WO2004056386A2 - Acides nucleiques intervenant dans la regulation de la barriere hemato-encephalique - Google Patents

Acides nucleiques intervenant dans la regulation de la barriere hemato-encephalique Download PDF

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WO2004056386A2
WO2004056386A2 PCT/NL2003/000915 NL0300915W WO2004056386A2 WO 2004056386 A2 WO2004056386 A2 WO 2004056386A2 NL 0300915 W NL0300915 W NL 0300915W WO 2004056386 A2 WO2004056386 A2 WO 2004056386A2
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WO2004056386A3 (fr
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Pieter Jaap Gaillard
Albertus Gerrit De Boer
Arjen Brink
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To-Bbb Holding B.V.
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Publication of WO2004056386A3 publication Critical patent/WO2004056386A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to nucleic acids and polypeptides encoded thereby, whose expression is modulated in brain microvascular endothelial cells undergoing dynamic changes in blood-brain barrier formation. These polypeptides are designated herein as "Pro-Barrier" polypeptides (PB polypeptides).
  • PB polypeptides Provided herein as "Pro-Barrier" polypeptides.
  • the invention further relates to methods useful for controlling blood-brain barrier properties in mammals in need of such biological effects. This includes the diagnosis and treatment of disturbances in the blood-brain/retina barrier, brain (including the eye) disorders, as well as peripheral vascular disorders.
  • the present invention further relates to the use of anti- PB polypeptide antibodies or ligands as diagnostic probes, as blood-brain barrier targeting agents or as therapeutic agents as well as the use of ligands or modulators of expression, activation or bioactivity of PB polypeptides as diagnostic probes, therapeutic agents or drug delivery enhancers.
  • astrocytes or astroglia
  • the central nervous system is also shielded from the general blood circulation by a number of blood-CNS barriers, i.e. the blood-brain barrier, blood- cerebral spinal fluid (CSF) barrier, pial vessel-CSF barrier, the ependyma and glia limitans, and also the blood-retina barrier, blood-nerve barrier, blood-spinal cord barrier.
  • CSF blood- cerebral spinal fluid
  • pial vessel-CSF barrier pial vessel-CSF barrier
  • the ependyma and glia limitans and also the blood-retina barrier, blood-nerve barrier, blood-spinal cord barrier.
  • the BBB regulates the trafficking of ions (Na + , K + , Ca 24 ), water, nutrients, metabolites, neurotransmitters (glutamic acid, tryptophan), plasma proteins (albumin, fibrinogen, immunoglobulins), cells from the immune system and also xenobiotics (drugs) in and out of the brain.
  • the capillary endothelium in the brain has special properties when compared to peripheral capillaries. It has narrow tight-junctions, no fenestrae, low pinocytotic activity and a continuous basement membrane. The narrow tight-junctions result in a high electrical resistance of 1500-2000 Ohm.cm 2 .
  • the endothelial cells have a negative surface charge that repulses negatively charged compounds. They have many mitochondria and enzymes to break down compounds and various selective transport systems to actively transport nutrients and other compounds into and out of the brain. Under healthy conditions, the BBB not only regulates the entry of drugs or endogenous compounds into the brain, but also cellular infiltration is lower compared to peripheral organs.
  • the normal endothelial cell layer provides a thromboresistant surface that prevents platelet and leukocyte adhesion and activation of any coagulation system.
  • the highly specialised brain microvascular endotheUal cells form a tight barrier which isolates the brain from inrmune surveillance, and allow only a few mononuclear cells (such as activated T-cells) to migrate into the CNS.
  • the present understanding of the anatomical basis of the BBB is that it functions as a dynamically regulated organ, influenced by peripheral (e.g. cortisol, adrenaline) and local (e.g. cytokines, chemokines) hormones.
  • peripheral e.g. cortisol, adrenaline
  • local e.g. cytokines, chemokines
  • astrocytes e.g. adenosarcomasin, adenothelial growth factor, and the endothelium plays an important role.
  • the BBB can be regarded as an organ that serves to protect the homeostasis of the brain.
  • dysfunction of the BBB plays a central role in the vast majority of brain disorders.
  • Some examples are: i. Cerebral vasogenic edema is the result of disease (inflammation) induced leakage of plasma proteins and water from the blood into brain tissue. This is the principal cause of death and disabilities in disorders like stroke, cerebral infections, head trauma, brain tumors and multiple sclerosis. The edema causes the brain to swell witiiin the rigid environment of the scull.
  • the resulting elevation in intracranial pressure may subsequently lead to herniation of the brain followed by failure of essential brain functions like respiration and, if left untreated, results in severe disabilities, coma and even death, ii.
  • activated autoreactive T cells cross the activated BBB.
  • these T cells induce an inflammatory response targeted against myelin, which also causes a disruption of the BBB.
  • Autoantibodies and complement factors now cross the disrupted BBB, which leads to the process of demyelination.
  • myelin fragments also leak back into the periphery through the disrupted BBB, where it activates more autoreactive T cells and increases the production of more autoantibodies .
  • a proper functioning BBB is also essential to block or reduce the entry into the brain of lymphocytes, which mediate an immune response. The same holds for the entry into the brain of metastatic cancer cells.
  • novel PB polypeptides whose gene expression is modulated in brain microvascular endothelial cells undergoing dynamic changes in blood-brain barrier formation, will prove useful to meet these needs.
  • the BBB also limits the delivery of xenobiotics (such as drugs and diagnostic agents) to the brain, which complicates classical drug therapy (i.e. targeted against neurons) of brain disorders.
  • Introducing BBB properties in peripheral microvessels will be beneficial in conditions involving (micro)angiopathies, pathological angiogenesis, failure of blood-testis barrier or blood-placenta barrier, and conditions such as pulmonary edema, shock caused by bacterial endotoxins, hyperfibrinolysis and anaphylactic shock.
  • novel PB polypeptides whose gene expression is modulated in brain microvascular endothelial cells undergoing dynamic changes in blood-brain barrier formation, will also prove useful to meet these needs.
  • TEER is a sensitive measure to quantify the permeability of small ions through the tight junctions between BCEC. TEER thus represents the functionality of tight junctions, which are considered the major hallmark of the BBB. The absolute value of TEER is mainly dependent on the amount and complexity of tight junctions between the cells. Likewise, this is also the limiting factor for the paracellular transport of large and hydrophilic compounds.
  • astrocytes cultured on the bottom side of the filter insert 1) maintained (or (re-)induced) the expression of P-glycoprotein (Pgp, a drug efflux pump involved in multidrug resistance) on BCEC after the first passage (Gaillard et al., 2000, Pharm. Res.
  • An alteration of the activity or steady state level of a polypeptide herein means any detectable change in the biological activity exerted by the polypeptide or in the steady state level of the protein as compared the normal activity or steady-state in a healthy individual.
  • An agonist is herein defined as any molecule that mimics a biological activity, preferably the biological activity of a polypeptide, a receptor or its ligand.
  • An antagonist is any molecule that partially or fully blocks, inhibits or neutralises such a biological activity.
  • treatment of vascular disorders refers to, inter alia, reducing or alleviating one or more symptoms in an individual, preventing one or more symptoms from worsening or progressing, promoting recovery or improving prognosis, and/or preventing disease in an individual who is free therefrom as well as slowing or reducing progression of existing disease.
  • improvement in a symptom, its worsening, regression, or progression may be determined by an objective or subjective measure.
  • Efficacy of treatment may be measured as an improvement in morbidity or mortality (e.g., lengthening of survival curve, for a selected population).
  • Increased permeability of the endothelial/vascular barrier makes it more leaky (i.e., less tight, more permeable).
  • Decreased permeability of the endothelial/vascular barrier makes it more tight (i.e., less leaky, less permeable). Treating a vascular disorders thus means decreasing vascular permeability, whereas increasing drug delivery thus requires increased vascular permeability.
  • Up-regulated PB polypeptides of the invention are involved in decreased vascular permeability.
  • Down-regulated PB polypeptides of the invention are involved in decreased vascular permeability. Modulation of endothelial permeability
  • the invention in a first aspect relates to a method for modulating the permeability of endothelial cells.
  • the method comprises altering in the endothelial cells the activity or the steady-state level of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53. Sequence identities or similarities are herein defined as described below.
  • the endothelial cells are preferably vascular endothelial cells, more preferably microvascular endothelial cells. Most preferably the endothelial cells are microvascular endothelial cells that constitute or are part of one of the blood-central nervous system (CNS) barriers, such as the blood-brain barrier, blood-retina barrier, blood-nerve barrier, blood-spinal cord barrier, of which brain microvascular endothelial cells are most preferred.
  • CNS central nervous system
  • endothelial barrier cells may be characterised in situ, ex situ (i.e., in isolated capillaries) or in vitro by e.g. specific endothelial cell markers, specific barrier markers, but also by barrier functional assays. More specifically, endothelial cells may be characterised by their morphology in situ, i.e. a tube-like structure of with a diameter of about 10-20 micrometers, formed by single (or no more than three) continuously connected endothelial cells, surrounded by a continuous basal lamina, in which perivascular pericytes reside and astrocyte endfeet are projected upon.
  • barrier-like endothelial cells are between 1 and 5 micrometers thick, have many mitochondria, are connected by tight junctions, have no intercellular clefts, no fenestrations and very few pinocytotic vesicles, as can be observed by e.g., electron microscopy.
  • capillary structures may be characterised by their morphology in culture i.e., a tube-like structure with a diameter of about 10-20 micrometers, between 50 and 200 micrometers long.
  • endothelial cells may be characterised by their morphology in culture i.e., cobblestone shape (when growing in a cluster, e.g.
  • endothelial specific cluster of differentiation (CD) antigens VCAM (CD 106), CD31 , EN-4, ICAMs, E-Selectin, PECAM, RBA), cadherins, integrins, actin, vimentin, factor NIII related antigen (vWF), collagen I and IV, fibronectin, matrix metalloproteinases, tissue inhibitor of metalloproteinases; non-thrombogenicity; low leukocyte adherence; release of vasoactive compounds (nitric oxide, endothelin-1 and prostacyclins); uptake of Dil-labeled-acetylated low density lipoprotein (Dil-Ac-LDL); lectin binding; presence of angiotensin converting enzyme, alkaline phosphatase, monoamine oxidase and anionic sites, hr addition, typical barrier markers and functions may be used, like visualisation of tight junctions or tight junction-related proteins (ZO-1) and restricted paracellular transport of reference compounds (such
  • Evans blue (binds to albumin), mannitol, sucrose, fluorescein, dextrans, albumin, AJJ3); absence of vesicular transport; absence of non-barrier markers like PAL-E; expression of gamma-glutamyl-transpeptidase ( ⁇ -GTP); expression and functionality of P-glycoprotein (Pgp), multi-drug resistance proteins 1-7, glucose transporters, nucleoside transporters, organic anion transporters, large and neutral amino acid transporters; transferrin receptors, msulin-growth factor receptors, scavenger receptors; marginal F-actin localisation and expression of many mitochondria, although none of these are specific for endothelial cells.
  • markers may be determined by e.g., molecular biological, biochemical, (immuno)- histo(cyto)chemical techniques as well as by functional assays using known substrates, ligands and/or inhibitors.
  • These markers have been described and reviewed in international scientific journals (de Boer et al., 1999, Eur J Pharm Sci. 8(1): 1-4; Hofrnan et al., 2001, Invest Ophthalmol Vis Sci. 42(5): 895-901; Schlingemann et al., 1997, Ophthalmic Res. 29(3): 130-8; Schlingemann et al., 1999, Diabetologia. 42(5): 596-602; Vorbrodt et al., 1986, Brain Res. 394(1): 69-79; Dai et al, 2002, Brain Res. 954(2): 311-316).
  • the permeability of the endothelial cells is herein understood to mean the measure of the ease with which a compound (may that be ions (e.g., Na + , K + , Ca 24 ), water, nutrients (e.g., glucose, amino acids), metabolites, neurotransmitters (e.g., glutamic acid, tryptophan), hormones, peptides, plasma proteins (e.g., albumin, fibrinogen, immunoglobulins, cytokines, growth factors), cells and xenobiotics (e.g., drugs, diagnostic markers)) can diffuse across, or be (actively) transported into or across, an endothelial cell layer in the luminal to abluminal direction or visa versa.
  • ions e.g., Na + , K + , Ca 24
  • nutrients e.g., glucose, amino acids
  • neurotransmitters e.g., glutamic acid, tryptophan
  • hormones e.g., glutamic acid, try
  • Changes in permeability of the endothelial cells can also be the result of endothelial biotransformation of a given compound (may that be nutrients (e.g., glucose, amino acids), metabolites, neurotransmitters (e.g., glutamic acid, tryptophan), hormones, peptides, plasma proteins (e.g., albumin, fibrinogen, immunoglobulins, cytokines, growth factors), cells and xenobiotics (e.g., drugs, diagnostic markers)).
  • the modulation of the permeability includes both increases and decreases in permeability.
  • the permeability may conveniently be determined in vitro by determining the transendothelial electrical resistance (TEER) as described in the Examples.
  • TEER is a sensitive measure to quantify the permeability of ions through the tight junctions between cells.
  • a modulation of the permeability of the endothelial cells preferably is a modulation that results in a change of the TEER of at least 20, 50, 100, 300 or 1000% (Gaillard et al., 2000b, Eur J Pharm Sci. 12(2): 95- 102).
  • Other methods for determining the permeability include e.g.
  • Functional assays for restricted paracellular transport of reference compounds e.g., mannitol, sucrose, fluorescein, dextrans, albumin
  • polar and active and inhibitable with e.g., verapamil, PSC-833, temperature
  • Pgp-substrates rhodamine 123, vinblastine, etc.
  • transferrin across endothelial cell layers are indicative for changes in endothelial permeability (Gaillard et al., 2000, supra; Gaillard et al., 2001, supra).
  • the permeability of the endothelial cells may be determined by the demonstration of changes in (functional) expression of the endothelial/barrier markers that are involved in permeability control as described above for the in vitro situation (by e.g., molecular biological, biochemical, (immuno)-histo(cyto)chemical techniques or by functional assays using known substrates, ligands and/or inhibitors of transporter systems).
  • extravasation of endogenous (e.g., fibrinogen, IgG) or (fluorescence- or radiolabeled) exogenous (e.g., Evans blue (binds to albumin), mannitol, sucrose, fluorescein, dextrans, albumin, ATE) reference compounds may be determined by (immuno)-histo(cyto)chemical techniques or by several in vivo sampling methods, like brain uptake index (BUI, Oldendorf, 1970 Brain Res. 24(2): 372-376), brain efflux index (BEL Kakee et al., 1996 J Pharmacol Exp Therap. 277(3):1550-1559), in situ perfusion (Takasato et al., 1984 Am J Physiol.
  • the activity or steady-state level of the PB polypeptide may be altered at the level of the polypeptide itself, e.g. by providing the PB polypeptide to the endothelial cells from an exogenous source, or by adding an antagonist or inhibitor of the PB polypeptide to the endothelial cells, such as e.g. an antibody against the PB polypeptide.
  • an antagonist or inhibitor of the PB polypeptide to the endothelial cells, such as e.g. an antibody against the PB polypeptide.
  • the PB polypeptide may conveniently be produced by expression of a nucleic acid encoding the PB polypeptide in suitable host cells as described below. An antibody against the PB polypeptide may be obtained as described below.
  • the activity or steady-state level of the PB polypeptide may be altered by regulating the expression level of a nucleotide sequence encoding the polypeptide.
  • the expression level of a nucleotide sequence is regulated in the endothelial cells.
  • the expression level of the PB polypeptide may be up-regulated by introduction of an expression vector into the endothelial cells, whereby the expression vector comprises a nucleotide sequence encoding the PB polypeptide, and whereby the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in the endothelial cells.
  • the expression level of the PB polypeptide may also be up-regulated by introduction of an expression vector into the endothelial cells, whereby the expression vector comprises a nucleotide sequence encoding a factor capable of trans-activation of the endogenous nucleotide sequence encoding the PB polypeptide.
  • the expression level of the PB polypeptide may be down regulated by providing an antisense molecule to the cells, whereby the anisense molecule is capable of inhibiting the expression of the nucleotide sequence encoding the PB polypeptide.
  • the antisense molecule may be provided as such or it may be provided by introducing an expression vector into the endothelial cells, whereby the expression vector comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding the PB polypeptide, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in the endothelial cells.
  • the expression level of the PB polypeptide may also be down-regulated by introducing an expression vector into the endothelial cells, whereby the expression vector comprises a nucleotide sequence encoding a factor capable of trans-repression of the endogenous nucleotide sequence encoding the PB polypeptide.
  • PB polypeptides may thus be modified by:
  • an antisense nucleic acid molecule against a nucleotide sequence coding for a PB polypeptide (a) an antisense nucleic acid molecule against a nucleotide sequence coding for a PB polypeptide; (b) an antisense nucleic acid molecule against a nucleotide sequence coding for a PB polypeptide receptor; (c) an antisense nucleic acid molecule against a nucleotide sequence coding for a PB polypeptide receptor agonist; (d) an antisense nucleic acid molecule against a nucleotide sequence coding for a PB polypeptide receptor antagonist;
  • an expression or gene therapy vector in which an antisense nucleic acid sequence against a nucleotide sequence coding for a PB polypeptide receptor agonist is operably linked to a promoter;
  • an expression or gene therapy vector in which an antisense nucleic acid sequence against a nucleotide sequence coding for a PB polypeptide receptor antagonist is operably linked to a promoter.
  • a full or partial agonist of a PB polypeptide such e.g.: (i) a natural ligand;
  • a full or partial agonist of a PB polypeptide receptor such as e.g. : (i) a natural ligand;
  • Antagonist including e.g.:
  • a full or partial antagonists of a PB polypeptide such as e.g. : (i) a natural antagonist;
  • a partial or inverse agonist of a PB polypeptide receptor such as e.g.: (i) a natural ligand;
  • a full or partial antagonists of a PB polypeptide receptor i) a natural antagonist of a PB polypeptide receptor; (ii) a PB polypeptide fragment;
  • the permeability of the endothelial cells is preferably decreased by increasing the activity or the steady-state level of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 6, 8, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 - 45, and 52.
  • the permeability is decreased by increasing the activity or the steady-state level of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 4, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 and 44.
  • the permeability is decreased by increasing the activity or the steady-state level of a PB polypeptide selected from the groups consisting of up- regulated secreted factors (SEQ ID NO.'s 3, 16, 24, 25, 27, 31), up-regulated signal transduction pathways (SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44), up-regulated receptors and adhesion molecules(SEQ ID NO.'s 2, 11-12), and up-regulated metabolic enzymes (SEQ ID NO.'s 4, 20-21).
  • the activity or the steady-state level of the PB polypeptide may be increased by any of the means described above, e.g.
  • the expression vector comprises a nucleotide sequence encoding the PB polypeptide, and whereby the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in the endothelial cells.
  • the permeability of the endothelial cells may be decreased by decreasing the activity or the steady-state level of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 1, 7, 9, 18, 22, 26, 28 - 30, 33 - 37, 46 - 51, and 53.
  • the permeability is decreased by decreasing the activity or the steady-state level of a PB polypeptide selected from the groups consisting of down-regulated secreted factors (SEQ ID NO.'s 7, 33-37, 47-51), down-regulated signal transduction pathways (SEQ ID NO.'s 1, 9, 22, 26, 53), down-regulated receptors and adhesion molecules (SEQ ID NO.'s 28-30, 46), and a down-regulated metabolic enzyme (SEQ ID NO. 18).
  • the activity or the steady-state level of the PB polypeptide may be decreased by any of the means described above, e.g.
  • the activity or the steady-state level of the PB polypeptide may be decreased by introducing an expression vector into the endothelial cells, whereby the expression vector comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding the PB polypeptide, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in the endothelial cells.
  • the permeability of the endothelial cells may be increased by increasing the activity or the steady-state level of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 1, 7, 9, 18, 22, 26, 28 - 30, 33 - 37, 46 - 51, and 53.
  • the permeability is increased by increasing the activity or the steady-state level of a PB polypeptide selected from the groups consisting of down-regulated secreted factors (SEQ ID NO.'s 7, 33-37, 47-51), down-regulated signal transduction pathways (SEQ ID NO.'s 1, 9, 22, 26, 53), down-regulated receptors and adhesion molecules (SEQ ID NO.'s 28-30, 46), and a down-regulated metabolic enzyme (SEQ ID NO. 18).
  • the activity or the steady-state level of the PB polypeptide may be increased by any of the means described above, e.g.
  • the expression vector comprises a nucleotide sequence encoding the PB polypeptide, and whereby the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in the endothelial cells.
  • the permeability of the endothelial cells may be increased by decreasing the activity or the steady-state level of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 6, 8, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 - 45, and 52.
  • the permeability is increased by decreasing the activity or the steady-state level of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 4, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 and 44.
  • the permeability is increased by decreasing the activity or the steady-state level of a PB polypeptide selected from the groups consisting of up- regulated secreted factors (SEQ ID NO.'s 3, 16, 24, 25, 27, 31), up-regulated signal transduction pathways (SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44), up-regulated receptors and adhesion molecules(SEQ ID NO.'s 2, 11-12), and up-regulated metabolic enzymes (SEQ ID NO.'s 4, 20-21).
  • the activity or the steady-state level of the PB polypeptide may be decreased by any of the means described above, e.g.
  • the activity or the steady-state level of the PB polypeptide may be decreased by introducing an expression vector into the endothelial cells, whereby the expression vector comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding the PB polypeptide, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in the endothelial cells.
  • the invention in another aspect, relates to a method for treating or preventing a microvascular permeability-modifying disorder in a subject.
  • the method comprises pharmacologically altering the activity or the steady-state level in the subject's microvascular endothelial cells, of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53.
  • the alteration is sufficient to reduce the symptoms of the microvascular permeability-modifying disorder.
  • the method preferably comprises administering to the subject in a therapeutically effective amount, a pharmaceutical composition comprising a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53, or a nucleic acid molecule comprising a nucleotide sequence encoding the PB polypeptide or another entitity that is effective in modifying the activity or steady state level of a PB polypeptide as listed herein above.
  • the PB polypeptide is a polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 6, 8, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 - 45, and 52. More preferably the amino acid sequence is selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 4, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 and 44.
  • the amino acid sequence is selected from the groups consisting of the amino acid sequences of up-regulated secreted factors (SEQ ID NO.'s 3, 16, 24, 25, 27, 31), up-regulated signal transduction pathways (SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44), up-regulated receptors and adhesion molecules (SEQ ID NO.'s 2, 11-12), and up-regulated metabolic enzymes (SEQ ID NO.'s 4, 20-21).
  • the nucleic acid molecule preferably is a gene therapy vector, in which the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in endothelial cells, preferably microvascular endothelial cells.
  • the method of treatment is a method comprising the step of administering to the subject in a therapeutically effective amount, a pharmaceutical composition comprising an antagonist of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 1, 7, 9, 18, 22, 26, 28 - 30, 33 - 37, 46 - 51, and 53, whereby preferably the antagonist is an antibody against the PB polypeptide.
  • amino acid sequence is selected from the groups consisting of amino acid sequences of down-regulated secreted factors (SEQ ID NO.'s 7, 33-37, 47-51), down-regulated signal transduction pathways (SEQ ID NO.'s 1, 9, 22, 26, 53), down-regulated receptors and adhesion molecules (SEQ ID NO.'s 28-30, 46), and a down-regulated metabolic enzyme (SEQ ID NO. 18).
  • SEQ ID NO.'s 7, 33-37, 47-51 down-regulated signal transduction pathways
  • SEQ ID NO.'s 1, 9, 22, 26, 53 down-regulated receptors and adhesion molecules
  • SEQ ID NO. 18 down-regulated metabolic enzyme
  • the gene therapy vector preferably comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 1, 7, 9, 18, 22, 26, 28 - 30, 33 - 37, 46 - 51, and 53, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in endothelial cells, preferably microvascular endothelial cells.
  • amino acid sequence is selected from the groups consisting of amino acid sequences of down-regulated secreted factors (SEQ ID NO.'s 7, 33-37, 47-51), down-regulated signal transduction pathways (SEQ ID NO.'s 1, 9, 22, 26, 53), down-regulated receptors and adhesion molecules (SEQ ID NO.'s 28-30, 46), and a down-regulated metabolic enzyme (SEQ ID NO. 18).
  • the microvascular permeability disorder preferably is selected from the group consisting of neurodegenerative disorders, such as cerebrovascular accidents (CVA), Alzheimer's disease (AD), vascular-related dementia, Creutzfeldt- Jakob disease (CJD), bovine spongiform encephalopathy (BSE), Parkinson's disease (PD), brain trauma, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington's chorea; peripheral disorders with a CNS component, such as septic shock, hepatic encephalopathy, (diabetic) hypertension, diabetic microangiopathy, sleeping sickness, Whipple disease, Duchenne muscular dystrophy (DMD) and (pre)eclampsia; neuropsychiatric disorders, such as depression, autism, anxiety attention deficit hyperactivity disorder (ADHD), bipolar disorder, schizophrenia and other psychoses; other CNS disorders, such as brain tumors, epilepsy, migraine, narcolepsy, insomnia, chronic fatigue syndrome
  • CVA cerebrovascular accidents
  • the invention relates to a method for reversibly increasing the microvascular permeability in a subject.
  • This method comprises the step of administering to the subject, a pharmaceutical composition comprising a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53, or a nucleic acid molecule comprising a nucleotide sequence encoding the PB polypeptide, or another entitity that is effective in modifying the activity or steady state level of a PB polypeptide as listed herein above, in an amount effective to increase the microvascular permeability.
  • the PB polypeptide is a polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 1, 7, 9, 18, 22, 26, 28 - 30, 33 - 37, 46 - 51, and 53. More preferably the amino acid sequence is selected from the groups consisting of amino acid sequences of down-regulated secreted factors (SEQ ID NO. ' s 7, 33-37, 47-51), down-regulated signal transduction pathways (SEQ ID NO.'s 1, 9, 22, 26, 53), down-regulated receptors and adhesion molecules (SEQ ID NO.'s 28-30, 46), and a down-regulated metabolic enzyme (SEQ ID NO. 18).
  • SEQ ID NO. 's 7, 33-37, 47-51 down-regulated signal transduction pathways
  • SEQ ID NO.'s 1, 9, 22, 26, 53 down-regulated receptors and adhesion molecules
  • SEQ ID NO. 18 down-regulated metabolic enzyme
  • the nucleic acid molecule is a gene therapy vector, in which the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in endothelial cells, preferably microvascular endothelial cells.
  • a promoter capable of driving expression of the nucleotide sequence in endothelial cells preferably is an inducible promoter. More preferably, the inducible promoter is a promoter that may be induced by the administration of small organic or inorganic compounds (see below).
  • method for reversibly increasing the microvascular permeability in a subject may also be a method comprising the step of administering to the subject in a therapeutically effective amount, a pharmaceutical composition comprising an antagonist of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 6, 8, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 - 45, and 52, whereby the antagonist preferably is an antibody against the PB polypeptide.
  • amino acid sequence is selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 4, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 and 44.
  • amino acid sequence is selected from the groups consisting of the amino acid sequences of up-regulated secreted factors (SEQ ID NO.'s 3, 16, 24, 25, 27, 31), up-regulated signal transduction pathways (SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44), up-regulated receptors and adhesion molecules (SEQ ID NO.'s 2, 11-12), and up-regulated metabolic enzymes (SEQ ID NO.'s 4, 20-21).
  • the method may also comprise the step of administering to the subject in a therapeutically effective amount, a pharmaceutical composition comprising a gene therapy vector, whereby the gene therapy vector comprises an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 6, 8, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 - 45, and 52, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in endothelial cells, preferably microvascular endothelial cells.
  • amino acid sequence is selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 4, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 and 44.
  • amino acid sequence is selected from the groups consisting of the amino acid sequences of up-regulated secreted factors (SEQ ID NO.'s 3, 16, 24, 25, 27, 31), up-regulated signal transduction pathways (SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44), up-regulated receptors and adhesion molecules (SEQ ID NO.'s 2, 11-12), and up-regulated metabolic enzymes (SEQ ID NO.'s 4, 20-21).
  • the methods for reversibly increasing the microvascular permeability in a subject may advantageously be applied when one wants to deliver blood-borne, membrane- impermeant drugs to the brain.
  • the drug may be any pharmaceutically, veterinarily or diagnostically useful compound or composition of compounds, which is normally impermeant to the blood-brain or other physiological barrier or at least insufficiently permeant.
  • the pharmacological nature of the drug is otherwise unimportant.
  • the invention is therefore useful in the delivery of a wide range of drugs across physiological barriers such as the blood-brain barrier.
  • anti-tumor compounds such as methotrexate, adriamycin and cisplatin
  • growth factors such as NGF, RDNF and CNTF, which are used to treat neurodegenerative disease
  • imaging agents especially those that are antibody based
  • neurotransmitter antagonists or agonists which do not penetrate the blood-brain barrier such as certain NMDA receptor blockers
  • the invention relates to various uses of the compounds of the invention for the manufacture of a medicament for treating or preventing a microvascular permeability-modifying disorder.
  • the invention relates to the use of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53, or a nucleic acid molecule comprising a nucleotide sequence encoding the PB polypeptide, or another entitity that is effective in modifying the activity or steady state level of a PB polypeptide as listed herein above, for the manufacture of a composition for treating or preventing a microvascular permeability-modifying disorder.
  • the PB polypeptide is a polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 6, 8, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 - 45, and 52. More preferably the amino acid sequence is selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 4, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 and 44.
  • the amino acid sequence is selected from the groups consisting of the amino acid sequences of up-regulated secreted factors (SEQ ID NO.'s 3, 16, 24, 25, 27, 31), up- regulated signal transduction pathways (SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44), up-regulated receptors and adhesion molecules (SEQ ID NO.'s 2, 11-12), and up- regulated metabolic enzymes (SEQ ID NO.'s 4, 20-21).
  • the nucleic acid molecule preferably is a gene therapy vector comprising the nucleotide sequence, whereby the nucleotide sequence is under control of a promoter capable of driving expression of the nucleotide sequence in endothelial cells.
  • An antagonist of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 1, 7, 9, 18, 22, 26, 28 - 30, 33 - 37, 46 - 51, and 53, may also be used for the manufacture of a composition for treating or preventing a microvascular permeability-modifying disorder, whereby the antagonist preferably is an antibody against the PB polypeptide.
  • amino acid sequence is selected from the groups consisting of amino acid sequences of down-regulated secreted factors (SEQ ID NO.'s 7, 33-37, 47- 51), down-regulated signal transduction pathways (SEQ ID NO.'s 1, 9, 22, 26, 53), down-regulated receptors and adhesion molecules (SEQ ID NO.'s 28-30, 46), and a down-regulated metabolic enzyme (SEQ ID NO. 18).
  • a gene therapy vector comprising an antisense nucleotide sequence that is capable of inliibiting the expression of the nucleotide sequence encoding a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 1, 7, 9, 18, 22, 26, 28 - 30, 33 - 37, 46 - 51, and 53, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in endothelial cells, preferably microvascular endothelial cells, may be used for the manufacture of a composition for treating or preventing a microvascular permeability-modifying disorder.
  • amino acid sequence is selected from the groups consisting of amino acid sequences of down-regulated secreted factors (SEQ ID NO.'s 7, 33-37, 47-51), down-regulated signal transduction pathways (SEQ ID NO.'s 1, 9, 22, 26, 53), down- regulated receptors and adhesion molecules (SEQ ID NO.'s 28-30, 46), and a down- regulated metabolic enzyme (SEQ ID NO. 18).
  • the disorder preferably is a microvascular permeability-modifying disorder as described above.
  • the invention relates to various uses of the compounds of the invention for the manufacture of a medicament or composition for reversibly increasing the microvascular permeability in a subject.
  • the compound is a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53, or a nucleic acid molecule comprising a nucleotide sequence encoding the PB polypeptide or another entitity that is effective in modifying the activity or steady state level of a PB polypeptide as listed herein above.
  • the PB polypeptide is a polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 1, 7, 9, 18, 22, 26, 28 - 30, 33 - 37, 46 - 51, and 53.
  • the amino acid sequence is selected from the groups consisting of amino acid sequences of down-regulated secreted factors (SEQ ID NO.'s 7, 33-37, 47-51), down-regulated signal transduction pathways (SEQ ID NO.'s 1, 9, 22, 26, 53), down-regulated receptors and adhesion molecules (SEQ ID NO.'s 28-30, 46), and a down-regulated metabolic enzyme (SEQ ID NO. 18).
  • the nucleic acid molecule preferably is a gene therapy vector, in which the nucleotide sequence encoding the PB polypeptide is under control of a promoter capable of driving expression of the nucleotide sequence in endothelial cells, preferably microvascular endothelial cells.
  • an antagonist of a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 6, 8, 10 - 17, 19 - 21 , 23 - 25, 27, 31 - 32, 38 - 45, and 52 may be used for the manufacture of a composition for reversibly increasing the microvascular permeability in a subject, whereby preferably the antagonist is an antibody against the PB polypeptide. More preferably the amino acid sequence is selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 4, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 and 44.
  • amino acid sequence is selected from the groups consisting of the amino acid sequences of up-regulated secreted factors (SEQ ID NO.'s 3, 16, 24, 25, 27, 31), up-regulated signal transduction pathways (SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44), up-regulated receptors and adhesion molecules (SEQ ID NO.'s 2, 11-12), and up-regulated metabolic enzymes (SEQ ID NO.'s 4, 20-21).
  • up-regulated secreted factors SEQ ID NO.'s 3, 16, 24, 25, 27, 31
  • up-regulated signal transduction pathways SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44
  • up-regulated receptors and adhesion molecules SEQ ID NO.'s 2, 11-12
  • up-regulated metabolic enzymes SEQ ID NO.'s 4, 20-21.
  • a gene therapy vector comprising an antisense nucleotide sequence that is capable of inhibiting the expression of the nucleotide sequence encoding a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 6, 8, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 - 45, and 52, and whereby the antisense nucleotide sequence is under control of a promoter capable of driving expression of the antisense nucleotide sequence in endothelial cells, preferably microvascular endothelial cells, may be used for the manufacture of a composition for reversibly increasing the microvascular permeability in a subject.
  • amino acid sequence is selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 4, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 and 44.
  • amino acid sequence is selected from the groups consisting of the amino acid sequences of up-regulated secreted factors (SEQ ID NO.'s 3, 16, 24, 25, 27, 31), up-regulated signal transduction pathways (SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44), up-regulated receptors and adhesion molecules (SEQ ID NO.'s 2, 11-12), and up- regulated metabolic enzymes (SEQ ID NO.'s 4, 20-21).
  • the gene therapy vector is a vector for transient expression (see below) and/or the promoter preferably is an inducible promoter. More preferably, the inducible promoter is a promoter that may be induced by the administration of small organic or inorganic compounds (see below).
  • the invention involves a method of treating or diagnosing a CNS or microvascular disorder by administering a therapeutic or diagnostic agent, e.g. a neuroactive agent, by targeting that agent, or its pharmaceutically acceptable carrier, to a selectively upregulated PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53, to a patient suffering from or at risk for developing the CNS or microvascular disorder.
  • a therapeutic or diagnostic agent e.g. a neuroactive agent
  • the neuroactive agent or its carrier is targeted to an upregulated PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 2 - 6, 8, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 - 45, and 52. More preferably the amino acid sequence is selected from the group consisting of the amino acid sequences depicted in SEQ ID NO.'s 2 - 4, 10 - 17, 19 - 21, 23 - 25, 27, 31 - 32, 38 and 44.
  • the amino acid sequence is selected from the groups consisting of the amino acid sequences of up-regulated secreted factors (SEQ ID NO.'s 3, 16, 24, 25, 27, 31), up-regulated signal transduction pathways (SEQ ID NO.'s 10, 13-16, 17, 19, 23, 32, 38, 44), up-regulated receptors and adhesion molecules (SEQ ID NO.'s 2, 11-12), and up-regulated metabolic enzymes (SEQ ID NO.'s 4, 20-21). Still more preferably an upregulated PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 2 or 11.
  • the targeting agent can be an antibody to a PB polypeptide, a peptide, a PB polypeptide agonist, a PB polypeptide antagonist, a peptidomimetic, a small molecule, or another compound that specifically binds to a PB polypeptide.
  • the term "specific binding” means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity, for example, a peptide of similar size that lacks a specific binding sequence.
  • Specific binding is present if the molecule has measurably higher affinity for the PB polypeptide than the control molecule. Specificity of binding can be determined, for example, by competition with a control molecule that is known to bind to a target.
  • the term "specific binding," as used herein, includes both low and high affinity specific binding. Specific binding can be exhibited, e.g., by a low affinity targeting agent having a Kd of at least about 10 "4 M. E.g., if a PB polypeptide has more than one binding site for a targeting agent, a targeting agent having low affinity can be useful for targeting the microvascular endothelium. Specific binding also can be exhibited by a high affinity targeting agents, e.g.
  • the targeting agent is preferably conjugated to the therapeutic agent or its pharmaceutically acceptable carrier.
  • a "conjugate” is herein defined as consisting of two entities that are covalently coupled together.
  • the first entity will usually be a targeting agent as herein defined above, whereas the second entity may be a therapeutic or diagnostic moiety, such as a molecule or structure, for use in the treatment or diagnosis of a CNS or microvascular disorder.
  • Such therapeutic or diagnostic moieties may e.g.
  • anti-tumor compounds such as methotrexate, adriamycin and cisplatin
  • growth factors such as NGF, RDNF and CNTF, which are used to treat neurodegenerative disease
  • imaging agents especially those that are antibody based
  • neurotransmitter antagonists or agonists which do not penetrate the blood-brain barrier such as certain NMD A receptor blockers.
  • a large variety of methods for conjugation of targeting agents with therapeutic or diagnostic moieties are known in the art. Such methods are e.g. described by Hermanson (1996, Bioconjugate Techniques, Academic Press), in U.S. 6,180,084 and U.S. 6,264,914 and include e.g. methods used to link haptens to carriers proteins as routinely used in applied immunology (see Harlow and Lane, 1988, “Antibodies: A laboratory manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). It is recognised that, in some cases, a targeting agent or a therapeutic or diagnostic moiety may lose efficacy or functionality upon conjugation depending, e.g., on the conjugation procedure or the chemical group utilised therein.
  • Suitable methods for conjugation of a targeting agent with a therapeutic or diagnostic moiety include e.g. carbodiimide conjugation (Bauminger and Wilchek, 1980, Meth. Enzymol. 70: 151-159).
  • a moiety can be coupled to a targeting agent as described by Nagy et al., Proc. Natl. Acad. Sci. USA 93:7269-7273 (1996); and Nagy et al., Proc. Natl. Acad. Sci.
  • Another method for conjugating that may suitable be used are e.g. sodium periodate oxidation followed by reductive alkylation of appropriate reactants and glutaraldehyde crosslinking.
  • a particularly advantageous method of conjugation may be applied when both the targeting agent and the therapeutic moiety are (poly)peptides.
  • the two entities may be synthesised as a single (poly)peptide chain comprising the amino acid sequences of both the targeting agent and the therapeutic peptide.
  • the conjugate may be synthesised by solid phase peptide synthesis as herein described above.
  • the single (poly)peptide chain comprising the targeting agent and the therapeutic peptide may be produced by recombinant expression techniques as outlined herein below. In such instances e.g.
  • the two nucleic acid sequences encoding each the targeting agent and the therapeutic peptide may be operably linked in frame to form a single open reading frame.
  • the nucleic acid sequence containing the single open reading frame may then be inserted in a suitable expression vector for expression in a suitable host from which the conjugate may then be recovered and optionally further purified as herein described below.
  • targeting peptides may be placed on either or both ends of the therapeutic peptide, or may be inserted within the amino acid sequence of the therapeutic peptide in one or more positions that do not disturb the function or efficacy of the respective peptides. Using routine methods the skilled person can establish the optimal position of the targeting peptide(s) with respect to the therapeutic peptide. Diagnosis of microvascular permeability
  • the invention relates to methods for diagnosing the status of the microvascular permeability in a subject.
  • a method preferably comprises the steps of: (a) determining the expression level of a nucleic acid sequence encoding a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53 in the subject's microvascular endothelium; and, (b) comparing the expression level of the nucleic acid sequence with a reference value for expression level of the nucleic acid sequence, the reference value preferably being the average value for the expression level in the microvascular endothelium of healthy individuals.
  • the expression level of the nucleic acid sequence may be determined indirectly by quantifying the amount of the PB polypeptide encoded by the nucleic acid sequence.
  • the expression level of more than one nucleic acid sequences are compared. When more than one nucleic acid sequence is analysed this may conveniently be done using microarrays comprising complementary nucleic acids as described below and in the Examples.
  • the expression level may be determined ex vivo in a sample obtained from the subject.
  • the method preferably is a method for diagnosing a microvascular permeability disorder or for diagnosing a susceptibility to a microvascular permeability disorder, whereby the microvascular permeability may be as described above.
  • the method may also be used to assess the efficacy of a treatment for restoration of the microvascular permeability.
  • the invention relates to methods for identification of substances capable of modulating the permeability of microvascular endothelial cells.
  • the method preferably comprises the steps of: (a) providing a test cell population capable of expressing one or more nucleic acid sequences encoding a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53; (b) contacting the test cell population with a composition comprising a substance to be tested; (c) determining the expression level of a nucleic acid sequence encoding a PB polypeptide having an amino acid sequence with at least 90% identity with an amino acid sequence as depicted in SEQ ID NO.'s 1 - 53, in the test cell population contacted with the substance; (d) comparing the expression of the nucleic acid sequence with the expression level of the nucleic acid sequence in a test cell population that is not contacted with the substance; and, (a) providing a test cell population capable of expressing one or more nucle
  • the expression level of the nucleic acid sequence may be determined indirectly by quantifying the amount of the PB polypeptide encoded by the nucleic acid sequence.
  • the expression level of more than one nucleic acid sequence may be compared.
  • the test cell population comprises endothelial cells, preferably vascular endothelial cells, more preferably microvascular endothelial cells, most preferably brain microvascular endothelial cells.
  • the cells in the test cell population are preferably mammalian cells, preferably human cells.
  • the test cell population that is contacted with the substance and the test cell population that is not contacted with the substance are derived from one cell population, preferably from one cell line, more preferably from one cell.
  • the test cell population is co-cultured with a helper cell population, whereby the test cell population is cultured on one side of the filter and the helper cell population is cultured on the other side of the filter, and whereby the helper cell population preferably comprises astrocytes.
  • PB polypeptides are involved in several different types of mechanisms involved in the control of blood-brain barrier functionality. These include secreted factors, cytoskeletal- and extracellular matrix factors, signal transduction pathways, receptors and adhesion molecules, and metabolic enzymes. PB polypeptides and these mechanisms are discussed in greater detail below. If known or applicable, for each PB polypeptide the following information is given:
  • ⁇ agonistic small molecules, or other drugs ⁇ antagonistic PB polypeptide fragment(s);
  • LGALS2 gene (SEQ ID NO. 3, PB03), or the human HL14 gene, encoding a soluble beta-galactoside binding protein (galectin 2), is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on LGALS2 expression was found (Table 2). The mouse homologue of the LGALS2 gene has been reported to act as a cell growth inhibitory factor.
  • Beta-galactoside-binding protein is an autocrine regulator of cell proliferation with a role in the maintenance of GO and in the control of G2 traverse.
  • the surprising finding that the LGALS2 gene, or galectin 2 was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier has not been reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. Therefore, any agent that changes the biological activity of the LGALS2 gene product (galectin-2) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention.
  • Galectin-2 activity may be conveniently decreased by antisense inhibition of the LGALS2 gene, while galectin-2 activity may be conveniently increased by introduction of the LGALS2 gene into the cell or, as exemplified in Example 5, by exposure of the endothelial cells to exogenous galectin-2.
  • Joubert et al. (1989, Dev Neurosci., 11 6): 397-413) developed useful antibodies against galectin-2, which may be used for diagnostic or treatment purposes. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • ESM1 gene (SEQ ID NO. 7, PB07), encoding endothelial cell-specific molecule 1 (endocan), is downregulated in BCEC by physical co-culture with astrocytes (Table 1). Downregulated PB polypeptides are involved in decreased vascular permeability. In addition, ESM1 is upregulated by LPS in BCEC-astrocyte cocultures and unaltered in BCEC monolayers (Table 2), indicating that astrocytes are essential in the regulation of ESM1 expression in BCEC. This gene encodes a secreted protein that is mainly expressed in the endothelial cells in human lung and kidney tissues.
  • ESM1 endothelial cell-specific molecule 1
  • ESM1 gene activity may be conveniently decreased by antisense inhibition of the ESM1 gene, while ESM1 activity may be conveniently increased by introduction of the ESM1 gene into the endothelial cell, or by exposure to the gene product endothelial cell-specific molecule 1, in order to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention.
  • Tsai et al. 2002, J Vase Res., 39(2): 148-159
  • THBS1 gene SEQ ID NO. 24, PB19
  • thrombospondin 1 is upregulated in BCEC by physical co-culture with astrocytes (Table 1).
  • Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on THBS1 expression was found (Table 2).
  • THBS1 is a multimodular secreted protein that associates with the extracellular matrix and possesses a variety of biologic functions, including a potent angiogenic activity.
  • THBS1 is a homotrimeric glycoprotein with disulfide-linked subunits of MW 180,000.
  • THBS1 was first described as a component of the alpha-granule of platelets, released on platelet activation. It is associated with the platelet membrane in the presence of divalent cations and has a role in platelet aggregation.
  • THBS1 is not limited to platelets, however. It is synthesised and secreted for incorporation into the extracellular matrix by a variety of cells including endothelial cells, fibroblasts, smooth muscle cells, and type II pneumocytes.
  • THBS1 binds heparin, sulfatides, fibrinogen, fibronectin, plasminogen, and type V collagen. THBS1 derive specificity for remodeling vessels from the dependence on Fas/Fas ligand-mediated apoptosis to block angiogenesis. THBS1 upregulated FasL on endothelial cells. Expression of the essential partner of FasL, Fas receptor, was low on quiescent endothelial cells and vessels but greatly enhanced by inducers of angiogenesis, thereby specifically sensitising the stimulated cells to apoptosis by inhibitor-generated FasL.
  • THBS 1 antiangiogenic activity of THBS 1 both in vitro and in vivo was dependent on this dual induction of Fas and FasL and the resulting apoptosis.
  • THBS1 gene, or thrombospondin 1 was upregulated by astrocytes and not by LPS in the cells that constitute the blood- brain barrier was not reported earlier and offers new opportanities to modify or monitor blood-brain barrier functionality, as well as to target drugs to the blood-brain barrier.
  • any ligand that specifically binds to the THBS 1 -induced FasL e.g., Fas receptor
  • Fas receptor any ligand that specifically binds to the THBS 1 -induced FasL is useful to target drugs to the blood-brain barrier in the embodiments of the present invention.
  • THBS 1 gene activity may be conveniently decreased by antisense inhibition of the THBS1 gene.
  • THBS1 gene activity may be conveniently increased by introduction of the THBS1 gene into, or exposure of the gene product thrombospondin 1 to, the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. Lawler et al. (1998, J Clin Invest., 101(5): 982- 992) developed a THBS1 knockout mouse, which may be useful to specifically investigate the functionality of the blood-brain barrier in the embodiments of the present invention.
  • IGFBP2 insulin-like growth factor binding protein 2
  • BCEC BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on IGFBP2 expression was found (Table 2). Insulinlike growth factors I and II are complexed to specific binding proteins in plasma. Two major classes of IGF-BPs have been identified in man. IGFBP2 is the quantitatively predominant form in plasma and has a molecular weight of about 150 kD. It contains an IGF-binding subunit (IGF-BP53) with an apparent molecular mass of 53 kD.
  • IGF-BP53 IGF-binding subunit
  • IGFBP2 binds to and modulates insulin-like growth factor activity.
  • the surprising finding that the IGFBP2 gene, or insulin-like growth factor binding protein 2, was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality.
  • IGFBP2 gene activity may be conveniently decreased by antisense inhibition of the IGFBP2 gene, or by any of the available (R&D Systems Europe Ltd., UK) neutralising antibodies directed against IGFBP2.
  • IGFBP2 gene activity may be conveniently increased by introduction of the IGFBP2 gene into, or exposure of the gene product insulin-like growth factor binding protein 2 to, the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • LOXL1 The LOXL1 gene (SEQ ID NO. 27, PB22), encoding lysyl oxidase-like 1, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on LOXL1 expression was found (Table 2). LOXL1 is similar to the C- terminus of lysyl oxidase (LOX, protein-lysine 6-oxidase; EC 1.4.3.13).
  • the JAG1 gene (SEQ ID NO. 31, PB24), encoding jagged 1, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on JAG1 expression was found (Table 2).
  • the jagged 1 protein encoded by JAG1 is the human homologue of the Drosophilia jagged protein. Human jagged 1 is the ligand for the receptor notch 1, the latter a human homologue of the Drosophilia jagged receptor notch. Mutations that alter the jagged 1 protein cause Alagille syndrome.
  • CNS manifestations of this syndrome include intracranial bleeding, stroke, vascular malformation, mental retardation, developmental delay, school dysfunction, abnormal visual, auditory and somatosensory evoked potentials.
  • mutations that alter the Notch signaling pathway i.e., mutations in Notch3 gene
  • CADASIL Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy
  • Jagged 1 signaling through notch 1 has also been shown to play a role in hematopoiesis.
  • JAGl gene activity may be conveniently decreased by antisense inhibition of the JAGl gene.
  • JAGl gene activity may be conveniently increased by introduction of the JAGl gene into, or exposure of the gene product jagged 1 (or other Notch receptor ligands including JAG2, Delta-like- 1, -2, and -3) to, the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • Xue et al. (1999, Hum Mol Genet., 8(5): 723-730) constructed a jagged 1 knockout mouse, which may be useful to specifically investigate the blood-brain barrier in the embodiments of the present invention.
  • the CASPl gene (SEQ ID NO.'s 33-37, PB26), encoding caspase 1, an apoptosis-related cysteine protease (or interleukin 1 beta convertase (ICE), is downregulated in BCEC by physical co-culture with astrocytes (Table 1).
  • PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on CASPl expression was found (Table 2).
  • This gene encodes a protein that is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis.
  • Caspases exist as inactive proenzymes that undergo proteolytic processing at conserved aspartic residues to produce 2 subunits, large and small, that dimerise to form the active enzyme.
  • This gene was identified by its ability to proteolytically cleave and activate the inactive precursor of interleukin- 1, a cytokine involved in the processes such as inflammation, septic shock, and wound healing. This gene has been shown to induce cell apoptosis and may function in various developmental stages. Studies of the similar gene in mouse suggested its role in the pathogenesis of Huntington disease. The surprising finding that the CASPl gene, or caspase 1, was downregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality.
  • CASPl gene or biological activity may be conveniently decreased by antisense inhibition of the CASPl gene or by exposure to erythropoietin (Chong et al., 2002, Circulation, 106(23): 2973-2979), Ac-WEHD-CHO (N-acetyl-Tyr-Glu-His-Asp-aldehyde) or YVAD (N-acetyl-Tyr- Val- Ala-Asp- aldehyde), two specific caspase- 1 (ICE) inhibitors, or ZVAD (N-benzyloxycarbonyl- Val-Ala-Asp-fluoromethyl ketone), a pan-caspase inhibitor (Suschek et al., 2002, Mol Pharmacol., 62(4): 936-946; Hayashi et al., 2001, Brain Res., 893(1-2): 113-120) or nitric oxide (NO)-releasing NSAIDs (Fiorucci e
  • CASPl gene or biological activity may be conveniently increased by introduction of the CASPl gene into, or exposure of the gene product caspase 1 to, the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • TMPRSS3 gene (SEQ ID NO.'s 47-51, PB33), encoding transmembrane serine protease 3, is downregulated in BCEC by physical co-culture with astrocytes (Table 1). Downregulated PB polypeptides are involved in decreased vascular permeability.
  • TMPRSS3 is downregulated by LPS in BCEC monolayers, presumably as a positive feedback mechanism to this stimulus (Table 2).
  • This gene encodes a protein that belongs to the serine protease family.
  • the encoded protein contains a serine protease domain, a transmembrane domain, a LDL receptor-like domain, and a scavenger receptor cysteine-rich domain.
  • Serine proteases are known to be involved in a variety of biological processes, whose malfunction often leads to human diseases and disorders. This gene was identified by its association with both congenital and childhood onset autosomal recessive deafness.
  • TMPRSS3 gene is expressed in fetal cochlea and many other tissues, and is thought to be involved in the development and maintenance of the inner ear or the contents of the perilymph and endolymph. This gene was also identified as a tumor associated gene that is overexpressed in ovarian tumors.
  • TMPRSS3 gene, or transmembrane serine protease 3 was downregulated by astrocytes and LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality.
  • TMPRSS3 gene or biological activity may be conveniently decreased by antisense inhibition of the TMPRSS3 gene, or by exposure to any TMPRSS3 inhibitor.
  • TMPRSS3 gene or biological activity may be conveniently increased by introduction of the TMPRSS3 gene into the endothelial cell, or by exposure to any TMPRSS3 activator, to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • Structural changes in polypeptides involved in the formation of the cytoskeleton (and hence motility of cells) and the extracellular matrix are ideal candidates to specifically (i.e., astrocyte-induced) monitor the permeability of the blood-brain barrier in the embodiments of the present invention.
  • aortic smooth muscle actin alpha 2 regulatory light polypeptide myosin, vimentin, fibronectin 1, light polypeptide 6 myosin, type III, alpha 1 collagen and gamma 1 actin.
  • the ACTA2 gene (SEQ ID NO. 5, PB05), encoding the human aortic smooth muscle actin alpha 2 gene, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Most probably, the ACTA2 gene is only expressed in the small contaminating fraction of pericytes which are present in the BCEC sample.
  • PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on ACTA2 expression was found (Table 2). Actin alpha 2 is one of six different actin isoforms, which have been identified. Actins are highly conserved proteins that are involved in cell motility, structure and integrity. Alpha actins are a major constituent of the contractile apparatus. The surprising finding that the ACTA2 gene, or actin alpha 2, was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to monitor blood-brain barrier functionality. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • the MLCB gene (SEQ ID NO. 6, PB06), or the non-sarcomeric regulatory light polypeptide myosin, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on MLCB expression was found (Table 2).
  • the MLCB gene is very strongly similar to rat RLC-A and may regulate myosin head ATPase activity in smooth muscle at rest.
  • VIM gene (SEQ ID NO. 8, PB08), encoding vimentin, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on VIM expression was found (Table 2).
  • Vimentin is an intermediate filament protein; a member of a family of intermediate filament proteins. Along with the microfilaments (actins) and microtubules (tubulins), the intermediate filaments represent a third class of well-characterised cytoskeletal elements.
  • the FN1 gene (SEQ ID NO. 39, PB28), encoding fibronectin 1, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on FN1 expression was found (Table 2). This gene encodes fibronectin, a glycoprotein present in a soluble dimeric form in plasma, and in a dimeric or multimeric form at the cell surface and in extracellular matrix. Fibronectin is involved in cell adhesion and migration processes including embryogenesis, wound healing, blood coagulation, host defense, and metastasis.
  • the MYL6 gene (SEQ ID NO.'s 40-43, PB29), encoding the alkali smooth muscle and non-muscle light polypeptide 6 myosin, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on MYL6 expression was found (Table 2).
  • Myosin is a hexameric ATPase cellular motor protein. It is composed of two heavy chains, two nonphosphorylatable alkali light chains, and two phosphorylatable regulatory light chains. This gene encodes a myosin alkali light chain that is expressed in smooth muscle and non-muscle tissues.
  • MYL6 gene or the alkali smooth muscle and non-muscle light polypeptide 6 myosin, was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to monitor blood-brain barrier functionality. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • the ACTGlgene (SEQ ID NO. 52, PB34), encoding gamma 1 actin, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on ACTG1 expression was found (Table 2). Actins are highly conserved proteins that are involved in various types of cell motility, and maintenance of the cytoskeleton. In vertebrates, three main groups of actin isoforms, alpha, beta and gamma have been identified. The alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus.
  • Polypeptides involved in intracellular signal transduction pathways are ideal candidates to specifically (i.e., astrocyte-induced) modulate the permeability of the blood-brain barrier in the embodiments of the present invention.
  • We have identified several novel of such specifically differentially expressed polypeptides including v- yes-1, SI 00 calcium binding protein A5, v-myc, alpha stimulating guanine nucleotide binding protein, growth factor receptor-bound protein 10, heme-regulated initiation factor 2-alpha kinase, mago-nashi homologue, calmodulin 1, cellular retinol binding protein 1, thyroid hormone receptor interactor 6, adaptor-related protein complex 4, light polypeptide ferritin and Kritl . These are discussed in greater detail below.
  • the gene YES1 (SEQ ID NO. 1, PB01), encoding v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1, is downregulated in BCEC by physical co-culture with astrocytes (Table 1). Downregulated PB polypeptides are involved in decreased vascular permeability. In addition, YESl is downregulated by LPS in BCEC monolayers, presumably as a positive feedback mechanism to this stimulus (Table 2). This gene is the cellular homolog of the Yamaguchi sarcoma virus oncogene, Yesl. The encoded protein has tyrosine kinase activity and belongs to the src family of proteins.
  • any agent which changes the biological activity of the YESl gene product is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention.
  • YESl activity may be conveniently decreased by antisense inhibition of the YESl gene (as exemplified in Example 3), while YES 1 activity may be conveniently temporarily increased (and antagonised by filipin) by exposure to Shiga toxin (Katagiri et al., 1999, J Biol Chem. 274(49): 35278-35282; or Shiga-like toxin II (Zhao et al, 2002, Brain Res.
  • S 100A5 gene (SEQ ID NO. 9, PB09), encoding S 100 calcium binding protein A5, is downregulated in BCEC by physical co-culture with astrocytes (Table 1). Downregulated PB polypeptides are involved in decreased vascular permeability. In addition, S100A5 is downregulated by LPS in BCEC monolayers, presumably as a positive feedback mechanism to this stimulus (Table 2).
  • the protein encoded by this gene is a member of the SI 00 family of proteins containing 2 EF-hand calcium-binding motifs.
  • SI 00 proteins are localised in the cytoplasm and/or nucleus of a wide range of cells, and involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation.
  • SI 00 genes include at least 13 members which are located as a cluster on chromosome lq21. This protein has a Ca2+ affinity 20- to 100- fold higher than the other SI 00 proteins studied under identical conditions. This protein also binds Zn2+ and Cu2+, and Cu2+ strongly which impairs the binding of Ca2+. This protein is expressed in very restricted regions of the adult brain.
  • S100A5 gene or SI 00 calcium binding protein A5
  • S100A5 activity may be conveniently decreased by antisense inhibition of the S100A5 gene, while S100A5 activity may be increased by introduction of the S100A5 gene into the endothelial cell, or by exposure to the gene product SI 00 calcium binding protein A5.
  • Coppens et al. (2001, Brain Res Dev Brain Res., 126(2): 191-199) developed polyclonal antisera raised against the human recombinant S100A5 protein, which may be used for diagnostic or treatment purposes.
  • the MYCN gene (SEQ ID NO. 10, PB10), encoding the neuroblastoma derived v-myc myelocytomatosis viral related oncogene, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. In addition, MYCN is downregulated by LPS in BCEC-astrocyte cocultures and unaltered in BCEC monolayers (Table 2), indicating that astrocytes are essential in the regulation of MYCN expression in BCEC.
  • the MYCN gene is a member of the Myc proto-oncoprotein family and contains a DNA binding domain.
  • LIF leukemia inhibitory factor
  • IL-6 interleukin-6
  • p73 protein a protein with growth suppressing properties which induces neuronal differentiation
  • MYCN gene activity may be conveniently decreased by antisense inhibition of the MYCN gene, while MYCN activity may be conveniently increased by introduction of the MYCN gene into the endothelial cell, or by exposure to the gene product v-myc myelocytomatosis viral related oncogene, or endothelial exposure to glucocorticoids, in order to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention.
  • Sun et al, (2002, Peptides, 23(9): 1557-1565) developed antisense peptide nucleic acids (conjugated to somatostatin analogs) targeted at the n-myc oncogene, which may be used for diagnostic or drug delivery purposes.
  • the GNAS gene (SEQ ID NO.'s 13-16, PB12), encoding the GNAS (guanine nucleotide binding protein, alpha stimulating) complex locus, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on GNAS expression was found (Table 2).
  • This gene has a highly complex imprinted expression pattern. It encodes maternally, paternally, and biallelically expressed proteins, which are derived from alternatively spliced transcripts with alternate 5' exons. Each of the upstream exons is within a differentially methylated region, commonly found in imprinted genes.
  • NESP55 neuroendocrine secretory protein
  • XL-alpha-s paternally (XL-alpha-s), and biallelically (Gs-alpha) derived proteins
  • GNAS biallelically derived proteins
  • NESP55 a novel member of the cliromogranin family, is a protein of yet unknown function.
  • XL alpha s is specifically associated with the tr ⁇ r ⁇ -Golgi network and occurs selectively in cells containing both the regulated and the constitutive pathway of protein secretion.
  • GNAS activity may be conveniently decreased by antisense inhibition of (portions of) the GNAS gene, while GNAS activity may be conveniently increased by introduction of (portions of) the GNAS gene into the cell or by exposure to NESP55.
  • the GRB10 gene (SEQ ID NO. 17, PB13), encoding growth factor receptor- bound protein 10, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on GRB 10 expression was found (Table 2).
  • GRB 10 is a member of the GRB7 family of adapter proteins lacking intrinsic enzymatic function and encodes functional domains including a pleckstrin homology (PH) domain and an SH2 domain.
  • GRB 10 interacts with tyrosine-phosphorylated receptors including the insulin receptor kinase (IRK), insulin-like growth factor 1 receptor (IGF1R), platelet-derived growth factor receptor beta (PDGFRB), epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) receptor KDR.
  • IGF1R insulin receptor kinase
  • PDGFRB platelet-derived growth factor receptor beta
  • EGFR epidermal growth factor receptor
  • VEGF vascular endothelial growth factor
  • GRB 10 was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality.
  • any agent which changes the biological activity of the GRB 10 gene product (growth factor receptor-bound protein 10) is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention.
  • GRB 10 activity may be conveniently decreased by antisense inhibition of the GRB 10 gene, while GRB 10 activity may be conveniently increased by introduction of the GRB 10 gene into the cell or by exposure to growth factor receptor- bound protein 10. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • the HRI gene (SEQ ID NO. 19, PB15), encoding heme-regulated initiation factor 2-alpha kinase, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on HRI expression was found (Table 2). Studies in rat and rabbit suggest that the HRI gene product phosphorylates the alpha subunit of eukaryotic initiation factor 2. Its kinase activity is induced by low levels of heme and inhibited by the presence of heme.
  • HRI gene activity may be conveniently decreased by antisense inhibition of the HRI gene, while HRI activity may be conveniently increased by introduction of the HRI gene into the endothelial cell, or by exposure to the gene product heme- regulated initiation factor 2-alpha kinase, in order to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • the MAGOH gene (SEQ ID NO.
  • PB 17 encoding the proliferation- associated mago-nashi homolog
  • BCEC BCEC by physical co-culture with astrocytes
  • PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on MAGOH expression was found (Table 2).
  • Drosopbila that have mutations in their mago nashi (grandchildless) gene produce progeny with defects in germplasm assembly and germline development. This gene encodes the mammalian mago nashi homolog. In mammals, mRNA expression is not limited to the germ plasm, but is expressed ubiquitously in adult tissues and can be induced by serum stimulation of quiescent fibroblasts.
  • MAGOH gene activity may be conveniently decreased by antisense inhibition of the MAGOH gene.
  • MAGOH gene activity may be conveniently increased by introduction of the MAGOH gene, or the gene product mago-nashi homolog, into the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • CALM1 gene (SEQ ID NO. 23, PB18), encoding calmodulin 1, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on CALM1 expression was found (Table 2).
  • Calmodulin is the archetype of the family of calcium-modulated proteins of which nearly 20 members have been found. They are identified by their occurrence in the cytosol or on membranes facing the cytosol and by a high affinity for calcium. Calmodulin contains 149 amino acids and has 4 calcium-binding domains. Its functions include roles in growth and the cell cycle as well as in signal transduction and the synthesis and release of neurotransmitters.
  • CALM1 gene or calmodulin 1
  • astrocytes and not by LPS in the cells that constitute the blood-brain barrier
  • CALMl gene activity may be conveniently decreased by antisense inhibition of the CALMl gene or by agents that modulate the biological activity of CALMl, such as described by Hamano et al.
  • CALMl gene activity may be conveniently increased by introduction of the CALMl gene, or the gene product calmodulin 1, into the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • the RBPl gene (SEQ ID NO.
  • PB21 encoding cellular retinol binding protein 1
  • BCEC BCEC by physical co-culture with astrocytes
  • PB polypeptides are involved in decreased vascular permeability.
  • RBPl is the intracellular carrier involved in intracellular movement of retinol (vitamin A alcohol).
  • Vitamin A is a fat-soluble vitamin necessary for growth, reproduction, differentiation of epithelial tissues, and vision.
  • RBPl gene activity may be conveniently decreased by antisense inhibition of the RBPl gene.
  • RBPl gene activity may be conveniently increased by introduction of the RBPl gene into, or exposure of the gene product cellular retinol binding protein 1 to, the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • Ghyselinck et al. (1999, EMBO J., 18(18):4903-4914) developed a RBPl knockout mouse, which may be useful to specifically investigate the functionality of the blood-brain barrier in the embodiments of the present invention.
  • the TRIP6 gene (SEQ ID NO. 32, PB25), encoding thyroid hormone receptor interactor 6, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on LOXLl expression was found (Table 2).
  • the thyroid hormone receptors (TRs) are hormone-dependent transcription factors that regulate expression of a variety of specific target genes. A number of different TR interacting proteins, or TRIPs were recovered. One of the proteins, TRIP6, contains 2 LIM domains and is similar to zyxin. TRIP6 interacts with TR-beta only in the presence of thyroid hormone.
  • TRIP6 The zyxin family of proteins function in integrin- mediated signaling at focal adhesions and along actin filaments, where they are involved in cell motility: overexpression of TRIP6 slows cell migration (Yi et al., 2002, J Biol Chem., 277(11): 9580-9589).
  • TRIP6 gene, or thyroid hormone receptor interactor 6 was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality. TRIP6 gene activity may be conveniently decreased by antisense inhibition of the TRIP6 gene.
  • TRIP6 gene activity may be conveniently increased by introduction of the TRIP6 gene into, or exposure of the gene product thyroid hormone receptor interactor 6 to, the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. Yi et al. (2002, supra) developed an expression vector and polyclonal antisera against TRIP6, which may be useful to specifically investigate the blood-brain barrier in the embodiments of the present invention.
  • the AP4S 1 gene (SEQ ID NO.
  • PB27 encoding the sigma 1 subunit of adaptor-related protein complex 4
  • BCEC BCEC by physical co-culture with astrocytes (Table 1).
  • Upregulated PB polypeptides are involved in decreased vascular permeability.
  • AP4S1 is upregulated by LPS in BCEC monolayers and downregulated by LPS in BCEC-astrocyte cocultures (Table 2).
  • AP-4 complex is expressed, in low abundance, in all tissues and cells examined. It is largely associated with the tr ⁇ r ⁇ -Golgi network. Unlike the AP-1 and 2 complex, AP-4 is most probably part of a non-clathrin coat.
  • the surprising finding that the AP4S1 gene, or caspase 1, was upregulated by astrocytes and differentially modified by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality.
  • AP4S1 gene or biological activity may be conveniently decreased by antisense inhibition of the AP4S1 gene or by exposure to brefeldin A (Dell'Angelica et al, 1999, J Biol Chem., 274(11): 7278-7285).
  • AP4S1 gene or biological activity may be conveniently increased by introduction of the AP4S1 gene into the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • Dell'Angelica et al. (1999, supra) described antibodies directed against AP-4 complex which may be useful to specifically investigate the blood-brain barrier in the embodiments of the present invention.
  • the FTL gene (SEQ ID NO. 44, PB30), encoding the light polypeptide ferritin, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on FTL expression was found (Table 2). Ferritin is the major intracellular iron storage protein in all organisms. It has the shape of a hollow sphere that permits entry of a variable amount of iron for storage as ferric hydroxide phosphate complexes.
  • the surprising finding that the FTL gene, or light polypeptide ferritin, was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opporftinities to modify or monitor blood-brain barrier functionality. FTL gene activity may be conveniently decreased by antisense inhibition of the FTL gene.
  • FTL gene activity may be conveniently increased by introduction of the FTL gene into, or exposure of the gene product light polypeptide ferritin to the endothelial cell, to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • the CCM1 gene (SEQ ID NO. 53, PB35), encoding cerebral cavernous malformations 1 (or Kritl), is downregulated in BCEC by physical co-culture with astrocytes (Table 1). Downregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on CCM1 expression was found (Table 2).
  • Cerebral cavernous malformations are found in 0.1% to 0.5% of the population and represent approximately 10% to 20% of cerebral vascular lesions. These vascular malformations are characterised by abnormally enlarged capillary cavities without intervening brain parenchyma. They occur as single or multiple malformations that lead to focal neurologic signs, hemorrhagic strokes, or seizures. The vascular malformations can arise sporadically or may be dominantly inherited.
  • the Krev Interaction Trapped 1 (Kritl) protein contains three ankyrin repeats and a FERM domain.
  • the ankyrin repeats are thought to interact with Krev- 1 /rap 1 a, a member of the Ras family of GTPases, whereas the FERM domain of the erzin/radixin moesin protein family is thought to regulate cytoskeletal/plasma membrane interactions.
  • CCM1 gene or cerebral cavernous malformations 1, was downregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality.
  • CCM1 gene or biological activity may be conveniently decreased by antisense inhibition of the CCM1 gene.
  • CCM1 gene or biological activity may be conveniently increased by introduction of the CCM1 gene into the endothelial cell, or by exposure to extracellular Kritl protein, to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • Polypeptides functioning as membrane (signaling) receptors or (signaling) adhesion molecules are ideal candidates to specifically (i.e., astrocyte-induced) modulate the permeability of the blood-brain barrier in the embodiments of the present invention.
  • astrocyte-induced polypeptides including E-selectin, stromal cell derived factor receptor 1, apolipoprotein E receptor 2 and G protein-coupled receptor 48. These are discussed in greater detail below.
  • SELE SEQ ID NO. 2, PB02
  • selectin E E-selectin
  • endothelial adhesion molecule 1 encoding selectin E (E-selectin), or endothelial adhesion molecule 1
  • PB02 Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on SELE expression was found (Table 2).
  • ELAM-1 endothelial leukocyte adhesion molecule- 1 (ELAM-1) is expressed by cytokine-stimulated endothelial cells. It is thought to be responsible for the accumulation of blood leukocytes at sites of inflammation by mediating the adhesion of cells to the vascular lining.
  • Adhesion molecules participate in the interaction between leukocytes and the endothelium and appear to be involved in the pathogenesis of atherosclerosis.
  • E-selectin Upregulated E-selectin was successfully used to target drugs into (inflammation) activated peripheral endothelial cells (Jutila et al., 2002, J Immunol., 169: 1768-1773; Everts et al., 2002, J immunol., 168: 883-889).
  • the surprising finding that E-selectin was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to target drugs to the blood-brain barrier.
  • any ligand that specifically binds to E-selectin is useful to target drugs to the blood-brain barrier in the embodiments of the present invention.
  • E-selectin may be conveniently targeted by e.g., P-selectin glycoprotein ligand 1 (PSGL-1), the HECA-452 antibody, the porcine E- selectin/Ig chimera PI 1.4, L-selectin (CD62L), the Ab hEse i (mouse anti-human E- selectin) monoclonal antibody produced by the HI 8/7 mAb-producing hybridoma or the Ab mEse i (rat anti-mouse E-selectin) monoclonal antibody produced by the 10E9 mAb-producing hybridoma (Jutila et al., 2002, supra; Everts et al., 2002, supra). Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • PSGL-1 P-selectin glycoprotein ligand 1
  • the HECA-452 antibody the porcine E- selectin/Ig chimera PI
  • SDFR1 The SDFR1 gene (SEQ ID NO.'s 11-12, PB11), encoding stromal cell derived factor receptor 1, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on SDFR1 expression was found (Table 2). SDFR1 is a type I transmembrane protein belonging to the lg superfamily. The protein is involved in cell-cell interactions or cell-substrate interactions. The alpha and beta transcripts show differential localisation within the brain.
  • the SDFR1 gene is, surprisingly, completely unrelated to the G-protein coupled chemokine receptor, CXCR4, for which stromal cell-derived factor 1 (CXCL12) is the natural ligand.
  • CXCL12 stromal cell-derived factor 1
  • the surprising finding that SDFR1 was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality, as well as to target drugs to the blood- brain barrier. For the latter, any ligand that specifically binds to SDFR1 is useful to target drugs to the blood-brain barrier in the embodiments of the present invention.
  • any agent which changes the biological activity of the SDFR1 gene product is useful to specifically modulate the permeability of the blood-brain barrier in the embodiments of the present invention.
  • SDFR1 activity may be conveniently decreased by antisense inhibition of the SDFR1 gene, while SDFR1 activity may be conveniently increased by introduction of the SDFR1 gene into the cell or, as exemplified in example 5, by exposure to stromal cell- derived factor 1 (CXCL12) or any other SDFR1 agonist. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • the LRP8 gene (SEQ ID NO.'s 28-30, PB23), encoding low-density lipoprotein receptor-related protein 8 (or apolipoprotein E receptor 2, or ApoER2), is downregulated in BCEC by physical co-culture with astrocytes (Table 1). Downregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on LRP8 expression was found (Table 2). This gene encodes an apolipoprotein E receptor, a member of the low-density lipoprotein receptor (LDLR) family. Gene polymorphisms in ApoER2 are associated with Alzheimer's disease (Ma et al., 2002, Neurosci Lett., 332(3): 216-218).
  • Apolipoprotein E is a small lipophilic plasma protein and a component of lipoproteins such as chylomicron remnants, very low-density lipoprotein (VLDL), and high-density lipoprotein (HDL).
  • the apolipoprotein E receptor is involved in cellular recognition and internalisation of these lipoproteins.
  • ApoER2 is also a signaling receptor (located in caveola) which is stimulated by Reelin, an extracellular matrix protein secreted by neurons, which results in the phosphorilation of a cytosolic protein that activates a tyrosine kinase called disabled (or DAB1) and in the suppression of tau phosphorylation, a microtubule-stabilising protein.
  • the secreted soluble form of ApoER2 acts as an ApoER2 antagonist and inhibits Reelin signaling.
  • LRP8 gene or apolipoprotein E receptor 2
  • LRP8 gene or biological activity may be conveniently decreased by antisense inhibition of the LRP8 gene, or by exposure to the secreted soluble form of ApoER2.
  • LRP8 gene or biological activity may be conveniently increased by introduction of the LRP8 gene into the endothelial cell, or by exposure to extracellular Reelin, to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention or to target drugs (bound to ApoER2 ligands) to the blood-brain barrier. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention. Riddell et al. (1999, J Lipid Res., 40(10): 1925-1930), developed anti-peptide antisera against LRP8, which may be useful to specifically investigate the blood-brain barrier in the embodiments of the present invention.
  • mice deficient in Reelin, DABl and ApoER2 have been described, which may be useful to specifically investigate the blood-brain barrier in the embodiments of the present invention.
  • the GPR48 gene (SEQ ID NO. 46, PB32), encoding the G protein-coupled receptor 48, is downregulated in BCEC by physical co-culture with astrocytes (Table 1). Downregulated PB polypeptides are involved in decreased vascular permeability. In addition, GPR48 is downregulated by LPS in BCEC monolayers, presumably as a positive feedback mechanism to this stimulus (Table 2). Glycoprotein hormone receptor-like G protein-coupled receptor. It is highly expressed in the adult human pancreas, and moderately expressed in placenta, kidney, brain, and heart. Additionally, this receptor is expressed as early as 7 days post coitus in the mouse, indicating its potential involvement in development.
  • GPR48 gene or G protein-coupled receptor 48
  • GPR48 gene or biological activity may be conveniently decreased by antisense inhibition of the GPR48 gene, or by exposure to any GPR48 antagonist.
  • GPR48 gene or biological activity may be conveniently increased by introduction of the GPR48 gene into the endothelial cell, or by exposure to any GPR48 agonist, to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • Polypeptides functioning as metabolic enzymes are ideal candidates to specifically (i.e., astrocyte-induced) modulate or monitor the permeability of the blood- brain barrier in the embodiments of the present invention.
  • the GLUDl gene (SEQ ID NO. 4, PB04), encoding for L-glutamate dehydrogenase 1 (EC 1.4.1.3), is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on GLUDl expression was found (Table 2). GLUDl has a central role in nitrogen metabolism in plants and animals. Glutamate dehydrogenase is found in all organisms and catalyses the oxidative deamination of l-glutamate to 2-oxoglutarate. Glutamate, the main substrate of GLUD, is present in brain in concentrations higher than in other organs.
  • GLUD appears to function in both the synthesis and the catabolism of glutamate and perhaps in ammonia detoxification.
  • the surprising finding that the GLUDl gene, or L- glutamate dehydrogenase, was upregulated by astrocytes and not by LPS in the cells that constitute the blood-brain barrier was not reported earlier and offers new opportunities to modify or monitor blood-brain barrier functionality.
  • GLUDl gene activity may be conveniently increased by introduction of the GLUDl gene, or the gene product L-glutamate dehydrogenase, into the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention.
  • the UBE2G1 gene (SEQ ID NO. 18, PB14), encoding ubiquitin-conjugating enzyme E2G 1, is downregulated in BCEC by physical co-culture with astrocytes (Table 1). Downregulated PB polypeptides are involved in decreased vascular permeability. In addition, UBE2G1 is downregulated by LPS in BCEC monolayers, presumably as a positive feedback mechanism to this stimulus (Table 2).
  • UBE2G1 catalyses ubiquitination of cellular proteins and marks them for degradation. Protein modification via covalent attachment of ubiquitin has emerged as one of the most common regulatory processes in all eukaryotes.
  • UBE2G1 gene activity may be conveniently decreased by antisense inhibition of the UBE2G1 gene, while UBE2G1 activity may be conveniently increased by introduction of the UBE2G1 gene into the endothelial cell, or by exposure to the gene product ubiquitin-conjugating enzyme E2G 1, in order to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • the ASNS gene (SEQ ID NO.'s 20-21, PB16), encoding for asparagine synthetase, is upregulated in BCEC by physical co-culture with astrocytes (Table 1). Upregulated PB polypeptides are involved in decreased vascular permeability. No effect of two hours LPS exposure on ASNS expression was found (Table 2). The protein encoded by this gene is involved in the synthesis of asparagine. ASNS catalyses the glutamine- and ATP-dependent conversion of aspartic acid to asparagine. ASNS appears to function in both the synthesis and the catabolism of aspartate and perhaps in ammonia detoxification.
  • ASNS gene activity may be conveniently decreased by antisense inhibition of the ASNS gene.
  • ASNS gene activity may be conveniently increased by introduction of the ASNS gene, or the gene product asparagine synthetase, into the endothelial cell to specifically modulate the functionality of the blood-brain barrier in the embodiments of the present invention. Changes in expression of this gene may be used for diagnostic purposes of vascular permeability status in the embodiments of the present invention.
  • Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to deteimine identity and similarity between two sequences include e.g.
  • BLAST Manual Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990).
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4.
  • a program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI.
  • the aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).
  • amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.
  • Recombinant techniques and methods for recombinant production of polypeptides are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He
  • Polypeptides for use in the present invention can be prepared using recombinant techniques, in which a nucleotide sequence encoding the polypeptide of interest is expressed in suitable host cells.
  • the present invention thus also concerns the use of a vector comprising a nucleic acid molecule or nucleotide sequence as defined above.
  • the vector is a replicative vector comprising on origin of replication (or autonomously replication sequence) that ensures multiplication of the vector in a suitable host for the vector.
  • the vector is capable of integrating into the host cell's genome, e.g. through homologous recombination or otherwise.
  • a particularly preferred vector is an expression vector wherein a nucleotide sequence encoding a polypeptide as defined above, is operably linked to a promoter capable of directing expression of the coding sequence in a host cell for the vector.
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a “constitutive” promoter is a promoter that is active under most physiological and developmental conditions.
  • an “inducible” promoter is a promoter that is regulated depending on physiological or developmental conditions.
  • a “tissue specific” promoter is only active in specific types of differentiated cells/tissues.
  • Expression vectors allow the PB polypeptides as defined above to be prepared using recombinant techniques in which a nucleotide sequence encoding the PB polypeptide of interest is expressed in suitable cells, e.g. cultured cells or cells of a multicellular organism, such as described in Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley-Interscience, New York (1987) and in Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entirety. Also see, Kunkel (1985) Proc.
  • nucleic acids encoding the desired polypeptides are used in expression vectors.
  • expression vector generally refers to nucleotide sequences that are capable of effecting expression of a gene in hosts compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression can also be used as described herein.
  • DNA constructs prepared for introduction into a particular host typically include a replication system recognised by the host, the intended DNA segment encoding the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment.
  • a DNA segment is "operably linked" when it is placed into a functional relationship with another DNA segment.
  • a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence.
  • DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide.
  • DNA sequences that are operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading phase.
  • enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.
  • the selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g. Sambrook and Russell, 2001, supra).
  • the transcriptional regulatory sequences typically include a heterologous enhancer or promoter that is recognised by the host.
  • promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Russell, 2001, supra).
  • Expression vectors include the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment can be employed. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and in Metzger et al. (1988) Nature 334: 31-36.
  • suitable expression vectors can be expressed in, yeast, e.g.
  • S.cerevisiae e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli.
  • the host cells may thus be prokaryotic or eukarotic host cells.
  • the host cell may be a host cell that is suitable for culture in liquid or on solid media.
  • the host cells are used in a method for producing a PB polypeptide as defined above. The method comprises the step of culturing a host cell under conditions conducive to the expression of the polypeptide.
  • the method may comprise recovery the polypeptide.
  • the polypeptide may e.g. be recovered from the culture medium by standard protein purification techniques, including a variety of chromatography methods known in the art per se.
  • the host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal, preferably a non-human animal.
  • a fransgenic plant comprises in at least a part of its cells a vector as defined above.
  • Methods for generating transgenic plants are e.g. described in U.S. 6,359,196 and in the references cited therein.
  • Such transgenic plants may be used in a method for producing a PB polypeptide as defined above, the method comprising the step of recovering a part of a transgenic plant comprising in its cells the vector or a part of a descendant of such transgenic plant, whereby the plant part contains the polypeptide, and, optionally recovery of the polypeptide from the plant part.
  • the transgenic animal comprises in its somatic and germ cells a vector as defined above.
  • the transgenic animal preferably is a non-human animal.
  • Methods for generating transgenic animals are e.g. described in WO 01/57079 and in the references cited therein.
  • Such transgenic animals may be used in a method for producing a PB polypeptide as defined above, the method comprising the step of recovering a body fluid from a transgenic animal comprising the vector or a female descendant thereof, wherein the body fluid contains the polypeptide, and, optionally recovery of the polypeptide from the body fluid.
  • the body fluid containing the polypeptide preferably is blood or more preferably milk.
  • polypeptides synthesised in vitro translation typically do not contain the post- translation modifications present on polypeptides synthesised in vivo, although due to the inherent presence of microsomes some post-translational modification may occur.
  • Methods for synthesis of polypeptides by in vitro translation are described by, for example, Berger & Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, CA, 1987.
  • Some aspects of the invention concern the use of expression vectors comprising the nucleotide sequences as defined above, wherein the vector is a vector that is suitable for gene therapy.
  • Vectors that are suitable for gene therapy are described in Anderson 1998, Nature 392: 25-30; Walther and Stein, 2000, Drugs 60: 249-71; Kay et al., 2001, Nat. Med. 7: 33-40; Russell, 2000, J. Gen. Virol. 81: 2573-604; Amado and Chen, 1999, Science 285: 674-6; Federico, 1999, Curr. Opin. Biotechnol.lO: 448-53; Vigna andNaldini, 2000, J. Gene Med. 2: 308-16; Marin et al., 1997, Mol. Med.
  • Particularly suitable gene therapy vectors include Adenoviral and Adeno- associated virus (AAV) vectors. These vectors infect a wide number of dividing and non-dividing cell types.
  • Adenoviral vectors are capable of high levels of transgene expression.
  • these viral vectors are most suited for therapeutic applications requiring only transient expression of the transgene (Russell, 2000, J. Gen. Virol. 81: 2573-2604) as indicated above.
  • Preferred adenoviral vectors are modified to reduce the host response as reviewed by Russell (2000, supra).
  • gene therapy vectors will be as the expression vectors described above in the sense that they comprise the nucleotide sequence encoding the PB polypeptide to be expressed, whereby the nucleotide sequence is operably linked to the appropriate regulatory sequences as indicated above.
  • Such regulatory sequence will at least comprise a promoter sequence.
  • Suitable promoters for expression of the nucleotide sequence encoding the polypeptide from gene therapy vectors include e.g.
  • CMV cytomegalovirus
  • LTRs viral long terminal repeat promoters
  • MMLV murine moloney leukaemia virus
  • HTLV-1 hematoma virus
  • SV 40 herpes simplex virus thymidine kinase promoter
  • inducible promoter systems have been described that may be induced by the administration of small organic or inorganic compounds.
  • Such inducible promoters include those controlled by heavy metals, such as the metallothionine promoter (Brinster et al. 1982 Nature 296: 39-42; Mayo et al. 1982 Cell 29: 99-108), RU-486 (a progesterone antagonist) (Wang et al. 1994 Proc. Natl. Acad. Sci. USA £1: 8180-8184), steroids (Mader and White, 1993 Proc. Natl. Acad. Sci. USA 90: 5603-5607), tetracycline (Gossen and Bujard 1992 Proc. Natl. Acad. Sci.
  • tTAER system that is based on the multi-chimeric transactivator composed of a tetR polypeptide, as activation domain of VP16, and a ligand binding domain of an estrogen receptor (Yee et al., 2002, US 6,432,705).
  • the gene therapy vector may optionally comprise a second or one or more further nucleotide sequence coding for a second or further protein.
  • the second or further protein may be a (selectable) marker protein that allows for the identification, selection and/or screening for cells containing the expression construct. Suitable marker proteins for this purpose are e.g.
  • the fluorescent protein GFP and the selectable marker genes HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection on G418), and dihydrofolate reductase (DHFR) (for selection on methotrexate), CD20, the low affinity nerve growth factor gene.
  • HSV thymidine kinase for selection on HAT medium
  • bacterial hygromycin B phosphotransferase for selection on hygromycin B
  • Tn5 aminoglycoside phosphotransferase for selection on G418)
  • DHFR dihydrofolate reductase
  • the second or further nucleotide sequence may encode a protein that provides for fail-safe mechanism that allows to cure a subject from the transgenic cells, if deemed necessary.
  • a nucleotide sequence often referred to as a suicide gene, encodes a protein that is capable of converting a prodrug into a toxic substance that is capable of killing the transgenic cells in which the protein is expressed.
  • Suitable examples of such suicide genes include e.g.
  • the gene therapy vectors are preferably formulated in a pharmaceutical composition comprising a suitable pharmaceutical carrier as defined below.
  • Some aspects of the invention concern the use of an antibody or antibody- fragment that specifically binds to a PB polypeptide of the invention as defined above.
  • Methods for generating antibodies or antibody-fragments that specifically bind to a given polypeptide are described in e.g. Harlow and Lane (1988, Antibodies: A
  • binding includes both low and high affinity specific binding. Specific binding can be exhibited, e.g., by a low affinity antibody or antibody-fragment having a Kd of at least about 10 "4 M.
  • Specific binding also can be exhibited by a high affinity antibody or antibody- fragment, for example, an antibody or antibody-fragment having a Kd of at least about of 10 "7 M, at least about 10 "8 M, at least about 10 "9 M, at least about 10 "10 M, or can
  • 1 1 1 have a Kd of at least about 10 " M or 10 " M or greater.
  • Peptide-like molecules referred to as peptidomimetics
  • non-peptide molecules that specifically bind to a PB polypeptide or a PB polypeptide receptor and that may be applied in any of the methods of the invention as defined herein may be identified using methods known in the art per se, as e.g. described in detail in US 6,180,084 which incorporated herein by reference. Such methods include e.g. screening libraries of peptidomimetics, peptides, DNA or cDNA expression libraries, combinatorial chemistry and, particularly useful, phage display libraries. These libraries may be screened for agonists and antagonsist of PB polypeptides or receptors thereof by contacting the libraries with substantially purified PB polypeptides, PB polypeptide receptors, fragments thereof or structural analogues thereof.
  • the invention further relates to a pharmaceutical preparation comprising as active ingredient a PB polypeptide, an antibody or a gene therapy vector as defined above.
  • the composition preferably at least comprises a pharmaceutically acceptable carrier in addition to the active ingredient.
  • the polypeptide or antibody of the invention as purified from mammalian, insect or microbial cell cultures, from milk of transgenic mammals or other source is administered in purified form together with a pharmaceutical carrier as a pharmaceutical composition.
  • a pharmaceutical carrier as a pharmaceutical composition.
  • the pharmaceutical carrier can be any compatible, non-toxic substance suitable to deliver the polypeptides, antibodies or gene therapy vectors to the patient.
  • Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier.
  • compositions may also be incorporated into the pharmaceutical compositions.
  • the concentration of the PB polypeptides or antibodies of the invention in the pharmaceutical composition can vary widely, i.e., from less than about 0.1% by weight, usually being at least about 1% by weight to as much as 20% by weight or more.
  • the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • Active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.
  • inactive ingredients examples include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like.
  • Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration can contain colouring and flavouring to increase patient acceptance.
  • the PB polypeptides, antibodies or gene therapy vectors are preferably administered parentally.
  • the polypeptide, antibody or vector for preparations for parental administration must be sterile. Sterilisation is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilisation and reconstitution.
  • the parental route for administration of the PB polypeptide, antibody or vector is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial or infralesional routes.
  • the polypeptide, antibody or vector is administered continuously by infusion or by bolus injection.
  • a typical composition for intravenous infusion could be made up to contain 10 to 50 ml of sterile 0.9% NaCl or 5% glucose optionally supplemented with a 20% albumin solution and 1 to 50 ⁇ g of the PB polypeptide, antibody or vector.
  • a typical pharmaceutical composition for intramuscular injection would be made up to contain, for example, 1 - 10 ml of sterile buffered water and 1 to 100 ⁇ g of the PB polypeptide, antibody or vector of the invention.
  • Methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Science (15th ed., Mack Publishing, Easton, PA, 1980) (incorporated by reference in its entirety for all purposes).
  • the pharmaceutical compositions are administered to a patient suffering from a microvascular permeability disorder in an amount sufficient to reduce the severity of symptoms and/or prevent or arrest further development of symptoms.
  • An amount adequate to accomplish this is defined as a "therapeutically-" or “prophylactically-effective dose”.
  • Such effective dosages will depend on the severity of the condition and on the general state of the patient's health.
  • a therapeutically- or prophylactically-effective dose preferably is a dose, which restores the microvascular permeability to the average levels found in normal unaffected healthy individuals.
  • the PB polypeptide or antibody is usually administered at a dosage of about 1 ⁇ g/kg patient body weight or more per week to a patient. Often dosages are greater than 10 ⁇ g/kg per week. Dosage regimes can range from 10 ⁇ g/kg per week to at least 1 mg/kg per week. Typically dosage regimes are 10 ⁇ g/kg per week, 20 ⁇ g/kg per week, 30 ⁇ g/kg per week, 40 ⁇ g/kg week, 60 ⁇ g/kg week, 80 ⁇ g/kg per week and 120 ⁇ g/kg per week. In preferred regimes 10 ⁇ g/kg, 20 ⁇ g/kg or 40 ⁇ g/kg is adniinistered once, twice or three times weekly. Treatment is preferably administered by parenteral route.
  • microarrays or other high throughput screening devices
  • a microarray is a solid support or carrier containing one or more immobilised nucleic acid or polypeptide fragments for analysing nucleic acid or amino acid sequences or mixtures thereof (see e.g. WO 97/27317, WO 97/22720, WO 97/43450, EP 0 799 897, EP 0 785 280, WO 97/31256, WO 97/27317, WO 98/08083 and Zhu and Snyder, 2001, Curr. Opin. Chem. Biol. 5: 40-45).
  • Microarrays comprising the nucleic acids may be applied e.g. in methods for analysing genotypes or expression patterns as indicated above.
  • Microarrays comprising polypeptides may be used for detection of suitable candidates of substrates, ligands or other molecules interacting with the polypeptides.
  • Microarrays comprising antibodies may be used for in methods for analysing expression patterns of the polypeptides as indicated above.
  • Figure 1 is a schematically detailed representation of a filter insert with BCEC- ACM monolayers (panel a) and with BCEC-ASTROCYTES cocultures (panel b).
  • Figure 2 is a diagram showing the astrocyte-induced increase in TEER across BCEC cell layers, expressed in Ohm . cm 2 (mean +/- standard error, panel a) and as % of BCEC-ACM monolayers (mean +/- standard error, panel b).
  • FIG. 3 is a schematically detailed representation of the event that occurs at the BBB in vitro after exposure to LPS.
  • BCEC were cultured as monolayers in 50 % ACM or co- cultured with astrocytes.
  • Astrocytes increased in vitro BBB performance (phase 1).
  • Disease-induction by lipopolysaccharide (LPS) disrupted BCEC monolayers (phase 2), while BCEC+ astrocyte co-cultures were able to recover (phase 3).
  • LPS lipopolysaccharide
  • phase 2 disrupted BCEC monolayers
  • BCEC+ astrocyte co-cultures were able to recover
  • This recovery process involves de novo protein synthesis, since cycloheximide (CHX) was able to completely inhibit the recovery phase.
  • CHX cycloheximide
  • Figure 4 is a diagram showing the effect on TEER across BCEC-ACM monolayers of 2 hours exposure to LPS, expressed in Ohm . cm (mean +/- standard error, panel a) and as % of control (i.e., untreated BCEC-ACM monolayers, mean +/- standard error, panel b).
  • Figure 5 is a diagram showing the effect on TEER across BCEC-ASTROCYTES cocultures of 2 hours exposure to LPS, expressed in Ohm . cm 2 (mean +/- standard error, panel a) and as % of control (i.e., untreated BCEC-ASTROCYTES cocultures, mean +/- standard error, panel b).
  • the DMEM formulated with high D-glucose (4.5 g/1), NaHCO 3 (3.7 g/1) and HEPES (25 mM), contained extra MEM non-essential amino acids, L-glutamine (2 mM), streptomycin sulfate (0.1 g/1) and penicillin G sodium (100000 U/l).
  • Blood vessel fragments were prepared by manual homogenisation using a Wheaton homogeniser and subsequently trapped on 150 ⁇ m nylon meshes.
  • the blood vessels were digested in coUagenase CLS3 (210 U/ml), trypsin TRL (91 U/ml) and DNAse I (170 U/ml, final concentrations) in DMEM+S for 1 hour at 37°C and subsequently filtered through a 200 ⁇ m nylon mesh.
  • the brain capillary fraction was resuspended in freeze mix (fetal calf serum (FCS) with 10% (v/v) DMSO) and stored at -80°C.
  • FCS fetal calf serum
  • Astrocytes were isolated from newborn Wistar rat pups (Harlan BN., Zeist, The Netherlands). Isolated cortices were fragmented and incubated with 0.016% (w/v) trypsin-EDTA (final concentration) in DMEM (fully HEPES buffered (50 mM), without NaHCO 3 ) in a shaking waterbath (80 rpm, 30 minutes) at 37°C. The suspension was filtered through a 120 and 45 ⁇ m nylon mesh, respectively.
  • the cell- suspension was cultured for 3 days in DMEM+S in 250 ml plastic tissue culture flasks (Greiner BN., Alphen a/d Rijn, The Netherlands) in a humidified incubator (Napco Scientific Company, Tualatin, OR, USA) at 37°C in a mixture of air with 10% CO 2 . Thereafter, the medium was refreshed every other day. After 7 days of culturing, other cells than astrocytes were removed by shaking the cultures in a shaking waterbath (80 rpm) overnight at room temperature.
  • Brain capillaries were seeded in collagen (human placenta type IV, 10 ⁇ g/ml solution in 0.1% (v/v) acetic acid for 2 hours and washed with PBS 3 times) and human plasma fibronectin (10 ⁇ g/ml solution in PBS, 30 minutes) coated 250 ml plastic tissue culture flasks and allowed to adhere for 4 hours in the incubator. Thereafter, the culture medium was replaced with growth medium (DMEM+S with 50% (v/v) astrocyte conditioned medium, supplemented with 125 ⁇ g/ml heparin) and the outgrowing cells, predominately BCEC and some pericytes, were cultured at 37°C, 10% CO 2 .
  • collagen human placenta type IV, 10 ⁇ g/ml solution in 0.1% (v/v) acetic acid for 2 hours and washed with PBS 3 times
  • human plasma fibronectin (10 ⁇ g/ml solution in PBS, 30 minutes) coated 250 ml plastic tissue culture flasks and
  • the in vitro BBB model was prepared on collagen coated (as above) Transwell polycarbonate filters (surface area: 0.33 cm 2 , pore-size: 0.4 ⁇ m, Corning Costar, Cambridge, MA, USA).
  • BCEC were passaged with trypsin-EDTA for endothelial cells (500 BAEE units porcine trypsin and 180 ⁇ g EDTA per ml) for approximately 1 minute, leaving the majority of pericytes still adhered to the substratum.
  • BCEC and astrocyte co- cultures were prepared with astrocytes seeded on the bottom of the filter at a density of 45000 astrocytes per filter.
  • BCEC Astrocytes were allowed to adhere to the bottom of the filter for 8 minutes, 2 or 3 days before BCEC were passaged. BCEC were seeded at a density of 30000 BCEC per filter. BCEC+astrocyte co-cultures were cultured to tight monolayers in DMEM+S supplemented with 125 ⁇ g/ml heparin for the first 2 days and in DMEM+S for the last 2 days. BCEC monolayers were cultured accordingly, but with 50%) (v/v) astrocyte conditioned medium added to the culture medium.
  • coli DNA polymerase I (Life Technologies, Rockville, MD, USA).
  • the double-stranded cDNA was then purified using phenol/chloroform extraction (utilising phase lock gels (Eppendorf AG, Hamburg, Germany)) followed by precipitation with ammonium acetate and ethanol.
  • Biotinylated cRNA was synthesised by in vitro transcription from cDNA using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY, USA), by incubation at 37 °C for 5 hours.
  • the labeled cRNA was then purified using the RNA cleanup protocol of the RNeasy mini kit (Qiagen, Hilden, Germany).
  • fragmentation buffer 40 mM Tris- acetate (pH 8.1), 125 mM KOAc, 30 mM MgOAc.
  • U133A array (Affymetrix, Santa Clara, CA, USA) under conditions recommended by the manufacturer.
  • cRNA was first hybridised to a Test2Chip (Affymetrix), to ensure the quality of the preparation.
  • hybridisation mix (lx MES hybridisation buffer, 100 ⁇ g/ml herring sperm, 50 ⁇ g/ml acetylated BSA, control oligonucleotide B2 and eukaryotic hybridisation controls) denatured and then hybridised for 16 hours at 45 °C at 60 rpm.
  • hybridisation were washed and stained with streptavidin-phycoerythrin using the Affymetrix Genechip® Fluidics Station 400. Fluorescent signals on the arrays were measured using the Hewlett-Packard Affymetrix GeneArray® scanner.
  • TEER Ohm . cm 2
  • TEER Ohm . cm 2
  • RNA isolation labeling of cRNA and hybridisation protocol was performed in triplo and all samples were analysed on both the HG- U95Av2 and HG-U133A arrays.
  • Affymetrix Microarray Suite 5.0 and Affymetrix Data Mining Tool 2.0 were used for primary analysis of the acquired intensity data.
  • Microsoft Excel Microsoft, USA
  • Global scaling where the data of each chip is scaled to a user-defined target intensity, was performed to make experiments comparable. Genes that were designated as "absent" by Affymetrix Microarray Suite 5.0 in all samples were eliminated from further analysis.
  • the identified genes are designated herein as "Pro-Barrier” genes, and coded accordingly (PB01-PB35) and presented in Table 1. Included are the fold change values, the gene symbol, a title name for the gene, and the accession codes for reference in publicly accessible databases (Unigene, OMIM, LocusLink and RefSeq). The general function of each PB gene is detailed in the description herein above. To date, there are no publications linking these PB genes to astrocyte-factor-induced BBB properties.
  • Example 2 Identification of Pro Barrier genes involved in BBB restoration after an LPS challenge
  • astrocytes and inflammatory processes display opposing effects in our dynamic co-culture model of the BBB.
  • LPS lipopolysaccharide
  • astrocytes increase barrier functionality, whereas LPS decreases it.
  • astrocytes bring about a recovery process from LPS which was not observed without the physical presence of astrocytes (i.e., in BCEC-ACM monolayers).
  • this recovery process was dependent on protein synthesis, which indicates that specific gene transcription is involved.
  • this experimental approach is schematically detailed.
  • RNA isolation, labeling of cRNA and hybridisation protocol was performed in triplo and all samples were analysed on both the HG-U95Av2 and HG-U133A arrays.
  • Affymetrix Microarray Suite 5.0 and Affymetrix Data Mining Tool 2.0 were used for primary analysis of the acquired intensity data.
  • Microsoft Excel (Microsoft, USA) was used for further analysis.
  • LPS responsive PB genes were PB: 1, 7, 9, 10, 14, 27, 32 and 33. For each identified PB gene, the specific result is presented in Table 2.
  • Example 3 Modulation of the biological activity of PB01 (YESl) in BCEC-ASTROCYTES cocultures
  • BCEC cultured from primary isolated brain capillaries from calf brain were used as monolayers on filter inserts with primary isolated newborn rat brain astrocytes cultured on the bottom side of the filter insert (Figure lb: BCEC- ASTROCYTES, as detailed in Gaillard et al., 2001, supra, which is included as a reference and briefly herein in "1.1 Cell Culture”).
  • BCEC were transfected with 0.031 microgram antisense probe (sequence: 5-end TGTTCCCATCCACACTTC 3-end) directed against the YESl gene in 12.5 microliter Saint mix (Synvolux Therapeutics B.V., Groningen, The Netherlands) at the apical chamber for three consecutive days. Control cells were incubated with either Saint mix alone or with an equal concentration of mismatch antisense probe. Every day after transfection, BBB functionality was assessed by TEER across the filters using an electrical resistance system (ERS) with a current-passing and voltage-measuring electrode (Millicell-ERS, Millipore Corporation, Bedford, MA, USA). TEER (Ohm .
  • ERS electrical resistance system
  • Millicell-ERS Millipore Corporation, Bedford, MA, USA
  • cm 2 was calculated from the displayed electrical resistance on the readout screen by subtraction of the electrical resistance of a collagen coated filter without cells and a correction for filter surface area.
  • TEER across collagen coated filters with only astrocytes on the bottom was close to zero (Gaillard et al., 2001, supra). Effects on TEER were normalized for the initial value of the filter prior to the transfection and represented as such. No effects on TEER were observed by Saint mix and the mismatch antisense probe.
  • TEER increased to 131 ⁇ 8 (mean ⁇ SD, % of initial value) at day 1, 128 ⁇ 11 (mean ⁇ SD, % of initial value) at day 2 and 126 ⁇ 13 (mean ⁇ SD, % of initial value) at day 3. After a single transfection on day 0 with 0.063 microgram of the same probe, these effects were no longer detectable on day 3. These results indicate that inhibition of the YESl gene increases the barrier function of the BBB.
  • TEER Ohm . cm 2
  • TEER Ohm . cm 2
  • TEER Ohm . cm 2
  • TEER across collagen coated filters only was close to zero (Gaillard et al, 2001, supra). Effects on TEER were normalized for control treated filters and represented as such. No effects on TEER were observed by BSA.
  • TEER increased to 116 ⁇ 4 (mean ⁇ SD, % of control) within 2 hours after treatment, up to 122 ⁇ 10 (mean ⁇ SD, % of control) at day 1, 123 ⁇ 13 (mean ⁇ SD, % of control) at day 2 and 121 ⁇ 21 (mean ⁇ SD, % of control) at day 3.
  • Trp Asp Asp Met Glu Lys lie Trp His His Ser Phe Tyr Asn Glu Leu 85 90 95
  • Trp Pro Phe Leu Gly Ile Leu Ala Glu Ile Ile Ile Leu Val Val Ile 225 230 235 240
  • Ser Glu Thr Glu Ser Glu lie Glu Ser Glu Thr Asp Phe Glu Thr Glu 115 120 125 Pro Glu Thr Ala Pro Thr Thr Glu Pro Glu Thr Glu Pro Glu Asp Asp 130 135 140
  • Gly Gin Thr Gly Arg Val lie Glu Asn Pro Ala Glu Ala Gin Ser Ala 435 440 445
  • Pro Pro Glu Met Arg lie Pro Lys Asn Gly Ile Glu Lys His Leu Leu 435 440 445

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Abstract

L'invention concerne des acides nucléiques et des polypeptides codés par ces derniers, dont l'expression est modulée dans les cellules endothéliales microvaculaires du cerveau subissant des modifications dynamiques lors de la formation de la barrière hémato-encéphalique. Ces peptides sont dénommés ici « polypeptides pro-barrière » (PB). Ces acides nucléiques et ces polypeptides peuvent être utilisés dans des méthodes permettant une régulation des propriétés de la barrière hémato-encéphalique chez les mammifères nécessitant de tels effets biologiques. Ces méthodes comprennent le diagnostic et le traitement des perturbations de la barrière hémato-encéphalique/rétinienne, des troubles cérébraux (yeux compris), ainsi que des troubles vasculaires périphériques. L'invention concerne en outre l'utilisation d'anticorps anti-polypeptides PB ou de ligands en tant que sondes de diagnostic, agents dirigés sélectivement sur la barrière hémato-encéphalique ou agents thérapeutiques, ainsi que l'utilisation de ligands ou de modulateurs de l'expression, de l'activation ou de la bioactivité des polypeptides PB en tant que sondes de diagnostic, agents thérapeutiques ou facilitateurs du transport de médicaments.
PCT/NL2003/000915 2002-12-19 2003-12-19 Acides nucleiques intervenant dans la regulation de la barriere hemato-encephalique WO2004056386A2 (fr)

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WO2008151637A3 (fr) * 2007-06-12 2009-02-19 Copenhagen University Peptides dérivés de la neuroplastine
US7897575B2 (en) 2000-05-24 2011-03-01 The United States Of America As Represented By The Department Of Health And Human Services Treatment and prevention of vascular dementia
WO2014097875A1 (fr) * 2012-12-20 2014-06-26 国立大学法人鳥取大学 Développement de cellules souches pluripotentes à l'aide d'un nouveau procédé d'induction d'une dédifférenciation
US9989539B2 (en) 2012-06-26 2018-06-05 Temple University—Of the Commonwealth System of Higher Education Method for detecting injury to the brain
WO2021113512A1 (fr) * 2019-12-04 2021-06-10 The Board Of Trustees Of The Leland Stanford Junior University Amélioration du transport de médicament à travers la barrière hémato-encéphalique par ciblage de régulateurs endogènes

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7897575B2 (en) 2000-05-24 2011-03-01 The United States Of America As Represented By The Department Of Health And Human Services Treatment and prevention of vascular dementia
US8940700B2 (en) 2000-05-24 2015-01-27 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, National Institutes Of Health E-selectin compositions and use thereof for inducing E-selectin tolerance
WO2008045488A2 (fr) * 2006-10-09 2008-04-17 Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services National Institutes Of Health Traitement de l'inflammation, de la démyélinisation et de la perte neuronale/axonale
WO2008045488A3 (fr) * 2006-10-09 2008-10-23 Us Gov Health & Human Serv Traitement de l'inflammation, de la démyélinisation et de la perte neuronale/axonale
WO2008151637A3 (fr) * 2007-06-12 2009-02-19 Copenhagen University Peptides dérivés de la neuroplastine
US9989539B2 (en) 2012-06-26 2018-06-05 Temple University—Of the Commonwealth System of Higher Education Method for detecting injury to the brain
WO2014097875A1 (fr) * 2012-12-20 2014-06-26 国立大学法人鳥取大学 Développement de cellules souches pluripotentes à l'aide d'un nouveau procédé d'induction d'une dédifférenciation
WO2021113512A1 (fr) * 2019-12-04 2021-06-10 The Board Of Trustees Of The Leland Stanford Junior University Amélioration du transport de médicament à travers la barrière hémato-encéphalique par ciblage de régulateurs endogènes

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