WO2012107724A1 - Mir-30 destiné à être utilisé dans la modulation de l'angiogenèse - Google Patents

Mir-30 destiné à être utilisé dans la modulation de l'angiogenèse Download PDF

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Publication number
WO2012107724A1
WO2012107724A1 PCT/GB2012/000131 GB2012000131W WO2012107724A1 WO 2012107724 A1 WO2012107724 A1 WO 2012107724A1 GB 2012000131 W GB2012000131 W GB 2012000131W WO 2012107724 A1 WO2012107724 A1 WO 2012107724A1
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mir
polynucleotide
dll4
nucleotide sequence
family
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PCT/GB2012/000131
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English (en)
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Chris Boshoff
Gemma BRIDGE
Victoria EMUSS
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Ucl Business Plc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the invention relates to a microRNA (miRNA) family for use in the treatment of cancer and other conditions, particularly those in which angiogenesis plays a role. Also provided are methods for producing the miRNA, molecules which affect the interaction of the miRNA and its target and the use of such molecules.
  • miRNA microRNA
  • Delta-like 4 is a membrane-bound ligand belonging to the Notch signaling family, which plays a fundamental role in vascular development and angiogenesis (Gridley, 2010; Phng and Gerhardt, 2009). DLL4 haploinsufficiency results in extensive arterial defects and embryonic lethality (Gale et al., 2004) indicating that the developing vasculature is sensitive to minor alterations in DLL4 dosage. Its expression is mainly restricted to the endothelium of nascent vessels, particularly the tip cells, where it maintains stalk cell identity in neighbouring cells, thereby regulating vessel sprouting and branching in response to angiogenic stimuli (Hellstrom et al., 2007).
  • dll4 deficient zebrafish When dll4 deficient zebrafish are examined at 2.5 days post-fertilisation (dpf) they have reduced circulation in the dorsal aorta (DA) and post cardinal vein (PCV) and blood flow is almost undetectable in the majority of ISVs (Leslie et al., 2007). Closer examination of these morphant embryos revealed that an aberrant network of vessels had replaced the normal T-junction between each ISV and the dorsal longitudinal anastomotic vessel (DLAV). The regulatory role fulfilled by DLL4 during physiological angiogenesis is also relevant in pathological settings (Lobov et al., 2007).
  • DLL4 expression is increased in human tumors, often in association with markers of inflammation, hypoxia and angiogenesis (Jubb, 2009; Jubb, 2010; Martinez, 2009; Patel et al., 2005; Patel, 2006), and inhibition of DLL4 in several tumor models blocks tumor growth by promoting non-productive, deregulated angiogenesis (Haller et al., 2010; Noguera-Troise et al., 2006; Oishi et al., 2010; Ridgway et al., 2006).
  • KSHV Kaposi's sarcoma herpesvirus
  • MiRNAs are small, non-coding RNAs that influence target gene expression through mRNA degradation and translation inhibition (Carthew and Sontheimer, 2009). Implicated in many key cellular and physiological processes, miRNAs are known to play a role in angiogenesis and cancer (Croce, 2009; Wang and Olson, 2009). The inventors have previously described the miRNA signature in LEC following KSHV infection (Lagos et al., 2010). Further examination of these data indicated significant downregulation of members of the miR-30 miRNA family after infection; we also observed that this family is amongst the most robustly expressed in LEC, suggesting suppression by KSHV could have functional implications.
  • miR-30 Encoded by six genes and expressed from four different transcripts across the human genome, the members of the miR-30 family share an identical seed sequence and hence have common predicted targets (Grimson et al., 2007).
  • miR-30 especially miR- 30b and miR-30c, target DLL4 both in vitro and in vivo.
  • overexpression of these miRNAs in the developing zebrafish embryo partially phenocopies dll4 knockdown and the described phenotypes have been shown to occur by way of dll4 targeting.
  • the inventors realised that the miR-30 family can be used to control angiogenesis and to treat conditions in which angiogenesis plays a role.
  • the invention provides a polynucleotide comprising a nucleotide sequence having substantial homology to the nucleotide sequence ACAAAUGU for use in the modulation of angiogenesis.
  • the polynucleotide is preferably a RNA molecule.
  • the polynucleotide could be a shRNA, a siRNA, but is preferably a miRNA.
  • the polynucleotide may be a polynucleotide which can processed to produce a miRNA, such as a pri-miRNA or pre-miRNA.
  • polynucleotide is also considered, herein, to encompass any molecule which has a base sequence with a structure similar to that of DNA or RNA so that the base sequence of the molecule can base pair with a complementary base sequence such as an oligodeoxyribonucleotide or an oligoribonucleotide, a phosphorodiamidate morpholino oligonucleotide (PMO), a 2'-0-methyl (2'OMe) oligonucleotide, a locked nucleic acid (LNA) or a peptide nucleic acid (PNA), oligonucleotides containing phosporothioate bonds, 2'-fluoro oligonucleotides, hexitol nucleic acid, 2'-0-methoxyethyl oligonucleotide, 2'-0-allyl oligonucleotide, 2'-0-propyl oligonucleotide, 2'-0-p
  • the polynucleotide is preferably between 20 and 30 bases in length and more preferably between 21 and 26 bases in length. It is particularly preferred that the polynucleotide is 21, 22 or 23 bases in length. In this embodiment, the polynucleotide is designed to be structurally similar to a naturally occurring miRNA.
  • the polynucleotide may be double stranded and thereby comprise a complementary second base sequence.
  • the two sequences may or may not be connected by a stem-loop like structure in the polynucleotide. If the two sequences are connected by a stem- loop structure the polynucleotide will have a similar structure to pre-miRNA or pri-miRNA. As a result, the polynucleotide can be processed to form a miRNA-like molecule by the Drosha, Pasha (DCGR8) and Dicer proteins. If the polynucleotide has a structure which is similar to pre-miRNA, the polynucleotide will preferably be between about 60 and about 80 bases in length.
  • the polynucleotide has a structure which is similar to pri-miRNA, the polynucleotide will preferably be between about 500 and about 2 kb in length and more preferably, between about 800 and about 1.2 kb in length. Alternatively, the two sequences may simply be held together by the base pairing between the sequences but are not connected in any way. This structure is similar to a miRNA/miRNA* duplex ("*" denotes the antisense or complementary sequence). If the polynucleotide has a structure which is similar to a miRNA/miRNA* duplex, each strand of the molecule will preferably be between 20 and 30 bases in length.
  • the polynucleotide comprises a nucleotide sequence having substantial homology to the sequence ACAAAUGU. In particular, it preferably comprises that nucleotide sequence, or one which differs by only one or two nucleotides.
  • the nucleotide sequence ACAAAUGU is preferably found at one end of the polynucleotide molecule, within the first or last 12 nucleotides, more preferably within the first or last 10 nucleotides. Most preferably it provides the first or last 8 nucleotides of the polynucleotide. It may be found at the 5' or 3' terminal, but is preferably at the 5' terminal such that the polynucleotide comprises a U as its final nucleotide at the 5' terminal.
  • the nucleotide sequence ACAAAUGU comprises the seed sequence of miR-30 and binds to a region within DLL4 mRNA.
  • the seed sequence of miR- 30 is CAAAUG, and it is particularly preferred that the polynucleotide comprises this sequence.
  • the polynucleotide preferably comprises additional nucleotides found in the sequences of miR-30 family.
  • the polynucleotide preferably comprises one or more of the nucleotides which flank the ACAAAUGU sequence in the miR-30 family sequences. Such nucleotides are preferably found at equivalent positions within the polynucleotide of the invention.
  • the polynucleotide preferably comprises the sequence CCU immediately 3' of the ACAAAUGU homologous sequence.
  • the polynucleotide may comprise additional nucleotides from the miR30 family sequences, again in corresponding locations.
  • the polynucleotide preferably comprises one or both of the nucleotides found at positions 12 and 15 in the miR-30 family sequences, especially of miR-30a, c, d or e, at a position an equivalent distance from the ACAAAUGU homologous sequence.
  • the nucleotide is preferably U or C.
  • the nucleotide is preferably A.
  • polynucleotide of the invention preferably comprises one of the following sequences:
  • X is any nucleotide.
  • the polynucleotide is indicated as containing the base uracil (U) since the sequences in these tables relate to miRNAs. However, as will be appreciated by one skilled in the art, U can be replaced with the base thymine (T). Whether the base T or U is selected will depend on the type of molecule containing the sequence. For example, if the molecule is a DNA molecule or a PMO, the base may be T whereas if the molecule is a RNA molecule, the base may be U.
  • the molecule of the invention is not limited to a sequence containing U but can also comprise a sequence containing T since the function of the base at these positions is to bind to the base A, a function which both U and T can fulfil.
  • the polynucleotide of the invention can be used to bind to DLL4 mRNA and to suppress its activity. It binds in a manner similar to the binding of the miR-30 family.
  • the polynucleotide is preferably based on the nucleotide sequence of the miR-30 family.
  • It preferably comprises, and may consist of, a nucleotide sequence having at least 50%, more preferably at least 55%, preferably at least 57%, more preferably at least 60%, even more preferably at least 70%, more preferably at least 80%, more preferably at least 90% homology with one of the miR-30 family sequences, particularly miR-30b or miR-30c, especially miR-30c, which are provided in figure 1C.
  • the polynucleotide preferably comprises at least 3, more preferably at least 4, 5, 6 or 7 nucleotides that are complementary to the nucleotides in DLL4 mRNA, outside of the seed sequence binding region.
  • the polynucleotide may comprise or consist of the nucleotide sequence of one of miR-30 a, b, c, d or e.
  • the polynucleotide preferably shows similar binding affinity, such as at least 65%, more preferably at least 70%, more preferably at least 75% even more preferably at least 80% of the binding affinity of any of the miR-30 family for DLL4 mRNA. It particularly preferably shows similar binding affinity to that of miR-30b or c for DLL4 mRNA.
  • the polynucleotide preferably comprises the nucleotides required to direct RISC to bind the DLL4 mRNA, as occurs with the miR-30 family.
  • complementary means that the majority of the bases in a first sequence are complementary to a second sequence. However, the two sequences will still be able to base pair if there are a small number of mismatched bases or a small "bulge" of non-paired bases in the first sequence. For example, if there are five or fewer mismatched bases or a bulge of five or fewer bases, the two base sequences should still be able to base pair. Preferably, there is no "bulge" of non-paired bases. Preferably, there are four or fewer mismatched bases, more preferably, three or fewer mismatched bases, even more preferably, two or fewer mismatched bases, more preferably still, one or fewer mismatched bases and, most preferably, no mismatched bases. Any mismatched bases etc are preferably found outside the seed region.
  • the polynucleotide is isolated so that it is substantially free from other compounds or contaminants.
  • the polynucleotide may be conjugated to or complexed with an entity, especially an entity which helps target the polynucleotide to the required site of action.
  • a vector comprising a polynucleotide as previously described, for use in the modulation of angiogenesis.
  • the vector may comprise components required for expression of the polynucleotide in a mammalian cell.
  • a vector comprising a promotor or repressor of miR-30 for use in the modulation of angiogenesis. Any appropriate vector can be used, including, for example, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector.
  • a cell comprising the vector of the invention, especially a mammalian, bacterial or insect cell. The cell is preferably not a human embryonic stem cell.
  • compositions comprising one or more of the polynucleotides or one or more or the vectors described previously and a pharmaceutically acceptable carrier or excipient.
  • the composition may comprise a carrier which enables the polynucleotide to be delivered to the relevant site for use.
  • the carrier may target a particular site or otherwise improve delivery to that site.
  • the pharmaceutical composition comprises a polynucleotide, it may also comprise an excipient which stabilises the polynucleotide.
  • Such stabilisers are well known in the art. Any appropriate stabiliser may be used.
  • compositions of this invention comprise any of the molecules of the present invention, and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the pharmaceutical compositions are administered orally or by injection.
  • the pharmaceutical compositions may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the route of administration of the composition is transdermal or intrathecal administration.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol.
  • suitable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions.
  • carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavouring and/or colouring agents may be added.
  • the pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration.
  • compositions can be prepared by mixing a molecule of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
  • Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application.
  • the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier.
  • Carriers for topical administration of the molecules of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention.
  • compositions of this invention may be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilising or dispersing agents known in the art.
  • an agent which increases the expression of one or more members of the miR-30 family for use in the modulation of angiogenesis.
  • a related aspect of the invention provides a method of modulating angiogenesis comprising administering one or more of a polynucleotide, a vector, another agent that alters the expression of one or more members of the miR-30 family or a pharmaceutical composition as described to a subject.
  • an agent that reduces the expression of one or more members of the miR-30 family or interferes with the interaction between one or more members of the miR-30 family and DLL4 mRNA for use in the modulation of angiogenesis.
  • Agents that increase or reduce the expression of one or more members of the miR-30 family include agents that increase or reduce transcription activators or inhibitors of miR-30. Also encompassed are agents that increase or reduce the processing of precursors of miR-30 family members into mature miRNA
  • Any agent that reduces the expression of one or more members of the miR-30 family may be used, such as a vector as described previously comprising a repressor of one or more members of the miR-30 family.
  • An agent that interferes with the interaction between one or more members of the miR-30 family and DLL4 mRNA is any agent that reduces the actual interaction between one or more members of the miR-30 family and DLL4 mRNA or any agent that reduces the effectiveness of such interaction.
  • agents include agents that compete with the miR-30 family for DLL4 mRNA binding, such as other polynucleotides that bind to DLL4 mRNA without suppressing DLL4 mRNA activity.
  • a target protector polynucleotide which binds to DLL4 mRNA without suppressing its activity.
  • Such a target protector preferably comprises at least 22, more preferably at least 23, more preferably at least 24, more preferably at least 25 nucleotides, including the nucleotide sequence TGTAAACA.
  • it preferably comprises the sequence TGTAAACA and at least 15, 16 or 17 other nucleotides which specifically bind to DLL4 mRNA.
  • it preferably comprises 25 contiguous nucleotides found in the following sequence:
  • the target protect may comprise the sequence: TGTAAACAATGCAGAAGGAAGGTC or ACCCATCCAGGATGCAATGTAAACA.
  • agents that interfere with the interaction between one or more members of the miR-30 family and DLL4 mRNA include agents that bind to one or more members of the miR-30 family or otherwise target one or more members of the miR-30 family to prevent binding to DLL4 mRNA.
  • agents might include polynucleotides that bind specifically to one or more members of the miR-30 family, such as anti-miRNA oligonucleotides; other binding molecules such as antibodies against one or more members of the miR-30 family; and agents that break down one or more members of the miR-30 family.
  • the agent may act as a sponge to mop up one or more members of the miR-30 family and reduce the interaction between one or more members of the miR-30 family and DLL4 mRNA.
  • a further aspect provides a method of modulating angiogenesis comprising administering an agent that reduces expression of one or more members of the miR-30 family or interferes with the interaction between one or more members of the miR-30 family and DLL4 mRNA to a subject.
  • polynucleotides, vectors and agents described herein are useful for the modulation of angiogenesis. This allows them to be used in the treatment of various conditions, including cancer and vascular conditions, such as schema related conditions.
  • Any cancer can be treated, particularly cancers in which a solid tumour is present or likely to develop.
  • Leukaemias may also be treated.
  • Vascular conditions include conditions in which the vasculature is disordered, whether because of the presence of abnormal vessels or an excessive number of vessels or due to the loss of normal vessels or their function.
  • Vascular conditions and ischemia related conditions can occur in various parts of the body. It is particularly relevant to treat eye disorders such as retinopathy of prematurity, ischemic retinopathy, retinal vein or artery occlusion, diabetic retinopathy, choroidal neovascularization, age related macular degeneration, corneal neovascularization, neovascular glaucoma or corneal transplantation. More broadly, though, treatment of any condition caused by or related to disordered vasculature or ischemia is envisaged.
  • any ischemic disease or condition caused by insufficient blood supply due to blood vessel loss and/or poor perfusion for example ischemic injury, cerebral ischemia, cardiac ischemia, ischemic conditions affecting the limbs and other organs or tissues, arteriovenous malformations, wound healing, organ or tissue transplantation, placental insufficiency, arterial narrowing and occlusion, atherosclerosis, and systemic or pulmonary hypertension is provided by the invention.
  • It is preferable to treat cancer and ischemic retinopathy by administering one of the polynucleotides of the invention, one of the vectors of the invention which increases expression of one of the polynucleotides of the invention or by increasing the expression of miR-30.
  • angiogenesis may be modulated as appropriate for the condition in question.
  • the polynucleotides, vectors and agents described herein may be for administration with other agents that complement or enhance their activity.
  • agents and polynucleotides that are for use in suppressing DLL4 activity can be administered with DLL4 antibodies or other antagonists. Alternatively they may be administered with agents that otherwise modulate angiogenesis, such as agents that modulate VEGF.
  • miR-30 when used herein, it may refer to any member of the miR-30 family, miR-30a, b, c, d or e. It can preferably mean miR-30a, c, d or e, especially miR-30c.
  • KSHV regulates expression of the miR-30 family, which is predicted to target
  • DLL4 DLL4.
  • A Heatmap representing relative changes in expression of hsa-miR-30 family members in LEC following KSHV infection. miRNAs are ordered alphabetically. Original GEM data from (Lagos et al., 2010) **, £ ⁇ 0.01 ; ***, gO.001. Black and white denote low and high expression respectively.
  • B Downregulation of miR-30b and miR-30c in KLEC, confirmed by qRT-PCR. Columns are the average of three independent experiments, expression is relative to LEC. Values are shown as mean + standard error of the mean (SEM). **, PO.01.
  • C Complementarity between miR-30 family members and the DLL4 3'UTR.
  • Solid lines indicate canonical Watson and Crick base-pairing, dashed lines indicate G:U wobbles.
  • the predicted target site within the DLL4 3'UTR, positions 59-66, is shown in bold, the miR-30 seed region is shown in bold italics.
  • FIG. 1 miR-30b and miR-30c target DLL4.
  • A Mean fold-change in DLL4 mRNA in LEC transfected with hsa-miR-30b or hsa-miR-30c mimics. The unrelated mRNA, E- selectin, is used as a control for global mRNA suppression. Columns are the average of four independent experiments. Expression measured by qRT-PCR relative to non-targeting control (NTC) mimic. ***, PO.001.
  • FIG. 3 Regulation of DLL4 by miR ⁇ 30b and miR-30c has relevance in pathophysiological settings.
  • A Mean fold-change in DLL4 mRNA in LEC or KLEC transduced with hsa-miR-30b- and hsa-miR-30c-expressing lentiviruses. Columns are the average of three independent experiments. *, PO.05; **, PO.01; ***, PO.001.
  • B Left- hand panels, representative photograph at indicated time points of HUVEC spheroids embedded in Matrigel, taken at 5x magnification in phase contrast. HUVEC were transfected with mimic before being induced to form spheroids through the hanging drop method.
  • FIG. 4 miR-30b and miR-30c overexpression induce aberrant endothelial cell behaviour in vivo.
  • dll4 and dre-miR-30b and dre-miR-30c expression correlate during the period immediately prior to, and during intersegmental vessel formation in the developing zebrafish embryo. Time points are shown as hours post fertilisation (hpf). Expression is relative to the shield stage of development (6hpf). RNA was obtained from whole embryos at each time point.
  • C Graph describing the relative size of the dorsal aorta (DA) in zebrafish embryos injected with dll4 MO or miR-30 mimic compared to wt embryos. Column values are the average of three embryos per condition. Six DA measurements were made for each embryo. ***, PO.001. Error bars indicate column values + SEM.
  • (A) Downregulation of DLL4 mRNA by miR-30 mimics can be titrated. Triangles indicate increasing concentrations of mimics: lOnM, 20nm, 40nM, 80nM and lOOnM. Columns are the average of three independent experiments. Expression is relative to NTC mimic at the same concentration. **, PO.01, ***, O.001.
  • LEC were purchased from PromoCell and cultured as previously described (Lagos et al., 2007).
  • HUVEC were purchased from PromoCell and cultured in MV2 (PromoCell).
  • BCBL-1 cells latently infected with recombinant GFP-KSHV (Vieira et al., 2001) were cultured as previously described (Vart et al., 2007).
  • 293T and human fibroblast cells were grown in Dulbecco modified Eagle medium (Invitrogen), supplemented with 10% FBS.
  • LEC, HUVEC or 293T were seeded in 6- or 12-well plates respectively, 16 h prior to transfection using Oligofectamine (Invitrogen), as per the manufacturer's instructions.
  • miRIDIAN miRNA Mimics for hsa-miR-30b, hsa-miR-30c and the Negative Control #1 (non-targeting control) (Thermo Fischer Scientific) were transfected at ⁇ , unless otherwise specified. Cells were harvested, utilised for the hanging drop assay or transfected with luciferase reporter plasmids 48h post-mimic transfection.
  • miRIDIAN miRNA Inhibitors (ThermoScientific) were transfected into cells ( ⁇ ) following the same protocol as mimic transfection.
  • LEC or HUVEC were lysed in Pierce M-PER buffer (ThermoScientific). Protein was quantified using Pierce BCA Protein Assay (ThermoScientific) and equal concentrations of protein were resolved on a 10% polyacrylamide gel. Antibodies against DLL4 (#2589, Cell Signaling Technology) and GAPDH (Monoclonal 6C5, Advacned Immunochemical Inc) were detected with HRP-conjugated secondary antibodies and were quantified using ECL or ECL Plus (GE Healthcare). Lentivirus production and infection of LEC
  • Genomic fragments containing pre-miR-30b and pre-miR-30c-l were cloned from LEC and were expressed using a modified pSIN-MCS lentiviral vector as described (Vart et al., 2007). The number of lentiviral copies per cell was determined by qPCR and miRNA expression was confirmed by RT-PCR. Experiments were performed 2-3 days post-lentivirus infection. qPCR and qRT-PCR
  • Genomic DNA for qPCR was extracted using the QIAamp DNA mini-kit (Qiagen). The number of lentiviral copies per cell (c/c) was determined as described previously (Vart et al., 2007).
  • Total RNA was extracted using miRNeasy mini-kit (Qiagen) and subjected to DNase I treatment (Qiagen). About 50 to 1,000 ng of total RNA was used for cDNA synthesis using the Superscript II reverse transcriptase (Invitrogen). GAPDH (housekeeping reference gene) and E-selectin mRNA levels were quantified by qRT-PCR using optimized forward and reverse primers and SYBR Green PCR Master Mix (Applied Biosystems).
  • qRT-PCR quantification of DLL4, dll4, bactinl, hsa-miR-30b and hsa-miR-30c was performed using Taqman Gene Expression or Taqman MicroRNA assays (Applied Biosystems). Quantification of pre-miR-30c-l and pre-miR-30c-2 was performed using miRNA qRT-PCR Kit and Primer Set (GenoExplorer).
  • the reporter plasmids 50ng, either empty vector (pEZX-MTOl) or the DLL4 3'UTR containing plasmid (pEZX-DLL4), were transfected into 293T cells, 48h post-transfection with miRNA mimic. Cells were harvested 24h post-transfection according to the Dual- Luciferase Reporter Assay System (Promega). Luciferase activity was measured using a Fluoroskan Ascent FL luminometer (ThermoScientific). Firefly activity was normalised to internal renilla luciferase levels. Spheroid-based sprouting angiogenesis assay
  • HUVEC were transfected with miRNA mimic (ThermoScientific) and after 24h spheroids were generated as previously described ⁇ Alajati, 2008 77 /id ⁇ .
  • miRNA mimic ThermoScientific
  • Zebrafish embryos were obtained by natural spawning of adult zebrafish. Embryos were raised and maintained at 28.5°C in system water and staged as described ⁇ Westerfield, 1993 283 id ⁇ . Tg(kdrkGFP) ⁇ Beis, 2005 285 /id ⁇ zebrafish were used to examine the developing vasculature. Antisense morpholinos (MOs) (GeneTools) and miRNA mimics (ThermoScientific) were injected into 1- to 4- cell stage embryos.
  • MOs Antisense morpholinos
  • miRNA mimics ThermoScientific
  • the MOs used in this work were dlI4-M01 ⁇ Siekmann, 2007 8 /id ⁇ (5ng) and dll4-TP miR - 30 ( Figure S4D) (lOng). miRNA mimics were injected in the quantities stated. In situ hybridization was performed as described ⁇ Gering, 2005 284 /id ⁇ . RNA probes were labelled with digoxigenin (Roche) and detected using BM Purple (Roche). Endothelial cells were visualised in Tg(kdrl:GFP) embryos using UV light.
  • KSHV regulates expression of a miRNA family predicted to target DLL4
  • the human miR-30 family members are encoded by six genes and expressed from four transcripts, but share an identical seed sequence and therefore have common predicted targets (Grimson et al., 2007).
  • TargetScan software To establish a role for miR-30 in LEC the inventors used TargetScan software to identify these targets and ranked them according to total context score (Grimson et al., 2007). They analyzed this list with respect to genes significantly altered in LEC and discovered that the 3'UTR of the Notch ligand DLL4, one of the most significantly upregulated genes in KLEC (Emuss et al., 2009), scored favorably (total Context Score - 0.39).
  • the inventors repeated the TargetScan analysis using the DLL4 3'UTR sequence and discovered that the miR-30 family are the best scoring miRNAs for this 3'UTR.
  • the family members demonstrate a full 8mer target sequence with additional 3' pairing that is located close to the start of the 3'UTR (Grimson et al., 2007) ( Figure 1C).
  • This miR-30 target site is absolutely conserved within the DLL4 3'UTR from 24 species and the inventors' alignment indicates that the surrounding sequence is also highly conserved (Figure ID).
  • miRNAs have been shown to target Notch components during tumor development (Zhiwei et al., 2010), but a function for this cross-talk during pathogenesis is unclear and no miRNAs targeting DLL4 have been identified.
  • DLL4 is one of the most significantly upregulated genes in KLEC (Emuss et al., 2009) and the corresponding suppression of a regulatory miRNA in response to KSHV infection suggests a functional relationship between DLL4 and miR-30 in these
  • miR-30b and miR-30c target DLL4
  • the inventors' GEM experiments show that miR-30b and miR-30c are amongst the most significantly suppressed members of the miR-30 family in KLEC.
  • the inventors transfected synthetic miR-30b and miR-30c mimics into LEC and measured DLL4 expression.
  • DLL4 mRNA levels were significantly reduced in LEC expressing either miR-30b or miR-30c mimics ( Figure 2A) and this corresponded to a significant decrease in DLL4 protein expression ( Figure 2B).
  • the inventors also generated lentiviruses expressing miR-30b or miR-30c by cloning 500bp fragments surrounding the pre-miRNA sequence of each miRNA from LEC genomic DNA (Lagos et al., 2010). They confirmed expression of mature miR-30b and miR-30c in LEC transduced with these viruses and a corresponding suppression of DLL4 (Figure 2C and S2B); these effects could be titrated with increasing copy number of virus per cell ( Figure S2B). Conversely, transfection of hairpin inhibitors against miR-30b and miR-30c into LEC led to an increase in DLL4 protein levels (Figure 2D). Taken together these data indicate that the miR-30 family may play a role in endogenous DLL4 regulation.
  • the inventors mutated the predicted miR-30 target site in the DLL4 3'UTR (Figure S2C) to prevent miRNA association (DLL4 3'UTR_mut) which significantly increased luciferase activity to near-baseline levels ( Figure 2D).
  • the original MT01 vector maintained its luciferase activity in the presence of exogenous miR-30, suggesting that the changes in activity were due to the effects of miR-30 on the DLL4 3'UTR.
  • these data suggest that miR-30 can influence the expression of DLL4 in LEC by targeting a predicted site in its 3'UTR.
  • endothelial cells expressing DLL4 stimulate signaling in adjacent cells. These signal-generating cells are specified as "tip" cells and localise to the tip of the developing sprout; the signal-receiving cells are excluded from the tip of the angiogenic sprout and contribute to the body of the developing vessel. Suppression of Notch signaling, such as through loss of ligand expression, leads to excessive sprouting and multiple vessel branches because the tip cell phenotype is not restricted. These vessels have aberrant morphology and are non-functional. The inventors utilised an in vitro model of sprouting angiogenesis to investigate whether DLL4 targeting by miR-30b could affect normal tip cell specification (Weber et al., 2008).
  • HUVEC Human umbilical vein endothelial cells
  • NTC or miR-30b mimics were transfected with either NTC or miR-30b mimics and then induced to form spheroids (Alajati et al., 2008; Korff and Augustin, 1998; Korff et al., 2004) which were subsequently embedded in matrigel.
  • Spheroids comprised of miR-30b overexpressing HUVEC displayed an increased propensity to form sprouts which was maintained for 5 days (Figure 3B). Furthermore, the sprouts in the miR-30b expressing spheroids were significantly longer. This indicates that miR-30 overexpression disturbs endothelial tip cell specification.
  • dll4 levels decrease between 18hpf and 24hpf as miR-30 expression increases; at 30hpf, dll4 levels return to 18hpf levels coincident with a drop in miR-30 expression ( Figure 4A and S4A).
  • This fluctuation corresponds with the temporal window during which the primary wave of angiogenic sprouting occurs, and the sharp increase in dll4 observed corresponds to the initiation of the secondary wave where ISV formation is consolidated and strict controls of endothelial branching are required (Ellertsdottir et al., 2010).
  • the inventors also visualized the embryos at 72hpf, once ISV formation is established ( Figure 4B, right panels, white arrows). Concurring with previous work, dll4 MO injection caused aberrant branching of the ISV (arrows) and the inventors observed aberrant branching in embryos expressing miR-30b and miR-30c mimics.
  • Kaposi's sarcoma a model of both malignancy and chronic inflammation. Panminerva Medica 49, 119-138.
  • KSHV Manipulates Notch Signaling by DLL4 and JAG1 to Alter Cell Cycle Genes in Lymphatic Endothelia.
  • Haller,B.K. Brave,A., Wallgard,E., Roswall,P., Sunkari.V.G., Mattson,U., Hallengard,D., Catrina,S.B., Hellstrom,M., and Pietras,K. (2010).
  • Harrington,L.S. Sainson,R.C.A., Williams,C.K., Taylor,J.M., Shi,W., LiJ.L., and Harris,A.L. (2008). Regulation of multiple angiogenic pathways by D114 and Notch in human umbilical vein endothelial cells. Microvascular Research 75, 144-154.
  • Korff,T. and Augustin,H.G. (1998). Integration of Endothelial Cells in Multicellular Spheroids Prevents Apoptosis and Induces Differentiation. The Journal of Cell Biology 143, 1341 -1352. KorffjT., Krauss,T. s and Augustin,H.G. (2004). Three-dimensional spheroidal culture of cytotrophoblast cells mimics the phenotype and differentiation of cytotrophoblasts from normal and preeclamptic pregnancies. Experimental Cell Research 297, 415-423.
  • Kaposi sarcoma herpesvirus- induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma. Nat Genet 36, 687-693.

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Abstract

L'invention concerne l'utilisation de micro-ARN dans le traitement des affections liées à l'angiogenèse, notamment le cancer.
PCT/GB2012/000131 2011-02-09 2012-02-08 Mir-30 destiné à être utilisé dans la modulation de l'angiogenèse WO2012107724A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003070910A2 (fr) * 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Inhibition induite par interference d'arn du facteur de croissance endothelial vasculaire et expression genetique du recepteur de facteur de croissance endothelial vasculaire au moyen d'acides nucleiques interferents courts (sina)
WO2005013901A2 (fr) * 2003-07-31 2005-02-17 Isis Pharmaceuticals, Inc. Composes oligomeres et compositions utilisables pour moduler des petits arn non-codants
WO2009008990A2 (fr) * 2007-07-06 2009-01-15 Intradigm Corporation Procédés et compositions de traitement du cancer et d'autres maladies associées à l'angiogenèse
WO2009137807A2 (fr) * 2008-05-08 2009-11-12 Asuragen, Inc. Compositions et procédés liés à la modulation de miarn de néovascularisation ou d’angiogenèse
WO2010005850A1 (fr) * 2008-07-08 2010-01-14 The J. David Gladstone Institutes Procédés et compositions de modulation de l’angiogenèse

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003070910A2 (fr) * 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Inhibition induite par interference d'arn du facteur de croissance endothelial vasculaire et expression genetique du recepteur de facteur de croissance endothelial vasculaire au moyen d'acides nucleiques interferents courts (sina)
WO2005013901A2 (fr) * 2003-07-31 2005-02-17 Isis Pharmaceuticals, Inc. Composes oligomeres et compositions utilisables pour moduler des petits arn non-codants
WO2009008990A2 (fr) * 2007-07-06 2009-01-15 Intradigm Corporation Procédés et compositions de traitement du cancer et d'autres maladies associées à l'angiogenèse
WO2009137807A2 (fr) * 2008-05-08 2009-11-12 Asuragen, Inc. Compositions et procédés liés à la modulation de miarn de néovascularisation ou d’angiogenèse
WO2010005850A1 (fr) * 2008-07-08 2010-01-14 The J. David Gladstone Institutes Procédés et compositions de modulation de l’angiogenèse

Non-Patent Citations (50)

* Cited by examiner, † Cited by third party
Title
ALAJATI,A.; LAIB,A.M.; WEBER,H.; BOOS,A.M.; BARTOL,A.; IKENBERG,K.; KORFF,T.; ZENTGRAF,H.; OBODOZIE,C.; GRAESER,R.: "Spheroid-based engineering of a human vasculature in mice", NAT METH, vol. 5, 2008, pages 439 - 445
BRAUN,J.; HOANG-VU,C.; DRALLE,H.; HUTTELMAIER,S.: "Downregulation ofmicroRNAs directs the EMT and invasive potential of anaplastic thyroid carcinomas", ONCOGENE, vol. 29, 2010, pages 4237 - 4244, XP055027241, DOI: doi:10.1038/onc.2010.169
CARTHEW,R.W.; SONTHEIMER,E.J.: "Origins and Mechanisms of miRNAs and siRNAs", CELL, vol. 136, 2009, pages 642 - 655, XP055091221, DOI: doi:10.1016/j.cell.2009.01.035
CHEN,P.Y.; MANNINGA,H.; SLANCHEV,K.; CHIEN,M.; RUSSO,J.J.; JU,J.; SHERIDAN,R.; JOHN,B.; MARKS,D.S.; GAIDATZIS,D.: "The developmental miRNA profiles of zebrafish as determined by small RNA cloning", GENES & DEVELOPMENT, vol. 19, 2005, pages 1288 - 1293
CHOI,W.Y.; GIRALDEZ,A.J.; SCHIER,A.F.: "Target Protectors Reveal Dampening and Balancing of Nodal Agonist and Antagonist by miR-430", SCIENCE, vol. 318, 2007, pages 271 - 274, XP055199883, DOI: doi:10.1126/science.1147535
CROCE,C.M.: "Causes and consequences ofmicroRNA dysregulation in cancer", NAT REV GENET, vol. 10, 2009, pages 704 - 714
DATABASE EMBL [Online] 16 January 2001 (2001-01-16), "RC2-CI0166-201100-018-a06 CI0166 Homo sapiens cDNA, mRNA sequence.", XP002676027, retrieved from EBI accession no. EMBL:BF812099 Database accession no. BF812099 *
DOUGLAS,J.; GUSIN,J.; DEZUBE,B.J.; PANTANOWITZ,J.; MOSES,A.: "Kaposi's sarcoma: a model of both malignancy and chronic inflammation", PANMINERVA MEDICA, vol. 49, 2007, pages 119 - 138
E!LERTSD6TTIR,E.; LENARD,A.; BLUM,Y.; KRUDEWIG,A.; HERWIG,L.; AFFOLTER,M.; BELTING,H.G.: "Vascular morphogenesis in the zebrafish embryo", DEVELOPMENTAL BIOLOGY, vol. 341, 2010, pages 56 - 65, XP026996105
EMUSS,V.; LAGOS,D.; PIZZEY,A.; GRATRIX,F.; HENDERSON,S.R.; BOSHOFF,C.: "KSHV Manipulates Notch Signaling by DLL4 and JAG1 to Alter Cell Cycle Genes in Lymphatic Endothelia", PLOS PATHOG, vol. 5, 2009, pages EL000616
F YU ET AL: "Mir-30 reduction maintains self-renewal and inhibits apoptosis in breast tumor-initiating cells", ONCOGENE, vol. 29, no. 29, 22 July 2010 (2010-07-22), pages 4194 - 4204, XP055027242, ISSN: 0950-9232, DOI: 10.1038/onc.2010.167 *
GALE,N.W.; DOMINGUEZ,M.G.; NOGUERA,I.; PAN,L.; HUGHES,V.; VALENZUELA,D.M.; MURPHY,A.J.; ADAMS,N.C.; LIN,H.C.; HOLASH,J.: "Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 101, 2004, pages 15949 - 15954, XP002432725, DOI: doi:10.1073/pnas.0407290101
GRIDLEY,T.: "Current Topics in Developmental Biology Notch Signaling", 2010, ACADEMIC PRESS, article "Notch Signaling in the Vasculature", pages: 277 - 309
GRIMSON; ANDREW; FARH; KYLE KAI-HOW; JOHNSTON; WENDY K.; GARRETT-ENGELE; PHILIP; LIM, LEE P.; BARTEL: "MicroRNA Targeting Specificity in Mammals: Determinants beyond Seed Pairing", MOLECULAR CELL, vol. 27, no. 1, pages 91 - 105, XP002624728, DOI: doi:10.1016/j.molcel.2007.06.017
HALLER,B.K.; BRAVE,A.; WALLGARD,E.; ROSWALL,P.; SUNKARI,V.G.; MATTSON,U.; HALLENGARD,D.; CATRINA,S.B.; HELLSTROM,M.; PIETRAS,K.: "Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma", ONCOGENE, vol. 29, 2010, pages 4276 - 4286
HARRINGTON,L.S.; SAINSON,R.C.A.; WILLIAMS,C.K.; TAYLOR,J.M.; SHI,W.; LI,J.L.; HARRIS,A.L.: "Regulation of multiple angiogenic pathways by D114 and Notch in human umbilical vein endothelial cells", MICROVASCULAR RESEARCH, vol. 75, 2008, pages 144 - 154, XP022487503, DOI: doi:10.1016/j.mvr.2007.06.006
HELLSTROM,M.; PHNG,L.K.; HOFMANN,J.J.; WALLGARD,E.; COULTAS,L.; LINDBLOM,P.; ALVA,J.; NILSSON,A.K.; KARLSSON,L.; GAIANO,N.: "D114 signalling through Notchl regulates formation of tip cells during angiogenesis", NATURE, vol. 445, 2007, pages 776 - 780, XP002613317, DOI: doi:10.1038/nature05571
KHAN,A.A.; BETEL,D.; MILLER,M.L.; SANDER,C.; LESLIE,C.S.; MARKS,D.S.: "Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs", NAT BIOTECH, vol. 27, 2009, pages 549 - 555
KORFF,T.; AUGUSTIN,H.G.: "Integration of Endothelial Cells in Multicellular Spheroids Prevents Apoptosis and Induces Differentiation", THE JOURNAL OF CELL BIOLOGY, vol. 143, 1998, pages 1341 - 1352
KORFF,T.; KRAUSS,T.; AUGUSTIN,H.G.: "Three-dimensional spheroidal culture of cytotrophoblast cells mimics the phenotype and differentiation of cytotrophoblasts from normal and preeclamptic pregnancies", EXPERIMENTAL CELL RESEARCH, vol. 297, 2004, pages 415 - 423, XP029305472, DOI: doi:10.1016/j.yexcr.2004.03.043
LAGOS,D.; POLLARA,G.; HENDERSON,S.; GRATRIX,F.; FABANI,M.; MILNE,R.S.B.; GOTCH,F.; BOSHOFF,C.: "miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator", NAT CELL BIOL, vol. 12, 2010, pages 513 - 519, XP009141493, DOI: doi:10.1038/ncb2054
LAGOS,D.; TROTTER,M.W.B.; VART,R.J.; WANG,H.W.; MATTHEWS,N.C.; HANSEN,A.; FLORE,O.; GOTCH,F.; BOSHOFF,C.: "Kaposi sarcoma herpesvirus-encoded vFLIP and vIRFl regulate antigen presentation in lymphatic endothelial cells", BLOOD, vol. 109, 2007, pages 1550 - 1558
LAKSHMANANE BOOMINATHAN: "The guardians of the genome (p53, TA-p73, and TA-p63) are regulators of tumor suppressor miRNAs network", CANCER AND METASTASIS REVIEWS, KLUWER ACADEMIC PUBLISHERS, DO, vol. 29, no. 4, 5 October 2010 (2010-10-05), pages 613 - 639, XP019856569, ISSN: 1573-7233, DOI: 10.1007/S10555-010-9257-9 *
LESLIE,J.D.; ARIZA-MCNAUGHTON,L.; BERMANGE,A.L.; MCADOW,R.; JOHNSON,S.L.; LEWIS,J.: "Endothelial signalling by the Notch ligand Delta-like 4 restricts angiogenesis", DEVELOPMENT, vol. 134, 2007, pages 839 - 844
LI,J.L.; SAINSON,R.C.A.; SHI,W.; LEEK,R.; HARRINGTON,L.S.; PREUSSER,M.; BISWAS,S.; TURLEY,H.; HEIKAMP,E.; HAINFELLNER,J.A.: "Delta-like 4 Notch Ligand Regulates Tumor Angiogenesis, Improves Tumor Vascular Function, and Promotes Tumor Growth In vivo", CANCER RES, vol. 67, 2007, pages 11244 - 11253, XP002523940, DOI: doi:10.1158/0008-5472.CAN-07-0969
LIU,R.; LI,X.; TULPULE,A.; ZHOU,Y.; SCEHNET,J.S.; ZHANG,S.; LEE,J.S.; CHAUDHARY,P.M.; JUNG,J.; GILI,P.S.: "KSHV-induced notch components render endothelial and mural cell characteristics and cell survival", BLOOD, vol. 115, 2010, pages 887 - 895
LOBOV,I.B.; RENARD,R.A.; PAPADOPOULOS,N.; GALE,N.W.; THURSTON,G.; YANCOPOULOS,G.D.; WIEGAND,S.J.: "Delta-like ligand 4 (D114) is induced by VEGF as a negative regulator of angiogenic sprouting", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 104, 2007, pages 3219 - 3224, XP002457726, DOI: doi:10.1073/pnas.0611206104
MARTINEZ,J.C., NUCLEAR AND MEMBRANE EXPRESSION OF THE ANGIOGENESIS REGULATOR DELTA-LIKE LIGAND 4 (DLL4) IN NORMAL AND MALIGNANT HUMAN TISSUES, 2009
MESRI,E.A.; CESARMAN,E.; BOSHOFF,C.: "Kaposi's sarcoma and its associated herpesvirus", NAT REV CANCER, vol. 10, 2010, pages 707 - 719
NITIN PATEL ET AL: "Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1 by placental growth factor in human pulmonary endothelial cells", BIOCHEMICAL JOURNAL, vol. 434, no. 3, 22 December 2010 (2010-12-22), pages 473 - 482, XP055027038, ISSN: 0264-6021, DOI: 10.1042/BJ20101585 *
NOGUERA-TROISE,!.; DALY,C.; PAPADOPOULOS,N.J.; COETZEE,S.; BOLAND,P.; GALE,N.W.; CHIEH LIN,H.; YANCOPOULOS,G.D.; THURSTON,G.: "Blockade of D114 inhibits tumour growth by promoting non-productive angiogenesis", NATURE, vol. 444, 2006, pages 1032 - 1037, XP002489427, DOI: doi:10.1038/nature05355
OISHI,H.; SUNAMURA,M.; EGAWA,S.; MOTOI,F.; UNNO,M.; FURUKAWA,T.; HABIB,N.A.; YAGITA,H.: "Blockade of Delta-Like Ligand 4 Signaling Inhibits Both Growth and Angiogenesis of Pancreatic Cancer", PANCREAS, vol. 39, 2010, pages 897 - 903
PATEL,N.S.: "Up-Regulation of Endothelial Delta-like 4 Expression Correlates with Vessel Maturation", BLADDER CANCER, 2006
PATEL,N.S.; LI,J.L.; GENERALI,D.; POULSOM,R.; CRANSTON,D.W.; HARRIS,A.L.: "Up- regulation of Delta-like 4 Ligand in Human Tumor Vasculature and the Role of Basal Expression in Endothelial Cell Function", CANCER RES, vol. 65, 2005, pages 8690 - 8697
PHNG,L.K.; GERHARDT,H.: "Angiogenesis: A Team Effort Coordinated by Notch", DEVELOPMENTAL CELL, vol. 16, 2009, pages 196 - 208
R. AGRAWAL ET AL: "The miR-30 miRNA family regulates Xenopus pronephros development and targets the transcription factor Xlim1/Lhx1", DEVELOPMENT, vol. 136, no. 23, 1 December 2009 (2009-12-01), pages 3927 - 3936, XP055027063, ISSN: 0950-1991, DOI: 10.1242/dev.037432 *
RIDGWAY,J.; ZHANG,G.; WU,Y.; STAWICKI,S.; LIANG,W.C.; CHANTHERY,Y.; KOWALSKI,J.; WATTS,R.J.; CALLAHAN,C.; KASMAN,I.: "Inhibition of D114 signalling inhibits tumour growth by deregulating angiogenesis", NATURE, vol. 444, 2006, pages 1083 - 1087
SIEKMANN,A.F.; LAWSON,N.D.: "Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries", NATURE, vol. 445, 2007, pages 781 - 784
SODHI,A.; MONTANER,S.; PATEL,V.; ZOHAR,M.; BAIS,C.; MESRI,E.A.; GUTKIND,J.S.: "The Kaposi's Sarcoma-associated Herpes Virus G Protein-coupled Receptor Up-Regulates Vascular Endothelial Growth Factor Expression and Secretion through Mitogen-activated Protein Kinase and p38 Pathways Acting on Hypoxia-inducible Factor l+", CANCER RES, vol. 60, 2000, pages 4873 - 4880, XP002443609
VART,R.J.; NIKITENKO,L.L.; LAGOS,D.; TROTTER,M.W.B.; CANNON,M.; BOURBOULIA,D.; GRATRIX,F.; TAKEUCHI,Y.; BOSHOFF,C.: "Kaposi's Sarcoma-Associated Herpesvirus-Encoded Interleukin-6 and G-Protein-Coupled Receptor Regulate Angiopoietin-2 Expression in Lymphatic Endothelial Cells", CANCER RES, vol. 67, 2007, pages 4042 - 4051
VICTORIA EMUSS ET AL: "KSHV Manipulates Notch Signaling by DLL4 and JAG1 to Alter Cell Cycle Genes in Lymphatic Endothelia", PLOS PATHOGENS, vol. 5, no. 10, 1 October 2009 (2009-10-01), pages E1000616 - E1000616, XP055027040, ISSN: 1553-7366, DOI: 10.1371/journal.ppat.1000616 *
VIEIRA,J; O'HEAM,P.; KIMBALL,L.; CHANDRAN,B.; COREY,L.: "Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus", J. VIROL., vol. 75, 2001, pages 1378 - 1386
VOLINIA,S.; GALASSO,M.; COSTINEAN,S.; TAGLIAVINI,L.; GAMBERONI,G.; DRUSCO,A.; MARCHESINI,J.; MASCELLANI,N.; SANA,M.E.; ABU JAROUR,: "Reprogramming of miRNA networks in cancer and leukemia", GENOME RESEARCH, vol. 20, 2010, pages 589 - 599
WANG Z ET AL: "Cross-talk between miRNA and Notch signaling pathways in tumor development and progression", CANCER LETTERS, NEW YORK, NY, US, vol. 292, no. 2, 28 June 2010 (2010-06-28), pages 141 - 148, XP027013756, ISSN: 0304-3835, [retrieved on 20100416] *
WANG,H.W.; TROTTER,M.W.B.; LAGOS,D.; BOURBOULIA,D.; HENDERSON,S.; MAKINEN,T.; ELLIMAN,S.; FLANAGAN,A.M.; ALITALO,K.; BOSHOFF,C.: "Kaposi sarcoma herpesvirus- induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma", NAT GENET, vol. 36, 2004, pages 687 - 693
WANG,S.; OLSON,E.N.: "AngiomiRs--Key regulators of angiogenesis", CURRENT OPINION IN GENETICS & DEVELOPMENT, vol. 19, 2009, pages 205 - 211, XP026171103, DOI: doi:10.1016/j.gde.2009.04.002
WEBER,H.; CLAFFEY,J.; HOGAN,M.; PAMPILL6N,C.; TACKE,M.: "Analyses of Titanocenes in the spheroid-based cellular angiogenesis assay", TOXICOLOGY IN VITRO, vol. 22, 2008, pages 531 - 534, XP022442253, DOI: doi:10.1016/j.tiv.2007.09.014
WIENHOLDS,E.; KLOOSTERMAN,W.P.; MISKA,E.; ALVAREZ-SAAVEDRA,E.; BEREZIKOV,E.; DE BRUIJN,E.; HORVITZ,H.R.; SAKARI,K.; PLASTERK,R.H.A: "MicroRNA Expression in Zebrafish Embryonic Development", SCIENCE, vol. 309, 2005, pages 310 - 311, XP002386061, DOI: doi:10.1126/science.1114519
YU,F.; DENG,H.; YAO,H.; LIU,Q.; SU,F.; SONG,E.: "Mir-30 reduction maintains self- renewal and inhibits apoptosis in breast tumor-initiating cells", ONCOGENE, vol. 29, 2010, pages 4194 - 4204, XP055027242, DOI: doi:10.1038/onc.2010.167
ZHIWEI; WANG; YIWEI; LI; DEJUAN; KONG; AAMIR; AHMAD; SANJEEV; BANERJEE: "Cross-talk between miRNA and Notch signaling pathways in tumor development and progression", CANCER LETTERS, vol. 292, no. 2, 28 June 2010 (2010-06-28), pages 141 - 148, XP027013756

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