WO2015143254A1 - Compositions pharmaceutiques comprenant des mutants d'occludine, et procédés d'inhibition de l'angiogenèse à l'aide celles-ci - Google Patents

Compositions pharmaceutiques comprenant des mutants d'occludine, et procédés d'inhibition de l'angiogenèse à l'aide celles-ci Download PDF

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WO2015143254A1
WO2015143254A1 PCT/US2015/021635 US2015021635W WO2015143254A1 WO 2015143254 A1 WO2015143254 A1 WO 2015143254A1 US 2015021635 W US2015021635 W US 2015021635W WO 2015143254 A1 WO2015143254 A1 WO 2015143254A1
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occludin
pharmaceutical composition
seq
angiogenesis
mutant
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David A. ANOTONETTI
Xuwen LIU
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University of Michigan System
University of Michigan Ann Arbor
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University of Michigan System
University of Michigan Ann Arbor
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14171—Demonstrated in vivo effect

Definitions

  • compositions comprising mutant variants of occludin protein, peptide fragments thereof, nucleic acids encoding such proteins and peptides, and methods of inhibiting angiogenesis and/or treating or preventing angiogenic disorders therewith.
  • occludin proteins, polypeptides, and/or peptides comprising the S490A mutation are provided for the inhibition of angiogenesis (e.g., VEGF- dependent angiogenesis) and the treatment or prevention of angiogenic disorders.
  • Angiogenesis contributes to a host of diseases of the central nervous system including brain tumors such as glioblastoma multiformate and eye diseases such as diabetic retinopathy, ocular tumors and age related macular degeneration. Preventing or reversing pathological angiogenesis may provide an important therapeutic option particularly for diseases such as aggressive brain tumors like glioblastoma multiformate for which there a limited therapeutic options.
  • compositions comprising mutant variants of occludin protein, peptide fragments thereof, nucleic acids encoding such proteins and peptides, and methods of inhibiting angiogenesis (e.g., of the central nervous system) and/or treating or preventing angiogenic disorders (e.g., of the central nervous system) therewith.
  • angiogenesis e.g., of the central nervous system
  • angiogenic disorders e.g., of the central nervous system
  • occludin proteins, polypeptides, and/or peptides comprising the S490A mutation are provided for the inhibition of angiogenesis (e.g., VEGF-dependent angiogenesis) and the treatment or prevention of angiogenic disorders.
  • the present invention provides pharmaceutical compositions comprising a polypeptide having 50% or greater (e.g., 50%... 60%... 70%... 75%... 80%...
  • sequence identity with SEQ ID NO: 2, or fragments thereof (e.g., 6-400 amino acids in length (e.g., >6, >8, >10, >12, >14, >16, >18, >20, >25, >30, >40, >50, >75, >100, >150, >200, >300; and/or ⁇ 400, ⁇ 350, ⁇ 300, ⁇ 250, ⁇ 200, ⁇ 150, ⁇ 100, ⁇ 80, ⁇ 60, ⁇ 50, ⁇ 40, ⁇ 30, ⁇ 25, ⁇ 21, ⁇ 20, ⁇ 18, ⁇ 16, ⁇ 14, ⁇ 12, ⁇ 10)).
  • 6-400 amino acids in length e.g., >6, >8, >10, >12, >14, >16, >18, >20, >25, >30, >40, >50, >75, >100, >150, >200, >300; and/or ⁇ 400, ⁇ 350, ⁇ 300, ⁇ 250, ⁇ 200, ⁇ 150,
  • the polypeptide or fragment thereof comprises an alanine residue at the position corresponding to 490 of SEQ ID NO: 2. In some embodiments, the polypeptide or fragment exhibits the characteristic of inhibiting angiogenesis (e.g., VEGF-mediated angiogenesis) in cells or in vivo.
  • angiogenesis e.g., VEGF-mediated angiogenesis
  • the present invention provides pharmaceutical compositions comprising nucleic acids that encode a polypeptide having 50% or greater (e.g., 50%... 60%... 70%... 75%... 80%... 85%... 90%... 95%... 98%... 99%) sequence identity with SEQ ID NO: 2, or fragments thereof (e.g., 6-400 amino acids in length (e.g., >6, >8, >10, >12, >14, >16, >18, >20, >25, >30, >40, >50, >75, >100, >150, >200, >300; and/or ⁇ 400, ⁇ 350, ⁇ 300, ⁇ 250, ⁇ 200, ⁇ 150, ⁇ 100, ⁇ 80, ⁇ 60, ⁇ 50, ⁇ 40, ⁇ 30, ⁇ 25, ⁇ 21, ⁇ 20, ⁇ 18, ⁇ 16, ⁇ 14, ⁇ 12, ⁇ 10), or any ranges therein).
  • SEQ ID NO: 2 or fragments thereof (e.g., 6-400 amino acids in length (e.g
  • the polypeptide or fragment thereof encoded by the nucleic acids comprises an alanine residue at the position corresponding to 490 of SEQ ID NO: 2. In some embodiments, the polypeptide or fragment thereof encoded by the nucleic acids exhibits the characteristic of inhibiting angiogenesis (e.g., VEGF-mediated
  • nucleic acids comprise
  • nucleic acids comprise a vector (e.g., comprising elements necessary for the expression of the polypeptide or peptide fragment).
  • the pharmaceutical compositions further comprise, or are coadministered with, an anti-cancer agent.
  • the anti-cancer agent is selected from the group consisting of: rapamycin, Alkylating agents (e.g., Cisplatin and carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil), Antimetabolites (e.g., azathioprine, or mercaptopurine), Plant alkaloids and terpenoids (e.g., vinca alkaloids and taxanes), Vinca alkaloids (e.g, Vincristine, Vinblastine, Vinorelbine, and Vindesine), Podophyllotoxin, Taxanes (e.g., paclitaxel), Topoisomerase inhibitors (e.g., camptothecins: irinotecan and topotecan, amsacrine, etoposide, etopo
  • the pharmaceutical compositions further comprise, or are coadministered with, a second anti-angiogenic agent.
  • the anti- angiogenic agent is selected from the group consisting of: soluble VEGFR-1, NRP-1, Angiopoietin 2, TSP-1, TSP-2, angiostatin and related molecules, endostatin, vasostatin, calreticulin, platelet factor-4, TIMP, CDAI, Meth-1, Meth-2, IFN-a, - ⁇ and - ⁇ , CXCL10, IL- 4, IL-12 IL-18, prothrombin (kringle domain-2), antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, and restin.
  • the pharmaceutical compositions further comprise a physiological tolerable buffer.
  • the present invention provides methods of inhibiting angiogenesis, comprising: contacting a tissue (e.g., exhibiting angiogenesis, at risk for angiogenesis, etc.) with a composition comprising an isolated polypeptide having 70% or greater (e.g., 70%... 75%... 80%... 85%... 90%... 95%... 98%...
  • sequence identity with SEQ ID NO: 2, or fragments thereof (e.g., 6-400 amino acids in length (e.g., >6, >8, >10, >12, >14, >16, >18, >20, >25, >30, >40, >50, >75, >100, >150, >200, >300; and/or ⁇ 400, ⁇ 350, ⁇ 300, ⁇ 250, ⁇ 200, ⁇ 150, ⁇ 100, ⁇ 80, ⁇ 60, ⁇ 50, ⁇ 40, ⁇ 30, ⁇ 25, ⁇ 21, ⁇ 20, ⁇ 18, ⁇ 16, ⁇ 14, ⁇ 12, ⁇ 10)) (e.g., under conditions such that angiogenesis is decreased in the tissue).
  • 6-400 amino acids in length e.g., >6, >8, >10, >12, >14, >16, >18, >20, >25, >30, >40, >50, >75, >100, >150, >200, >300; and/or ⁇ 400, ⁇
  • the present invention provides methods of inhibiting angiogenesis, comprising: contacting a tissue (e.g., exhibiting angiogenesis, at risk for angiogenesis, etc.) with a composition comprising nucleic acids that encode a polypeptide having 70% or greater (e.g., 70%... 75%... 80%... 85%... 90%... 95%... 98%...
  • nucleic acids comprise oligonucleotides.
  • nucleic acids comprise a vector (e.g., comprising elements necessary for the expression of the polypeptide or peptide fragment).
  • the methods further comprise the step of administering a second agent to the tissue (e.g., anti-cancer agent, anti-macular degeneration agent, anti- angiogenic agent, etc.).
  • a second agent e.g., anti-cancer agent, anti-macular degeneration agent, anti- angiogenic agent, etc.
  • the tissue is in a subject.
  • the subject has an angiogenesis-associated or mediated disease or condition (e.g., ocular disease or condition (e.g., age-related macular degeneration, retinopathy (e.g., diabetic retinopathy, retinopathy of prematurity, etc.), ocular tumors, etc.), diseases or disorders affecting the brain (e.g., gliomas, glioblastoma multiformate or any brain tumor), cancer (e.g., brain cancer (e.g., Glioblastoma multiforme), ocular cancer, etc.), etc.
  • angiogenesis-associated or mediated disease or condition e.g., ocular disease or condition (e.g., age-related macular degeneration, retinopathy (e.g., diabetic retinopathy, retinopathy of prematurity, etc.), ocular tumors, etc.), diseases or disorders affecting the brain (e.g., gliomas, glioblastoma multi
  • the methods further comprise a step of administering a second agent to the tissue.
  • the second agent is anti-angiogenic.
  • the second agent is anti-cancerous.
  • agents are co- administered.
  • agents are co-formulated.
  • the present invention provides the use of mutant occludin (e.g., S490A) in the manufacture of a medicament for the inhibition of angiogenesis. In some embodiments, the present invention provides the use of mutant occludin (e.g., S490A) in the manufacture of a medicament for the treatment of angiogenic disorders.
  • mutant occludin e.g., S490A
  • FIG 1A-D Inhibition of occludin S490 phosphorylation by AAV2-S490A occludin reduces VEGF-induced BREC tube formation, as demonstrated by (A) Western blot of occludin expression after viral transduction; (B) Quantification of Western blot; (C)
  • FIG. 2A-C Occludin S490A inhibits retinal angiogenesis in vivo, as demonstrated by: (A) Anit-GFP pAB(green), Alexa Fluor 594 isolectin GS-IB4 conjugate (red) and DAPI (blue) demonstration of viral transduction of GFP in retinal endothelium.
  • AAV2 virus with VECadherin promoter provides endothelial specific expression of GFP 3 -weeks after introduction of virus;
  • B BREC tubes were lysed for Western blot analysis after dissolving the gels with collagenase.
  • C Quantification of occludin pS490 from three independent experiments.
  • E Quantification of the content of occludin pS490 and
  • F histone H3 (pS IO) from three independent experiments.
  • VEGF (50ng/mL) was added and proliferation rates were measured 24hr later.
  • FIG. 5A-D S490A occludin prevents VEGF-induced endothelial cell proliferation, migration, and tube formation.
  • BREC were transfected with EV, WT Occ, and S490A
  • FIG. 6A-D Occludin S490A has little effect in VEGF-induced VEGFR2 and MAPK pathways in BREC.
  • BREC were transfected with empty vector, WT occludin, and occludin S490A for 18 hrs in MCDB complete medium followed by step down to 1% FBS medium for 4 hrs. The cells were then treated with vehicle or VEGF
  • BREC were transfected with scramble siRNA (siOccl) or sham transfection (Vehicle) and subjected to tube assays, (A) tubes longer than 100 ⁇ were quantified, (B) DNA synthesis with quantification of 3 independent experiments and (C) Western blot analysis.
  • FIG. 8A-C knockdown of occludin increases the BREC tube formation and proliferation.
  • BREC were transfected with scramble siRNA (100 nM) or siOcc2 (100 nM) compared to vehicle.
  • BREC grown in 3D bovine type I collagen gel matrices were cultured without or with VEGF (50 ng/ml) in 1% FBS step down medium. At 24 hours cells were stained with Calcein AM prior to fixing and imaging.
  • A The vessel area was measured using ImageJ software, and the number of tubes greater than 100 ⁇ was measured and counted using MetaMorph software. Quantification of three independent experiments.
  • B BREC cell proliferation assay at 8 hours after VEGF addition was performed according to the protocol of Click-iT EdU cell proliferation assay. Quantification of three independent experiments.
  • C Transfected BREC were lysed for Western blot analysis.
  • FIG. 9A-C Occludin S490 is phosphorylated during retinal angiogenesis.
  • VEGF was induced from rods by delivery of doxycycline to rho/rtTA- TRE/VEGF double-transgenic mice.
  • A Western blot analysis of occludin pS490 and
  • B quantification from 3 independent experiments.
  • C Quantification of pS490 at 48h after VEGF induction.
  • TRE/VEGF(+) mice after induction of VEGF for 48h were analyzed by IHC for occludin, IB4, and Ki67 in both superficial plexus and deep plexus.
  • A Staining of occludin at the tight junction border was quantified by a rank scoring system, (see legend). For each condition, images are from 4 random fields from 6 eyes, analysis by Chi-square test.
  • B Sulfo-NHS- biotin staining was quantified from images of 4 random fields from 6 eyes.
  • C FITC pixel intensity was quantified from images of 4 fields next to optic nerve region in serial sections from 6 eyes.
  • FIG. 11 VEGF-induced angiogenesis alters the distribution of ZO-1. Staining of ZO-1 at the tight junction border was quantified by a rank scoring system.
  • Occludin S490A inhibits angiogenesis in vivo.
  • AAV2-GFP, AAV2-Wt Occ, or AAV2-S490A Occ was delivered to Rho/rtTA(+) TRE-VEGF(+) mice through subretinal injection. After 3 weeks, VEGF in mouse retinas was induced by administration of Dox for 3 days. Retinas were isolated and prepared for imaging. Vessel area was quantified.
  • angiogenesis refers to the development of new blood vessels, including capillary vessels. It can take place in healthy tissue or diseased tissue, such as, for example, cancer and macular degeneration.
  • the term includes neovascularization, revascularization, angiopoiesis, and vasculogenesis.
  • New blood vessel growth typically results from stimulation of endothelial cells by angiogenic factors which may be active in proliferative conditions, such as in cancer or macular degeneration.
  • An "angiogenic factor” is one that promotes angiogenesis.
  • angiogenic disorders refers to conditions in which there is inappropriate development of new blood vessels (e.g., cancer).
  • the term "pharmaceutically acceptable carrier” refers to non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent for administration to a subject.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction.
  • an effective amount refers to the amount of a composition (e.g., pharmaceutical composition) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • the term "administration" refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., pharmaceutical compositions of the present invention) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs.
  • exemplary routes of administration to the human body can be through the eyes (e.g., intraocularly, intravitrealy, periocularly, ophthalmic, etc.), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.
  • co-administration refers to the administration of at least two agent(s) (e.g., mutant occludin and one or more additional agents (e.g., anti-angiogenesis agent, anti-cancer agent, etc.)) or therapies to a subject.
  • the co-administration of two or more agents or therapies is concurrent (e.g., in the same or separate formulations).
  • a first agent/therapy is administered prior to a second agent/therapy.
  • formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art.
  • agents or therapies when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone.
  • co-administration is especially desirable in embodiments where the coadministration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s).
  • vector refers to a polynucleotide that is used to express a polypeptide of interest in a host cell.
  • a vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., ⁇ -galactosidase).
  • regulatory sequences such as, for example, promoters and/or enhancers
  • selectable marker genes such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., ⁇ -galactosidase.
  • the terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition (e.g., angiogenesis), disease state (e.g., cancer), or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state.
  • the terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof).
  • treating angiogenesis refers to reducing the amount or severity of angiogenesis occurring in a subject presently experiencing or afflicted with angiogenesis.
  • a composition or method need only reduce the severity or amount of angiogenesis or symptoms thereof, not completely abolish all angiogenesis in the subject.
  • Treatment encompasses any administration or application of a therapeutic or technique for a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its development, relieving the disease, causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process. Treatment may be achieved with surgery, radiation, and/or administration of one or more molecules, including, but not limited to, small molecules and polymers, such as polypeptides.
  • prevent refers to reducing the likelihood of a particular condition (e.g., angiogenesis) or disease state (e.g., cancer) from occurring in a subject not presently experiencing or afflicted with the condition or disease state.
  • the terms do not necessarily indicate complete or absolute prevention.
  • preventing angiogenesis refers to reducing the likelihood of angiogenesis occurring in a subject not presently experiencing or afflicted with angiogenesis.
  • a composition or method need only reduce the likelihood of angiogenesis, not completely block any possibility thereof.
  • prevention encompasses any administration or application of a therapeutic or technique to reduce the likelihood of a disease developing (e.g., in a mammal, including a human).
  • the term "gene therapy” refers to the transfer of genetic material (e.g., DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition.
  • the genetic material of interest encodes a product (e.g., a protein (e.g., mutant occludin (e.g., S490A)) or polypeptide or peptide (e.g., occludin fragment), the production of which is desired in vivo.
  • a product e.g., a protein (e.g., mutant occludin (e.g., S490A)) or polypeptide or peptide (e.g., occludin fragment), the production of which is desired in vivo.
  • wild-type occludin refers to a gene or gene product (e.g., protein, polypeptide, peptide) that has a sequence corresponding to that which is most frequently observed in nature (e.g., SEQ ID NO: 1).
  • mutant occludin and variant occludin refer to a gene or gene product (e.g., protein, polypeptide, peptide) that exhibits modifications in sequence (e.g., deletions, substitutions, insertions, etc.) when compared to the wild-type gene or gene product (e.g., occludin S490A). It is noted that mutants may be naturally occurring (e.g., a "natural occluding variant") or man-made (e.g., a "synthetic occluding variant").
  • similarity refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into
  • compositions comprising mutant variants of occludin protein, peptide fragments thereof, nucleic acids encoding such proteins and peptides, and methods of inhibiting angiogenesis and/or treating or preventing angiogenic disorders therewith.
  • occludin proteins, polypeptides, and/or peptides comprising the S490A mutation are provided for the inhibition of angiogenesis (e.g., VEGF- dependent angiogenesis) and the treatment or prevention of angiogenic disorders.
  • the S490A occludin variant inhibits (e.g., completely) VEGF-induced angiogenesis (e.g., in cell culture models and in vivo).
  • VEGF-induced angiogenesis e.g., in cell culture models and in vivo.
  • phosphorylation on Ser490 of occludin facilitates VEGF-induced neovascularization in retinal endothelial cells expressing tight junctions.
  • VEGF induces occludin phosphorylation within 15-30 minutes and is required for increased permeability of confluent endothelial monolayers (Murakami et al.
  • transfection with an occludin S490A point mutant inhibited tube formation, proliferation and migration in primary endothelial cells after VEGF treatment.
  • Transduction of occludin S490A mutant in endothelial cells by sub-retinal delivery using a viral expression system blocked VEGF-induced retinal neovascularization in vivo.
  • occludin is inhibitory to VEGF-induced neovascularization, which may be relieved by occludin knockdown or physiologically by occludin Ser490 phosphorylation.
  • occludin Ser490 phosphorylation in neovascularization likely relates to its centrosomal localization; although the embodiments described herein are not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice such embodiments.
  • the experiments conducted during development of embodiments described herein reveal that occludin localizes to centrosomes with an increase in Ser490 phosphorylation during mitosis.
  • Ser490 phosphorylated occludin localizes to centrosomes of proliferating endothelial cells, retinal vasculature of mice with VEGF- induced angiogenesis and epiretinal membranes from human PDR patients.
  • Centrosomes contribute to the maintenance of cell polarity during migration, tissue growth, and homeostasis and are critical for signal transduction (Bornens (2012) Science 335(6067):422-426.; Dormoy V, Tormanen K, & Sutterlin C (2013) Journal of cell science 126(Pt 3):860-870).; herein incorporated by reference in their entireties).
  • the centriole linker complex that bridges the mother and daughter centrosomes contains the adherens junction protein ⁇ -catenin, which participates in the reorganization of the linker and centrosome separation in a NEK2 kinase dependent manner (Bahmanyar et al.
  • occludin contributes to angiogenesis in addition to a centrosomal function; although the present invention is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention. Since alterations in cell adhesion have been implicated in cell migration in the angiogenic tube (Bentley K, et al. (2014) Nat Cell Biol 16(4): 309-321.; herein incorporated by reference in its entirety) the changes in occludin phosphorylation related to permeability may contribute to cell migration.
  • occludin also regulates directional migration of epithelial cells through organization of the atypical PKC, Par3, PATJ polarity complex at the cell leading edge as demonstrated by gene knockdown experiments (Du D, et al. (2010) Dev Cell 18(l):52-63.; herein incorporated by reference in its entirety). These studies demonstrate a role for occludin Tyr 474 (human) phosphorylation in PI 3- kinase activation and migration. It is noteworthy that the S490A mutation inhibited both proliferation as measured by DNA synthesis and migration towards VEGF.
  • mice with doxycycline inducible VEGF expression in photoreceptors develop neovascularization that originates from the deep capillary bed of the retina and causes outer retinal folds followed by total retinal detachment in 3 to 4 days after administration of doxycycline (Ohno-Matsui K, et al. (2002) Am J Pathol 160(2):71 1-719.; herein incorporated by reference in its entirety).
  • This mouse model allowed investigation of occludin Ser490 phosphorylation from vasculature with pre-formed tight junctions. Two days after VEGF induction, organization of tight junction proteins occludin and ZO-1 were dramatically altered in the deep capillary bed.
  • occludin organization was altered more severely than ZO- 1 in the deep capillary bed but remained intact in superficial vessels where ZO-1 organization was also partially altered. Further, loss of BRB integrity and increased permeability were observed, specifically in the deep capillary bed in association with loss of occludin border staining, increased occludin phosphorylation and phospho- occludin staining at centrosomes. This neovascularization was blocked by viral delivery of S490A occludin.
  • Ser490 phosphorylation of occludin facilitates VEGF-induced permeability of the endothelial monolayer.
  • the Ser490 phosphorylation allows a subsequent ubiquitination of occludin and permeability of the endothelium.
  • S490A acts in a dominant negative manner to inhibit VEGF induced-permeability.
  • the present invention provides administration of occludin variants (e.g., phosphorylation deficient (e.g., S490A)) or fragments thereof for the treatment and/or prevention of angiogenesis.
  • the present invention provides administration of occludin variants (e.g., phosphorylation deficient (e.g., S490A)) or fragments thereof for the treatment and/or prevention of VEGF-induced permeability.
  • mutant occludin protein (e.g., S490) or peptide fragments thereof are administered.
  • nucleic acids encoding mutant occludin protein e.g., S490
  • are administered e.g., gene therapy).
  • the present invention provides administration of
  • antiangiogenesis compositions e.g. nucleic acids encoding mutant occludin (e.g., S490A) or polypeptide or peptide fragments thereof.
  • the present invention provides administration of nucleic acids encoding peptides and/or polypeptides (e.g. mutant occludin (e.g., S490A) fragments thereof) which inhibit angiogenesis.
  • the present invention provides administration of nucleic acids which encode peptides or polypeptides (e.g. mutant occludin (e.g., S490A) fragments thereof) which inhibit angiogenesis or treat or prevent angiogenic disorders.
  • compositions inhibit a VEGF-mediated pathway.
  • nucleic acids encoding mutant occludin e.g., S490A
  • nucleic acids encoding a fragment (e.g., peptide, polypeptide) of a mutant occludin are administered.
  • a nucleic acid encoding a polypeptide with at least 50% homology to WT occludin is administered (e.g.
  • a nucleic acid encoding a polypeptide with at least 50% homology to occludin S490A is administered (e.g. at least 60% homology, at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology, at least 99% homology, etc.).
  • a nucleic acid encoding a peptide or polypeptide having the S490A mutation is administered.
  • administering a nucleic acid encoding a peptide or polypeptide related to a mutant occludin e.g., S490A
  • administering a nucleic acid encoding a peptide or polypeptide related to a mutant occludin inhibits angiogenesis, and/or treats or prevents angiogenic disorders or diseases related to angiogenesis.
  • the present invention contemplates the delivery of exogenous nucleic acids encoding mutant occludin proteins or peptide fragments thereof, or the delivery of proteins themselves (e.g., recombinant mutant occludin, synthetic mutant occludin, etc.) to a subject via any suitable method.
  • nucleic acids are delivered within suitable vectors.
  • the present invention is not limited to any particular vector. Indeed, a variety of vectors may be used to deliver the nucleic acids.
  • the nucleic acids are delivered via an adenovirus vector.
  • an adenovirus vector See e.g., Westfall et al, Meth. Cell Biol. 32:307-322 (1998); and U.S. Pat. Nos. 6,451,596, 6,083,750, 6,063,622, 6,057, 158, or 5,994,132, all of which are herein incorporated by reference).
  • a nucleic acid encoding a mutant occludin protein or fragment thereof is delivered via an adeno-associated vector (AAV).
  • AAV vector integrates into the genome of the cells to which it is administered (e.g., a patient's cells (e.g., endothelial cells)).
  • a number of AAV vectors which have been developed for gene therapy are useful in the present invention (See e.g., U.S. Pat. Nos. 5,173,414; 5, 139,941 ; and 5,843,742; PCT publications WO92/01070 and WO93/03769; Lebkowski et al, Mol. Cell. Biol. 8:3988-3996 (1988); Carter, Curr. Opin. Biotech. 3 :533-39, (1992);
  • a mutant occludin protein or fragment thereof is incorporated into an AAV2 vector in which expression is driven by a vascular endothelial-cadherin promoter (Investigative Ophthalmology & Visual Science, February 2012, Vol. 53, No. 2; pp. 574- 585.; herein incorporated by reference in its entirety).
  • recombinant adenovirus vectors are constructed by
  • the full-length adenovirus DNA is provided from pJM17 which is a 0-100 map unit (m.u.) derivative of adenovirus serotype (Ad5) that contains a partial deletion in the E3 region and a 4.3-kb pBRX insert at 3.7 m.u. (See e.g., Graham and Prevec, Manipulation of Adenovirus Vectors, in Gene Transfer and Expression Protocols, E. J.
  • a shuttle vector comprises 0-1 m.u. and 9-16 m.u. of the Ad5 genome flanking an expression cassette containing the nucleic acid encoding a mutant occludin protein.
  • the present invention is not limited by the type of AAV or the methods of construction thereof.
  • the nucleic acid encoding a mutant occludin protein or fragment thereof is delivered via a liposome or naked DNA plasmid.
  • the liposome is a cationic liposome (See e.g., U.S. Pat. Nos. 5,908,777 and 5,676,954 each incorporated herein by reference in their entireties; Hug and Sleight, Biochim. Biophys. Acta. 1097: 1-17, (1991); Straubinger et al., in Methods of Enzymology, Vol. 101 pp.
  • vector comprising nucleic acid encoding mutant occludin protein or a fragment thereof further includes a suitable promoter (e.g., cell specific promoter, e.g., endothelial specific promoter (e.g., VEcadherin promoter, etc.), etc.), etc.) and/or enhancer, and also any necessary ribosome binding sites,
  • a suitable promoter e.g., cell specific promoter, e.g., endothelial specific promoter (e.g., VEcadherin promoter, etc.), etc.
  • enhancer e.g., VEcadherin promoter, etc.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required non- transcribed genetic elements.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (e.g., promoter) to direct mRNA synthesis.
  • the promoter is the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • an inducible promoter system is used.
  • an endothelial specific promoter e.g., VEcadherin promoter
  • an endothelial restricted promoter e.g., Tie2 promoter
  • Other promoters useful in embodiments of the present invention include, but are not limited to, the LTR or SV40 promoter, the E. coli.
  • recombinant expression vectors include selectable markers permitting transformation of the host cell (e.g. dihydrofolate reductase or neomycin resistance for eukaryotic cell culture).
  • the promoter is a tissue specific and/or inducible promoter.
  • transcription of the DNA encoding peptides and/or polypeptides described herein by higher eukaryotes is increased by inserting an enhancer sequence into the vector.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription;
  • Enhancers useful in the present invention include, but are not limited to, the SV40 enhancer (e.g., 100 to 270 base pairs on the late side of the replication origin), a cytomegalovirus early promoter enhancer, the polyoma enhancer (e.g., on the late side of the replication origin), and adenovirus enhancers.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector includes appropriate sequences for amplifying expression.
  • the present invention provides administration of
  • antiangiogenesis compositions e.g. mutant occludin (e.g., S490A) or polypeptide or peptide fragments thereof.
  • the present invention provides administration of peptides and/or polypeptides (e.g. mutant occludin (e.g., S490A) fragments thereof) which inhibit angiogenesis and/or treat or prevent angiogenic disorders.
  • the present invention provides administration of nucleic acids which encode peptides or polypeptides (e.g. mutant occludin (e.g., S490A) fragments thereof) which inhibit angiogenesis.
  • administered compositions inhibit a VEGF-mediated pathway.
  • mutant occludin e.g., S490A
  • a fragment e.g., peptide, polypeptide
  • a polypeptide with at least 50% homology to WT occludin is administered (e.g. at least 60% homology, at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology, at least 99% homology, etc.).
  • a peptide or polypeptide having the S490A mutation is administered.
  • a polypeptide with at least 50% homology to occludin S490A (SEQ ID NO:2) is administered (e.g. at least 60% homology, at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology, at least 99% homology, etc.).
  • administering a peptide or polypeptide related to a mutant occludin (e.g., S490A) to a subject or cell inhibits angiogenesis, and/or treats or prevents angiogenic disorders or diseases related to angiogenesis.
  • polypeptides of the present invention are isolated and/or purified (or substantially isolated and/or substantially purified). Accordingly, the invention provides polypeptide in substantially isolated form. In some embodiments, polypeptides are isolated from other polypeptides as a result of solid phase protein synthesis, for example. Alternatively, polypeptides can be substantially isolated from other proteins after cell lysis from recombinant production. Standard methods of protein purification (e.g., HPLC) can be employed to substantially purify polypeptides.
  • HPLC Standard methods of protein purification
  • the present invention provides a preparation of polypeptides in a number of formulations, depending on the desired use.
  • the polypeptide can be formulated in a suitable medium solution for storage (e.g., under refrigerated conditions or under frozen conditions).
  • suitable medium solution for storage e.g., under refrigerated conditions or under frozen conditions.
  • Such preparations may contain protective agents, such as buffers, preservatives, cryprotectants (e.g., sugars such as trehalose), etc.
  • the form of such preparations can be solutions, gels, etc., and the inventive polypeptide can, in some embodiments, be prepared in lyophilized form.
  • such preparations can include other desired agents, such as small molecules or even other polypeptides and proteins, if desired.
  • the invention provides such a preparation comprising a mixture of different embodiments of the inventive polypeptide (e.g., a plurality of polypeptide species as described herein).
  • the present invention also provides a pharmaceutical composition comprising of one or more polypeptides (e.g., mutant occludin) of nucleic acids (e.g., vector) encoding mutant occludin, and a pharmaceutically acceptable carrier.
  • a pharmaceutical carrier which can supply a polypeptide and/or nucleic acid without destroying the vector within the carrier is a suitable carrier, and such carriers are well known in the art.
  • the composition can be formulated for ocular, parenteral, oral, or topical administration.
  • a parenteral formulation consists of a prompt or sustained release liquid preparation, dry powder, emulsion, suspension, or any other standard formulation.
  • An oral formulation of the pharmaceutical composition for example, is a liquid solution, such as an effective amount of the composition dissolved in diluents (e.g., water, saline, juice, etc.), suspensions in an appropriate liquid, or suitable emulsions.
  • diluents e.g., water, saline, juice, etc.
  • an oral formulation is delivered in tablet form, and includes excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • a topical formulation includes compounds to enhance absorption or penetration of the active ingredient through the skin or other affected areas, such as dimethylsulfoxide and related analogs.
  • the pharmaceutical composition is delivered topically using a transdermal device, such as a patch, which could include the composition in a suitable solvent system with an adhesive system, such as an acrylic emulsion, and a polyester patch.
  • compositions are delivered via eye drops or other topical eye delivery method.
  • compositions are delivered intraocularly, anywhere in the eye including, for example, the vitreous cavity, the anterior chamber, etc.
  • compositions are delivered intravitrealy as is commonly done with intravitreal injections of Lucentis (ranabizumab), Avastin (bevazizumab), triamcinolone acetonide, antibiotics, etc.
  • compositions are delivered periocularly (e.g. to the tissue around the eyeball (globe) but within the bony orbit).
  • compositions are delivered via intraocular implant (e.g. gancyclovir implant, fluocinolone implant, etc.).
  • intraocular implant delivery devices containing compositions of the present invention are surgically implanted (e.g. within the vitreous cavity), and the pharmaceutical composition is released into the eye (e.g. at a predetermined rate).
  • compositions are administered using encapsulated cell technology (e.g. by Neurotech) in which genetically modified cells are engineered to produce and secrete compositions of the present invention (e.g. mutant occludin).
  • compositions are delivered via transcleral drug delivery using a device sutured or placed next to the globe that would slowly elute the drug, which would then diffuse into the eye.
  • the methods of the present invention are employed in vivo.
  • polypeptides and/or nucleic acids are delivered to a human or animal subject in an amount and at a location sufficient to inhibit or attenuate angiogenesis within the patient (e.g., within desired tissue).
  • Polypeptides and/or nucleic acids can be formulated into a suitable pharmaceutical composition (e.g., as described above or as otherwise known to those of ordinary skill in the art) for delivery into the subject.
  • the delivery can be local (e.g., by injection or implantation within the desired tissue to be treated) or systemic (e.g., by intravenous or parenteral injection).
  • the present invention provides a method for treating patients suffering ocular and/or retinal disorders, cancer, a central nervous system disorder, etc. and in need of treatment.
  • a pharmaceutical composition comprising at least one polypeptide and/or nucleic acids of the present invention is delivered to such a patient in an amount and at a location sufficient to treat the disorder or disease.
  • polypeptides and/or nucleic acids of the present invention can be delivered to the patient systemically or locally, and it will be within the ordinary skill of the medical professional treating such patient to ascertain the most appropriate delivery route, time course, and dosage for treatment.
  • inventive method of treating a patient most preferably substantially alleviates or even eliminates such symptoms; however, as with many medical treatments, application of the inventive method is deemed successful if, during, following, or otherwise as a result of the inventive method, the symptoms of the disease or disorder in the patient subside to a degree ascertainable.
  • the present invention provides mutant occludin proteins, polypeptides, and peptides that retain the activity (e.g., angiogenesis inhibitory activity) or tertiary folding of occludin S490.
  • an occludin protein, polypeptide, or peptide is at least 50% sequence identical (e.g., 50%... 60%... 70%... 75%... 80%... 85%... 90%... 95%... 98%... 99%) to SEQ ID NO:2 and comprises an alanine at the position corresponding to position 490 of SEQ ID NO: 2.
  • a mutant occludin protein, polypeptide, or peptide is at least 50% sequence similar (e.g., 50%... 60%... 70%...
  • a mutant occludin protein, polypeptide, or peptide is at least 75% sequence similar (e.g., 75%... 80%... 85%... 90%... 95%... 98%... 99%) at least 50% sequence identical (e.g., 50%... 60%... 70%... 75%... 80%... 85%... 90%... 95%... 98%... 99%)to SEQ ID O:2.
  • the present invention further provides variants of SEQ ID NO:2 that maintain the identity of positon 490, comprising amino acid variations that do not significantly alter one or more or any therapeutically relevant properties of the protein. Examples of these variants are set forth in more detail herein.
  • a leucine with an isoleucine or valine an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e., conservative mutations) will not have a major effect on the biological activity of the resulting molecule.
  • some embodiments of the present invention provide variants of occludin, or fragments thereof, containing conservative replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally be grouped separately as aliphatic - hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,
  • a variant includes "nonconservative" changes (e.g., replacement of a glycine with a tryptophan).
  • Analogous minor variations can also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs (including, but not limited to, FADE (Mitchell et al, (2004). Molec. Simul. 30, 97-106); MAPS (Ban et al, Proceedings of the 8th Annual International Conference on Research in Computational Molecular Biology, 2004, 205-212), SYBYL (Tripos, Inc, St.
  • mutant occludin polypeptides and peptide comprise the sequence of SEQ ID NO:2, or at least 50% sequence identity to SEQ ID NO:2, with an N- terminal and/or C-terminal deletion (e.g., deletion of 1-500 amino acids).
  • a fragment of SEQ ID NO: 2 comprising the position corresponding to position 490 is provided.
  • Occludin Ser490 phosphorylation facilitates VEGF- induced neovascularization.
  • Occludin phosphorylated on Ser490 was detected in centrosomes of proliferating endothelial cells, in vivo in mouse retinal whole mounts with proliferating vessels, and in human surgical samples of retinal neovascularization from patients with PDR.
  • Occludin Ser490 phosphorylation increases as endothelial cells are induced to form tubes in the presence of VEGF and expression of S490A occludin completely inhibits endothelial cell proliferation, migration and tube formation induced by VEGF.
  • occludin acts to inhibit angiogenesis that may be relieved by occludin knockdown or Ser490 phosphorylation. Further, increased occludin Ser490 phosphorylation is associated with pathologic retinal angiogenesis in mice as observed using doxycycline-inducible VEGF expression from photoreceptors and subsequent
  • Occludin S490A inhibits retinal angiogenesis in vivo
  • AAV2-GFP, AAV2-Wt Occ, or AAV2- S490A Occ was delivered to 4-weeks VEGF inducible mice through subretinal injection. After three weeks, expression of VEGF in mice retinas were achieved by administration of doxycycline ( or water as control ) for 3 days. Mice retinas were then isolated and prepared for retinal flat mounts or cryostat sections.
  • AAV2 delivery to retina was confirmed by Immunno-fluorescence staining of anti-GFP pAb (green), Alexa Fluor 594 isolectin GS-IB4 conjugate (red) and DAPI (blue) on retinal flat mounts (Figure 2A).
  • Occludin Ser490 phosphorylation regulates vascular endothelial growth factor-induced retinal neovascularization
  • Occludin Ser490 is phosphorylated during mitosis and co-localizes with centrosomes in proliferating endothelial cells and human retinal neovascular tufts.
  • Immunofluorescence analysis using a phosphospecific antibody targeting pSer490 occludin was used to examine the localization of phospho-occludin in proliferating endothelial cells.
  • Primary bovine retinal endothelial cells (BREC) were grown on bovine Type I collagen film for 6 hours followed by step down to medium with 1% FBS for 24 hours. Mitotic cells were identified based on nuclear condensation patterns representative of each stage of mitosis observed by Hoechst staining using confocal microscopy.
  • Epiretinal fibrovascular membranes were obtained from 6 patients with active PDR during pars plana vitrectomy for the removal of vitreous hemorrhage and/or the repair of tractional retinal detachment. Thin sections of these membranes were immunostained with Ki67 antibody to identify proliferative cells, ⁇ - tubulin antibody to identify centrosomes and occludin pSer490-specific antibody.
  • Occludin Ser490 is phosphorylated during VEGF-induced BREC tube formation.
  • occludin Ser490 contributes to the regulation of endothelial cell proliferation and retinal vascular angiogenesis
  • the amount of occludin Ser490 phosphorylation was monitored during VEGF-induced BREC tube formation.
  • Primary BREC were subjected to a classic tube formation assay in 3D collagen matrix in the presence or absence of VEGF. Clear evidence of tube formation was observed during a 24h time-course, with tube growth occurring between 12 and 24 hours (Fig. 3 A).
  • Immunoblotting cell lysates for pSer490 revealed two clear bands, one at just over 50kDa (the expected MW of occludin) and a prominent band at ⁇ 66kDa previously shown by peptide blocking and immunoprecipitation experiments to represent Ser490 phosphorylated occludin (Fig. 3B) (Sundstrom JM, et al. (2009) Journal of proteome research 8(2):808-817.; herein incorporated by reference in its entirety). Quantification revealed that the amount of pSer490 occludin (66kDa) relative to total occludin significantly increased by 12 hours and 24 hours as tube formation proceeds (Fig. 3C). Claudin 5 did not change significantly over the course of the experiment.
  • BREC were used to examine the effects of expression of the occludin S490A mutant in well- established models of angiogenesis including proliferation, migration, and tube formation.
  • Primary BREC were transfected with Wt Occ, S490A Occ or empty vector expressing GFP only by using the Amaxa Nucleofector System.
  • Measures of endothelial proliferation in response to VEGF were made by either 3H thymidine incorporation on 2D culture (Fig. 4) or Click-iT EdU incorporation in 3D culture (Fig. 5A).
  • occludin expression was reduced by transfecting small interfering (siRNA) targeting occludin in BREC.
  • Tube formation assays demonstrated that the occludin knockdown significantly promoted endothelial tube formation, both basally and after VEGF-induction in BREC (Fig 7A; Fig. 8A).
  • Click-iT EdU DNA synthesis assay in 3D culture revealed that occludin knockdown promoted BREC proliferation by -100% in the absence of VEGF (Fig. 7B; Fig. 8B) as compared with vehicle control and scramble siRNA transfected BREC.
  • Occludin Ser490 is phosphorylated during VEGF-induced retinal angiogenesis.
  • Fig. 9A neovascularization by 3 days
  • Fig. 9B Blotting for occludin pSer490 revealed a clear increase at 24 and 48 hours (Fig. 9B-C). Since the rd8 mutation was observed in some mice in the inducible VEGF strain, the mutation was bred out of the line and the experiment was further verified in mice in the absence of rd8 mutation with the same response observed (Fig. 5D). Further, increased occludin Ser490
  • VEGF induced neovascularization was measured.
  • retinas in mice with doxycycline-inducible VEGF expression from photoreceptors were removed after 48 hours administration of doxycycline or water as a negative control.
  • Immunofluorescence staining of the retinal whole mounts revealed Ki67-positive proliferative cells were present only in vessels in the deep capillary plexus.
  • BRB permeability was also characterized by intravenous injection of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) followed by digital image analysis of retinal sections. After VEGF induction, retinal fluorescence intensity was increased by 28% (IPL), 59% (INL), 31% (OPL), and 32% (ONL), respectively (Fig. 10F).
  • Wt occludin and S490A mutant occludin expression specifically targeted to vascular endothelial cells was achieved using recombinant AAV serotype 2 (AAV2) quadruple tyrosine to phenylalanine (quadYF) capsid mutant vectors containing cDNA under control of the vascular endothelial cadherin promoter (20).
  • AAV2 AAV serotype 2
  • Recombinant human VEGF165 was from R&D Systems (Minneapolis, MN).
  • Bovine Type I collagen solution BREC grown on 2D bovine type I collagen film were arrested in prometaphase with 200 ng/niL nocodazole for 16 hours followed by release into fresh media and harvesting at the indicated time points for Western blot analysis.
  • Ser490 of human occludin (Occ) in pENTR221 was substituted to alanine (S490A) or aspartic acid (S490D) and cDNA of these mutants or wildtype human occludin (WT-Occ) were transferred into pmaxFP-Green-C expression vector (Lonza, Walkersville, MD). All constructs were confirmed by sequencing.
  • BREC transfected with occludin or mutants were resuspended in 1% FBS step down medium and seeded at 5 x 103 cells /well in Transwell upper chambers with both top and bottom of the membranes coated with (50 ⁇ g/mL) type I collagen.
  • Cells were induced to migrate by addition of VEGF (50 ng/ml), placed in the bottom chamber, for 6 hours.
  • Cells that migrated to the bottom of the chamber were counted after SYT013 Green Fluorescent Nucleic Acid-staining.
  • Three different fields per well were randomly chosen and photographed using a model Eclipse TE300 inverted phase microscope (Nikon, Tokyo, Japan). The values are expressed as number of migrated cells per 10X power field.
  • a recombinant AAV serotype 2 quadruple tyrosine to phenylalanine (Y-F) capsid mutant containing the vascular endothelial cadherin (VEC) promoter was used to drive human occludin cDNA or Occludin S490A mutant expression.
  • AAV vectors were packaged and purified.
  • Sub-retinal injections were performed using a tapered pulled glass pipette inserted into a sclerotomy and connected to a nano injector (Nano injector II, Drummond Scientific Company), which allowed for a slow injection of 1 ⁇ of virus at 2.0 x 1013 genome copies per mL.
  • the sub-retinal injection was performed with direct visualization through a dissecting microscope.
  • mice were deeply anesthetized and perfused with 10 ml of Sulfo-NHS-LC-Biotin (Thermo Scientific) at 0.5 mg/ml in PBS by transcardiac perfusion, followed by flushing with 1% paraformaldehyde (PFA) in PBS. Eyes were postfixed in 1% PFA at 4 °C for 6 hours prior to retina dissection. BRB permeability was measured by FITC-BSA accumulation. FITC-BSA (100 mg/kg body weight) was injected in femoral vein and allowed to circulate for 10 min.
  • Eyes were enucleated and fixed in 4% PFA. Retinas were dissected and subjected to cryosectioning and subsequent imaging analysis. Whole mount retinas were incubated with rat anti-Ki67 (eBiosciences) antibody and Isolectin GS-IB4 Alexa Fluor 647 (Life Technologies).
  • Epiretinal membranes of human proliferative diabetic retinopathy Epiretinal membranes of human proliferative diabetic retinopathy.
  • Epiretinal fibrovascular membranes were obtained from patients during vitrectomy for PDR.
  • the Institutional Review Board of the University of Michigan approved the protocol.
  • the severity of retinal neovascular activity was graded clinically at the time of vitrectomy. Partly active PDR was present in all patients used.
  • Membranes were fixed in 4% PFA for 15 minutes and 5 ⁇ cryostat sections were prepared for immunostaining.
  • neovascularization is associated with reduction of superoxide and proinflammatory cytokines.
  • beta-Catenin is a Nek2 substrate involved in centrosome separation. Genes & development 22(1):91-105.
  • Tranilast inhibits protein kinase C-dependent signalling pathway linked to angiogenic activities and gene expression of retinal microcapillary endothelial cells. British journal of pharmacology 127(2):537-545.
  • Aiello LP et al. (1994) Vascular Endothelial Growth Factor in Ocular Fluid of Patients With Diabetic Retinopathy and Other Retinal Disorders. N. Eng. J. Med.

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Abstract

L'invention fournit des compositions pharmaceutiques comprenant des variants mutants de la protéine occludine, des fragments peptidiques de ceux-ci, des acides nucléiques codant pour ces protéines et peptides, et des procédés d'inhibition de l'angiogenèse et/ou de traitement ou de prévention des troubles angiogéniques à l'aide de ces compositions. En particulier, des protéines occludines, des polypeptides et/ou des peptides comprenant la mutation S490A sont fournis pour l'inhibition de l'angiogenèse (p.ex. l'angiogenèse VEGF-dépendante) et pour le traitement ou la prévention des troubles angiogéniques.
PCT/US2015/021635 2014-03-21 2015-03-20 Compositions pharmaceutiques comprenant des mutants d'occludine, et procédés d'inhibition de l'angiogenèse à l'aide celles-ci Ceased WO2015143254A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997033605A1 (fr) * 1996-03-15 1997-09-18 Yale University Occludine humaine, ses utilisations et amelioration de l'absorption de medicaments a l'aide d'inhibiteurs d'occludine
WO2000062815A2 (fr) * 1999-04-15 2000-10-26 Glaxo Group Limited Nouvelle composition pharmaceutique utilisable en therapie genique
WO2012149452A1 (fr) * 2011-04-29 2012-11-01 Regents Of The University Of Michigan Composés, formulations et procédés d'inhibition de la protéine kinase c

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997033605A1 (fr) * 1996-03-15 1997-09-18 Yale University Occludine humaine, ses utilisations et amelioration de l'absorption de medicaments a l'aide d'inhibiteurs d'occludine
WO2000062815A2 (fr) * 1999-04-15 2000-10-26 Glaxo Group Limited Nouvelle composition pharmaceutique utilisable en therapie genique
WO2012149452A1 (fr) * 2011-04-29 2012-11-01 Regents Of The University Of Michigan Composés, formulations et procédés d'inhibition de la protéine kinase c

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GARIANO, RF ET AL.: "Retinal Angiogenesis In Development And Disease.", NATURE ., vol. 438, 15 December 2005 (2005-12-15), pages 960 - 966, XP055227743 *
LIU, X ET AL.: "Ocdudin Ser490 Phosphorylation Regulates Endothelial Cell Growth Control And Angiogenesis.", INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE ., vol. 54, no. 15, June 2013 (2013-06-01), pages 1 - 5, XP055227738 *

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