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WO2018149978A1 - Activation alternative de macrophages (cellules m2) par l'intermédiaire d'un axe polyamine-eif5a-hypusine - Google Patents

Activation alternative de macrophages (cellules m2) par l'intermédiaire d'un axe polyamine-eif5a-hypusine Download PDF

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Publication number
WO2018149978A1
WO2018149978A1 PCT/EP2018/053913 EP2018053913W WO2018149978A1 WO 2018149978 A1 WO2018149978 A1 WO 2018149978A1 EP 2018053913 W EP2018053913 W EP 2018053913W WO 2018149978 A1 WO2018149978 A1 WO 2018149978A1
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cells
elf5a
compound
activation
macrophages
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PCT/EP2018/053913
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English (en)
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Erika PEARCE
Daniel PULESTON
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO2018149978A1 publication Critical patent/WO2018149978A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5055—Cells of the immune system involving macrophages
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells

Definitions

  • the present invention relates to a method for determining whether a compound has the capability of inhibiting elF5a activation, comprising (a) contacting a sample comprising cells being capable of differentiating into M2 macrophages with the compound under conditions that allow for the differentiation of cells into M2-type macrophages in the absence of the compound; and (b) quantifying in the sample the cells having a M2-phenotype, wherein a reduction of the number of cells having a M2-phenotype as compared to a control sample not contacted with the compound indicates that the compound has the capability of inhibiting el F5a activation.
  • the polyamine spermidine is needed to hypusinate the eukaryotic translation elongation factor 5A (elF5A) 1 .
  • the polyamine family of molecules is comprised of small polycationic metabolites, the most well described being putrescine, spermidine and spermine. They are formed by a series of enzymatic reactions that begin with the conversion of the amino acid ornithine to putrescine by ornithine decarboxylase (ODC) (figure 1 ).
  • ODC ornithine decarboxylase
  • a notable feature of the polyamine pathway is its role in the activation of the translation elongation factor elF5a.
  • elF5a is an essential cellular protein required for the production of a certain subset of proteins. Its function is dependent on the two enzymes deoxyhypusine hydroxylase (DOHH) and deoxyhypusine synthase (DHPS) that use the metabolite spermidine to mediate the activation of elF5a.
  • DOHH deoxyhypusine hydroxylase
  • DHPS deoxyhypusine synthase
  • the present invention aims at providing novel screening methods and therapeutic uses encompassing to interfere with the activation of elF5a.
  • the present invention therefore relates in a first aspect to a method for determining whether a compound has the capability of inhibiting elF5a activation, comprising (a) contacting a sample comprising cells being capable of differentiating into M2 macrophages with the compound under conditions that allow for the differentiation of cells into M2-type macrophages in the absence of the compound; and (b) quantifying in the sample the cells having a M2-phenotype, wherein a reduction of the number of cells having a M2-phenotype as compared to a control sample not contacted with the compound indicates that the compound has the capability of inhibiting elF5a activation
  • elF5a refers to the protein eukaryotic translation initiation factor 5A-1 that is encoded by the EIF5A gene.
  • the compound may interfere with any step in the polyamine pathway and the subsequent hypusine-elF5a formation as shown in Figures 1 and 2 as all these steps are required for the final activation of elF5a.
  • activated elF5a is hypusine-elF5a.
  • Hypusine is the amino acid N (e)-(4-amino-2-hydroxybutyl)-lysine.
  • the compound having the capability of inhibiting elF5a activation may interfere with any one of the precursors of hypusine-elF5a, e.g. spermidine or deoxyhypusine-elF5a, so that hypusine-elF5a cannot be generated as one or more of the required precursors are absent or not processible.
  • the compound having the capability of inhibiting elF5a activation may also interfere with one or more of the enzymes in the pathway, e.g.
  • the method of the invention may be performed in high through-put format for testing several compounds at the same time.
  • High-throughput methods are capable of screening up to several thousand compounds in parallel.
  • High-throughput assays independently of being biochemical, cellular or other assays, generally may be performed in wells of microtiter plates, wherein each plate may contain 96, 384 or 1536 wells. Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact of test compounds with the assay mixture is preferably effected by one or more computer-controlled robotic systems including pipetting devices.
  • mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well.
  • said mixture of test compounds may be deconvolved to identify the one or more test compounds in said mixture giving rise to the inhibition of elF5a activation.
  • the compound having the capability of inhibiting elF5a activation may either act on the DNA level (e.g. a siRNA against the mRNA encoding DHS or DOHH) or on the protein or precursor level (e.g. an antibody against the DHS or DOHH enzyme or an antibody against spermidine). If follows that the compound having the capability of inhibiting elF5a activation may be (i) a compound inhibiting the expression of an mRNA encoding an enzyme being required for the activation of elF5a, or (ii) a compound inhibiting an enzyme or a precursor being required for the activation of elF5a.
  • Compounds under option (i) include compounds interfering with the transcriptional machinery and/or its interaction with the promoter of said gene and/or with expression control elements remote from the promoter such as enhancers.
  • Compounds under option (i) also include compounds interfering with the translational machinery.
  • the compound inhibiting the expression of a mRNA encoding an enzyme being required for the activation of elF5a inhibits the expression and/or translation of said mRNA, for example, by specifically interfering with the promoter region controlling the expression of the mRNA or leading to the silencing of the mRNA.
  • the transcription and/or translation of the mRNA is reduced by at least 50%, more preferred at least 75% such as at least 90% or 95%, even more preferred at least 98% and most preferred by about 100% (e.g., as compared to the same experimental set up in the absence of the compound).
  • the compound preferably specifically inhibits the activity of the enzyme or precursor.
  • the activity of the enzyme is its capability of catalyzing a chemical reaction being involved in the pathway leading to hypsuine-elF5a.
  • the activity of the precursor is that it can be processed into another precursor further upstream in the pathway of elF5a activation or hypsuine-elF5a itself.
  • the activity of the enzyme or precursor is reduced by at least 50%, more preferred at least 75% such as at least 90% or 95%, even more preferred at least 98%, and most preferably about 100% (e.g., as compared to the same experimental set up in the absence of the compound).
  • the capability of the compound of inhibiting elF5a is determined by comparing the amount of cells having an M2-phenotype in the presence of the compound to that in the absence of the compound. In case the amount of cells having an M2-phenotype is reduced in the presence of the compound the compound is capable of inhibiting elF5a activation.
  • a compound is preferably qualified as being capable of inhibiting elF5a activation in case the amount of cells having a M2-phenotype is reduced by at least 50%, more preferred at least 75% such as at least 90% or 95%, even more preferred at least 98% and most preferred by about 100% (e.g., as compared to the same experimental set up in the absence of the compound).
  • a sample comprising cells being capable of differentiating into M2 macrophages is contacted with the test compound.
  • a cell being capable of differentiating into M2 macrophages may be a precursor cell of a M2 macrophage.
  • Preferred examples are hemoblasts, common myeloid progenitor cells, myeloblasts and monocytes.
  • the most preferred precursor cells of macrophages are monocytes or other progenitor cells derived from the bone marrow, such as the common myeloid progenitor, that can differentiate into macrophages under the control of factors such as colony stimulating factor 1 (CFS-1 , otherwise known as macrophage-colony stimulating factor 1 ).
  • CFS-1 colony stimulating factor 1
  • Monocytes are the direct precursor cells of macrophages in the hematopoietic cell lineage.
  • unactivated macrophages i.e. M0 cells
  • M2 macrophages e.g. when cultured in the presence of IL-4.
  • M1 and M2 macrophage activation represent two possible, distinct fates that unactivated macrophages can differentiate into.
  • the sample comprising cells being capable of differentiating into M2 macrophages preferably comprises unactivated macrophages (MO macropahges) and/or monocytes.
  • the sample can be a body sample or in vitro cultured cells.
  • Preferred body samples are a tissue sample (e.g. bone marrow derived macrophages or organ biopsy), including a tumor sample derived from a biopsy or resection, a blood sample (e.g. whole blood, plasma or serum), spinal fluid, and fluid from bronchoalveolar lavage.
  • Non-limiting examples of in vitro cultured cells are the human macrophage or monocytic cell lines THP-1 (ATCC TIB-202), U937 (ATCC CRL-1593.2), KG-1 (KG-1 ATCC CCL-246), MV-4-11 (ATCC CRL-959), SC (ATCC CRL-9855), MD (ATCC CRL-9850), Mono Mac 6 (DSM ACC 124) or hMo-PB cells (commercially available human "untouched" monocytes isolated from fresh peripheral blood, Cat.No. CSC-C1671 ).
  • the M1 macrophage phenotype is characterized by the production of high levels of pro-inflammatory cytokines, an ability to mediate resistance to pathogens, strong microbicidal properties, high production of reactive nitrogen and oxygen intermediates, and promotion of Th1 responses.
  • M2 macrophages are characterized by their involvement in parasite control, tissue remodeling, immune regulation, tumor promotion and efficient phagocytic activity.
  • the sample comprising cells being capable of differentiating into M2 macrophages is cultured under conditions that allow for the differentiation of the precursor cells into M2- type macrophages in the absence of the compound. Conditions that can be used in order to differentiate monocytes and/or macrophages into M2 macrophages are known in the art.
  • Preferred examples are the differentiation of monocytes and/or unactivated macrophages (MO cells) into M2 macrophages with (i) IL-4, (ii) IL-10, (iii) glucocorticoids (GC), or (iv) GC plus TGF-beta plus.
  • a more preferred example is the differentiation of monocytes and/or unactivated macrophages (MO cells) into M2 macrophages with IL- 4, preferably in conjunction with M-CSF.
  • IL-4 at a concentration of about 10ng/mL and, if present, M-CSF at a concentration of about 20ng/m.
  • Macrophages designates a cell of myeloid origin. Macrophages are large white blood cells, occurring principally in connective tissue and in the bloodstream. They ingest foreign particles and infectious microorganisms by phagocytosis andhave the capacity for antigen presentation. Macrophages are classified in the art into MO, M1 and M2 macrophages.
  • MO macrophages are also termed unactivated macrophages. They express the classical macrophage markers CD11b and CD68 but exhibit low expression of the activation markers MHC class II molecules, CD80, and CD86. They are moreover negative for cytokine expression in the form of IL-12, IL-10 and IL-1 beta.
  • a MO cell is preferably a cell expressing CD11b and CD68 but not expressing IL-12, IL-10 and IL-1 beta. More preferably, a MO cell is a cell expressing CD11b and CD68 but not expressing IL-12, IL-10 and IL-1 beta, and expressing low amounts of MHC class II molecules, CD80, and CD86.
  • a M1 macrophage is defined herein as a macrophage that was activated in the presence of a bacterium, virus, or a bacterial or viral compound (such as and preferably LPS) and optionally also in the presence of IFN-gamma. Accordingly, these cells can be denoted as M(LPS) or M(LPS+IFN) macrophages. M(LPS) and M(LPS+IFN) macrophages express the classical macrophage markers CD68 and CD11 b. Moreover these cells express - in contrast to MO cells - high amounts of MHC class II molecules, CD80 and CD86 expression. These cells also express the cytokines IL-1 beta, IL-12, and TNFalpha.
  • a M1 cell is preferably a cell expressing CD11 b and CD68, IL-1beta, IL-12, and TNFalpha. More preferably, a M1 cell is a cell expressing CD11 b, CD68, IL-12, IL-10 and IL-1 beta and expressing high amounts of MHC class II molecules, CD80, and CD86.
  • a M2 cell is also designated as alternatively activated macrophage in the art and hence as a macrophage that was activated in the absence of a bacterium, a virus, or a bacterial or viral compound (such as and preferably LPS) and IFN-gamma.
  • a M2 macrophage as used herein refers to a macrophage cell that was activated in the presence of a compound selected from IL-4, IL-10, glucocorticoids (GC),and GC plus TGF-beta plus, or any combination of these compounds.
  • M2 cells are also designated M(IL-4), M(IL-10), M(GC) or M(GC+TGF-beta) cells.
  • the M2 cells herein are preferably M(IL-4), M(IL-10), M(GC) or M(GC+TGF-beta) cells and are most preferably M(IL- 4) cells.
  • M(IL-4) cells express CD68, GATA3, IRF4, SOCS1 , CCL4, CCL13, CCL17, CCL18, MRC1 , STAB1 , F13A1 , TGFB1 , MMP12, TGM2, ALOX15, CD200R, but do not express MARCO and CD163.
  • M(IL-10) cells express CD68, SOCS3 and IL-4Ra.
  • M(GC) cells express CD68, CD163, STAB1 , MARCO, TGFBR2, ADORA3.
  • M(GC+TGF-beta) cells express CD68, ID3, RGS1 , pSMAD2, TGFBR2, ALOX5AP, IL17RB.
  • M(IL-4), M(IL-10), M(GC) and M(GC+TGF-beta) cells are preferably identified by the presence or absence of at least three of the respective markers listed in Table 1.
  • M(IL-10) cells are preferably identified by the presence of CD68, SOCS3 and IL- 4Ra.
  • M(IL-4)) cells are, for example, preferably identified by the presence of CCL17 and CCL18, and the absence of MARCO.
  • M(IL-4), M(GC) and M(GC+TGF-beta) cells are more preferably identified by the presence or absence of at least six of the markers listed in Table 1.
  • (M(GC)) cells are more preferably identified by the presence of CD68, CD163, STAB1 , MARCO, TGFBR2, ADORA3.
  • (M(IL-4)) cells are, for example, preferably identified by the presence of MRC1 , STAB1 , F13A1 , TGFB1 and MMP12, and the absence of CD163.
  • (M(GC+TGF)) cells are most preferably identified by the presence of CD68, ID3, RGS1 , pSMAD2, TGFBR2, ALOX5AP, IL17RB.
  • M(GC+TGF) cells are most preferably identified by the presence of CD68, GAT A3, IRF4, SOCS1 , CCL4, CCL13, CCL17, CCL18, MRC1 , STAB1 , F13A1 , TGFB1 , MMP12, TGM2, ALOX15 and CD200R, and the absence of CD163 and MARCO.
  • CD206 is a general marker of M2 cells in vivo.
  • Hypusination of elF5A is a conserved process critical for TCA (i.e. tricarboxylic acid) cycle integrity and mitochondrial respiration.
  • Hypusinated elF5A enables translation of a subset of TCA cycle and mitochondrial electron transport chain (ETC) enzymes that mediate stable TCA cycle flux and oxidative phosphorylation (OXPHOS).
  • ETC mitochondrial electron transport chain
  • OXPHOS oxidative phosphorylation
  • Inhibition of deoxyhypusine synthase (DHPS) or deoxyhypusine hydroxylase (DOHH) the enzymes that hypusinate elF5A 2,3 , leads to a break in the TCA cycle that is defined by the loss of elF5AH-regulated enzymes and associated metabolic flux into the TCA cycle.
  • DHPS deoxyhypusine synthase
  • DOHH deoxyhypusine hydroxylase
  • elF5AH facilitates efficient production of proteins with difficult to translate mitochondrial targeting sequences (MTS), an activity that is consistent with a known function of elF5AH in overcoming ribosome stalling 4,5 .
  • MMS mitochondrial targeting sequences
  • elF5a regulates the differential activation of macrophages (M2 cells), a cell type whose divergent functional fates are defined by a metabolic switch between respiration and glycolysis 6 .
  • quantifying the cells having a M2-phenotype comprises measuring the amounts of at least one marker molecule selected from the group consisting of GAT A3, IRF4, SOCS1 , CCL4, CCL13, CCL17, CCL18, MRC1 , STAB1 , F13A1 , TGFB1 , MMP12, TGM2, ALOX15, CD200R, SOCS3, IL-4Ra, CD163, STAB1 , MARCO, TGFBR2, ADORA3, ID3, RGS1 , pSMAD2, TGFBR2, ALOX5AP, CD206 and IL17RB.
  • marker molecule selected from the group consisting of GAT A3, IRF4, SOCS1 , CCL4, CCL13, CCL17, CCL18, MRC1 , STAB1 , F13A1 , TGFB1 , MMP12, TGM2, ALOX15, CD200R, SOCS3, IL-4Ra, CD163, STAB1 , MARCO, TGF
  • M2 macrophages are phenotypically characterized by the expression of a specific set of markers.
  • markers are GAT A3, IRF4, SOCS1 , CCL4, CCL13, CCL17, CCL18, MRC1 , STAB1 , F13A1 , TGFB1 , MMP12, TGM2, ALOX15, CD200R, SOCS3, IL-4Ra, CD163, STAB1 , MARCO, TGFBR2, ADORA3, ID3, RGS1 , pSMAD2, TGFBR2, ALOX5AP, CD206 and IL17RB.
  • One or more of these markers can be used in order to quantify the cells having a M2-phenotype is step (b) of the method of the invention.
  • Using more than one cell surface markers may increase the reliability of determining the cells having a M2-phenotype. With increasing preference at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 and at least 10 markers are measured.
  • the specific set of cell surface markers on M2 macrophages depends on the conditions used to allow for the differentiation into M2 macrophages.
  • IL-4 GATA3, IRF4, SOCS1 , CCL4, CCL13, CCL17, CCL18, MRC1 , STAB1 , F13A1 , TGFB1 , MMP12, TGM2, ALOX15 and CD200R are expressed as markers.
  • step (b) quantifying the cells having a M2-phenotype in step (b) preferably comprises measuring the amounts of at least one marker molecule selected from the group consisting of GATA3, IRF4, SOCS1 , CCL4, CCL13, CCL17, CCL18, MRC1 , STAB1 , F13A1 , TGFB1 , MMP12, TGM2, ALOX15 and CD200R.
  • at least 2 at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 and at least 10 of these markers are measured. Most preferably all of these markers are measured.
  • step (a) of the claimed method comprise IL-10 quantifying the cells having a M2-phenotype in step (b) preferably comprises measuring the amounts of at least one marker molecule selected from the group consisting of SOCS3 and IL-4Ra. More preferably both of these markers are measured.
  • step (a) of the claimed method comprise GC quantifying the cells having a M2-phenotype in step (b) preferably comprises measuring the amounts of at least one marker molecule selected from the group consisting of CD163, STAB1 , MARCO, TGFBR2 and ADORA3. With increasing preference at least 2, at least 3 and at least 4 of these markers are measured. Most preferably all five of these markers are measured.
  • step (a) of the claimed method comprise GC plus TGF-beta quantifying the cells having a M2-phenotype in step (b) preferably comprises measuring the amounts of at least one marker molecule selected from the group consisting of ID3, RGS1 , pSMAD2, TGFBR2, ALOX5AP and IL17RB. With increasing preference at least 2, at least 3, at least 4 and at least 5 of these markers are measured. Most preferably all six of these markers are measured.
  • the absence of a particular marker can further help to define cells having a M2-phenotype.
  • Non-limiting examples are phosphorylated STAT1 and IL-12.
  • IL-4 is used in the conditions allowing for the differentiation into M2 macrophages furthermore the absence of CD163 and MARCO can further help to define cells having a M2-phenotype.
  • quantifying the cells having a M2-phenotype comprises flow cytometry analysis, quantitative PCR, and/or western blot analysis.
  • the skilled person is aware of several methods being suitable for quantifying cells having a M2-phenotype. Non-limiting preferred examples of such methods are cytometry analysis, quantitative PCR, and/or western blot analysis. These methods are employed depending on whether nucleic acid or protein is measured.
  • Quantitative PCR qPCR or real-time PCR or RT-PCR uses the linearity of DNA amplification to determine absolute or relative amounts of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation.
  • Quantitative PCR DNA amplification may be monitored at each cycle of PCR. Quantitative PCR also lends itself to relative studies. A reaction may be performed using primers unique to each region to be amplified and tagged with different fluorescent dyes. Several commercially available quantitative thermal cyclers include multiple detection channels. In this multiplex system, the amount of target DNA/cDNA can be compared to the amount of a housekeeping sequence e.g. GAPDH or ⁇ -actin. Two types of detection chemistries are most commonly used for quantitative PCR. The first uses an intercalating dye that incorporates into double-stranded DNA. Of these fluorescent dyes, SYBR ® Green I dye is the most common one used.
  • the second detection method uses a primer or oligonucleotide specific to the target of interest, as in TaqMan ® probes, Molecular BeaconsTM, or Scorpion primers.
  • the oligonucleotide is labeled with a fluorescent dye and quencher.
  • the oligonucleotide itself has no significant fluorescence, but fluoresces either when annealed to the template (as in molecular beacons) or when the dye is clipped from the oligonucleotide during extension (as in TaqMan probes). Multiplex PCR is possible by using dyes with different fluorescent emissions for each primer.
  • a gene encoding a marker selected from the group consisting of GATA3, IRF4, SOCS1 , CCL4, CCL13, CCL17, CCL18, MRC1 , STAB1 , F13A1 , TGFB1 , MMP12, TGM2, ALOX15, CD200R, SOCS3, IL-4Ra, CD163, STAB1 , MARCO, TGFBR2, ADORA3, ID3, RGS1 , pSMAD2, TGFBR2, ALOX5AP, CD206 and IL17RB is amplified.
  • Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1 ) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. Western blots can be carried out in a quantitative manner. The quantification may be based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots (see, for example, Tyloer and Posh, BioMed Research International, Volume 2014 (2014), Article ID 361590).
  • Flow cytometry is an analytical cell-biology technique that utilizes light to count and profile cells in a heterogenous fluid mixture. Flow cytometry is a particularly and preferred powerful method because it allows a researcher to rapidly, accurately, and simply collect data related to many parameters from a heterogeneous fluid mixture containing live cells.
  • flow cytometry is used to identify, separate, and characterize various immune cell subtypes by virtue of their size and morphology.
  • antibodies tagged with fluorescent dyes, and raised against highly specific cell surface antigens e.g. clusters of differentiation or CD markers
  • the compound is an antisense molecule, siRNA, shRNA, antibody, ribozyme, aptamer, protein drug or small molecule.
  • the aptamer, ribozyme, antibody, small molecule, protein drug, siRNA, a shRNA or an antisense oligonucleotide of this embodiment specifically binds to / interacts with a compound being involved in the activation of elF5a thereby inhibiting the compound.
  • the compounds are the precursor and enzymes (including the mRNA encoding the enzymes) as shown in Figures 1 and 2 and the respective nucleic acid molecules encoding them.
  • the enzymes ornithine decarboxylase, spermidine synthase, spermine synthase, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) are involved in the activation of elF5a.
  • the mRNA of human ornithine decarboxylase is shown in SEQ ID NO: 1 , of human spermidine synthase in SEQ ID NO: 3, of human spermine synthase in SEQ ID NO: 5, of human DOHH in SEQ ID NO: 7 and of human DDHPS in SEQ ID NO: 9.
  • amino acid sequence of human ornithine decarboxylase is shown in SEQ ID NO: 2, of human spermidine synthase in SEQ ID NO: 4, of human spermine synthase in SEQ ID NO: 6, of human DOHH in SEQ ID NO: 8 and of human DHPS in SEQ ID NO: 10.
  • SEQ ID NOs 7 and 9 and within the amino acid sequences SEQ ID NOs 8 and 10 are preferred.
  • the mRNAs of SEQ ID NOs 7 and 9 encode the enzymes catalyzing the last two chemical reactions being required for the activation of elF5a and the amino acid sequences of SEQ ID NOs 8 and 10 are the enzymes catalyzing the last two chemical reactions being required for the activation of elF5a.
  • These enzymes catalyze the deoxyhypusination (i.e. the addition of deoxyhypusine) and the hypusination (i.e. the addition of hypusin) of elF5a.
  • a compound targeting the mRNA of SEQ ID NOs 7 or 9 or the amino acid sequence of SEQ ID NOs 8 of 10 is expected to prevent unwanted off target effects.
  • the term "aptamer” in accordance with the present invention refers to DNA or RNA molecules being either in the natural D-conformation or in the L-conformation ("aptamer") that usually have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms. Hence, apatamer can be designed against the precursors, enzymes and the mRNA encoding the enzymes.
  • aptamers can be classified as DNA or RNA aptamers or peptide aptamers. Whereas the former consist of (usually short) strands of oligonucleotides, the latter consist of a short variable peptide domain, attached at both ends to a protein scaffold.
  • Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.
  • Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecules, in particular antibodies.
  • aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamer's inherently low molecular weight.
  • the rapid clearance of aptamers can be an advantage in applications such as in vivo diagnostic imaging.
  • ribozymes refers to RNA molecules that act as enzymes in the absence of proteins. These RNA molecules act catalyticly or autocatalyticly and are capable of cleaving e.g. other RNAs at specific target sites but they have also been found to catalyze the aminotransferase activity of the ribosome. Hence, the ribozymes may act on the mRNA encoding the enzymes being involved in elF5a activation. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Zaher and Unrau (2007), RNA, 13 (7): 1017-1026.
  • RNAs examples include the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead- dependent ribozymes.
  • the principle of catalytic self-cleavage has become well established in the last 10 years.
  • the hammerhead ribozymes are characterized best among the RNA molecules with ribozyme activity. Since it was shown that hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, it appears that catalytic sequences for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site.
  • RNA which contains the GUC (or CUC) triplet
  • GUC GUC
  • CUC CUC
  • Molecules of this type were synthesized for numerous target sequences. They showed catalytic activity in vitro and in some cases also in vivo. The best results are usually obtained with short ribozymes and target sequences.
  • the aptamers and ribozymes may comprise modified nucleotides, such as locked nucleic acids (LNAs).
  • LNAs locked nucleic acids
  • antibody as used in accordance with the present invention comprises, for example, polyclonal or monoclonal antibodies. Furthermore, also derivatives or fragments thereof, which still retain the binding specificity, are comprised in the term "antibody”. Antibody fragments or derivatives comprise, inter alia, Fab or Fab' fragments, Fd, F(ab') 2 , Fv or scFv fragments, single domain V H or V-like domains, such as VhH or V-NAR-domains, as well as multimeric formats such as minibodies, diabodies, tribodies, tetrabodies or chemically conjugated Fab'-multimers (see, for example, Altshuler et al., Biochemistry (Mosc).
  • antibody also includes embodiments such as chimeric (human constant domain, non- human variable domain), single chain and humanized (human antibody with the exception of non-human CDRs) antibodies.
  • chimeric human constant domain, non- human variable domain
  • single chain humanized antibodies.
  • Various techniques for the production of antibodies and fragments thereof are well known in the art and described, e.g. in Altshuler et al., Biochemistry (Mosc). 2010 Dec; 75(13): 1584-605.
  • polyclonal antibodies can be obtained from the blood of an animal following immunisation with an antigen in mixture with additives and adjuvans and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples for such techniques are described, e.g. Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999 and include the hybridoma technique originally described by Kohler and Milstein, Nature 256 (1975), 495-497, the trioma technique, the human B-cell hybridoma technique (see e.g.
  • recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display.
  • a suitable system for the expression of the recombinant (humanized) antibodies or fragments thereof may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US patent 6,080,560; Holliger and Hudson, Nat Biotechnol., 2005; 23(9):1126-36). Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies specific for the target of this invention. Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies. Antibodies may be directed to proteins or small organic molecules.
  • the antibody used by the present invention may act on one of the enzymes or precursors being involved in elF5a activation.
  • the antibody thus preferably specifically binds to an amino acid sequence being selected from SEQ ID NOs 2, 4, 6, 8 and 10, preferably from SEQ ID NOs 8 and 10, or a precursor being selected from ornithine, spermidine, spermine, and elF5a-deoxyhypusine.
  • the antibody inhibits the activity of elF5a by inhibiting an enzyme or a precursor being required for the activation of elF5a.
  • the antibody more preferably directly targets elF5a- hypusine thereby inhibiting its activity.
  • protein drug designates designer drugs that are derivatives of human proteins. These proteins are used as scaffold to create a protein drug by well-established screening procedures (see Tomlinson et al (2004), Nature Biotechnology, 22(5): 521-522).
  • Non-limiting examples of human proteins which serve as a scaffold for designing protein drugs are transferrin, C-type lectins, trinectins, domain antibodies, kunitz domains, lipocalins and the Fyn SH3 domain.
  • the protein drug used by the present invention may act on one of the enzymes or precursors being involved in elF5a activation.
  • small molecule designates a low molecular weight organic compound that may have or has the capability to inhibit elF5a activation.
  • the upper molecular weight limit for a small molecule is preferably about 900 Daltons. Small molecule of this molecular weight can in general rapidly diffuse across cell membranes, so that they can reach intracellular sites of action. As lower molecular weight a cut-off of 500 Daltons is preferred. This is because clinical attrition rates may be significantly reduced if the molecular weight is kept is below 500 Daltons.
  • the antisense technology for the downregulation of RNA is well-established and widely used in the art to treat various diseases.
  • the basic idea of the antisense technology is the use of oligonucleotides for silencing a selected target mRNA through the extraordinar specificity of complementary-based pairing (Re, Ochsner J. 2000 Oct; 2(4): 233-236).
  • oligonucleotides for silencing a selected target mRNA through the extraordinar specificity of complementary-based pairing (Re, Ochsner J. 2000 Oct; 2(4): 233-236).
  • compound classes of siRNAs, shRNAs and antisense oligonucleotides will be provided.
  • antisense oligonucleotides are single stranded antisense constructs while siRNAs and shRNAs are double stranded antisense constructs with one strand comprising an antisense oligonucleotide sequence (i.e. the so-called antisense strand). All these compound classes may be used to achieve downregulation or inhibition of a target RNA.
  • the target of antisense constructs in general as well as all the specific classes of antisense constructs being described herein is preferably an mRNA of an enzyme being involved in the activation of elF5a.
  • the target is preferably a nucleic acid sequence selected from one or more of SEQ ID NOs 1 , 3, 5, 7 and 9. Within SEQ ID NOs 1 , 3, 5, 7 and 9, SEQ ID NOs are 7 and 9 are preferred. It is a matter of routine in the field of antisense technology to design an antisense construct with a sufficient number of nucleotide mismatches to any off-target in order to ensure that no off- targets become down-regulated.
  • siRNA in accordance with the present invention refers to small interfering RNA, also known as short interfering RNA or silencing RNA.
  • siRNAs are a class of 12 to 30, preferably 18 to 30, more preferably 20 to 25, and most preferred 21 to 23 or 21 nucleotide- long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome.
  • RNAi RNA interference
  • siRNAs have a well defined structure: a short double-strand of RNA (dsRNA), advantageously with at least one RNA strand having a 5' or 3' overhang. Each strand typically has a 5' phosphate group and a 3' hydroxyl (-OH) group.
  • dsRNA short double-strand of RNA
  • Each strand typically has a 5' phosphate group and a 3' hydroxyl (-OH) group.
  • This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs.
  • siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest. Thus, any gene of which the sequence is known can in principle be targeted based on sequence complementarity with an appropriately tailored siRNA.
  • the double-stranded RNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and/or DNA methylation.
  • one or both ends of the double-strand have a 3'-overhang from 1-5 nucleotides, more preferably from 1-3 nucleotides and most preferably 2 nucleotides.
  • the most efficient silencing was so far obtained with siRNA duplexes composed of 21 -nt sense and 21 -nt antisense strands, paired in a manner to have 2-nt 3'- overhangs.
  • the sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair (Elbashir et al. Nature. 2001 May 24; 41 1 (6836):494-8).
  • 2'-deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant.
  • the siRNA used in the invention preferably comprises an antisense strand which comprises or consists of a sequence which is with increasing preference complementary to at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, or at least 21 nucleotides of one or more sequences selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably SEQ ID NOs 7 and 9.
  • a preferred example of a siRNA is an endoribonuclease-prepared siRNA (esiRNA).
  • esiRNA is a mixture of siRNA oligos resulting from cleavage of a long double-stranded RNA (dsRNA) with an endoribonuclease such as Escherichia coli RNase III or dicer.
  • esiRNAs are an alternative concept to the usage of chemically synthesized siRNA for RNA interference (RNAi).
  • RNAi RNA interference
  • a cDNA of an mRNA template may be amplified by PCR and tagged with two bacteriophage-promotor sequences. RNA polymerase is then used to generate long double-stranded RNA that is complentary to the target-gene cDNA.
  • This complementary RNA may be subsequently digested with RNase III from Escherichia coli to generate short overlapping fragments of siRNAs with a length between 18-25 base pairs.
  • This complex mixture of short double-stranded RNAs is similar to the mixture generated by dicer cleavage in vivo and is therefore called endoribonuclease-prepared siRNA or short esiRNA.
  • esiRNA are a heterogeneous mixture of siRNAs that all target the same mRNA sequence. esiRNAs lead to highly specific and effective gene silencing.
  • a “shRNA” in accordance with the present invention is a short hairpin RNA, which is a sequence of RNA that makes a (tight) hairpin turn that can also be used to silence gene expression via RNA interference.
  • shRNA preferably utilizes the U6 promoter for its expression.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the shRNA that is bound to it.
  • RISC RNA-induced silencing complex
  • the shRNA used in the invention preferably comprises an antisense strand which comprises or consists of a sequence which is with increasing preference complementary to at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably SEQ ID NOs 7 and 9.
  • antisense oligonucleotide in accordance with the present invention preferably refers to a single-stranded nucleotide sequence being complementary by virtue of Watson- Crick base pair hybridization to an mRNA selected from SEQ ID NOs 1 , 3, 5, 7 and 9 whereby the respective mRNA is blocked and cannot be translated into protein.
  • the antisense oligonucleotides may be unmodified or chemically modified. In general, they are relatively short (preferably between 13 and 25 nucleotides). Moreover, they are specific for one of SEQ ID NOs 1 , 3, 5, 7 and 9, i.e. they hybridize to a unique sequence in the total pool of targets present in the target cells/organism.
  • the antisense oligonucleotide according to the invention comprises or consists a sequence which is with increasing preference complementary to at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9.
  • At least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides are preferably complementary to a contiguous part of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9, i.e. the nucleotides are consecutive in the respective SEQ ID NO.
  • the antisense oligonucleotide is preferably a LNA-GapmeR, an Antagomir, or an antimiR.
  • GapmeRs LNA-GapmeRs or simply GapmeRs are potent antisense oligonucleotides used for highly efficient inhibition of mRNA function. GapmeRs function by RNase H dependent degradation of complementary RNA targets. They are an excellent alternative to siRNA for knockdown of mRNA. They are advantageously taken up by cell without transfection reagents. GapmeRs contain a central stretch of DNA monomers flanked by blocks of LNAs. The GapmeRs are preferably 14-16 nucleotides in length and are optionally fully phosphorothioated. The DNA gap activates the RNAse H-mediated degradation of targeted RNAs and is also suitable to target transcripts directly in the nucleus.
  • the LNA-GapmeR used in the invention preferably comprises a sequence which is with increasing preference complementary to at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9.
  • These at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides are preferably complementary to a contiguous part of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9, i.e. the nucleotides are consecutive in the respective SEQ ID NO.
  • LNA-GapmeRs are routinely designed using established algorithms. LNA-GapmeRs to a selected target are commercially available including positive and negative controls, for example, from Exiqon.
  • AntimiRs are oligonucleotide inhibitors that were initially designed to be complementary to a miRNA. AntimiRs against miRNAs have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. As used herein, the AntimiRs are preferably designed to be complementary to a sequence selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9. AntimiRs are preferably 14 to 23 nucleotides in length.
  • An AntimiR according to the invention more preferably comprises or consists a sequence which is with increasing preference complementary to at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, or at least 23 nucleotides of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9.
  • At least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, or at least 23 nucleotides are preferably complementary to a contiguous part of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9, i.e. the nucleotides are consecutive in the respective SEQ ID NO.
  • AntimiRs are preferably AntagomiRs.
  • AntagomiRs are synthetic 2-O-methyl RNA oligonucleotides, preferably of 21 to 23 nucleotides which are preferably fully complementary to the selected target RNA. While AntagomiRs were initially designed against miRNAs they may also be designed against mRNAs.
  • the AntagomiRs used according to the invention therefore preferably comprise a sequence being complementary to 21 to 23 nucleotides of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably within SEQ ID NOs 7 and 9.
  • nucleotides are preferably complementary to a contiguous part of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9, i.e. the nucleotides are consecutive in the respective SEQ ID NO.
  • AntagomiRs are preferably synthesized with 2'-OMe modified bases (2'-hydroxyl of the ribose is replaced with a methoxy group), phosphorothioate (phosphodiester linkages are changed to phosphorothioates) on the first two and last four bases, and an addition of cholesterol motif at 3' end through a hydroxyprolinol modified linkage.
  • the addition of 2'-OMe and phosphorothioate modifications improves the bio-stability whereas cholesterol conjugation enhances distribution and cell permeation of the AntagomiRs.
  • Antisense molecules are preferably chemically synthesized using a conventional nucleic acid synthesizer.
  • Suppliers of nucleic acid sequence synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).
  • antisense molecules including antisense oligonucleotides, such as LNA- GapmeR, an Antagomir, an antimiR
  • siRNA and shRNA to potently, but reversibly, silence or inhibit a target mRNA in vivo makes these molecules particularly well suited for use in the medical applications of the invention being further described herein below.
  • Ways of administering siRNA to humans are described in De Fougerolles et al., Current Opinion in Pharmacology, 2008, 8:280-285. Such ways are also suitable for administering other small RNA molecules like antisense oligonucleotides or shRNAs.
  • compositions may be administered directly formulated as a saline, via liposome based and polymer-based nanoparticle approaches, as conjugated or complexation pharmaceutical compositions, or via viral delivery systems.
  • Direct administration comprises injection into tissue, intranasal and intratracheal administration.
  • Liposome based and polymer-based nanoparticle approaches comprise the cationic lipid Genzyme Lipid (GL) 67, cationic liposomes, chitosan nanoparticles and cationic cell penetrating peptides (CPPs).
  • Conjugated or complexation pharmaceutical compositions comprise PEI-complexed antisense molecules (including antisense oligonucleotides), siRNA, or shRNA.
  • viral delivery systems comprise influenza virus envelopes and virosomes.
  • the antisense molecules may comprise modified nucleotides such as locked nucleic acids (LNAs).
  • LNAs locked nucleic acids
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes.
  • LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired.
  • nucleic acid sequences as defined in items (a) to (c) of this preferred embodiment comprise or consist of sequences being complementary to nucleotides of the amino acids encoding the enzymes required for the activation of elF5a as defined by SEQ ID NOs 1 , 3, 5, 7 and 9, preferably by SEQ ID NOs 7 and 9.
  • the nucleic acid sequences as defined in items (a) to (c) comprise or are antisense nucleic acid sequences.
  • the nucleic acid sequence according to item (a) of this further preferred embodiment of the invention comprises or consists of a sequence which is with increasing preference complementary to at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides of one or more sequences selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9.
  • At least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, or at least 21 nucleotides are preferably complementary to a contiguous part of one or more selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9.
  • the format of the nucleic acid sequence according to item (a) is not particularly limited as long as it comprises or consists of at least 12 continuous nucleotides being complementary to a nucleic acid sequence selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9.
  • the nucleic acid sequence according to item (a) reflects the above- mentioned basic principle of the antisense technology which is the use of an oligonucleotide for silencing a selected target RNA through the extraordinar specificity of complementary-based pairing. Therefore, it is to be understood that the nucleic acid sequence according to item (a) is preferably in the format of an siRNA, shRNA or an antisense oligonucleotide as defined herein above.
  • the antisense oligonucleotides are preferably LNA-GapmeRs, AntagomiRs, or antimiRs as defined herein above.
  • a nucleic acid sequence according to item (b) of the above preferred embodiment of the invention is capable of interacting with, more specifically hybridizing with the target mRNA being selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9. By formation of the hybrid the function of the mRNA being selected from SEQ ID NOs 1 , 3, 5, 7 and 9 is reduced or blocked.
  • sequence identity of the molecule according to item (b) in connection with a sequence selected from SEQ ID NOs 1 , 3, 5, 7 and 9, preferably from SEQ ID NOs 7 and 9 is with increasing preference at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99% and 100%.
  • sequence identity in connection with each of SEQ ID NOs 1 , 3, 5, 7 and 9 can be individually selected. For instance, a non-limiting example is at least 85% in connection with SEQ ID NO: 7 and at least 90% in connection with SEQ ID NO: 9. Means and methods for determining sequence identity are known in the art.
  • the BLAST (Basic Local Alignment Search Tool) program is used for determining the sequence identity with regard to one or more of SEQ ID NOs 1 , 3, 5, 7 and 9.
  • nucleic acid sequences which comprise a nucleotide sequence which is at least 70% identical to the complementary strand of one or more of SEQ ID NOs 1 , 3, 5, 7 and 9 are the complementary strands of SEQ ID NOs 1 , 3, 5, 7 and 9.
  • the nucleotide sequences may be RNA or DNA.
  • RNA or DNA encompasses chemically modified RNA nucleotides or DNA nucleotides.
  • RNA comprises the nucleotide U while DNA comprises the nucleotide T.
  • the inhibitor may also be an expression vector or host, respectively being capable of producing an nucleic acid sequence as defined in any one of items (a) to (c).
  • An expression vector may be a plasmid that is used to introduce a specific transcript into a target cell. Once the expression vector is inside the cell, the inhibitor of the activation of elF5a being encoded by the expression vector is produced by the cellular-transcription.
  • the plasmid is in general engineered to contain regulatory sequences that act as enhancer and/or promoter regions and lead to efficient transcription of the transcript.
  • the expression vector preferably contains a macrophage-specific promoter. Macrophage-specific promoters are known in the art, for example, from Levin et a., Gene Ther., 2012; 19(11 ):1041-7. Using a macrophage-specific promoter ensures that the nucleic acid sequence is only expressed in macrophages and may avoid potential undesired side effects by expression in other cell types.
  • Non-limiting examples of expression vectors include prokaryotic plasmid vectors, such as the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMCI neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1 , pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, plZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-H
  • Examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pA0815, pPIC9K and pPIC3.5K (all Intvitrogen).
  • a suitable vector is selected in accordance with good manufacturing practice.
  • Such vectors are known in the art, for example, from Ausubel et al, Hum Gene Ther. 201 1 Apr; 22(4):489-97 or Allay et al., Hum Gene Ther. May 2011 ; 22(5): 595-604.
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Moreover, elements such as origin of replication, drug resistance gene, regulators (as part of an inducible promoter) may also be included.
  • the lac promoter is a typical inducible promoter, useful for prokaryotic cells, which can be induced using the lactose analogue isopropylthiol-b-D-galactoside ("IPTG").
  • IPTG lactose analogue isopropylthiol-b-D-galactoside
  • the polynucleotide of interest may be ligated between e.g.
  • PelB leader signal which directs the recombinant protein in the periplasm and the gene III in a phagemid called pHEN4 (described in Ghahroudi et al, 1997, FEBS Letters 414:521 -526). Additional elements might include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from retroviruses, e.g., RSV, HTLVI, HIVI, and the early promoter of the cytomegalovirus (CMV).
  • LTRs long terminal repeats
  • CMV cytomegalovirus
  • cellular elements can also be used (e.g., the human actin promoter).
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • the inhibitor can be expressed in stable cell lines that contain the gene construct integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
  • the transfected nucleic acid can also be amplified to express large amounts of the encoded (poly)peptide.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the nucleic acid molecule encoding the inhibitor.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al.1991 , Biochem J. 227:277-279; Bebbington et al. 1992, Bio/Technology 70:169-175). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected.
  • the expression vectors will preferably include at least one selectable marker.
  • vectors can contain one or more origins of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
  • origins of replication include, for example, the Col E1 , the SV40 viral and the M 13 origins of replication.
  • the sequences to be inserted into the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can be carried out using established methods.
  • Transcriptional regulatory elements parts of an expression cassette
  • These elements comprise regulatory sequences ensuring the initiation of the transcription (e.g., translation initiation codon, promoters, enhancers, and/or insulators), internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci.
  • nucleotide sequence as defined in item (a) of the above preferred embodiment of the invention is operatively linked to such expression control sequences allowing expression in prokaryotic or eukaryotic cells.
  • the host may be a prokaryotic or eukaryotic cell.
  • a suitable eukaryotic host may be a mammalian cell, an amphibian cell, a fish cell, an insect cell, a fungal cell or a plant cell.
  • Representative examples of bacterial cells are E. coli, Streptomyces and Salmonella typhimurium cells; of fungal cells are yeast cells; and of insect cells are Drosophila S2 and Spodoptera Sf9 cells. It is preferred that the cell is a mammalian cell such as a human cell.
  • Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1 , Cos 7 and CV1 , quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the cell may be a part of a cell line, preferably a human cell line. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • the host is preferably a host cell and more preferably an isolated host cell.
  • the host is also preferably a non-human host.
  • the compound is directed against ornithine decarboxylase, spermidine synthase, spermine synthase, deoxyhypusine synthase (DHPS) or deoxyhypusine hydroxylase (DOHH) and is preferably directed against DHPS or DOHH.
  • ornithine decarboxylase, spermidine synthase, spermine synthase, DHPS and DOHH are enzymes being required for the activation of elF5a.
  • the compound is directed against the mRNA encoding one of the enzymes or one of the enzymes in their protein from.
  • siRNA or shRNA it may be directed against the mRNA encoding one of the enzymes.
  • the compound is an antibody it may be directed against one of the enzymes in their protein form.
  • the compounds are preferably directed against the human mRNAs and enzymes.
  • the mRNA of human ornithine decarboxylase is shown in SEQ ID NO: 1 , of human spermidine synthase in SEQ ID NO: 3, of human spermine synthase in SEQ ID NO: 5, of human DOHH in SEQ ID NO: 7 and of human DDHPS in SEQ ID NO: 9.
  • the amino acid sequence of human ornithine decarboxylase is shown in SEQ ID NO: 2, of human spermidine synthase in SEQ ID NO: 4, of human spermine synthase in SEQ ID NO: 6, of human DOHH in SEQ ID NO: 8 and of human DHPS in SEQ ID NO: 10.
  • mRNAs SEQ ID NOs 7 and 9 and within the amino acid sequences SEQ ID NOs 8 and 10 are the preferred targets. This is because in the polyamine synthesis pathway the mRNAs of SEQ ID NOs 7 and 9 encode the enzymes catalyzing the last two chemical reactions being required for the activation of elF5a and the amino acid sequences of SEQ ID NOs 8 and 10 are the enzymes catalyzing the last two chemical reactions being required for the activation of elF5a. These enzymes catalyze the deoxyhypusiantion and the hypusination of elF5a.
  • the efficacy of a compound for inhibiting elF5a activation in a patient is determined, wherein the sample of step (a) and the control sample are samples that have been obtained from the patient, and wherein a reduction of the number of cells having a M2-phenotype in the sample as compared to the control sample not contacted with the compound indicates that the compound is effective for inhibiting elF5a activation in the patient.
  • the compound to be tested for its capability of inhibiting elF5a activation may also be a compound for which it is already known that it has the capability of inhibiting elF5a activation and in this case the method can determine how much inhibition is achieved by the compound.
  • This option of performing the claimed method is particularly suitable for determining the efficacy of a compound for inhibiting elF5a activation in a patient.
  • the first aspect of the present invention also encompasses a method for determining the efficacy of a compound for inhibiting elF5a activation in a patient, comprising (a) obtaining a sample comprising cells being capable of differentiating into M2 macrophages from a patient, (a) contacting the obtained sample with the compound under conditions that allow for the differentiation of cells into M2-type macrophages in the absence of the compound; and (b) quantifying in the sample the cells having a M2-phenotype, wherein a reduction of the number of cells having a M2-phenotype as compared to a control sample not contacted with the compound indicates that the compound has the capability of inhibiting elF5a activation.
  • a compound for inhibiting elF5a activation can be used to treat several diseases in a patient.
  • determining the efficacy of a compound for inhibiting elF5a activation in a patient allows to predict whether this compound will be effective to treat or prevent a disease in the patient, in particular a disease being mediated by M2 macrophages, or that can be treated by inhibiting cellular differentiation into M2 macrophages.
  • the patient is afflicted with a hyperproliferative disorder, a fibrotic disorder or the macrophage activation syndrome.
  • a hyperproliferative disorder is any disease comprising an abnormally high rate of proliferation of cells by rapid cell division.
  • Non-limiting examples are psoriasis, psoriatic arthritis, rheumatoid arthritis, cutaneous hyperkeratoses, inflammatory bowel disease tumors and cancers.
  • a fibrotic disorder is any disorder comprising the formation of excess fibrous connective tissue, e.g. in an organ or tissue in a reparative or reactive process.
  • Non-limiting examples of fibrotic disorders will be provided herein below.
  • MAS macrophage activation syndrome
  • SoJIA systemic-onset juvenile idiopathic arthritis
  • MAS has been described in association with systemic lupus erythematosus (SLE), Kawasaki disease, and adult-onset Still's disease. It is thought to be closely related and pathophysiologically very similar to reactive (secondary) hemophagocytic lymphohistiocytosis (HLH).
  • the hallmark clinical and laboratory features include high fever, hepatosplenomegaly, lymphadenopathy, pancytopenia, liver dysfunction, disseminated intravascular coagulation, hypofibrinogenemia, hyperferritinemia, and hypertriglyceridemia.
  • ESR erythrocyte sedimentation rate
  • the low ESR helps to distinguish the disorder from a flare of the underlying rheumatic disorder, in which case the ESR is usually elevated.
  • a bone marrow biopsy or aspirate usually shows hemophagocytosis.
  • M2 macrophages are known to secrete large amounts of pro-fibrotic factors, such as TGF- ⁇ and Galactin-3 (Braga et I. (2015), Front Immunol; 6:602 and Wermuth and Jimenez (2015), Clin Transl Med; 4: 2.). M2 macrophages are also known to promote cell proliferation (Mills (2012), Crit Rev Immunol.; 32(6):463-88.). It follows that M2 macrophages play an important role in promoting the formation or hyperproliferative or fibrotic disorders and interfering with the formation of M2 macrophages is a suitable means for treating or preventing a hyperproliferative or fibrotic disorders. In connection with the method of the invention it is particularly useful to determine the efficacy of a compound for inhibiting elF5a activation in a patient being afflicted with a hyperproliferative or fibrotic disorder.
  • pro-fibrotic factors such as TGF- ⁇ and Galactin-3
  • the sample is a tissue sample or a blood sample.
  • the sample has to be a sample that has been obtained from the body of the patient and has to comprise cells being M2 macrophages or capable of differentiating into M2 macrophages.
  • a sample is preferably a tissue sample or a blood sample. Macrophages are known to be distributed in tissues throughout the body, e.g. in the brain, liver, lung, spleen, intestine, skin, heart, kidney and peritoneum (Epelman et al.
  • tissue sample is preferably obtained from such a patient.
  • Macrophages are likewise found in the blood, including the peripheral blood.
  • the blood sample may be a whole blood sample, serum or plasma.
  • the compound is guanyl-1 ,7-diaminoheptane (GC7), L-mimosine, ciclopirox, deferiprone, hydralazine, agent I or CNI-1493.
  • GC7 guanyl-1 ,7-diaminoheptane
  • L-mimosine L-mimosine
  • ciclopirox deferiprone
  • hydralazine agent I or CNI-1493.
  • GC7 Guanyl-1 ,7-diaminoheptane (GC7), L-mimosine, ciclopirox, deferiprone, hydralazine, agent I and CNI-1493 are non-limiting examples of compounds interfering with an enzyme being involved in elF5a activation.
  • GC-7 Guanyl-1 ,7-diaminoheptane (CAS 150333-69-0) is an efficient inhibitor of deoxyhypusine synthase. As a competitive inhibitor it binds to the active site of deoxyhypusine synthase and prevents the first step in the post-translational conversion of a single lysine to hypusine in elF5A. GC-7 is used in the examples of the application.
  • L-mimosine (CAS 500-44-7) is a plant non-protein amino acid, chemically similar to tyrosine. The synthesis of the amino acid hypusine by deoxyhypusine hydroxylase is blocked by L- mimosine, further noting that hypusine is necessary for the activation of elF-5A.
  • the drugs ciclopirox (CAS 29342-05-0) and deferiprone (CAS 30652-1 1-0) are inhibitors of deoxyhypusine hydroxylase and hence the posttranslational modification by hypusine being requiring for the activation of elF-5A (Memin et al al. (2014), Cancer Res. 2014 Jan 15;74(2):552-62).
  • Hydralazine (CAS 86-54-4) inhibits the posttranslational hydroxylation of deoxyhypusine (Paz et al. (1984), Biochem Pharmacol, 33: 779-785).
  • Agent I Hoechst 768159, [2-(4-hydroxytoluene-3-yl)-4,5-dihydro-4- carboxythiazole] is an iron chelator that blocks the function of deoxyhypusine hydroxylase and hence the hypusination of elF5a; see Hanauske-Abel et al. (1994) Biochimica et Biophysica Acta 1221 115-124.
  • CNI-1493 or semapimod; CAS 164301-51 -3) is an inhibitor of deoxyhypusine synthase (Specht et al. (2008), Parasitol Res; 102(6): 1 177-84).
  • the present invention relates to a compound inhibiting elF5a activation for use in the treatment or prevention of a disease being mediated by M2 macrophages, wherein the disease is preferably a hyperproliferative, a fibrotic disorder or the macrophage activation syndrome.
  • the present invention likewise relates to a method for treating or preventing a disease being mediated by M2 macrophages, wherein the disease is preferably a hyperproliferative, a fibrotic disorder or the or the macrophage activation syndrome comprising administering a therapeutically effective amount of a compound inhibiting elF5a activation to a subject in need thereof.
  • the present invention relates to a compound inhibiting elF5a activation for use in the treatment or prevention of a hyperproliferative, a fibrotic disorder or the macrophage activation syndrome by inhibiting cellular differentiation into M2 macrophages.
  • the present invention also relates to a method for treating or preventing a hyperproliferative disorder, a fibrotic disorder or the macrophage activation syndrome by inhibiting cellular differentiation into M2 macrophages comprising administering a therapeutically effective amount of a compound inhibiting elF5a activation to a subject in need thereof, thereby inhibiting the cellular differentiation into M2 macrophages in the subject.
  • the subject to be treated in accordance with the second and third aspect of the invention is preferably a mammal, more preferably a primate and most preferably a human.
  • the compounds inhibiting elF5a activation are preferably admixed with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition.
  • a pharmaceutically acceptable carrier or excipient is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type (see also Handbook of Pharmaceutical Excipients 6ed. 2010, Published by the Pharmaceutical Press).
  • the compounds inhibiting elF5a activation or the pharmaceutical composition may be administered, for example, orally, parenterally, such as subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathecally, transdermally, transmucosally, subdurally, locally or topically via iontopheresis, sublingually, by inhalation spray, aerosol or rectally and the like in dosage unit formulations optionally comprising conventional pharmaceutically acceptable carriers or excipients.
  • parenterally such as subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathecally, transdermally, transmucosally, subdurally, locally or topically via iontopheresis, sublingually, by inhalation spray, aerosol or rectally and the like in dosage unit formulations optionally comprising conventional pharmaceutically acceptable carriers or excipients.
  • the compounds inhibiting elF5a activation may be formulated as vesicles, such as liposomes.
  • Liposomes have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. Liposomal delivery systems have been used to effectively deliver nucleic acids, such as siRNA in vivo into cells (Zimmermann et al. (2006) Nature, 441 :11 1-114). Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall.
  • Non-cationic liposomes although not able to fuse as efficiently with the cell wall, are phagocytosed by macrophages and other cells in vivo.
  • the compounds inhibiting elF5a activation can be administered to the subject at a suitable dose.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The therapeutically effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 g to 5 g units per day.
  • a more preferred dosage is in the range of 0.01 mg to 100 mg, even more preferably 0.01 mg to 50 mg and most preferably 0.01 mg to 10 mg per day.
  • said compound comprises or is an nucleic acid molecule, such as an siRNA
  • the total pharmaceutically effective amount of pharmaceutical composition administered will typically be less than about 75 mg per kg of body weight, such as for example less than about 70, 60, 50, 40, 30, 20, 10, 5, 2, 1 , 0.5, 0.1 , 0.05, 0.01 , 0.005, 0.001 , or 0.0005 mg per kg of body weight.
  • the amount will be less than 2000 nmol of nucleic acid molecule per kg of body weight, such as for example less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075 or 0.00015 nmol per kg of body weight.
  • the length of treatment needed to observe changes and the interval following treatment for responses to occur vary depending on the desired effect.
  • the particular amounts may be determined by conventional tests which are well known to the person skilled in the art. Suitable tests are, for example, described in Tamhane and Logan (2002), Journal of the American statistical association, 97(457):1-9.
  • M2 macrophages play an important role in promoting the formation or hyperproliferative or fibrotic disorders as well as the macrophage activation syndrome and therefore these diseases can be treated or prevented by interfering with the formation of M2 macrophages.
  • a compound inhibiting elF5a activation can be used to treat or prevent a disease being mediated by M2 macrophages or a hyperproliferative or fibrotic disorder or the macrophage activation syndrome by inhibiting cellular differentiation into M2 macrophages.
  • the finding that the formation of M2 macrophages can be inhibited by inhibiting the activation of elF5a also revealed that diseases that are mediated by M2 macrophages can be treated or prevented by inhibiting the activation of elF5a.
  • the compound targets ornithine decarboxylase, spermidine synthase, spermine synthase, deoxyhypusine synthase (DHPS) or deoxyhypusine hydroxylase (DOHH) and preferably DHPS or DOHH.
  • ornithine decarboxylase, spermidine synthase, spermine synthase, DHPS and DOHH are enzymes being involved in the activation of elF5a. Further details on how the compounds of the invention target these enzymes are provided herein above in connection with the corresponding embodiment of the first aspect of the invention. These details apply mutatis mutandis to the second and third aspect of the invention.
  • the compound is an antisense molecule, siRNA, shRNA, antibody, ribozyme, aptamer, or protein drug, small molecule.
  • the compound inhibiting elF5a activation may be any one of an antisense molecule, siRNA, shRNA, antibody, ribozyme, aptamer, protein drug and small molecule as defined herein above in connection with the first aspect of the invention.
  • the details on the compounds of the invention as provided herein above in connection with the corresponding embodiment of the first aspect of the invention apply mutatis mutandis to the second and third aspect of the invention.
  • the compound is guanyl-1 ,7-diaminoheptane (GC7), L-mimosine, ciclopirox, deferiprone, hydralazine, agent I or CNI-1493.
  • GC7 guanyl-1 ,7-diaminoheptane
  • L-mimosine L-mimosine
  • ciclopirox deferiprone
  • hydralazine agent I or CNI-1493.
  • the hyperproliferative disorder is a neoplasm, tumor or cancer and is preferably selected from cancer of the breast, lung, prostate, kidney, skin, neural, ovary, uterus, liver, pancreas, epithelial, gastric, intestinal, exocrine, endocrine, lymphatic, hematopoietic system or a head and neck tissue; and/or (ii) the fibrotic disorder is selected from sarcoidosis, renal fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, liver fibrosis, cardiac fibrosis, endomyocardial fibrosis, atrial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, chronic kidney disease, nephrogenic systemic fibrosis, Chron's disease, hypertrophic scarring, keloid
  • a neoplasm or tumor is an abnormal benign or malignant new growth of tissue that possesses no physiological function and arises from uncontrolled usually rapid cellular proliferation.
  • the WHO classifies neoplasms into four main groups: benign neoplasms, in situ neoplasms, malignant neoplasms, and neoplasms of uncertain or unknown behavior.
  • a malignant neoplasm is also referred to as cancer.
  • each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from.
  • a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I
  • the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C,
  • FIG. 1 The polyamine synthesis pathway. Polyamine synthesis begins with the production of putrescine from the amino acid ornithine, catalysed by ornithine decarboxylase. Spermidine and spermine are then synthesised from putrescine from spermidine synthase and spermine synthase, respectively.
  • DHS deoxyhypusine synthase
  • DOHH deoxyhypusine hydroxylase
  • FIG. 3 The deoxyhypusine synthase inhibitor GC7 blocks M2 macrophage differentiation
  • BMDMs Bone marrow-derived macrophages
  • IL-4 IL-4 in the presence or absence of 10 uM GC7.
  • M2 phenotype was assessed by flow cytometry analysis of CD301 and RELMa expression, two prototypic markers of M2 cells
  • BMDMs were differentiated into M1 cells with LPS and IFN-y in the presence or absence of 10 uM GC7. 18 hours later, the expression of nitric oxide synthase 2 (NOS2), a hallmark of M1 cells, was assessed by flow cytometry.
  • NOS2 nitric oxide synthase 2
  • GC7 disrupts oxidative phosphorylation in M2 macrophages.
  • BMDCs were polarised toward a M2 phenotype, as described above, in the presence or absence of 10 uM GC7.
  • oxygen consumption rate OCR - an indicator of OXPHOS was measured using a Seahorse Analyser.
  • the polyamine biosynthesis pathway regulates OXPHOS via hypusinated elF5A.
  • the polyamine pathway comprises the cationic metabolites putrescine, spermidine, and spermine that are synthesized downstream of the amino acid ornithine.
  • Spermidine acts as a substrate for the hypusination of elF5A, catalyzed by DHPS and DOHH.
  • DFMO inhibits ODC
  • DENSPM induces polyamine catabolism.
  • Both GC7 and CPX act as inhibitors of hypusination.
  • Relative ECAR of (c) GC7- and (d) CPX-treated MEFs (e) MEFs transduced with an IPTG-inducible E/ ' f5a-inducible shRNA; (f) Dhps itoxinox MEFs expressing a 4-OHT-inducible Cre-ER;
  • S2 cells D. melanogaster
  • MDCK C. familiaris
  • MCF-7 H. sapiens
  • FIG. 8 (a) ECAR of E/f5a-shRNA transduced MEFs at baseline, or in response to 2- deoxyglucose (2-DG), Oligo, FCCP, and R+A. (b) Fold increase in cell number of Eif5a- shRNA-expressing MEFs over 5 days of culture. Relative ECAR of (c) GC7- and (d) CPX- treated M(IL-4); (e-g) and M(IL-4) transduced with indicated shRNA. (h) Immunoblot analysis of specified proteins in M(IL-4) expressing control, Eif5a, Dhps or EG7-OVA cells expressing control or Dohh-shRNA.
  • Hypusinated elF5A maintains TCA cycle and ETC integrity in macrophages.
  • FIG. 10 (a) 13 C-palmitate, (b) D- 13 C-glucose, and (c) 3 C-glutamine GCMS trace analysis of specified metabolites in M(IL-4) ⁇ GC7 for 24 hours, (c-e) Isotopologue evaluation of indicated metabolites in 13 C-glutamine-labelled M(IL-4) ⁇ GC7, where d,e indicate oxidative and reductive metabolism, respectively. Red X indicates TCA cycle breaks from proteomics data. All data are mean ⁇ SEM (p* ⁇ 0.05, p** ⁇ 0.005, p** * ⁇ 0.0005). (a) Represents one experiment, (b-f) representative of two experiments.
  • FIG. 11 Immunoblot analysis of specified proteins in (a, b) M(IL-4) ⁇ 10 ⁇ GC7 for 22 hours; (c) M0, M(L/y), M(IL-4) (left panel) and M(IL-4) (right panel) ⁇ GC7; (d) EG7-OVA cells ⁇ DFMO/DENSPM for 48 hours; (e) M(IL-4) ⁇ DFMO for 48 hours; (f) M(IL-4) ⁇ 20 ⁇ CPX for 24 hours; (g) M(IL-4) expressing control, Dhps, or Eif5a shRNA. (h) OCR in M(IL-4) treated for 24 hours with GC7 ⁇ dimethylsuccinate.
  • FIG. 13 Expression of markers associated with alternative activation in M(IL-4) (a) ⁇ CPX, (b) ⁇ GC7, (c) expressing control or £/r ⁇ 5a-shRNA, (d) expressing control or Dhps-s R A, (e) expressing control or Dohft-shRNA. (f, g) Western blots of indicated proteins in M0, M(LJy), M(IL-4) ⁇ GC7 and (g) analysis of NOS2 expression by flow cytometry in M0, M(L/y) ⁇ GC7 (right panel).
  • Figure 14 (a) Activated and control OT-I CD8 + T cells ⁇ the indicated concentration of GC7 were assessed for proliferation on day 3 of culture, (b) CFSE proliferation analysis of OT-I CD8 + T cells treated with GC7 for the indicated time periods, (a-b) representative of one to three experiments.
  • FIG. 15 Mitochondrial metabolism is controlled by hypusinated elF5A-dependent translation of metabolic machinery, (a) Relative mRNA expression of indicated genes in GC7-treated M(IL-4). (b) HA-tagged elF5A construct was retrovirally transduced into MEFs and bound mRNAs were immunoprecipitated and submitted for microarray analysis against total RNA.
  • FIG. 16 (a) IDH2 MTS and MCM MTS target sequences fused to mCherry. (b) Representative histograms of indicated constructs ⁇ GC7, representative of six independent experiments.
  • elF5a and the polyamine pathway could be important for the alternative activation of macrophages (M2 cells) given that they express arginase, the enzyme that converts the amino acid arginine into ornithine, the precursor to polyamines.
  • M2 cells macrophages
  • arginase the enzyme that converts the amino acid arginine into ornithine, the precursor to polyamines.
  • GC7 N1-Guanyl-1 ,7-diaminoheptane
  • M2 macrophage differentiation is impaired in the absence of functional elF5a, unlike M1 cells which are unaffected (figure 3).
  • the action of GC7 also had profound effects on macrophage metabolism.
  • elF5a M2 cells engage oxidative phosphorylation (OXPHOS) to help drive their differentiation
  • OXPHOS oxidative phosphorylation
  • GC7 treatment significantly decreased OXPHOS in M2 cells (figure 4).
  • M1 metabolism was unaffected in the presence of GC7 (data not shown).
  • hypusinated elF5a was found to be an important factor in regulating macrophage differentiation and respiration.
  • the ability of compounds to inhibit elF5a hypusination can be determined by analyzing the quantity of elF5a-hypusine by western blot in untreated control cells versus treated cells.
  • figure 5 an example of this is shown - here, kidney cells were incubated with the deoxyhypusine synthase inhibitor GC7 and the quantity of elF5a-hypusine determined by western blot. GC7-treated cells showed a significant reduction in elF5a-hypusine levels.
  • Example 3 Exposure of murine embryonic fibroblasts (MEFs) to 2-difluromethylornithine (DFMO) and to diethylnorspermine (DENSPM)
  • Polyamines are synthesized in metabolically active cells 7 (Fig. 6a). To investigate how this pathway contributes to metabolism murine embryonic fibroblasts (MEFs) were exposed to 2- difluromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor 8 , and to diethylnorspermine (DENSPM), which activates the spermidine catabolising enzyme SSAT 9 .
  • DFMO 2- difluromethylornithine
  • ODC ornithine decarboxylase
  • DESPM diethylnorspermine
  • OCR oxygen consumption rates
  • ECAR an indicator of aerobic glycolysis
  • LCMS analysis of treated cells confirmed a drop in spermidine levels, whereas the upstream metabolite ornithine was unaffected (Fig. 6c).
  • An important cellular function of spermidine is to provide substrate for DHPS, the rate-limiting enzyme during elF5A H formation (Fig 6a).
  • M(IL-4) and LPS/IFN-y-activated macrophages [M(LPS/IFN-y)] do not proliferate to any appreciable extent in v/ ' fro 13,14 , it was possible to assess the role of polyamine biosynthesis on respiration dissociated from its known ability to regulate proliferation (Fig. 8b) 15 .
  • Inhibiting elF5A H either pharmacologically or genetically, blocked respiration in M(IL-4) (Fig. 9b-f, Fig. 8h), while again having differential effects on ECAR (Fig. 8c-g).
  • metabolites were analyzed by LCMS.
  • GC7-treated M(IL-4) (Fig. 9g, h) and £/75a-shRNA-expressing MEFs (Fig. 8i) displayed decreased metabolites associated with the first half of the TCA cycle, as well as additional alterations in other metabolic pathways (Fig. 8j, k).
  • M(IL-4) were cultured with 13 C-labeled glucose, glutamine, or palmitate and carbons were traced from these substrates into metabolites.
  • GC7-treated M(IL-4) incorporated significantly less carbon from 13 C-glucose into TCA cycle metabolites compared to control cells (Fig. 9i), indicating that DHPS function, and thus elF5A H , regulates TCA cycle engagement.
  • Subgroup column6 M IL4 is 2
  • TCA cycle enzymes in GC7-treated M(IL-4), as well as in resting macrophages (MO) and M(LPS/IFN-y) were assessed, by western blot.
  • TCA cycle enzymes in GC7-treated M(IL-4), as well as in resting macrophages (MO) and M(LPS/IFN-y) were assessed, by western blot.
  • succinyl-CoA synthetase Suclgl
  • SDH succinate dehydrogenase
  • CS citrate synthase
  • IDH isocitrate dehydrogenase
  • MCM methylmalonyl-CoA mutase
  • FIG. 11 b Other enzymes identified by proteomics analysis that feed substrates into the TCA cycle, such as methylmalonyl-CoA mutase (MCM), were also diminished after GC7 treatment (Fig. 11 b), while expression of many enzymes in glycolysis, fatty acid synthesis, and the aspartate-arginosuccinate shunt remained stable (Fig. 11c). Exposing M(IL-4) or lymphoma cells to polyamine synthesis inhibitors also resulted in TCA cycle enzyme deficiencies, as did treatment with CPX or expression of Eif5a and Dhps-s RNA (Fig. 11d- g)-
  • Proteomics data of GC7-treated M(IL-4) confirmed the down-regulation of many proteins associated with alternative activation, including CD301 (Table 2).
  • GC7 also diminished accumulation in vivo of IL-4 complex (IL-4c)-elicited macrophages in the peritoneal cavity (Fig. 12d).
  • Proteins in the polyamine-elF5A H axis were enriched in M(IL-4) compared to M(LPS/IFN-y) (Fig. 12e), supporting the notion that this pathway is dynamically regulated in these cells.
  • GC7 in vitro generated memory T cells cultured with GC7 augmented ECAR in response to restimulation, but could not increase OXPHOS (Fig. 12i, Fig. 13k).
  • GC7 also blocked T cell proliferation in a dose- dependent manner when present during naive T cell activation (Fig. 14a), but this effect was mitigated when GC7 was introduced days later (Fig. 14b), consistent with an initial requirement for OXPHOS to induce clonal expansion during T cell activation 17,18 .
  • LCMV-specific P14 T cells (gp33 + ) were transduced with a retrovirus expressing £/T5a-shRNA, which was introduced after activation, and adoptively transferred these cells into LCMV-infected recipients.
  • Donor cells after infection were tracked and it was found that while both control and £/75a-shRNA-transduced cells participated in the primary effector T cell response 8 days after infection, the frequency of £/75a-shRNA-transduced donor cells was decreased in the weeks after infection compared to control cells, indicating that the cells expressing £/T5a-shRNA were less able to form memory T cells (Fig 3j).
  • elF5A H regulates the phenotype of immune cells that rely on mitochondrial metabolism.
  • RNA-IP immunoprecipitated bound mRNAs
  • elF5A H facilitates translation elongation of difficult to translate proteins or motifs, such as those with proline, glycine, or charged amino acids, which can lead to ribosome stalling 4,5 .
  • mice specific for LCMV were purchased from Jackson Laboratories. All mice were bred and maintained under specific pathogen free conditions under protools approved by the Animal Welfare Committee of the Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany. Mice used in all in vitro and in vivo experiments were 6-10 weeks of age and were age/sex matched. For lymphocytic choriomeningitis virus (LCMV) infections, mice were immunised with 2x10 5 plaque forming units (PFU) LCMV Armstrong strain by intraperitoneal injection.
  • LCMV lymphocytic choriomeningitis virus
  • Bone marrow cells were differentiated for 7 days into bone marrow macrophages ( ⁇ ) by culturing in complete medium (RPMI 1640 media supplemented with 10% FCS, 2mM L- glutamine, 100 U/mL penicillin/streptomycin) with 20 ng/mL macrophage colony-stimulating factor (M-CSF; PeproTech).
  • M(IL-4) were generated with 20 ng/mL IL-4 overnight from day 7 of culture;
  • M(LPS/IFNy) were generated with 20 ng/mL LPS (Sigma) and 50 ng/mL IFN- ⁇ (R&D Systems) overnight from day 7 of culture. All drug treatments on ⁇ began from day 7 of culture.
  • N1-guanyl-1 7-diaminoheptane (GC7; Enzo Life Sciences) and ciclopirox (Sigma) were typically used at 10 ⁇ and 20 ⁇ , respectively, unless otherwise stated.
  • DHPS FIOXROX Cre-ER MEFs generated as previously described 31 , were cultured in complete DMEM (DMEM supplemented with 10% FCS, 2mM L-glutamine, 100 U/mL penicillin/streptomycin) and generated as previously described 31 , Cre-ER expression was induced with 1 ⁇ 4-OHT (Sigma) for the indicated time period.
  • NIH3T3 MEFs purchased from ATCC
  • E/ " f5a-shRNA expression was induced with 100 ⁇ isopropyl ⁇ -D-1 thiogalactopyranoside (IPTG, Sigma) for the indicated period of time.
  • Madin-Darby Kidney Canine (MDCK) cells were grown in complete DMEM, as was the human breast adenocarcinoma line MCF-7 but with 0.01mg/ml recombinant human insulin.
  • D. melanogaster Schneider 2 (S2) cells were cultured without C0 2 at 28°C in complete Schneider's Drosophila medium (Gibco; supplemented with 10% FCS, 50 U/mL penicillin/streptomycin, 25% conditioned complete Schneider's medium).
  • OTI splenocytes were activated with OVA peptide (SINFEKL, New England Peptide), or with anti- CD3 and anti-CD28 antibodies, with IL-2 (100 U/mL) in T cell media (RPMI 1640 media supplemented with 10% FCS, 2mM L-glutamine, 100 U/mL penicillin/streptomycin and 55 ⁇ ⁇ -mercaptoethanol) for the indicated length of time.
  • OVA peptide SINFEKL, New England Peptide
  • IL-2 100 U/mL
  • T cell media RPMI 1640 media supplemented with 10% FCS, 2mM L-glutamine, 100 U/mL penicillin/streptomycin and 55 ⁇ ⁇ -mercaptoethanol
  • OT-I splenocytes were activated with OVA-peptide and IL-2 (100 U/mL) for 3 days and subsequently cultured in the presence of either IL-2 or IL-15 (10 ng/mL), respectively, for an additional 3 days in TCM.
  • Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using the Seahorse XFe Bioanalyser (Seahorse Bioscience). 8x10 4 ⁇ were added to seahorse 96 well plates on day 7 of culture and analysed in XF media (non-buffered RPMI 1640 containing 25 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate) the following day after cytokine and drug treatment. For T cells, 2x10 5 were spun down on a poly-D-lysine-coated seahorse 96 well plate.
  • MEFs were plated at 4x10 4 cells per well of a 96 well seahorse plate in XF. Prior to analysis, cells were incubated for a minimum of 45 minutes at 37°C in the absence of C0 2 .
  • OCR and ECAR were measured under basal conditions, after restimulation with 50 ng/mL phorbal 12-myristate 13-acetate (PMA) and 0.5 ⁇ g/mL ionomycin, or after the addition of the following drugs: 1 ⁇ oligomycin, 1.5 ⁇ fluoro- carbonyl cyanide phenylhydrazone (FCCP), 100 nM rotenone, and 1 ⁇ antimycin A (all Sigma). Measurements were taken using a 96 well Extracellular Flux Analyser (Seahorse Bioscience).
  • Isotopomer distributions were measured using a DB5-MS GC column in a 7890 GC system (Agilent Technologies) combined with a 5977 MS system (Agilent Technologies). Correction for natural isotope abundance and calculation of fractional contribution was performed as described elsewhere 32 .
  • Metabolites were quantified by LC-MS using HILIC Chromatography on an Acquity UPLC BEH Amide column 1.7 ⁇ , 2.1x100 mm (polyamines) or a Luna NH2 column (all other metabolites) on a 1290 Infinity II UHPLC system (Agilent Technologies) combined with targeted detection in a 6495 MS system (Agilent Technologies). Peak areas were normalized to 13 C labelled internal standard (ISOtopic Solutions).
  • cells were washed with ice cold PBS and lysed in 1 x Cell Signaling lysis buffer (20 mM Tris-HCI, [pH 7.5], 150 mM NaCI, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ⁇ - glycerophosphate, 1 mM Na 3 V0 4 , 1 g/mL leupeptin (Cell Signaling Technologies), supplemented with 1 mM PMSF. Samples were frozen and thawed 3 times followed by centrifugation at 20,000 x g for 10 min at 4°C.
  • 1 x Cell Signaling lysis buffer 20 mM Tris-HCI, [pH 7.5], 150 mM NaCI, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -
  • All antibodies were from Cell Signaling Technologies except for anti-ABAT, Anti-ACC, anti- Aconitase 1 , anti-ASL, anti-DLD, anti-DOHH, anti-DHPS, anti-MCM, anti-ODC (Abeam), anti- elF5A (BD Bioscience), anti-hypusine (Merck-Millipore). Electron transport chain complexes were probed with the Total OXPHOS Rodent WB Antibody Cocktail (Abeam).
  • activated P14 splenocytes were transduced with luciferase (empty vector) or £ 5a-expressing retrovirus by centrifugation for 90 minutes in media containing hexadimethrine bromide (8 pg/mL; Sigma) and IL-2 (100 U/mL).
  • GFP was used as a marker for retroviral expression.
  • bone marrow cells exposed to M-CSF were transduced with luciferase (empty vector) or Eif5a-,Dhps-, or ⁇ -expressing retrovirus by centrifugation for 90 minutes in media containing hexadimethrine bromide on day 2 of culture.
  • Transduced cells were subsequently drug or cytokine-treated on day 7 of culture and assayed on day 8, sometimes following sorting on day 6.
  • GFP was used as a marker for transduction in these cells.
  • a G-block construct (IDT) containing mCherry fused to the degron from ODC 33 (HGFPPEVEEQDDGTLPMSCAQESGMDRH*) (mCherry* 39 ) was constructed to reduce half- life of the mCherry fusion protein. Between the cloning sites and mCherry a Gly-Ser-Gly-Ser- Gly flexible linker was included, to allow correct and independent folding of the introduced sequences and mCherry.
  • the mCherry deg was cloned into MSCV-I-GFP using Xhol and EcoRI.
  • MTS, NLS, or control sequences were ordered as G-blocks (IDT) or as phosphorylated oligos containg Xhol and BamHI compatible overhangs and cloned into MSCV-mCherry de9 -l-GFP using Xhol and BamHI.
  • the targeted sequences were as follows: MTS-IDH
  • MPKKKRKV (SEQ ID NO: 4) PP (MPPPP) (SEQ ID NO: 5)
  • IPTG-inducible MISSION shRNA lentiviral vector pl_KO-puro-IPTG-3xl_acO was purchased either with a shRNA against the 3 " -UTR of the murine elF5A mRNA sequence (custom-made from #SHCLND-NM181582-TRCN0000125229; Sigma) or a corresponding non-target shRNA control (#SHC332-1 EA; Sigma).
  • Stable transduction of the lentiviral was performed as previously described 34 using HEK293T cells, the packaging plasmids: pMDLg/pRRE (Gag/Pol), pRSV-Rev (Rev) and phCMV-VSV-G (envelope) as well as the ProFection Mammalian Transfection System Calcium Phosphate Kit (Promega). Positive cells were selected using puromycin.
  • MS raw files were analyzed by MaxQuant software and peak lists were searched against the mouse Uniprot FASTA database (concatenated with a database containing common contaminants) by the Andromeda search engine embedded in MaxQuant 38,39 .
  • MS1 -based label free quantification (LFQ) was done using maxLFQ algorithm 40 .
  • Perseus platform 41 was used to perform data filtering and statistical testing.
  • step 1 contaminant hits, reverse identification hits, and proteins "only identified by site” were removed from the dataset.
  • LFQ intensities were log 2 transformed.
  • Step 3 involved removal of missing quantitative data points to minimize the number of missing values in the dataset and this was followed by missing value data imputation using a normal distribution simulating the distribution of low abundant proteins in the dataset.
  • Student's T-test was utilized to define differentially expressed proteins employing a two-fold change as a cut-off at a 5% FDR.
  • Flow cytometric staining was performed as previously described 42 . All fluorochrome- conjugated monoclonal antibodies were from (eBioscience), except for anti-CD301 (BioRad). Both NOS2 and RELMa protein levels were quantified after fixation and permeabilisation using the transcription buffer staining set (eBioscience) and monoclonal antibodies against NOS2 (Santa Cruz) and RELMa (Peprotech). Cells were stained with Live/Dead viability dye (Thermo) prior to antibody staining. Cells were labelled with CFSE as described 43 .
  • P14 TCR transgenic T cells were identified in vivo and ex vivo using the congenic marker CD45.1 and H-2D b GP 33 -4i MHC Class I tetramer.
  • Cells were collected on LSR II and Fortessa flow cytometers (BD Biosciences) and analysed using FlowJo (TreeStar) software. Cells were sorted using a FACS Aria II. Cells were imaged using a Zeiss spinning disk confocal microscope with an Evolve (EMCCD) camera. Cells were kept in a humidified incubation chamber at 37°C with 5% C0 2 during image collection. Images were deconvolved and analysed using ImageJ (NIH).
  • EMCD Evolve
  • RNA isolations were done by using the RNeasy kit (Qiagen) and single-strand cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). All RT-PCR was performed with Taqman primers using an Applied Biosystems 7000 sequence detection system. The expression levels of mRNA were normalized to the expression of a housekeeping gene ( ⁇ -actin). For RNA-Seq, total RNA samples were extracted using RNeasy isolation kit (Qiagen).
  • Murine elF5A-1 open reading frame were PCR-amplified, fused with a C-terminal HA-tag and cloned into the pMSCV-puro vector (Clontech, pMSCV-elF-5A1-HA-puro).
  • the following primers were used for cloning:
  • NIH3T3 cells Ecotropic retroviruses for the transduction of NIH3T3 cells were obtained by transient calcium-phosphate-mediated transfection of the retroviral vectors into the packaging cell line Phoenix eco.
  • NIH-3T3 cells were transduced by adding the filtered retrovirus-containing supernatant and selected with puromycin.
  • 3.5x10 6 transduced cells were seeded in triplicates on a 100-mm 3 culture dish the day before.
  • Cells were homogenized in pre-cooled lysis buffer containing 100 mM KCI, 5 mM MgCI 2 , 10 mM HEPES, 0.5 % Nonidet-P40, 1 mM DTT, 100 U/mL Ribolock (Invitrogen) and 25 ⁇ /mL Protease Inhibitor Cocktail (Sigma). Lysates were incubated for 15 minutes, cellular debris was pelleted at 20,000 g for 15 minutes and the supernatants were incubated with 50 pL magnetic anit-HA-MicroBeads (Miltenyi Biotec) for 30 minutes on ice in the dark.
  • lysis buffer containing 100 mM KCI, 5 mM MgCI 2 , 10 mM HEPES, 0.5 % Nonidet-P40, 1 mM DTT, 100 U/mL Ribolock (Invitrogen) and 25 ⁇ /mL Protease Inhibitor Cocktail (Sigma). Lysates were incubated
  • RNA-protein- anti-HA-MicroBead complexes were eluted with lysis buffer containing 0.1 % SDS and 0.3 pg/pL proteinase K and incubated for 35 minutes at 50 C. Then the anti-HA-MicroBeads were removed by applying the solution to the ⁇ Columns and eluting the RNA containing fraction with DEPC-treated water. The RNA was cleaned and concentrated with the NucleoSpin RNA XS Kit (Macherey-Nagel).
  • RNA from the same cells were isolated as back ground control. RNA quantity and quality were evaluated by Nanodrop ND1000 Spectrophotometer and Agilent 2100 Bioanalyzer measurement. Procedures for cDNA synthesis, labelling and hybridisation were carried out according to the manufacturer's protocol (Affymetrix 3'-IVT Express Kit) starting with 100 ng of each RNA sample (three samples from immunoprecipitation and three samples of total RNA without immunoprecipitation). The experiments were performed using Affymetrix Mouse Genome 430 2.0 GeneChip. All reactions were performed in triplicates. The signals were processed with a target value of 300 using Affymetrix GeneChip Operating Software 1.4.
  • P14 TCR transgenic CD45.1 T cells were activated in vitro with gp33-41 peptide and transduced with E/ " f5a-shRNA.
  • a day later 5x10 5 T cells were transferred into CD45.2 C57BL/6 congenic recipient mice on day one of infection. Blood samples were collected at the indicated time points and analysed by flow cytometry.
  • MaxLFQ normalization and maximal peptide ratio extraction

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Abstract

La présente invention concerne un procédé permettant de déterminer si un composé a la capacité d'inhiber l'activation de l'elF5a, comprenant (a) la mise en contact d'un échantillon comprenant des cellules susceptibles de se différencier en macrophages M2 avec le composé dans des conditions qui permettent la différenciation de cellules en macrophages de type M2 en l'absence du composé ; et (b) la quantification dans l'échantillon des cellules présentant un phénotype M2, une réduction du nombre de cellules présentant un phénotype M2 par comparaison avec un échantillon témoin non mis en contact avec le composé indiquant que le composé a la capacité d'inhiber l'activation elF5a.
PCT/EP2018/053913 2017-02-16 2018-02-16 Activation alternative de macrophages (cellules m2) par l'intermédiaire d'un axe polyamine-eif5a-hypusine WO2018149978A1 (fr)

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WO2020227385A1 (fr) * 2019-05-07 2020-11-12 The Regents Of The University Of California Composés et méthodes de traitement ou de prévention de la fibrose
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WO2020227385A1 (fr) * 2019-05-07 2020-11-12 The Regents Of The University Of California Composés et méthodes de traitement ou de prévention de la fibrose
CN113133995A (zh) * 2021-05-09 2021-07-20 河南牧业经济学院 抑制剂cni-1493在猪繁殖与呼吸综合征中的应用
RU2810558C1 (ru) * 2023-05-02 2023-12-27 федеральное государственное автономное образовательное учреждение высшего образования "Казанский (Приволжский) федеральный университет" (ФГАОУ ВО КФУ) Способ подбора лекарственных средств для реализации фармакологической индукции митохондриальной дисфункции в макрофагах для противоопухолевой терапии

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