WO2016168615A1 - Methods of treating or preventing demyelination using thrombin inhibitors and methods of detecting demyelination using neurofascin 155 - Google Patents

Methods of treating or preventing demyelination using thrombin inhibitors and methods of detecting demyelination using neurofascin 155 Download PDF

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WO2016168615A1
WO2016168615A1 PCT/US2016/027776 US2016027776W WO2016168615A1 WO 2016168615 A1 WO2016168615 A1 WO 2016168615A1 US 2016027776 W US2016027776 W US 2016027776W WO 2016168615 A1 WO2016168615 A1 WO 2016168615A1
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casprl
thrombin
seq
antibody
antigen binding
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PCT/US2016/027776
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French (fr)
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Richard Douglas FIELDS
Dipankar Jyoti DUTTA
Dong Ho Woo
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 123,299 Byte ASCII (Text) file named "723232_ST25.TXT,” dated April 15, 2016.
  • Myelin is the electrical insulation on nerve fibers (axons) that is involved in the normal transmission of electrical impulses.
  • Many neurological disorders are the result of myelin damage (e.g., demyelination), for example multiple sclerosis (MS), cerebral palsy, and many other leucodystrophies resulting from toxic effects on the cells that make myelin (oligodendrocytes); for example, hypoxia, ischemia, viral infection, premature birth, and autoimmune disorders.
  • demyelination there exists a need for improved compositions and methods for treating, preventing, and detecting demyelination.
  • An embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a thrombin inhibitor to the mammal in an amount effective to treat or prevent the demyelination in the mammal.
  • Another embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a compound to the mammal in an amount effective to stimulate astrocytes in the mammal to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or (v) any combination of (i)-(iv) in an amount effective to treat or prevent the demyelination in the mammal.
  • inventions provide isolated or purified antibodies, or antigen binding fragments thereof, having antigenic specificity for neurofascin 155 (NF155) amino acid sequences and methods of detecting the presence of demyelination in a mammal using one or more of the antibodies.
  • NF155 neurofascin 155
  • Another embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the mouse Casprl 1-38 o amino acid sequence of SEQ ID NO: 33, the mouse Casprl 3 8 i -138 5 amino acid sequence of SEQ ID NO: 34, the mouse Casprl 1-947 amino acid sequence of SEQ ID NO: 35, the mouse Casprl9 48 _i 385 amino acid sequence of SEQ ID NO: 36, the mouse Casprl 38 i-9 47 amino acid sequence of SEQ ID NO: 39, the human Casprl 1-379 amino acid sequence of SEQ ID NO: 41, the human Casprl 38 o-i 384 amino acid sequence of SEQ ID NO: 42, the human Casprl 1-946 amino acid sequence of SEQ ID NO: 43, the human Casprl 94 7-138 4 amino acid sequence of SEQ ID NO: 44, or the human Casprl 38 o-94 6 amino acid
  • Figure 1 is a graph showing the clinical score of EAE mice following treatment with fondaparinux sodium (circles) or PBS (squares) each day up to 30 days after induction of EAE (post-immunization).
  • Figure 2 is a Kaplan-Meier survival curve showing the percentage of EAE mice surviving following treatment with fondaparinux sodium (solid line) or PBS (dotted line) each day up to 30 days after induction of EAE (post-immunization).
  • Figure 3 is a graph (incidence curve) showing the percentage of EAE incidence in EAE mice treated with fondaparinux sodium (solid line) or PBS (dotted line) at various time points (days) after induction of EAE (days elapsed).
  • Figure 4 is a graph showing the amount of PN-1 released from mouse astrocytes in cell culture via SNARE-dependent exocytosis, relative to total protein (pg ⁇ g) measured in cultures of astrocytes from WT mice without mastoparan (MP) stimulation, WT with (MP) stimulation, astrocytes from DOX-on mice after MP stimulation, and astrocytes from DOX- off mice after MP stimulation.
  • MP induces exocytosis by stimulating GTPase activity (activating Gi and Go proteins).
  • ANOVA F 3,22 14.13 pO.0001 ; Posthoc Tukey's
  • Myelin is attached to the axon membrane by the cell adhesion molecule neurofascin 155 in the region adjacent to the node of Ranvier (paranodal region). It has been discovered that perinodal astrocytes regulate enzymatic cleavage of NF155 at a thrombin cleavage site, allowing detachment of the myelin from the axon. Thrombin cleaves NF155 into two fragments: NF125 and NF30. Full-length human NF155 has the amino acid sequence of SEQ ID NO: 24 (Genbank Acession No. NP_001153803.1).
  • the thrombin cleavage site of human NF155 (as well as mouse NF155) has the amino acid sequence of GRG (human NF1559 24- 9 26 ; SEQ ID NO: 31), with the cleavage occurring after the arginine in GRG (SEQ ID NO: 31).
  • Human NF125 has the amino acid sequence of SEQ ID NO: 25
  • human NF30 has the amino acid sequence of SEQ ID NO: 26.
  • Casprl axonal adhesion molecule contactin-associated protein
  • Full-length mouse Casprl has the amino acid sequence of SEQ ID NO: 32.
  • Full-length human Casprl has the amino acid sequence of SEQ ID NO: 40.
  • thrombin cleaves Casprl into two fragments: the amino acid sequence of SEQ ID NO: 41 (human Casprl 1 -37 9) and the amino acid sequence of SEQ ID NO: 42 (human Casprl 38 o-i 38 4).
  • the thrombin- cleaved fragments are the amino acid sequence of SEQ ID NO: 33 (mouse Casprl i -38 o) and the amino acid sequence of SEQ ID NO: 34 (mouse Casprl 38i-i38s)-
  • the thrombin cleavage site of human and mouse Casprl has the amino acid sequence of RRG (mouse Casprl 38 o- 38 2; human Casprl 37 9 -381 ; SEQ ID NO: 37), with the cleavage occurring between the arginine residues in RRG (SEQ ID NO: 37).
  • factor Xa cleaves Casprl into two fragments: the amino acid sequence of SEQ ID NO: 43 (human Casprl !_9 46 ) and the amino acid sequence of SEQ ID NO: 44 (human Casprl 94 7- i 384 ).
  • the factor Xa-cleaved fragments are the amino acid sequence of SEQ ID NO: 35 (mouse Casprl 1,947) and the amino acid sequence of SEQ ID NO: 36 (mouse Casprlg ⁇ -nss).
  • the factor Xa cleavage site of human and mouse Casprl has the amino acid sequence of LEGR (mouse Casprl 957.940; human Casprl 943-940; SEQ ID NO: 38), with the cleavage occurring after the arginine residue in LEGR (SEQ ID NO: 38).
  • an embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a thrombin inhibitor to the mammal in an amount effective to treat or prevent the
  • an embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a factor Xa inhibitor to the mammal in an amount effective to treat or prevent the demyelination in the mammal.
  • the thrombin inhibitor can be any agent that inhibits the biological activity of thrombin.
  • Thrombin is secreted as the inactive prothrombin, which is then converted into active thrombin by the enzyme Factor Xa.
  • the biological activity of thrombin may be inhibited in any manner, e.g., by inhibiting the conversion of prothrombin to thrombin (e.g., by inhibiting the activity of enzyme Factor Xa); by inhibiting the expression of one or both of thrombin mRNA and thrombin protein; by inhibiting the binding of thrombin to NF155; by inhibiting thrombin-mediated cleavage of NF155; by inhibiting the binding of thrombin to Casprl , and/or by inhibiting thrombin-mediated cleavage of Casprl, as compared to that which is observed in the absence of the thrombin inhibitor.
  • the biological activity may be inhibited to any degree that realizes a beneficial therapeutic effect.
  • the biological activity may be completely inhibited (i.e., prevented), while in other embodiments, the biological activity may be partially inhibited (i.e., reduced).
  • thrombin encompasses thrombin and prothrombin in any form (e.g., mRNA or protein) and from any species (e.g., human, rat, or mouse).
  • the factor Xa inhibitor can be any agent that inhibits the biological activity of factor Xa.
  • the biological activity of factor Xa may be inhibited in any manner, e.g., by inhibiting the expression of one or both of factor Xa mRNA and factor Xa protein; by inhibiting the binding of factor Xa to Casprl ; by inhibiting factor Xa-mediated cleavage of Casprl ; and/or by inhibiting the binding of factor Xa to Casprl , as compared to that which is observed in the absence of the factor Xa inhibitor.
  • the biological activity may be inhibited to any degree that realizes a beneficial therapeutic effect, as described herein with respect to other aspects of the invention.
  • the thrombin inhibitor is an agent that inhibits thrombin-mediated cleavage of one or both of Casprl and NF155.
  • the thrombin inhibitor may be an agent that binds to the thrombin protein or the NF155 protein, thereby reducing or preventing thrombin-mediated cleavage of NF155.
  • the thrombin inhibitor may be an agent that binds to the thrombin protein or the Casprl protein, thereby reducing or preventing thrombin-mediated cleavage of Casprl .
  • the agent that inhibits thrombin-mediated cleavage of one or both of NF155 and Casprl can be any of the antibodies or antigen binding fragments thereof, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide (or
  • the factor Xa inhibitor is an agent that inhibits factor Xa-mediated cleavage of Casprl .
  • the factor Xa inhibitor may be an agent that binds to the factor Xa protein or the Casprl protein, thereby reducing or preventing factor Xa-mediated cleavage of Casprl .
  • the factor Xa inhibitor may be an agent that binds to the factor Xa protein or the Casprl protein, thereby reducing or preventing factor Xa-mediated cleavage of Casprl .
  • the agent that inhibits factor Xa-mediated cleavage of Casprl can be any of the antibodies or antigen binding fragments thereof, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide (or polypeptide) inhibitor) described herein.
  • the thrombin inhibitor is an agent that inhibits the binding of thrombin to one or both of Casprl and NF155.
  • the thrombin inhibitor may be an agent that binds to thrombin or NF155, thereby reducing or preventing the binding of the thrombin protein to the NF155 protein and inhibiting the ability of thrombin to cleave NF155.
  • the thrombin inhibitor may be an agent that binds to thrombin or Casprl , thereby reducing or preventing the binding of the thrombin protein to the Casprl protein and inhibiting the ability of thrombin to cleave Casprl .
  • the thrombin inhibitor may be an agent that competes with the thrombin protein for the native thrombin binding or cleavage site of the NF155 protein or an agent that competes with the NF155 protein for the native NF155 binding site of the thrombin protein.
  • the thrombin inhibitor may be an agent that competes with the thrombin protein for the native thrombin binding or cleavage site of the Casprl protein or an agent that competes with the Casprl protein for the native Casprl binding site of the thrombin protein.
  • the agent that inhibits the binding of thrombin to one or both of Caspr 1 and NF155 can be any of the antibodies or antibody fragments, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide inhibitor) described herein.
  • the factor Xa inhibitor is an agent that inhibits the binding of factor Xa to Casprl .
  • the factor Xa inhibitor may be an agent that binds to factor Xa or Casprl , thereby reducing or preventing the binding of the factor Xa protein to the Casprl protein and inhibiting the ability of factor Xa to cleave Casprl .
  • the factor Xa inhibitor may be an agent that competes with the factor Xa protein for the native factor Xa binding or cleavage site of the Casprl protein or an agent that competes with the Casprl protein for the native Casprl binding site of the factor Xa protein.
  • the agent that inhibits the binding of factor Xa to Caspr 1 can be any of the antibodies or antibody fragments, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide inhibitor) described herein.
  • the thrombin inhibitor is an antibody, or an antigen binding fragment thereof, having antigenic specificity for thrombin, Casprl, or NF155.
  • the factor Xa inhibitor is an antibody, or an antigen binding fragment thereof, having antigenic specificity for factor Xa or Casprl .
  • Anti- factor Xa, anti-thrombin, anti-Casprl , and anti-NF155 antibodies and antigen binding fragments thereof can be monoclonal or polyclonal.
  • Anti-factor Xa, anti-thrombin, anti- Casprl, and anti-NF155 antibodies and antigen binding fragments thereof can be prepared using the factor Xa, thrombin, Casprl , and NF155 proteins disclosed herein and routine techniques.
  • antibodies or antigen binding fragments thereof are described herein with respect to other aspects of the invention and include those which specifically bind to a functional fragment of the factor Xa protein, the Casprl protein, the NF155 protein, or the thrombin protein, e.g., the native factor Xa binding or cleavage site of the Casprl protein, the native Casprl binding site of the factor Xa protein, the native thrombin binding or cleavage site of the Casprl protein, the native Casprl binding site of the thrombin protein, the native thrombin binding or cleavage site of the NF155 protein or the native NF155 binding site of the thrombin protein.
  • the functional fragment of the thrombin or NF155 protein can comprise any contiguous part of the thrombin or NF155 protein that retains a relevant biological activity of the thrombin or NF155 protein, e.g., binds to NF1 5 or thrombin, respectively, and/or participates in thrombin-mediated cleavage of NF155.
  • the functional fragment of the thrombin or Casprl protein can comprise any contiguous part of the thrombin or Casprl protein that retains a relevant biological activity of the thrombin or Casprl protein, e.g., binds to Casprl or thrombin, respectively, and/or participates in thrombin-mediated cleavage of Casprl .
  • the functional fragment of the factor Xa or Casprl protein can comprise any contiguous part of the factor Xa or Casprl protein that retains a relevant biological activity of the factor Xa or Casprl protein, e.g., binds to Casprl or factor Xa, respectively, and/or participates in factor Xa-mediated cleavage of Casprl .
  • Any given fragment of a factor Xa, thrombin, Casprl , or NF155 protein can be tested for such biological activity using methods known in the art.
  • the functional fragment can comprise, consist essentially of, or consist of the factor Xa binding portion of the Casprl protein, the Casprl binding portion of the factor Xa protein, the thrombin binding portion of the Casprl protein, the Casprl binding portion of the thrombin protein, the thrombin binding portion of the NF155 protein, or the NF155 binding portion of the thrombin protein.
  • the functional fragment preferably comprises, for instance, about 10% or more, 25% or more, 30% or more, 50% or more, 60% or more, 80% or more, 90% or more, or even 95% or more of the parent factor Xa, thrombin, Casprl, or NF155 protein, respectively.
  • Chemical inhibitors of thrombin include small molecules and peptides or polypeptides that inhibit thrombin-mediated cleavage of NF155, bind the thrombin protein or the NF155 protein or functional fragment of thrombin or NF155, or compete with the thrombin or NF155 protein or functional fragment of thrombin or NF155 for its native binding or cleavage site on the NF155 protein or its native binding site on the thrombin protein, respectively.
  • chemical inhibitors of thrombin include small molecules and peptides or polypeptides that inhibit thrombin- mediated cleavage of Casprl , bind the thrombin protein or the Casprl protein or functional fragment of thrombin or Casprl , or compete with the thrombin or Casprl protein or functional fragment of thrombin or Casprl for its native binding or cleavage site on the Casprl protein or its native binding site on the thrombin protein, respectively.
  • chemical inhibitors of factor Xa include small molecules and peptides or polypeptides that inhibit factor Xa-mediated cleavage of Casprl, bind the factor Xa protein or the Casprl protein or functional fragment of factor Xa or Casprl , or compete with the factor Xa or Casprl protein or functional fragment of factor Xa or Casprl for its native binding or cleavage site on the Casprl protein or its native binding site on the factor Xa protein, respectively.
  • Suitable inhibitors can include, for example, chemical compounds or a non-active fragment or mutant of a factor Xa protein, thrombin protein, Casprl protein, or a NF155 protein.
  • the thrombin inhibitor is a mutated thrombin, a mutated Casprl, or a mutated NF155.
  • the factor Xa inhibitor is a mutated factor Xa or a mutated Casprl .
  • the mutation may include any insertions, deletions, and/or substitutions of one or more amino acids in any position of the thrombin, Casprl , or NF155 protein that effectively inhibits thrombin biological activity (e.g., one or more of thrombin-mediated cleavage of NF155, binding of NF155 to thrombin, thrombin-mediated cleavage of Casprl, and binding of Casprl to thrombin) or any insertions, deletions, and/or substitutions of one or more amino acids in any position of the factor Xa or Casprl protein that effectively inhibits factor Xa biological activity.
  • the chemical inhibitor can bind to one or more of factor Xa, Casprl, thrombin, and bind to NF155.
  • the thrombin inhibitor is a mutant of a thrombin protein that binds NF155 protein but does not cleave it. Such a mutant thrombin protein would competitively inhibit NF155 cleavage by wild-type (non-mutated) thrombin.
  • the thrombin inhibitor is a mutant of a thrombin protein that binds Casprl protein but does not cleave it.
  • the factor Xa inhibitor is a mutant of a factor Xa protein that binds Casprl protein but does not cleave it. Such a mutant factor Xa protein would competitively inhibit Casprl cleavage by wild-type (non-mutated) factor Xa.
  • the thrombin inhibitor or factor Xa inhibitor may be a chemical inhibitor.
  • the thrombin inhibitor inhibits thrombin-mediated cleavage of one or both of Casprl and NF155, as described herein.
  • the factor Xa inhibitor inhibits factor Xa-mediated cleavage of Casprl , as described herein.
  • Chemical inhibitors of thrombin or factor Xa can be identified using routine techniques. For example, chemical inhibitors can be tested in assays to identify molecules and peptides (or polypeptides) that bind to one or more of factor Xa, thrombin, Casprl, and NF155 with sufficient affinity to inhibit thrombin biological activity (e.g., one or more of binding of thrombin to NF155, thrombin-mediated cleavage of NF155, binding of thrombin to Casprl, and thrombin-mediated cleavage of Casprl) or to inhibit factor Xa biological activity.
  • thrombin biological activity e.g., one or more of binding of thrombin to NF155, thrombin-mediated cleavage of NF155, binding of thrombin to Casprl, and thrombin-mediated cleavage of Casprl
  • competition assays can be performed to identify small-molecules and peptides (or polypeptides) that inhibit thrombin-mediated cleavage of NF155 or compete with thrombin or NF155 (or a functional fragment thereof) for binding to its native binding or cleavage site of NF155 or its native binding site of thrombin, respectively.
  • Competition assays can be performed to identify small-molecules and peptides (or polypeptides) that inhibit thrombin-mediated cleavage of Casprl or compete with thrombin or Casprl (or a functional fragment thereof) for binding to its native binding or cleavage site of Casprl or its native binding site of thrombin, respectively.
  • Competition assays can be performed to identify small-molecules and peptides (or polypeptides) that inhibit factor Xa-mediated cleavage of Casprl or compete with factor Xa or Casprl (or a functional fragment thereof) for binding to its native binding or cleavage site of Casprl or its native binding site of factor Xa, respectively.
  • Such techniques could be used in conjunction with mutagenesis of the thrombin protein, factor Xa protein, Casprl protein, or the NF155 protein, or a functional fragment thereof, and/or with high- throughput screens of known chemical inhibitors.
  • the chemical thrombin inhibitor may be a direct thrombin inhibitor or an indirect thrombin inhibitor.
  • the direct thrombin inhibitor may be a bivalent, univalent, or an allosteric inhibitor.
  • the thrombin inhibitor is a catalyst that activates native thrombin inhibitors like antithrombin III (such as, for example, heparin).
  • thrombin inhibitors may include, but are not limited to, hirudin, bivalirudin, lepirudin, desirudin, argatroban, melagatran, ximelagatran, dabigatran, DNA aptamers, benzofuran dimers, benzofuran trimers, polymeric lignins, sulfated ⁇ -04 lignin (Sb04L), heparin (including, for example, low molecular weight heparins (LMWHs)), warfarin, fondaparinux, and pharmaceutically acceptable salts and derivatives thereof.
  • the thrombin inhibitor is a thrombin inhibitor that is released by astrocytes.
  • thrombin inhibitors that are released by astrocytes include antithrombin III (Dowell et al., J. Proteome Res., 8(8): 4135-43 (2009)), protease nexin-1 (PN1), plasminogen activator inhibitor-1 (PAI-1) (Hultman et al., J. Neurosci. Res., 2441-9 (2010)), thrombomodulin (Pindon et al., Glia, 19(3): 259-68 (1997)), and pharmaceutically acceptable salts and derivatives thereof.
  • antithrombin III Dowell et al., J. Proteome Res., 8(8): 4135-43 (2009)
  • protease nexin-1 PN1
  • PAI-1 plasminogen activator inhibitor-1
  • thrombomodulin Pindon et al., Glia, 19(3): 259-68 (1997)
  • the thrombin inhibitor is fondaparinux, antithrombin III, a LMWH, or a pharmaceutically acceptable salt or derivative thereof (such as, for example, fondaparinux sodium).
  • Fondaparinux inhibits the enzyme Factor Xa- mediated conversion of prothrombin to thrombin.
  • the chemical factor Xa inhibitor may be a direct factor Xa inhibitor or an indirect factor Xa inhibitor.
  • the thrombin inhibitor is any suitable agent that inhibits the expression of one or both of thrombin mRNA and thrombin protein.
  • the factor Xa inhibitor is any suitable agent that inhibits the expression of one or both of factor Xa mRNA and factor Xa protein.
  • the thrombin inhibitor can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding one or both of thrombin mRNA and thrombin protein or a complement thereof.
  • the thrombin inhibitor may be introduced into a host cell, wherein the cell is capable of expressing one or both of thrombin mRNA and thrombin protein, in an effective amount for a time and under conditions sufficient to interfere with expression of one or both of thrombin mRNA and thrombin protein, respectively.
  • the factor Xa inhibitor can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding one or both of factor Xa mRNA and factor Xa protein or a complement thereof.
  • the factor Xa inhibitor may be introduced into a host cell, wherein the cell is capable of expressing one or both of factor Xa mRNA and factor Xa protein, in an effective amount for a time and under conditions sufficient to interfere with expression of one or both of factor Xa mRNA and factor Xa protein, respectively.
  • RNA interference RNAi
  • the thrombin inhibitor or factor Xa inhibitor may comprise an RNAi agent.
  • the RNAi agent may comprise a small interfering RNA (siRNA), a short hairpin miRNA (shMIR), a microRNA (miRNA), or an antisense nucleic acid.
  • RNAi agent e.g., siRNA, shRNA, miRNA, and/or antisense nucleic acid
  • RNA interference nucleic acids employed can be at least about 19, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 220, at least about 240, from about 19 to about 250, from about 40 to about 240, from about 60 to about 220, from about 80 to about 200, from about 60 to about 180, from about 80 to about 160, and/or from about 100 to about 140 nucleotides in length.
  • the RNAi agent e.g., siRNA or shR A
  • a nucleotide sequence included in a cassette e.g., a larger nucleic acid construct such as an appropriate vector.
  • vectors include lentiviral and adenoviral vectors, as well as other vectors described herein with respect to other aspects of the invention.
  • the resulting nucleic acid can be longer than the comprised RNAi nucleic acid, e.g., greater than about 70 nucleotides in length.
  • the RNAi agent employed cleaves the target mRNA.
  • the RNAi agent employed does not cleave the target mRNA.
  • the thrombin inhibitor or the factor Xa inhibitor is a GENEART CRISPR-Cas9 system for genome-editing (available from Life Technologies, Carlsbad, CA).
  • the antisense nucleic acid comprises a nucleotide sequence complementary to at least about 8, at least about 15, at least about 19, or from about 19 to about 22 nucleotides of a nucleic acid encoding (i) one or both of thrombin mRNA and thrombin protein or a complement thereof or (ii) one or both of factor Xa mRNA and thrombin protein or a complement thereof.
  • the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat-associated siRNAs (rasiRNAs).
  • the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).
  • the thrombin inhibitor may inhibit or downregulate to some degree the expression of the protein encoded by a thrombin gene, e.g., at the DNA, RNA, or other level of regulation.
  • a host cell comprising a thrombin inhibitor expresses none of one or both of thrombin mRNA and thrombin protein or lower levels of one or both of thrombin mRNA and thrombin protein as compared to a host cell that lacks a thrombin inhibitor.
  • the inhibitor such as an RNAi agent, such as a shMIR, can target a nucleotide sequence of a thrombin gene or mRNA encoded by the same.
  • the thrombin sequence is a human thrombin sequence.
  • human prothrombin is assigned Gene NCBI Entrez Gene ID No. 2147, and an Online Mendelian Inheritance in Man (OMIM) No. 176930.
  • the human thrombin gene is found on chromosome 1 1 at 1 lpl 1.2.
  • a human thrombin mRNA transcript includes mRNA GenBank Accession No: NM_000506.3 (SEQ ID NO: 27), with corresponding protein sequence GenBank Accession No: NP_000497.1 (SEQ ID NO: 28).
  • Human genomic thrombin sequences include GenBank Accession Nos: NG_008953.1 , NC _00001 1.10, AC1 15088.6, AF478696.1 , AF493953.1 , AJ5441 14.1 , AMYH0202471 1.1, CH471064.2, CS355181.1, M17262.1 , and S50162.1.
  • Human thrombin mRNA sequences also include Genbank Accession Nos: AJ972449.1 , A 222775.1 , A 222777.1 , AK293326.1 ,
  • Human thrombin amino acid sequences include Genbank Accession Nos: AAL77436.1 , AAM1 1680.1 , CAD80258.1 , EAW67977.1, EAW67978.1, EAW67979.1 , CAL24231.1 , AAC63054.1 , AAB24476.1 , CAJOl 369.1, BAD96495.1, BAD96497.1, BAG56844.1 , BAG64719.1 , BAG35804.1 , AAR08142.1, AAR08143.1, AAH51332.1, AAA60220.1 , CAA23842.1 , ACE87755.1 , and ACE87074.1.
  • Other human thrombin sequences, as well as other thrombin species can be employed in accordance with the invention.
  • the thrombin sequence is a mouse sequence.
  • mouse thrombin is assigned Gene NCBI Entrez Gene ID No. 14061.
  • the mouse thrombin gene is found on chromosome 2 at 2E1.
  • a transcript includes mRNA Genbank Accession No.: NM_010168.3 (SEQ ID NO: 29), with corresponding protein sequence NP_034298.1 (SEQ ID NO: 30).
  • Mouse genomic thrombin sequences include Genbank Accession Nos: AAHY01018393.1, AL691489.21 , and CH466519.1.
  • Mouse thrombin mRNA sequences also include Genbank Accession Nos: AK050056.1, A 149367.1, AK167532.1, BC013662.1 , BY705183.1 , M81394.1, and X52308.1.
  • Mouse thrombin amino acid sequences include Genbank Accession Nos: EDL27567.1 , BAE39601.1 , AAHl 3662.1 , AAA40435.1, and CAA36548.1.
  • Other mouse thrombin sequences, as well as other thrombin species can be employed in accordance with the invention.
  • the thrombin inhibitor such as an RNAi agent, such as a shMIR
  • RNAi agent such as a shMIR
  • the thrombin inhibitor can target a nucleotide sequence selected from the group consisting of the 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), and the coding sequence of thrombin, complements thereof, and any combination thereof.
  • Any suitable thrombin target sequence can be employed.
  • the sequences of the thrombin inhibitor can be designed against a human thrombin with
  • the sequences of the thrombin inhibitor can be designed against a mouse thrombin with Accession No. NM_010168.3 (SEQ ID NO: 29).
  • RNAi agents can be designed against any appropriate thrombin mRNA sequence.
  • the thrombin inhibitor is an NFl 55 thrombin binding site/Fc fusion protein.
  • the NFl 55 thrombin binding site/Fc fusion protein is a soluble variation of the native NFl 55 which binds thrombin protein, thereby competing with the native NF155 for binding to thrombin.
  • the NF155 thrombin binding site/Fc fusion protein may inhibit the binding of thrombin to the native NFl 55.
  • the NFl 55 thrombin binding site/Fc fusion protein may also inhibit the thrombin-mediated cleavage of NFl 55.
  • the NFl 55 thrombin binding site/Fc fusion protein may be from any mammal.
  • the NFl 55 thrombin binding site/Fc fusion protein is a mouse NFl 55 thrombin binding site/Fc fusion protein or a human NFl 55 thrombin binding site/Fc fusion protein.
  • the thrombin inhibitor is a thrombin NFl 55 binding site/Fc fusion protein.
  • the thrombin NFl 55 binding site/Fc fusion protein is a soluble variation of the native thrombin which binds NFl 55 protein, thereby competing with the native thrombin for binding to NFl 55. Accordingly, the thrombin NFl 55 binding site/Fc fusion protein may inhibit the binding of thrombin to the native NFl 55.
  • the thrombin NFl 55 binding site/Fc fusion protein may also inhibit the thrombin-mediated cleavage of NFl 55.
  • the thrombin NFl 55 binding site/Fc fusion protein may be from any mammal.
  • the thrombin NFl 55 binding site/Fc fusion protein is a mouse thrombin NFl 55 binding site/Fc fusion protein or a human thrombin NFl 55 binding site/Fc fusion protein.
  • the thrombin inhibitor is a Casprl thrombin binding site/Fc fusion protein.
  • the Casprl thrombin binding site/Fc fusion protein is a soluble variation of the native Casprl which binds thrombin protein, thereby competing with the native Casprl for binding to thrombin. Accordingly, the Casprl thrombin binding site/Fc fusion protein may inhibit the binding of thrombin to the native Casprl .
  • the Casprl thrombin binding site/Fc fusion protein may also inhibit the thrombin-mediated cleavage of Casprl .
  • the Casprl thrombin binding site/Fc fusion protein may be from any mammal.
  • the Casprl thrombin binding site/Fc fusion protein is a mouse Casprl thrombin binding site/Fc fusion protein or a human Casprl thrombin binding site/Fc fusion protein.
  • the thrombin inhibitor is a thrombin Casprl binding site/Fc fusion protein.
  • the thrombin Casprl binding site/Fc fusion protein is a soluble variation of the native thrombin which binds Casprl protein, thereby competing with the native thrombin for binding to Casprl . Accordingly, the thrombin Casprl binding site/Fc fusion protein may inhibit the binding of thrombin to the native Casprl .
  • the thrombin Casprl binding site/Fc fusion protein may also inhibit the thrombin-mediated cleavage of Casprl .
  • the thrombin Casprl binding site/Fc fusion protein may be from any mammal.
  • the thrombin Casprl binding site/Fc fusion protein is a mouse thrombin Casprl binding site/Fc fusion protein or a human thrombin Casprl binding site/Fc fusion protein.
  • the factor Xa inhibitor is a Casprl factor Xa binding site/Fc fusion protein.
  • the Casprl factor Xa binding site/Fc fusion protein is a soluble variation of the native Casprl which binds factor Xa protein, thereby competing with the native Casprl for binding to factor Xa. Accordingly, the Casprl factor Xa binding site/Fc fusion protein may inhibit the binding of factor Xa to the native Casprl .
  • the Casprl factor Xa binding site/Fc fusion protein may also inhibit the factor Xa-mediated cleavage of Casprl .
  • the Casprl factor Xa binding site/Fc fusion protein may be from any mammal.
  • the Casprl factor Xa binding site/Fc fusion protein is a mouse Casprl factor Xa binding site/Fc fusion protein or a human Casprl factor Xa binding site/Fc fusion protein.
  • the factor Xa inhibitor is a factor Xa Casprl binding site/Fc fusion protein.
  • the factor Xa Casprl binding site/Fc fusion protein is a soluble variation of the native factor Xa which binds Casprl protein, thereby competing with the native factor Xa for binding to Casprl . Accordingly, the factor Xa Casprl binding site/Fc fusion protein may inhibit the binding of factor Xa to the native Casprl .
  • the factor Xa Casprl binding site/Fc fusion protein may also inhibit the factor Xa-mediated cleavage of Casprl .
  • the factor Xa Casprl binding site/Fc fusion protein may be from any mammal.
  • the factor Xa Casprl binding site/Fc fusion protein is a mouse factor Xa Casprl binding site/Fc fusion protein or a human factor Xa Casprl binding site/Fc fusion protein.
  • the thrombin inhibitor or the factor Xa inhibitor can be obtained by methods known in the art.
  • thrombin inhibitors or factor Xa inhibitors that are peptides or polypeptides can be obtained by de novo synthesis.
  • thrombin inhibitors or factor Xa inhibitors can be recombinantly produced using standard recombinant methods. See, for instance, Green et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2012.
  • the thrombin inhibitor or factor Xa inhibitor 75 can be isolated and/or purified from a natural source, e.g., a human. Methods of isolation and purification are well-known in the art.
  • the thrombin inhibitors may be exogenous and can be synthetic, recombinant, or of natural origin.
  • the thrombin inhibitors or factor Xa inhibitors that are peptides or polypeptides can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
  • Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
  • mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids
  • organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
  • the methods of the invention can comprise administering two or more thrombin inhibitors, or two or more factor Xa inhibitors, any of which may be the same or different from one another.
  • the thrombin inhibitor or factor Xa inhibitor can be provided as part of a larger polypeptide construct.
  • the thrombin inhibitor or factor Xa inhibitor can be provided as a fusion protein comprising a thrombin inhibitor or factor Xa inhibitor along with other amino acid sequences or a nucleic acid encoding same.
  • the thrombin inhibitor or factor Xa inhibitor also can be provided as part of a conjugate or nucleic acid encoding same. Conjugates, as well as methods of synthesizing conjugates in general, are known in the art.
  • the thrombin inhibitor or factor Xa inhibitor can be administered to the mammal by administering a nucleic acid encoding the thrombin inhibitor or factor Xa inhibitor to the mammal.
  • Nucleic acid as used herein includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • Nucleic acids encoding the thrombin inhibitor or factor Xa inhibitor can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green et al., supra.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
  • the nucleic acids can be incorporated into a recombinant expression vector.
  • the term "recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA or polypeptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA or polypeptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA or polypeptide expressed within the cell.
  • the vectors are not naturally- occurring as a whole. However, parts of the vectors can be naturally-occurring.
  • the recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturally-occurring or non- naturally-occurring internucleotide linkages, or both types of linkages.
  • the non- naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
  • the recombinant expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can be of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif.).
  • Bacteriophage vectors such as GTIO, ⁇ 1 , ⁇ ZapII (Stratagene), EMBL4, and ⁇ 149, also can be used.
  • plant expression vectors include pBIOl , pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).
  • the recombinant expression vector is a viral vector, e.g., a retroviral vector.
  • the recombinant expression vectors can be prepared using standard recombinant DNA techniques described in, for example, Green et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
  • the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • regulatory sequences such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • the recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts.
  • Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
  • Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • the recombinant expression vector can comprise a native or nonnative promoter and/or stop codon operably linked to the nucleotide sequence encoding the thrombin inhibitor or factor Xa inhibitor, or to the nucleotide sequence which is complementary to the nucleotide sequence encoding the thrombin inhibitor or factor Xa inhibitor.
  • the selection of stop codons and promoters e.g., strong, weak, inducible, tissue- specific and developmental- specific, is within the ordinary skill of the artisan.
  • the combining of a nucleotide sequence with a stop codon and a promoter is also within the skill of the artisan.
  • the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
  • CMV cytomegalovirus
  • the thrombin inhibitors, factor Xa inhibitors and nucleic acids encoding them can be of synthetic or natural origin, and can be isolated or purified to any degree.
  • isolated and purified means having been removed from its natural environment.
  • purified or isolated means having been increased in purity and does not require absolute purity or isolation; rather, it is intended as a relative term.
  • the purity can be at least about 50%, can be greater than about 60%, about 70% or about 80%, or can be about 100%.
  • astrocytes at the node of Ranvier encase the node, stabilize the node, and maintain the physical structure of myelin.
  • demyelination may originate at the node of Ranvier.
  • immune cells may cause autoimmune demyelinating disorders, such as MS, by attacking at or near the node of Ranvier.
  • stimulating the astrocyte to reduce or prevent immune cell access to the paranodal junctions and maintain the physical structure of the node may reduce or prevent demyelination.
  • Astrocytes release thrombin inhibitors and also physically stabilize the node of Ranvier. Accordingly, it is also believed that stimulating astrocytes to release a thrombin inhibitor may reduce or prevent demyelination.
  • an embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a compound to the mammal in an amount effective to stimulate astrocytes in the mammal to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or (v) any combination of (i)-(iv) in an amount effective to treat or prevent the demyelination in the mammal.
  • the thrombin inhibitor release by the astrocytes may be any thrombin inhibitor released by astrocytes.
  • the thrombin inhibitor released by astrocytes is antithrombin III, PN1 , PAI-1 , or thrombomodulin.
  • the compound that stimulates astrocytes in the mammal to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or (v) any combination of (i)-(iv) may be any suitable compound that stimulates astrocytes in the mammal to carry out one or more of (i)-(v).
  • Compounds can be tested in assays to identify agents that stimulate astrocytes to carry out one or more of (i)-(v).
  • agents that stimulate astrocytes may include adenosine triphosphate (ATP), adenosine, glutamate, cytokines, nitric oxide, chemokines, growth factors, and factors regulating GFAP (glial fibrillary acidic protein).
  • the demyelination may be associated with (e.g., caused by) a demyelinating disease.
  • the method may comprise treating or preventing a demyelinating disease in the mammal.
  • the demyelinating disease may be any disorder that involves myelin loss (e.g., damage, and/or impairment), regardless of the cause (e.g., a demyelinating disease).
  • a demyelinating disease is any disorder that results in deficient or abnormal myelination (e.g., destruction of myelin).
  • the pathology of the demyelinating disease can have autoimmune, inflammatory, neurodegenerative, or other components.
  • the disease is classified as an inflammatory demyelinating disease or an autoimmune/inflammatory demyelinating disease.
  • the demyelinating disease can be, for example, a leucodystrophy, multiple sclerosis (MS), cerebral palsy, optic neuritis, Devic's disease (neuromyelitis optica), transverse myelitis, acute MS (Marburg variant), Balo's concentric sclerosis, acute disseminated encephalomyelitis (ADEM),
  • adrenoleukodystrophy adrenomyeloneuropathy
  • Gulf War Illness combined central and peripheral demyelination (CCPD)
  • CCPD central and peripheral demyelination
  • the disease is multiple sclerosis (MS).
  • the disease is Guillain-Barre syndrome (GBS).
  • GBS involves demyelination in the peripheral nervous system (PNS).
  • PNS peripheral nervous system
  • Schwann cells produce myelin in the PNS, and Casprl and NF155 are present at the nodes in the PNS.
  • GBS may also be treated by reducing or preventing thrombin biological activity, e.g., thrombin-mediated cleavage of one or both of Casprl and NF155, and it is believed that GBS may also be treated by reducing or preventing factor Xa biological activity, e.g., factor Xa-mediated cleavage of Casprl .
  • the demyelination may be associated with (e.g., caused by) damage to or death of the cells that make myelin (oligodendrocytes).
  • Demyelination may be caused by any of a variety of events including, but not limited to, any one or more of white matter injury, difficult birth, hypoxia, ischemia, viral infection, premature birth, any of the demyelinating diseases described herein, and autoimmune disorders.
  • the demyelination may include injury-related demyelination, e.g., noise-induced hearing loss.
  • the factor Xa inhibitor, thrombin inhibitor or compound that stimulates astrocytes to may be administered to the mammal in any suitable manner.
  • the anti-demyelination material is administered parenterally (e.g., subcutaneously, intravenously, intraarterially, intramuscularly,
  • the anti- demyelination material is administered in combination with a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active anti-demyelination material, and by the route of administration.
  • Pharmaceutically acceptable carriers for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active anti-demyelination material and one which has no detrimental side effects or toxicity under the conditions of use.
  • the amount or dose of the anti-demyelination material administered should be sufficient to effect a desired response, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame.
  • the dose of the anti-demyelination material should be sufficient to reduce or prevent cleavage of one or both of Casprl and NF155, reduce or prevent demyelination, or treat or prevent a demyelinating disease (e.g., demyelinating disease progression) in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer.
  • the dose will be determined by the efficacy of the particular anti-demyelination material and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated.
  • an administered dose may be determined in vitro (e.g., cell cultures) or in vivo (e.g., animal studies). For example, an administered dose may be determined by determining the ICs 0 (the dose that achieves a half-maximal inhibition of signs of disease), LD 5 o (the dose lethal to 50% of the population), the ED 50 (the dose therapeutically effective in 50% of the
  • the therapeutic index is the ratio of LD 50 to ED 5 o (i.e., LD 5 0/ED 5 0).
  • the dose of the anti-demyelination material also may be determined by the existence, nature, and extent of any adverse side effects that might accompany the
  • the attending physician will decide the dosage of the anti-demyelination material with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, anti-demyelination material to be administered, route of administration, and the severity of the condition being treated.
  • the dose of the anti-demyelination material can be about 0.001 to about 1000 mg/kg body weight of the subject being treated/day, from about 0.01 to about 100 mg/kg body weight/day, about 0.01 mg to about 50 mg/kg body weight/day, from about 1 to about to about 1000 mg/kg body weight/day, from about 5 to about 500 mg/kg body weight/day, from about 5 to about 250 mg/kg body weight/day, about 5 to about 150 mg/kg body weight/day, about 8 to about 32 mg/kg body weight/day, about 10 mg/kg body weight/day, about 2 mg/kg body weight/day to about 5 mg/kg body weight/day, or about 4 mg/kg body weight/day.
  • inventive methods can provide any amount or any level of treatment or prevention of demyelination or a
  • the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions, symptoms, or signs of the demyelinating disease, e.g., MS, or demyelination being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom, sign, or condition thereof.
  • administering the anti-demyelination material to the mammal reduces or prevents any one or more of (i) cleavage of NF155; (ii) detachment of myelin from neuronal axons; (iii) an increase in nodal gap length; (iv) dispersion (e.g., decrease in density) of neuronal sodium channels; and (v) cleavage of Casprl .
  • administering the anti-demyelination material to the mammal stimulates astrocytes to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or any combination of (i)-(iv).
  • administering the anti-demyelination material to the mammal promotes remodeling or myelin.
  • the inventive methods may, advantageously, improve visual acuity in a mammal suffering from demyelination.
  • NF155 is cleaved into two cleaved fragments (NF125 and NF30) and that this cleavage results in detachment of the myelin from the axon.
  • thrombin cleaves mouse Casprl into two cleaved fragments (Casprl i_ 380 (SEQ ID NO: 33) and Casprl 3 8 i -i 385 (SEQ ID NO: 34)) and human Casprl into two cleaved fragments (Casprl i_ 37 g (SEQ ID NO: 41) and Casprl 3 0 - i 38 4 (SEQ ID NO: 42)). It is believed that this thrombin-mediated cleavage of Casprl may result in detachment of the myelin from the axon. It has also been discovered that factor Xa cleaves mouse Casprl into two cleaved fragments (Casprl 1.947 (SEQ ID NO: 35) and
  • Casprl 94 8- i 3 8 5 (SEQ ID NO: 36)) and human Casprl into two cleaved fragments (Casprl 1.946 (SEQ ID NO: 43) and Casprl 947- i 3 84 (SEQ ID NO: 44)). It is believed that this factor Xa- mediated cleavage of Casprl may result in detachment of the myelin from the axon.
  • thrombin-mediated cleavage of NF155 causes one or both of the NF125 and NF30 fragments to detach from the oligodendrocyte and enter the blood or CSF;
  • thrombin-mediated cleavage of Casprl causes one or more of the Casprl fragments mouse Casprl i_ 38 o (SEQ ID NO: 33),mouse Casprl 38 i-i 385 (SEQ ID NO: 34), human Casprl i -379 (SEQ ID NO: 41), and human Casprl 38 o-i 384 (SEQ ID NO: 42) to detach from the oligodendrocyte and enter the blood or CSF;
  • factor Xa-mediated cleavage of Casprl causes one or more of the Casprl fragments mouse Casprl 1.947 (SEQ ID NO:
  • Casprl i. 94 6 (SEQ ID NO: 43), human Casprl 947-1384 (SEQ ID NO: 44), and human Casprl 38 o- 94 6 (SEQ ID NO: 45) may be useful biomarkers for detecting demyelination.
  • mouse Casprl 3 8 i.i385 (SEQ ID NO: 34), mouse Casprl 1.947 (SEQ ID NO: 35), human Casprl 380 -i 38 4 (SEQ ID NO: 42), and human Casprl 1.946 (SEQ ID NO: 43), and an NF155 amino acid sequence (e.g., one or both of NF125 and NF30) in blood and/or CSF indicates the presence of demyelination in a mammal.
  • an NF155 amino acid sequence e.g., one or both of NF125 and NF30
  • the inventive detecting methods may provide many advantages. For example, the inventive detecting methods may be less expensive and/or faster than magnetic resonance imaging (MRI) brain imaging. In addition, detecting methods using blood or CSF as the biological sample can be more easily performed on newborn children and other types of patients. In addition, because the node of Ranvier is one of the first locations of attack in demyelination (e.g., demyelinating diseases), the inventive detecting methods may be particularly useful for early detection of demyelination, for example, before demyelinating lesions can be detected by MRI, or before physical symptoms start to appear.
  • demyelination e.g., demyelinating diseases
  • MRI can only detect demyelination after it has progressed to the point where it is visible as lesions using myelin dyes.
  • the inventive detecting methods may also be useful for predicting a relapse in relapsing-remitting MS.
  • the inventive methods may also be useful for detecting myelin damage such as, for example, myelin damage in a newborn infant that results after a difficult delivery.
  • an embodiment of the invention provides an isolated or purified antibody, or antigen binding fragment thereof, having antigenic specificity for an NF155 amino acid sequence.
  • antigenic specificity as used herein with respect to an anti-NF155 antibody or antigen binding portion thereof, means that the particular antibody, or antigen binding fragment thereof, under consideration binds with measurably higher affinity to only one of the NF155 amino acid sequences of SEQ ID NOs: 21 -23 (in the alternative), than to other molecules.
  • inventive antibodies, and antigen binding fragments thereof are hereinafter collectively referred to as "anti-NF155 antibodies.”
  • Each of the inventive anti- NF155 antibodies described herein advantageously binds to NF155 but does not bind to other neurofascin protein family members which lack the thrombin recognition sequence GRG in the third FNIII domain such as, for example, NF186, NF180, NF166 and NF140. Therefore, the inventive anti-NF155 antibodies are able to distinguish between NF155 amino acid sequences and those of other neurofascin family members.
  • an embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21 ).
  • the amino acid sequence of SEQ ID NO: 21 is positioned upstream of the NF155 thrombin cleavage site and is unique to NF155.
  • SEQ ID NO: 21 is present in both NF155 and the cleaved NF125. Accordingly, the antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21 ) recognizes and binds to either NF155 or NF125.
  • Another embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22).
  • the amino acid sequence of SEQ ID NO: 22 is positioned immediately upstream of the thrombin cleavage site.
  • SEQ ID NO: 22 is present in both NF155 and the cleaved NF125. Accordingly, the antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of
  • LEMVVVNGR (SEQ ID NO: 22), recognizes and binds to either NF155 or NF125.
  • Another embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23).
  • SEQ ID NO: 23 includes the NF155 thrombin cleavage site. The entire amino acid sequence of SEQ ID NO: 23 is, therefore, present in NF155 but is not present in NF30 or NF125. Accordingly, the antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23) recognizes and binds to NF155.
  • Another embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the mouse Casprl 1-380 amino acid sequence of SEQ ID NO: 33, the mouse Casprl 3 8i-i 3 8 5 amino acid sequence of SEQ ID NO: 34, the mouse Casprl i _9 47 amino acid sequence of SEQ ID NO: 35, the mouse Casprl 9 48 -i 38 5 amino acid sequence of SEQ ID NO: 36, the mouse Casprl 38 i.94 7 amino acid sequence of SEQ ID NO: 39, the human Casprl 1.379 amino acid sequence of SEQ ID NO: 41, the human Casprl 3 8o-i384 amino acid sequence of SEQ ID NO: 42, the human Casprl 1.946 amino acid sequence of SEQ ID NO: 43, the human Casprl 9 47- i 3 8 4 amino acid sequence of SEQ ID NO: 44, or the human Casprl 3 8o-9
  • an embodiment of the invention provides an isolated or purified antibody, or antigen binding fragment thereof, having antigenic specificity for a Caspr 1 amino acid sequence.
  • antigenic specificity as used herein with respect to an anti-Casprl antibody or antigen binding portion thereof, means that the particular antibody, or antigen binding fragment thereof, under consideration binds with measurably higher affinity to only one of the Casprl amino acid sequences of SEQ ID NOs: 33-36 and 39 (in the alternative), than to other molecules.
  • inventive antibodies, and antigen binding fragments thereof are hereinafter collectively referred to as "anti-Casprl antibodies.”
  • inventive anti-NF155 antibodies may, advantageously, detect a mouse, rat, or human NF155 (or Casprl) amino acid sequence.
  • Methods of testing antibodies, or antigen binding fragments thereof, for the ability to recognize antigen and for antigen specificity are known in the art. Examples of such methods may include immunoprecipitation, immunonephelometry, radioimmunoassay (RIA), immunohistochemistry, enzyme immunoassay (EIA), fluorescent immunoassay (FIA), enzyme-linked immunosorbent assay (ELISA), and the like.
  • inventive anti-NF155 antibodies and anti-Casprl antibodies can be of any type.
  • the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc.
  • the antibody can be monoclonal or polyclonal.
  • the antibody can be a naturally-occurring antibody, e.g., an antibody isolated and/or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.
  • the antibody can be a genetically- engineered antibody, e.g., a humanized antibody or a chimeric antibody.
  • the antibody can be in monomeric or polymeric form.
  • the antigen binding fragment of the antibody can be any fragment of the antibody that has at least one antigen binding site.
  • the antigen binding fragment is a Fab fragment (Fab), F(ab')2 fragment, diabody, triabody, tetrabody, single-chain variable region fragment (scFv), or disulfide-stabilized variable region fragment (dsFv).
  • Fab Fab fragment
  • F(ab')2 fragment diabody, triabody, tetrabody
  • scFv single-chain variable region fragment
  • dsFv disulfide-stabilized variable region fragment
  • a single- chain variable region fragment (scFv) which is a truncated Fab fragment including the variable (V) domain of an antibody heavy chain linked to a V domain of a light antibody chain via a synthetic peptide, can be generated using routine recombinant DNA technology techniques (see, e.g., Murphy et al.
  • dsFv disulfide-stabilized variable region fragments
  • inventive antibodies and antigen binding fragments thereof can be isolated and/or purified, as described herein with respect to other aspects of the invention.
  • an embodiment of the invention provides a method of detecting the presence of demyelination in a mammal, the method comprising: (a) contacting a biological sample comprising blood and/or CSF with at least one of any of the inventive antibodies, or antigen binding fragments thereof, described herein, thereby forming a complex, and (b) detecting the complex, wherein detection of the complex is indicative of the presence of demyelination in the mammal.
  • the demyelination may be as described herein with respect to other aspects of the invention.
  • Detection of the complex can occur through any number of ways known in the art.
  • the inventive antibodies, or antigen binding fragments thereof, described herein can be labeled with a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).
  • a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).
  • the method comprises (a) contacting the biological sample with a first antibody, or antigen binding fragment thereof, selected from the group consisting of (i) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21), (ii) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22), or (iii) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23), thereby forming a first complex; (b) contacting the first complex with a second antibody, or antigen binding fragment thereof, selected from the group consisting of (i) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21), (
  • the first antibody, or antigen binding fragment thereof is the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21)
  • the second antibody, or antigen binding fragment thereof is the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22).
  • the first antibody, or antigen binding fragment thereof is the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23) and the second antibody, or antigen binding fragment thereof, is the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22).
  • the biological sample may be as described herein with respect to other aspects of the invention.
  • the blood is umbilical cord blood.
  • the method comprises washing any unbound first antibody and any unbound antigen from the first complex.
  • the method may further comprise washing any unbound second antibody and any unbound antigen from the second complex.
  • the method further comprises detecting the second complex.
  • the second complex may be detected in any suitable manner known in the art.
  • detecting the second complex comprises contacting the second complex with a detecting agent.
  • the detecting agent may comprise, for example, a third antibody or antigen binding fragment thereof that specifically binds to the second complex (hereinafter referred to collectively as "detection antibody” or “detection antibodies”).
  • the detection antibody binds to the second complex with measurably higher affinity to the second complex than to other molecules.
  • the detection antibody specifically binds to the second antibody.
  • the detection antibody comprises a detectable label.
  • the detectable label may be any suitable detectable label that provides a detectable signal.
  • detectable labels include enzymes (e.g., alkaline phosphatase, horseradish peroxidase), a radioisotope, fluorescent molecules (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), luminescent molecules, dyes, biotin, and element particles (e.g., gold particles).
  • the detectable label is an enzyme
  • the method may further comprise adding a substrate to the second complex that is catalyzed by the enzyme to produce a detectable signal.
  • the detectable signal may be amplified and/or visually detected. For example, acetylcholinesterase-catalyzed hydrolysis may be useful for colorimetric detection through gold or silver nanoparticle aggregation.
  • Another embodiment of the invention provides a method of detecting the presence of demyelination in a mammal, the method comprising: (a) contacting a biological sample comprising blood and/or cerebral spinal fluid (CSF) with an agent, thereby forming a complex between (i) the agent and (ii) an antibody, or antigen binding fragment thereof, having antigenic specificity for the agent, and (b) detecting the complex, wherein detection of the complex is indicative of demyelination in the mammal, wherein the agent is NF125, NF30, the mouse Casprl ⁇ .
  • CSF cerebral spinal fluid
  • the contacting and detecting may be carried out as described herein with respect to other aspects of the invention.
  • Casprl 3 8 o-i 38 4 amino acid sequence of SEQ ID NO: 42, the human Casprl 1.946 amino acid sequence of SEQ ID NO: 43, the human Casprl 94 7 _i 384 amino acid sequence of SEQ ID NO: 44, and the human Casprl 380 -9 46 amino acid sequence of SEQ ID NO: 45 may be as described herein with respect to other aspects of the invention.
  • the method comprises (a) contacting a biological sample comprising blood and/or CSF with NF125 and NF30, thereby forming a first complex with an antibody, or antigen binding fragment thereof, having antigenic specificity for NF125 and forming a second complex with an antibody, or antigen binding fragment thereof, having antigenic specificity for NF30 and (b) detecting the first and second complexes, wherein detection of the complexes is indicative of demyelination in the mammal.
  • Another embodiment of the invention provides a method of detecting
  • demyelination in a mammal comprising (a) contacting a biological sample comprising blood and/or cerebral spinal fluid (CSF) with an antibody having antigenic specificity for mouse Casprl 38 i -9 47 (SEQ ID NO: 39) or human Casprl 38 o-946 (SEQ ID NO: 45) and (b) detecting the complex, wherein detection of the complex is indicative of demyelination in the mammal.
  • the method may, for example, use a sandwich enzyme-linked immunosorbent assay (ELISA) system.
  • the sandwich ELISA system may use two antibodies, each antibody having antigenic specificity for Factor Xa cleaved non-membrane anchored fragments of Casprl comprising mouse Casprl 3 ⁇ -947 (SEQ ID NO: 39).
  • the sandwich ELISA system may use two antibodies, each antibody having antigenic specificity for Factor Xa cleaved non- membrane anchored fragments of Casprl comprising human Casprl 380-946 (SEQ ID NO: 45).
  • Epitopes to generate the antibodies may be derived from mouse Casprl 38 1 -947 (SEQ ID NO: 39) . Such antibodies will only detect the full-length mouse Casprl (SEQ ID NO: 32) and the Factor Xa cleaved non-membrane anchored fragments of mouse Casprl, i.e., mouse Casprl ⁇ . ⁇ (SEQ ID NO: 35) and will not detect the membrane-bound fragment of Casprl , i.e., mouse Casprl 948-1385 (SEQ ID NO: 36). Epitopes to generate the antibodies may be derived from human Casprl 38 o-946 (SEQ ID NO: 45).
  • Such antibodies will only detect the full-length human Casprl (SEQ ID NO: 40) and the Factor Xa cleaved non-membrane anchored fragments of human Casprl , i.e., human Casprl 1.946 (SEQ ID NO: 43) and will not detect the membrane-bound fragment of Casprl , i.e., human Casprl 9 47- 1 3 84 (SEQ ID NO: 44).
  • the contacting can take place in vitro or in vivo with respect to the mammal.
  • the contacting is in vitro.
  • the detecting methods comprise detecting the presence of a demyelinating disease in the mammal.
  • the demyelinating disease may be as described herein with respect to other aspects of the invention.
  • the demyelination is associated with white matter injury.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
  • n 10, 180 nodes of t-test frequency of 4
  • N 2 Dox-off mice for 5 day intensities of 3 I Work in EGFP in
  • Example corpus callosum N 3 Dox off mice P ⁇ 0.001 animal
  • n number of fields.
  • Table 1 shows the experimental design, sampling size, and statistical analysis for the major experiments in the Examples. Statistical analysis was based on unpaired Student's t-test for two group comparisons of continuous variables that were normally distributed.
  • Two nerves per animal ware analyzed from multiple animals. Sampling was evenly balanced among different animals (4 microscope fields/animal; 3-4 mice for the data in Example 3 (measuring nodal length in optic nerve of Dox-on and Dox-off mice), for example. For example; 22,400 nodes were measured for this experiment, (400 nodes X 56 fields in Dox On), but only an "n” of 56 was used for the statistical analysis, and this sampling was balanced evenly among 4 animals.
  • Astrocyte specific glial fibrillary acidic protein (GFAP) promoter drives the expression of the "tet-OFF" tetracycline transactivator: GFAP.tTA (Ye et al., J. Neurosci Res., 78: 472-484 (2004)).
  • Another transgenic line expresses a tet operator (tetO)-regulated dnVAMP2 domain and enhanced green fluorescent protein (EGFP) and LacZ reporter genes: tetO.VAMP2.
  • tetO tet operator
  • EGFP enhanced green fluorescent protein
  • LacZ reporter genes tetO.VAMP2. Details of producing GFAP.tTA and tet0.dnVAMP2 lines are as described elsewhere (Pascual et al., Science, 310: 1 13-1 16 (2005)). These lines were maintained in a heterozygous state and backcrossed onto a C57B16/J genetic background (Jackson
  • mice were housed under specific pathogen-free conditions under a 12-hour (h) light/dark cycle with access to food and water ad libitum according to a protocol approved by National Institutes of Health (NIH) Animal Care and Use Committee.
  • NASH National Institutes of Health
  • Astrocyte-specific expression was confirmed in optic nerve, retina, corpus callosum, and visual cortex by immunocytochemistry and by western blot for optic nerve and cortex. No transgene expression in cells other than astrocytes could be detected in these areas.
  • Western blot and immunocytochemistry were used to determine the time-course of gene regulation by doxycycline treatment (Example 3). The transgene was fully suppressed within 5 days of adding Dox to the diet (Example 5). After removing Dox from the diet, approximately 14 days are required to clear Dox from the system and reach maximum transgene expression (Example 3).
  • Dox was removed from the mother's diet after fertilization. To inhibit transgene expression, Dox was supplied continuously to the mothers during gestation and until weaning, and then supplied directly to pups after weaning.
  • mice were deeply anesthetized with isoflurane and perfused with 4%
  • OHg2 (rabbit polyclonal antibody, 1 :400, IBL, Takasaki, Japan) was used as a pan-oligodendrocyte marker.
  • Anti-GFAP antibody (rabbit polyclonal IgG, 1 : 100, Invitrogen, Camarillo, CA, US) was applied to identify astrocytes. Images were captured by confocal laser microscope (LSM510, Zeiss, Germany). Nodal length measurement
  • mice were deeply anesthetized and perfused with 2.5% gluteraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4. Optic nerves were immediately removed from animals, then post-fixed in the same solution for 2 hours (hr). Samples were post-fixed for 1 hr in 1 % osmium tetroxide in 0.1 M sodium cacodylate, pH 7.4. Samples were washed two times in 0.05 M sodium acetate, pH 5.0, and stained with 1% uranyl acetate in 0.05 M sodium acetate, pH 5.0, for 2 hr at 4 °C.
  • Optic nerves were fixed, embedded and stained according to protocols provided by Renovo Neural Inc. (Cleveland, OH). Both nerves from each animal were cut into 3-4 segments and mixed together for microscopic analysis. Serial block face imaging was performed with 15 nm/pixel, 10 nm/pixel, 7.6 nm/pixel, and 5 nm/pixel resolution on 75 nm, 80nm, and 135 nm thick slices, and photographed using 16bit digitization. Field of view typically ranges from 40-46 ⁇ , at a magnification of 13,800 to 15,200 X. Beam voltage of 2 kV was typically used and approximately 100 slices imaged in z-series stacks.
  • Imaging was also performed at 5.4k, 6.1 , 13k, 15k, 20k, 30k, 99k.
  • Serial block face electron microscopy was performed at Renovo Neural, Inc, without knowledge of experimental condition and returned to the lab for analysis and quantitation.
  • Serial 3D reconstruction was done in the laboratory and at Renovo, Inc.
  • Serial image stacks were registered and cropped using ImageJ/FIJI. Axons, compact myelin, paranodal loops and astrocyte processes were traced manually using RECONSTRUCT software and meshes exported and rendered using BLENDER software (Blender.org).
  • NF155, NF125, NF30, NF186 Neuron specific Enolase (NSE), Casprl, myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and
  • GPDH glyceraldehyde 3 -phosphate dehydrogenase
  • PNGase F Peptide-N-Glycosidase F
  • PNGase F Peptide-N-Glycosidase F
  • Membranes were probed with appropriate antibodies in TBS-T and 5% BSA overnight at 4 °C. Primary antibodies were visualized with HRP- conjugated secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) at 1 :5000 dilution and enhanced chemiluminescence. Nonsaturated immunoblots were quantified using IMAGE J and/or METAMORPH software and normalized to GAPDH and/or Enolase.
  • GAPDH Endometrial phosphatidylase
  • MBP Microsomal Protein
  • GFAP Zymed Laboratories San Francisco, CA
  • EGFP Abeam, Cambridge, MA
  • NF186 and NF30 Abeam, Cambridge, MA
  • NSE Abeam, Cambridge, MA
  • Casprl Santa Cruz Biotech., Santa Cruz, CA
  • RNA expression analysis 2 ig of total RNA was reverse-transcribed using SUPERSCRIPT II and oligo-dT, and polymerase chain reaction (PCR) performed on a Roche LIGHTCYCLER instrument (Roche, Minneapolis, MN) using FASTSTART DNA Master SYBR Green 1 PCR reaction mix. Relative expression levels were quantified. Primer sequences were as follows:
  • GLT-1 TTGACTCCCAACACCGAATGC (SEQ ID NO: 7),
  • KCNJ10 Kir4.1: TGCTGGAGCCCTTCCTTTTCC (SEQ ID NO: 1 1)
  • AACACTCATGCCCGTGACC (SEQ ID NO: 14), • GAPDH: AATGCATCCTGCACCACCAAC (SEQ ID NO: 15),
  • MBP GCCAAATGTCACCATCTCTCC, ACTCCTGCAGTCCCACTTCC.
  • ExPASy PEPTIDECUTTER tool (Wilkins et al., Methods Mol. Biol, 112: 531-52 (1999)) was used to look for potential protease sites in Neurofascin 155 (NF155).
  • Universal Protein Resource (UniProt) was used to assess the cellular localization of the Thrombin cleavage site in NF155. Hydrophilicity, surface probability and solvent accessibility of domains within NF155, including the third Fibronectin Type III domain containing the Thrombin cleavage site, were assessed by Kyte-Doolittle hydropathy analysis ( yte and Doolittle., J. Mol.
  • I-TASSER Intelligent Threading ASSEmbly Refinement
  • CASP Critical Assessment of Techniques for Protein Structure Prediction
  • LC-MS Liquid chromatography-mass spectrometry
  • Subcortical white matter from Dox-OFF animals was run in a SDS-PAGE gel with size- appropriate protein ladders. Bands on the gel corresponding to 155KDa, 125KDa and 30KDa were excised and sent to the LC-MS facility, NIH for downstream analysis.
  • Fondaparinux Sodium is a synthetic pentasaccharide that mimics the minimum necessary pentasaccharide sequence in Heparin which binds and activates Antithrombin-III (AT-III). It is an ultra-low molecular weight selective Factor Xa inhibitor that can cross placental and blood-brain barriers (Hoppensteadt et al., Hematol. Oncol. Clin. North Am., 17(1): 313-341 (2003)). Factor Xa converts prothrombin to thrombin and AT-III inhibits this reaction by competitively binding to the catalytic site of Factor Xa.
  • microcontinuum composite cable model consisting of an infinite array of nodes of Ranvier separated by myelinated internodal segments (Basser et al., Medical & Biological
  • this macroscopic model can be used to predict the macroscopic or aggregate current and electrical potential distributions along the axon from individual electrical properties and microscopic dimensions of the nodal and myelinated regions.
  • Effective speed of propagation, c as defined in Basser, J. Integr. Neurosci., 3(2): 227-44 (2004), can be written as:
  • the ratio of the effective space and time constants has the unit of speed, while the ratio of the square of the effective space constant and time constant can be interpreted as an electrical diffusivity. Both quantities can help assess how efficiently a disturbance in the transmembrane potential is transported along the myelinated axon. Therefore, this framework allowed prediction as to how changes in the width of the node or nodal capacitance will affect how electrical disturbances will migrate, with the caveat that the model does not predict the actual propagation speed which is much larger and would depend on many other factors not studied here. It is not expected to be a good indicator of the relative changes in the speed, in terms of the changes in the nodal region width, delta ( ⁇ ), and the G-ratio.
  • Optic nerves were dissected from adult (2-4-month-old) mice in which vesicle fusion in astrocytes was inhibited (Dox-off) or transgene expression was inhibited by doxycycline (Dox-on). Optic nerves (4-5 mm of length) were dissected free and cut at the optic chiasm and behind the orbit.
  • the preparation was placed in an interface perfusion chamber (Medical Systems), maintained at 35 °C and superfused with artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 4 KC1, 25 NaHC0 3 , 1.25 NaH 2 P0 4 , 2.5 CaCl 2 , 1.5 MgCl 2 , and 25 glucose, pH 7.4.
  • the chamber was continuously aerated with humidified gas mixture of 95% 0 2 /5% C0 2 .
  • ACSF ran continuously at 2-3 ml/min. Following dissection, optic nerves were allowed to equilibrate for about 30 minutes (min) before the experiment was started.
  • Suction electrode back filled with ACSF was attached to the rostral end (retina side) and stimulated every 10 seconds (s) (A365R stimulus isolator, WPI).
  • a recording suction electrode filled with ACSF was attached to the caudal end (adjacent to the chiasm) of the nerve to record CAPs. The distance between the electrodes was measured with a calibrated ocular reticule. Evoked potentials were amplified with Axoclamp 2B (Molecular Devices, Sunnyvale, CA) and acquired on-line (Digidata 1200 A, Molecular Devises, Sunnyvale, CA) using PCLAMP software (Molecular Devices, Sunnyvale, CA).
  • Strength-duration curves were determined by recording responses to a series of square pulses of 0.05 to 0.2 mA intensities of 0.01 to 0.2 ms stimulus durations, and were used to determine rheobase and chronaxie; data in Example 5 are from supramaximal stimulation using 0.05 ms duration stimuli.
  • CAP compound action potential
  • B(t) is the part of the CAP attributed to the stimulus artifacts
  • ) is the residual of the fit.
  • the features of B(t) are the initial pair of sharp peaks, which is a capacitive response to the application of a square pulse, and a slight shift in the baseline that persists until after 6 ms.
  • each individual curve was fit separately (single curve fitting, SF), and in the other, multiple curves, corresponding to a set of n c strongest stimulus strengths, were fit simultaneously (multiple curve fitting, MF).
  • MF relies on the fact that the strength of the stimulus did not have a major influence on the position or the width of the peaks, but changes only their amplitude.
  • this type of center-of-mass (COM) estimate can be made independent of the fitting procedure.
  • the COM was calculated for each data curve, by weighting each point in time series by its amplitude, obtaining essentially the arrival mean time, if the curve is interpreted as a probability density function of spike time arrivals.
  • the COM was calculated for each data curve, by weighting each point in time series by its amplitude, obtaining essentially the arrival mean time, if the curve is interpreted as a probability density function of spike time arrivals.
  • the precise choice of the limits is not important as long as the artifact peak, located in most curves between 0.13 and 0.15 ms, is excluded.
  • PLSE ⁇ % for virtually all CAPs with reasonable stimulus strength.
  • Example 5 The PISE for the examples described in Example 5 were 0.65% for Dox-on and 1% for Dox-off (the worst fit obtained for Dox-off). Since the fits were not perfect, a number of other more robust measures of latency, but less specific, were used. In Example 5, the comparisons were summarized using the bar charts and using a number of different measures of temporal latency. It was observed that the Dox-off time latency is larger no matter what measure is used. Averaged over all measures Dox-on times were 14% ⁇ 1% shorter. This difference was further quantified using hypothesis tests, a non-parametric Mann-Whitney- Wilcoxon (MWW) test, and a Mest which relies on normality of the data.
  • MWW non-parametric Mann-Whitney- Wilcoxon
  • VEP and ERG electrophysiology were performed using the ESPION e2 software from Diagnosys, LLC.
  • the mice were anesthetized with an intraperitoneal injection of xylazine-ketamine (10 mL/kg) in normal saline. Body temperature was maintained at 37 °C with a heating pad. Pupils were dilated with atropine (1%).
  • ERG recordings a gold wire electrode was placed on the corneal surface of each eye and reference to a gold wire in the mouth.
  • VEP recordings 2 needle electrodes were inserted subcutaneously to touch the skull above the visual cortex. A needle electrode in the base of the tail served as the ground. Animals were light-adapted for all recordings. Responses were obtained to 6 different light intensities, covering a range eliciting threshold to supramaximal responses.
  • Exocytosis of ATP from astrocytes is inhibited approximately 50%, without measurable effects on glutamate release, in transgenic mice expressing a doxycycline (Dox) regulated dominant-negative VAMP2-fragment, vesicle associated membrane protein 2 (dnVAMP2), which is expressed specifically in astrocytes by the astrocyte-specific promoter of GFAP (Pascual et al., Science, 310: 113-116 (2005);
  • Dox doxycycline
  • dnVAMP2 vesicle associated membrane protein 2
  • GFAP cytoskeletal filaments observed by confocal microscopy were more diffuse in optic nerve astrocytes in the Dox-off condition consistent with the formation of robust bundles of GFAP cytoskeleton in astrocytes treated with adenosine or ATP.
  • Ultrastructural analysis by transmission electron microscopy (EM) and serial block-face scanning EM revealed fine structural changes in glial intermediate filaments in optic nerve astrocytes, and the morphology of perinodal astrocytes changed after inhibiting exocytosis in astrocytes.
  • EM Transmission electron microscopy
  • perinodal astrocytes partly ensheath the nodal gap and associate with axons through blunt terminals in close contact with the axon in the nodal region (Black et al., Glia, 1 : 169-83 (1988); Raine et al., J. Neurocytology, 13: 21 -27 (1984)) as shown in the Dox-off condition.
  • TEM analysis showed that perinodal astrocytes withdrew from the node and contacted the axon through long filopodial processes that lacked accumulations of intermediate filaments when exocytosis from astrocytes was inhibited.
  • the normal morphology (blunt) was seen in the majority of nodes of Ranvier in animals on Dox (45%), but the majority of nodes in the Dox off condition (80%) had filopodial contacts with the axon.
  • paranodal lopes are cytoplasmic pockets of uncompacted myelin membrane attached to the axon through a tri-molecular complex of cell adhesion molecules: Neurofascin 155 (NF155) in the paranodal myelin membrane, Casprl in paranodal axon membrane, and axonal Contactinl interacting with these two molecules form septate-like axo-glial junctions.
  • Neurofascin 155 NF155
  • Casprl in paranodal axon membrane
  • axonal Contactinl interacting with these two molecules form septate-like axo-glial junctions.
  • Bioinformatic analysis revealed a potential thrombin cleavage site in the extracellular domain of NF155 at AA924 in mouse (AA910 in rat), a region critical for interacting with Contactin-1.
  • Thrombin participates in activity-dependent synapse elimination at neuromuscular junctions (Liu et al., Nature Neurosci., 15: 1621-1623 (1994)), and cleavage of NF 155 at AA924 would disrupt its binding to Contactin-1 , breaking the axo- glial junction attaching paranodal loops to the axon.
  • Validity of the putative thrombin binding site on NF155 was determined by thrombin treatment of subcortical white matter extract.
  • Two antibodies were used to provide independent confirmation of cleavage of NF155 into two fragments: a long 125 kDa fragment, Neurofascin 125 (NF125), and a short 30 kDa fragment, Neurofascin 30 (NF30).
  • One antibody binds with amino acid sequences that are N- terminal to the thrombin cleavage site, thus recognizing the full length NF155 and the longer cleaved fragment, NF125, but not the shorter NF30 fragment.
  • the second antibody binds amino acid sequences C-terminal (intracellular) to the thrombin cleavage site, a domain that is conserved among all neurofascin family members, (NF186, 180, 166, and 155), but it will not recognize the NF125 fragment.
  • perinodal astrocytes may also regulate enzymatic cleavage of the cell adhesion molecules forming the junctions between paranodal loops and the axolemma.
  • Astrocytes in culture release antithrombin III, which inhibits thrombin activity, (Deschepper et al., Brain Res.
  • Dox-on animals showed the typical ultrastructure of regularly arranged paranodal loops of myelin attached to the axon by intercellular septate junctions.
  • paranodal loops of myelin were often irregular and probed by astrocyte filopodia and in some cases, paranodal loops were dislodged from the axon adjacent to astrocyte filopodia after inhibiting exocytosis from astrocytes in Dox-off animals.
  • many of the nodal gap lengths exceeded the maximal length measured in the Dox-on condition as evidenced by cumulative probability plots. This is evident by the two nodes shown in the serial block face EM section taken from optic nerve of a Dox-off animal.
  • the increased nodal gap length resulting from detachment of the paranodal loops adjacent to the perinodal astrocyte should be accompanied by reduced myelin thickness if the outer layer of myelin associated with these loops is resorbed back into the oligodendrocyte.
  • the mean increase in nodal gap length in the Dox-off condition corresponds to a loss of approximately 2 paranodal loops of myelin. This would predict a decrease in myelin sheath thickness corresponding to two wraps of compact myelin.
  • mature myelin is structurally dynamic and regulated by exocytosis in astrocytes.
  • Perinodal astrocytes acting through VAMP2-dependent exocytosis, regulate nodal structure and myelin thickness dynamically by regulating thrombin-dependent cleavage of axo-glial junctions attaching the outermost paranodal loops of myelin to the axon membrane.
  • mice were housed in the dark and optic nerves were analyzed. Nodal gap length in wild-type animals dark-reared from P21 to P60 was not significantly different from animals reared under normal conditions. Thus, the widening of the nodal gap in the Dox-off animals was not produced by perturbing normal functional activity. In the absence of normal sensory input, myelination appeared normal. However, functional activity was found to promote the recovery process of restoring normal gap length after the nodal gap widening produced by inhibiting exocytosis from astrocytes.
  • VEP latency might have resulted from inhibiting VAMP2- dependent exocytosis of neurotransmitters from astrocytes associated with synapses in gray matter was tested.
  • Western blot and immunocytochemistry confirmed that expression of the dnVAMP2 gene was blocked, thus restoring exocytosis from astrocytes, and thereby any hypothetical effects of gliotransmitters on retinal synapses.
  • ATP release from axons firing action potentials also increases myelination of unmyelinated axons as oligodendrocyte precursor cells (OPCs) differentiate to a
  • LIF cytokine leukemia inhibitory factor
  • myelin is normal in LIF -/- animals, but myelin structure is altered by exocytosis of ATP from astrocytes throughout life.
  • EAE autoimmunity against myelin
  • MOG peptide which is a component of myelin, and pertussis toxin.
  • the MOG peptide induces an autoimmune response to myelin.
  • Pertussis toxin over-activates the immune system in general. The day of EAE induction was day zero and all time elapsed was measured from that date.
  • EAE was induced in mice, and the EAE mice were injected with either fondaparinux sodium or PBS (control), as described in Example 6. The incidence of disease was measured daily. The results are shown in Figure 3.
  • dnSNARE is a genetic mouse model that selectively interferes with astrocyte signaling via conditional expression of a dominant-negative SNARE protein under control of the GFAP promoter. In the dnSNARE mouse, myelin thickness is reduced and the nodal gap is increased.
  • the animal has a normal (wild-type) phenotype when the animal is fed doxycycline (Dox) (Dox-ON condition).
  • Dox doxycycline
  • doxycycline is removed from the diet, (Dox-OFF condition), vesicular release from astrocytes is partially inhibited.
  • Mastoparan (MP) is a drug that induces vesicular release.
  • MP was administered to astrocytes in cell culture from wild-type (WT) mice, transgenic mice in the Dox-ON condition, and transgenic mice in the Dox-OFF condition to induce vesicular release (including PN1 release).
  • the amount of PN-1 released into the culture medium was measured and normalized to total protein. The results are shown in Figure 4.
  • NF155 is cleaved by thrombin in the Dox- OFF condition (causing septate junctions to break, paranodal loops to come off, nodal gap to increase, and myelin to thin).
  • this cleavage by thrombin is facilitated in the Dox-OFF condition because astrocytes in the Dox-OFF condition (that is, with partially inhibited vesicular release) secrete less Protease-Nexin 1 (PN1), a potent thrombin inhibitor.
  • PN1 has been shown to be released via vesicles from astrocytes. Accordingly, perinodal astrocytes have a role in modulating myelin thickness by regulating thrombin dependent cleavage of NF155 via PN-1.
  • Casprl has a thrombin cleavage site (RRG) (SEQ ID NO: 37) at amino acid positions 380-382. Three dimensional protein structure modeling showed that the thrombin cleavage site is located on the surface of the folded protein. Cleavage of Casprl by thrombin was expected to yield a short thrombin-cleaved Casprl fragment of about 60 kDa and a longer thrombin-cleaved Casprl fragment of about 120 kDa.
  • RRG thrombin cleavage site
  • a sample of full-length Casprl alone or samples of Casprl which had been contacted in vitro with 0.04, 0.02, 0.01 , or 0.005 units of thrombin were placed on a gel and analyzed by Western blot.
  • a band corresponding to the expected 60 kDa thrombin-cleaved Casprl fragment was observed for those Casprl samples that were contacted with thrombin in vitro.
  • No band corresponding to the 60 kDa Casprl fragment was observed for the Casprl sample which was not contacted with thrombin.
  • Casprl samples were obtained from transgenic mice in the Dox-ON or Dox-OFF condition. The samples were placed on a gel and analyzed by Western blot. Enolase was used as a loading control.
  • Casprl has a factor Xa cleavage site (LEGR) (SEQ ID NO: 38) at amino acid positions 937-940. Three dimensional protein structure modeling showed that the factor Xa cleavage site is located on the surface of the folded protein. Cleavage of Casprl by factor Xa was expected to yield a short factor Xa-cleaved Casprl fragment of about 50 kDa and a longer factor Xa-cleaved Casprl fragment of about 130 kDa.
  • LGR factor Xa cleavage site
  • Casprl samples were obtained from transgenic mice in the Dox-ON or Dox-OFF condition. The samples were placed on a gel and analyzed by Western blot. Enolase was used as a loading control.

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Abstract

Disclosed are methods of treating or preventing demyelination in a mammal, the method comprising administering to the mammal a (1) thrombin inhibitor or (2) compound that stimulates astrocytes in the mammal to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or (v) any combination of (i)-(iv) in an amount effective to treat or prevent the demyelination in the mammal. Also disclosed are isolated or purified antibodies, or antigen binding fragments thereof, having antigenic specificity for neurofascin 155 (NF155) or Caspr1 amino acid sequences and methods of detecting the presence of demyelination in a mammal using one or more of the antibodies.

Description

METHODS OF TREATING OR PREVENTING DEMYELINATION USING THROMBIN INHIBITORS AND METHODS OF DETECTING DEMYELINATION USING
NEUROFASCIN 155
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/148,802, filed April 17, 2015, which is incorporated by reference in its entirety herein.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED
ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a computer-readable
nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 123,299 Byte ASCII (Text) file named "723232_ST25.TXT," dated April 15, 2016.
BACKGROUND OF THE INVENTION
[0003] Myelin is the electrical insulation on nerve fibers (axons) that is involved in the normal transmission of electrical impulses. Many neurological disorders are the result of myelin damage (e.g., demyelination), for example multiple sclerosis (MS), cerebral palsy, and many other leucodystrophies resulting from toxic effects on the cells that make myelin (oligodendrocytes); for example, hypoxia, ischemia, viral infection, premature birth, and autoimmune disorders. Despite advancements in the detection and treatment of
demyelination, there exists a need for improved compositions and methods for treating, preventing, and detecting demyelination.
BRIEF SUMMARY OF THE INVENTION
[0004] An embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a thrombin inhibitor to the mammal in an amount effective to treat or prevent the demyelination in the mammal.
[0005] Another embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a compound to the mammal in an amount effective to stimulate astrocytes in the mammal to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or (v) any combination of (i)-(iv) in an amount effective to treat or prevent the demyelination in the mammal.
[0006] Other embodiments of the invention provide isolated or purified antibodies, or antigen binding fragments thereof, having antigenic specificity for neurofascin 155 (NF155) amino acid sequences and methods of detecting the presence of demyelination in a mammal using one or more of the antibodies.
[0007] Another embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the mouse Casprl 1-38o amino acid sequence of SEQ ID NO: 33, the mouse Casprl 38i-1385 amino acid sequence of SEQ ID NO: 34, the mouse Casprl 1-947 amino acid sequence of SEQ ID NO: 35, the mouse Casprl948_i385 amino acid sequence of SEQ ID NO: 36, the mouse Casprl38i-947 amino acid sequence of SEQ ID NO: 39, the human Casprl 1-379 amino acid sequence of SEQ ID NO: 41, the human Casprl 38o-i384 amino acid sequence of SEQ ID NO: 42, the human Casprl 1-946 amino acid sequence of SEQ ID NO: 43, the human Casprl 947-1384 amino acid sequence of SEQ ID NO: 44, or the human Casprl38o-946 amino acid sequence of SEQ ID NO: 45.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0008] Figure 1 is a graph showing the clinical score of EAE mice following treatment with fondaparinux sodium (circles) or PBS (squares) each day up to 30 days after induction of EAE (post-immunization).
[0009] Figure 2 is a Kaplan-Meier survival curve showing the percentage of EAE mice surviving following treatment with fondaparinux sodium (solid line) or PBS (dotted line) each day up to 30 days after induction of EAE (post-immunization).
[0010] Figure 3 is a graph (incidence curve) showing the percentage of EAE incidence in EAE mice treated with fondaparinux sodium (solid line) or PBS (dotted line) at various time points (days) after induction of EAE (days elapsed).
[0011] Figure 4 is a graph showing the amount of PN-1 released from mouse astrocytes in cell culture via SNARE-dependent exocytosis, relative to total protein (pg^g) measured in cultures of astrocytes from WT mice without mastoparan (MP) stimulation, WT with (MP) stimulation, astrocytes from DOX-on mice after MP stimulation, and astrocytes from DOX- off mice after MP stimulation. MP induces exocytosis by stimulating GTPase activity (activating Gi and Go proteins). ANOVA F3,22=14.13 pO.0001 ; Posthoc Tukey's
***P<0.001.
DETAILED DESCRIPTION OF THE INVENTION
[0012] Myelin is attached to the axon membrane by the cell adhesion molecule neurofascin 155 in the region adjacent to the node of Ranvier (paranodal region). It has been discovered that perinodal astrocytes regulate enzymatic cleavage of NF155 at a thrombin cleavage site, allowing detachment of the myelin from the axon. Thrombin cleaves NF155 into two fragments: NF125 and NF30. Full-length human NF155 has the amino acid sequence of SEQ ID NO: 24 (Genbank Acession No. NP_001153803.1). The thrombin cleavage site of human NF155 (as well as mouse NF155) has the amino acid sequence of GRG (human NF155924-926; SEQ ID NO: 31), with the cleavage occurring after the arginine in GRG (SEQ ID NO: 31). Human NF125 has the amino acid sequence of SEQ ID NO: 25, and human NF30 has the amino acid sequence of SEQ ID NO: 26.
[0013] NF155 clusters at the paranodal regions of the myelin sheath where it localizes in apposition to the axonal adhesion molecule contactin-associated protein (Casprl), which is a constituent of the septate junction-like axo-glial adhesion zone. Full-length mouse Casprl has the amino acid sequence of SEQ ID NO: 32. Full-length human Casprl has the amino acid sequence of SEQ ID NO: 40. It has also been discovered that thrombin cleaves Casprl into two fragments: the amino acid sequence of SEQ ID NO: 41 (human Casprl 1 -379) and the amino acid sequence of SEQ ID NO: 42 (human Casprl38o-i384). In mice, the thrombin- cleaved fragments are the amino acid sequence of SEQ ID NO: 33 (mouse Casprl i-38o) and the amino acid sequence of SEQ ID NO: 34 (mouse Casprl 38i-i38s)- The thrombin cleavage site of human and mouse Casprl has the amino acid sequence of RRG (mouse Casprl 38o-382; human Casprl379-381 ; SEQ ID NO: 37), with the cleavage occurring between the arginine residues in RRG (SEQ ID NO: 37).
[0014] It has also been discovered that factor Xa cleaves Casprl into two fragments: the amino acid sequence of SEQ ID NO: 43 (human Casprl !_946) and the amino acid sequence of SEQ ID NO: 44 (human Casprl 947-i384). In mice, the factor Xa-cleaved fragments are the amino acid sequence of SEQ ID NO: 35 (mouse Casprl 1,947) and the amino acid sequence of SEQ ID NO: 36 (mouse Casprlg^-nss). The factor Xa cleavage site of human and mouse Casprl has the amino acid sequence of LEGR (mouse Casprl 957.940; human Casprl 943-940; SEQ ID NO: 38), with the cleavage occurring after the arginine residue in LEGR (SEQ ID NO: 38).
[0015] Without being bound to a particular theory or mechanism, it is believed that reducing or preventing thrombin biological activity, e.g., thrombin-mediated cleavage of one or both of Casprl and NF155, may reduce or prevent demyelination and/or promote recovery after myelin damage. Accordingly, an embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a thrombin inhibitor to the mammal in an amount effective to treat or prevent the
demyelination in the mammal.
[0016] Without being bound to a particular theory or mechanism, it is believed that reducing or preventing factor Xa biological activity, e.g., factor Xa-mediated cleavage of Casprl, may reduce or prevent demyelination and/or promote recovery after myelin damage. Accordingly, an embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a factor Xa inhibitor to the mammal in an amount effective to treat or prevent the demyelination in the mammal.
[0017] The thrombin inhibitor can be any agent that inhibits the biological activity of thrombin. Thrombin is secreted as the inactive prothrombin, which is then converted into active thrombin by the enzyme Factor Xa. The biological activity of thrombin may be inhibited in any manner, e.g., by inhibiting the conversion of prothrombin to thrombin (e.g., by inhibiting the activity of enzyme Factor Xa); by inhibiting the expression of one or both of thrombin mRNA and thrombin protein; by inhibiting the binding of thrombin to NF155; by inhibiting thrombin-mediated cleavage of NF155; by inhibiting the binding of thrombin to Casprl , and/or by inhibiting thrombin-mediated cleavage of Casprl, as compared to that which is observed in the absence of the thrombin inhibitor. The biological activity may be inhibited to any degree that realizes a beneficial therapeutic effect. For example, in some embodiments, the biological activity may be completely inhibited (i.e., prevented), while in other embodiments, the biological activity may be partially inhibited (i.e., reduced). As used herein, unless stated otherwise, the term "thrombin" encompasses thrombin and prothrombin in any form (e.g., mRNA or protein) and from any species (e.g., human, rat, or mouse).
[0018] The factor Xa inhibitor can be any agent that inhibits the biological activity of factor Xa. The biological activity of factor Xa may be inhibited in any manner, e.g., by inhibiting the expression of one or both of factor Xa mRNA and factor Xa protein; by inhibiting the binding of factor Xa to Casprl ; by inhibiting factor Xa-mediated cleavage of Casprl ; and/or by inhibiting the binding of factor Xa to Casprl , as compared to that which is observed in the absence of the factor Xa inhibitor. The biological activity may be inhibited to any degree that realizes a beneficial therapeutic effect, as described herein with respect to other aspects of the invention.
[0019] In an embodiment of the invention, the thrombin inhibitor is an agent that inhibits thrombin-mediated cleavage of one or both of Casprl and NF155. For example, the thrombin inhibitor may be an agent that binds to the thrombin protein or the NF155 protein, thereby reducing or preventing thrombin-mediated cleavage of NF155. In another embodiment of the invention, the thrombin inhibitor may be an agent that binds to the thrombin protein or the Casprl protein, thereby reducing or preventing thrombin-mediated cleavage of Casprl . By way of illustration, the agent that inhibits thrombin-mediated cleavage of one or both of NF155 and Casprl can be any of the antibodies or antigen binding fragments thereof, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide (or
polypeptide) inhibitor) described herein.
[0020] In an embodiment of the invention, the factor Xa inhibitor is an agent that inhibits factor Xa-mediated cleavage of Casprl . For example, the factor Xa inhibitor may be an agent that binds to the factor Xa protein or the Casprl protein, thereby reducing or preventing factor Xa-mediated cleavage of Casprl . In another embodiment of the invention, the factor Xa inhibitor may be an agent that binds to the factor Xa protein or the Casprl protein, thereby reducing or preventing factor Xa-mediated cleavage of Casprl . By way of illustration, the agent that inhibits factor Xa-mediated cleavage of Casprl can be any of the antibodies or antigen binding fragments thereof, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide (or polypeptide) inhibitor) described herein.
[0021] In an embodiment, the thrombin inhibitor is an agent that inhibits the binding of thrombin to one or both of Casprl and NF155. In this regard, the thrombin inhibitor may be an agent that binds to thrombin or NF155, thereby reducing or preventing the binding of the thrombin protein to the NF155 protein and inhibiting the ability of thrombin to cleave NF155. In another embodiment of the invention, the thrombin inhibitor may be an agent that binds to thrombin or Casprl , thereby reducing or preventing the binding of the thrombin protein to the Casprl protein and inhibiting the ability of thrombin to cleave Casprl . The thrombin inhibitor may be an agent that competes with the thrombin protein for the native thrombin binding or cleavage site of the NF155 protein or an agent that competes with the NF155 protein for the native NF155 binding site of the thrombin protein. In another embodiment of the invention, the thrombin inhibitor may be an agent that competes with the thrombin protein for the native thrombin binding or cleavage site of the Casprl protein or an agent that competes with the Casprl protein for the native Casprl binding site of the thrombin protein. By way of illustration, the agent that inhibits the binding of thrombin to one or both of Caspr 1 and NF155 can be any of the antibodies or antibody fragments, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide inhibitor) described herein.
[0022] In an embodiment, the factor Xa inhibitor is an agent that inhibits the binding of factor Xa to Casprl . In this regard, the factor Xa inhibitor may be an agent that binds to factor Xa or Casprl , thereby reducing or preventing the binding of the factor Xa protein to the Casprl protein and inhibiting the ability of factor Xa to cleave Casprl . The factor Xa inhibitor may be an agent that competes with the factor Xa protein for the native factor Xa binding or cleavage site of the Casprl protein or an agent that competes with the Casprl protein for the native Casprl binding site of the factor Xa protein. By way of illustration, the agent that inhibits the binding of factor Xa to Caspr 1 can be any of the antibodies or antibody fragments, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide inhibitor) described herein.
[0023] In an embodiment of the invention, the thrombin inhibitor is an antibody, or an antigen binding fragment thereof, having antigenic specificity for thrombin, Casprl, or NF155. In an embodiment of the invention, the factor Xa inhibitor is an antibody, or an antigen binding fragment thereof, having antigenic specificity for factor Xa or Casprl . Anti- factor Xa, anti-thrombin, anti-Casprl , and anti-NF155 antibodies and antigen binding fragments thereof can be monoclonal or polyclonal. Anti-factor Xa, anti-thrombin, anti- Casprl, and anti-NF155 antibodies and antigen binding fragments thereof can be prepared using the factor Xa, thrombin, Casprl , and NF155 proteins disclosed herein and routine techniques. Examples of such antibodies or antigen binding fragments thereof are described herein with respect to other aspects of the invention and include those which specifically bind to a functional fragment of the factor Xa protein, the Casprl protein, the NF155 protein, or the thrombin protein, e.g., the native factor Xa binding or cleavage site of the Casprl protein, the native Casprl binding site of the factor Xa protein, the native thrombin binding or cleavage site of the Casprl protein, the native Casprl binding site of the thrombin protein, the native thrombin binding or cleavage site of the NF155 protein or the native NF155 binding site of the thrombin protein. [0024] The functional fragment of the thrombin or NF155 protein can comprise any contiguous part of the thrombin or NF155 protein that retains a relevant biological activity of the thrombin or NF155 protein, e.g., binds to NF1 5 or thrombin, respectively, and/or participates in thrombin-mediated cleavage of NF155. In another embodiment of the invention, the functional fragment of the thrombin or Casprl protein can comprise any contiguous part of the thrombin or Casprl protein that retains a relevant biological activity of the thrombin or Casprl protein, e.g., binds to Casprl or thrombin, respectively, and/or participates in thrombin-mediated cleavage of Casprl . In another embodiment of the invention, the functional fragment of the factor Xa or Casprl protein can comprise any contiguous part of the factor Xa or Casprl protein that retains a relevant biological activity of the factor Xa or Casprl protein, e.g., binds to Casprl or factor Xa, respectively, and/or participates in factor Xa-mediated cleavage of Casprl . Any given fragment of a factor Xa, thrombin, Casprl , or NF155 protein can be tested for such biological activity using methods known in the art. For example, the functional fragment can comprise, consist essentially of, or consist of the factor Xa binding portion of the Casprl protein, the Casprl binding portion of the factor Xa protein, the thrombin binding portion of the Casprl protein, the Casprl binding portion of the thrombin protein, the thrombin binding portion of the NF155 protein, or the NF155 binding portion of the thrombin protein. In reference to the parent factor Xa, thrombin, Casprl, or NF155 protein, the functional fragment preferably comprises, for instance, about 10% or more, 25% or more, 30% or more, 50% or more, 60% or more, 80% or more, 90% or more, or even 95% or more of the parent factor Xa, thrombin, Casprl, or NF155 protein, respectively.
[0025] Chemical inhibitors of thrombin include small molecules and peptides or polypeptides that inhibit thrombin-mediated cleavage of NF155, bind the thrombin protein or the NF155 protein or functional fragment of thrombin or NF155, or compete with the thrombin or NF155 protein or functional fragment of thrombin or NF155 for its native binding or cleavage site on the NF155 protein or its native binding site on the thrombin protein, respectively. In another embodiment of the invention, chemical inhibitors of thrombin include small molecules and peptides or polypeptides that inhibit thrombin- mediated cleavage of Casprl , bind the thrombin protein or the Casprl protein or functional fragment of thrombin or Casprl , or compete with the thrombin or Casprl protein or functional fragment of thrombin or Casprl for its native binding or cleavage site on the Casprl protein or its native binding site on the thrombin protein, respectively. In another embodiment of the invention, chemical inhibitors of factor Xa include small molecules and peptides or polypeptides that inhibit factor Xa-mediated cleavage of Casprl, bind the factor Xa protein or the Casprl protein or functional fragment of factor Xa or Casprl , or compete with the factor Xa or Casprl protein or functional fragment of factor Xa or Casprl for its native binding or cleavage site on the Casprl protein or its native binding site on the factor Xa protein, respectively. Suitable inhibitors can include, for example, chemical compounds or a non-active fragment or mutant of a factor Xa protein, thrombin protein, Casprl protein, or a NF155 protein. In this regard, in an embodiment of the invention, the thrombin inhibitor is a mutated thrombin, a mutated Casprl, or a mutated NF155. In an embodiment of the invention, the factor Xa inhibitor is a mutated factor Xa or a mutated Casprl . The mutation may include any insertions, deletions, and/or substitutions of one or more amino acids in any position of the thrombin, Casprl , or NF155 protein that effectively inhibits thrombin biological activity (e.g., one or more of thrombin-mediated cleavage of NF155, binding of NF155 to thrombin, thrombin-mediated cleavage of Casprl, and binding of Casprl to thrombin) or any insertions, deletions, and/or substitutions of one or more amino acids in any position of the factor Xa or Casprl protein that effectively inhibits factor Xa biological activity. For example, the chemical inhibitor can bind to one or more of factor Xa, Casprl, thrombin, and bind to NF155. In an embodiment of the invention, the thrombin inhibitor is a mutant of a thrombin protein that binds NF155 protein but does not cleave it. Such a mutant thrombin protein would competitively inhibit NF155 cleavage by wild-type (non-mutated) thrombin. In another embodiment of the invention, the thrombin inhibitor is a mutant of a thrombin protein that binds Casprl protein but does not cleave it. Such a mutant thrombin protein would competitively inhibit Casprl cleavage by wild-type (non-mutated) thrombin. In another embodiment of the invention, the factor Xa inhibitor is a mutant of a factor Xa protein that binds Casprl protein but does not cleave it. Such a mutant factor Xa protein would competitively inhibit Casprl cleavage by wild-type (non-mutated) factor Xa. In this regard, the thrombin inhibitor or factor Xa inhibitor may be a chemical inhibitor. In a preferred embodiment, the thrombin inhibitor inhibits thrombin-mediated cleavage of one or both of Casprl and NF155, as described herein. In a preferred embodiment, the factor Xa inhibitor inhibits factor Xa-mediated cleavage of Casprl , as described herein.
[0026] Chemical inhibitors of thrombin or factor Xa can be identified using routine techniques. For example, chemical inhibitors can be tested in assays to identify molecules and peptides (or polypeptides) that bind to one or more of factor Xa, thrombin, Casprl, and NF155 with sufficient affinity to inhibit thrombin biological activity (e.g., one or more of binding of thrombin to NF155, thrombin-mediated cleavage of NF155, binding of thrombin to Casprl, and thrombin-mediated cleavage of Casprl) or to inhibit factor Xa biological activity. Also, competition assays can be performed to identify small-molecules and peptides (or polypeptides) that inhibit thrombin-mediated cleavage of NF155 or compete with thrombin or NF155 (or a functional fragment thereof) for binding to its native binding or cleavage site of NF155 or its native binding site of thrombin, respectively. Competition assays can be performed to identify small-molecules and peptides (or polypeptides) that inhibit thrombin-mediated cleavage of Casprl or compete with thrombin or Casprl (or a functional fragment thereof) for binding to its native binding or cleavage site of Casprl or its native binding site of thrombin, respectively. Competition assays can be performed to identify small-molecules and peptides (or polypeptides) that inhibit factor Xa-mediated cleavage of Casprl or compete with factor Xa or Casprl (or a functional fragment thereof) for binding to its native binding or cleavage site of Casprl or its native binding site of factor Xa, respectively. Such techniques could be used in conjunction with mutagenesis of the thrombin protein, factor Xa protein, Casprl protein, or the NF155 protein, or a functional fragment thereof, and/or with high- throughput screens of known chemical inhibitors.
[0027] For example, the chemical thrombin inhibitor may be a direct thrombin inhibitor or an indirect thrombin inhibitor. The direct thrombin inhibitor may be a bivalent, univalent, or an allosteric inhibitor. In a preferred embodiment, the thrombin inhibitor is a catalyst that activates native thrombin inhibitors like antithrombin III (such as, for example, heparin). Examples of thrombin inhibitors may include, but are not limited to, hirudin, bivalirudin, lepirudin, desirudin, argatroban, melagatran, ximelagatran, dabigatran, DNA aptamers, benzofuran dimers, benzofuran trimers, polymeric lignins, sulfated β-04 lignin (Sb04L), heparin (including, for example, low molecular weight heparins (LMWHs)), warfarin, fondaparinux, and pharmaceutically acceptable salts and derivatives thereof. In an embodiment of the invention, the thrombin inhibitor is a thrombin inhibitor that is released by astrocytes. Examples of thrombin inhibitors that are released by astrocytes include antithrombin III (Dowell et al., J. Proteome Res., 8(8): 4135-43 (2009)), protease nexin-1 (PN1), plasminogen activator inhibitor-1 (PAI-1) (Hultman et al., J. Neurosci. Res., 2441-9 (2010)), thrombomodulin (Pindon et al., Glia, 19(3): 259-68 (1997)), and pharmaceutically acceptable salts and derivatives thereof. Preferably, the thrombin inhibitor is fondaparinux, antithrombin III, a LMWH, or a pharmaceutically acceptable salt or derivative thereof (such as, for example, fondaparinux sodium). Fondaparinux inhibits the enzyme Factor Xa- mediated conversion of prothrombin to thrombin. In an embodiment of the invention, the chemical factor Xa inhibitor may be a direct factor Xa inhibitor or an indirect factor Xa inhibitor.
[0028] In an embodiment of the invention, the thrombin inhibitor is any suitable agent that inhibits the expression of one or both of thrombin mRNA and thrombin protein. In an embodiment of the invention, the factor Xa inhibitor is any suitable agent that inhibits the expression of one or both of factor Xa mRNA and factor Xa protein. The thrombin inhibitor can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding one or both of thrombin mRNA and thrombin protein or a complement thereof. The thrombin inhibitor may be introduced into a host cell, wherein the cell is capable of expressing one or both of thrombin mRNA and thrombin protein, in an effective amount for a time and under conditions sufficient to interfere with expression of one or both of thrombin mRNA and thrombin protein, respectively. The factor Xa inhibitor can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding one or both of factor Xa mRNA and factor Xa protein or a complement thereof. The factor Xa inhibitor may be introduced into a host cell, wherein the cell is capable of expressing one or both of factor Xa mRNA and factor Xa protein, in an effective amount for a time and under conditions sufficient to interfere with expression of one or both of factor Xa mRNA and factor Xa protein, respectively. In some embodiments, RNA interference (RNAi) is employed. In this regard, the thrombin inhibitor or factor Xa inhibitor may comprise an RNAi agent. In an embodiment, the RNAi agent may comprise a small interfering RNA (siRNA), a short hairpin miRNA (shMIR), a microRNA (miRNA), or an antisense nucleic acid. The RNAi agent, e.g., siRNA, shRNA, miRNA, and/or antisense nucleic acid can comprise overhangs. That is, not all nucleotides need bind to the target sequence. RNA interference nucleic acids employed can be at least about 19, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 220, at least about 240, from about 19 to about 250, from about 40 to about 240, from about 60 to about 220, from about 80 to about 200, from about 60 to about 180, from about 80 to about 160, and/or from about 100 to about 140 nucleotides in length. [0029] The RNAi agent, e.g., siRNA or shR A, can be encoded by a nucleotide sequence included in a cassette, e.g., a larger nucleic acid construct such as an appropriate vector. Examples of such vectors include lentiviral and adenoviral vectors, as well as other vectors described herein with respect to other aspects of the invention. When present as part of a larger nucleic acid construct, the resulting nucleic acid can be longer than the comprised RNAi nucleic acid, e.g., greater than about 70 nucleotides in length. In some embodiments, the RNAi agent employed cleaves the target mRNA. In other embodiments, the RNAi agent employed does not cleave the target mRNA. In an embodiment of the invention, the thrombin inhibitor or the factor Xa inhibitor is a GENEART CRISPR-Cas9 system for genome-editing (available from Life Technologies, Carlsbad, CA).
[0030] Any type of suitable siRNA, miRNA, and/or antisense nucleic acid can be employed. In an embodiment, the antisense nucleic acid comprises a nucleotide sequence complementary to at least about 8, at least about 15, at least about 19, or from about 19 to about 22 nucleotides of a nucleic acid encoding (i) one or both of thrombin mRNA and thrombin protein or a complement thereof or (ii) one or both of factor Xa mRNA and thrombin protein or a complement thereof. In an embodiment, the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat-associated siRNAs (rasiRNAs). In another embodiment, the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).
[0031] In an embodiment of the invention, the thrombin inhibitor may inhibit or downregulate to some degree the expression of the protein encoded by a thrombin gene, e.g., at the DNA, RNA, or other level of regulation. In this regard, a host cell comprising a thrombin inhibitor expresses none of one or both of thrombin mRNA and thrombin protein or lower levels of one or both of thrombin mRNA and thrombin protein as compared to a host cell that lacks a thrombin inhibitor. In accordance with an embodiment of the invention, the inhibitor, such as an RNAi agent, such as a shMIR, can target a nucleotide sequence of a thrombin gene or mRNA encoded by the same.
[0032] In an embodiment, the thrombin sequence is a human thrombin sequence. For example, human prothrombin is assigned Gene NCBI Entrez Gene ID No. 2147, and an Online Mendelian Inheritance in Man (OMIM) No. 176930. The human thrombin gene is found on chromosome 1 1 at 1 lpl 1.2. A human thrombin mRNA transcript includes mRNA GenBank Accession No: NM_000506.3 (SEQ ID NO: 27), with corresponding protein sequence GenBank Accession No: NP_000497.1 (SEQ ID NO: 28). Human genomic thrombin sequences include GenBank Accession Nos: NG_008953.1 , NC _00001 1.10, AC1 15088.6, AF478696.1 , AF493953.1 , AJ5441 14.1 , AMYH0202471 1.1, CH471064.2, CS355181.1, M17262.1 , and S50162.1. Human thrombin mRNA sequences also include Genbank Accession Nos: AJ972449.1 , A 222775.1 , A 222777.1 , AK293326.1 ,
A 303747.1, A 312965.1 , AY344793.1, AY344794.1 , BC051332.1 , CB156997.1, DB183734.1, M33031.1 , and V00595.1. Human thrombin amino acid sequences include Genbank Accession Nos: AAL77436.1 , AAM1 1680.1 , CAD80258.1 , EAW67977.1, EAW67978.1, EAW67979.1 , CAL24231.1 , AAC63054.1 , AAB24476.1 , CAJOl 369.1, BAD96495.1, BAD96497.1, BAG56844.1 , BAG64719.1 , BAG35804.1 , AAR08142.1, AAR08143.1, AAH51332.1, AAA60220.1 , CAA23842.1 , ACE87755.1 , and ACE87074.1. Other human thrombin sequences, as well as other thrombin species can be employed in accordance with the invention.
[0033] In another embodiment, the thrombin sequence is a mouse sequence. For example, mouse thrombin is assigned Gene NCBI Entrez Gene ID No. 14061. The mouse thrombin gene is found on chromosome 2 at 2E1. A transcript includes mRNA Genbank Accession No.: NM_010168.3 (SEQ ID NO: 29), with corresponding protein sequence NP_034298.1 (SEQ ID NO: 30). Mouse genomic thrombin sequences include Genbank Accession Nos: AAHY01018393.1, AL691489.21 , and CH466519.1. Mouse thrombin mRNA sequences also include Genbank Accession Nos: AK050056.1, A 149367.1, AK167532.1, BC013662.1 , BY705183.1 , M81394.1, and X52308.1. Mouse thrombin amino acid sequences include Genbank Accession Nos: EDL27567.1 , BAE39601.1 , AAHl 3662.1 , AAA40435.1, and CAA36548.1. Other mouse thrombin sequences, as well as other thrombin species can be employed in accordance with the invention.
[0034] In accordance with an embodiment of the invention, the thrombin inhibitor, such as an RNAi agent, such as a shMIR, can target a nucleotide sequence selected from the group consisting of the 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), and the coding sequence of thrombin, complements thereof, and any combination thereof. Any suitable thrombin target sequence can be employed. In an embodiment of the invention, the sequences of the thrombin inhibitor can be designed against a human thrombin with
Accession No. NM_000506.3 (SEQ ID NO: 27). In still another embodiment, the sequences of the thrombin inhibitor can be designed against a mouse thrombin with Accession No. NM_010168.3 (SEQ ID NO: 29). RNAi agents can be designed against any appropriate thrombin mRNA sequence. [0035] In another embodiment, the thrombin inhibitor is an NFl 55 thrombin binding site/Fc fusion protein. The NFl 55 thrombin binding site/Fc fusion protein is a soluble variation of the native NFl 55 which binds thrombin protein, thereby competing with the native NF155 for binding to thrombin. Accordingly, the NF155 thrombin binding site/Fc fusion protein may inhibit the binding of thrombin to the native NFl 55. The NFl 55 thrombin binding site/Fc fusion protein may also inhibit the thrombin-mediated cleavage of NFl 55. The NFl 55 thrombin binding site/Fc fusion protein may be from any mammal. In a preferred embodiment, the NFl 55 thrombin binding site/Fc fusion protein is a mouse NFl 55 thrombin binding site/Fc fusion protein or a human NFl 55 thrombin binding site/Fc fusion protein.
[0036] In another embodiment, the thrombin inhibitor is a thrombin NFl 55 binding site/Fc fusion protein. The thrombin NFl 55 binding site/Fc fusion protein is a soluble variation of the native thrombin which binds NFl 55 protein, thereby competing with the native thrombin for binding to NFl 55. Accordingly, the thrombin NFl 55 binding site/Fc fusion protein may inhibit the binding of thrombin to the native NFl 55. The thrombin NFl 55 binding site/Fc fusion protein may also inhibit the thrombin-mediated cleavage of NFl 55. The thrombin NFl 55 binding site/Fc fusion protein may be from any mammal. In a preferred embodiment, the thrombin NFl 55 binding site/Fc fusion protein is a mouse thrombin NFl 55 binding site/Fc fusion protein or a human thrombin NFl 55 binding site/Fc fusion protein.
[0037] In another embodiment, the thrombin inhibitor is a Casprl thrombin binding site/Fc fusion protein. The Casprl thrombin binding site/Fc fusion protein is a soluble variation of the native Casprl which binds thrombin protein, thereby competing with the native Casprl for binding to thrombin. Accordingly, the Casprl thrombin binding site/Fc fusion protein may inhibit the binding of thrombin to the native Casprl . The Casprl thrombin binding site/Fc fusion protein may also inhibit the thrombin-mediated cleavage of Casprl . The Casprl thrombin binding site/Fc fusion protein may be from any mammal. In a preferred embodiment, the Casprl thrombin binding site/Fc fusion protein is a mouse Casprl thrombin binding site/Fc fusion protein or a human Casprl thrombin binding site/Fc fusion protein.
[0038] In another embodiment, the thrombin inhibitor is a thrombin Casprl binding site/Fc fusion protein. The thrombin Casprl binding site/Fc fusion protein is a soluble variation of the native thrombin which binds Casprl protein, thereby competing with the native thrombin for binding to Casprl . Accordingly, the thrombin Casprl binding site/Fc fusion protein may inhibit the binding of thrombin to the native Casprl . The thrombin Casprl binding site/Fc fusion protein may also inhibit the thrombin-mediated cleavage of Casprl . The thrombin Casprl binding site/Fc fusion protein may be from any mammal. In a preferred embodiment, the thrombin Casprl binding site/Fc fusion protein is a mouse thrombin Casprl binding site/Fc fusion protein or a human thrombin Casprl binding site/Fc fusion protein.
[0039] In another embodiment, the factor Xa inhibitor is a Casprl factor Xa binding site/Fc fusion protein. The Casprl factor Xa binding site/Fc fusion protein is a soluble variation of the native Casprl which binds factor Xa protein, thereby competing with the native Casprl for binding to factor Xa. Accordingly, the Casprl factor Xa binding site/Fc fusion protein may inhibit the binding of factor Xa to the native Casprl . The Casprl factor Xa binding site/Fc fusion protein may also inhibit the factor Xa-mediated cleavage of Casprl . The Casprl factor Xa binding site/Fc fusion protein may be from any mammal. In a preferred embodiment, the Casprl factor Xa binding site/Fc fusion protein is a mouse Casprl factor Xa binding site/Fc fusion protein or a human Casprl factor Xa binding site/Fc fusion protein.
[0040] In another embodiment, the factor Xa inhibitor is a factor Xa Casprl binding site/Fc fusion protein. The factor Xa Casprl binding site/Fc fusion protein is a soluble variation of the native factor Xa which binds Casprl protein, thereby competing with the native factor Xa for binding to Casprl . Accordingly, the factor Xa Casprl binding site/Fc fusion protein may inhibit the binding of factor Xa to the native Casprl . The factor Xa Casprl binding site/Fc fusion protein may also inhibit the factor Xa-mediated cleavage of Casprl . The factor Xa Casprl binding site/Fc fusion protein may be from any mammal. In a preferred embodiment, the factor Xa Casprl binding site/Fc fusion protein is a mouse factor Xa Casprl binding site/Fc fusion protein or a human factor Xa Casprl binding site/Fc fusion protein.
[0041] The thrombin inhibitor or the factor Xa inhibitor can be obtained by methods known in the art. For example, thrombin inhibitors or factor Xa inhibitors that are peptides or polypeptides can be obtained by de novo synthesis. Also, thrombin inhibitors or factor Xa inhibitors can be recombinantly produced using standard recombinant methods. See, for instance, Green et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2012. Further, the thrombin inhibitor or factor Xa inhibitor 75 can be isolated and/or purified from a natural source, e.g., a human. Methods of isolation and purification are well-known in the art. In this respect, the thrombin inhibitors may be exogenous and can be synthetic, recombinant, or of natural origin.
[0042] The thrombin inhibitors or factor Xa inhibitors that are peptides or polypeptides can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
[0043] Of course, the methods of the invention can comprise administering two or more thrombin inhibitors, or two or more factor Xa inhibitors, any of which may be the same or different from one another. Furthermore, the thrombin inhibitor or factor Xa inhibitor can be provided as part of a larger polypeptide construct. For instance, the thrombin inhibitor or factor Xa inhibitor can be provided as a fusion protein comprising a thrombin inhibitor or factor Xa inhibitor along with other amino acid sequences or a nucleic acid encoding same. The thrombin inhibitor or factor Xa inhibitor also can be provided as part of a conjugate or nucleic acid encoding same. Conjugates, as well as methods of synthesizing conjugates in general, are known in the art.
[0044] The thrombin inhibitor or factor Xa inhibitor can be administered to the mammal by administering a nucleic acid encoding the thrombin inhibitor or factor Xa inhibitor to the mammal. "Nucleic acid" as used herein includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
[0045] Nucleic acids encoding the thrombin inhibitor or factor Xa inhibitor (and degenerate nucleic acid sequences encoding the same amino acid sequences), can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green et al., supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
[0046] The nucleic acids can be incorporated into a recombinant expression vector. For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA or polypeptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA or polypeptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA or polypeptide expressed within the cell. The vectors are not naturally- occurring as a whole. However, parts of the vectors can be naturally-occurring. The recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring or non- naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non- naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
[0047] The recombinant expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as GTIO, λΘΤΙ 1 , λZapII (Stratagene), EMBL4, and λΝΜΙ 149, also can be used. Examples of plant expression vectors include pBIOl , pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, e.g., a retroviral vector.
[0048] The recombinant expression vectors can be prepared using standard recombinant DNA techniques described in, for example, Green et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like.
[0049] Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
[0050] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
[0051] The recombinant expression vector can comprise a native or nonnative promoter and/or stop codon operably linked to the nucleotide sequence encoding the thrombin inhibitor or factor Xa inhibitor, or to the nucleotide sequence which is complementary to the nucleotide sequence encoding the thrombin inhibitor or factor Xa inhibitor. The selection of stop codons and promoters, e.g., strong, weak, inducible, tissue- specific and developmental- specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a stop codon and a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
[0052] The thrombin inhibitors, factor Xa inhibitors and nucleic acids encoding them can be of synthetic or natural origin, and can be isolated or purified to any degree. The terms "isolated" and "purified," as used herein, means having been removed from its natural environment. The term "purified" or "isolated" means having been increased in purity and does not require absolute purity or isolation; rather, it is intended as a relative term. For example, the purity can be at least about 50%, can be greater than about 60%, about 70% or about 80%, or can be about 100%.
[0053] In accordance with another embodiment of the invention, it has also been discovered that astrocytes at the node of Ranvier encase the node, stabilize the node, and maintain the physical structure of myelin. Without being bound to a particular theory or mechanism, it is believed that demyelination may originate at the node of Ranvier. For example, it is believed that immune cells may cause autoimmune demyelinating disorders, such as MS, by attacking at or near the node of Ranvier. Accordingly, it is believed that stimulating the astrocyte to reduce or prevent immune cell access to the paranodal junctions and maintain the physical structure of the node may reduce or prevent demyelination.
Astrocytes release thrombin inhibitors and also physically stabilize the node of Ranvier. Accordingly, it is also believed that stimulating astrocytes to release a thrombin inhibitor may reduce or prevent demyelination.
[0054] Accordingly, an embodiment of the invention provides a method of treating or preventing demyelination in a mammal, the method comprising administering a compound to the mammal in an amount effective to stimulate astrocytes in the mammal to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or (v) any combination of (i)-(iv) in an amount effective to treat or prevent the demyelination in the mammal.
[0055] The thrombin inhibitor release by the astrocytes may be any thrombin inhibitor released by astrocytes. In an embodiment of the invention, the thrombin inhibitor released by astrocytes is antithrombin III, PN1 , PAI-1 , or thrombomodulin.
[0056] The compound that stimulates astrocytes in the mammal to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or (v) any combination of (i)-(iv) may be any suitable compound that stimulates astrocytes in the mammal to carry out one or more of (i)-(v).
Compounds can be tested in assays to identify agents that stimulate astrocytes to carry out one or more of (i)-(v). Examples of such compounds may include adenosine triphosphate (ATP), adenosine, glutamate, cytokines, nitric oxide, chemokines, growth factors, and factors regulating GFAP (glial fibrillary acidic protein).
[0057] In an embodiment of the invention, the demyelination may be associated with (e.g., caused by) a demyelinating disease. In this regard, the method may comprise treating or preventing a demyelinating disease in the mammal. The demyelinating disease may be any disorder that involves myelin loss (e.g., damage, and/or impairment), regardless of the cause (e.g., a demyelinating disease). A demyelinating disease is any disorder that results in deficient or abnormal myelination (e.g., destruction of myelin). The pathology of the demyelinating disease can have autoimmune, inflammatory, neurodegenerative, or other components. In an embodiment, the disease is classified as an inflammatory demyelinating disease or an autoimmune/inflammatory demyelinating disease. The demyelinating disease can be, for example, a leucodystrophy, multiple sclerosis (MS), cerebral palsy, optic neuritis, Devic's disease (neuromyelitis optica), transverse myelitis, acute MS (Marburg variant), Balo's concentric sclerosis, acute disseminated encephalomyelitis (ADEM),
adrenoleukodystrophy, adrenomyeloneuropathy, Gulf War Illness, combined central and peripheral demyelination (CCPD), chronic inflammatory demyelinating
polyradiculoneuropathy (CIDP) (Kawamura et al., Neurology, 81(8): 714-22 (2013)), a psychiatric disorder, or a learning disability. In a preferred embodiment, the disease is multiple sclerosis (MS).
[0058] In an embodiment of the invention, the disease is Guillain-Barre syndrome (GBS). GBS involves demyelination in the peripheral nervous system (PNS). Schwann cells produce myelin in the PNS, and Casprl and NF155 are present at the nodes in the PNS. Without being bound to a particular theory or mechanism, it is believed that GBS may also be treated by reducing or preventing thrombin biological activity, e.g., thrombin-mediated cleavage of one or both of Casprl and NF155, and it is believed that GBS may also be treated by reducing or preventing factor Xa biological activity, e.g., factor Xa-mediated cleavage of Casprl .
[0059] In an embodiment of the invention, the demyelination may be associated with (e.g., caused by) damage to or death of the cells that make myelin (oligodendrocytes).
Demyelination may be caused by any of a variety of events including, but not limited to, any one or more of white matter injury, difficult birth, hypoxia, ischemia, viral infection, premature birth, any of the demyelinating diseases described herein, and autoimmune disorders. The demyelination may include injury-related demyelination, e.g., noise-induced hearing loss.
[0060] The factor Xa inhibitor, thrombin inhibitor or compound that stimulates astrocytes to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin (hereinafter collectively referred to as "anti-demyelination material(s)") may be administered to the mammal in any suitable manner. In an embodiment of the invention, the anti-demyelination material is administered parenterally (e.g., subcutaneously, intravenously, intraarterially, intramuscularly,
intradermally, interperitoneally, or intrathecally) intranasally, or orally. Preferably, the anti- demyelination material is administered in combination with a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active anti-demyelination material, and by the route of administration. Pharmaceutically acceptable carriers, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active anti-demyelination material and one which has no detrimental side effects or toxicity under the conditions of use.
[0061] For purposes of the invention, the amount or dose of the anti-demyelination material administered should be sufficient to effect a desired response, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame. For example, the dose of the anti-demyelination material should be sufficient to reduce or prevent cleavage of one or both of Casprl and NF155, reduce or prevent demyelination, or treat or prevent a demyelinating disease (e.g., demyelinating disease progression) in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular anti-demyelination material and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated.
[0062] Many assays for determining an administered dose are known in the art. An administered dose may be determined in vitro (e.g., cell cultures) or in vivo (e.g., animal studies). For example, an administered dose may be determined by determining the ICs0 (the dose that achieves a half-maximal inhibition of signs of disease), LD5o (the dose lethal to 50% of the population), the ED50 (the dose therapeutically effective in 50% of the
population), and the therapeutic index in cell culture, animal studies, or combinations thereof. The therapeutic index is the ratio of LD50 to ED5o (i.e., LD50/ED50).
[0063] The dose of the anti-demyelination material also may be determined by the existence, nature, and extent of any adverse side effects that might accompany the
administration of a particular anti-demyelination material. Typically, the attending physician will decide the dosage of the anti-demyelination material with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, anti-demyelination material to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of the anti-demyelination material can be about 0.001 to about 1000 mg/kg body weight of the subject being treated/day, from about 0.01 to about 100 mg/kg body weight/day, about 0.01 mg to about 50 mg/kg body weight/day, from about 1 to about to about 1000 mg/kg body weight/day, from about 5 to about 500 mg/kg body weight/day, from about 5 to about 250 mg/kg body weight/day, about 5 to about 150 mg/kg body weight/day, about 8 to about 32 mg/kg body weight/day, about 10 mg/kg body weight/day, about 2 mg/kg body weight/day to about 5 mg/kg body weight/day, or about 4 mg/kg body weight/day.
[0064] The terms "treat" and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount or any level of treatment or prevention of demyelination or a
demyelinating disease in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions, symptoms, or signs of the demyelinating disease, e.g., MS, or demyelination being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom, sign, or condition thereof. In an embodiment of the invention, administering the anti-demyelination material to the mammal reduces or prevents any one or more of (i) cleavage of NF155; (ii) detachment of myelin from neuronal axons; (iii) an increase in nodal gap length; (iv) dispersion (e.g., decrease in density) of neuronal sodium channels; and (v) cleavage of Casprl . In an embodiment of the invention, administering the anti-demyelination material to the mammal stimulates astrocytes to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or any combination of (i)-(iv). In an embodiment of the invention, administering the anti-demyelination material to the mammal promotes remodeling or myelin. In an embodiment of the invention, the inventive methods may, advantageously, improve visual acuity in a mammal suffering from demyelination.
[0065] As described above, it has also been discovered that NF155 is cleaved into two cleaved fragments (NF125 and NF30) and that this cleavage results in detachment of the myelin from the axon. It has also been discovered that thrombin cleaves mouse Casprl into two cleaved fragments (Casprl i_380 (SEQ ID NO: 33) and Casprl 38 i -i 385 (SEQ ID NO: 34)) and human Casprl into two cleaved fragments (Casprl i_37g (SEQ ID NO: 41) and Casprl 3 0- i384 (SEQ ID NO: 42)). It is believed that this thrombin-mediated cleavage of Casprl may result in detachment of the myelin from the axon. It has also been discovered that factor Xa cleaves mouse Casprl into two cleaved fragments (Casprl 1.947 (SEQ ID NO: 35) and
Casprl 948-i385 (SEQ ID NO: 36)) and human Casprl into two cleaved fragments (Casprl 1.946 (SEQ ID NO: 43) and Casprl 947-i384 (SEQ ID NO: 44)). It is believed that this factor Xa- mediated cleavage of Casprl may result in detachment of the myelin from the axon. Full- length Casprl , mouse Casprl 38M385 (SEQ ID NO: 34), mouse Casprl 1.947 (SEQ ID NO: 35), human Casprl 380.1384 (SEQ ID NO: 42), human Casprl i.946 (SEQ ID NO: 43), NF155, NF125, and NF30 are not present in the blood or cerebral spinal fluid (CSF) of healthy individuals. Without being bound to a particular theory or mechanism, it is believed that (i) thrombin-mediated cleavage of NF155 causes one or both of the NF125 and NF30 fragments to detach from the oligodendrocyte and enter the blood or CSF; (ii) thrombin-mediated cleavage of Casprl causes one or more of the Casprl fragments mouse Casprl i_38o (SEQ ID NO: 33),mouse Casprl 38i-i385 (SEQ ID NO: 34), human Casprl i-379 (SEQ ID NO: 41), and human Casprl 38o-i384 (SEQ ID NO: 42) to detach from the oligodendrocyte and enter the blood or CSF; (iii) factor Xa-mediated cleavage of Casprl causes one or more of the Casprl fragments mouse Casprl 1.947 (SEQ ID NO: 35), mouse Casprl 948-1385 (SEQ ID NO: 36), human Casprl 1.945 (SEQ ID NO: 43), and human Casprl 947-1384 (SEQ ID NO: 44) to detach from the oligodendrocyte and enter the blood or CSF. Therefore, it is contemplated that one or more of NF125, NF30, mouse Casprl i-380 (SEQ ID NO: 33), mouse Casprl38i-i385 (SEQ ID NO: 34), mouse Casprl 1.947 (SEQ ID NO: 35), mouse Casprl 948-i385 (SEQ ID NO: 36), human Casprl 1.379 (SEQ ID NO: 41), human Casprl 38o-i384 (SEQ ID NO: 42), human
Casprl i.946 (SEQ ID NO: 43), human Casprl 947-1384 (SEQ ID NO: 44), and human Casprl38o- 946 (SEQ ID NO: 45) may be useful biomarkers for detecting demyelination. In particular, it is contemplated that the presence of one or more of mouse Casprl 38i.i385 (SEQ ID NO: 34), mouse Casprl 1.947 (SEQ ID NO: 35), human Casprl 380-i384 (SEQ ID NO: 42), and human Casprl 1.946 (SEQ ID NO: 43), and an NF155 amino acid sequence (e.g., one or both of NF125 and NF30) in blood and/or CSF indicates the presence of demyelination in a mammal.
[0066] The inventive detecting methods may provide many advantages. For example, the inventive detecting methods may be less expensive and/or faster than magnetic resonance imaging (MRI) brain imaging. In addition, detecting methods using blood or CSF as the biological sample can be more easily performed on newborn children and other types of patients. In addition, because the node of Ranvier is one of the first locations of attack in demyelination (e.g., demyelinating diseases), the inventive detecting methods may be particularly useful for early detection of demyelination, for example, before demyelinating lesions can be detected by MRI, or before physical symptoms start to appear. In contrast to the inventive methods, MRI can only detect demyelination after it has progressed to the point where it is visible as lesions using myelin dyes. The inventive detecting methods may also be useful for predicting a relapse in relapsing-remitting MS. The inventive methods may also be useful for detecting myelin damage such as, for example, myelin damage in a newborn infant that results after a difficult delivery.
[0067] Accordingly, an embodiment of the invention provides an isolated or purified antibody, or antigen binding fragment thereof, having antigenic specificity for an NF155 amino acid sequence. The phrase "antigenic specificity," as used herein with respect to an anti-NF155 antibody or antigen binding portion thereof, means that the particular antibody, or antigen binding fragment thereof, under consideration binds with measurably higher affinity to only one of the NF155 amino acid sequences of SEQ ID NOs: 21 -23 (in the alternative), than to other molecules. The inventive antibodies, and antigen binding fragments thereof, are hereinafter collectively referred to as "anti-NF155 antibodies." Each of the inventive anti- NF155 antibodies described herein advantageously binds to NF155 but does not bind to other neurofascin protein family members which lack the thrombin recognition sequence GRG in the third FNIII domain such as, for example, NF186, NF180, NF166 and NF140. Therefore, the inventive anti-NF155 antibodies are able to distinguish between NF155 amino acid sequences and those of other neurofascin family members.
[0068] For example, an embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21 ). The amino acid sequence of SEQ ID NO: 21 is positioned upstream of the NF155 thrombin cleavage site and is unique to NF155. SEQ ID NO: 21 is present in both NF155 and the cleaved NF125. Accordingly, the antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21 ) recognizes and binds to either NF155 or NF125.
[0069] Another embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22). The amino acid sequence of SEQ ID NO: 22 is positioned immediately upstream of the thrombin cleavage site. SEQ ID NO: 22 is present in both NF155 and the cleaved NF125. Accordingly, the antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of
LEMVVVNGR (SEQ ID NO: 22), recognizes and binds to either NF155 or NF125.
[0070] Another embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23). SEQ ID NO: 23 includes the NF155 thrombin cleavage site. The entire amino acid sequence of SEQ ID NO: 23 is, therefore, present in NF155 but is not present in NF30 or NF125. Accordingly, the antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23) recognizes and binds to NF155.
[0071] Another embodiment of the invention provides an isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the mouse Casprl 1-380 amino acid sequence of SEQ ID NO: 33, the mouse Casprl38i-i 385 amino acid sequence of SEQ ID NO: 34, the mouse Casprl i _947 amino acid sequence of SEQ ID NO: 35, the mouse Casprl 948-i385 amino acid sequence of SEQ ID NO: 36, the mouse Casprl38i.947 amino acid sequence of SEQ ID NO: 39, the human Casprl 1.379 amino acid sequence of SEQ ID NO: 41, the human Casprl 38o-i384 amino acid sequence of SEQ ID NO: 42, the human Casprl 1.946 amino acid sequence of SEQ ID NO: 43, the human Casprl 947-i384 amino acid sequence of SEQ ID NO: 44, or the human Casprl 38o-946 amino acid sequence of SEQ ID NO: 45.
[0072] Accordingly, an embodiment of the invention provides an isolated or purified antibody, or antigen binding fragment thereof, having antigenic specificity for a Caspr 1 amino acid sequence. The phrase "antigenic specificity," as used herein with respect to an anti-Casprl antibody or antigen binding portion thereof, means that the particular antibody, or antigen binding fragment thereof, under consideration binds with measurably higher affinity to only one of the Casprl amino acid sequences of SEQ ID NOs: 33-36 and 39 (in the alternative), than to other molecules. The inventive antibodies, and antigen binding fragments thereof, are hereinafter collectively referred to as "anti-Casprl antibodies."
[0073] The inventive anti-NF155 antibodies (or anti-Casprl antibodies) described herein may, advantageously, detect a mouse, rat, or human NF155 (or Casprl) amino acid sequence. Methods of testing antibodies, or antigen binding fragments thereof, for the ability to recognize antigen and for antigen specificity are known in the art. Examples of such methods may include immunoprecipitation, immunonephelometry, radioimmunoassay (RIA), immunohistochemistry, enzyme immunoassay (EIA), fluorescent immunoassay (FIA), enzyme-linked immunosorbent assay (ELISA), and the like. [0074] The inventive anti-NF155 antibodies and anti-Casprl antibodies can be of any type. For instance, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal or polyclonal. The antibody can be a naturally-occurring antibody, e.g., an antibody isolated and/or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc. Alternatively, the antibody can be a genetically- engineered antibody, e.g., a humanized antibody or a chimeric antibody. The antibody can be in monomeric or polymeric form.
[0075] The antigen binding fragment of the antibody can be any fragment of the antibody that has at least one antigen binding site. In an embodiment, the antigen binding fragment is a Fab fragment (Fab), F(ab')2 fragment, diabody, triabody, tetrabody, single-chain variable region fragment (scFv), or disulfide-stabilized variable region fragment (dsFv). A single- chain variable region fragment (scFv), which is a truncated Fab fragment including the variable (V) domain of an antibody heavy chain linked to a V domain of a light antibody chain via a synthetic peptide, can be generated using routine recombinant DNA technology techniques (see, e.g., Murphy et al. (eds.), Murphy 's Immunobiology, 7th Ed., Garland Science, New York, NY (2008)). Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology. The antigen binding fragments of the invention, however, are not limited to these exemplary types of antibody fragments.
[0076] The inventive antibodies and antigen binding fragments thereof can be isolated and/or purified, as described herein with respect to other aspects of the invention.
[0077] It is contemplated that the inventive antibodies and antigen binding fragments thereof may be useful in methods of detecting the presence of demyelination in a mammal. In this regard, an embodiment of the invention provides a method of detecting the presence of demyelination in a mammal, the method comprising: (a) contacting a biological sample comprising blood and/or CSF with at least one of any of the inventive antibodies, or antigen binding fragments thereof, described herein, thereby forming a complex, and (b) detecting the complex, wherein detection of the complex is indicative of the presence of demyelination in the mammal. The demyelination may be as described herein with respect to other aspects of the invention.
[0078] Detection of the complex can occur through any number of ways known in the art. For instance, the inventive antibodies, or antigen binding fragments thereof, described herein, can be labeled with a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).
[0079] In another embodiment of the invention, the method comprises (a) contacting the biological sample with a first antibody, or antigen binding fragment thereof, selected from the group consisting of (i) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21), (ii) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22), or (iii) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23), thereby forming a first complex; (b) contacting the first complex with a second antibody, or antigen binding fragment thereof, selected from the group consisting of (i) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21), (ii) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22), or (iii) the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23), thereby forming a second complex; (c) detecting the second complex, wherein detection of the second complex is indicative of the presence of demyelination in the mammal, and wherein the second antibody, or antigen binding fragment thereof, is different from the first antibody, or antigen binding fragment thereof.
[0080] Preferably, the first antibody, or antigen binding fragment thereof, is the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of NPYNDSSLRNHPD (SEQ ID NO: 21), and the second antibody, or antigen binding fragment thereof, is the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22). In another preferred embodiment, the first antibody, or antigen binding fragment thereof, is the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23) and the second antibody, or antigen binding fragment thereof, is the antibody, or antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22).
[0081] The biological sample may be as described herein with respect to other aspects of the invention. In an embodiment of the invention, the blood is umbilical cord blood. [0082] In an embodiment of the invention, the method comprises washing any unbound first antibody and any unbound antigen from the first complex. The method may further comprise washing any unbound second antibody and any unbound antigen from the second complex.
[0083] The method further comprises detecting the second complex. The second complex may be detected in any suitable manner known in the art. In an embodiment of the invention, detecting the second complex comprises contacting the second complex with a detecting agent. The detecting agent may comprise, for example, a third antibody or antigen binding fragment thereof that specifically binds to the second complex (hereinafter referred to collectively as "detection antibody" or "detection antibodies"). The detection antibody binds to the second complex with measurably higher affinity to the second complex than to other molecules. In an embodiment of the invention, the detection antibody specifically binds to the second antibody.
[0084] In an embodiment of the invention, the detection antibody comprises a detectable label. The detectable label may be any suitable detectable label that provides a detectable signal. Non-limiting examples of detectable labels include enzymes (e.g., alkaline phosphatase, horseradish peroxidase), a radioisotope, fluorescent molecules (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), luminescent molecules, dyes, biotin, and element particles (e.g., gold particles). When the detectable label is an enzyme, the method may further comprise adding a substrate to the second complex that is catalyzed by the enzyme to produce a detectable signal. In an embodiment of the invention, the detectable signal may be amplified and/or visually detected. For example, acetylcholinesterase-catalyzed hydrolysis may be useful for colorimetric detection through gold or silver nanoparticle aggregation.
[0085] Another embodiment of the invention provides a method of detecting the presence of demyelination in a mammal, the method comprising: (a) contacting a biological sample comprising blood and/or cerebral spinal fluid (CSF) with an agent, thereby forming a complex between (i) the agent and (ii) an antibody, or antigen binding fragment thereof, having antigenic specificity for the agent, and (b) detecting the complex, wherein detection of the complex is indicative of demyelination in the mammal, wherein the agent is NF125, NF30, the mouse Casprl ι.380 amino acid sequence of SEQ ID NO: 33, the mouse Casprl38 i _ 13S5 amino acid sequence of SEQ ID NO: 34, the mouse Casprl 1.94-7 amino acid sequence of SEQ ID NO: 35, the mouse Casprl 94 -i 385 amino acid sequence of SEQ ID NO: 36, the mouse Casprl 38 i _947 amino acid sequence of SEQ ID NO: 39, the human Casprl 1.379 amino acid sequence of SEQ ID NO: 41 , the human Casprl38o-i384 amino acid sequence of SEQ ID NO: 42, the human Casprl 1-946 amino acid sequence of SEQ ID NO: 43, the human Casprl 947_i384 amino acid sequence of SEQ ID NO: 44, or the human Casprl 38o-946 amino acid sequence of SEQ ID NO: 45. The contacting and detecting may be carried out as described herein with respect to other aspects of the invention. The biological sample, the antibody, or antigen binding fragment thereof, the NF125, the NF30, the mouse Casprl 1.330 amino acid sequence of SEQ ID NO: 33, the mouse Casprl 38i-i385 amino acid sequence of SEQ ID NO: 34, the mouse Casprl 1.94·? amino acid sequence of SEQ ID NO: 35, the mouse Casprl 948-1385 amino acid sequence of SEQ ID NO: 36, the mouse Casprl38i.947 amino acid sequence of SEQ ID NO: 39, the human Casprl i-3 9 amino acid sequence of SEQ ID NO: 41 , the human
Casprl 38o-i384 amino acid sequence of SEQ ID NO: 42, the human Casprl 1.946 amino acid sequence of SEQ ID NO: 43, the human Casprl 947_i384 amino acid sequence of SEQ ID NO: 44, and the human Casprl 380-946 amino acid sequence of SEQ ID NO: 45 may be as described herein with respect to other aspects of the invention. In an embodiment of the invention, the method comprises (a) contacting a biological sample comprising blood and/or CSF with NF125 and NF30, thereby forming a first complex with an antibody, or antigen binding fragment thereof, having antigenic specificity for NF125 and forming a second complex with an antibody, or antigen binding fragment thereof, having antigenic specificity for NF30 and (b) detecting the first and second complexes, wherein detection of the complexes is indicative of demyelination in the mammal.
[0086] Another embodiment of the invention provides a method of detecting
demyelination in a mammal comprising (a) contacting a biological sample comprising blood and/or cerebral spinal fluid (CSF) with an antibody having antigenic specificity for mouse Casprl 38i-947 (SEQ ID NO: 39) or human Casprl38o-946 (SEQ ID NO: 45) and (b) detecting the complex, wherein detection of the complex is indicative of demyelination in the mammal. The method may, for example, use a sandwich enzyme-linked immunosorbent assay (ELISA) system. The sandwich ELISA system may use two antibodies, each antibody having antigenic specificity for Factor Xa cleaved non-membrane anchored fragments of Casprl comprising mouse Casprl 3^-947 (SEQ ID NO: 39). The sandwich ELISA system may use two antibodies, each antibody having antigenic specificity for Factor Xa cleaved non- membrane anchored fragments of Casprl comprising human Casprl 380-946 (SEQ ID NO: 45).
[0087] Epitopes to generate the antibodies may be derived from mouse Casprl 381 -947 (SEQ ID NO: 39) . Such antibodies will only detect the full-length mouse Casprl (SEQ ID NO: 32) and the Factor Xa cleaved non-membrane anchored fragments of mouse Casprl, i.e., mouse Casprl \.^η (SEQ ID NO: 35) and will not detect the membrane-bound fragment of Casprl , i.e., mouse Casprl 948-1385 (SEQ ID NO: 36). Epitopes to generate the antibodies may be derived from human Casprl 38o-946 (SEQ ID NO: 45). Such antibodies will only detect the full-length human Casprl (SEQ ID NO: 40) and the Factor Xa cleaved non-membrane anchored fragments of human Casprl , i.e., human Casprl 1.946 (SEQ ID NO: 43) and will not detect the membrane-bound fragment of Casprl , i.e., human Casprl 947- 1384 (SEQ ID NO: 44).
[0088] For purposes of the inventive detecting methods, the contacting can take place in vitro or in vivo with respect to the mammal. Preferably, the contacting is in vitro.
[0089] In an embodiment of the invention, the detecting methods comprise detecting the presence of a demyelinating disease in the mammal. The demyelinating disease may be as described herein with respect to other aspects of the invention. In an embodiment of the invention, the demyelination is associated with white matter injury.
[0090] As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
[0091] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLES
[0092] The following materials and methods were employed in the experiments set forth in Examples 1-5. Experimental design and statistical analysis
TABLE 1
Experimental Experimental
Experimental n P value
description manipulation
One-way
For Adenosine treatment ANOVA
C
n=105 Morphometric
Time course of change in Dunnette's
For 2MeSATP analyses
Example cell morphology
n=104 post test
1
P<0.0001
8 fields of view, optic
F
nerves, n=3 Dox-on mice t-test
Morphological changes in Measurement optic nerve astrocytes p=0.003 of GFAP area
7 fields of view, optic
nerves, n=3 Dox-off mice
Chi-square analysis of
C
n=10, 180 nodes of t-test frequency of 4
Perinodal astrocyte
Ranvier p<0.0001 morphologies morphology
of perinodal astrocytes
Example n=23 fields of view,
1
N= 3 Dox-on mice
D
t-test
Extracellular space in nodal TEM images area n=25 fields of view, p< 0.001 analyzed
N= 3 Dox-off mice
D Analysis of
Subcortical white matter Work in NF155/125
N=3
from Dox-on mice treated progress bands from with or without thrombin immunoblots
E
ELISA for Antithrombinlll ELISA for
Work in
release from primary N=7 Antithrombin progress
astrocytes from Dox-on and III Dox-off mice
Example
G Analysis of 2
Subcortical white matter Work in NF155/125
N=6
from progress bands from
Dox-on and Dox-off mice immunoblots
I
Subcortical white matter Analysis of from Work in NF155/125
N=8
Dox-off mice injected with progress bands from Fondaparinux sodium or immunoblots
PBS
D N = 7 Dox-on mice One-way IHC confocal
Example
Nodal length P60 optic N= 7 Dox-off mice
3 ANOVA images: 4 fields nerve N= 3 Recovery mice
Figure imgf000032_0001
Experimental Experimental
Experimental n P value
description manipulation
N=17 Dox-off mice VEP traces
Measurement
I N=7 Dox-on mice t-test
of optomotor
Visual acuity (in- vivo) N=6 Dox-off mice P=0.009
reflex
A N=6 Dox-on to on mice t-test Measurement VEP N=8 Dox-off to on mice n.s. of latency
24 fields n=6 Dox-on mice One-way
Example ANOVA
24 fields n=6 Dox-off mice IHC confocal 5 B Dunnette's
images: 4 fields 39 fields n=10 Dox-on to on post test
Nodal length (63x) per p>0.0001
mice animal
* P<0.05
30 fields n=8 Dox-off to on
H N=2 Dox-on mice t-test
Normalised intensity N=2 Dox-off mice P=0.01
Ratio of
N=4 Dox-on mice
normalized
Example
N=2 Dox-off mice for 5 day intensities of 3 I Work in EGFP in
Normalised intensity incubation progress immunoblots
N=2 Dox-off mice for 14
day incubation
Example E N=3 Dox-on mice One-way
4 ERG N=4 Dox-off mice ANOVA
n.s.
Electron
Example Size frequency distribution N=5 Dox-on mice t-test
microscopy 1 of axon diameters N=5 Dox-off mice P=0.126
analyses
Nodal length P60 optic N =4 Dox on mice t-test IHC confocal nerve N =4 Dox off mice P <0.001 images: 4 fields
Nodal length P60 N =3 Dox on mice t-test (63X) per
Example corpus callosum N =3 Dox off mice P <0.001 animal;
average of all 3 Nodal length P21 optic N =4 Dox on mice t-test
nodes in field nerve N =4 Dox off mice P <0.001
used in
Nodal length P21 N =4 Dox on mice t-test statistical corpus callosum N =4 Dox off mice P <0.001 analysis,
Experimental Experimental
Experimental n P value
description manipulation
n=number of fields.
N=7 Dox-on mice
One-way
N=7 Dox-off mice ANOVA
Dunnette's
Example Nodal length P60 optic N=3 Recovery mice
post test
3 nerve N=3 Recovery dark mice P<0.0001
N=4 Wild-type dark mice * P<0.05
N=3 adult onset mice
One-way
N=7 Dox-on mice ANOVA
B
Example N=7 Dox-off mice Dunnette's
Nodal length P60 optic post test
5
nerve N=3 LIF -/- mice P<0.0001
*** P<0.001
[0093] Table 1 shows the experimental design, sampling size, and statistical analysis for the major experiments in the Examples. Statistical analysis was based on unpaired Student's t-test for two group comparisons of continuous variables that were normally distributed.
ANOVA was used for multiple comparisons followed by Dunnett post-hoc test for
comparison with controls and Fischer's Exact test for multiple comparisons. Chi-square test was applied for analysis of discontinuous variables. Linear regression was by least squares fitting. Calculations were performed using MINITAB, MATHEMATICA, SIGMAPLOT, and GRAPHPAD software. Values are expressed as mean ± standard error of the mean
(SEM).
[0094] The sample size "n" used for statistical analysis was derived from experimental sampling designs that ensure that the sample size is not artificially inflated in the statistical calculations and that the results are well replicated. The approach that was used was highly conservative: "n = 1" typically represents the mean value of 300-500 separate measurements. Measurements were made on all nodes of Ranvier in a microscope field (300-500) and the average value taken as n = 1 for the quantitative analysis. Two nerves per animal ware analyzed from multiple animals. Sampling was evenly balanced among different animals (4 microscope fields/animal; 3-4 mice for the data in Example 3 (measuring nodal length in optic nerve of Dox-on and Dox-off mice), for example. For example; 22,400 nodes were measured for this experiment, (400 nodes X 56 fields in Dox On), but only an "n" of 56 was used for the statistical analysis, and this sampling was balanced evenly among 4 animals.
[0095] For experiments using very high power magnification (63 X objective lens with a 4X zoom by confocal microscopy), the smaller microscope field contained approximately 20 nodes of Ranvier. Measurements were made on all nodes in the microscope field and the average value used as n = 1 in statistical calculations.
[0096] A similar approach was taken in measuring myelin thickness (g-ratio). The thickness of the axon sheath and axon diameter was determined for every axon in the field of each transmission electron micrograph, and the mean value for all the axons was calculated and used as "n = 1" in the statistical analysis. For example, the data in Example 3 (measuring myelin thickness recovered after restoring vesicle release in astrocytes) use a sample size of 92 for the statistical analysis by ANOVA, but this represents measurements on 1 135 axons. Four measurements were made on every axon: the major and minor axes with and without the myelin sheath for each axon in cross section. This was done to more accurately determine diameter, as axons in cross section are not perfect circles. Both optic nerves from each animal were cut into 3-4 segments and mixed together for analysis by TEM.
Transgenic mouse production and maintenance
[0097] A line of transgenic mice - dominant-negative VAMP2 mice (dnVAMP2) - in which there is astrocyte-specific expression of an inducible dominant-negative form of VAMP2 that inhibits the release of ATP, by inhibiting VAMP2-dependent vesicle fusion, was used. Astrocyte specific glial fibrillary acidic protein (GFAP) promoter drives the expression of the "tet-OFF" tetracycline transactivator: GFAP.tTA (Ye et al., J. Neurosci Res., 78: 472-484 (2004)). Another transgenic line expresses a tet operator (tetO)-regulated dnVAMP2 domain and enhanced green fluorescent protein (EGFP) and LacZ reporter genes: tetO.VAMP2. Details of producing GFAP.tTA and tet0.dnVAMP2 lines are as described elsewhere (Pascual et al., Science, 310: 1 13-1 16 (2005)). These lines were maintained in a heterozygous state and backcrossed onto a C57B16/J genetic background (Jackson
Laboratories, Bar Harbor, ME) for more than 20 generations. Bigenic offspring of these two lines (dnVAMP2) were maintained on 200 mg/kg Dox containing food (Bioserv,
Frenchtown, NJ) to block transgene expression as control mice, whereas dnVAMP2 animals were fed regular diet to allow transgene expression in astrocytes. Previous studies (Pascual et al., Science, 310: 113-116 (2005)) and these data show that both dnVAMP2 and EGFP tetO transgenes co-inherit, implying that they are likely co-integrated on the same chromosome. This co-integration and consequent co-inheritance means that the highly sensitive reporter gene EGFP can be very reliably used as a surrogate reporter for the dnVAMP2 transgene in these studies. All mice were housed under specific pathogen-free conditions under a 12-hour (h) light/dark cycle with access to food and water ad libitum according to a protocol approved by National Institutes of Health (NIH) Animal Care and Use Committee.
[0098] Astrocyte-specific expression was confirmed in optic nerve, retina, corpus callosum, and visual cortex by immunocytochemistry and by western blot for optic nerve and cortex. No transgene expression in cells other than astrocytes could be detected in these areas. Western blot and immunocytochemistry were used to determine the time-course of gene regulation by doxycycline treatment (Example 3). The transgene was fully suppressed within 5 days of adding Dox to the diet (Example 5). After removing Dox from the diet, approximately 14 days are required to clear Dox from the system and reach maximum transgene expression (Example 3). Therefore to inhibit exocytosis from astrocytes during the period of myelination (late prenatal and early postnatal periods), Dox was removed from the mother's diet after fertilization. To inhibit transgene expression, Dox was supplied continuously to the mothers during gestation and until weaning, and then supplied directly to pups after weaning.
Immunohistochemistry
[0099] Mice were deeply anesthetized with isoflurane and perfused with 4%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Optic nerves and brains were collected, frozen, and cut in longitudinal and sagittal sections, respectively. Immuno staining with anti-pan sodium channel (rabbit polyclonal IgG, 1 : 100, Sigma, Saint Louis, MO, US) and anti-Casprl antibody (mouse monoclonal IgG, 1 :50, NeuroMab, Davis, CA, US) was conducted to visualize nodes of Ranvier in optic nerves and corpus callosum. To visualize oligodendrocyte lineage cells, OHg2 (rabbit polyclonal antibody, 1 :400, IBL, Takasaki, Japan) was used as a pan-oligodendrocyte marker. Anti-GFAP antibody (rabbit polyclonal IgG, 1 : 100, Invitrogen, Camarillo, CA, US) was applied to identify astrocytes. Images were captured by confocal laser microscope (LSM510, Zeiss, Germany). Nodal length measurement
[0100] Nodes of Ranvier were visualized by immunohistochemical staining of voltage- gated sodium channels, which are concentrated at the nodes of Ranvier, together with contactin-associated protein (Casprl), a cell adhesion molecule expressed in the paranodal region flanking the nodal gap. The length of nodes of Ranvier was measured using NIH IMAGE J software. For statistical analysis, all nodes of Ranvier in a microscope field at 63 X, magnification (approximately 300) were measured and averaged. N (sample size) in the statistical analysis represents the mean value of all nodes in each microscope field to avoid artificially inflating the sample size. Similarly, for very high magnification confocal imaging (100X with 4X zoom), the measurements were made on all nodes of Ranvier in the field (approximately 20) and the average taken for n = 1 in the statistical analysis.
Electron microscopy and measuring G-ratio
[0101] Mice were deeply anesthetized and perfused with 2.5% gluteraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4. Optic nerves were immediately removed from animals, then post-fixed in the same solution for 2 hours (hr). Samples were post-fixed for 1 hr in 1 % osmium tetroxide in 0.1 M sodium cacodylate, pH 7.4. Samples were washed two times in 0.05 M sodium acetate, pH 5.0, and stained with 1% uranyl acetate in 0.05 M sodium acetate, pH 5.0, for 2 hr at 4 °C. Samples were then dehydrated through a graded series of ethanol solutions and infiltrated with Epon. Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate and examined by transmission electron microscopy. G-ratio was determined by the numerical ratio between the diameter of the axon proper and the outer diameter of the myelinated fiber measured using NIH Image J software. Diameters were determined by measuring the major and minor axis of the axons in cross-section to correct for any deviation from circularity, and calculating the area of an ellipse to derive the diameter for a circle of equivalent area. Quantitative analysis of paranodal astrocyte morphology was determined without knowledge of the experimental condition.
Serial block face scanning electron microscopy and 3D reconstruction
[0102] Optic nerves were fixed, embedded and stained according to protocols provided by Renovo Neural Inc. (Cleveland, OH). Both nerves from each animal were cut into 3-4 segments and mixed together for microscopic analysis. Serial block face imaging was performed with 15 nm/pixel, 10 nm/pixel, 7.6 nm/pixel, and 5 nm/pixel resolution on 75 nm, 80nm, and 135 nm thick slices, and photographed using 16bit digitization. Field of view typically ranges from 40-46 μιη, at a magnification of 13,800 to 15,200 X. Beam voltage of 2 kV was typically used and approximately 100 slices imaged in z-series stacks. Imaging was also performed at 5.4k, 6.1 , 13k, 15k, 20k, 30k, 99k. Serial block face electron microscopy was performed at Renovo Neural, Inc, without knowledge of experimental condition and returned to the lab for analysis and quantitation. Serial 3D reconstruction was done in the laboratory and at Renovo, Inc. Serial image stacks were registered and cropped using ImageJ/FIJI. Axons, compact myelin, paranodal loops and astrocyte processes were traced manually using RECONSTRUCT software and meshes exported and rendered using BLENDER software (Blender.org).
Immunoblotting
[0103] For detection of NF155, NF125, NF30, NF186, Neuron specific Enolase (NSE), Casprl, myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and
glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), cells and brains were lysed in loading buffer with or without protease inhibitors. Cytosolic protein fractionation was done using PROTEOEXTRACT kit (Calbiochem, Billerica, MA) according to manufacturer instructions to probe for VAMP2 expression using VAMP2 antibody (Synaptic Systems, Gottingen, Germany) at 1 :1000.
[0104] For Peptide-N-Glycosidase F (PNGase F) treatment, subcortical white matter from Dox-OFF animals, post denaturation at 100 °C for 10 minutes, was incubated with PNGaseF for 1 hour at 37 °C as per manufacturer instructions (New England Biolabs, Ipswich, MA). PNGase F is an amidase that cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins, thereby removing N-linked glycosylated side-chains without affecting the amino acids linked to it (Maley et al., Anal. Biochem., 180 (2): 195-204 (1989)).
[0105] Then total protein was resolved by sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE) on 4-12% NUPAGE Bis-Tris gels and/or 3-8% Tris-Acetate gels (Life Technologies, Carlsbad, CA), transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA) and blocked in TBS (10 Mm Tris-Cl, pH 7.5, 0.9% NaCl) containing 0.1% (v/v) Triton X-100 (TBST) and 5% (w/v) bovine serum albumin for 1 hr at room temperature. Membranes were probed with appropriate antibodies in TBS-T and 5% BSA overnight at 4 °C. Primary antibodies were visualized with HRP- conjugated secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) at 1 :5000 dilution and enhanced chemiluminescence. Nonsaturated immunoblots were quantified using IMAGE J and/or METAMORPH software and normalized to GAPDH and/or Enolase.
Primary antibodies used for immunoblotting: GAPDH (Encor, Gainesville, FL) used at 1 :4000 dilution, MBP (Millipore, Billerica, MA) used at 1 :1000, GFAP (Zymed Laboratories San Francisco, CA) used at 1 :500, EGFP (Abeam, Cambridge, MA) used at 1 :2000, NF186 and NF30 (Abeam, Cambridge, MA) used at 1 :1000, NF155 and NF125 (Millipore, Billerica, MA) used at 1 : 1000, NSE (Abeam, Cambridge, MA) used at 1 :2000 and Casprl (Santa Cruz Biotech., Santa Cruz, CA) used at 1 : 1000.
Semi-quantitative RT-PCR
[0106] For mRNA expression analysis, 2 ig of total RNA was reverse-transcribed using SUPERSCRIPT II and oligo-dT, and polymerase chain reaction (PCR) performed on a Roche LIGHTCYCLER instrument (Roche, Minneapolis, MN) using FASTSTART DNA Master SYBR Green 1 PCR reaction mix. Relative expression levels were quantified. Primer sequences were as follows:
• GFAP: ACATCGAGATCGCCACCTAC (SEQ ID NO: 1),
TGCTTCGACTCCTTAATGACC (SEQ ID NO: 2)
• AQP4: TTCTCTTCGGTGCTAGGAAAC (SEQ ID NO: 3),
AGGAAGCTTATGTCTCTGGTG (SEQ ID NO: 4),
• GLAST: ACTTGATGAGCAATTATCAGTTACC (SEQ ID NO: 5),
TGGATGAGACAAGGCTCACTC (SEQ ID NO: 6),
• GLT-1: TTGACTCCCAACACCGAATGC (SEQ ID NO: 7),
AGGAATGGGAAAGGTACCTTGC (SEQ ID NO: 8)
• S100: CTCAAGTCTCTTCTTCCACAGTG (SEQ ID NO: 9),
TTGATTCTCGGTCGTGAGTTAG (SEQ ID NO: 10),
• KCNJ10 (Kir4.1): TGCTGGAGCCCTTCCTTTTCC (SEQ ID NO: 1 1),
TCCATCCAGTCACATGGTCCTC (SEQ ID NO: 12),
• EN02: TCCTGCTGTTCTTGGCTTCC (SEQ ID NO: 13),
AACACTCATGCCCGTGACC (SEQ ID NO: 14), • GAPDH: AATGCATCCTGCACCACCAAC (SEQ ID NO: 15),
TGGATGCAGGGATGATGTTCTG (SEQ ID NO: 16),
• NF-H: CCTCCGTAAGAAGAAACACTGC (SEQ ID NO: 17),
GTAGCGTTCAGCATACATCACG (SEQ ID NO: 18), and
• MBP: GCCAAATGTCACCATCTCTCC, ACTCCTGCAGTCCCACTTCC.
Bioinformatic analyses of paranodal cell adhesion molecules
[0107] ExPASy PEPTIDECUTTER tool (Wilkins et al., Methods Mol. Biol, 112: 531-52 (1999)) was used to look for potential protease sites in Neurofascin 155 (NF155). Universal Protein Resource (UniProt) was used to assess the cellular localization of the Thrombin cleavage site in NF155. Hydrophilicity, surface probability and solvent accessibility of domains within NF155, including the third Fibronectin Type III domain containing the Thrombin cleavage site, were assessed by Kyte-Doolittle hydropathy analysis ( yte and Doolittle., J. Mol. Biol, 157: 105-132 (1982)), Emini analysis (Emini et al., J. Virol, 55(3): 836-839 (1985)) and PREDICTPROTEIN analysis (Yachdav et al., Nucleic Acids Res., 42 (Web Server issue), W337-W343 (2014)), respectively. MULTALIN tool (Corpet, Nucleic Acids Res., 16(22): 10881-10890 (1988)) was used to align alternatively spliced isoforms of the Neurofascin family expressed in the nodal apparatus; NF186, in the node of Ranvier and NF155 in the paranode.
Protein structure prediction
[0108] The three dimensional structure of NF155 was predicted using I-TASSER (Iterative Threading ASSEmbly Refinement), a computer algorithm for protein structure and function predictions (Yang et al., Nature Methods, 12: 7-8 (2015); Roy et al., Nature Protocols, 5: 725-738 (2010)). I-TASSER has been ranked as the best algorithm in the world for protein structure prediction in the five most recent CASP (Critical Assessment of Techniques for Protein Structure Prediction) experiments; CASP7, CASP8, CASP9, CASPIO and CASP 1 1 (Roy et al., Nature Protocols, 5: 725-738 (2010)). Each CASP experiment is a biennial, world-wide evaluation of structure prediction methods, with approximately 100 participating laboratories. The predicted NF-155 structure was then rendered and annotated in POLYVIEW-3D tool (Porollo et al., BMC Bioinformatics, 8: 316 (2007)). Liquid chromatography-mass spectrometry (LC-MS) analyses
[0109] Liquid chromatography-mass spectrometry (LC-MS) facility was used to identify NF125 and NF30 bands in immunoblots as cleaved products of NF155 (Example 2).
Subcortical white matter from Dox-OFF animals was run in a SDS-PAGE gel with size- appropriate protein ladders. Bands on the gel corresponding to 155KDa, 125KDa and 30KDa were excised and sent to the LC-MS facility, NIH for downstream analysis.
Fondaparinux Sodium treatment
[0110] Fondaparinux Sodium is a synthetic pentasaccharide that mimics the minimum necessary pentasaccharide sequence in Heparin which binds and activates Antithrombin-III (AT-III). It is an ultra-low molecular weight selective Factor Xa inhibitor that can cross placental and blood-brain barriers (Hoppensteadt et al., Hematol. Oncol. Clin. North Am., 17(1): 313-341 (2003)). Factor Xa converts prothrombin to thrombin and AT-III inhibits this reaction by competitively binding to the catalytic site of Factor Xa. However, in the absence of catalysts like Fondaparinux, only 1 in 400 AT-III molecules (0.25%) are in the right conformation to sterically inhibit conversion of prothrombin to thrombin via Factor Xa (Langdown et al., J. Biol. Chem., 279(45): 47288-47297 (2004)). Fondaparinux Sodium was administered daily to 8 Dox-OFF mice via subcutaneous injection at a concentration of 10 mg/kg for 20 days, following which the animals were euthanized and subcortical white matter harvested for immunoblotting and optic nerves for immunohistochemistry. Control Dox-off animals were injected with phosphate buffered saline (PBS) instead.
Mathematical modeling of conduction velocity
[0111] A macroscopic homogenized cable equation describing the passive, linear ("electrotonic") response of a myelinated axo, was previously derived from a
microcontinuum composite cable model consisting of an infinite array of nodes of Ranvier separated by myelinated internodal segments (Basser et al., Medical & Biological
Engineering & Computing, 31 : S87-92 (1993)). The effective space and length constants of this macroscopic cable equation were functionally related to the space and length constants of the individual nodal and myelinated segments (see Eqs. 32 and 33 in Basser et al., Medical & Biological Engineering & Computing, 31 : S87-92 (1993)). This macroscopic model appropriately balances the contributions from the nodal membranes and the insulated myelinated portions of the axon to predict the response of the entire myelinated axon.
Specifically, this macroscopic model can be used to predict the macroscopic or aggregate current and electrical potential distributions along the axon from individual electrical properties and microscopic dimensions of the nodal and myelinated regions. Effective speed of propagation, c, as defined in Basser, J. Integr. Neurosci., 3(2): 227-44 (2004), can be written as:
Figure imgf000042_0001
where the parameters are as follows:
* g: g-ratio (variable)
» i: width of node of Ranvier (variable)
• L: '.distance ..between nodes of Ranvier (sw 100 /an - varies with S)
• pm: resistivity of myelin 7 A 10s kO cm
• pn: resistivity of membrane 6,9 10s Ml cm
* pa. resistivity of axopiasin 5.47 10"2 Ml em
* ίίπι: dielectric constant of myelin (7)
# κ.η: dielectric1 constant of membrane (7)
# (£¾: permittivity of vacuum (8.85 ICr8 μψ/cm)
• h,'thickiiass of the membrane (5 ntxt)
» <¾: inner diameter of the axon membrane (5 μιη)
• e a factor by which inner diameter increases at the nodal region
[0112] The ratio of the effective space and time constants has the unit of speed, while the ratio of the square of the effective space constant and time constant can be interpreted as an electrical diffusivity. Both quantities can help assess how efficiently a disturbance in the transmembrane potential is transported along the myelinated axon. Therefore, this framework allowed prediction as to how changes in the width of the node or nodal capacitance will affect how electrical disturbances will migrate, with the caveat that the model does not predict the actual propagation speed which is much larger and would depend on many other factors not studied here. It is not expected to be a good indicator of the relative changes in the speed, in terms of the changes in the nodal region width, delta (δ), and the G-ratio.
Electrophysiological recording of compound action potential
[0113] An ex vivo optic nerve preparation was used to study compound action potentials (CAPs). Optic nerves were dissected from adult (2-4-month-old) mice in which vesicle fusion in astrocytes was inhibited (Dox-off) or transgene expression was inhibited by doxycycline (Dox-on). Optic nerves (4-5 mm of length) were dissected free and cut at the optic chiasm and behind the orbit. The preparation was placed in an interface perfusion chamber (Medical Systems), maintained at 35 °C and superfused with artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 4 KC1, 25 NaHC03, 1.25 NaH2P04, 2.5 CaCl2, 1.5 MgCl2, and 25 glucose, pH 7.4. The chamber was continuously aerated with humidified gas mixture of 95% 02/5% C02. ACSF ran continuously at 2-3 ml/min. Following dissection, optic nerves were allowed to equilibrate for about 30 minutes (min) before the experiment was started. Suction electrode back filled with ACSF was attached to the rostral end (retina side) and stimulated every 10 seconds (s) (A365R stimulus isolator, WPI). A recording suction electrode filled with ACSF was attached to the caudal end (adjacent to the chiasm) of the nerve to record CAPs. The distance between the electrodes was measured with a calibrated ocular reticule. Evoked potentials were amplified with Axoclamp 2B (Molecular Devices, Sunnyvale, CA) and acquired on-line (Digidata 1200 A, Molecular Devises, Sunnyvale, CA) using PCLAMP software (Molecular Devices, Sunnyvale, CA). Strength-duration curves were determined by recording responses to a series of square pulses of 0.05 to 0.2 mA intensities of 0.01 to 0.2 ms stimulus durations, and were used to determine rheobase and chronaxie; data in Example 5 are from supramaximal stimulation using 0.05 ms duration stimuli.
Compound action potential analysis
[0114] To compare the signal conduction properties between Dox-on and Dox-off mice, compound action potential (CAP) responses from both were measured. The CAP time series were obtained using fixed duration of 0.05 ms (a value established using standard stimulus strength-duration curve procedure), and the strength was incremented in small steps (usually, 50 or 100 μΑ) until multiple peaks started to appear. After that point, additional 7 to 10 stimulus increments were collected to assure supramaximal stimulation (maximal strength applied in each case varied between 0.5 and 1 mA). Several approaches were used to obtain measures of temporal latency in the CAPs. The central approach involved decomposing the obtained CAPs into a sum of Gaussians, in which the number of the Gaussians, ^ , is fixed a priori. Hence, the model used for fitting (with ^g x ^ parameters) can be written as,
Ne (f fl, )2
C4P( =∑c,.e 2b<~ + B(t) + (t)
(SI) where a/A>c/ are the mean, standard deviation and the amplitude of the z'th Gaussian, B(t) is the part of the CAP attributed to the stimulus artifacts, and ) is the residual of the fit. The features of B(t) are the initial pair of sharp peaks, which is a capacitive response to the application of a square pulse, and a slight shift in the baseline that persists until after 6 ms. B(t) was eliminated from the model by (a) truncating the negative portion of the peak (and using the time of this negative peak to define t=0), (b) fitting the positive peak as just another Gaussian in the model, and (c) empirically determining the persistent baseline (average over the last 50 points) and subtracting it. Since the initial and the final baselines were different, the subtracted signal was smoothly (exponentially) ramped up from zero to the given measured persistent baseline between t=0 and the time of the arrival of the first peak. Thus obtained curves were fit to a sum of Gaussians in two different manners. In the first, each individual curve was fit separately (single curve fitting, SF), and in the other, multiple curves, corresponding to a set of nc strongest stimulus strengths, were fit simultaneously (multiple curve fitting, MF). MF relies on the fact that the strength of the stimulus did not have a major influence on the position or the width of the peaks, but changes only their amplitude.
Hence, the parameters a\ and are shared between different curves and the total number of parameters used to fit nc curves was + n ^g _ Since fitting the Gaussians can be very sensitive to the choice of initial conditions, in both SF and MF, a randomized search for the best initial conditions was performed until a fit with the overall smallest least square error was obtained, expressed as the percent of the total square magnitude in the data,
plSE = 00%∑r* /∑y >
(Pajevic et al., PLoS ONE, 8(1): e54095 (2013)) where r\ and J>/ are the residual and the magnitude of the zth point in the time series. The first Gaussian (Gl) was deemed contaminated by the stimulus artifact and only the remaining three Gaussians (G2, G3, and G4) were used in the analysis. Since closely spaced Gaussians are difficult to resolve and the estimate of one could influence the nearby Gaussians, a weighted time estimate between 2 or 3 slowest peaks, i.e., each Gaussian's peak time was weighted by its area-under-curve, which is robust to such interactions, was also used. Ultimately, this type of center-of-mass (COM) estimate can be made independent of the fitting procedure. To do so, the COM was calculated for each data curve, by weighting each point in time series by its amplitude, obtaining essentially the arrival mean time, if the curve is interpreted as a probability density function of spike time arrivals. To avoid influence of the early artifact, only the data points between 0.3 ms and 5 ms were considered, but it was found that the precise choice of the limits is not important as long as the artifact peak, located in most curves between 0.13 and 0.15 ms, is excluded.
[0115] The measured CAPs were fit using different number of Gaussians ( ~ 4, or
5), and it was found that for ~ ^ most parsimonious fits were able to be obtained with
PLSE<\ % for virtually all CAPs with reasonable stimulus strength. For example, for all the data and the strongest five stimuli PISE range from 0.28% to 1.01% (mean: 0.62+/-0.21 %) for Dox-on, and range from 0.14% to 1.00% (mean: 0.56+/-0.28) for Dox-off. No difference in terms of the quality of the fits was detected between Dox-on and Dox-off (p=0.35 using a
Mest). The PISE for the examples described in Example 5 were 0.65% for Dox-on and 1% for Dox-off (the worst fit obtained for Dox-off). Since the fits were not perfect, a number of other more robust measures of latency, but less specific, were used. In Example 5, the comparisons were summarized using the bar charts and using a number of different measures of temporal latency. It was observed that the Dox-off time latency is larger no matter what measure is used. Averaged over all measures Dox-on times were 14% ± 1% shorter. This difference was further quantified using hypothesis tests, a non-parametric Mann-Whitney- Wilcoxon (MWW) test, and a Mest which relies on normality of the data. All tests were performed as one-sided tests with null hypothesis that the Dox-on and Dox-off temporal latencies are equal vs. the alternative hypothesis that Dox-on temporal latency is smaller. Results in Example 5 showed that Dox-on signal conduction is declared faster for all measures and in all tests at the statistical significance level =0.05, except for the Mest on COM measure, for which p = 0.0525 (but still declared significant using a non-parametric MWU test at p = 0.023 and Mest with p = 0.038). Electrophysiological recording of visual evoked potential (VEP) and electroretinogram (ERG)
[0116] VEP and ERG electrophysiology were performed using the ESPION e2 software from Diagnosys, LLC. The mice were anesthetized with an intraperitoneal injection of xylazine-ketamine (10 mL/kg) in normal saline. Body temperature was maintained at 37 °C with a heating pad. Pupils were dilated with atropine (1%). For ERG recordings, a gold wire electrode was placed on the corneal surface of each eye and reference to a gold wire in the mouth. For VEP recordings, 2 needle electrodes were inserted subcutaneously to touch the skull above the visual cortex. A needle electrode in the base of the tail served as the ground. Animals were light-adapted for all recordings. Responses were obtained to 6 different light intensities, covering a range eliciting threshold to supramaximal responses.
Visual acuity measurement
[0117] Visual acuity was performed with a commercially available optomotor system designed for mice (OPTOMOTRY, CerebralMechanics, Lethbridge, Canada). Mice were placed on the elevated platform in the enclosed environment that displayed moving vertical stripes on 4 computer monitors surrounding the animal. Optomotor responses were recorded simultaneously by two observers monitoring the mouse via video camera as the size of the rotating vertical bars on the striped drum assay were varied automatically according to a computerized program, and varied systematically to determine the threshold response indicating maximal visual acuity. A complete series of measurements was made with the stripes rotating in the clock-wise direction and then repeated with rotation in the opposite direction.
EXAMPLE 1
[0118] This example demonstrates morphological changes in perinodal astrocytes by purinergic signaling.
[0119] Confirming previous reports (Abbracchio et al., Prog. Brain Res., 120: 333-342 (1999); Abe and Saito, Brain Res., 804: 63-71 (1998); Neary et al., Acta Neuropathol., 87: 8- 13 (1994)), application of 2MeSATP (a non-hydrolysable analog of ATP) or adenosine caused accumulation of GFAP cytoskeletal filaments and changed the shape of astrocytes to assume a more condensed stellate morphology in cell culture. The morphological changes in astrocytes and bundling of GFAP cytoskeletal filaments induced by adenosine and 2 MesATP were evident within 10 minutes. Therefore, the hypothesis that perinodal astrocytes undergo similar changes in vivo was tested. ATP is released from astrocytes in part by VAMP2- dependent exocytosis and then rapidly hydrolyzed to adenosine (Parpura et al., Neurochem. International, 57: 451-459 (2010); Pangrsic et al., J. Biol. Chem., 282: 28749-28758 (2007); Coco et al., J. Biol. Chem., 278: 1354-1362 (2003); Nadjar et al., Glia, 61 : 724-731 (2013); Lalo et al., PLoS Biol. 12(1): el001747 (2014)). Exocytosis of ATP from astrocytes is inhibited approximately 50%, without measurable effects on glutamate release, in transgenic mice expressing a doxycycline (Dox) regulated dominant-negative VAMP2-fragment, vesicle associated membrane protein 2 (dnVAMP2), which is expressed specifically in astrocytes by the astrocyte-specific promoter of GFAP (Pascual et al., Science, 310: 113-116 (2005);
Marpegan et al., J. Neurosci., 31 : 8342-8350 (201 1)). Expression of the dnVAMP2 protein is induced by removing Dox from the diet (Dox-off). This can be monitored by the EGFP and LacZ reporters, and was further confirmed by Western blot analysis of the transgene. Using this method, exocytosis was inhibited specifically from astrocytes and the structure of nodes of Ranvier and myelin were examined. Optic nerve was chosen for analysis because it is comprised of myelinated axons and astrocytes, without complications from synapses or other neurons, and the structure and circuitry of the retina is well known and easily manipulated by light exposure. Expression of the transgene in optic nerve and retina was strictly exclusive to astrocytes and only expressed in the Dox-off condition. No changes in astrocyte number, number of oligodendrocyte lineage cells (01ig2+) (Table 2), or axon diameter were found in Dox-on or Dox-off animals. Electrophysiological recordings showed normal retinal function in animals on and off Dox. The shape, amplitude, and latency of the electoretinogram (ERG) are highly sensitive to retinal and optic nerve function (Miura et al., Exp. Eye Res., 89: 49-62 (2009)). The ERG was normal in dnVAMP2 animals, and no differences were found between animals on or off Dox. TABLE 2
Figure imgf000048_0001
[0120] GFAP cytoskeletal filaments observed by confocal microscopy were more diffuse in optic nerve astrocytes in the Dox-off condition consistent with the formation of robust bundles of GFAP cytoskeleton in astrocytes treated with adenosine or ATP. Ultrastructural analysis by transmission electron microscopy (EM) and serial block-face scanning EM revealed fine structural changes in glial intermediate filaments in optic nerve astrocytes, and the morphology of perinodal astrocytes changed after inhibiting exocytosis in astrocytes. Transmission electron microscopy (EM) showed perinodal astrocytes (astro) forming close associations with the axolemma at the node when the dnVAMP2 transgene was suppressed (Dox-on). Glial filaments (gf) accumulated in the Dox-on condition.
[0121] Typically, perinodal astrocytes partly ensheath the nodal gap and associate with axons through blunt terminals in close contact with the axon in the nodal region (Black et al., Glia, 1 : 169-83 (1988); Raine et al., J. Neurocytology, 13: 21 -27 (1984)) as shown in the Dox-off condition. TEM analysis showed that perinodal astrocytes withdrew from the node and contacted the axon through long filopodial processes that lacked accumulations of intermediate filaments when exocytosis from astrocytes was inhibited. A chi-square test was performed to determine whether the two morphologies of perinodal astrocytes (blunt and filopodial) were equally represented at nodes of Ranvier in animals on and off Dox, and the null hypothesis was rejected with a high probability, χ (2, n = 130) = 55.9, p < 0.0001. The normal morphology (blunt) was seen in the majority of nodes of Ranvier in animals on Dox (45%), but the majority of nodes in the Dox off condition (80%) had filopodial contacts with the axon. Morphology of perinodal astrocytes returned to normal after restoring exocytosis from astrocytes by resupplying Dox to the diet at postnatal day 21 and examining the nerves at 60 days of age (P60). Only 7% of nodes had astrocytes with the filopodial morphology after restoring vesicular release, compared with 80%> of nodes in the Dox-off condition, χ2 = (2, n = 1 15) = 10.6, p < 0.005. In all conditions 18-20 % of nodes either lacked a perinodal astrocyte or the morphology of the astrocyte could not be classified.
[0122] Perinodal astrocytes having filopodial contacts with the node are typical of early stages of myelination or remyelination, where the filopodial contacts have been suggested to play an unknown role in nodal development (Raine et al., J. Neurocytology, 13: 21-27 (1984)). Quantitative analysis by TEM showed that the extracellular space surrounding the node increased markedly as astrocytes withdrew from the nodal membrane (p < 0.001, n=48, /-test), but extracellular space did not change in non-nodal regions (p = 0.35, Dox on vs. off, /-test, n=62) indicating an effect localized specifically to the nodes of Ranvier. These ultrastructural features were documented in three-dimensional detail by three dimensional (3D) serial reconstruction from serial sections obtained by serial block-face scanning electron microscopy. Note that the nodal gap is tightly ensheathed by imbricating globular-shaped astrocytes in the Dox-on condition, but when exocytosis from astrocytes was inhibited (Dox- off), the astrocytes withdrew and contacted the node through slender filopodial processes.
EXAMPLE 2
[0123] This example demonstrates that perinodal astrocytes regulate node of Ranvier and myelin thickness.
[0124] Astrocytes that are closely associated with synapses stabilize spine morphology (Nishida et al., J. Neurosci., 27: 331 -340 (2007)), suggesting that the reduction in GFAP bundling and withdrawal of perinodal astrocytes might permit structural dynamics at the node of Ranvier. Layers of myelin membrane wrapped around the axon flank the nodal region to form a spiral, which appears as a series of paranodal loops when seen by TEM in long- section through the axon. These paranodal lopes are cytoplasmic pockets of uncompacted myelin membrane attached to the axon through a tri-molecular complex of cell adhesion molecules: Neurofascin 155 (NF155) in the paranodal myelin membrane, Casprl in paranodal axon membrane, and axonal Contactinl interacting with these two molecules form septate-like axo-glial junctions. In NF155 and Casprl knock-out animals, these junctions are disrupted and paranodal loops become detached, resulting in mislocalization of ion channels in the nodal and paranodal regions (Sherman et al., Neuron, 48(5):737-742 (2005); Pillai et al, J. Neurosci Res., 87(8): 1773-1793 (2009); Bhat et al, Neuron, 30(2): 369-383 (2001)). Withdrawal of the perinodal astrocyte in the Dox-off condition could expose these paranodal junctions to enzymatic cleavage and thus permit detachment of the outermost paranodal loops of myelin flanking the nodal gap.
[0125] Bioinformatic analysis revealed a potential thrombin cleavage site in the extracellular domain of NF155 at AA924 in mouse (AA910 in rat), a region critical for interacting with Contactin-1. Thrombin participates in activity-dependent synapse elimination at neuromuscular junctions (Liu et al., Nature Neurosci., 15: 1621-1623 (1994)), and cleavage of NF 155 at AA924 would disrupt its binding to Contactin-1 , breaking the axo- glial junction attaching paranodal loops to the axon. Validity of the putative thrombin binding site on NF155 was determined by thrombin treatment of subcortical white matter extract. This resulted in cleavage of NF155 at AA924, which was blocked by co-treatment with antithrombin III, a specific inhibitor of thrombin enzymatic activity, as well by general protease inhibitors. Amino acid sequencing by Matrix Assisted Laser Desorption/Ionization (MALDI)-Time of Flight (TOF) confirmed the identity of NF155 and the cleaved fragment bands on the immunoblots. The cleaved fragments have the correct molecular weights expected from cleavage at the putative thrombin cleavage site on NF155 at AA924. Two antibodies were used to provide independent confirmation of cleavage of NF155 into two fragments: a long 125 kDa fragment, Neurofascin 125 (NF125), and a short 30 kDa fragment, Neurofascin 30 (NF30). One antibody binds with amino acid sequences that are N- terminal to the thrombin cleavage site, thus recognizing the full length NF155 and the longer cleaved fragment, NF125, but not the shorter NF30 fragment. The second antibody binds amino acid sequences C-terminal (intracellular) to the thrombin cleavage site, a domain that is conserved among all neurofascin family members, (NF186, 180, 166, and 155), but it will not recognize the NF125 fragment. [0126] In addition to structural plasticity of perinodal astrocytes in stabilizing and destabilizing the node of Ranvier, perinodal astrocytes may also regulate enzymatic cleavage of the cell adhesion molecules forming the junctions between paranodal loops and the axolemma. Astrocytes in culture release antithrombin III, which inhibits thrombin activity, (Deschepper et al., Brain Res. Mol. Brain Res., (3-4): 355-358 (1991); Dowell et al., J. Proteome Res., 8(8): 4135-4143 (2009)). Release of antithrombin II from astrocytes was verified by enzyme-linked immuno assay (ELISA) of antithrombin III concentration in conditioned medium from cultures of cortical astrocytes, indicating 1.2 nM of antithrombin III in culture medium 1 hour after a medium change. Consistent with the hypothesis that withdrawal of the perinodal astrocyte promotes cleavage of NF155, Western blot analysis of subcortical white matter extracts from Dox-off animals revealed that NF155 was cleaved when exocytosis of ATP from astrocytes was inhibited. No changes in other cell adhesion molecules at the node (NF186, NF166), were observed. Moreover, cleavage of NF155 in vivo was protected in Dox-off animals by daily subcutaneous injections of the thrombin inhibitor Fondaparinux, demonstrating thrombin-dependent cleavage of NF155 occurs in animals when exocytosis is inhibited in astrocytes.
EXAMPLE 3
i
[0127] This example demonstrates the structural plasticity of the node of Ranvier and myelin thickness.
[0128] Withdrawal of the perinodal astrocyte surrounding the node and cleavage of NF155 might allow modification of the outer wrapping of myelin. Structural support of the nodal apparatus would weaken as perinodal astrocytes lose bundles of GFAP filaments and withdraw from the node. Reduced protection from extracellular thrombin, which derives from many cellular sources in the CNS (Sokolova and Reiser, Thromb Haemost., 100(4): 576-81 (2008)), and a reduction in astrocyte-secreted antithrombin III in close proximity to the paranodal loops, would facilitate thrombin-dependent cleavage of NF155 observed when exocytosis in astrocytes is inhibited. Detachment of the outermost paranodal loop from the axon and resorption of this outer layer of myelin membrane back into the oligodendrocyte cell body would increase the nodal gap length so that the myelin sheath would thin slightly. All of these structural changes were observed, as explained in more detail below.
[0129] High resolution EM and 3D serial reconstruction from serial block-face scanning EM showed filopodia from perinodal astrocytes in the Dox-off condition probing the paranodal loops of myelin. In many cases the outermost paranodal loops of myelin adjacent to astrocyte filopodial lacked septate junctions and were detached from the axon when exocytosis from astrocytes was inhibited. This was evident both by high-resolution EM, and 3D reconstruction from serial block-face scanning EM. There was a marked change from the normal globular and imbricating layers of perinodal astrocytes tightly ensheathing the node in Dox-on animals to withdrawal of perinodal astrocytes forming long filopodial processes when exocytosis from astrocytes was inhibited in Dox-off animals. Dox-on animals showed the typical ultrastructure of regularly arranged paranodal loops of myelin attached to the axon by intercellular septate junctions. In contrast, the outermost paranodal loops of myelin were often irregular and probed by astrocyte filopodia and in some cases, paranodal loops were dislodged from the axon adjacent to astrocyte filopodia after inhibiting exocytosis from astrocytes in Dox-off animals.
[0130] Detachment of the paranodal loops of myelin adjacent to the perinodal astrocyte should increase the size of the nodal gap. This was evident in electron micrographs as astrocytes withdrew from the node in the Dox-off condition. The nodal gap lengthening was apparent by confocal microscopy and analyzed quantitatively following immunocytochemical staining of sodium channels marking the nodal gap, and the cell adhesion molecule Casprl marking the paranodal regions. Nodal gap length in optic nerves from animals treated with Dox was not different from normal (Rosenbluth et al., J. Neurosci. Res., 87: 3250-3258 (2009); 0.55 ± 0.032 vs. 0.52 ± 0.01 1 μ ι nodal gap length in optic nerve from wild type and Dox-off mice respectively (n=24, 3 mice), but it increased to 0.72 ± 0.019 μιη when VAMP2- dependent exocytosis from astrocytes was inhibited (Dox-off). Similarly, nodal gap length was significantly larger in the Dox-off condition in the corpus callosum (p<0.001 , t-test, n=32 for optic nerve (P21 and P60), p < O.OOx, t-test, n=32 for corpus callosum (P21), p < 0.001 , t- test, n=24 for corpus callosum (P60)). In the Dox-off condition, many of the nodal gap lengths exceeded the maximal length measured in the Dox-on condition as evidenced by cumulative probability plots. This is evident by the two nodes shown in the serial block face EM section taken from optic nerve of a Dox-off animal.
[0131] The increased nodal gap when exocytosis was inhibited in astrocytes was evident at all ages (P25, P60, P I 00), indicating a persistent effect that was not restricted to the period of active myelination, or a consequence of failure to maintain normal morphology with age. Importantly, the increase in nodal gap length after inhibiting exocytosis from astrocytes in adult mice (Dox-off) was prevented by daily subcutaneous injection of the thrombin inhibitor Fondaparinux (10 mg/ g) for three weeks, consistent with the proposed mechanism of thrombin-mediated cleavage of NF155 widening the nodal gap when exocytosis in astrocytes is inhibited. Immunoblot analysis confirmed that NF155 cleavage in subcortical white matter was inhibited in these same animals that were treated with the thrombin inhibitor.
[0132] Voltage-sensitive sodium channel distribution in the nodal gap changed as a consequence of nodal remodeling regulated by exocytosis in perinodal astrocytes acting through a thrombin-dependent mechanism. The spatial distribution of sodium channels within the node were determined by quantitative line scans of sodium channel abundance within the nodal gap (fluorescence intensity of sodium channel immunostaining, using high- resolution confocal microscopy). Sodium channel distributions were well-fitted by a Laplace distribution in the Dox-on condition (where the nodal gap length was normal) (r2 = 0.978, p < 0.0001 ; f = 0.161 + 0.783 * exp (-0.5*abs ((χ-χ0)/0.178)Λ1.01). The distribution of sodium channels was less sharply concentrated in the node when exocytosis from astrocytes was inhibited (Dox-off), following a distribution that was better fit by a generalized Gaussian function (r2 = 0.994, /? < 0.0001, f=0.0679+0.858*(-0.5*((x-x0)/0.366)A2). Treating Dox-off animals with the thrombin inhibitor, Fondaparinux, prevented the dispersion of sodium channels, and was best fit by a Laplace distribution. The distribution was indistinguishable from the Dox-on condition (r2 = 0.995; p < 0.0001, f = 0.0707+0.847*exp(-0.5*abs((x- x0)/0.255)A1.48). The lateral dispersion of sodium channels was significantly different comparing the width of the sodium channel density functions in all three conditions at the half-maximal point, 0.477±0.0445, 0.797±0.041 1 , 0.544±0.0239 ran respectively for Dox-on, Dox-off, and Dox-off treated with Fondaparinux; One-way ANOVA, F2;78 = 20.5, p < 0.0001. There was no significant difference in sodium channel dispersion between Dox-on and Dox- off treated with Fondaparinux. Thus, the density distribution of sodium channel inside the node of Ranvier is influenced by structural changes in the node regulated by exocytosis in astrocytes, and the density distribution is restored to normal by thrombin inhibition in Dox- off animals.
[0133] By hypothesis, the increased nodal gap length resulting from detachment of the paranodal loops adjacent to the perinodal astrocyte should be accompanied by reduced myelin thickness if the outer layer of myelin associated with these loops is resorbed back into the oligodendrocyte. The mean increase in nodal gap length in the Dox-off condition corresponds to a loss of approximately 2 paranodal loops of myelin. This would predict a decrease in myelin sheath thickness corresponding to two wraps of compact myelin. Quantitative TEM analysis of axons in ultrathin cross-sections showed that myelin sheath thickness was reduced significantly (p < 0.001 , /-test, n=56) when exocytosis in astrocytes was inhibited. The difference in myelin sheath thickness on and off Dox for all axon diameters was equivalent to a mean loss of 2.58 ± 1.55 wraps of myelin in the Dox-off condition, which is not significantly different from the predicted value of 1.99 ± 1.40 wraps (p =0.106), if the myelin thinning was caused by the observed increase in nodal gap length resulting from an average of two paranodal loops becoming detached. The interlamellar distance within the compact myelin sheath did not differ and no delamination or degeneration of the myelin was seen, indicating normal compaction of myelin. Paranodal loops more distal to the perinodal astrocyte had normal morphology and prominent septate junctions.
[0134] This remodeling of myelin structure was reversible. Perinodal astrocyte morphology, nodal gap length, and myelin thickness recovered when exocytosis in astrocytes was restored by re-supplying Dox in the diet at P21 to inhibit the transgene (n=24 for Dox on and off; n=28 for recovery; n=40 for adult onset; all p < 0.001). This process could be induced in the adult also by removing Dox from the diet after the optic nerve is myelinated. In these adult-onset experiments, Dox was removed from the diet at P21, which would inhibit the transgene two weeks later when the optic nerve is fully myelinated, and nodal gap length was quantified at P60. Thus mature myelin is structurally dynamic and regulated by exocytosis in astrocytes. Perinodal astrocytes, acting through VAMP2-dependent exocytosis, regulate nodal structure and myelin thickness dynamically by regulating thrombin-dependent cleavage of axo-glial junctions attaching the outermost paranodal loops of myelin to the axon membrane.
EXAMPLE 4
[0135] This example demonstrates the influence of functional activity on myelin plasticity.
[0136] To investigate the possible effects of functional activity on structural plasticity of the node of Ranvier, mice were housed in the dark and optic nerves were analyzed. Nodal gap length in wild-type animals dark-reared from P21 to P60 was not significantly different from animals reared under normal conditions. Thus, the widening of the nodal gap in the Dox-off animals was not produced by perturbing normal functional activity. In the absence of normal sensory input, myelination appeared normal. However, functional activity was found to promote the recovery process of restoring normal gap length after the nodal gap widening produced by inhibiting exocytosis from astrocytes. After restoring VAMP2- dependent exocytosis in astrocytes by resupplying Dox to the diet, the nodal gap length only partially recovered in animals housed in the dark, in contrast to animals reared under normal conditions (n=56 for Dox ON and OFF; n=40 for recovery, recovery dark and adult onset; n=56 for WT; all p<0.001). This is consistent with different processes involved in widening the nodal gap (and thinning the myelin sheath), and narrowing the nodal gap (and increasing myelin sheath thickness), and that the two processes are influenced differently by sensory deprivation. Together these studies indicate that functional activity in axons promotes increased myelin thickness and decreases nodal gap length after these structural features have been modified by perinodal astrocytes.
[0137] Secondly, the sensory deprivation experiments showing normal nodal morphology in the absence of visual input is inconsistent with a possible alternative hypothesis that the nodal gap widening and myelin sheath thinning seen in the Dox-off condition could have resulted from the dnVAMP2 gene inhibiting neuronal activity. Hypothetically, reduced exocytosis from astrocytes could have perturbed astrocyte modulation of synaptic function in the retina, but there is no evidence for this. Even complete sensory deprivation fails to produce similar effects on myelin structure. Additional evidence against this alternative is that histological structure of the retina and electrophysiological responses were normal in animals with exocytosis inhibited in astrocytes (Dox-off).
EXAMPLE 5
[0138] This example demonstrates the functional consequences of astrocyte regulation of nodes of Ranvier and myelin thickness.
[0139] The structural modifications identified here are relatively modest in comparison with the structural modifications seen in demyelinating conditions, raising the question of whether these changes have functional consequences. Theoretical and experimental evidence show that these changes in myelin and nodal structure do have significant functional effects.
[0140] Mathematical modeling based on passive cable properties predicts a conduction velocity decrease of approximately 15% for the observed decrease in myelin thickness and nodal gap increase seen in dnVAMP2-expressing animals. Electrophysiological recordings from excised optic nerve confirmed these predictions. The conduction times were
significantly slower in Dox-off animals for all three components of the compound action potential that could be measured. (Interference by the stimulus artifact, which partially obscures responses of the most rapidly conducting population of fibers, precluding analysis of this population.)
[0141] To determine whether this 15% conduction delay could alter the latency and synchrony of cortical processing of visual input, visually-evoked potential (VEP)
electrophysiological responses were recorded from the visual cortex in response to visual stimulation. The results showed a longer latency to peak VEP and slower rise-time of VEP in animals with lengthened nodal gap and thinner myelin as a consequence of inhibiting exocytosis in astrocytes. In parallel with this longer-latency and reduced synchrony of visual input to the cortex, visual acuity was reduced significantly as measured by the optomotor reflex in a computerized striped-drum assay modulating the spatial frequency of vertical stripes. These data are consistent with loss of visual acuity resulting from suboptimal impulse conduction in axons as occurs in multiple sclerosis (MS), where visual acuity decrement is an early diagnostic for myelin damage.
[0142] An alternative hypothesis that VEP latency might have resulted from inhibiting VAMP2- dependent exocytosis of neurotransmitters from astrocytes associated with synapses in gray matter was tested. When vesicular release of neurotransmitters from astrocytes was restored for 3-5 days by re-introducing Dox into the feed, VEP latency did not change (n=14, paired t-test, n.s.). Western blot and immunocytochemistry confirmed that expression of the dnVAMP2 gene was blocked, thus restoring exocytosis from astrocytes, and thereby any hypothetical effects of gliotransmitters on retinal synapses. The nodal gap length in these same animals was examined and found to still be increased because structural remodeling of myelin requires much longer than restoring neurotransmitter release from astrocytes at synapses (p<0.0001, n=l 17). This finding supports the conclusion that the increased VEP latency results from the observed 15% slower axonal conduction velocity and the associated remodeling of myelin. The conduction velocity was measured in excised optic nerve, which lacks synapses; thus the conduction delay measured in nerves excised from animals in which exocytosis is inhibited in astrocytes, cannot involve retinal or cortical synapses. Together the results support the conclusion that a purinergic autocrine signaling pathway in astrocytes alters GFAP bundling and cell shape, and promotes NF155 cleavage at a thrombin proteolytic site to remodel myelin.
[0143] ATP release from axons firing action potentials also increases myelination of unmyelinated axons as oligodendrocyte precursor cells (OPCs) differentiate to a
premyelinating stage, by stimulating release of the cytokine leukemia inhibitory factor (LIF) from astrocytes. This differs from the present findings in several respects. Nodal gap length was not altered in LIF knock-out animals. Also, NF155 was not cleaved in LIF knock-outs. Cytokines are released by multiple VAMP2-independent mechanisms (Eder et al.,
Immunobiology, 214: 543-53 (2009)), so LIF secretion from astrocytes during development would not be blocked in the dnVAMP2 animals. Secondly, LIF promotes OPC
differentiation to a premyelinating stage during a precise developmental window. By two weeks of age, myelin is normal in LIF -/- animals, but myelin structure is altered by exocytosis of ATP from astrocytes throughout life.
EXAMPLE 6
[0144] This example demonstrates that the thrombin inhibitor, fondaparinux sodium, provides a therapeutic benefit for the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis.
[0145] It was sought to determine whether daily injections of the thrombin inhibitor, ARIXTRA (fondaparinux sodium), would provide a therapeutic benefit for the EAE animal model of multiple sclerosis. EAE (autoimmunity against myelin) was induced in mice by injecting the mice with MOG peptide, which is a component of myelin, and pertussis toxin. The MOG peptide induces an autoimmune response to myelin. Pertussis toxin over-activates the immune system in general. The day of EAE induction was day zero and all time elapsed was measured from that date.
[0146] The EAE mice were injected with either fondaparinux sodium or saline (control). The mice were clinically scored for motor impairment in a blinded manner daily. The results are shown in Figure 1.
[0147] As shown in Figure 1 , EAE mice injected with fondaparinux sodium exhibited significantly lower levels of disability. The results were highly significantly different (p < 0.001 by ANOVA). Animals receiving the fondaparinux sodium treatment demonstrated a reduced severity of motor impairment (mean clinical score of 3.3) as compared to animals receiving the control saline treatment (mean clinical score of 4.65) at the end of 30 days post- immunization. Animals with a clinical score of 5 died.
EXAMPLE 7
[0148] This example demonstrates that fondaparinux sodium treatment significantly reduces mortality in the EAE animal model of multiple sclerosis. [0149] EAE was induced in mice, and the EAE mice were injected with either fondaparinux sodium or PBS (control), as described in Example 6. The number of mice surviving at various time points (days) after induction of EAE (post-immunization) was measured daily. The results are shown in Figure 2.
[0150] As shown in Figure 2, a significantly lower mortality rate was observed in EAE mice injected with fondaparinux sodium as compared to EAE mice treated with PBS. The results were significantly different (p =0.006 by chi square test).
EXAMPLE 8
[0151] This example demonstrates that animals treated with fondaparinux sodium demonstrate a lower rate of EAE incidence as compared to animals treated with PBS.
[0152] EAE was induced in mice, and the EAE mice were injected with either fondaparinux sodium or PBS (control), as described in Example 6. The incidence of disease was measured daily. The results are shown in Figure 3.
[0153] As shown in Figure 3, the first disease symptom (clinical score 0.5, flaccid tail/tail paralysis) appeared at a later point in time in the fondaparinux sodium-treated animals as compared to the control-treated animals (incidence of disease) (p=0.08).
EXAMPLE 9
[0154] This example demonstrates that the thrombin inhibitor, fondaparinux sodium, improves visual acuity in the dnSNARE mouse model.
[0155] dnSNARE is a genetic mouse model that selectively interferes with astrocyte signaling via conditional expression of a dominant-negative SNARE protein under control of the GFAP promoter. In the dnSNARE mouse, myelin thickness is reduced and the nodal gap is increased.
[0156] dnSNARE mice were treated with ARIXTRA (fondaparinux sodium) or saline (control), and visual acuity was measured. The results showed a highly significant improvement in visual acuity in the mice treated with fondaparinux sodium injections as compared to mice treated with control injections (p < 0.007). EXAMPLE 10
[0157] This example demonstrates that the release of protease nexin 1 (PN1) is inhibited in DOX-off animals.
[0158] In the Dox-ON/OFF transgenic mouse model, the animal has a normal (wild-type) phenotype when the animal is fed doxycycline (Dox) (Dox-ON condition). When
doxycycline is removed from the diet, (Dox-OFF condition), vesicular release from astrocytes is partially inhibited. Mastoparan (MP) is a drug that induces vesicular release. Here, MP was administered to astrocytes in cell culture from wild-type (WT) mice, transgenic mice in the Dox-ON condition, and transgenic mice in the Dox-OFF condition to induce vesicular release (including PN1 release). The amount of PN-1 released into the culture medium was measured and normalized to total protein. The results are shown in Figure 4.
[0159] As shown in, for example, Example 2, NF155 is cleaved by thrombin in the Dox- OFF condition (causing septate junctions to break, paranodal loops to come off, nodal gap to increase, and myelin to thin). As shown in Figure 4, this cleavage by thrombin is facilitated in the Dox-OFF condition because astrocytes in the Dox-OFF condition (that is, with partially inhibited vesicular release) secrete less Protease-Nexin 1 (PN1), a potent thrombin inhibitor. PN1 has been shown to be released via vesicles from astrocytes. Accordingly, perinodal astrocytes have a role in modulating myelin thickness by regulating thrombin dependent cleavage of NF155 via PN-1.
EXAMPLE 1 1
[0160] This example demonstrates that thrombin cleaves Casprl in vitro.
[0161] Casprl has a thrombin cleavage site (RRG) (SEQ ID NO: 37) at amino acid positions 380-382. Three dimensional protein structure modeling showed that the thrombin cleavage site is located on the surface of the folded protein. Cleavage of Casprl by thrombin was expected to yield a short thrombin-cleaved Casprl fragment of about 60 kDa and a longer thrombin-cleaved Casprl fragment of about 120 kDa.
[0162] A sample of full-length Casprl alone or samples of Casprl which had been contacted in vitro with 0.04, 0.02, 0.01 , or 0.005 units of thrombin were placed on a gel and analyzed by Western blot. A band corresponding to the expected 60 kDa thrombin-cleaved Casprl fragment was observed for those Casprl samples that were contacted with thrombin in vitro. No band corresponding to the 60 kDa Casprl fragment was observed for the Casprl sample which was not contacted with thrombin.
EXAMPLE 12
[0163] This example demonstrates that partial inhibition of vesicular release from astrocytes results in an increase in thrombin-mediated cleavage of Casprl .
[0164] Casprl samples were obtained from transgenic mice in the Dox-ON or Dox-OFF condition. The samples were placed on a gel and analyzed by Western blot. Enolase was used as a loading control.
[0165] A band corresponding to the expected 60 kDa thrombin-cleaved Casprl fragment was observed for mice in the Dox-ON and Dox-OFF conditions. However, an increase in the quantity of the expected 60 kDa thrombin-cleaved Casprl fragment was observed for mice in the Dox-OFF condition as compared to mice in the Dox-ON condition. These results were confirmed by mass spectrometry (MS). These results suggested that partial inhibition of vesicular release from astrocytes results in an increase in thrombin-mediated cleavage of Casprl .
EXAMPLE 13
[0166] This example demonstrates that the enzyme factor Xa cleaves Casprl in vitro.
[0167] Casprl has a factor Xa cleavage site (LEGR) (SEQ ID NO: 38) at amino acid positions 937-940. Three dimensional protein structure modeling showed that the factor Xa cleavage site is located on the surface of the folded protein. Cleavage of Casprl by factor Xa was expected to yield a short factor Xa-cleaved Casprl fragment of about 50 kDa and a longer factor Xa-cleaved Casprl fragment of about 130 kDa.
[0168] A sample of full-length Casprl alone or samples of Casprl which had been contacted in vitro with 0.1 , 0.05, 0.025, or 0.0125 units of factor Xa were placed on a gel and analyzed by Western blot. A band corresponding to the expected 50 kDa factor Xa-cleaved Casprl fragment was observed for those Casprl samples that were contacted with factor Xa in vitro. No band corresponding to the 50 kDa Casprl fragment was observed for the Casprl sample which was not contacted with factor Xa. EXAMPLE 14
[0169] This example demonstrates that partial inhibition of vesicular release from astrocytes results in an increase in factor Xa-mediated cleavage of Casprl .
[0170] Casprl samples were obtained from transgenic mice in the Dox-ON or Dox-OFF condition. The samples were placed on a gel and analyzed by Western blot. Enolase was used as a loading control.
[0171] A band corresponding to the expected 50 kDa factor Xa-cleaved Casprl fragment was observed for mice in the Dox-ON and Dox-OFF conditions. However, an increase in the quantity of the expected 50 kDa factor Xa-cleaved Casprl fragment was observed for mice in the Dox-OFF condition as compared to mice in the Dox-ON condition. These results were confirmed by mass spectrometry (MS). These results suggested that partial inhibition of vesicular release from astrocytes results in an increase in factor Xa-mediated cleavage of Casprl .
[0172] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0173] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly
contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0174] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):
1. A thrombin inhibitor for use in treating or preventing demyelination in a mammal.
2. The thrombin inhibitor for the use of claim 1, wherein the thrombin inhibitor is a chemical inhibitor.
3. The thrombin inhibitor for the use of claim 1 , wherein the thrombin inhibitor is fondaparinux, antithrombin III, PN1, or a pharmaceutically acceptable salt or derivative thereof.
4. The thrombin inhibitor for the use of claim 1, wherein the thrombin inhibitor is an agent that inhibits the expression one or both of thrombin mRNA and thrombin protein.
5. The thrombin inhibitor for the use of claim 1 , wherein the thrombin inhibitor is a neurofascin 155 (NF155) thrombin binding site/Fc fusion protein, a thrombin NF155 binding site/Fc fusion protein, a contactin-associated protein 1 (Casprl) thrombin binding site/Fc fusion protein, or a thrombin Casprl binding site/Fc fusion protein.
6. The thrombin inhibitor for the use of claim 1, wherein the thrombin inhibitor is an antibody or an antigen binding fragment thereof having antigenic specificity for Casprl, NF155 or thrombin.
7. The thrombin inhibitor for the use of claim 1 , wherein the thrombin inhibitor is a mutated thrombin, a mutated NF155, or a mutated Casprl .
8. A compound for use in treating or preventing demyelination in a mammal by stimulating astrocytes in the mammal to (i) release a thrombin inhibitor, (ii) encase the node of Ranvier, (iii) stabilize the node of Ranvier, (iv) maintain the physical structure of myelin, or (v) any combination of (i)-(iv).
9. The compound for the use of claim 8, wherein the thrombin inhibitor is antithrombin III, PN1 , PAI-1 , thrombomodulin, or a pharmaceutically acceptable salt or derivative thereof.
10. The thrombin inhibitor or the compound for the use of any one of claims 1-9, wherein the thrombin inhibitor or the compound reduces or prevents cleavage of Casprl or neurofascin 155 (NF155) in the mammal.
1 1. The thrombin inhibitor or the compound for the use of any one of claims 1-10, wherein the thrombin inhibitor or the compound reduces or prevents detachment of myelin from neuronal axons in the mammal.
12. The thrombin inhibitor or the compound for the use of any one of claims 1-1 1 , wherein the thrombin inhibitor or the compound reduces or prevents an increase in nodal gap length in the mammal.
13. The thrombin inhibitor or the compound for the use of any one of claims 1-12, wherein the thrombin inhibitor or the compound t reduces or prevents dispersion of neuronal sodium channels in the mammal.
14. The thrombin inhibitor or the compound for the use of any one of claims 1-13, for further use in treating or preventing a demyelinating disease in the mammal.
15. The thrombin inhibitor or the compound for the use of claim 14, wherein the disease is multiple sclerosis (MS), cerebral palsy, optic neuritis, Devic's disease
(neuromyelitis optica), transverse myelitis, Balo's concentric sclerosis, acute disseminated encephalomyelitis (ADEM), adrenoleukodystrophy, adrenomyeloneuropathy, Gulf War Illness, combined central and peripheral demyelination (CCPD), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), a psychiatric disorder, a learning disability, or Guillain-Barre syndrome (GBS).
16. An isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the neurofascin 155 (NF155) amino acid sequence of
NPYNDSSLRNHPD (SEQ ID NO: 21).
17. An isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of LEMVVVNGR (SEQ ID NO: 22).
18. An isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the NF155 amino acid sequence of MVVVNGRGDGPRSE (SEQ ID NO: 23).
19. An isolated or purified antibody, or an antigen binding fragment thereof, having antigenic specificity for the mouse Casprl 1-38o amino acid sequence of SEQ ID NO: 33, the mouse Casprl38i.i385 amino acid sequence of SEQ ID NO: 34, the mouse Casprl 1-94-7 amino acid sequence of SEQ ID NO: 35, the mouse Casprl 948-1385 amino acid sequence of SEQ ID NO: 36, or mouse Casprl38i-947 amino acid sequence of SEQ ID NO: 39, the human Casprl \. 379 amino acid sequence of SEQ ID NO: 41, the human Casprl 38o-i 384 amino acid sequence of SEQ ID NO: 42, the human Casprl 1 -940 amino acid sequence of SEQ ID NO: 43, the human Casprl 947_1384 amino acid sequence of SEQ ID NO: 44, or the human Casprl 3 o-946 amino acid sequence of SEQ ID NO: 45.
20. A method of detecting the presence of demyelination in a mammal, the method comprising:
(a) contacting a biological sample comprising blood and/or cerebral spinal fluid (CSF) with at least one antibody, or antigen binding fragment thereof, of any one of claims 16-19, thereby forming a complex, and
(b) detecting the complex, wherein detection of the complex is indicative of demyelination in the mammal.
21. The method of claim 20, comprising
(a) contacting the biological sample with a first antibody, or antigen binding fragment thereof, selected from the group consisting of
(i) the antibody, or antigen binding fragment thereof, of claim 16,
(ii) the antibody, or antigen binding fragment thereof, of claim 17, or
(iii) the antibody, or antigen binding fragment thereof, of claim 18, thereby forming a first complex; (b) contacting the first complex with a second antibody, or antigen binding fragment thereof, selected from the group consisting of
(i) the antibody, or antigen binding fragment thereof, of claim 16,
(ii) the antibody, or antigen binding fragment thereof, of claim 17, or
(iii) the antibody, or antigen binding fragment thereof, of claim 18, thereby forming a second complex;
(c) detecting the second complex, wherein detection of the second complex is indicative of the presence of demyelination in the mammal,
wherein the second antibody, or antigen binding fragment thereof, is different from the first antibody, or antigen binding fragment thereof.
22. The method of claim 21 , wherein the first antibody, or antigen binding fragment thereof, is the antibody, or antigen binding fragment thereof, of claim 16 and the second antibody, or antigen binding fragment thereof, is the antibody, or antigen binding fragment thereof, of claim 17.
23. The thrombin inhibitor or the compound for the use of claim 15, wherein the first antibody, or antigen binding fragment thereof, is the antibody, or antigen binding fragment thereof, of claim 18 and the second antibody, or antigen binding fragment thereof, is the antibody, or antigen binding fragment thereof, of claim 17.
24. A method of detecting the presence of demyelination in a mammal, the method comprising:
(a) contacting a biological sample comprising blood and/or cerebral spinal fluid (CSF) with an agent, thereby forming a complex between (i) the agent and (ii) an antibody, or antigen binding fragment thereof, having antigenic specificity for the agent, and
(b) detecting the complex, wherein detection of the complex is indicative of demyelination in the mammal,
wherein the agent is NF125, NF30, the mouse Casprl38i .i3 5 amino acid sequence of SEQ ID NO: 34, the mouse Casprl i_947 amino acid sequence of SEQ ID NO: 35, the mouse Casprl 381 -947 amino acid sequence of SEQ ID NO: 39, the human Casprl 3 o-i384 amino acid sequence of SEQ ID NO: 42, the human Casprl i .94 amino acid sequence of SEQ ID NO: 43, or the human Casprl 3 o-946 amino acid sequence of SEQ ID NO: 45.
25. The method of claim 24, wherein the method comprises
(a) contacting a biological sample comprising blood and/or CSF with NF125 and NF30, thereby forming a first complex with an antibody, or antigen binding fragment thereof, having antigenic specificity for NF125 and forming a second complex with an antibody, or antigen binding fragment thereof, having antigenic specificity for NF30, and
(b) detecting the first and second complexes, wherein detection of the complexes is indicative of demyelination in the mammal.
PCT/US2016/027776 2015-04-17 2016-04-15 Methods of treating or preventing demyelination using thrombin inhibitors and methods of detecting demyelination using neurofascin 155 WO2016168615A1 (en)

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CN108611411A (en) * 2018-05-09 2018-10-02 中国医科大学 Caspr1 memebrane proteins are as the purposes prepared in neonatal meningitis drug
CN109734791A (en) * 2019-01-17 2019-05-10 武汉明德生物科技股份有限公司 People NF186 antigen, people's NF186 antibody assay kit and the preparation method and application thereof
CN109810184A (en) * 2019-01-17 2019-05-28 武汉明德生物科技股份有限公司 People NF155 antigen, people's NF155 antibody assay kit and the preparation method and application thereof
CN109810184B (en) * 2019-01-17 2022-07-12 武汉明德生物科技股份有限公司 Human NF155 antigen, human NF155 antibody detection kit, preparation method and application thereof
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