Classifications

• • 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
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
  • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
  • C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering N.A.
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates
• • 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
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/321—2'-O-R Modification
• • 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
  • C12N2310/30—Chemical structure
  • C12N2310/33—Chemical structure of the base
  • C12N2310/334—Modified C
  • C12N2310/3341—5-Methylcytosine
• • 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
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
  • C12N2310/341—Gapmers, i.e. of the type ===---===
• • 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
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
  • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

• E2-EPF5 a Novel Therapeutic Protein and Target
• the present invention pertains to therapeutic methods and pharmaceutical compositions for the inhibition of VEGF-dependent vascularisation, and in particular of tumor related vascularisation.
• VEGF Vascular endothelial growth factor
• hypoxia is a reduction in the normal levels of tissue oxygen tension homeostasis and occurs during acute and vascular disease, pulmonary disease and cancer.
• HIF-1 hypoxia-inducible factor-1
• hypoxia is a reduction in the normal levels of tissue oxygen tension homeostasis and occurs during acute and vascular disease, pulmonary disease and cancer.
• tumor cells adapt to hypoxia by inducing HIF-1.
• HIF-1 mediates adaptive responses to changes in tissue oxygenation and activates the transcription of a variety of genes involved in multiple processes including cell proliferation, cell survival, invasion and metastasis, apoptosis, angiogenesis, glucose and iron metabolism.
• Hif-1 is overexpressed in numerous human cancers and is associated with poor prognosis and increased patient mortality. Recent studies have indicated that HIF-1 mediates resistance to chemotherapy and radiation due to hypoxia, oncogene activation and mutations. Inhibition of HIF-1 activity may therefore represent an important component of anti-angiogenesis therapies.
• E2-EPF5 is a member of the ubiquitin-conjugating enzyme (E2) family that does conjugate ubiquitin to cellular proteins.
• Ubiquitin and ubiquitin-like modifiers are processed and attached to substrate proteins by a mechanistically conserved enzymatic cascade, which includes ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2 or UBCs) and ubiquitin ligases (E3).
• E1 ubiquitin activating enzymes
• E2 or UBCs ubiquitin conjugating enzymes
• E3 ubiquitin ligases
• the present invention now provides a new role for ubiquitin-conjugating enzyme E2-EPF5 in hypoxia, and, in particular, in the building up of proteins regulated by transcription factor HIF- 1 in response to hypoxia.
• the invention provides a method for the treatment of diseases related to aberrant neo-vascularisation comprising administering an effective amount of an agent inhibiting the expression of the gene encoding ubiquitin conjugating enzyme E2-EPF5 or inhibiting an activity of E2-EPF5 gene product.
• the neo- vascularisation is angiogenesis in tumors, synovial angiogenesis in rheumatoid arthritis, ocular neo-vascularisation as observed in diabetic retinopathy, skin angiogenesis in psoriasis, or hypoxia-induced angiogenesis in liver cirrhosis.
• the neo-vascularisation is VEGF-dependent tumor angiogenesis.
• the agent is an inhibitory nucleic acid capable of specifically inhibiting ubiquitin conjugating enzyme E2-EPF5, preferably an antisense oligonucleotide compound, more preferably an siRNA compound.
• the agent is an antibody specifically binding to ubiquitin conjugating enzyme E2-EPF5.
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising an effective amount of an agent inhibiting the expression of the gene encoding ubiquitin conjugating enzyme E2-EPF5 or inhibiting the an activity of E2-EPF5 gene product.
• the E2-EPF5 inhibitor is an antisense oligonucleotide or an siRNA or an antibody specifically binding E2-EPF5.
• the present invention relates to methods for identifying compounds useful for treatment of VEGF-dependent vascularisation comprising: (a) contacting a E2- EPF5 polypeptide with a test compound (b) detecting modulation of E2-EPF5 biological activity.
• E2-EPF5 biological activity is reduced.
• the present invention relates to a method for reducing the amount or to inhibiting the activity of one or more polypeptides regulated by HIF-1 in response to hypoxia comprising inhibiting the expression of ubiquitin conjugating relating enzyme E2- EPF5.
• the HIF-1 regulated genes or proteins are selected from the group consisting of GLUT-1 , GLUT-3, HK2, EPO, NOS2, VEGF, TGF-alpha, TGF-beta, VEGFR-2, C-Met, UPAR, CXCR4, carbonic anhydrase IX (CAIX).
• the present invention is based on the surprising discovery that inhibition of ubiquitin conjugating enzyme E2-EPF5 interferes with the HIF-1 dependent protein expression in response to hypoxia.
• E2-EPF5 inhibiting E2-EPF5 was found to interfere with the building-up of VEGF in response to hypoxia.
• Proangiogenic VEGF is required for the proliferation and migration of the endothelial cells that constitute the first blood vessels.
• VEGF is a 34-45 kDa glycoprotein with a wide range of activities that besides promotion of angiogenesis also includes enhancement of vascular permeability, a crucial feature of inflammations.
• VEGF not only acts as a growth factor for endothelial cells, but also on several other cells including HIV-associated Kaposi's sarcoma cells and other tumor and leukemia cells.
• Neoplastic cells frequently express elevated levels of VEGF, which not only is thought to be related to stimulation of angiogenesis but also to activation of proliferation- stimulating autocrine signalling pathways.
• VEGF also induces indirect effects.
• the receptor Notchi is up- regulated by VEGF allowing also an enhanced response to the ligands of this cell-surface receptor too.
• VEGF expression has been associated with several pathological states such as tumor angiogenesis, hypoxia-related angiogenesis, several forms of blindness (e.g. diabetic retinopathy, proliferative diabetic retinopathy, age related macular degeneration), rheumatoid arthritis, psoriasis and wound healing and others.
• a number of studies have demonstrated that elevated levels of VEGF alone are sufficient to induce neovascularization. For instance, it has been demonstrated that a single injection of VEGF augmented collateral vessel development in a rabbit model of ischemia (Takashita et al., 1995 J; Clin. Invest. 93, 662).
• VEGF also can induce neovascularization when injected into the cornea.
• the present invention now provides methods for reducing the amount or activity of one or more HIF-1 regulated polypeptides, and in particular of VEGF, which is built up in response to hypoxia.
• a number genes encoding a proteins with a variety of functions are regulated by HIF-1 under hypoxia is known, including but not limited to the following genes: GLUT-1 , GLUT-3, HK2, CAIX (involved e.g. in metabolic adaptation); EPO, NOS2 (involved e.g. in Apoptosis resistance); VEGF, TGF-alpha, TGF-beta, VEGFR-2 (involved e.g.
• HIF-1 regulated polypeptide in the context of the present invention includes any protein or polypeptide, the expression of which is controlled or influenced by the transcription factor HIF-1 , e.g. by binding of HIF-1 to a transcription regulatory element of the gene encoding said protein or polypeptide.
• the protein expression is activated by the activity of HIF-1 transcription factor, i.e. more protein is expressed in response to HIF-1 activation.
• HIF-1 is a heterodimer consisting of one of three alpha subunits (HIF-1 ⁇ , HIF-2 ⁇ or HIF-3 ⁇ ) and a beta subunit (HIF-1 ⁇ , also known as the Aryl Hydrocarbon Nuclear Translocator, or ARNT).
• HIF-Ib is constitutively expressed, whereas the expression of the alpha subunits is highly regulated.
• the level of the alpha subunits is determined by the rates of protein synthesis and protein degradation. Synthesis of the HIF1 alpha subunits is regulated via oxygen-independent mechanisms, whereas degradation is regulated primarily via O 2 -dependent mechanisms.
• the genes for the alpha subunits are mostly continuously transcribed and translated, the alpha subunit proteins is maintained at very low levels due to rapid destruction via proteasomal degradation. This destruction is inhibited under hypoxic conditions and this is the major mechanism of induction of HIFIa and the genes dependent on this transcription factor.
• E2-EPF5 encodes a 25 kDa class Il ubiquitin-conjugating enzyme with a 65aa-long basic C- terminal extension of low sequence complexity. E2-EPF5 was first discovered in a patient suffering from a skin disease called endemic pemphigus foliaceus (EPF) (Liu et al., 1992, JBC 267, 15829).
• EPF endemic pemphigus foliaceus
• E2-EPF5 is functionally distinct from other characterized E2 isoenzymes since it catalyzes multiubiquitin chain formation through lysine residue K11 and not through the K48 residue, which is the mechanism that is considered to mediate proteolytic events (Bach and Ostendorff, Trends in Biochemical Sciences (2003), 28(4), 189-195). E2-EPF5 was also found to support auto-ubiquitination suggesting a possible autoregulatory model for E2-EPF5. Substrates for E2-EPF5 have not been reported so far but its highly basic carboxy-terminal extension domain, which is unique within the E2 family members may indicate specificity to acidic proteins (Liu et al., J Biol Chem. 271 , 2817- 2822).
• the E2-EPF5 term (biological) activity includes, within the context of the present invention, besides its ubiquitin related activities also interference with HIF-1 mediated induction of hypoxia-regulated genes.
• E2-EPF5 The sequence of the human E2-EPF5 is available from public databases (GenBank Accession M91670, GM81915, SwissProt entry Q16763).
• the cDNA sequence is set forth as SEQ ID NO:1.
• the amino acid sequence is set forth as SEQ ID NO:2.
• E2-EPF5 also includes any homologous or orthologous sequences, variants and fragments as long as they keep the biological activity of E2-EPF5 herein described.
• the percentage of homology between the homologous sequence and the reference sequence desirably is at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.
• a "fragment” means any polypeptide molecule having at least 5, 10, 15 or optionally at least 25,35, or 45 contiguous amino acids of E2- EPF5. Further possible fragments include the catalytic site or domain including the recognition sites, ubiquitin binding sites, sites important for subunit interaction, and sites important for carrying out the other functions of the ubiquitin conjugating enzyme. Such domains or motifs can be identified by means of routine computerized homology searching procedures.
• Fragments can extend in one or both directions from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or up to 100 amino acids. Also encompassed in the term fragment are for instance E2-EPF5 epitopes.
• An E2-EPF5 epitope represents a site on the polypeptide against which an antibody may be produced and to which the antibody binds. Therefore, polypeptides comprising the amino acid sequence of a E2-EPF5 epitope are useful for making antibodies to E2-EPF5 polypeptide.
• an epitope comprises a sequence of at least 5, more preferably at least 10, 15, 20, 25, or 50 amino acid residues in length.
• the invention provides methods for the treatment of aberrant neo- vascularisation comprising administering an effective amount of an agent inhibiting ubiquitin conjugating relating enzyme E2-EPF5 activity.
• the present invention provides the use of agent inhibiting E2-EPF5 activity for the manufacture of a medicament for the treatment of a pathological state related to VEGF-dependent vascularisation, in particular to tumor angiogenesis.
• agent inhibiting E2-EPF5 activity for the manufacture of a medicament for the treatment of a pathological state related to VEGF-dependent vascularisation, in particular to tumor angiogenesis.
• the term "aberrant neo-vascularisation” as used herein means a vascularisation which does not normally occur in a healthy organism and is related to a abnormal or disease state.
• the aberrant neo-vascularisation is preferably controlled or influenced by the activity of VEGF, i.e. "VEGF-dependent vascularisation”.
• the aberrant neo-vascularisation is VEGF-dependent tumor angio
• VEGF-dependent vascularisation refers to any generation of new blood vessels upon stimulation by VEGF and includes without limitation angiogenesis in tumors, synovial angiogenesis in rheumatoid arthritis, ocular neo-vascularisation as observed in diabetic retinopathy and some other eye diseases, skin angiogenesis in psoriasis, or hypoxia-induced angiogenesis in liver cirrhosis.
• the present invention provides a method for inhibiting a HIF-1 regulated gene in a cell comprising inhibiting the expression or activity of ubiquitin conjugating relating enzyme E2-EPF5.
• the inhibition of the HIF-1 regulated gene may for instance be achieved by lowering the amount of HIF-1 by ubiquitin dependent protein degradation e.g. by interference with the synthesis or stabilization of HIF-Ia protein or the HIF-Ia transactivation.
• the present invention provides a method for reducing the amount of a HIF-1 regulated polypeptide comprising inhibiting the expression or activity of ubiquitin conjugating relating enzyme E2-EPF5.
• the HIF-1 regulated gene is VEGF.
• the present invention further provides anti-angiogenic methods.
• methods are provided for the inhibition of angiogenesis, including tumor angiogenesis, comprising inhibiting the expression or activity of ubiquitin conjugating relating enzyme E2-EPF5.
• the term "inhibition” means a reduction of the gene expression or protein activity. Preferably, such a reduction is at least 20%, more preferably at least 50%, 60%, 70%, 80%, 90% or 95% as compared to the level of expression or activity without inhibition.
• Gene or protein inhibition may be achieved by any suitable technique.
• the skilled person knows a variety of methods and techniques how to inhibit gene expression or protein activity.
• E2-EPF5 can be inhibited by RNA interference or antisense technologies or using LMW compounds that interfere with the function of E2-EPF5 or by any agent that lowers ubiquitin conjugating relating enzyme E2- EPF5.
• An agent inhibiting the ubiquitin conjugating relating enzyme E2-EPF5 activity can be any substance that reduces the biological activity of E2-EPF5.
• the agent may, for instance, inhibit the expression of an E2-EPF5 gene or an enzymatic activity of E2-EPF5, may induce degradation of E2-EPF5 polypeptides or may interfere with the biological activity of E2-EPF5 in any other way.
• the inhibitory agent is a low molecular weight compound or an inhibitory nucleic acid or an antibody.
• inhibitory nucleic acid refers to nucleic acid compounds capable of producing gene-specific inhibition of gene expression.
• Typical inhibitory nucleic acids include, but are not limited to, antisense oligonucleotides, triple helix DNA, RNA aptamers, ribozymes and siRNAs.
• knowledge of a nucleotide sequence may be used to design siRNA or an antisense molecules which potently inhibit the expression of ubiquitin conjugating relating enzyme E2-EPF5.
• ribozymes can be synthesized to recognize specific nucleotide sequences of a gene and cleave it. Techniques for the design of such molecules for use in targeted inhibition of gene expression is well known to one of skill in the art.
• Inhibitory nucleic acid compounds of the present invention may be synthesized by conventional means on a commercially available automated DNA synthesizer, e.g. an Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA synthesizer, or like instrument. Phosphoramidite chemistry may be employed.
• the inhibitory nucleic acid compounds of the present invention may also be modified, for instance, nuclease resistant backbones such as e.g. phosphorothioate, phosphorodithioate, phosphoramidate, or the like, described in many references may be used. The length of the inhibitory nucleic acid has to be sufficient to ensure that the biological activity is inhibited.
• the antisense oligonucleotides of the invention have lengths in the range of about 15 to 40 nucleotides.
• the oligonucleotide moieties have lengths in the range of about 18 to 25 nucleotides.
• Double-stranded RNA i.e. sense-antisense RNA, also termed small interfering RNA (siRNA) molecules, can also be used to inhibit the expression of nucleic acids for E2-EPF5.
• RNA interference is a method in which exogenous, short RNA duplexes are administered where one strand corresponds to the coding region of the target mRNA (Elbashir et al., Nature 2001 , 411 : 494-498).
• siRNA molecules Upon entry into cells, siRNA molecules cause not only degradation of the exogenous RNA duplexes, but also of single-stranded RNAs having identical sequences, including endogenous messenger RNAs. Accordingly, siRNA may be more potent and effective than traditional antisense RNA methodologies since the technique is believed to act through a catalytic mechanism.
• Preferred siRNA molecules are typically from 19 to 25 nucleotides long, preferably about 21 nucleotides in length and comprise the sequence of a nucleic acid for E2-EPF5. Effective strategies for delivering siRNA to target cells include, for example, transduction using physical or chemical transfection.
• siRNAs may be expressed in cells using, e.g., various PoIIII promoter expression cassettes that allow transcription of functional siRNA or precursors thereof. See, for example, Scherr et al., Curr. Med. Chem. 2003, 10(3):245-256; Turki et al., Hum. Gene Ther. 2002, 13(18):2197-2201 ; Georgia et al., Nat. Struct. Biol. 2003, 10(2):91-92.
• the invention also covers other small RNAs capable of mediating RNA interference (RNAi) such as for instance micro-RNA (miRNA) and short hairpin RNA (shRNA).
• RNAi RNA interference
• miRNA micro-RNA
• shRNA short hairpin RNA
• the agent inhibiting the ubiquitin conjugating relating enzyme E2-EPF5 activity is an antibody.
• Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
• mAbs monoclonal antibodies
• humanized or chimeric antibodies single chain antibodies
• Fab fragments fragments
• F(ab') 2 fragments fragments produced by a Fab expression library
• anti-Id anti-idiotypic antibodies
• Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof.
• an antigen such as target gene product, or an antigenic functional derivative thereof.
• host animals may be immunized by injection with E2-EPF5 polypeptides, derivatives or fragments, supplemented with suitable adjuvants.
• Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture.
• Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
• the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
• chimeric antibodies i.e. a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived from a murine mAb and a human immunoglobulin constant region
• techniques developed for the production of single chain antibodies can be adapted to produce E2-EPF5 antibodies.
• Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Such techniques are known in the art.
• the E2-EPF5 antibody is a "humanized antibody.”
• Techniques useful for the production of "humanized antibodies” can be adapted to produce antibodies to theE2-EPF5 polypeptides, fragments, derivatives, and functional equivalents disclosed herein. Such techniques are disclosed in U.S. Patent Nos, 5,932, 448; 5,693,762; 5,693,761 ; 5,585,089; 5,530,101; 5,910,771 ; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,545,580; 5,661 ,016; and 5,770,429, the disclosures of all of which are incorporated by reference herein in their entirety.
• Antibody fragments which recognize specific E2-EPF5 epitopes may be generated by known techniques.
• such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
• Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
• Suitable antibodies to E2-EPF5 proteins may be obtained from a commercial source or produced according to conventional methods. Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, single domain antibodies, Fv fragments, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
• mAbs monoclonal antibodies
• humanized or chimeric antibodies single chain antibodies
• single domain antibodies single domain antibodies
• Fv fragments fragments
• Fab fragments fragments
• F(ab') 2 fragments fragments produced by a Fab expression library
• anti-Id anti-idiotypic antibodies
• antibody/ immunoglobulin frameworks or scaffolds can be employed so long as the resulting polypeptide includes one or more binding region which is specific for the E2-EPF5 protein.
• Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, or fragments thereof (such as those disclosed elsewhere herein), and include immunoglobulins of other animal species, preferably having humanized aspects. Single heavy-chain antibodies such as those identified from camelids are of particular interest in this regard. Novel frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.
• non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the E2-EPF5 protein.
• Known non- immunoglobulin frameworks or scaffolds include Adnectins (fibronectin) (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd (Cambridge, MA) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), Small modular immuno- pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc. (Mountain View, CA)), Protein A (Affibody AG, Sweden) and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).
• Adnectins fibronectin
• ankyrin Molecular
• the anti-E2-EPF5 antibody or fragment thereof, or the polypeptide comprising a E2-EPF5-specific binding region may be bound, either covalently or non-covalently, to an additional moiety.
• the additional moiety may be a polypeptide, an inert polymer such as PEG, small molecule, radioisotope, metal, ion, nucleic acid or other type of biologically relevant molecule.
• Such a construct which may be known as an immunoconjugate, immunotoxin, or the like, is also included in the meaning of antibody, antibody fragment or polypeptide comprising a E2-EPF5-specific binding region, as used herein.
• the inhibitory agent is a small molecule (e.g., organic or inorganic molecules which are less than about 2 kDa in molecular weight, are more preferably less than about 1 kDa in molecular weight, and/or are able to cross the blood-brain barrier or gain entry into an appropriate cell) which affect E2-EPF5 expression or the activity of E2-EPF5 polypeptide.
• a small molecule compound may be identified by the screening methods as described below.
• E2-EPF5 gene and gene product as drug target for the development of therapeutics for use in treatment of individuals suffering from a disease as described above.
• screening methods for identifying compounds that may be used to treat diseases as described herein comprise, in preferred embodiments, contacting a test compound to a reaction mixture that contains an E2-EPF5 polypeptide.
• E2-EPF5 is a polypeptide having an amino acid sequence which may comprise the sequence set forth in SwissProt entry Q16763. However, any E2-EPF5 homolog, ortholog, variant, etc.
• E2-EPF5 polypeptide may have an amino acid sequence that is substantially homologous (e.g., at least 75%, 80%, 85%, 90%, 95% or 99% identical) to SwissProt entry Q16763.
• E2-EPF5 is provided as novel target for the screening for therapeutics useful in the treatment of diseases in which VEGF-dependent angiogenesis plays a role and, in particular, in tumor angiogenesis or ocular vascularisation or hypoxia-induced angiogenesis.
• the present invention provides methods for identifying a compound useful for the inhibition of VEGF-dependent vascularisation comprising (a) contacting a E2-EPF5 polypeptide with a test compound (b) detecting a modulation of E2- EPF5 biological activity.
• the modulation is usually detected with respect to a control reaction lacking the test compound.
• Modulation as used herein refers to an increase or reduction of the biological activity, preferably by at least 10%, at least 20%, at least 30%, at least 50% or at least 100%.
• the present invention provides a method of identifying a compound useful for treatment of a disease related to VEGF-dependent vascularisation, e.g. tumor vascularisation, comprising i) contacting a test compound with a E2-EPF5 polypeptide under sample conditions permissive for E2-EPF5 biological activity; ii) determining the level of said at least one E2-EPF5 biological activity; iii) comparing said level to that of a control sample lacking said test compound; and, optionally, iv) selecting a test compound which causes said level to change for further testing as a compound for the prophylactic and/or therapeutic treatment of a disease related to VEGF- dependent vascularisation.
• a disease related to VEGF-dependent vascularisation e.g. tumor vascularisation
• Compound screening assays may include cell-based or cell-free systems.
• Cell-based systems can be native, i.e., cells that normally express the ubiquitin conjugating enzyme E2- EPF5, as a biopsy or expanded in cell culture. In one embodiment, however, cell- based assays involve recombinant host cells expressing the ubiquitin conjugating enzyme. Determining the ability of the test compound to interact with the ubiquitin conjugating enzyme E2-EPF5 can also comprise determining the ability of the test compound to preferentially bind to the polypeptide as compared to the ability of a known binding molecule (e.g., ubiquitin) to bind to the polypeptide.
• a known binding molecule e.g., ubiquitin
• the polypeptides can be used to identify compounds that modulate ubiquitin ubiquitin conjugating activity.
• Such compounds can increase or decrease affinity for ubiquitinated protein substrate, or ubiquitinated protein substrate remnants.
• Such compounds could also, for example, increase or decrease the rate of binding to these components.
• Such compounds could also compete with these components for binding to the ubiquitin conjugating enzyme or displace these components bound to the ubiquitin conjugating enzyme.
• Such compounds could also affect interaction with other components, such as ATP, other subunits, such as E1 activating enzymes or E3 ligases.
• Ubiquitin conjugating enzyme E2-EPF5 derivatives and fragments can be used in fast screening methods e.g. automated high-throughput screens (HTS) to assay candidate compounds for the ability to bind to the ubiquitin conjugating enzyme.
• fast screening methods e.g. automated high-throughput screens (HTS) to assay candidate compounds for the ability to bind to the ubiquitin conjugating enzyme.
• HTS high-throughput screens
• Numerous suitable fast screening assays are known to the skilled person.
• E2-EPF5 functional ubiquitin conjugating enzyme
• E2-EPF5 ubiquitin conjugating enzyme
• Modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject.
• the E2-EPF5 polypeptides of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the E2-EPF5 protein and a target molecule that normally interacts with the E2-EPF5 protein.
• the target can be ubiquitin, ubiquitinated substrate, or polyubiquitin or another component of the pathway with which the ubiquitin conjugating enzyme protein normally interacts (for example E1 or E3 proteins).
• the assay includes the steps of combining the E2-EPF5 protein with a candidate compound under conditions that allow the E2-EPF5 protein or fragment to interact with the target molecule, and to detect the formation of a complex between the E2-EPF5 protein and the target or to detect the biochemical consequence of the interaction with E2-EPF5 and the target. Any of the associated effects of ubiquitin conjugating function can be assayed. This includes the production of ubiquinated substrates, proteolysis, decrease of free polyubiquitin, stability of the substrate.
• Determining the ability of the ubiquitin conjugating enzyme to bind to a target molecule can also be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA).
• BiA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore®). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
• test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound 1 library method; and synthetic library methods using affinity chromatography selection.
• biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in DeWitt et al. (1993) Proc. Natl. Acad. Sci.
• Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84; Houghten et al.
• Suitable assays for measuring activity of ubiquitin conjugating relating enzyme activity are well known in the art. These assays include, but are not limited to, the appearance of substrate, including increase in the amount of polyubiquitin or ubiquitinated substrate protein or protein remnant, appearance of intermediate and end products, such as disappearance of free ubiquitin monomers, general protein turnover, specific protein turnover, ubiquitin binding, binding to ubiquitinated substrate protein, subunit interaction, interaction with ATP, interaction with cellular components such as trans- acting regulatory factors, stabilization of specific proteins, and the like.
• compounds identified by the screening methods in accordance with the present invention are provided.
• Such compounds are preferably low molecular weight compounds or antibodies, in particular monoclonal antibodies, or inhibitory nucleic acids.
• the compounds have preferably anti-angiogenic activity, i.e. they inhibit the growth of blood vessel in general and in particular aberrant neo-vascularisation.
• the anti-angiogenic activity of such a can be determined by methods known in the art. Such methods include for instance microscopic assessment of angiogenesis in tumors, in vivo matrigel migration and angiogenesis assays, alginate microbead release assay of angiogenesis, disc angiogenesis assay, sponge implant model of angiogenesis, hollow fiber assay for tumor angiogenesis or corneal assay for angiogenesis.
• Such assays are for instance described in "Angiogenesis Protocols", March 2001 , ISBN: 0-89603-698-7, Series: Methods in Molecular Medicine, Volume #: 46.
• In vivo techniques consist of the cornea pocket andiris implant in the eye, the rabbit ear chamber, the dorsal skinfold chamber, the cranial window, the hamster cheek pouch window, the sponge implant assay.the fibrin clots, the sodium alginate beads and the Matrigel plugs, the ratmesenteric window, the chick embryo chorioallantoic membrane and the airsac in mice and rats (1).
• the avascular cornea assay we will discuss the avascular cornea assay, and the advantages and disadvantages of using this assay in different species.
• the cornea assay is based on the placement of an angiogenic inducer(tumor tissue, cell suspension, growth factor) into a corneal pocket in order toevoke vascular outgrowth from the peripherally located limbal vasculature. Incomparison to other in vivo assays, this assay has the advantage of measuringonly new blood vessels, because the cornea is initially avascular.
• An additional aspect of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
• Such pharmaceutical compositions comprise an effective amount of an agent inhibiting the expression of the gene encoding ubiquitin conjugating enzyme E2-EPF5 or inhibiting the an activity of E2-EPF5 gene product.
• compositions may for instance comprise antibodies, mimetics, agonists, antagonists, or inhibitory nucleic acids of ubiquitin conjugating enzyme E2-EPF5 in accordance with the present invention.
• the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
• the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
• compositions encompassed by the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra ⁇ articular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
• these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
• compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
• Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
• the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
• the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0. 1%- 2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
• compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
• administration labeling would include amount, frequency, and method of administration.
• compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
• the determination of an effective dose is well within the capability of those skilled in the art.
• the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
• the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
• a therapeutically effective dose refers to that amount of active ingredient, fragments thereof, antibodies, agonists, antagonists or inhibitors of the ubiquitin conjugating enzyme E2-EPF5. which ameliorates the symptoms or condition.
• Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
• Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
• Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
• Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
• Antisense (ASO) and siRNA oligonucleotides siRNAs are available from a number of commercial suppliers such as e.g. Qiagen,
• Oligoribonucleotides can be synthesized using TOM-phosphoramidite chemistry, as described by the manufacturer (Xeragon) and purified by RP-HPLC. Purity is assessed by capillary gel electrophoresis. Quantification is carried out by UV according to the extinction coefficient at 260 nM. Annealing of double-stranded RNA (dsRNA) is performed as described elsewhere (Elbashir et al., 2001).
• Modified antisense oligodeoxyribonucleotides are synthesized using phosphoramidite chemistry purified by HPLC, analyzed and characterized by electrospray mass spectrometry and capillary gel electrophoresis. Quantification is carried out by UV according to the extinction coefficient at 260 nM.
• AII oligonucleotides are designed against the human E2-EPF5 sequence (GenBank Accession M91670, Gl number, Gl:181915, SwissProt entry Q16763) and checked by BLAST against the Refseq and UNIGENE databases to maximize the level of specificity.
• a series of ASOs and siRNAs targeting E2-EPF5 are screened in a dual-color reporter assay as described previously (H ⁇ sken, D. et al, 2003).
• a vector containing the coding sequence of E2-EPF5 is recombined with a destination vector that contains the CFP- and YFP reporter sequences.
• the resulting dual reporter plasmid pNAS-X generates a fusion mRNA where the E2-EPF5 target sequence is inserted into the 3'-UTR region of the expressed YFP- reporter gene.
• E2-EPF5 ASO and siRNA activity is measured by the degree of inhibition of the YFP reporter protein.
• All cell culture reagents are from Gibco-BRL-lnvitrogen AG (Basel. Switzerland). Basal culture medium used are Dulbecco's Modified Eagle Medium (DMEM) for HeLa cells and RPMI1640 for NCI-H1299 cells. Media are supplemented with 10% fetal bovine serum (FBS) and 1% L-Glutamine plus optionally 50 microgram/ml Gentamycine antibiotic. For routine maintenance cells are split 1 : 4 twice a week.
• DMEM Dulbecco's Modified Eagle Medium
• FBS fetal bovine serum
• L-Glutamine fetal bovine serum
• Gentamycine antibiotic For routine maintenance cells are split 1 : 4 twice a week.
• cells are trypsinized, resuspended in medium with FBS but without antibiotics, and plated either into 24-well culture plates at a density of 1.5-2 x 104 cells per 0.5 ml per well or into 6-well plates at a density of 5-1O x 104 cells per 2 ml per well. The plates are incubated for 24 hours at 37 0 C in 5% CO2 at high humidity.
• siRNAs and desferrioxamine (DFO) and cobalt chloride treatment Transfection of siRNAs is performed using Oligofectamine according to the protocols provided by the manufacturer (Invitrogen). Briefly, siRNAs are diluted in Optimem to 1.2 ⁇ M siRNA per ⁇ l. Separately, Oligofectamine is diluted to 0.25 ⁇ l Oligofectamine per ⁇ l Optimem. Equal amounts of these siRNA and Oligofectamine solutions are mixed and incubated 20-25 minutes at room temperature to allow complex formation to proceed. Next, the siRNA/oligofectamine complexes are diluted in standard medium with 10 % FBS without antibiotics to the final desired concentration, typically 20-80 nM siRNA.
• the old medium is removed from the cells and replaced with the siRNA/Oligofectamine suspension: for 24-well plates 0.5 ml per well and for 6-well plates 2 ml per well.
• the media with the siRNAs are replaced with fresh media with 10 % FBS either without inducer or containing 150 ⁇ M deferrioxamine (abbreviated: DFO) or CoCI 2 (both from Sigma; from freshly prepared aqueous stock solutions).
• DFO deferrioxamine
• CoCI 2 both from Sigma; from freshly prepared aqueous stock solutions.
• ASOs are stored at 1 mM concentration in TE (10 mM Tris pH 8.0, 1 mM EDTA). Prior to the experiment, a 10 ⁇ M working solution is prepared in Optimem (Life Sciences Inc.). ASOs are diluted to 2 x the final concentration in Optimem, mixed with Lipofectin or Effectene also diluted at 2 x the final concentration and left at room temperature for 30 minutes. The standard medium is removed from a 30-50 % confluent T24 cell culture and the cell monolayer is once with serum-free OptiMEMI (Gibco BRL).
• the ASO/Lipofectin mix is then added to the cell monolayer which is then incubated 4 h at 37 0 C in the CO2 incubator, after which the medium is removed and replaced with standard medium with 10 % FBS. After 24- 72 hr incubation in a humidified CO2 incubator, the media are replaced with fresh media with 10 % FBS either without inducer or containing 150 DM deferrioxamine (abbreviated : DFO) or CoCI2 (both from Sigma; from freshly prepared aqueous stock solutions). After 6 h at 37°C the medium of the cells is collected for determination of secreted VEGF whereas of the cell monolayer total RNA is prepared or whole cell protein extract for Western blot analysis.
• DFO DM deferrioxamine
• CoCI2 both from Sigma
• RNA is prepared using the RNeasy 96 kit (Qiagen #74183) following the manufacturer's instructions.
• Primer pairs and FAM-labelled TaqMan probes for real time PCR are designed using the Primer Express v1.0 program (ABI PRISM, PE Biosystems) and purchased from Microsynth (Switzerland), Qiagen or Applied Biosystems ("Assays-on- demand"). The following primer sequences are used:
• E2-EPF5 (Ace. Nr GI 181915): Reverse primer: 5'-AAAGACCTTGATGCCATCGG-3 I (SEQ ID NO: 27)
• TaqMan probe 5'-FAM-TGACGACACTGACCGCAGACCCA-TAMRA-3 I (SEQ ID NO: 29)
• VEGF (Ace. Nr): NM_003376
• Reverse primer ⁇ '-CACATTTGTTGTGCTGTAGGAAGC-S' (SEQ ID NO: 30)
• TaqMan probe 5'-FAM-CCATGCAGATTATGCGGATCAACCTCA-TAMRA-3 l (SEQ ID NO:
• Hexokinase 2 (Ace. Nr): NM_000189.
• Reverse primer 5'-TGTCTTGAGCCGCTCTGAGAT-S' (SEQ ID NO: 33)
• TaqMan probe 5'-FAM-CCAAGCGTGGACTGCTCTTCCGAG-TAMRA-3 • (SEQ ID NO: 35)
• Reverse primer ⁇ '-TAATGTCACGCACGATTTCCC-S' (SEQ ID NO: 36)
• TaqMan probe ⁇ '-FAM-CAGCTTCACCACCACGGCCGA-TAMRA-S' (SEQ ID NO: 38)
• Reverse primer ⁇ '-GGTCGCATTGTGGCCTTT-S' (SEQ ID NO: 39)
• TaqMan probe ⁇ '-FAM-ACAGTGCCCCGCCGAAGGAGA-TAMRA-S' (SEQ ID NO: 41)
• CYPA Cyclophilin-A, Ace. Nr: NM_021130
• Reverse primer ⁇ '-TCGAGTTGTCCACAGTCAGCA-S' (SEQ ID NO: 42)
• Forward primer ⁇ '-GCGCTTTGGGTCCAGGA-S' (SEQ ID NO: 43)
• TaqMan probe 5'-FAM-TGGCAAGACCAGCAAGAAGATCACCA-TAMRA-S' (SEQ ID NO: 44)
• RNA in 6 ⁇ l water is mixed with 0.33 ⁇ l 5' and 3' primers (10 ⁇ M each), 0.33 ⁇ l TaqMan probe (5 ⁇ M), 6.33 ⁇ l RT PCR Master Mix Kit (Eurogentec, # RT-QRT-032X) in a total volume of 13 ⁇ l following the manufacturer's instructions.
• Reverse transcription and real time PCR is performed in a ABI PRISM sequence detector 5700 or 7700 (Applied Biosystems) as follows: 30 minutes reverse transcription at 48°C, 10 minutes denaturation at 95 0 C followed by 50 cycles of denaturation for 15 sec. at 95°C and annealing and elongation for 1 min at 60 0 C.
• the relative quantitation of gene expression is calculated using the delta-Ct method as described in the ABI PRISM 5700 user bulletin #2.
• Cell lysates are prepared in M-PER mammalian protein extraction reagent according the protocol provided by the manufacturer (Pierce, Rockford, IL). 10 ⁇ g total protein extract is loaded on 8% premade acrylamide gels (NOVEX, San Diego, CA) and after electrophoretic separation transferred onto PVDF membranes. After saturation of the membrane in TBST-5 % milk powder, proteins are immunostained with the relevant primary antibodies diluted in TBST-5 % milk powder. Next, the membrane is rinsed 3 x with TBST and for 30 in incubated with the appropriate secondary antibody also dissolved in TBST-5 % milk powder. Next, the membrane is rinsed 3 x with TBST and ECL Western blotting detection reagents (Amersham Biosciences) are used to visualise the immunostaining according to the manufacturer's protocol.
• Dilutions used for primary antibodies are 1 :250 for mouse anti-HIF-1a (BD Biosciences/Pharmingen 610958), 1 : 100.000 for mouse anti- ⁇ -actin (Sigma A5441), 1 : 2000 for rabbit anti-ARNT (Aryl hydrocarbon Receptor Nuclear Translocator , Novus NB 730- H), 1 : 500 for rabbit anti-GLUT1 (Glucose Transporter ABCAM ab652), 1 : 1 '000 for rabbit anti- HIF2a-EPAS1 (Novus ab199 Cat. No. 730-H).
• VEGF secretion As secondary antibodies are used : goat anti-mouse IgG (Fab specific)-Peroxidase antibody (Sigma A2304) at a dilution of 1 : 1 : 5'000-20'00O or Goat polyclonal antibody to Rabbit IgG (HRP conjugated) Novus Catalog Number NB 730-H at a dilution of 1 : 20O00 7. Quantification of VEGF secretion
• VEGF is determined by using an enzyme linked immunoassay (ELISA) kit for human VEGF (R& D Systems) according to the manufacture's instructions.
• ELISA enzyme linked immunoassay
• E2-EPF5 specific gene inhibitors In order to characterize E2-EPF5 specific gene inhibitors, NCI-H1299 and HeLa cells are exposed to E2-EPF5 siRNAs and the inhibition of E2-EPF5 mRNA is studied by TaqMan RT- PCR. E2-EPF5 mRNA is reduced 70 to 90% after 48 h, respectively 72 h siRNA incubation in both cell lines. Mismatch and an unrelated control siRNAs do not affect E2-EPF5 gene expression.
• NCI-H1299 cells expressing recombinant E2-EPF5 tagged with a HIS-FLAG epitope at the N-terminus are exposed to E2-EPF5 siRNAs and controls. All match siRNAs decrease E2-EPF5 protein levels specifically compared to the controls with 17828 showing the strongest reduction.
• NCI-H 1299, HeLa and DU-145 cell lines which have been reported to be amenable to transfection of oligonucleotides are exposed to hypoxia-simulating compounds: desferrioxamine (DFO), cobalt chloride (CoCI2) and N-Oxalylglycine.
• DFO desferrioxamine
• CoCI2 cobalt chloride
• N-Oxalylglycine The results demonstrate that in NCI-H1299 and HeIa cells, DFO consistently induced VEGF mRNA 4-7- fold and VEGF protein 2-3-fold after 6 and 24 h stimulation. The extent of induction by CoCI2 is similar, but less consistent, declining after prolonged stimulation.
• N-Oxalylglycine significantly induces VEGF expression especially after 6 h.
• the basal level of expression of VEGF mRNA and protein (2 ng secreted VEGF protein per h per million cells) is found to be about 10-fold higher (2160 ⁇ 120 pg VEGF/h, 106 cells) in DU-145 than in the two other cell lines tested (NCI-H1299: 318 ⁇ 40 pg VEGF/h, 106 cells; HeLa: 301 ⁇ 60 pg VEGF/h, 106 cells).
• Significant VEGF induction 2-3-fold at the RNA and 1.3-1.5-fold at the protein level can only be demonstrated with all inducers but only after 6 not after 24 h stimulation.
• E2-EPF5 mRNA is not found to be significantly regulated in neither of the cell lines investigated.
• VEGF vascular endothelial growth factor
• Table 4 Effect of siRNA-mediated E2-EPF5 inhibition on the secretion of VEGF.
• NCI-H1299 cells are transfected with 60 nM siRNAs against E2-EPF5, corresponding mismatch controls and an unrelated control siRNA 8548. 72 h later, cells are incubated with 150 ⁇ M DFO for 6 h in comparison with the untreated 8548 control.
• VEGF protein concentrations in conditioned media are determined by ELISA and expressed as pg/ml per the total amount of protein in each well.
• E2-EPF5 siRNA 17828 A further analysis applying the most potent E2-EPF5 siRNA 17828 reveals that in both NCI- HI 299 and HeLa cell lines, inhibition of E2-EPF5 reduces the hypoxia-induced expression of VEGF mRNA and the secretion of hypoxia-induced VEGF protein. Reversion of hypoxic VEGF mRNA and protein is nearly complete to the levels observed in normoxically cultured cells as this is the case by a control siRNA targeting the transcription factor HIF-Ia.
• E2-EPF5 downregulation inhibits another endogenous Hif-1 target gene, namely GLUT-1 (Glucose transporter 1) whereas Hif-1 mRNA is not significantly changed. This suggests that E2-EPF5 suppression inhibits Hif-1 mediated transcriptional activation of HRE-driven genes without affecting the transcription of Hif-1 itself.
• Table 5 siRNA-mediated inhibition of E2-EPF5 suppresses hypoxia-induced expression of GLUT-1 mRNA to a similar extent than an siRNA targeting HIF-Ia.
• HeLa cells are transfected with 40 nM siRNAs against E2-EPF5 (17828), HIF-Ia (25560), corresponding mismatch controls (E2-EPF5: 25296, Hif-1 ⁇ : 25584) and an unrelated control siRNA 8548. 72 h later, cells are incubated with 150 ⁇ M DFO for 6 h in comparison with the untreated 8548 control.
• GLUT-1 mRNA is assayed by TaqMan RT-PCR.
• E2-EPF5 encodes an ubiquitin-conjugating enzyme (E2).
• E2s attach ubiquitin to cellular proteins thereby targeting them for proteasomal degradation or modulate their function, similarly to phosphorylation.
• Ubiquitin pathways play a key role in the regulation of cell growth and proliferation by controlling the abundance of cell cycle proteins (Bashier et al, 2003) and many components of the ubiquitination machinery have been found to be disregulated, mutated or amplified in various cancers and/or correlate with a poor prognosis.
• the expression of E2-EPF5 in various human tissues by TaqMan real-time PCR using primers specific to E2-EPF5 is examined.
• E2-EPF5 is slightly upregulated in thymus and testis suggesting a potential relationship between cell proliferation and expression of E2- EPF5 (Table 6).
• E2-EPF5 mRNA expression of E2-EPF5 mRNA in various tissues (Clontech). The tissue distribution is determined by TaqMan real-time PCR using primers specific for E2-EPF5. 11. Cloning of human E2-EPF5 cDNA in vector PDONR201 E2-EPF5 cDNA is cloned by two sequential PCR reactions followed by insertion in a GatewayTM donor vector The first DNA amplification is done in the presence of 1 ng of Quick-CloneTM cDNA from human fetal liver tissue (Clontech) using 10 pmoles each of the E2-EPF5-specific PCR primers (forward: ATC GAA GGT CGT ATG AAC TCC AAC GTG GAG AAC CTA CCC CCG (SEQ ID NO: 45), reverse: TCA CTT GTC GTC GTC GTC CTT GTA GTC CAG CCG CCG CAG CGC CCG CAG CGC CCG (SEQ ID NO: 46)), 10 nmoles each of the
• the PCR cycling conditions are as follows: 94 0 C for 10 min, [94 0 C for 1 min, 62 0 C for 1 min, 72 0 C for 1 min] 30 cycles, 72 0 C for 10 min, then 10 0 C on hold.
• the PCR products were analysed by PAGE. DNA is eluted from agarose by the Gene Clean Il kit. Weak DNA bands are reamplified by the same protocol. A typical yield of amplified PCR product is about 8 ⁇ g DNA in 50 ⁇ l H2O.
• the PCR product is composed of 5 1 - FXa site-specific E2 sequence-FLAG tag- 3'.
• the second DNA amplification is done in the presence of 100 ng template DNA from the first PCR reaction, 100 pmoles each of PCR primers ATTB1 FXA2 (GGG ACA AGT TTG TAC AAA AAA GCA GGC TTA GCT GGT ATC GAA GGT CGT ATG (SEQ ID NO: 47)) and ATTB2FLAG (GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA TCA CTT GTC GTC GTC GTC CTT GTA GTC (SEQ ID NO: 48)), 20 nmoles each of the dNTPs, 2 mM MgSO4, 10 % DMSO, 2.5 U of Pwo DNA polymerase in 100 ⁇ l buffer (10 mM Tris-HCI pH 8.8, 25 mM KCI, 5 mM (NH4)SO4), overlaid with 100 ⁇ l mineral oil, in a thermocycler block.
• 100 ⁇ l buffer 10 mM Tris-HCI pH 8.8, 25 mM
• the PCR cycling conditions are as follows: 94 0 C 2 min, [94 0 C for 1 min, 65 0 C for 1 min, 72 0 C for 1 min] 2 cycles, [94 0 C for 1 min, 60 0 C for 1 min, 72 0 C for 1 min] 2 cycles, [94 0 C for 1 min, 55 0 C for 1 min, 72 0 C for 1min] 30 cycles, 72 0 C for 2 min, then 10 0 C on hold..
• the att-PCR products are analysed by PAGE. DNA was purified for cloning with the PCR Purification Kit (Qiagen).Typically, the yield of amplified att-PCR products is 7-8 ⁇ g DNA in 50 ⁇ l H2O.
• the att-PCR products are composed of 5 1 - ATTBI-FXa site-specific E2 sequence-FLAG tag- ATTB2 - 3'.
• the att-PCR product is cloned in the GatewayTM donor vector pDONR201.
• the BP ClonaseTM enzyme mix catalyses a site-specific and orientation-specific in vitro recombination reaction via the attB1/B2 (PCR product) and attP1/P2 (vector) sites.
• the reaction mix (20 ⁇ L) contains BP buffer (as provided), 200 ng att-PCR DNA, 300 ng pDONR201 vector and 4 ⁇ L BP ClonaseTM enzyme mix.
• proteinase K (2 ⁇ g/ ⁇ L) is added and samples are incubated for 10 min at 37 0 C.
• 1 ⁇ L is used to transform competent DH5a E. coli cells. Transformants are selected on LB plates with kanamycin (50 mg/L). Clones are characterized using restriction enzymes and by DNA sequencing. A suitable clone is designated as pBM2537/NPL002981.
• the ClonaseTM LR enzyme mix mediates the GATEWAY LR recombination reaction via the attL1/L2 (pBM2537 entry clone) and attR1/R2 (pDEST12.2 vector) sites.
• the reaction mix (20 ⁇ L) contains LR buffer (as provided by the manufacturer), 200 ng entry clone (pBM2537) DNA, 300 ng expression vector (pDEST12.2, GIBCO-BRL-lnvitrogen Corp) and 4 ⁇ L LR ClonaseTM enzyme mix. After 1 h at 25 0 C proteinase K (2 ⁇ g/ ⁇ L) was added and samples are incubated for 10 min at 37 0 C.
• fetal calf serum (FCS), Versene, Lipofectin, Geneticin are purchased from Life Sciences Inc..
• NCI-H 1299 cells (CRL- 5803) are obtainable from the American Type Culture Collection (ATCC). The cells are maintained at 37°C in humidified atmosphere with 5% CO2 in RPM11640 with 10% FCS and 60 ⁇ g/ml Gentamycin. To propagate the culture, cells are split weekly: i.e. rinsed twice with Versene, treated for 5 min.
• NCI-H1299 cells are transfected using Lipofectamine Plus reagent (Life Sciences-lnvitrogen Corp.) Briefly, cells are seeded in 6-well multidishes at 1x105 per 6-well and grown for 1 day.
• 2 ⁇ g plasmid DNA (pDEST-12.2 or pDEST-EPF5) is mixed with 100 ⁇ l of Optimem and 15 ⁇ l of PLUS reagent, and incubated at room temperature for 15 min.
• 10 ⁇ l of Lipofectamine is mixed with 100 ⁇ l of Optimem medium and also incubated for 15 min at room temperature.
• the DNA mixtures are added to the Lipofectamine solution, mixed well, and again incubated at room temperature for 15 min.
• the FCS-containing medium is aspired from the cells, the cells are rinsed with 1 ml/well Optimem and then 1 ml of Optimem is added to each well.
• the cells are trypsinized as described above, resuspended in 3 ml RPMI1640 medium with FCS and 300, 150, 60 or 30 ⁇ l of the cell suspension are plated in 8-cm Petri dishes with 10 ml of RPM11640 with 10% FCS and 60 ⁇ g/ml Gentamycin.
• the next day 1.0 mg/ml Geneticin is added. Twice weekly, the selective medium (RPM11640 + 10 % FCS + 1 mg/ml Geneticin) is replaced by fresh medium. Colonies appear after 2-3 weeks.
• This RT-PCR measures the combined level of the recombinant E2-EPF5 mRNA from the integrated copy (or copies) of pDEST-EPF5 and of the native endogenous E2-EPF5 mRNA.
• the combined E2-EPF5 mRNA is more than 2.5 x higher than in native H1299 cells or cells transformed with pDEST12.2.
• Three independent NCI-H 1299/pDEST-EPF5 clones are further amplified (nrs 1 , 5, and 15).
• Table 7 VEGF protein levels in 6 h conditioned medium of pDEST12.2 and pDEST-EPF5- transformed NCI-H1299 cells compared to the level in parental NCI-H1299 cells
• RNA levels in pDEST-EPF5 NCI-H 1299 cell lines Using the total RNA isolated from plasmid-transformed NCI-H 1299 cells (example 13), the levels of several mRNAs species are determined by RT-PCR (example 5).
• the E2-EPF5 mRNA level (recombinant + native E2-EPF5 mRNA) is in cells transformed with pDEST- EPF5 2.3-3-fold higher than in the parental NCI-H1299 cells (Table 8).
• VEGF mRNA 38 ⁇ 10 % increased
• hexokinase-2 mRNA HK2, 4.9 ⁇ 2.5 -fold increased
• GLUT-1 mRNA 3.8 ⁇ 0.2 -fold increased
• basal levels of the mRNAs of a number of genes not regulated by HIF are not significantly changed ( ⁇ -actin, Bcl-xL, CYPA and HIFIa).
• H1299/vector pool 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
• the insert from plasmid pBM2537/NPL002981 (example 11) is recombined according to the GatewayTM system manual with vector pDEST17 (Invitrogen Corp) to give plasmid E2-12- flag-pDEST17/ NPL006782.
• the encoded amino acid sequence of the recombinant E2- EPF5 protein is :
• This plasmid is used to express recombinant E2-EPF5 in E. coli.
• E. coli Through the N-terminal 6- HIS tag it can be purified by standard metal-chelate affinity chromatography and this results in a more than 95 % pure preparation of tagged E2-EPF5. It is active in an in vitro autoubiquitination assay and can be detected using antibodies to the C-terminal FLAG-tag peptide sequence (DYKDDDDK).
• E2EPF5 (example 15) was covalently coupled to Anti-FLAG Sepharose using dimethyl pimelimidate in borate buffer as described in: Harlow, E. and Lane, D. Using Antibodies: A Laboratory Manual, (1999) Cold Spring Harbor NY Cold Spring Harbor Press, pp. 522-523.
• E2-EPF5-specific antibodies 1.5 ml crude rabbit anti-E2-EPF5 serum is applied to 150 microliter packed beads equilibrated with PBS. Unbound antibodies are removed with an excess of PBS.
• Anti-E2-EPF5 antibodies are eluted using Glycine-HCI 0.2M (pH 2.5), immediately neutralized by addition of 1/10 volume of Tris-HCI 1 M pH 9 and stored at 4 0 C.
• Tumor used for staining are from B16BL6 melanomas grown in C57BL/6 mice.
• Female black, C57BL/6 mice, weighing between 17 to 20 g, are obtained from lffa Credo (L'Arbresle, France) animal breeding facility. They are identified via tail markings and kept in groups (6-7 animals per cage) under normal conditions and observed daily. Six mice are used per treatment group.
• the melanin producing murine melanoma tumor cell line B16/BL6 is derived from a spontaneous tumor of C57BL/6 mice, has been extensively characterized and has been obtained from Dr. renowned J.Fidler, Texas Medical Center, Houston, USA.
• the cultured tumor cells used in all experiments are free of Mycoplasma.
• MEM MEM EBS, AMIMED, Allschwil
• glutamine supplemented with 5% fetal calf serum, 1% sodium pyruvate, 1% non-essential amino acids and 2% vitamins and grown until confluency.
• they are detached with 0.25% trypsin-0.02% EDTA (2 min at 37 0 C), and then processed. Viability is assessed by trypan blue exclusion, and only suspensions with >90% viability are used.
• the tumor cells are resuspended in Hanks Buffer and a suspension of 5 x 10 4 cells/ ⁇ l is prepared for intradermal (i.d.) injection into the ears of immuno-competent syngeneic C57BL/6 female mice.
• anesthesia is induced by a 3% lsoflurane (Forene r , Abbott AG, Cham, Switzerland) inhalation.
• the animals are placed on a warmed operative field maintained at a temperature of 39°C and their ears are gently extended over a steel cone fitted with a double-sided sticker.
• a 3OG hypodermic needle is then inserted at the periphery of the ear and tunneled for 4-5 mm in a subcutaneous plane to allow delivery of the tumor cells to a site distal to the needle entry point.
• the injection site is always located on the dorsum of the ear between the first and second neurovascular bundle.
• 1 ⁇ l of tumor cells suspension (5 x 104 cells ) are injected into the subcutaneous plane of the mouse ear forming a 2 x 2 mm sub-dermal blister.
• the primary tumor starts to develop and a black dot can be easily seen in the middle of the ear.
• Primary tumor size was monitored at day 7, 14 and 21. After three weeks (Day 21) the animals are killed by CO 2 inhalation, the cervical lymph nodes weighed, fixed ' in 4.2 % formaldehyde, and embedded in paraffin.
• paraffin sections prepared with a Microtome, MICROM
• SuperFrost+ (Menzel) glas slides dried over night at 37oC, and heated for 5 minutes at 59oC on a hot plate.
• Sections are dewaxed 2x in Xylene, rehydrated in decreasing ethanol solutions (100%, 95%, 90%, 70%), rinsed in dd water and subsequently subjected to a high temperature antigen unmasking technique.
• Sections are microwaved (14 minutes heat up to 98oC, 10min hold at 98oC; Milestone #T/TMEGA) in 0.1 m Na-citrate pH 6.0.
• Sections are cooled down to room temperature, rinsed in double-distilled water (ddH20) and immersed in PBS.
• ddH20 double-distilled water
• sections are incubated for 30 minutes in 0.3% H 2 O 2 in PBS and rinsed in PBS.
• sections are incubated for 20 minutes with PBS containing 1.5% goat serum (Vector Laboratories) at room temperature in a humid chamber. The blocking serum is blotted off, and the sections are incubated for 1 hour with the primary antibody (0.5 - 1.0 ⁇ g/ml polyclonal rabbit anti-EPF5) diluted in PBS containing 0.1% Tween-20.
• the primary antibody 0.5 - 1.0 ⁇ g/ml polyclonal rabbit anti-EPF5
• Sections are rinsed 3x 2 minutes in PBS and subsequently incubated with the secondary link antibody (biotinylated goat anti-rabbit IgG, Vector Laboratories) diluted in PBS containing 1.5% blocking serum. Sections are rinsed 3x2 minutes in PBS and incubated for 30 minutes at room temperature with avidin horseradish H complex (VECTASTAIN Elite ABC kit PK-6101). The sections are washed 3x2 minutes in PBS and stained for 5 to 10 minutes with Vector NovaRed (substrate kit for peroxidase, Vector Laboratories #SK-480) and rinsed 3x2 minutes in ddH2O.
• the secondary link antibody biotinylated goat anti-rabbit IgG, Vector Laboratories
• the sections are washed 3x2 minutes in PBS and stained for 5 to 10 minutes with Vector NovaRed (substrate kit for peroxidase, Vector Laboratories #SK-480) and rinsed 3x2 minutes in ddH2O.
• Sections are then counterstained for 30 seconds in Mayer's Hematoxylin (Fluka #51275), rinsed for 5 minutes in running tap water, rinsed in ddH2O and air dried.
• the sections are mounted with Eukitt (Fluka #03989) and analyzed by brightfield microscopy.
• Ischemic retinopathy is produced in C57/BL6J mice (Smith et al.1994, Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 35:101-111.). Briefly, seven-day-old mice and their mothers are placed in an airtight incubator and exposed to an atmosphere of 75 + 3% oxygen for 5 days (hyperoxia). Incubator temperature is maintained at 23 ⁇ 2°C, and oxygen is measured every 8 hours with an oxygen analyzer.
• mice After 5 days, the mice are removed from the incubator, placed in room air, and after 5 days at P17, the mice are sacrificed, eyes are rapidly removed and frozen in optimum cutting temperature embedding compound (OCT; Miles Diagnostics, Elkhart, IN) or fixed in 4% phosphate-buffered formaldehyde and embedded in paraffin.
• OCT optimum cutting temperature embedding compound
• Endogenous tissue peroxidase activity is blocked by incubating the sections 30 minutes in 0.3% H 2 O 2 in PBS. The sections are rinsed and incubated for 20 minutes with PBS containing 1.5% goat serum. All subsequent immunostaining steps are performed as described in 17. Immunostaining of EPF5 in paraffin sections is performed as described in17. Examination of the tissue slides reveals significantly higher levels of E2-EPF5 staining in the areas of pathological neovascularization in ROP eyes including the blood vessel cells themselves, but not in the corresponding areas of control eyes. The observation of elevated levels of E2-EPF5 protein in a tissue reacting to hypoxia is consistent with a functional role of E2-EPF5 in the cellular hypoxia response.
• ROP retinal angiogenesis model

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Genetics & Genomics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Zoology (AREA)
• Microbiology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Epidemiology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Physical Education & Sports Medicine (AREA)
• Gastroenterology & Hepatology (AREA)
• Ophthalmology & Optometry (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Rheumatology (AREA)
• Virology (AREA)
• Mycology (AREA)
• Dermatology (AREA)
• Cell Biology (AREA)
• Plant Pathology (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=36203455

Family Applications (1)

Country Status (9)

Families Citing this family (7)

Family Cites Families (2)

• 2005
  • 2005-10-12 WO PCT/US2005/036505 patent/WO2006044366A2/en active Application Filing
  • 2005-10-12 RU RU2007117771/14A patent/RU2007117771A/ru not_active Application Discontinuation
  • 2005-10-12 CN CNA2005800353498A patent/CN101039694A/zh active Pending
  • 2005-10-12 EP EP05810594A patent/EP1802343A2/en not_active Withdrawn
  • 2005-10-12 JP JP2007536798A patent/JP2008516953A/ja active Pending
  • 2005-10-12 CA CA002580883A patent/CA2580883A1/en not_active Abandoned
  • 2005-10-12 KR KR1020077008125A patent/KR20070083640A/ko not_active Application Discontinuation
  • 2005-10-12 BR BRPI0518132-1A patent/BRPI0518132A/pt not_active Application Discontinuation
  • 2005-10-12 AU AU2005295863A patent/AU2005295863A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events