WO2008068046A1 - Microarn ciblant aml1 - Google Patents

Microarn ciblant aml1 Download PDF

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WO2008068046A1
WO2008068046A1 PCT/EP2007/011073 EP2007011073W WO2008068046A1 WO 2008068046 A1 WO2008068046 A1 WO 2008068046A1 EP 2007011073 W EP2007011073 W EP 2007011073W WO 2008068046 A1 WO2008068046 A1 WO 2008068046A1
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mir
amll
seq
cells
use according
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Cesare Peschle
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Istituto Superiore di Sanità
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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
    • C12N2310/14—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.
  • MiRs are a recently discovered class of small ( ⁇ 22nt) RNAs, which play 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 certain cancers of the blood.
  • miRs 17-5p, 20a and 106a target the 3'UTR of AMLl (human acute myeloid leukaemia protein). This has a downstream effect on M- CSFR, which controls blast cell proliferation. Therefore, miR can each inhibit or block translation or activity of AMLl mRNA.
  • microRNA in the miR-17-5p/l 06a/ 106b clusters is capable of interacting with the 3' untranslated region of AMLl protein microRNA is useful in stimulating haematopoietic blast proliferation, and inhibiting differentiation into monocytes and dendritic cells.
  • MicroRNA and inhibitors thereof are also useful in the inhibitory and stimulatory control of in vivo and ex vivo production of monocytes and dendritic cells, as wells as in treating tumours related to enhanced microRNA level.
  • the present invention provides the use of antisense RNA specific for AMLl, and inhibitors of said antisense RNA, in therapy.
  • the present Micro RNA is capable of interacting with the 3' untranslated region of AMLl protein mRNA and is useful in stimulating haematopoietic blast proliferation, and inhibiting differentiation into monocytes and dendritic cells.
  • Micro RNA and inhibitors therefor are also useful in the inhibitory and stimulatory control of in vivo and ex vivo expansion or production of monocytes and dendritic cells, as wells as in treating monocyte- and dendritic cell-related tumours.
  • treatment or prophylaxis of neoplasia is also envisaged, most preferably by the inhibitors or suppressors (antagomirs) of the present invention.
  • the antisense RNA is miR 17-5p, miR 20a or miR 106a, or combinations thereof.
  • the present invention is preferably in methods of blood cell replacement, especially Mo and DC cells, preferably replacing defective Mo and DC cells.
  • the miRs are used in a method of ex vivo production or expansion of primitive Mo cells, preferably in a method of ex vivo production of primitive DCs.
  • the present invention also provides use of AMLl anti-miRs or antagomirs, inhibitors of antisense RNA specific for AMLl, in therapy.
  • AMLl anti-miRs or antagomirs As the antisense RNA sequences are implicated in the promotion of cycle cycling and differentiation, the use of sense inhibitors or suppressors of these antisense RNA sequences (antagomirs) are envisaged to be useful in controlling or limiting cycle cycling and/or differentiation. This is discussed further in the Discussion section of the Examples.
  • tumours show enhanced levels of miR- 17/20/ 106 ("oncomiRs"), where these antisense RNA sequences play an important role in oncogenesis and tumour progression, for instance in pediatric neuroblastoma. Therefore, treatment with sense inhibitors or suppressors, such as antagomirs, may well prove beneficial, as indicated in the Discussion.
  • sense inhibitors or suppressors such as antagomirs
  • 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 oncomirs, miR- 17/20/106), 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.
  • anti-miRs is in a method of ex vivo differentiation of Mo cells, preferably in a method of ex vivo differentiation of DCs.
  • the present invention especially the anti-miRs can be used in the treatment of Mo or DC-dependent cancers, or more preferably for controlling or reducing excessive production of Mo and DC cells.
  • miRs of the present invention are useful in stimulating expansion or production of primitive cells but can also reduce levels of differentiation of mature cells.
  • the anti-mirs, or miR inhibitors, of the present invention can be used to reduce the degree of expansion of primitive cells, but also enhance differentiation in mature cells.
  • a combination of both miRs and miR- inhibitors (anti-miRs or antagomirs) of the present invention can be used at different times or in relation to different cell types: i.e. in a spatio-, temporal or spatio-temporal pattern.
  • This is particularly preferred in expansion or cell production methods where levels of control can be exerted on different cell types. This may include, for instance, provision of miR sequences to primitive cells, separation of mature from more primitive cells and application of anti-miRs to the mature cells to enhance differentiation.
  • expansion and differentiation can be "fine tuned.”
  • miR microRNA 17-5p, 20a and 106a in monocytic differentiation-maturation.
  • Mo monocytic differentiation-maturation.
  • these miRs are downmodulated, whereas the transcription factor AMLl is upmodulated at protein but not mRNA level.
  • miR-17/20/106 bind the AMLl mRNA 3'UTR, their decline “unblocks” or "de-represses” AMLl translation.
  • miR-17/20/106 transfection suppresses AMLl protein expression, leading to M-CSF receptor (M-CSFR) downmodulation, which, in turn, stimulates blast proliferation and inhibits Mo differentiation-maturation.
  • M-CSFR M-CSF receptor
  • anti-miR- 17/20/106 oligonucleotides similar to the in vivo utilized "antagomirs" causes opposite effects to those described above. Knockdown of AMLl by siRNA mimics the action of miR-17/20/106 on monocytopoiesis, indicating that AMLl is the major target of miR-17/20/106. What is particularly surprising is that AMLl also binds the miR- 17 cluster promoter and transcriptionally inhibits miR- 17/20 expression, providing a negative feedback loop.
  • miR-17/20/106 function as a master gene complex interlinked with AMLl in a mutual negative feedback loop. Therefore, said miRs and antagomirs are particularly useful in controlling the growth of Monocytes (Mo) and Dendritic Cells (DC), for instance in an in vivo, and most preferably in an ex vivo, expansion method. Such methods are useful for replacing cells damaged or lost due to injury or chemotherapy, for instance, or to produce these crucial white blood cells "on demand,” for instance to produce recombinant proteins, especially antibodies.
  • the antagomirs can be used to control or reduce expansion, whilst the miRs can be used to promote expansion.
  • Monocytes are produced by the bone marrow from haematopoietic stem cell precursors called monoblasts. Monocytes are responsible for phagocytosis (ingestion) of foreign substances in the body. Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibodies, termed antibody-mediated cellular cytotoxicity. Vacuolization may be present in a cell that has recently phagocytised foreign matter.
  • Macrophages are responsible for protecting tissues from foreign substances but are also suspected to be the predominant cells involved in triggering atherosclerosis. They are cells that possess a large smooth nucleus, a large area of cytoplasm and many internal vesicles for processing foreign material.
  • the present invention can be used in the prophylaxis or treatment of monocytosis, the state of excess monocytes in the peripheral blood.
  • Monocytosis may be indicative of various disease states.
  • Preferred examples of conditions treatable include chronic inflammation, stress response, hyperadrenocorticism, immune mediated disease, pyogranulomatous disease, necrosis, and red cell regeneration.
  • Dendritic cells form part of the mammalian immune system. Their main function is to process antigen material and present it on their surface to other cells of the immune system. Dendritic cells are present in small quantities in tissues that are in contact with the external environment, mainly the skin (where they are often called Langerhans cells) and the inner lining of the nose, lungs, stomach and intestines. They can also be found at an immature state in the blood. Once activated, they migrate to the lymphoid tissues where they interact with T cells and B cells to initiate and shape the immune response.
  • DC Dendritic cells
  • Dendritic cells constantly sample the surroundings for pathogens such as viruses and bacteria. This is done through pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs).
  • PRRs pattern recognition receptors
  • TLRs toll-like receptors
  • DCs act as antigen-presenting cells: they activate helper T-cells and killer T-cells as well as B-cells by presenting them with antigens derived from the pathogen, alongside non-antigen specific costimulatory signals.
  • Dendritic cells There are three types of Dendritic cells: Lymphoid dendritic cells, Myeloid dendritic cells (also called Plasmacytoid dendritic cells) and Follicular dendritic cell, any of which are preferred.
  • the DC are Myeloid Dendritic cells (MDC), which are most similar to monocytes.
  • MDC are made up of at least two subsets, the more common MDC-I (which is a major stimulator of T cells) and the rare MDC-2 (which may have a function in fighting wound infection).
  • the DC may be Plasmacytoid Dendritic cells (PDC), which can produce high amounts of interferon-alpha.
  • PDC Plasmacytoid Dendritic cells
  • the DC are produced in vitro, and are preferably Mo-DC or MDDC (referring to cells matured from monocytes) or HP-DC (referring to cells derived from hematopoietic progenitor cells).
  • DC are known to play a role in HIV
  • a preferred use of the present invention is in the treatment of HIV.
  • the Dendritic Cells are also known to play a major or even key role in allergy and autoimmune diseases like lupus erythematosus.
  • the present invention can also be in modulating either of said conditions in a patient.
  • particularly preferred conditions for treatment include blood cancers and, most preferably, Mo cancers.
  • the antagomirs of the present invention are particularly useful in the control of Mo cell proliferation, preferably cancer cell proliferation, especially given the surprising feedback system.
  • Particularly preferred are any conditions, especially Mo cancers, associated with AMLl expression and its effect on M-CSFR.
  • the feedback loop leads to a "differentiation blockade” induced by elevated levels of the oncomirs (miR- 17/20/106), as well as by siRNA-mediated AMLl silencing. This is associated with downmodulation of both AMLl and M-CSFR.
  • MiRs 17-5p, 20a and 106a can be referred to as "oncomirs" because of their association with these Mo cancers.
  • 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 oncomirs.
  • This leads to greater AMLl activity de-repression of AMLl caused by removal of AMLl inhibition by said oncomirs
  • M-CSFR up-modulation thus controlling blast proliferation and stimulation of Mo differentiation and maturation.
  • Suitable sequences will include sufficient nucleotides to hybridise to the oncomir and induce an RNAi response.
  • 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 oncomirs of the present invention can be used to downmodulate M-CSFR, stimulating blast proliferation and inhibiting stimulation of Mo differentiation and maturation. Stimulating blast proliferation is useful, for instance in ex vivo expansion methods.
  • the antagomirs of the present invention can be used to control blast proliferation and stimulation of Mo differentiation and maturation. Control of blast proliferation is particularly useful in combating Mo cancers.
  • the antagomirs may be thought of as inhibitors or suppressors of miRs- 17/20/ 106.
  • 2'-O-Methyl antagomir oligonucleotides directed against miR- 17/20/ 106 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 3' untranslated regions (UTR) of human AMLl protein mRNA interacting, preferably binding under highly stringent conditions, with miR-17-5p, miR-20a and miR 106a are provided in SEQ NOS. 4, 5 and 6, respectively.
  • Antisense RNA may be specific for any part of the 3' UTR of AMLl 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 AMLl 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 or the inhibitory (antagomir or suppressor) 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/inhibitory 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.
  • 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 17-5p, miR 20a and miR 106a. Combinations such as miR 17- 5p and miR 20a, miR 17-5p and miR 106a, and miR 20a and miR 106a are also preferred. Similar combinations of antagomirs are also envisaged.
  • the mature miR sequences are shown hereinafter as SEQ. ID NO's 1, 2 and 3, and have a mature length of 23 or 24 bases.
  • 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 17-5p, miR 20a and miR 106a, and targeting either of these regions with antisense RNA substantially reduces translation of AMLl 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.
  • AMLl 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.
  • the AMLl 3'UTR from different species shows a 100% identity in a region pairing with the "seed" nucleotides of miR- 17/20/ 106 (nt 1936-1944 of the human AMLl 3'UTR (see box in Fig. 3a entitled "AMLl short”).
  • the miRs match the AMLl 3'UTR at nucleotide positions 1936-1944 of the human AMLl 3'UTR.
  • the antagomirs comprise or consist of the same sequence as SEQ ID Nos. 4-6.
  • the antagomirs comprise or consist of a sequence being exactly complementary to the miR (i.e. the complementary sequence to SEA ID Nos. 4-6). Therefore, it is preferred that the antagomirs comprise or consist of any of SEQ ID Nos. 38, 39 and 40 (anti-miR-17, anti-miR-20 and anti-miR-106, respectively).
  • 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 AMLl ORF.
  • the present invention provides a vector comprising the RNA or DNA encoding said RNA, or which encodes or comprises the mature form of the RNA, where the RNA is a micro RNA.
  • 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.
  • antisense 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).
  • microRNAs are known from WO 2005/013901, the patent specification of which alone is over 400 pages. However, no specific function is provided for the vast majority of these miRs.
  • RNA sequences in particular miR 17-5p, miR 20a and miR 106a, or inhibitors thereof, are in fact capable of modulating the expression of AMLl 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 AMLl protein mRNA in therapy.
  • antagomir RNAs of the present invention may be used in the treatment of Monocyte (Mo)-dependent cancers or tumours, such as Acute Myeloid Leukaemia.
  • 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
  • systemic administration may be appropriate, and RNA may be administered by injection in a suitable vehicle, for example.
  • antisense (oncomir) or sense (antagomir) 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 17-5p, miR 20a and miR 106a inhibitors, and their use in therapy. These are referred to as “sense inhibitors” or “antagomirs” in that they are complementary, at least in part, to the antisense miRNA of the present invention.
  • inhibitors or “antagomirs” can be delivered in much the same way as described above for the miR's themselves.
  • miR 17-5p, miR 20a and miR 106a are naturally occurring, and high levels of these micro RNAs inhibit Mo differentiation, and this effect can be undesirable, such as with cancer patients undergoing chemotherapy, which can repress Mo differentiation.
  • the present invention provides the use of a miR 17-5p, miR 20a and miR 106a inhibitor in therapy.
  • a sense or antisense polynucleotide according to 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 AMLl protein expression.
  • an inhibitor for miR 17-5p, miR 20a and for miR 106a in any such therapy, although combinations of just two are also preferred such as miR 17-5p and miR 20a, miR 17-5p and miR 106a, and miR 20a and miR 106a.
  • Suitable inhibitors for miR 17-5p, miR 20a and miR 106a 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 AMLl 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 17-5p, miR 20a or of miR 106a is entirely homologous for the corresponding length of miR 17-5p, miR 20a or miR 106a.
  • the length of the inhibitor need not be as long as miR 17-5p, miR 20a or miR 106a, provided that it interacts sufficiently at least to prevent either of these miRs interacting with the 3' UTR or AMLl mRNA, when so bound.
  • Conditions treatable by miR 17-5p, miR 20a and miR 106a inhibitors include, Mo and DC cancers, especially Acute Myeloid Leukaemia, and those described above.
  • the antisense miRNA of the present invention may also be used for the stimulation of blast proliferation or ex vivo expansion of primitive Mo or DC cells and for the inhibition of the differentiation and maturation effects of AMLl in Mo or DC cells, whether such cells be of a normal or abnormal phenotype.
  • the inhibitors or 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 AMLl de-repressed (relieving the miR repression of AMLl, leading to increased AMLl activity), then antagomirs of the present invention can also be administered.
  • 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.
  • the present invention is useful in methods of blood cell replacement, especially Mo cells and DCs, for instance after chemotherapy or due to other damage or cancer. It is also useful in methods or replacing defective cells or controlling excessive cell production. Such methods may be in vivo and are most preferably ex vivo in Mo and DC expansion.
  • test kit capable of testing the level of expression of the AMLl protein such that the physician or patient can determine whether or not levels of the AMLl 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.
  • the second element may have the function or a marker so that the location of microRNAs can be followed.
  • the cells of the present invention are not limited to Monocytes (Mo) or Dendritic Cells (DC), although these are particularly preferred, especially Monocytes that are macrophages.
  • Progenitor cells for these cells and cells derived from the Mo Macropahge or DCs are also particularly preferred, as long as they express AMLl. Methods of treating cancer and methods of cellular production or expansion are also provided.
  • the present invention will now be further illustrated by the following, non- limiting Examples.
  • MicroRNAs are a novel regulatory class of non coding, single-stranded RNAs of ⁇ 22 nucleotides, identified in plants and animals " .
  • MiRs repress protein expression at post-transcriptional level, mostly through base pairing to the 3' untranslated region (UTR) of the target mRNA, thus leading to its degradation and/or reduced translation 4 .
  • MiRs have been shown to control basic biological functions, such as cell proliferation and differentiation 5'7 .
  • miRs may function as "oncomirs” 8 .
  • miR-17-5p-92 is a cluster of seven miRs on chromosome 13, that are transcribed as a polycistronic unit 12 . This cluster is highly homologous to the miR-106a-92 and the miR-106b-25 clusters located on chromosome X and 7, respectively 13 . Recent data have pointed to a link between the miR-17-5p-92 cluster upregulation and cancer formation. Specifically, expression of this cluster is directly induced by the oncogene c-myc 14 , and some of its miRs are overexpressed in lung cancers 15 .
  • chromosome 13 including the miR-17-5p-92 cluster (C13Orf25) is amplified in human B-cell lymphomas 16 ' 17 , alveolar rhabdomyosarcoma 18 and liposarcoma 19 , while its overexpression accelerates the onset of malignant lymphomas 17 .
  • the acute myeloid leukaemia-1 protein (AMLl, also known as Runt-related transcription factor 1, Runxl) is the DNA-binding subunit of the hematopoietic transcription factor CBF, which controls the expression of several genes underlying myeloid differentiation, e.g., IL-3 20 , granulocyte-monocyte colony-stimulating factor (GM-CSF) 21 , myeloperoxidase 22 , as well as the expression of the M-CSF receptor (M- CSFR) 23 playing a specific pivotal role in monocytic-macrophage (Mo) differentiation- maturation 24 .
  • AMLl also known as Runt-related transcription factor 1, Runxl
  • mice for AMLl show an embryonic lethal phenotype characterized by absence of definitive hematopoiesis 25 ' 26 . Moreover, adult mice with induced deletion of AMLl develop progressive splenomegaly with an expansion of the myeloid compartment resulting from increased self-renewal of the myeloid progenitors 27 . Finally, loss of AMLl in ES cells culture impairs Mo differentiation, indicating that AMLl expression is required for monocytopoiesis 25 .
  • the serum-free medium of unilineage Mo cultures of CB HPCs includes a saturating dosage of M-CSF , i.e., the key growth factor for Mo differentiation and maturation 24 .
  • Mo cultures of CB HPCs are characterized by extensive cell growth of a > 95% pure Mo cell population: this is coupled with progressive monocytic differentiation and then terminal macrophage maturation through a 3-4-wk period, as indicated by cell morphology and expression of membrane antigens, including the Mo-specific CD 14 (Fig. Ia, b; see also results presented below).
  • AMLl may be a putative target of miR-17-5p, -18a, -20a and -106a. Further studies were selectively focused on miR-17-5p, -20a and -106a (briefly referred as miR-17/20/106). Since AMLl is involved in myeloid and monocytic differentiation 25 ' 33 , we hypothesized that these miRs may regulate AMLl expression at translational level during monocytopoiesis. In line with this hypothesis, Western blot analysis of AMLl in Mo cultures revealed that the protein, expressed at low levels in HPCs, increases during Mo differentiation- maturation (Fig. 2a); conversely, Real-Time PCR indicated that AMLl mRNA level is essentially stable, from HPCs throughout monocytopoiesis (Fig. 2b).
  • Analysis of M-CSFR expression by Real-Time PCR and flow cytometry in unilineage Mo culture showed that both mRNA and protein, absent in undifferentiated CD34+ cells, gradually increase during Mo differentiation and maturation (Fig. 2d, e), as observed for AMLl protein (Fig. 2a). These results confirm that AMLl induces the transcriptional upmodulation of M-CSFR in monocytopoiesis, as previously reported in cell lines 23 .
  • MiR-17-5p, -20a and -106a downmodulate AMLl expression at translational level in Jurkat cell line
  • anti-miR- or "antagomirs” 2'-0-Methyl oligonucleotides directed against miR- 17/20/106.
  • anti-miR oligonucleotides are chemically modified antisense oligonucleotides that bind and irreversibly inactivate miRs, providing a valuable tool to selectively suppress miRs function in vitro and in vivo 34 ' 35 .
  • the level of endogenous miR- 17/20/106 declined, as indicated by Northern blotting (see Fig. 10b): this was coupled with increased expression of AMLl at protein level, while the mRNA abundance was unaffected (see Fig. l ie, d).
  • MiR-17-5p, -20a and -106a directly interact with AMLl 3'UTR
  • the vectors were transfected in K562 cells, which express high levels of miR-17/20/106 (data not shown), and luciferase activity was measured 48 hr later.
  • Endogenous miR-17/20/106 caused an 80% decrease of luciferase activity in cells transfected with the "sensor vector", as compared to cells transfected with the control vector or the empty pGL3-Promoter vector (Fig. 3b).
  • inhibition of endogenous miR-17/20/106 with anti-miR, but not with a scrambled control anti-miR enhanced the luciferase activity of the sensor construct by 2-3-fold (Fig. 3c).
  • the AMLl 3'UTR from different species shows a 100% identity in a region pairing with the "seed" nucleotides of miR-17/20/106 (nt 1936-1944 of the human AMLl 3'UTR, box in Fig. 3a entitled “AMLlshort”).
  • MiR-17-5p, -20a and -106a control Mo differentiation-maturation through AMLl
  • RNA small-interfering RNA
  • AMLl binds the promoter of the miR-17 cluster and inhibits transcription of miR-17/20
  • 5kb of their upstream region located on chromosome 13 and X respectively, for putative AMLl-binding sites (5'-PUACCPUCA-S').
  • 5'-PUACCPUCA-S' putative AMLl-binding sites
  • chromatin immunoprecipitatio ⁇ ChIP
  • myeloid cell line U937 stably transfected with a Zn-inducible expression vector for AMLl.
  • Cells were treated with Zn for 3 and 6 hr and chromatin fragments were immunoprecipitated with an anti- AMLl antibody.
  • DNA from the immunoprecipitates was then amplified by PCR using three different pairs of oligonucleotides located in the three promoter regions encompassing the putative AMLl-binding sites (Fig. 7a). As shown in Fig. 7b, AMLl was bound to all these sites; furthermore, the signal was enhanced by increased AMLl expression upon Zn treatment (the rise of AMLl level was confirmed by Western blot, data not shown).
  • the fragment 3 includes the closest AMLl binding site to the miR-17 cluster, while in proximity to the previously identified c-myc binding site (Fig. 7a): we hence focused on this region of the promoter.
  • a 1423bp fragment containing the AMLl binding site included in fragment 3 was cloned upstream the luciferase gene in the reporter pGL4 vector (pGL4proml7) (Fig. 7a).
  • Transfection of this construct in K562 cells caused a 100-fold increase of luciferase activity, as compared to pGL4 vector (Fig. 7c).
  • co-transfection of pGL4proml7 with an expression vector for AMLl induced a sharp decrease of luciferase activity, whereas AMLl overexpression did not affect the luciferase activity of pGL4 (Fig. 7d).
  • AMLl negatively regulates miR-17 cluster transcription through recognition of binding sites present in this fragment of the promoter.
  • the activity of pGL4proml7 was not affected by overexpression of AMLIa, a form of AMLl truncated of both activating and inhibitory domains (Fig. 7d).
  • a pGL4proml7 mutant deleted of the indicated AMLl site showed a modest inhibition of the luciferase activity upon overexpression of AMLl (data non shown): we cannot exclude, therefore, the presence of other cryptic non-consensus AMLl binding sites in this promoter fragment.
  • miRs pertaining to the miR- 17- 5p-92 and -106a-92 clusters, play a pivotal role in human monocytopoiesis. These miRs are highly expressed in CD34+ HPCs and markedly downmodulated during Mo differentiation-maturation. Their functional role was demonstrated by overexpression and knockdown experiments: an enhanced level of miR- 17/20/106 promotes blast cell proliferation and inhibits Mo differentiation-maturation, whereas their knockdown leads to opposite effects, i.e., reduced proliferative activity and accelerated monocytopoiesis.
  • AMLl targeting As mentioned above, murine knock out studies indicate that AMLl plays a pivotal role in hematopoiesis 25 ' 26 and myelopoiesis 25 ' 27 . Particularly, AMLl expression is required for monocytopoiesis, since loss of AMLl in ES cells cultured in vitro impairs Mo differentiation 2 .
  • AMLl expression is translationally regulated by miR-17/20/106 through interaction with the 3'UTR of AMLl mRNA.
  • AMLl transactivates a number of myeloid and Mo-related target genes 20"23 .
  • a major AMLl target may be represented by the M- CSFR.
  • this receptor is required for monocyte/macrophage differentiation 24 , while its expression is activated at transcriptional level by AMLl 23 .
  • M-CSFR mRNA and protein levels rise in Mo culture, as observed for AMLl protein.
  • the differentiation blockade induced by overexpression of miR- 17/20/106, as well as by siRNA-mediated AMLl silencing is associated with downmodulation of both AMLl and M-CSFR.
  • the miR- 17/20/106 control of monocytopoiesis is mediated by AMLl via the M-CSFR.
  • AMLl functions in a context-dependent manner as either activator or repressor of transcription, depending on the availability of a tissue-specific co-factor .
  • AMLl binds to the miR- 17-92 cluster promoter and transcriptionally inhibits miR-17/20 expression.
  • MiR-17/20/106 and AMLl are hence interlinked by a mutual negative feedback loop, whereby miR-17/20/106 block AMLl translation and AMLl inhibits miR-17/20 (and possibly miR- 106) transcription.
  • this reciprocal inhibitory mechanism mediates the switch from the initial expression pattern (elevated miR-17/20/106 and low AMLl levels) to the more advanced one (low miR- 17/20/106 and elevated AMLl levels).
  • monocytopoiesis is controlled by a pivotal cascade, which involves downmodulation of miR-17/20/106 leading to increased AMLl and M-CSFR expression.
  • high levels of miR-17/20/106 repress AMLl translation and hence M-CSFR expression, thus allowing maintenance of an undifferentiated state.
  • miR-17/20/106 are initially downmodulated, thereby unblocking translation of AMLl, which further inhibits miR-17/20 (and possibly miR- 106) expression and transactivates the M-CSFR, thus promoting Mo differentiation- maturation.
  • miRs function as a master gene complex through the AML 1 and M-CSFR circuitry, while interlinked with AMLl in a mutual negative feedback loop (Fig. 8).
  • miR- 17/20/ 106 The expression pattern of miR- 17/20/ 106 is sharply different in Mo vs E culture: this implies that the transcription and/or processing of this miR complex is in part controlled by lineage-specific factors. Noteworthily, miR- 17/20/106 levels are not inversely correlated to those of AMLl protein in the E series, as observed in Mo culture. These findings suggest that regulation of AMLl translation by this miR complex may be modified by lineage-specific proteins, as postulated in cells subjected to stress 37 . Altogether our observations suggest that in hematopoiesis the function of this miR complex is lineage-specific, in that their expression as well as their action on AMLl mRNA is differentially controlled in the Mo and E intracellular environments.
  • AMLl is a potential target of miR-17/18a/20a/106a, but not of miR-19a; conversely, AML2 (a member of the RUNX family, also referred as RUNX3) is predicted as a target of miR 19a, but not of miR-17/18a/20a/ 106a.
  • AMLl is one of the most frequent target of chromosomal translocation associated with human acute myeloid leukemia (AML) 33 .
  • AMLl fuses to the transcription factor ETO to form an AMLl-ETO chimeric product: this fusion protein interferes with AMLl -dependent transactivation, resulting in altered transcriptional regulation of normal AMLl target genes 38 .
  • overexpression of AMLl-ETO in CD34+ HPCs promotes the expansion of immature cells 39 ' 40 and blocks monocytopoiesis by transcriptional silencing of the M-CSFR gene 41 .
  • miR- 17/20/106 induces a reduced AMLl protein level and therefore mimics the effects of AMLl-ETO fusion protein.
  • the effects of miR overexpression are pronounced of the phenotype of AML FAB-M2 and M4 subtypes associated with AMLl-ETO t(8,21) translocation, which is characterized by blockade of myeloid- monocytic differentiation coupled with extensive self-renewal capacity. It is noteworthy that miR overexpression does not cause a leukemogenic state.
  • AMLl-ETO "knock in” mice show abnormal maturation and proliferation of myeloid progenitor cells, but do not develop leukaemia 39 : leukemogenesis apparently requires multiple genetic changes, while AMLl-ETO expression may not be sufficient for its onset 42.
  • Human erytroleukemic K562, myelomonocytic U937 and T lymphocytic Jurkat cell lines (American Type Culture Collection, Manassas, VA) were grown in RPMI medium supplemented with 10% FCS (Gibco, Carlsbad, CA).
  • CB CD34+ FIPCs purification, unilineage Mo or E liquid cultures and clonogenic assays were performed as described 28 ' 29 ' 45 .
  • cells were smeared on glass slides by cytospin centrifugation, stained with May-Grunwald Giemsa and analyzed at 400X magnification under a microscope (Eclipse 1000, Nikon) equipped with a digital camera.
  • K562 cells were transfected with firefly luciferase vectors (0.8 ⁇ g), together with a Renilla luciferase vector (50 ng) and, where indicated, with anti-miR oligonucleotides (160 nM) (IDT).
  • IDT anti-miR oligonucleotides
  • pcDNA3 Promega
  • Firefly luciferase activity was measured 48 hr post-transfection by using Microlite TLXl (Dynatech Laboratoires, Chantilly, CA) and then normalized for Reni ⁇ la luciferase activity.
  • U937 cells were electroporated with pMT/AMLl and selected as described 46 . Protein expression was induced by treating the cells with 150 ⁇ M ZnSO4 for the indicated times.
  • a 1061 bp fragment of the AMLl 3'UTR (nt 1029-2090) was PCR amplified from human genomic DNA using the forward AMLl-Target.for
  • the fragment obtained was cloned by blunt ligation in the Xbal site of the pGL3- Promoter vector (Promega, Madison, WI), downstream the luciferase gene, thus generating the pGL3prom/AMLl-Target-3'UTR plasmid ("sensor vector").
  • a construct containing a polylinker downstream the luciferase gene was used to generate the pGL3prom/AMLl-Cont-3'UTR plasmid.
  • the polylinker region was removed from the pGL3 -Promoter vector by digestion with Kpnl and BgIII and the blunted vector was self-ligated, thus generating the pGL3prom/PLK- vector.
  • All the fragments of the miR-17 cluster promoter were PCR-amplified from genomic DNA, digested with BgIII and HindIII and cloned into the BgHI-Hindlll-digested pGL4.10 vector (Promega).
  • the oligonucleotides used were: prom 17. for (5'-GATAGATCTCCTGCCCGGTCTTCTGTTCCTAAAC-S' SEQ ID NO. 17) and proml7.rev (5'-GATAAGCTTGGTCACAATCTTCAGTTTTACAAGGTGATG-S' SEQ ID NO. 18) for the pGL4proml7 construct; proml7/l.for (5'-GATAGATCTGTGTCAATCCATTTGGGAGAG-S' SEQ ID NO.
  • AMLl cDNA is commercially available (OriGene).
  • the AMLIa cDNA was PCR amplified from the AMLl cDNA by introducing a stop codon at the amminoacid position 277.
  • the oligonucleotides used were: AMLla.for (5'- ATGGCTTCAGACAGCATATTTGAG-3 SEQ ID NO. 23') and AMLla.rev (5'-TCAGGATGGTTGGATCTGCCTTGTATC-S' SEQ ID NO. 24).
  • Both the cDNAs were subcloned into the EcoRI site of the pCDNA3 expression vector and used for transactivation experiments.
  • AMLl cDNA was also cloned into the Xbal site of pMT carrying a Zn inducible MTl promoter and a selectable neo r gene 46 , thus generating the pMT/AMLl vector.
  • RNA isolation was performed using the Acid Phenol-Guanidinium Thiocyanate- Cloroform protocol 11 .
  • RNA samples (10 micro-g each) were run on 15% acrylamide urea-denaturing precast gel (Invitrogen), and transferred onto Hybond-N+ membrane (Amersham Biosciences, Little Chalfont, UK). The hybridization was performed overnight with DNA probes, previously labelled with [ ⁇ - 32 P] ATP, at 37°C in 0.1% SDS / 6 X SSC. Membranes were washed at room temperature twice with 0.1% SDS / 2 X SSC. Human tRNA for initiator methionine (Met-tRNA) was used as loading control.
  • Method-tRNA Human tRNA for initiator methionine
  • the probes used were:
  • miR-18a 5'-TATCTGCACTAGATGCACCTT-S' (SEQ ID NO. 25); miR-19a, 5'-TCAGTTTTGCATAGATTTGCACA-S' (SEQ ID NO. 26); Met-tRNA, 5'-TGGTAGCAGAGGATGGTTTC GATCC ATCGACCTCTG-3' (SEQ ID NO. 27).
  • LNA locked nucleic acid
  • oligonucleotides were used as probes. Labelling and hybridization were performed as described using high stringency conditions 47 . Blots were stripped at 75°C in 0.05% SDS / 0.1 X SSPE and reprobed several times. Bands were quantified with Chemi Doc Software (Minneapolis, MN, USA).
  • AMLl protein expression was analyzed from total cell extracts in IX Laemmli buffer with a polyclonal anti-AMLl/Runxl (Active Motif, Rixensart, Belgium) and a secondary anti-rabbit IgG peroxidase conjugated antibodies (Biorad, Hercules, CA). Actin was detected with a mouse monoclonal antibody (Calbiochem, Darmstadt, Germany) followed by a secondary anti-mouse IgG peroxidase conjugated antibody (Biorad). Bands were quantified with Chemi Doc Software.
  • Membrane-bound M-CSFR and CDl 4 were FACS stained with PE-conjugated antibodies (BD PharMingen, San Diego, CA) and analyzed with FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, Oxford, UK).
  • RT Real-Time reverse transcriptase
  • Real-Time PCR was performed by TaqMan technology, using the ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems, Foster City, CA) according to standard procedures. Human Cyclophilin was used as endogenous control. Commercial ready-to-use primers/probe mixes were used (Applied bioystems).
  • CChhrroommaattiinn IImmmmuunnoopprreecciippiittaattiioonn wwsas performed as described using an anti-AMLl antibody (Santa Cruz- H-65) and amplifying the immunoprecipitated DNA by PCR with the following oligonucleotides derived from the genomic region upstream the rm ' R- 17 cluster: fragment 1 forward, 5'-TTGAGAAGGCTGTGTGTCTCA-S '(SEQ ID NO. 28) and reverse, 5'-TGAAGCTTAGCCAACTTGATG-S' (SEQ ID NO. 29); fragment 2 forward, 5'-ACCAGTGATATGTGCTTTGCA-S' (SEQ ID NO.
  • Control for non specific DNA immunoprecipitation was produced by amplifying a fragment of the ⁇ -actin gene (NM-001101) with the following oligonucleotides: forward, 5'-CTTCTA CAATGAGCTGCGTGTGG-3' (SEQ ID NO. 34) and reverse, 5'-ATGGCTGGGGT GTTGAAGGTGTC A-3' (SEQ ID NO. 35).
  • the oligonucleotides described aboved were used to PCR amplify the DNA immunoprecipitated by an unrelated antibody (anti-CD40).
  • the PCR products were Southern blotted on nylon filters and probed with oligonucleotides internal to the amplified sequences: fragment 1, 5'-TTGCAGAAGGCATTTGTTAGC-S' (SEQ ID NO. 36), fragment 2, 5'-TATGTTTCTGAGAATTCCGGA-S '(SEQ ID NO. 37), fragment 3, 5'-GTTCCTAAACTGCAGCAAAGG-3'(SEQ ID NO. 41), actin, 5'-CGAGAAGATGACCCAGA TCAT-3'(SEQ ID NO. 42).
  • microRNA miR-181 targets the homeobox protein Hox-Al 1 during mammalian myoblast differentiation. Nat Cell Biol 8, 278-84 (2006).
  • a minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis. Cell 123, 819- 31 (2005).
  • Testa U. et al. Expression of growth factor receptors in unilineage differentiation culture of purified hematopoietic progenitors. Blood 88, 3391-406 (1996).
  • Rhoades K.L. et al. Analysis of the role of AMLl-ETO in leukemogenesis, using an inducible transgenic mouse model. Blood 96, 2108-15 (2000). 41.
  • follows G. A. et al. Epigenetic consequences of AMLl-ETO action at the human c-FMS locus. Embo J 22, 2798-809 (2003).
  • Figure 3 rm ' R-17-5p, -20a and -106a directly bind AMLl 3'UTR.
  • (b) Luciferase activity resulting from transfection of K562 cells with pGL3-Promoter vectors. Firefly luciferase values, normalized for Renilla luciferase, are presented (mean ⁇ s.e.m., n 6).
  • FIG. 4 Overexpression of miR-17-5p, -20a and -106a (300 nM) in Mo cultures affects cell proliferation and differentiation-maturation. A representative experiment out of four is shown (except for panel g).
  • FIG. 5 Inhibition of AMLl expression by small-interfering RNA (siRNA) treatment affects monocytopoiesis.
  • siRNA small-interfering RNA
  • a representative experiment out of three is presented (except for panel f).
  • Scale bar represents 50 ⁇ m.
  • (d) FACS analysis of CD 14 expression in transfected cells on day 14 of Mo culture. Percent values of CD 14+ cells are presented, (e) Percent M-CSFR expression in transfected cells on day 14 of Mo culture, (f) AMLl mRNA expression by Real-Time PCR in transfected cells at 72 hr post-transfection (mean ⁇ s.d., n 3). (g) Western blot of AMLl protein expression in transfected Mo culture at 72 hr post-transfection. ° P ⁇ 0.001 when compared to control.
  • FIG. 7 AMLl binds the miR-17 cluster promoter and transcriptionally inhibits miRs expression
  • C- is a negative PCR control.
  • PCR amplification of Fr. 2 was also performed on chromatin immunoprecipitated with an unrelated antibody (UA). Control amplifications were carried out on either chromatin before IP with oligonucleotides amplifying the Fr. 2 (Input) or AMLl -immunoprecipitated chromatin with oligonucleotides amplifying the D-Actin gene (Actin).
  • FIG. 8 Model for the role of AMLl and miR-17/20 in Mo differentiation.
  • miR-17/20 bind the 3' UTR of AMLl mRNA and repress translation: low levels of AMLl allow elevated transcription of miR-17/20 and determine low M- CSFR expression.
  • M-CSF addition to the Mo culture downmodulation of miR- 17/20 unblocks AMLl protein expression, which in turn inhibits miR-17/20 transcription and transactivates M-CSFR, thus promoting monocytopoiesis.
  • FIG. 9 Expression of miR-17-5p, -20a and -106a and AMLl in E cultures, (a) Northern blot analysis of miR-17-5p, -20a and -106a expression in CD34+ HPCs (day 0) and unilineage E culture at sequential culture days. Representative experiments are presented, (b) Western blot analysis of AMLl expression in E culture. A representative experiment is shown.
  • FIG. 10 Overexpression of miR-17-5p, -20a and -106a or anti-miR oligonucleotides recognizing these miRs in Jurkat cell line, (a) Representative Northern blot analysis of RNA from Jurkat cells transfected with miR-17-5p, -20a and -106a or a control scrambled oligonucleotide (Cont) (160 nM) at 48 hr post-transfection. Met-tRNA was used as a loading control, (b) Northern blot analysis of RNA from Jurkat cells transfected with anti-miR oligonucleotides recognizing miR-17-5p, -20a and -106a (160 nM) at 48 hr post-transfection. A representative experiment is shown.
  • FIG. 11 AMLl expression is translationally regulated by miR-17-5p, -20a and -106a in Jurkat cell line,
  • (d) AMLl mRNA level in Jurkat cells transfected with anti-miR oligonucleotides (mean ⁇ s.d., n 3). ° P ⁇ 0.001 when compared to control group.
  • SEQ ID NO. 4 (AMLl sequence targeted by miR-17-5p) miR-17-5p 3 ' -UGAUG-G-ACGUGACAUUCGUGAAAC- ⁇ '
  • SEQ ID NO. 5 (AMLl sequence targeted by miR-20a) miR-20a 3 ' -GAUG-G- ACGUGAU AUUCGUGAAAU- 5 '
  • SEQ ID NO. 6 (AMLl sequence targeted by miR-106a) miR-106a 3 ' -CGAUG-G-ACGUGACAUUCGUGAAAA- ⁇ '
  • SEQ ID Nos. 7-37 and 40-42 are disclosed in the Examples.
  • SEQ ID No. 38 (anti-miR-17), 5'-ACUACCUGCACUGUAAGCACUUUG-S'; SEQ ID No. 39 (anti-miR-20), 5'- CUACCUGCACUAUAAGCACUUUA-3'; and SEQ ID No. 40 (anti-miR-106), 5'- GCUACCUGCACUGUAAGC ACUUUU -3'.

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Abstract

L'invention concerne un microARN dans les amas miR-17-5p/106a/106b capables d'interagir avec la région non traduite en 3' d'un microARN de protéine AML1, qui est utile pour la stimulation de la prolifération des blastes hématopoïétiques, et l'inhibition de la différenciation en monocytes et cellules dendritiques. Le microARN et les inhibiteurs de celui-ci sont également utiles dans le contrôle inhibiteur et stimulateur de la production in vivo et ex vivo de monocytes et cellules dendritiques, ainsi que dans le traitement de tumeurs apparentées à un taux de microARN augmenté.
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WO2010017510A1 (fr) * 2008-08-07 2010-02-11 University Of Southern California Système pour l'expression synergétique de multiples petits éléments d'arn fonctionnel
WO2010036939A3 (fr) * 2008-09-26 2010-06-17 University Of Southern California Système d'expression synergiste de petits éléments d'arn fonctionnels multiples
EP2638159A4 (fr) * 2010-11-11 2015-07-29 Univ Miami Compositions, kits et méthodes de traitement des maladies cardio-vasculaires, immunologiques et inflammatoires
US9248144B2 (en) 2010-11-11 2016-02-02 University Of Miami Compositions, kits and methods for treatment of cardiovascular, immunological and inflammatory diseases
WO2014199377A1 (fr) * 2013-06-10 2014-12-18 Yeda Research And Development Co. Ltd. Compositions et procédés de traitement d'une malignité hématologique associée à une activité ou une expression modifiée de runx1
US20160208246A1 (en) * 2013-06-10 2016-07-21 Yeda Research And Development Co. Ltd. Compositions and methods for treating a hematological malignancy associated with an altered runx1 activity or expression
WO2019020593A1 (fr) * 2017-07-25 2019-01-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pharmaceutiques pour la modulation de la monocytopoïèse
US11926664B2 (en) 2017-07-25 2024-03-12 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and pharmaceutical compositions for modulating monocytopoiesis

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