WO2009100955A1 - Arn antisens ciblant cxcr4 - Google Patents

Arn antisens ciblant cxcr4 Download PDF

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WO2009100955A1
WO2009100955A1 PCT/EP2009/001300 EP2009001300W WO2009100955A1 WO 2009100955 A1 WO2009100955 A1 WO 2009100955A1 EP 2009001300 W EP2009001300 W EP 2009001300W WO 2009100955 A1 WO2009100955 A1 WO 2009100955A1
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cxcr4
mir
plzf
expression
cells
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Cesare Peschle
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Istituto Superiore di Sanita ISS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—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 receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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 nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present invention relates to the use of antisense RNA.
  • MicroRNAs are a class of regulatory, single-stranded RNAs of about 22 nucleotides (Bartel, 2004; He and Harmon, 2004; Ambros, 2004). 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 reduced translation and/or degradation (Valencia- Sanchez et al., 2006). MiRs control basic biological functions, such as cell proliferation and differentiation (Zhao et al., 2005; Naguibneva et al., 2006; Chen et al., 2006).
  • miRs may function as "oncomirs" (Esquela-Kerscher and Slack, 2006).
  • oncomirs Esquela-Kerscher and Slack, 2006.
  • hematopoietic differentiation Chon et al., 2004; Fazi et al., 2005; Felli et al., 2005; Fontana et al., 2007.
  • the promyelocytic leukemia zinc finger protein, PLZF is a transcription factor involved in the regulation of development (Kelly and Daniel, 2006), hematopoietic proliferation and differentiation (Ball et al., 1999; Labbaye et al., 2002), leukemogenesis (Melnick and Licht, 1999; Parrado et al., 2000) and tumorigenesis (Felicetti et al., 2004).
  • PLZF The promyelocytic leukemia zinc finger protein
  • the chemokine receptor 4, CXCR4, and its ligand SDF-I are key molecules in the process of homing/mobilization of normal hematopoietic and leukemic cells (Peled et al., 1999; Gazitt, 2004; Lapidot et al., 2005; Ratajczak et al., 2006), particularly at stem cells level (Kucia et al., 2005; Burger and Kipps 2006).
  • SDF-I acts as a mobilization or retention signal in hematopoiesis (Cottier-Fox et al., 2003; Ruiz de Almodovar et al., 2006), while CXCR4 regulation by cytokines is an important step in myeloid cell mobilization (Kim et al., 2006).
  • CXCR4 acts as a transporter for internalization and secretion of SDF-I across the bone marrow-blood barrier (Dar et al., 2005).
  • CXCR4 Inhibition of CXCR4 blocks thrombopoiesis in vivo (Avecilla et al., 2004), indicating that the integrity of the SDF-1/CXCR4 pathway is indispensable for megakaryocyte proliferation and differentiation/maturation (Pang et al., 2005). Nevertheless, the mechanism controlling CXCR4 expression in angiogenesis and hematopoiesis is not fully understood.
  • miR-146a targets CXCR4 mRNA. Accordingly, by up-modulating miR146a, CXCR4 can be inhibited. Conversely, by down-modulating miR- 146a, CXCR4 inhibition can be lifted and upregulation of CXCR4 stimulates megakaryopoiesis.
  • PLZF represses miR-146a transcription.
  • HPC hematopoietic progenitor cell
  • Mk unilineage megakaryocytic
  • Functional studies showed that miR-146a or PLZF siRNA transfection similarly downmodulate CXCR4 translation and impair proliferation, differentiation and maturation of Mk cells, indicating that the PLZF/miR-146a/CXCR4 cascade controls normal megakaryopoiesis.
  • the present invention provides use of antisense RNA specific for all or part of the 3' untranslated region of CXCR4 protein mRNA in therapy.
  • Up-modulating miR-146a may be achieved, for instance, by administering or increasing expression of miR-146a, for instance administering it directly or in the form of a vector with a coding sequence for miR-146a preferably under the control of a suitable promoter, or by reducing antagomir (anti-miR 146a) levels. It may also preferably be achieved by decreasing the levels or expression of PLZF, for instance by increasing the levels of inhibitory antisense RNA targeted to PLZF. Down-modulation of CXCR4 leads to reduced megakaryopoiesis.
  • down-modulating miR-146a may be achieved, for instance, by increasing antagomir (anti- miR-146a) levels, increasing PLZF levels, for instance using a vector with a coding sequence for anti-miR-146a and/or PLZF, preferably under the control of a suitable promoter. It may also preferably be achieved by reducing the levels of inhibitory antisense RNA targeted to PLZF. Up-modulation of CXCR4 leads to increased megakaryopoiesis.
  • therapy is treatment of cancer, preferably tumour metastasis.
  • therapy is treatment of HIV, most preferably by prevention of HIV entry into host cells. It is also preferred that the therapy is treatment of Rheumatoid Arthritis.
  • the therapy is treatment of other CXCR4-dependent tumours or disease conditions, such as leukaemia and solid tumours.
  • the therapy is treatment of the therapy is the modulation of Megakaryopoiesis.
  • antisense RNA such as miR-146a
  • Megakaryopoiesis can be inhibited.
  • sense RNA such as an antagomir can stimulate Megakaryopoiesis.
  • the therapy is via CXCR4 receptor down-modulation or inhibition, particularly by posttranscriptional control of CXCR4 by miR-146a.
  • the antisense RNA is a micro RNA.
  • the antisense RNA has at least 60% homology with a selected region of the 3' untranslated region of CXCR4 protein mRNA.
  • the antisense RNA is between about 12 bases and 45 bases in length.
  • the antisense RNA is preferably a sequence having the same sequence as mature miR- 146a, which is preferably the RNA sequence of SEQ E) NO 1 (mature miR-146a 5'- ugagaacugaauuccauggguu-3 ' ) .
  • a sequence capable of inhibiting miR-146a is required (anti- miR-146a, antagomirs)
  • its sequence is preferably the RNA sequence of SEQ ID NO 2 (anti-sense miR-146a 5'- aacccatggaattcagttctca-3 ' ) .
  • the preferred coding sequence: 305 ..1375 is shown in bold and underlined above and in the Sequence Listing.
  • a preferred nucleotide sequence encoding PLZF is shown in SEQ ID NO. 5.
  • the 3' untranslated (UTR) region of human CXCR4 protein mRNA is provided as SEQ ID NO. 5.
  • the antisense RNA is preferably specific for all or part of the 3' UTR of CXCR4 protein mRNA. Preferably, it is specific for the full length 3' UTR of CXCR4 mRNA. Preferably, it is specific for at least one, and preferably both, of the following putative miR-146a binding sites: "Site 2" (from 1575 to 1596 bp); and "Site 3" (from 1800 to 1821 bp) as described in Didiano and Hobert, 2006, as shown above in bold and underlined text and also shown in Figure 3c.
  • the binding sites for the present polynucleotide on CXCR4 are preferably one or more of those shown in Figure 3 c, especially the highlighted portions thereof and above.
  • Preferred PLZF binding site sequences shown in Figures 3e and 3g are provided in SEQ ID NOs 7 and 8, where GTAC in the first sequence is mutated to CGGC.
  • an inhibitor or suppressor of miR-146a.
  • this is a sense RNA.
  • the invention provides for the use of this inhibitor or suppressor in therapy, preferably in mediation, and preferably stimulation, of megakaryopoiesis.
  • the sense RNA preferably hybridises to miR-146a under highly stringent conditions, such as 6 x SSC.
  • the invention also provides the use of PLZF to increase CXCR4 expression or reduce miR 146a-mediated inhibition of CXCR4, in therapy, preferably in mediation, and preferably stimulation, of Megakaryopoiesis.
  • RNA encodes or comprises the mature form of the RNA, where the RNA is a micro RNA.
  • the invention also provides a vector comprising nucleotides encoding PLZF.
  • the invention also provides a method of treating Cancer, HIV and/or Rheumatoid Arthritis, for example, or other CXCR4-dependent tumours or disease conditions, comprising administering to a patient an antisense RNA, preferably miR- 146a, and/or antisense RNA targeted to PLZF, to thereby inhibit CXCR4 expression, as discussed above.
  • an antisense RNA preferably miR- 146a, and/or antisense RNA targeted to PLZF
  • Antisense RNA may be specific for any part of the 3' UTR of CXCR4 protein mRNA, and it will be appreciated that the 3' UTR may vary slightly from individual to individual.
  • the invention also provides the use of PLZF, or inhibitors thereof, to modulate CXCR4 expression, for instance via miR- 146a, and for treatment of conditions associated with CXCR4.
  • siRNA targeted to PLZF, thereby inhibiting or downmodulating PLZF expression also has a similar effect to upmodualting miR- 146a, i.e. inhibition of CXCR4, as well as Mk cell proliferation and differentiation/maturation.
  • RNA preferably miR- 146a, and/or antisense RNA targeted to PLZF, to thereby inhibit CXCR4 expression.
  • a method of stimulating megakaryopoiesis comprising administering to a patient an antagomir of miR- 146a and/or PLZF or nucleotides encoding it, to thereby increase CXCR4 expression.
  • 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 CXCR4 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 methods for assessment of such homology include the BLAST program.
  • 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.
  • the preferred miR is miR- 146a, according to SEQ ID NO. 1. Its mature length is of 22 bases. Thus, a particularly preferred length is between 20 and 25 bases, and especially 22.
  • 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- 146a, and targeting either of these regions with antisense RNA substantially reduces translation of CXCR4 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. Other CXCR4 mRNA sequences, such as the coding region for instance, may also be targeted. It is preferred that 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 miR- 146a.
  • 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.
  • Particularly preferred stretches of the microRNA of the present invention correspond to the so-called "seed" sequences.
  • 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 CXCR4 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.
  • 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 therefor.
  • RNA sequences in particular miR- 146a, or inhibitors thereof, are in fact capable of modulating the expression of CXCR4 protein.
  • the present invention does not extend to these compounds per se.
  • the present invention extends to these and all other antisense RNAs provided by the present invention, for use in therapy and other processes. More particularly, the present invention provides the use of antisense RNA specific for all or part of the 3' untranslated region of CXCR4 protein mRNA in therapy.
  • antisense RNAs of the present invention may be used in the treatment of CXCR4-dependent tumours, as well as other CXCR4-dependent, such as HIV and RA.
  • Solid, non-diffuse tumours may be targeted by direct injection of the tumour with a transforming vector, such as lenti virus, or adenovirus.
  • a transforming vector such as lenti virus, 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.
  • 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-146a 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.
  • 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.
  • Suitable inhibitors for miR-146a 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 CXCR4 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-146a is entirely homologous for the corresponding length of miR-146a. The length of the inhibitor need not be as long as miR-146a, provided that it interacts sufficiently at least to prevent either of these miRs interacting with the 3' UTR or CXCR4 mRNA, when so bound.
  • Conditions treatable by miR-146a inhibitors include those associated with downmodulation of CXCR4, thereby requiring upregulation of CXCR4.
  • 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.
  • test kit capable of testing the level of expression of the CXCR4 protein such that the physician or patient can determine whether or not levels of the CXCR4 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.
  • the 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.
  • miR-146a and antagomirs thereof are useful in controlling or mediating expression of CXCR4 and controlling or mediating Megakaryopoiesis.
  • miR-146a masters Mk differentiation by targeting the CXCR4 receptor. This is surprising because miR-146a was only previously known in carcinogenesis (He et al., 2005) and in the regulation of the innate immune response (Taganov et al., 2006). However, our results now indicate that miR-146a plays a pivotal role in the control of CXCR4 expression in Mk differentiation. Although bioinformatic algorithms suggest that several miRs putatively target CXCR4 (see Results), their absence or specific expression profile in Mk cell lines suggests that they do not control CXCR4 protein levels in Megakaryopoiesis.
  • the CXCR4 receptor is involved in the process of cancer metastasis (Wang et al. 2006), mediates HIV entry into CD4+ lymphocytes (Littman DR, 1998) and plays a major role in the pathogenesis of rheumatoid arthritis (Nanki et al. 2000).
  • the interaction between SDF-Ia and CXCR4 has a pivotal role in the directional migration of hematopoietic and epithelial cancer cells during the metastatic process (Burger and Kipps, 2006): therefore, small CXCR4 antagonist molecules have been developed for potential use in metastasis therapy (Tamamura and Fujii, 2005).
  • anti-miR antisense oligonucleotides (“antagomirs”) to exert effective in vivo actions (Krutzfeldt et al 2005; Care et al., 2007), our results indicate that these anti-miR- 146a molecules may represent a key tool for molecular therapy in the above clinical settings.
  • Regulation loop among PLZF, miR-146a and CXCR4 protein expression in leukemic cells (e) Left panel: Real time PCR analysis of PLZF mRNA expression level; right panels: Western blot analysis of PLZF protein level, in PLZF-siRNAs transfected HEL cells (siR) as compared to control- siRN As transfected HEL cells (C). (f) Northern blot analysis of miR-146a expression in PLZF-siRNA transfected HEL cells (siR) as compared to control siRNA-transfected HEL cells (C).
  • Values represent the percentage of the luciferase activity obtained when co-transfecting the nontargeting oligonucleotide. Mean ⁇ SEM values from three separate experiments. ** P ⁇ 0.01 where compared to control group.
  • FIG. 8 MicroRNA expression profiling in leukemic cell lines Northern Blot analysis of human microRNAs expression in leukemic cell lines: K562-PLZF compared to K562-LXSN cells, HEL and Jurkat (JURK). MiRs expressed in the cell lines are shown (except for miR-9). MiR-Ib, 7b, 9, 122a, 124, 125a, 129, 133, 139*, 148a, 153, 183, 184, 191, 200b, 205, 217, 221, 222, 224*, 281-1, 281-2, 381*, 410* were not expressed in these cell lines. tRNA was used for RNA normalization. Representative results from three independent experiments are shown, * Putative miRs targeting CXCR4 mRNA (www.TargetScan.org ' ).
  • K562, HEL, Jurkat and Phoenix cells were cultured using standard methods. Transduced K562-LXSN and three selected clones 1, 2, 3 of K562-PLZF cells
  • PDB treatment of K562 cells was incubated for 4 days in the presence of PDB (Phorbol Dibutyrate, Sigma St Louis, USA) as described in 35 and analyzed for Mk membrane markers (CD9, CD41, CD61) expression by Flow cytometry analysis, as
  • Quantitative real-time (qRT)-PCR analysis of miR-146a was performed by TaqMan technology, using the kit ABI PRISM 7700 DNA Sequence Detection System specific for miR-146a reverse transcription and PCR analysis (Applied Biosystems, Foster City, CA) according to the manufacturer's procedure.
  • RT-PCR analysis for pre-miR-146a was performed using random primers-RT kit (Invitrogen), according to the manufacturer's procedure. PCR analysis was performed with the following pre-miR-146a primers: forward - SEQ ID NO. 9; Reverse - SEQ ID NO. 10. PCR products were analyzed by Southern blot using a miR-146a antisense 32 P-labelled probe: SEQ ID NO. 11. Normalization was performed using GAPDH RT-PCR analysis 32 . qRT-PCR analysis for GAPDH, PLZF (ZNF 145), CXCR4 was performed according to standard procedures 32 . Commercial ready-to-use primers/probe mixes were used (Assays on Demand Products, Applied Biosystems, Foster City, CA).
  • F£PCs were harvested on different days of Mk unilineage culture, transferred to glass slides by cytospin centrifugation and stained with May- Gr ⁇ nwald-Giemsa.
  • HEL cells were crosslinked by addition to the culture medium of formaldehyde at 1% final concentration and incubation for 10 min at 37°C. After sonication, ChIP assay kit (Upstate USA, Charlottesville, VA) was used according to the manufacturer's procedure and protein-DNA complexes were immunoprecipitated overnight with the anti-PLZF mAb, an anti c-abl mAb (Oncogene Research Products, Boston, MA) or protein-A sepharose only.
  • ChIP assay kit Upstate USA, Charlottesville, VA
  • protein-DNA complexes were immunoprecipitated overnight with the anti-PLZF mAb, an anti c-abl mAb (Oncogene Research Products, Boston, MA) or protein-A sepharose only.
  • a genomic fragment of 128 bp containing the PLZF binding site in the pre-miR-146a upstream region was amplified by PCR using primers flanking the PLZF site: forward SEQ ID NO. 12; reverse SEQ ID NO. 13.
  • PCR products were Southern blotted and hybridized with the internal probe 32 P-labeled oligonucleotide SEQ ID NO. 14.
  • a flanking 140 bp genomic region, not containing any PLZF site was amplified by PCR and analyzed using the following primers: sense SEQ ID NO. 15; antisense SEQ ID NO. 16 and internal probe SEQ ID NO. 17.
  • Non relevant cellular DNA sequences were detected by amplification of a GAPDH coding region using primers and PCR conditions as described 32 .
  • a 475 bp DNA fragment of Prom 146a was PCR-amplified from genomic DNA using the primers forward SEQ ID NO. 18 and reverse SEQ ID NO. 19, and cloned upstream to the luciferase gene into the pGL3Basic (pGL3Basic/Prom 146a) and pGL3 Promoter (pGL3 Prom/Prom 146a) vectors (Promega).
  • pGL3Basic pGL3Basic/Prom 146a
  • pGL3 Promoter pGL3 Prom/Prom 146a
  • mutagenesis of the PLZF site into the pGL3 Prom/Prom 146a vector using, according manufacturer's instructions, the QuickChange Site-Directed mutagenesis kit (Stratagene), we prepared the PLZF mutated Prom 146a vector (pGL3Prom/Mut. Prom 146a).
  • the pcDNA3/PLZF (PLZF) was
  • luciferase assay experiments Phoenix cells were transfected using Lipofectamine 2000 (Invitrogen), with a Renilla luciferase vector (50 ng), together with luciferase vectors described above (0,8 ug) and, where indicated, with the pcDNA3/PLZF. Luciferase activity was measured 48 hr post-transfection with the Dual Luciferase Reporter System (Promega, Madison, WI, USA) according to the manufacturer's instructions, by using Microlite TLXl (Dynatech Laboratoires, Chantilly, CA) and then normalized for Renilla Luciferase activity. Data are presented as mean values, obtained from at least three independents experiments. Silencing of PLZF or CXCR4 expression by siRNAs
  • PLZF-siRNA sequences Two double-stranded small interfering PLZF-siRNA sequences (NM_001018011), or CXCR4-siRNAs sequences (NM_003467) and the control C-siRNA sequence with no homology to the human genome were purchased from Dharmacon, Lafayette, CO. Cells were co-transfected with 90 nM PLZF-siRNA or CXCR4-siRNAs or C-siRNA and FITC- conjugated nontargeting oligonucleotide using Lipofectamine according to the manufacturer's instruction. After 48 hrs, cells were harvested and analysed for PLZF, miR- 146a and CXCR4 mRNA and protein expression as described above.
  • miR-146a Stability-enhanced miR-146a oligonucleotide (miR-146a), control nontargeting RNA oligonucleotide (Cont. oligo), antagomir-146a ( ⁇ -miR-146a), unrelated antagomir-133a (Cont. ⁇ -miR) and FITC-conjugated nontargeting oligonucleotide were purchased from Dharmacon. Cell lines were transfected using Lipofectamine and collected for analysis 48 hrs after transfection. HPCs were transfected at day 0 or 1 of Mk cultures and collected at sequential times through Mk differentiation/maturation.
  • Plasmids and constructs We used the psiCHECK 2- 3'UTR vector (Promega, Madison, WI USA) to clone, downstream to the Renilla luciferase gene, the full length 3' UTR of CXCR4 mRNA (FL wild type, wt vector) (NM_001008540, from position 1450 to 1862), which includes 3 putative miR-146a binding sites (Site 1 : from 1452 to 1473 bp; Site 2: from 1575 to 1596 bp; Site 3: from 1800 to 1821 bp).
  • CXCR4 mRNA fragments Sl (1450 to 1573 bp, including Site 1), S2 (1554 to 1775 bp, including Site 2), S3 (1756 to 1862 bp, including Site 3), S2/3 (1554 to 1862 bp, including both Sites 2 and Site 3) and Cont. (1621 to 1750 bp) a fragment without putative miR-146a binding site to use as a control.
  • mutagenesis QuickChange Site-Directed mutagenesis kit ,Stratagene
  • Site 2 and/or Site 3 in the reporter vectors FL wt and S2, S3, we prepared the constructs FL-Mutl, FL-Mut2, FL-Mut3, FL-Mut2/3 and MutS2, MutS3 respectively.
  • SEQ ID NO. 21 5'-GCAGGACCTGTGGCCAGCGGCCGCGTTGCTGTATGTCTCG- 3' (from 1573 to 1612); Site 3, SEQ ID NO. 22 5'- ATAGAAATGCTGGTTTGCGGCCGC7OGGAGTGGG TTGATTTC-3' (from 1795 to 1837).
  • Luciferase assay Phoenix cells were cotransfected with 75 ng of psiCHECK-2 reporter vectors and 60 pmol of either the nontargeting RNA oligonucleotide or stability- enhanced miR-146a oligonucleotide, using Lipofectamine. After 48 hrs, cells were lysed with Passive Lysis Buffer (Promega, Madison, WI USA), and their Renilla luciferase activity was measured by using the Femto-master FB 12 (Zylux, Oak Ridge, TN). The relative reporter activity was obtained by normalization to the psiCHECK-2- 3' UTR/nontargeting oligonucleotide, cotransfection.
  • K562 cells were cotransfected with 100 ng of pcDNA 3.1 empty vector, CXCR4-wt or CXCR4-Mut.2/3 vector and miR-146a vector, using Lipofectamine. After 48 hrs, cells were treated with PDB and analyzed 2/4 days later for CXCR4 and Mk markers expression, as compared to untreated cells.
  • PLZF, miR-146a and CXCR4 protein expression correlate in hematopoietic cell lines
  • CXCR4 mRNA expression is not significantly modulated, whereas CXCR4 protein level is upregulated, as indicated respectively by quantitative Real Time (qRT-) PCR (Fig. Ic, left panel) and FACS evaluation (Fig. Ic, middle and right panels).
  • qRT- quantitative Real Time
  • Fig. Ic left panel
  • FACS evaluation Fig. Ic, middle and right panels
  • CXCR4 mRNA is a putative target of other miRNAs, including miR-P, -93, -224, -381, -410.
  • miR-P, - 224, -381, -410 were not expressed in hematopoietic cell lines; in the case of miR-95, we found no correlation with the levels of CXCR4 protein expression (Fig. 8 and legend).
  • miR- 23b is not consistently regulated in different K562-PLZF clones (Fig.8), does not target CXCR4 3'UTR and does not modify the expression of CXCR4 protein when overexpressed in Jurkat cells (Fig 9).
  • PLZF positive cell line HEL to downmodulate PLZF expression by small interfering RNAs (siRNA), as shown by qRT-PCR (Fig 10a left panel) and Western blot analysis (Fig 10a middle and right panels), and examined the effects on miR-146a, and CXCR4 expression at mRNA and protein level.
  • PLZF siRNA siRNA
  • C control siRNA
  • Fig. 10b promoted miR-146a expression
  • Fig. 10c middle and right panels
  • K562 cells induced to Mk differentiation a model system to investigate the role of the PLZF/miR-146a/CXCR4 receptor pathway in megakaryopoiesis.
  • PLZF protein expression is gradually upregulated starting from day 1-3, as shown by anti-PLZF immunofluorescence and Western blot analysis (Fig. 2a).
  • pre-miR- 146a pre-miR-146a
  • miR-146a miR-146a expression by RT-PCR (Fig. 2b, left panel) and qRT- PCR (Fig. 2b, right panel) analysis respectively.
  • the data showed a high level of pre-miR- 146a and miR-146a expression in quiescent CD34 + cells (day 0), followed by a gradual, marked downmodulation in the day 1-3 period, which persisted thereafter.
  • Fig. 2d we observed an inverse correlation between PLZF protein and miR-146a expression levels during Mk differentiation/maturation
  • the CXCR4 mRNA level analysed by RT-PCR, showed a relatively mild fluctuation: specifically, it was elevated at day 0 and 1, lower at day 3-8 and then elevated again at day 10 (Fig. 2c, left panel). These fluctuations are seemingly mediated by transcriptional regulatory mechanisms. Conversely, FACS analysis showed that total CXCR4 protein expression was markedly upregulated, peaking at day 3 and sustainedly enhanced through day 10 (Fig. 2c, right panel). The rise of CXCR4 protein, in presence of a decreased mRNA, suggests that the sharp decline of miR-146a effectively unblocks CXCR4 mRNA translation.
  • CXCR4 is a direct target of miR-146a
  • each vector was transfected in the miR-146a negative Phoenix cell line, together with miR- 146a or a control non-targeting oligonucleotide (Cont. oligo).
  • the luciferase activity of the FL wt, FL-Mutl, FL-Mut2, FL-Mut3, and also of the S2, S3 or S2/3 reporter vectors was markedly diminished after miR-146a cotransfection (Fig.
  • PLZF is a transcriptional repressor of miR-146a expression
  • Protein-DNA complexes were immunoprecipitated with either the anti-PLZF mAb, or no antibody, or the unrelated anti-c-abl mAb used as negative control (Fig. 3f, ⁇ -PLZF, no-Ab, ⁇ -c-abl respectively).
  • the immunoprecipitates were then amplified by PCR using: (i) primers flanking the putative PLZF DNA-binding site in the Prom 146a region (Fig. 3f, Prom 146a); (ii) primers recognizing a region upstream to Prom 146a sequence, which does not contain any identifiable PLZF site (Fig. 3f, Control Prom).
  • Our ChIP assays indicated that in HEL cells PLZF is associated to the region surrounding the putative PLZF binding site into the Prom 146a genomic region.
  • Prom 146a into a reporter vector containing an artificial minimal promoter (pGL3 Promoter) and performed luciferase assays.
  • PLZF was able to repress the luciferase activity detected for the Prom 146a vector, but not the activity of the Mut. Prom 146a vector (Fig. 3g, right panel).
  • CD34 + cells seeded in unilineage Mk liquid suspension cultures, were co-transfected on day 1 with: (a) miR-146a or a nontargeting scrambled oligonucleotide and (b) a FITC- conjugated non-targeting oligonucleotide used as a tracer for FACS analysis. Transfection efficiency was 70% (data not shown).
  • miR-146a transfected cells
  • Control Mk Control Mk
  • Fig. 4a Analysis of CXCR4 mRNA expression by qRT- PCR (Fig. 4b, left panel) and CXCR4 protein level by FACS evaluation (Fig. 4b, right panel) indicated that miR-146a enforced expression induced protein downmodulation throughout megakaryopoiesis, without significantly affecting mRNA expression, indicating that CXCR4 is a target of miR-146a in the Mk lineage.
  • miR-146a-transfection induced a marked decrease in proliferation rate (Fig. 4a) and a marked reduction of cells with polylobated nuclei, suggesting a reduced Mk maturation (Fig. 4c).
  • Fig. 4a a marked decrease in proliferation rate
  • Fig. 4c a marked reduction of cells with polylobated nuclei
  • miR-146a By clonogenic Mk 35 and multilineage 37 progenitor assay, we observed that miR-146a overexpression reduced the number of Mk colonies to ⁇ 30% of control value (Fig. 4e, upper panel), whereas it did not affect the number of BFU-E and CFU-GM colonies (lower panels), thus indicating that the miR-146a effect is Mk-specific.
  • Mk cultures were also transfected with siRNAs that specifically blocked PLZF expression (PLZF-siRNA) or nontargeting control siRNA (C-siRNA), together with FITC- conjugated double-stranded RNA.
  • FITC positive cells were then sorted and cultured.
  • Silencing of PLZF mRNA i) enhanced pre-miR-146a expression (Fig. 5b, left panel), ii) decreased CXCR4 protein level as evaluated by FACS (Fig. 5c), iii) decreased cell proliferation (Fig. 5a), and iv) impaired Mk differentiation/maturation, as shown by morphology (Fig. 5d), reduced expression of the surface antigens CD61, CD9 (Fig.
  • MiR-146a suppression in unilineage Mk cultures increases CXCR4 protein expression and stimulates cell proliferation and differentiation/maturation
  • CD34 + cells seeded in unilineage Mk liquid cultures, were co-transfected after 10 hrs with: (i) antagomiR-146a oligonucleotide ( ⁇ -miR-146a) or control unrelated antagomir- 133a (Cont. ⁇ -miR) and (ii) a FITC-co ⁇ jugated non-targeting oligonucleotide used as a tracer for FACS analysis. We sorted the FITC-positive cells by FACS and seeded them again in unilineage Mk liquid culture. We next analyzed at sequential times antagomir-146a transfected cells ( ⁇ -miR- 146a), as compared to control (Cont.
  • CXCR4-siRNA siRNA targeting CXCR4 mRNA
  • C-siRNA nontargeting control siRNA
  • FITC-conjugated double-stranded RNA FITC positive cells were then sorted and cultured.
  • Silencing of CXCR4 mRNA decreased CXCR4 protein level as evaluated by FACS (Fig. 6c), decreased cell proliferation (Fig. 6a) and impaired Mk differentiation/maturation, as shown by morphology (Fig. 6d) and reduced expression of the surface antigens CD61, CD9 (Fig. 6e), CD41 and CD42b (not shown), as well as by clonogenic Mk and multilineage progenitor assay (Fig. 6f).
  • the rescue of miR-146a expression blocks CXCR4 translation and impairs Mk-like differentiation in K562-PLZF cells, thus confirming that the effects of PLZF are exerted through the miR-146a/CXCR4 pathway.
  • This regulatory pathway involves: (a) enhanced expression of PLZF, which inhibits miR-146a transcription; (b) miR-146a downmodulation, which causes an increased translation of the target CXCR4 mRNA.
  • miR-146a acts as an effector of the PLZF transcription factor, mediating its control activity on CXCR4 protein level and megakaryopoiesis .
  • MiRNAs are posttranscriptional regulators of gene expression, which play a key role in basic cell functions under normal and abnormal conditions 3 ' 38 . Although their role in the control of normal 9 and leukemic 39 hematopoiesis has been recognized, relatively little is known so far on the specific pathways involved, particularly in megakaryopoiesis. We have previously shown that miR-221 and -222 are critical regulators of erythropoiesis through targeting of the kit receptor 11 . In addition, enhanced expression of miR-223 repressing translation of the NFI-A transcription factor is necessary for granulocytic differentiation 10 .
  • this pathway comprises miR-17-5p/20a/106a, which target the transcription factor AML-I, activating in turn the expression of the M-CSF receptor 12 .
  • miR-146a masters Mk differentiation by targeting the CXCR4 receptor.
  • MiR- 146a is involved in carcinogenesis 40 and in the regulation of the innate immune response 41 .
  • Our results now indicate that miR- 146a plays a pivotal role in the control of CXCR4 expression in Mk differentiation.
  • CXCR4 mRNA region thereby inhibiting CXCR4 translation.
  • the CXCR4 receptor is upmodulated through all steps of the Mk differentiation pathway, from CFU-Mk through platelets (ref. 42 and our results).
  • chemokines, cytokines and adhesive interactions contribute to differentiation control by regulating both proliferation and the spatial relationships among precursor cells within the bone marrow microenvironment 30 ' 31 ' 42 .
  • previous reports 30 ' 31 confirmed by the hereby presented data, show that inhibition of CXCR4 blocks normal megakaryopoiesis and thrombopoiesis, indicating a major role for CXCR4 in these processes.
  • miR-146a/CXCR4 pathway is a major controller of both early and late phases of megakaryopoiesis, triggering the regulated expression of the Mk-specific gene program.
  • This program particularly in its late stages, may well involve the modulation of other miRNAs 43 .
  • the molecular pathway acting through the miR-146a/CXCR4 complex is controlled by the PLZF suppressor.
  • This transcription factor inhibits miR-146a transcription by interacting directly with a newly discovered PLZF binding site within the miR-146a promoter region.
  • ChIP experiments, luciferase-based promoter assays and PLZF silencing studies coherently indicate that the progressive increase of PLZF expression during megakaryopoiesis represses miR-146a transcription, thereby unblocking CXCR4 protein expression and stimulating megakaryopoiesis.
  • the effects of PLZF silencing on Mk differentiation mimic those observed upon either miR-146a overexpression or CXCR4 silencing.
  • rescue experiments indicate that the PLZF action on megakaryopoiesis is essentially mediated by miR-146a.
  • the PLZF/miR-146a/CXCR4 action may be in part mediated by regulation of the interactions of Mk precursor cells with other cells and, in vivo, with the extracellular matrix, hi line with this hypothesis, PLZF down-regulates the adhesion molecule VLA-4 involved in the mobilization of hematopoietic and leukemic cells 32 , while CXCR4 is an essential regulator of hematopoietic stem cells traffic in stem cells niches 22 and a mediator of myeloid cells homing/mobilization .
  • the CXCR4 receptor is involved in the process of cancer metastasis 44 , mediates HIV entry into CD4+ lymphocytes 45 and plays a major role in the pathogenesis of rheumatoid arthritis 46 . Therefore, the discovery of inhibitors of the SDF1/CXCR4 pathway is an important objective in molecular pharmacology 47 . In view of the capacity of miRNAs and anti-miRNAs oligonucleotides to exert effective in vivo actions 48 ' 49 , our results indicate that miR-146a may represent a key tool for the development of molecular therapy in these
  • miRNAs act as intermediate effectors through which transcription factors exert their control on differentiation and proliferation of precursor cells.
  • MiRNA-based regulatory mechanisms have been described in erythropoiesis l , granulopoiesis and monocytopoiesis 12 . All these control pathways target a key hematopoietic receptor:
  • c-kit in erythropoiesis CXCR4 in megakaryopoiesis
  • G-CSF receptor in granulopoiesis (our unpublished results)
  • M-CSF receptor in monocytopoiesis.
  • the receptor is either a direct miRNA target, as in erythropoiesis and megakaryocytopoiesis, or is regulated by a miRNA-controlled transcription factor, as in granulocytopoiesis and monocytopoiesis.
  • PZF promyelocyte leukemia zinc finger
  • CXCR4 a key receptor in the crosstalk between tumor cells and their microenvironment. Blood 107, 1761-1767.
  • RNA genes miR15 and miR16 at 13ql4 in chronic lymphocytic leukemia Proc. Natl. Acad.
  • Cottier-Fox M.H., Lapidot, T., Petit, L, Kollet, O., DiPersio, J.F., Link, D., and Devine, S.
  • Fazi F., Rosa, A., Fatica, A., Gelmetti, V., De Marchis, ML., Nervi, C, and Bozzoni, I.
  • a minicircuitry comprised of microRNA-223 and transcription factors NFI-A and
  • C/EBPalpha regulates human granulopoiesis.
  • MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation. Proc.
  • hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specific antibodies.
  • Stromal cell-derived factor l ⁇ increases polyploidization of megakaryocytes generated by human hematopoietic progenitor cells.
  • MicroRNAs small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522-531.
  • PLZF induces megakaryocyte development, activates Tpo receptor expression and interacts with GATAl protein.
  • microRNA miR-181 targets the homeobox protein Hox-Al 1 during mammalian myoblast differentiation. Nat. Cell. Biol. 8,

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Le MiR-146a et ses antagomires sont utiles dans le contrôle de l'expression de CXCR4 et de la mégacaryopoïèse, de la métastase tumorale, de l'entrée de HTV et/ou de la polyarthrite rhumatoïde.
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WO2016179417A3 (fr) * 2015-05-06 2016-12-15 The University Of Utah Research Foundation Administration d'exosomes de micro-arn
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US10670603B2 (en) 2011-09-02 2020-06-02 The Trustees Of Columbia University In The City Of New York Diagnosis and treatment of cancer expressing ILT3 or ILT3 ligand
WO2013036282A3 (fr) * 2011-09-07 2014-04-24 The Trustees Of Columbia University In The City Of New York Régulation à la baisse de microarn inflammatoires par l'ilt3
WO2013036993A1 (fr) * 2011-09-13 2013-03-21 Commonwealth Scientific And Industrial Research Organisation Détection d'une infection virale
US9657360B2 (en) 2011-09-13 2017-05-23 Commonwealth Scientific And Industrial Research Organisation Detection of viral infection
WO2016179417A3 (fr) * 2015-05-06 2016-12-15 The University Of Utah Research Foundation Administration d'exosomes de micro-arn
US11980663B2 (en) * 2015-07-08 2024-05-14 American Gene Technologies International Inc. HIV pre-immunization and immunotherapy
US10765742B2 (en) 2015-07-17 2020-09-08 The Trustees Of Columbia University In The City Of New York Methods of treating CD166-expressing cancer
US12090200B2 (en) 2016-02-08 2024-09-17 American Gene Technologies International Inc. Methods of producing cells resistant to HIV infection
US11612649B2 (en) 2016-07-08 2023-03-28 American Gene Technologies International Inc. HIV pre-immunization and immunotherapy
US11911458B2 (en) 2016-07-08 2024-02-27 American Gene Technologies International Inc. HIV pre-immunization and immunotherapy
US12370253B2 (en) 2016-07-08 2025-07-29 American Gene Technologies International Inc. Pre-immunization and immunotherapy
US20230175020A1 (en) * 2019-03-27 2023-06-08 Emendobio Inc. Compositions and methods for promoting gene editing of cxcr4 gene
US12031149B2 (en) * 2019-03-27 2024-07-09 Emendobio Inc. Compositions and methods for promoting gene editing of CXCR4 gene
CN110917359A (zh) * 2019-11-21 2020-03-27 武汉理工大学 离子键自组装制备PEG-P（Asp-AP）-ANTAGOMIR-RNA微球

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