated as previously described (34).
  • FCS liquid culture (10 5 cells/ml) supplemented with thrombopoietin (TPO 100 ng/ml) (Peprotech) (20).
  • CB progenitors cultured in megakaryocyte culture were transfected on day 1.
  • Cells were seeded (1.25xlO 5 /ml) in antibiotic-free media and transfected with 160 nM of either a miR control oligonucleotide or miR- 155, miR-221 or a miR-222 precursors (all pre-miRs were purchased from Ambion), all combined with Lipofectamine 2000 (Invitrogen).
  • Human small interfering RNA against Ets-1 were purchased by Dharmacon (siGENOME SMARTpool reagent M-003887-00-0020).
  • Cells were collected, washed in PBS, cytocentrifuged on glass slides and identified by morphological analysis after staining with May-Grunwald-Giemsa (Sigma). The number of poly-lobated nuclei cells was calculated by counting at least 600 cells in at least 10 randomly chosen fields.
  • HPCs were plated in methylcellulose medium containing saturating dosage of TPO as previously described (35). HPC colonies were scored on days 10-14.
  • RNA extraction Microarray, Northern Blot and Real-Time PCR.
  • miR-155 5' CCCCTATCACGATTAGC ATTAA 3' (SEQ ID NO. 7) miR-221 5' AGCTAC ATTGTCTGCTGGGTTTC 3' (SEQ ID NO. 8) miR-222 5' AGACCC AGTAGCC AGATGTAGCT 3' (SEQ ID NO. 9) Met-tRNA 5' TGGTAGC AGAGGATGGTTTCGA 3' (SEQ ID NO. 10)
  • Real Time RT-PCR analysis was performed using an ABI Prism 7700 Sequence Detector (Applied Biosystems). Commercial ready-to-use Taqman primers/probe mixes were used (Applied Byoystems).
  • the human Ets-1 3' UTR (NM_005238) segments containing the target sites for miR- 155 and miR-221, -222 were amplified by PCR from genomic DNA and cloned into pGL3 control vector (Promega) in the Xbal site immediately downstream from the stop codon of luciferase.
  • the following primers were used:
  • FIG. 1 Growth and differentiation of cord blood (CB) CD34+ hematopoietic progenitor cells (HPCs) in megakaryopoietic (MK) culture. Top panel. Growth curve of five indepedent experiments. Bottom. MK differentiation and maturation: percentage of CD41+ cells (left) and polylobated nuclei (right).
  • CB cord blood
  • HPCs hematopoietic progenitor cells
  • MK megakaryopoietic
  • FIG. 1 Expression of miR-155, -221, -222 and Ets-1 in CB CD34+ HPCs MK culture.
  • A Top panel. Normalized microarray results. Bottom. Northern blot analysis of miR-155, -221 and -222 expression. As loading controls, blots were reprobed for Met-tRNA (left). Normalized Northern blot results: a representative experiment out of three is shown (right).
  • B Analysis of Ets-1 expression in MK culture by Real time RT- PCR (top) and Western blot (bottom). Immunoblotting with actin antibody was performed to verify equal loading (bottom left). Actin-normalized Ets-1 values are reported (bottom right).
  • C Mean fluorescence intensity (MFI) of CD41 in MK cells transfected with pre-miRs and siEts-1 at day 10 of culture, as compared to isotype control. A representative experiment out of four is shown. Figure 5. miR-155, -221, -222 precursors and siEts-1 transfectio ⁇ in MK cells impairs megakaryocitic precursor differentiation-maturation and progenitor commitment.
  • FIG. 1 Top panel. Immunofluorescence analysis of CD41 and vWf in MK cells transfected with the indicated pre-miRs precursors at day 8 of culture. A representative experiment out of 4 is shown. CD41 and v Wf are depicted in green and red, respectively; nuclei are in blue. Original magnification, 100OX. Bottom. Normalized mean+ S.E.M. expression values from 4 independent experiments. **P ⁇ 0.01 when compared with scr transfected cells values before normalization.
  • a minicircuitry comprised of microRNA-223 and transcription factors NF-IA and C/EBPa regulates human granulopoiesis. Cell. 123:819-831.
  • Extracellular signal-regulated kinase induces the megakaryocyte GPIIb/CD41 gene through MafB/Kreisler. MoI. Cell. Biol. 24:4534-4545.
  • SEQ ID NOS 7-16 are the primers disclosed in the Examples above.
  • SEQ ID NOS 17-19 are the direct complementary sequences to SEQ ID NOS 1-3: SEQ ID NO 17 (anti-miR221): 5'-GAAACCCAGCAGACAAUGUAGCU-S'
  • SEQ ID NO 20 is a protein sequence of human Ets-1 available from NCBI (accession number J04101):

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Abstract

L'invention concerne un microARN capable d'interagir avec la région non traduite en 3' d'un ARN de protéine Ets-1 qui est utile dans le traitement de tumeurs dépendantes de Ets-1 et des inhibiteurs pour celui-ci qui sont utiles dans le traitement de la mégacaryopoïèse supprimée dans des patients atteints de cancer ou d'une mégacaryopoïèse anormale. Le microARN et des inhibiteurs pour celui-ci sont également utiles dans l'expansion in vivo et ex vivo des mégacaryocytes et des plaquettes.
PCT/EP2007/011074 2006-12-05 2007-12-05 Microarn ciblant ets1 WO2008068047A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9017940B2 (en) 2006-01-05 2015-04-28 The Ohio State University Methods for diagnosing colon cancer using MicroRNA signatures
US9017939B2 (en) 2006-01-05 2015-04-28 The Ohio State University Methods for diagnosing breast, colon, lung, pancreatic and prostate cancer using miR-21 and miR-17-5p
US9085804B2 (en) 2007-08-03 2015-07-21 The Ohio State University Research Foundation Ultraconserved regions encoding ncRNAs
EP2683387A1 (fr) * 2011-03-07 2014-01-15 The Ohio State University Activité mutatrice induite par l'inflammation des liaisons au microarn-155 (mir-155) et le cancer
EP2683387A4 (fr) * 2011-03-07 2014-09-03 Univ Ohio State Activité mutatrice induite par l'inflammation des liaisons au microarn-155 (mir-155) et le cancer
AU2012225506B2 (en) * 2011-03-07 2016-11-17 The Ohio State University Mutator activity induced by microRNA-155 (miR-155) links inflammation and cancer
US9249468B2 (en) 2011-10-14 2016-02-02 The Ohio State University Methods and materials related to ovarian cancer
US9481885B2 (en) 2011-12-13 2016-11-01 Ohio State Innovation Foundation Methods and compositions related to miR-21 and miR-29a, exosome inhibition, and cancer metastasis
US9434995B2 (en) 2012-01-20 2016-09-06 The Ohio State University Breast cancer biomarker signatures for invasiveness and prognosis
CN108025016A (zh) * 2015-09-16 2018-05-11 国立大学法人东北大学 核酸分子

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