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WO2012140234A1 - Modulation du miarn dans les maladies caractérisées par une angiogenèse aberrante - Google Patents

Modulation du miarn dans les maladies caractérisées par une angiogenèse aberrante Download PDF

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WO2012140234A1
WO2012140234A1 PCT/EP2012/056839 EP2012056839W WO2012140234A1 WO 2012140234 A1 WO2012140234 A1 WO 2012140234A1 EP 2012056839 W EP2012056839 W EP 2012056839W WO 2012140234 A1 WO2012140234 A1 WO 2012140234A1
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mir
angiogenesis
tumor
expression
mice
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Marc Tjwa
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Vib Vzw
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Definitions

  • the present application relates to the field of microRNAs, and more particular to the role of microRNAs in angiogenesis. It is demonstrated herein that modulation of specific microRNAs, such as miR-144, has therapeutic potential in diseases characterized by aberrant angiogenesis. This is particularly relevant in, for instance, tumour growth and metastasis, which are characterized by excessive angiogenesis and for which inhibition of miR-144 is a novel therapeutic strategy. On the other hand, diseases characterized by insufficient angiogenesis (e.g. associated with ischemia) may benefit from increasing miR-144 activity.
  • Cancer remains one of the major causes of mortality and morbidity in the Western society, and constitutes a prime target for drug development.
  • One of the prime leads is the development of inhibitors of blood vessel formation (anti-angiogenic drugs), based on the fact that cancer progression requires angiogenesis to support its growth and metastasis to distant organs (Carmeliet et al., 2009).
  • anti-angiogenic drugs based on the fact that cancer progression requires angiogenesis to support its growth and metastasis to distant organs.
  • tumoral blood vessels as tumors form blood vessels with abnormal structure and function
  • a milestone in the exploitation of this novel therapeutic paradigm was the clinical development of a monoclonal antibody against a prime regulator of angiogenesis, namely Vascular Endothelial Growth Factor (VEGF).
  • VEGF Vascular Endothelial Growth Factor
  • Avastin catalyzed the further development of a wide spectrum of additional anti-VEGF drugs, of which the class of receptor tyrosine kinase inhibitors (RTKi) such as Sunitinib (Sutent ® , Pfizer Inc) and Sorafenib (Nexavar ® , Bayer Corporation/Onyx Pharmaceuticals Inc) are most advanced into clinical trials (Carmeliet, 2005).
  • RTKi receptor tyrosine kinase inhibitors
  • VEGF is a prime regulator of endothelial cells in tumors but also in the normal vasculature
  • treatment with Avastin is endowed with great risk of affecting the non- tumoral endothelium, thereby causing various life-threatening side effects (e.g. hypertension, thrombosis, aggravation of cardiovascular disease, etc), and hence warranting a tight control of its administration and limitation of eligible patients (Carmeliet, 2005).
  • Deleterious side effects are especially present in the clinical experiences so far with TKi administration in patients, in part amplified by the non-specific targeting of additional tyrosine kinase receptors, expressed in normal endothelium and other cell types.
  • endothelial cell subtypes such as tip and stalk cells
  • endothelial branching vascular branching, maintenance and function
  • the cornerstone of vascular sprouting and branching is the initiation of endothelial tip cells, which, after progressive recruitment of stalk cells and a series of other events, leads to a functional branched vessel lined with quiescent endothelial cells (Carmeliet et al., 2009).
  • VEGF and jagged-1 have been identified as initiators of tip cell fate specification (Gerhardt et al., 2003; Benedito et al., 2009), but the cell-intrinsic regulation of tip cell initiation remains unclear.
  • current dogma states that tip cell initiation is regulated through a "permissive" model in which, amongst several candidate endothelial cells, one single tip cell is selected by negative effects on adjacent endothelial cells via lateral signaling pathways such as Notch (Hellstrom et al., 2007).
  • targeting tip cells might potentially result in novel antiangiogenic strategies to block angiogenesis in disease states including cancer.
  • the proof-of-principle for such strategies was recently shown in cancer studies in mice targeting established tip cell surface molecules such as Unc5B and DII4 (Noguera-Troise et al., 2006; Larrivee et al., 2007).
  • targeting tip cells may also allow developing angiogenic strategies in disorders where angiogenesis is impaired, such as limb ischemia.
  • Angiogenesis equals the formation of a hierarchically branched vascular network.
  • the cellular basis of vessel branching is formed by endothelial heterogeneity, i.e. endothelial subpopulations with specific fates and functions: amongst others the endothelial tip, stalk and quiescent cell (in tumors called phalanx cell) (Mazzone et al., 2009).
  • the cornerstone of vascular branching is the initiation of tip cells, which, after progressive recruitment of stalk cells and a series of other events, leads to a functional branched vessel lined with quiescent endothelial cells (Larrivee et al., 2009).
  • tip cell fate specification (Benedito et al., 2009; Gerhardt et al., 2003), but the cell-intrinsic regulation of tip cell initiation remains unclear. Indeed, current dogma states that tip cell initiation is regulated through a "permissive" model in which, amongst several candidate endothelial cells, one single tip cell is selected by negative effects on adjacent endothelial cells via lateral signaling pathways mediated by, for instance, Notch and Wnt family members (Hellstrom et al., 2007; Phng et al., 2009).
  • microRNAs represent a class of conserved non-coding small RNAs ( ⁇ 22 nt long), which repress gene expression post-transcriptionally by targeting 3'-untranslated regions (3'UTRs) of specific target gene mRNAs, leading to translational repression or mRNA degradation (Bartel, 2009).
  • miRNAs control cell fate of various cell types, involved in proliferation, differentiation, migration, and so on (Bartel, 2009).
  • miRNAs Nearly 700 miRNAs have been identified in humans so far, and are predicted to regulate a third of protein coding genes, suggesting that miRNAs are likely to play roles in almost all biological processes (Lewis et al., 2005). A few miRNAs ( ⁇ 2%) have been shown to control blood vessel formation (angiogenesis), both positively (e.g. miR-126, let-7f, the miR-17-92 cluster, miR-31, miR-130a, miR-296, miR-320) (Wang and Olson, 2009) and negatively (e.g. miR-92a, miR-221/222) (Bonauer et al., 2009), thereby affecting the progression of various diseases including malignancy, retinopathy and ischemic cardiovascular disease.
  • angiogenesis both positively (e.g. miR-126, let-7f, the miR-17-92 cluster, miR-31, miR-130a, miR-296, miR-320) (Wang and Olson, 2009) and negatively (e.
  • miRNAs currently identified as having a role in angiogenesis were predominantly expressed in tumor cells and affected tumor angiogenesis via paracrine mechanisms.
  • the identification of miRNAs primarily expressed by tumor endothelial cells remains an outstanding question.
  • microRNAs are currently envisioned as a major target for personalized medicine and (current) drug development in oncology. Indeed, not only it became clear that specific miRNA expression signatures in various tumors seem to be associated with defined disease states and/or specific cancers, also approaches in which tumor-derived miRNAs in the circulating blood plasma are assayed - as a novel biomarker - are currently extensively explored. Indeed, recent reports suggest the potential to use circulating miRNAs, analyzed in blood plasma, as a surrogate biomarker of tumor progression (Mitchell et al., 2008). It would be advantageous to have a novel pro- or anti-angiogenic therapeutic with selective efficacy on blood vessels, particularly one that does not target the VEGF pathway. Most particularly, an anti- angiogenic therapeutic selectively affecting tumoral blood vessels is highly desirable. If a target were identified that is specifically upregulated in these blood vessels, this molecule could also serve as a biomarker in cancer.
  • MicroRNAs critically fine-tune gene expression during blood vessel formation (angiogenesis), but their role in endothelial tip/stalk cell specification remains unknown.
  • endothelial deletion of Dicer limits the development of a branched vascular network in the yolk sac, and miRNA profiling suggested the potential involvement of particularly miR-144 herein.
  • Forced overexpression of miR-144 in endothelial cells stimulated vessel branching both in vitro and in vivo, and was sufficient to initiate an endothelial fate and gene signature, which is reminiscent of branch-inducing tip cells.
  • inhibition of miR-144 reduced vessel branching and the formation of endothelial tip cells.
  • miR-144 promotes (tumor) angiogenesis by instructing endothelial tip cell specification
  • miRNAs are identified herein that are potential drug targets in cancer.
  • This strategy does not aim to target the tumor cell directly, but rather the tumor endothelial (tip) cell.
  • inhibition of miR-144 in in vivo tumor models results in delayed and reduced tumor growth, increased survival and lower incidence of cancer- associated systemic syndrome - see Examples section.
  • inhibitors against specific miRNAs e.g. antagomir-144 represent a new class of angiogenesis inhibitors with a beneficial safety profile and efficacy.
  • miR-144 is upregulated in tumours in vivo, most particularly in tumour endothelial cells, the level of miR-144 expression can be used as a biomarker to detect cancer and/or tumour angiogenesis.
  • the modulators of miR-144 are for use in treatment of a disease characterized by aberrant angiogenesis. Accordingly, methods are provided for treating diseases characterized by aberrant angiogenesis, comprising:
  • the modulators of miR-144 are inhibitors of miR-144. These can be used in treatment of diseases characterized by excessive angiogenesis, as inhibition of miR-144 reduces vessel branching and tip cell formation.
  • the diseases characterized by excessive angiogenesis are selected from the group of cancer, diabetic retinopathy and age-related macular degeneration.
  • the inhibitors of miR-144 are particularly suited to reduce or slow down tumour growth, i.e. when an inhibitor of miR-144 is administered, the tumour may still grow, but will remain smaller than when left untreated.
  • the inhibitors of miR-144 reduce tumour metastasis.
  • the inhibitors of miR-144 reduce the incidence of a disease that is the consequence of the presence of cancer in the body, but is not due to the local presence of cancer cells (typically referred to as a paraneoplastic syndrome). Particularly, they can be used to treat cancer-associated systemic syndrome (CASS) or one or more of its symptoms (cachexia, anemia, increased chance of thrombosis).
  • CASS cancer-associated systemic syndrome
  • methods are provided for reducing the development or occurrence of CASS, by administering an inhibitor of miR-144. This way, the chances of survival (and thus also the life expectancy) is increased.
  • the inhibitor of miR-144 is an antagomir (e.g. antagomir-144) or a locked nucleic acid (LNA).
  • antagomir e.g. antagomir-144
  • LNA locked nucleic acid
  • the modulators of miR-144 are agents that mimic the biological action of miR-144, i.e. miR-144 mimetics or miR-144 itself.
  • These agents can be used in diseases where upregulation of miR-144 is desired, such as diseases characterized by impaired angiogenesis.
  • miR-144 (or a mimetic thereof) will increase vessel branching, allowing more vessels to be formed and a better perfusion of the tissue.
  • Typical diseases characterized by impaired angiogenesis are those characterized by ischemia, such as ischemic heart disease or, most particularly, ischemic limb disease.
  • the agents that mimic the biological action of miR-144 are administered to endothelial cells.
  • the agents that mimic the biological action of miR-144 are not administered in cardiomyocytes.
  • the effects of the agents that mimic the biological action of miR-144 are GATA4-independent.
  • miR-144 is provided for use as a biomarker. As it was found that miR- 144 is specifically upregulated in tumour endothelial cells, miR-144 can be used as a biomarker to detect cancer, i.e. increased levels of miR-144 correlate with tumour progression. As miR-144 is normally absent in many healthy tissues, the presence of miR-144 as such can already be used as a biomarker to indicate the presence of cancer.
  • the expression levels of miR-144 can be further correlated to the tumor size.
  • the expression levels can be correlated to the nature of the tumour (e.g. breast cancer, pancreatic cancer, etc. This is especially useful in the case of metastases that are not directly linked to a primary tumour) and/or the expression levels can be correlated to the aggressiveness of the tumour, i.e. the risk of metastasis can be derived from the miR-144 levels.
  • the treatment when miR-144 expression has been correlated to tumour size, nature and/or aggressiveness, the treatment may be adapted based on this information.
  • FIG. 1 Genotyping of offspring of male Tek Cre/+ :Dicer Aflox/+ and female Dicer flox/flox mice.
  • B Dicer expression in endothelium-selective Dicer-deficient (Tek Cre + :Dicer A flox ) mice versus control mice as evaluated by qPCR (top) and microarray (bottom).
  • miR-144 expression in HUVEC cells is dose-dependently increased upon transfection with miR- 144 duplex (left panel) or miR-144 precursor (right panel), both after 24 hours (top) and after 48 hours (bottom).
  • Triangles on Y axis indicate increase in dose; numbers on X axis are fold increase relative to a scrambled control miRNA.
  • FIG. 5 A, miR-144 overexpression increases sprout length, number of sprouts and number of branched sprouts in a spheroid model of sprouting angiogenesis (Bonauer et al., 2009). Left panel: transfection of miR-144 duplex versus scrambled control; right panel: transfection of miR-144 precursor versus scrambled control. B, Microscopic images showing filopodia formation of cells shown in A, left panel.
  • Figure 6 A, qPCR expression analysis of selected tip cell (Unc5B, VEGFR-1, NP-1, NP-2, PDGF-B, VEGFR-2, DII4, Notch-1, Notch-4, VEGFR-3) and stalk cell (Nrarp, Hesl, Robo4) genes in HUVEC cells transfected with miR-144 duplexes, shown as fold increase relative to control.
  • B co-transfection of control miRNA or miR-144 duplexes with a TP1 Notch signaling reporter construct shows similar Notch activity.
  • Figure 7 A, Microarray (top) and qPCR (bottom) analysis of predicted target genes of miR-144 that are upregulated in mutant yolk sacs.
  • B Microarray (left panel) and qPCR (right panel) analysis of FoxOl target genes that are upregulated in mutant yolk sacs.
  • C FoxOl protein levels in yolk sac extracts of normal or mutant (endothelial-specific Dicer-deficient) mice.
  • FIG. 8 A, Total protein levels of FoxOl in HUVEC cells treated with miR-144 duplex or scrambled control. Tubulin is shown as control.
  • B FoxOl-dependent luciferase activity in HUVEC cells treated with miR-144 duplex or scrambled control.
  • C mutating the 3'UTR binding site confirms that FoxOl is a target gene of miR-144. HUVEC cells are treated with miR-144 duplex or scrambled control.
  • FIG. 10 Expression of miR-144 in syngeneic pancreatic adenocarcinoma tumours of different sizes in C57BI6 mice, shown relative to expression in the cultured Panc02 cells.
  • FIG. 11 A, Tumor volume time course of C57BI6 mice subcutaneously inoculated with Panc02 cells and treated with one bolus i.v. injection of antagomir-144 or scrambled control at 8 mg/kg. B, Survival of the mice shown in A. Solid line: control; dashed line: mice injected with antagomir-144.
  • Figure 12 Weight of tumors of the two groups of mice shown in Figure 11A at the end of the experiment (day 36). Left column: control, right column: mice injected with antagomir-144.
  • FIG. 14 A, Tumor volume time course of C57BI6 mice subcutaneously inoculated with Panc02 cells and treated with one bolus i.v. injection of antagomir-144 or scrambled control at 40 mg/kg. B, Survival of the mice shown in A. Solid line: control; dashed line: mice injected with antagomir-144. Detailed description
  • miRNA refers to short (typically 20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding.
  • the primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and the minor (sometimes referred to as antisense) miRNA star (miRNA*) products.
  • pre-miRNA stem-loop precursor miRNA
  • miRNA* miRNA star
  • RISC RNA-induced silencing complex
  • miR-144 refers to the microRNA 144 RNA gene (in humans, also indicated as “hsa-mir-144", characterized by HGNC ID: 31531; Gene ID: 406936; MIMID: 612070; miRBase MI0000460; in mice (“mmu-mir-144”): GenelD: 387162; miRBase MI0000168).
  • the primary miRNA transcript (pri-miRNA) that encodes miR-144 also encodes miR-451 (Dore et al., 2008), although recent reports indicate they are differently regulated post-transcriptionally (Cheloufi et al., 2010; Cifuentes et al., 2010).
  • miR144 has been documented to be involved in hematopoiesis, particularly erythropoiesis (Dore et al., 2008; Fu et al., 2009; Rasmussen et al., 2010), but not in angiogenesis. miR-144 has also been implicated in neurodegeneration (An et al., 2010; Persengiev et al., 2010) and bipolar disorder (Dinan, 2010). WO2006/137941 suggests that miR-144 should be administered in cancer, particularly in colon cancer. WO2009/070653 mentions that miR-144 downregulation (amongst others) is associated with lung cancer. Guled et al.
  • miR-144* the minor miR* sequence of hsa-mir-144, is one of the miRNAs severely reduced in malignant mesothelioma (Guled et al., 2009). Similar to these publications on miR-144, downregulation of miR-451 has been reported to be associated with a worse prognosis in gastric cancer (Bandres et al., 2009) and is also associated with pre-B-ALL (Ju et al., 2009). Of note, all these studies in cancer report associations but no causative effects; the administration or inhibition of mir-144 has also not been tested in vivo.
  • a “modulator of miR-144" as used in the application refers to a compound that modulates the function, activity and/or functional effect of miR-144. Modulators can either inhibit or decrease miR-144 function, in which case they are referred to as “inhibitors of miR-144", or enhance or increase miR-144 function.
  • “Inhibitors of miR-144" are molecules that interfere with the function of miR-144, either at the DNA level (e.g. by interfering with transcription of miR-144) or at the RNA level (e.g. by interfering with the successive cleavage steps, through destabilization of the miRNA so that it is degraded, or typically, by interfering with the miRNA itself, e.g. through hybridization).
  • Inhibiting at the DNA level can for instance be done by inhibiting functional expression of the mi -144 RNA gene itself.
  • functional expression of the miR-144 gene it is meant the transcription of functional miR-144 gene product.
  • “Inhibition of functional expression” at the DNA level can e.g. be achieved by removing or disrupting the miR-144 gene, or preventing transcription to take place (in both instances preventing synthesis of the miR-144 gene product).
  • a "knock-out" can be a gene knockdown or the gene can be knocked out by a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques known in the art, including, but not limited to, retroviral gene transfer.
  • a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques known in the art, including, but not limited to, retroviral gene transfer.
  • Another way in which genes can be knocked out is by the use of zinc finger nucleases.
  • Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain.
  • Zinc finger domains can be engineered to target desired DNA sequences, which enables zinc-finger nucleases to target unique sequence within a complex genome. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.
  • An alternative genome customization tool that can be used are Transcription Activator-Like Effector Nucleases (TALENs), which are sequence-specific nucleases created by the fusion of transcription activator-like effectors (TALEs) to the catalytic domain of an endonuclease.
  • TALENs Transcription Activator-Like Effector Nucleases
  • TALEs transcription activator-like effectors
  • miRNA inhibitors that inhibit by hybridization are miRNA inhibitor molecules that are between 17 and 25 nucleotides in length and comprise a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature miRNA (particularly of miR-144).
  • antisense oligomers used to inhibit miR function may consist of DNA, RNA or other, synthetic, structures such as phosphorothiates, 2'-0-alkyl ribonucleotide chimeras, locked nucleic acid (LNA) (which will be discussed further), peptide nucleic acid (PNA), or morpholinos.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • antisense oligomers typically act in eukaryotic cells through the mechanism of RNase H-mediated target cleavage.
  • an antisense oligomer refers to an antisense molecule or anti-gene agent that comprises an oligomer of at least about 10 nucleotides in length. In particular embodiments an antisense oligomer comprises at least 15, 18, 20, 25, 30, 35, 40, or 50 nucleotides. Antisense approaches involve the design of oligonucleotides (either DNA or RNA, or derivatives thereof) that are complementary to the miRNA of choice, particularly miR-144.
  • Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery. This effect is therefore stoichiometric. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense polynucleotide sequences, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense polynucleotide sequence. Generally, the longer the hybridizing polynucleotide sequence, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • morpholino antisense oligonucleotides in zebrafish and frogs overcome the limitations of RNase H-competent antisense oligonucleotides, which include numerous non-specific effects due to the non target-specific cleavage of other mRNA molecules caused by the low stringency requirements of RNase H. Morpholino oligomers therefore represent an important new class of antisense molecule. Oligomers of the invention may be synthesized by standard methods known in the art. As examples, phosphorothioate oligomers may be synthesized by the method of Stein et al. (1988) Nucleic Acids Res.
  • methylphosphonate oligomers can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. USA. 85, 7448-7451). Morpholino oligomers may be synthesized by the method of Summerton and Weller U.S. Patent Nos. 5,217,866 and 5,185,444. Particularly envisaged molecules for inhibition of miRNAs are antagomirs. Antagomirs are chemically engineered oligonucleotides that can be used to silence endogenous microRNA.
  • antagomir is a small synthetic RNA that is perfectly complementary to the specific miRNA target with either mispairing at the cleavage site of Ago2 or some sort of base modification to inhibit Ago2 cleavage. Usually, antagomirs have some sort of modification to make it more resistant to degradation. Without being bound to a particular mechanism, it is believed that antagomirization (the process by which an antagomir inhibits miRNA activity) operates by irreversibly binding the miRNA. miRNA may also be inhibited using ribozymes instead of antisense RNA. Ribozymes are catalytic RNA molecules with enzyme-like cleavage properties that can be designed to target specific RNA sequences.
  • miR mimetics typically are molecules consisting of a double-stranded RNA strand. Often, the RNA contains modifications to make them more suitable for administration (e.g. by increasing stability). According to particular embodiments, the miR-144 mimetic does not contain modifications and is identical to the native miR-144 or one of its precursor molecules, but in isolated form. Thus, unless otherwise stated, the use of a miR-144 mimetic also envisages the use of miR-144.
  • isolated means altered or removed from the natural state through human intervention.
  • a miRNA naturally present in a living animal is not “isolated,” but a synthetic miRNA, or an miRNA partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated miRNA can exist in substantially purified form, or can exist in a non native environment such as, for example, a cell into which the miRNA has been delivered.
  • the miRNA modulators of the invention can comprise partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non nucleotide material, such as to the end(s) of the miRNA or to one or more internal nucleotides of the miRNA, including modifications that make the miRNA resistant to nuclease digestion.
  • the miRNA modulators described herein can be obtained using a number of techniques known to those of skill in the art.
  • the miRNA modulators can be chemically synthesized or recombinantly produced using methods known in the art.
  • the miRNA modulators of the invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • Commercial suppliers of synthetic miRNA modulators (i.e. inhibitors and mimetics) or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA) (e.g.
  • miRNA modulators have also been described in e.g. WO/2009/029690; and Olejniczak et al., 2010.
  • miRNA modulators can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • suitable promoters for expressing miRNA of the invention from a plasmid include, for example, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the miRNA modulator in a particular tissue or in a particular intracellular environment.
  • the miRNA modulators expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly, e.g. in muscle tissue or in tumours.
  • the miRNA modulators of the invention can also be expressed intracellularly from recombinant viral vectors.
  • the miR-144 modulators provided herein are used in treatment of diseases characterized by aberrant angiogenesis.
  • aberrant angiogenesis is used herein to refer to blood vessel growth that is either excessive (i.e. too much formation of new blood vessels) or impaired (not enough new blood vessels are formed).
  • Diseases characterized or caused by abnormal or excessive angiogenesis in one or more organs include, but are not limited to, cancer, infectious diseases, autoimmune disorders, vascular malformations (e.g. Tie-2 mutation), DiGeorge syndrome, HHT, cavernous hemangioma, atherosclerosis, transplant arteriopathy, obesity, psoriasis, warts, allergic dermatitis, scar keloids, pyogenic granulomas, blistering disease, Kaposi sarcoma in AIDS patients, persistent hyperplastic vitreous syndrome, diabetic retinopathy, retinopathy of prematurity, choroidal neovascularization, primary pulmonary hypertension, asthma, nasal polyps, inflammatory bowel and periodontal disease, ascites, peritoneal adhesions, endometriosis, uterine bleeding, ovarian cysts, ovarian hyperstimulation, arthritis, synovitis, osteomyelitis, and osteophyte formation (see Table 1 of Carmeliet
  • Diseases characterized or caused by insufficient or impaired angiogenesis or vessel regression in one or more organs include, but are not limited to, Alzheimer disease, amyotrophic lateral sclerosis (ALS), diabetic neuropathy, stroke, atherosclerosis, hypertension, diabetes, restenosis, gastric or oral ulcerations, Crohn disease, hair loss, skin purpura, telangiectasia and venous lake formation, pre-eclampsia, menorrhagia, neonatal respiratory distress, pulmonary fibrosis, emphysema, nephropathy, osteoporosis, and impaired bone fracture healing (see Table 2 of Carmeliet, 2003).
  • ALS amyotrophic lateral sclerosis
  • diabetic neuropathy diabetic neuropathy
  • stroke atherosclerosis
  • hypertension diabetes
  • restenosis gastric or oral ulcerations
  • Crohn disease Crohn disease
  • hair loss skin purpura
  • telangiectasia and venous lake formation pre-eclampsia
  • ischemic diseases characterized by impaired angiogenesis include, but are not limited to, limb ischemia or critical limb ischemia, chronic obstructive pulmonary disease, ischemia-reperfusion injury, post-operative ischemia, ischemic cardiovascular disease, restenosis, acute myocardial infarction, chronic ischemic heart disease, atherosclerosis, ischemic stroke, ischemic cerebral infarction, or ischemic bowel disease.
  • the modulation of the miR-144 gene function is limited to the tissue where the angiogenesis is aberrant.
  • a typical example hereof is in the case of cancer: inhibition of miR-144 may be restricted to the tissue where the tumour is located, and most particularly, the gene function knockout is limited to the tumour itself, and miR-144 is not inhibited in the host subject.
  • miR-144 function is only modulated in blood vessels and/or endothelial tissue.
  • the modulation may also be temporary (or temporally regulated).
  • Temporally and tissue-specific gene inactivation may be achieved through targeted administration of miR-144 modulators, or, in case of gene therapy, by using specific expression cassettes including specific promoters and/or enhancers to control expression of the miR-144 modulators.
  • the disease to be treated is characterized by excessive angiogenesis, particularly cancer.
  • miR-144 is an attractive target to specifically affect angiogenesis, most particularly tumour angiogenesis.
  • Inhibitors of miR-144 such as antagomirs against miR-144, have the potential to effectively and specifically inhibit tumor angiogenesis, thereby preventing tumor growth and metastasis.
  • an inhibitor of miR-144 gene function can be used to reduce tumour growth (i.e., methods of reducing tumour growth according to these embodiments involve administering an inhibitor of miR-144 to a subject in need thereof).
  • reducing tumour growth means that a tumour contacted or treated with the inhibitors described herein will grow less than an untreated tumour in the same time span. Particularly, growth will be 10% less, 20% less, 30% less, 40% less, 50% less, 75% less, 90% less, 95% less or 100% less (i.e., once treatment is started, the tumour does not grow any more). In particular cases, the tumour growth may even be reversed upon treatment, i.e. the tumour starts to shrink (either immediately or after a period of reduced growth). Tumour growth can be assessed by the skilled person using methods known in the art, typically by measuring tumour volume or size. Alternatively, the weight of the tumour can be estimated to assess tumour growth.
  • the inhibitor of miR-144 can be used to reduce tumour metastasis (or equivalent: methods of reducing tumour metastasis according to these embodiments involve administering an inhibitor of miR-144 to a subject in need thereof).
  • reducing tumour metastasis it is meant that a tumour (or subject) treated with the inhibitors described herein will develop less metastases (i.e. secondary or metastatic tumours) than an untreated tumour in the same time span.
  • metastases will be 10% less, 20% less, 30% less, 40% less, 50% less, 75% less, 90% less, 95% less or 100% less (i.e., once treatment is started, the tumour does not metastasise any more).
  • the inhibitor of miR-144 can be used to reduce the incidence of paraneoplastic syndrome (herein also referred to as cancer-associated systemic syndrome or CASS) (or equivalent: methods of reducing the incidence of paraneoplastic syndrome according to these embodiments involve administering an inhibitor of miR-144 to a subject in need thereof).
  • CASS cancer-associated systemic syndrome
  • reducing the incidence it is meant that the likelihood of developing CASS is reduced upon treatment (i.e., preventing new cases of CASS) or, if symptoms of CASS are already present, reducing the severity of at least one symptom of CASS.
  • Reducing the severity may refer to an improvement or may entail the complete disappearance of one or more symptoms (e.g. improving anemia may result in a less anemic state or result in a non-anemic state; improving cachexia may result in gain of lean body mass up to the point that body weight is restored to normal).
  • paraneoplastic syndrome refers to diseases or symptoms that are the consequence of the presence of cancer in the body, but is not due to the local presence of cancer cells. These phenomena are typically mediated by humoral factors (by hormones or cytokines) excreted by tumor cells or by an immune response against the tumor. Paraneoplastic syndromes are typical among middle aged to older patients, and they most commonly present with cancers of the lung, breast, ovaries or lymphatic system (a lymphoma). The most common symptoms of CASS include cachexia, anemia and an increased chance of thrombosis.
  • Cachexia or wasting syndrome is loss of weight, muscle atrophy, fatigue, weakness and significant loss of appetite in someone who is not actively trying to lose weight.
  • the formal definition of cachexia is the loss of body mass that cannot be reversed nutritionally: even if the affected patient eats more calories, lean body mass will be lost, indicating there is a fundamental pathology in place. Cachexia is often seen in end-stage cancer, and in that context is called "cancer cachexia.”
  • the inhibitors of mi -144 can be used in other diseases characterized by excessive angiogenesis, for example diabetic retinopathy.
  • Another advantage is that it has recently been shown that a single injection of miRNA inhibitors like antagomirs ensures an efficient reduction of the expression and function of a miRNA for at least 2 months. As the toxicity of antagomirs is little and very transient, these agents are very attractive candidate drugs in the clinic.
  • modulators of miR-144 can be used to treat diseases characterized by impaired angiogenesis, such as ischemic heart disease or ischemic limb disease (which is equivalent to saying that methods of treating diseases characterized by impaired angiogenesis according to these embodiments involve administering a miR-144 mimetic to a subject in need thereof).
  • miR-144 mimetics can effectively increase revascularization of ischemic hearts and limbs.
  • no molecule-based treatment to promote angiogenesis has made it to the clinic.
  • the capacity of miRNAs (such as miR-144) to target various downstream effectors might offer a therapeutic advantage to interfere with the complex modulation of vessel growth, maturation and functional maintenance.
  • an "effective amount" of the miR-144 modulators is an amount sufficient to obtain the desired effect (e.g. sufficient to hybridize to the target miRNA in case of antagomirs, or an amount sufficient to inhibit tumour growth in a subject; or, in the case of mimetics, an amount sufficient to increase blood flow in ischemic tissue).
  • an effective amount of the miRNA modulator to be administered to a given subject by taking into account factors such as the size and weight of the subject; the extent of the disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic.
  • an effective amount of the miRNA modulator of the invention comprises an intracellular concentration of from about 1 nanomolar (nM) to about 1 ⁇ , preferably from about 2 nM to about 100 nM, more preferably from about 2.5 nM to about 50 nM or to about 10 nM. It is contemplated that greater or lesser amounts of miRNA modulators can be administered.
  • miR-144 can be used as a biomarker.
  • endogenous miR-144 is meant.
  • a "biomarker", or biological marker, as used herein is a substance used as an indicator of a biological state. It can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. According to particular embodiments, the expression levels of endogenous miR-144 will be assessed. The way expression levels are measured is not vital to the invention, and the skilled person is well aware of methods to determine expression levels of a given miRNA, e.g. using realtime PCR such as TaqMan ® MicroRNA Assay (Ambion), or the microRNA Expression Profiling Assay (lllumina).
  • miR-144 expression levels can be used as a biomarker to detect diseases with excessive angiogenesis, most particularly cancer. Indeed, in subjects with cancer expression of miR-144 will be increased, so that the increased levels can be used to detect the presence of cancer. To this end, miR-144 expression levels will typically be determined in a sample from said subject, most particularly a sample suspected to contain a tumour. To determine whether miR-144 levels are increased or not, expression will typically be compared to a control sample. Choosing a relevant control is well within the reach of the skilled person, but typically will be a comparable sample (e.g. from the same tissue and/or in the same form (liquid, solid, ...)) in which it has been verified that no tumour is present.
  • a comparable sample e.g. from the same tissue and/or in the same form (liquid, solid, ...)
  • the mere presence of mi -144 may indicate the presence of disease, more particularly cancer (e.g. in tissues/samples where miR-144 is not normally expressed).
  • the expression levels of miR-144 can not only be correlated with the presence of cancer, but can also be correlated with the nature and/or the aggressiveness of the tumour.
  • miR-144 is not only a biomarker for the presence of a tumour, but can in these embodiments also be a biomarker to determine malignancy of a tumour and/or to determine the nature of the tumour (e.g. whether it is a breast tumour or not).
  • the expression levels of miR-144 can be used to assess pharmacologic responses to a therapeutic intervention (i.e. to check whether a given therapy works) and/or to determine a change in medication or treatment regime (e.g. a switch in therapy if the levels of miR-144 do not decrease after initiation of a given therapy).
  • a kit comprising material to detect expression levels of miR-144 (e.g. specific probes for miR-144), and/or materials (such as enzymes) needed to facilitate miR- 144 detection (e.g. buffers and enzymes to isolate miRNA, reverse transcriptase and primers to convert miR-144 to cDNA, primers and polymerase to perform PCR to quantify miR-144, or (possibly labeled) probes hybridizing to miR-144, optionally fixed to a substrate such as a microarray or chip).
  • material to detect expression levels of miR-144 e.g. specific probes for miR-144
  • materials such as enzymes
  • materials such as enzymes
  • needed to facilitate miR- 144 detection e.g. buffers and enzymes to isolate miRNA, reverse transcriptase and primers to convert miR-144 to cDNA, primers and polymerase to perform PCR to quantify miR-144, or (possibly labeled) probes hybridizing to miR-144
  • Example 1 Generation of endotheliumselective Dicer deficient mice
  • Dicer Dicer
  • Cre-LoxP Cre-LoxP system
  • vascular network formation in E13.5 extra-embryonic yolk sacs, as we aimed to study the role of miRNAs once vasculogenesis was completed and vascular network formation was ongoing (Gordon et al., 2008).
  • mice homozygous for the floxed Dicer alleles (Dicer flox/flox ) (Yi et al., 2006) were therefore bred with male transgenic mice expressing the Cre recombinase under the control of the endothelium-specific Tek promotor (Koni et al., 2001). Analogous to what is described in this reference (Koni et al., 2001), double-heterozygous male progenies (Tek Cre + :Dicer Aflox + ) were identified and mated with female Dicer flox flox mice to obtain endothelium-specific Dicer mutant mice.
  • Phenotypic analysis of the mutant yolk sacs revealed an incompletely established vascular network. Indeed, stereomicroscopic analysis of the mutants indicated that, compared to controls, the formation of a hierarchically branched vascular network - with ramifications into large and small branches covering the entire yolk sac - was disturbed (data not shown). These observations suggest impaired vascular branching and networking in the absence of endothelial Dicer and, as a result, large areas of the mutant yolk sacs appeared to lack any blood supply (data not shown). Whole mount staining for CD31 on intact control and mutant yolk sacs confirmed fewer blood vessels in parts of the yolk sacs (data not shown). Similar observations were made with whole mount staining for VE-cadherin (not shown).
  • the Dicer mutant embryos exhibited no general developmental delay, but suffered from various organ-specific defects.
  • Tek Cre + :Dicer A flox mutants we found incomplete and defective branching of the yolk sac vascular network.
  • miRNA-126 regulated in the Dicer mutants, was miR-126 (Table 1 and 2), which was recently shown to be an endothelium-selective miRNA (Fish et al., 2008; Wang et al., 2008).
  • qPCR analysis utilizing miRNA-specific primers we confirmed in the mutant yolk sacs the reduced expression of miR- 126 ( Figure 2).
  • miR-144 might be an interesting candidate to further investigate for its potential role in angiogenesis.
  • miR-144 promotes an endothelial tip cell fate, also the genetic program of these cells should change, as endothelial tip cells have a distinct gene signature (Larrivee et al., 2009).
  • miR-126 was already recognized as a pro-angiogenic miRNA (Fish et al., 2008), but the strongest regulated miRNA, miR-144, was only known for its hematopoietic involvement (Fu et al., 2009).
  • miR-144 was (lowly) expressed in HUVECs, and GOF studies with 3D spheroid assays, modified to allow fluorescent-based tracing in angiogenic sprouts (not shown), revealed that miR-144 prioritized endothelial cells to acquire the tip cell position, thereby promoting sprout branching in vitro.
  • miR-144 is primarily expressed in tumor endothelial cells in vivo
  • miR-144 might be an interesting anti-angiogenic target during tumor growth.
  • the expression of miR-144 progressively increased ⁇ 35-fold in Panc02 tumors in vivo, reaching a maximum around ⁇ 900-1000 mm 3 (see Figure 10).
  • Pilot FACS sorting and in situ hybridization experiments on these tumors showed that miR-144 was primarily expressed in tumor endothelial cells (not shown).
  • mice were inoculated subcutaneously with Panc02 cells.
  • panc02 cells When the tumors reached a size of ⁇ 100 mm3, mice were randomized to receive one bolus i.v. injection of specific antagomirs against miR-144 (antagomir-144) or a control scrambled version (antagomir-co) at 8 mg/kg (i.e.
  • pro-thrombotic status was characterized by increased platelet activation, decreased aPTT, lower circulating levels of fibrinogen, and elevated circulating levels of PAI-1 (known to be primarily derived from the endothelium (Fischer et al., 2007)), but with absence of systemic coagulopathy (e.g. normal FVII levels) or acute phase response (e.g. normal hepatic synthesis of fibrinogen).
  • antagomir-144 treatment protected mice from death and the development of CASS.
  • Example 9 Further validation of mi -144 as anti-tumor target
  • the therapeutic potential of antagomir-144 will be validated in various mouse tumor models.
  • Second phase we will use various other syngeneic and xenograft mouse tumor models to test the therapeutic potential of antagomir-144.
  • Antagomirs have medically seen enormous potential as it has been reported that a single bolus i.v. injection of antagomirs (80 mg/kg) efficiently silences the expression of a specific endogenous mi NA up to 8 weeks after administration (Krutzfeldt et al., 2005). In addition, toxicity is very little, with only documented findings on transiently (non-specific) elevated liver enzymes and disturbed levels of cholesterol, and a slight inflammatory response (Krutzfeldt et al., 2005).
  • mice we will inoculate 1 x 10 6 Panc02 cells in the dorsal flank of age- and gender-matched C57BI6 mice. When the tumors have reached a size of ⁇ 100 mm 3 , we will randomize the mice into groups which receive i.v. injections of antagomir-144 or control scrambled antagomir (both dissolved in saline):
  • antagomirs e.g. 8 mg/kg, 20 mg/kg, 40 mg/kg
  • Different doses of antagomirs will be further evaluated in a single bolus injection i.v. when the tumors reached a size of ⁇ 100 mm 3 ;
  • Mouse health and survival will be followed up daily, and tumor growth will be followed up daily by measurements of the length (L) and width (W) of tumors via an accurate electronic caliper, and by an investigator blinded to the treatment.
  • Mouse tumor volumetric size is calculated by the following formula: (L x (W 2 ) x ⁇ )/6 (Loges et al., 2010).
  • Mouse total body weights will be documented regularly.
  • tumors will be taken out at sizes around ⁇ 1000 mm 3 , or at the end of the experiment (day 36 after inoculation, or when the tumor size has reached the ethical limit of ⁇ 2500 mm 3 ).
  • Tumor tissue will be subjected to RNA extraction using previously optimized protocols (miRNAEasy, Qiagen), and the expression of miR-144 and the housekeeping small RNA U6 will be measured by Taqman qPCR (commercially available via Applied Biosystems). For specificity reasons, we will also measure the expression of related (miR-451) and unrelated (miR-126) miRNAs. Tests in various syngeneic and xenograft mouse tumor models
  • antagomir-144 will also be explored in various subcutaneous syngeneic and xenograft mouse tumor models, including the use of tumor models previously shown to be relatively resistant to anti-VEGF treatment (Loges et al., 2010):
  • Panc02 pancreas adenocarcinoma, C57BI6
  • 4T1 breast adenocarcinoma, BalbC
  • CT26 colon adenocarcinoma, BalbC
  • EL-4 T-cell lymphoma, C57BI6
  • MDA-MB-231 human breast adenocarcinoma
  • DanG human pancreas adenocarcinoma
  • mice age- and gender-matched recipient mice will be inoculated in the dorsal flank with 1 x 10 6 cells, and followed up for survival and tumor growth as described above (the duration of follow-up depends on tumor cell type).
  • We will first do an expression analysis for miR-144 by qPCR at different tumor sizes and in the different tumor cell lines, as previously performed in the subcutaneous Panc02 model ( Figure 10). Then, we will apply the optimal mode of administration of antagomir-144, and analyze, compared to control treated mice, the following parameters:
  • ex vivo analysis of vascular densities includes staining for endothelial markers (CD31, lectin) on paraffin sections.
  • endothelial markers CD31, lectin
  • the area of tumor cell proliferation will be determined by staining for proliferation markers (Ki-67, PCNA) or by pre-injection of BrdU 24.
  • the necrotic area will be determined by measuring the area of autofluorescence (Loges et al., 2010). Differences in the recruitment of inflammatory cells will be analyzed by staining for leukocyte (CD45) and macrophage (Mac3) markers (Loges et al., 2010). Differences in the cancer-associated stromal fibroblast compartment will be analyzed by staining for vimentin (Loges et al., 2010). The presence of thrombi will be analyzed by staining for fibrinogen and VWF (Loges et al., 2010).
  • tumor hypoxia will be detected at 120 min after injection of 60 mg/kg pimonidazole hydrochloride into tumor-bearing mice (Loges et al., 2010).
  • tumor sections will be immunostained with hypoxyprobe-l-Mabl (Chemicon) following the manufacturer's instructions.
  • blood will be repetitively sampled from tumor-bearing mice by puncturing the retro-orbital plexus under isoflurane anesthesia. Specific blood samples (plasma, serum) will be taken for mechanistic studies (see below). Full blood counts will be obtained via an automated blood counter (Abbott Cell-Dyn 3500), including mean platelet volume (MPV) as a surrogate marker of platelet activation.
  • MPV mean platelet volume
  • antagomir-144 might affect tumor angiogenesis.
  • miR-144 fl fl mice which, by intercrossing with endothelial-selective Cre deleter mice, will allow to generate cell-type specific genetic data to corroborate the role and potential of miR-144 as a novel anti-angiogenic target in cancer.
  • One of the goals is to identify the molecular mechanism by which antagomir-144 is capable of affecting tumor angiogenesis.
  • miR-144 targets the endothelial forkhead transcription factor FoxOl
  • Panc02 tumor-bearing Tek-YFP mice C57BI6 background; available
  • Tumor endothelial cells will be isolated from tumor cell suspensions with a FACS Aria Cell Sorter by positive staining for YFP and CD31, and negative staining for Macl/CDllb (operational).
  • RNA extraction of sorted cells using miRNAEasy will be performed to analyze the expression of miR-144, FoxOl and the FoxOl target gene eNOS (Taqman probes are commercially available with Applied Biosystems). Protein extracts of sorted cells will be used for western blotting of FoxOl and eNOS, using commercially available antibodies.
  • Sorted tumor endothelial cells will be used for the measurement of sgppl by qPCR (Taqman probe available via Applied Biosystems) and western blot using commercially available antibodies.
  • miR-144 targets FoxOl and sgppl with different biological read-out: miR-144 represses FoxOl expression to instruct endothelial tip cell fate, whereas it represses sgppl expression to increase its thrombogenicity.
  • miR-144-mediated repression of sgppl affects endothelial tip cell fate, we will use our previously established dual-labeled spheroid assay and analyze the role of sgppl in endothelial tip cell specification, using HUVECs transfected with sgppl siRNA. Vice versa, we will use HUVECs transfected with FoxOl siRNA to analyze aggregation of co-cultured platelets.
  • HUVECs are transfected with luciferase constructs in which the binding sequence of miR-144 to sgppl is cloned (operational).
  • antagomir-144 To analyze the specificity of antagomir-144 to affect platelet activation in tumor-bearing mice, we will also study the effect of antagomir-144 in non-tumor mouse thrombosis models. To this end, we will use a mouse model of pulmonary thrombo-embolism to test the effect of antagomir-144. C57BI6 mice are injected with antagomir-144 or control scrambled antagomir, and 3 days later with a mixture of collagen (0.5 mg/kg) and epinephrine (60 mg/kg) (Angelillo-Scherrer et al., 2005). Thrombo-emboli will be formed and the time of survival will be followed.
  • antagomir-144 protects against cancer-associated anemia.
  • the anemia in these mice was macrocytic of nature (i.e. increased MCV), and with normal serum iron levels.
  • CASS could be caused by abnormal circulating levels VEGF thereby causing regression of hepatic blood vessels and reduced levels of circulating Epo (Tarn et al., 2008; Xue et al., 2008), this appears not to be the case in our experiments (i.e. no differences in plasma levels of VEGF and Epo, absence of hepatic vessel regression).
  • antagomir-144 protects against anemia by preventing repression of erythropoiesis.
  • HUVECs we will transfect HUVECs with miR-144 duplexes and analyze the levels of (secreted) SCF by qPCR and ELISA. As we strongly believe that miR-144 targets FoxOl, sgppl and SCF with different biological readout (see also above), we will use our previously established dual-labeled spheroid assay and analyze the role of SCF in endothelial tip cell specification, using HUVECs transfected with SCF siRNA (Qiagen). To further establish sgppl as a target gene of miR-144, we will use state-of-the-art 3'UTR luciferase reporter assays: i.e. HUVECs are transfected with luciferase constructs in which the binding sequence of miR-144 to SCF is cloned (operational). Regulation of miR-144 expression
  • a key research tool will be the availability of miR-144 fl fl mice. We developed this unique mouse model via outsourcing to GenOway, and these mice are currently available.
  • mice can be used for cross-breedings.
  • We will generate and phenotype mice lacking miR-144 expression in a conditional cell-type specific fashion (i.e. VE-Cad-CreERT), to further explore its role in tumor angiogenesis using the syngeneic models as described above (Tamoxifen will be administered to induce genetic recombination in VE-Cad-expressing host endothelial cells). These mice will also be instrumental for our mechanistic studies as described above.
  • mice will also be instrumental to perform expression analysis and genetic lineage tracing. Therefore, we plan to cross-breed the miR-144 fl/fl mice with Rosa26 LacZ , Rosa26 YFP and/or Rosa26 RFP reporter mice (all available). Further intercrossing with VE-Cad-CreERT will allow to genetically trace down miR-144 expressing endothelial tip cells by confocal microscopy (see above), and to isolate them by FACS sorting for further mechanistic studies. Extension of clinical data
  • antagomir-144 will be studied in some clinically relevant settings, including safety/toxicity studies, studies on co-administration of antagomir-144 together with chemotherapy, studies on potential effects on metastasis and lymphangiogenesis, and studies in a mouse model of choroidal neovascularization (CNV).
  • CNV choroidal neovascularization
  • Imperative for potential further development of antagomir-144 as a novel anti-angiogenic drug will be the lack of toxicity in vivo.
  • Antagomirs have medically seen enormous potential as it has been reported that a single bolus i.v. injection of antagomirs (80 mg/kg) efficiently silences the expression of a specific endogenous mi NA for many weeks (Krutzfeldt et al., 2005).
  • toxicity is very little, with only documented findings on transiently (non-specific) elevated liver enzymes and disturbed levels of cholesterol, and a slight inflammatory response (Krutzfeldt et al., 2005).
  • antagomir-144 might synergistically amplify the efficacy of chemotherapy.
  • We will therefore test the efficacy of combined administration of antagomir-144 and chemotherapy in mice, subcutaneously inoculated with Panc02 cells, as done previously (Fischer et al., 2007).
  • We will administer antagomir-144 or scrambled control antagomir and/or the chemotherapeutic agent gemcitabine (125 mg/kg, 2x per week), and mouse survival and tumor growth will be followed as described above. Effects of antagomir-144 on metastasis
  • antagomir-144 might block tumor angiogenesis, it might also reduce the hematogenic metastasis of tumor cells to distant organs.
  • Age-matched female BalbC mice will be injected with 1 x 10 6 4T1 cells in the left mammary fat pad, and mouse survival and tumor growth will be followed as described above.
  • antagomirs will be administered in bolus i.v., when the primary tumor reached a size of ⁇ 100mm 3 .
  • primary tumors will be removed, and tumor weight and volume will be determined.
  • the incidence of tumor invasion into the lungs will be analyzed by tracheal instillation with Indian Ink, and macroscopic quantification of metastatic nodules on the lung surfaces (white after destaining), and histologically confirmed.
  • antagomirs will be administered in bolus i.v. at 2 days after inoculation (when the primary tumor reached a size of ⁇ 30mm 3 ). At day 11 after inoculation, primary tumors will be removed, and tumor weight and volume will be determined. The incidence of tumor invasion into adjacent organs, hemorrhagic ascites, and regional celiac and mesenteric lymph node metastasis will be recorded, and histologically confirmed. We will carefully analyze that potential differences are not due to differences in primary tumor size.
  • antagomir-144 might hold the potential to become a novel anti-angiogenic drug in diseases other than cancer, such as eye diseases.
  • CNV choroidal neovascularization
  • 3 laser burns will be placed with a 532 nm green laser at 9, 12, and 3 o'clock positions around the optic disk using a slit lamp delivery system in age- and gender-matched C57BI6 mice, anesthetized with Nembutal and pretreated with the pupil dilator Tropicamide.
  • a slit lamp delivery system in age- and gender-matched C57BI6 mice, anesthetized with Nembutal and pretreated with the pupil dilator Tropicamide.
  • First we will perform a time course study on the expression of mi -144 by qPCR in the lasered mouse eye.
  • FITC-conjugated dextran 50 mg/ml, Mr 2 x 106 Da; Sigma
  • FITC+ vascular area will be quantified on flat mount preparations using a Zeiss fluorescence microscope with automated macros.
  • antagomir-144 is an innovative angiogenesis inhibitor with high therapeutic potential, which will be further explored. Given the high efficacy of antagomirs, as well as their low toxicity with a single bolus injection, the benefits of this approach are clear. As far as we know, this is the first suggestion to use antagomirs as anti-angiogenic drug in cancer.
  • endothelial tip cells are critical for a better understanding of vascular branching and sprouting in health and disease, with potential clinical translation. Indeed, drug development focuses on specifically targeting these endothelial tip cells as anti-angiogenic strategy, as exemplified by the recent findings to target tip cell molecules such as Dl 14 and Unc5B.
  • the present approach allows specific targeting of endothelial tip cells in a hereto unrecognized manner.
  • MicroRNAs target recognition and regulatory functions. Cell 136, 215-233.
  • MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science (New York, NY 324, 1710-1713.
  • Cheloufi S Dos Santos CO, Chong MM, Hannon GJ. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature. 2010; 465(7298):584-9. Cifuentes D, Xue H, Taylor DW, Patnode H, Mishima Y, Cheloufi S, Ma E, Mane S, Hannon GJ, Lawson ND, Wolfe SA, Giraldez AJ. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science. 2010; 328(5986):1694-8.
  • miR-126 regulates angiogenic signaling and vascular integrity. Developmental cell 15, 272-284.
  • VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161, 1163-1177.
  • LYVE-1 hyaluronan receptor LYVE-1
  • LYVE-1 is not restricted to the lymphatic vasculature; LYVE-1 is also expressed on embryonic blood vessels.
  • Tie2-Cre transgenic mice a new model for endothelial cell-lineage analysis in vivo. Developmental biology 230, 230-242.
  • Noguera-Troise I Daly C, Papadopoulos NJ, Coetzee S, Boland P, Gale NW, Lin HC, Yancopoulos GD, Thurston G. Blockade of DII4 inhibits tumour growth by promoting non-productive angiogenesis. Nature. 2006; 444(7122):1032-7.
  • miR- 451 regulates zebrafish erythroid maturation in vivo via its target gata2. Blood 113, 1794-1804.
  • VEGF VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis. Nat Med. 2006; 12(7):793-800.
  • Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancer-associated systemic syndrome. Proc Natl Acad Sci U S A. 2008; 105(47):18513-8.

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Abstract

La présente demande concerne le domaine des micro-ARN, et plus particulièrement, le rôle des micro-ARN dans l'angiogenèse. Il est démontré ici que la modulation de micro-ARN spécifiques, tels que miR-144, a un pouvoir thérapeutique dans les maladies caractérisées par une angiogenèse aberrante. Ceci est particulièrement pertinent dans, par exemple, la croissance tumorale et la métastase, qui sont caractérisées par une angiogenèse excessive et pour lesquelles l'inhibition de miR-144 est une nouvelle stratégie thérapeutique. A l'inverse, les maladies caractérisées par une angiogenèse insuffisante (par e., associée à l'ischémie) peuvent bénéficier d'une augmentation de l'activité du miR-144.
PCT/EP2012/056839 2011-04-13 2012-04-13 Modulation du miarn dans les maladies caractérisées par une angiogenèse aberrante WO2012140234A1 (fr)

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WO2016022536A3 (fr) * 2014-08-04 2016-03-31 MiRagen Therapeutics, Inc. Inhibiteurs de myh7b et utilisations associées
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CN114632090A (zh) * 2015-09-08 2022-06-17 西伦蒂斯私人股份公司 siRNA及其在抑制NRARP基因表达的方法和组合物中的用途

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WO2014140911A3 (fr) * 2013-03-15 2015-02-19 The Hospital For Sick Children Méthodes de diagnostic et thérapeutique associées au microarn-144
US20160076026A1 (en) * 2013-03-15 2016-03-17 The Hospital For Sick Children Diagnostic and therapeutic methods relating to microrna-144
EP3068884A4 (fr) * 2013-11-13 2017-06-14 The Texas A&M University System Micro-arn modulant les voies de la lymphangiogenèse et de l'inflammation dans les cellules des vaisseaux lymphatiques
WO2016022536A3 (fr) * 2014-08-04 2016-03-31 MiRagen Therapeutics, Inc. Inhibiteurs de myh7b et utilisations associées
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