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WO2005094497A2 - Administration systemique d'agents therapeutiques au systeme nerveux central - Google Patents

Administration systemique d'agents therapeutiques au systeme nerveux central Download PDF

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Publication number
WO2005094497A2
WO2005094497A2 PCT/US2005/009714 US2005009714W WO2005094497A2 WO 2005094497 A2 WO2005094497 A2 WO 2005094497A2 US 2005009714 W US2005009714 W US 2005009714W WO 2005094497 A2 WO2005094497 A2 WO 2005094497A2
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Prior art keywords
neuron
neuroactive
fusion molecule
polypeptide
nervous system
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WO2005094497A3 (fr
Inventor
Shawn Hochman
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Emory University
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Emory University
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Publication of WO2005094497A3 publication Critical patent/WO2005094497A3/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
    • A61K49/0047—Green fluorescent protein [GFP]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • CNS central nervous system
  • an object of this invention is to provide methods and molecules to facilitate simple delivery of large therapeutic molecules across the BBB into the CNS.
  • Retrograde delivery of compounds to CNS neurons with fenestrated capillaries permits delivery of many substances en-mass to their cell bodies in the CNS.
  • Retrograde transport mechanisms can ferry proteins to CNS and peripheral nervous system (PNS) neurons with terminals outside the BBB and BNB.
  • TTC tetanus toxin
  • TTC tetanus toxin
  • BBB/BNB activity-dependent internalization and fast retrograde axonal transport from peripheral terminal fields.
  • large proteins fused to TTC are transported efficaciously with their biological activity intact. Fusion protein gene constructs can be engineered in plasmids and transformed into host cells to generate large quantities of fusion protein.
  • TTC can be delivered systemically to reach all central and peripheral neurons whose terminals have access to substances transported through fenestrated capillaries, such as motoneurons, autonomic pre-ganglionics, autonomic post-ganglionics, circumventricular organs (e.g. area postrema), select hypothalamic and brainstem reticular nuclei and primary sensory neurons.
  • TTC-protein conjugates can also be transported trans-synaptically to other neurons within nervous system circuits.
  • trans-synaptic transfer of TTC is activity-dependent, providing for selective targeting of therapeutic agents to active CNS circuits.
  • NMJ neuromuscular junction
  • the blood-brain and blood-nerve barrier strictly limit the transfer of molecules into brain and spinal cord compartments of the central nervous system, as well as the peripheral nervous system. Very few effective delivery systems exist for the delivery of therapeutic molecules across these barriers.
  • One effective delivery method comprises the use of retrograde transport across these barriers. However, this method is limited to intramuscular injection, and only targets motoneurons that project into the injected muscle.
  • the present invention in one aspect, provides novel methods and compositions which comprise the use of retrograde transport for the delivery of therapeutic molecules, for example, large therapeutic molecules, across the BBB, which are not so limited.
  • the present invention provides methods for delivering a neuroactive fusion molecule across the blood-brain or blood-nerve barrier comprising: (a) administering by non-intramuscular means to a subject a neuroactive fusion molecule comprising a therapeutic polypeptide and a delivery polypeptide, such that the bloodstream of said patient is capable of transporting said administered neuroactive fusion molecule to a fenestrated capillary of said patient; and (b) wherein, said administered fusion molecule is delivered across said fenestrated capillary and into a neuron.
  • the neuron can be, for example, a central nervous system neuron, a peripheral neuron, an enteric nervous system neuron, or an autonomic nervous system neuron.
  • the methods of this invention directly target therapeutic moieties to all neurons in the peripheral nervous system and in the central nervous system. They directly target all somatic and autonomic motoneurons, circumventricular organs, neurons in the hypothalamus and reticular formation, area postrema, hippocampus, and medial habenula. Other brain regions are targeted indirectly because they project to these systems and the therapeutic moiety is transferred trans-synaptically to them.
  • the methods further comprise trans-synaptically transferring the administered neuroactive fusion molecule from a peripheral or central neuron with access to fenestrated capillaries to at least one other neuron.
  • a method further comprises trans-synaptically transferring the administered neuroactive fusion molecule from the neuron to at least one other neuron, wherein the secondary targeting polypeptide causes said trans-synaptic transfer to selectively or non-selectively target the at least one other neuron.
  • the administration of the neuroactive fusion molecule can be accomplished by a variety of non-intramuscular means.
  • the administering of the neuroactive fusion molecule can be intravenous.
  • Such intravenous administration can be intermittent or continuous.
  • the administering of the neuroactive fusion molecule can also be, for example, intraperitoneal, subcutaneously, transdermally, or orally.
  • the present invention in another aspect, also provides neuroactive fusion molecules, as well as nucleic acids encoding such neuroactive fusion molecules, vectors, and host cells.
  • the neuroactive fusion molecule can comprise a therapeutic polypeptide and a delivery polypeptide.
  • the delivery polypeptide of the neuroactive fusion molecule can be selected from the group consisting of: wheat-germ agglutinin (and other related plant lectins like barley lectin), tetanus toxin (e.g., tetanus toxin C-fragment), and Thy-1.
  • the neuroactive fusion molecule can further comprise a secondary targeting polypeptide.
  • the therapeutic polypeptide of the neuroactive fusion molecule can be any polypeptide that is able to exert a therapeutic effect.
  • the therapeutic polypeptide can be a neurotrophic factor, an endocrine factor, a growth factor, a paracrine factor, a hypothalamic releasing factor, a neurotransmitter polypeptide, an antibody or antibody fragment which binds to a neurotrophic factor, an antibody or antibody fragment which binds to a neurotrophic factor receptor, a polypeptide antagonist, an agonist or antagonist for a receptor expressed by a CNS cell, and a polypeptide involved in modifying intracellular processes including signal transduction cascades, trafficking, synaptic function, changes in gene expression and intracellular organelle function (e.g.
  • the therapeutic moiety of the fusion molecule can also be any therapeutic molecule known to the art.
  • Conventional therapeutic molecules used for treatment of disorders of nervous function can be targeted preferentially to the nerves as opposed to other tissues where they may give rise to unwanted side effects.
  • the neuroactive fusion molecule comprises a diagnostic molecule.
  • Such neuroactive fusion molecules also can comprise a therapeutic molecule or secondary target molecule.
  • the diagnostic molecules allow the neuroactive fusion molecule to be detected by an imaging modality.
  • the diagnostic molecule is a molecule that is not a polypeptide, such as for example, a fluorophore or radiolabeled molecule.
  • the diagnostic molecule can be a polypeptide, such as for example, a fluorescent polypeptide such as green or yellow fluorescent protein, or a polypeptide carrying a radiolabel.
  • the diagnostic molecule can be a contrast agent.
  • the subject neuroactive fusion molecule can be of any molecular weight as is resultant from its composition, e.g. the sum of the weights of the delivery polypeptide, the therapeutic polypeptide, and optionally the secondary targeting polypeptide.
  • a subject neuroactive fusion molecule can have a molecular weight of, for example, at least about 1 kDa, at least about 10 kDa, at least about 20 kDa, at least about 30 kDa, at least about 40 kDa, at least about 50 kDa, at least about 60 kDa, at least about 70 kDa, at least about 80 kDa, at least about 90kDa, at least about 100 kDa, at least about 100 kDa, at least about 120 kDa, at least about 140 kDa, and so on, up to about 200 kDa or more.
  • the present invention also provides nucleic acids encoding the subject neuroactive fusion molecules, and vectors and host cells for expressing the
  • the present invention provides methods of treating a subject that a disorder of the nervous system comprising: (a) administering by non- intramuscular means to a subject a therapeutically effective amount of a neuroactive fusion molecule comprising a therapeutic polypeptide or other therapeutic molecule, and a delivery polypeptide; (b) wherein said administered neuroactive fusion molecule crosses the blood-brain or blood-nerve barrier.
  • the disorder can be, in certain embodiments, a peripheral nervous system disorder and the neuron a peripheral neuron, or a central nervous system disorder and the neuron a central nervous system neuron.
  • the disorder can be an autonomic nervous system disorder and the neuron an autonomic nervous system neuron, or the disorder can be an enteric nervous system disorder and the neuron an enteric nervous system neuron.
  • the disorder is a central nervous system disorder, wherein the neuron is a peripheral neuron, and wherein the method further comprises trans-synaptically transferring the administered fusion molecule from the peripheral neuron to a central nervous system neuron.
  • the neuroactive fusion molecule in certain embodiments, can further comprise a secondary targeting polypeptide.
  • the method of treating can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from the neuron to at least one other neuron, wherein the secondary targeting polypeptide causes the trans-synaptic transfer to target at least one other neuron.
  • the present invention also provides pharmaceutical compositions comprising the neuroactive fusion molecules.
  • the pharmaceutical composition comprises an isolated, purified neuroactive fusion molecule.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the neuroactive fusion molecule comprises a therapeutic polypeptide and a delivery polypeptide.
  • the neuroactive fusion molecule further comprises a secondary targeting polypeptide.
  • the invention provides devices for administering the pharmaceutical compositions, for example, devices for intravenous, intraperitoneal, or subcutaneous injection.
  • the present invention provides methods of diagnosing a disorder of the nervous system in a subject comprising: (a) administering by non- intramuscular means to a subject a neuroactive fusion molecule comprising a diagnostic molecule and a delivery polypeptide; and (b) wherein said administered neuroactive fusion molecule crosses the blood-brain barrier.
  • the diagnostic molecule can be a fluorophore, such as for example rhodamine.
  • the diagnostic molecule can be a polypeptide, such as for example, green fluorescent protein or yellow fluorescent protein.
  • diagnostic methods can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from a peripheral neuron to at least one other neuron.
  • the neuroactive fusion molecule can further comprise a secondary targeting polypeptide.
  • the diagnostic method can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from a neuron to at least one other neuron, wherein the secondary targeting polypeptide causes the trans- synaptic transfer to selectively target at least one other neuron.
  • the present invention provides methods of imaging a neuron in a subject comprising: (a) administering by non-intramuscular means to a subject a neuroactive fusion molecule comprising a diagnostic molecule and a delivery polypeptide; and (b) wherein said administered neuroactive fusion molecule crosses the blood-brain barrier or blood-nerve barrier.
  • the diagnostic molecule can be a contrast agent.
  • such diagnostic methods can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from a peripheral neuron to at least one other neuron.
  • the neuroactive fusion molecule can further comprise a secondary targeting moiety such as a polypeptide.
  • the imaging method can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from a neuron to at least one other neuron, wherein the secondary targeting polypeptide or other moiety causes the trans-synaptic transfer to selectively target at least one other neuron.
  • the present invention further provides a kit comprising compositions of the present invention, and optionally instructions for their use. Such kits can have a variety of uses, including, for example, therapy, diagnosis, imaging, vaccination and other applications.
  • Previously-known delivery systems are limited to intramuscular injection, which results in a restricted delivery of the substance to the nerve cells innervating that muscle and other cell populations that connect to it.
  • the methods of this invention provide systemic delivery, for example, via i.v. and i.p. injection, sublingual doses, e.g., in pill form, suppositories, transdermal routes and other means that reach all nerve cells that have access to the circulation and so can be delivered to about 1000 times as many nerve cells as can be targeted by i.m. injection. Delivery to all nerve cells in the brain is possible.
  • the inventors hereof have made the surprising discovery that nerve cells can pick up the fusion molecules of this invention from the circulation.
  • this invention provides methods for delivering therapeutic molecules to nerve cells that are not in the central nervous system - all the nerve cells of the peripheral nervous system including those involved in sensation (e.g. pain systems) the enteric (digestive) nervous system and the autonomic nervous system.
  • therapeutic fusion molecules via the circulatory system allows them to reach about a thousand times as many nerve cells as delivery via i.m. injection. This allows the molecules to spread trans-synaptically to neurons that do not have direct access to the circulatory system. Once the therapeutic fusion molecules reach the receptors that the therapeutic moieties are intended to affect, these receptors are affected, e.g., by binding to the therapeutic moiety or otherwise as known to the art.
  • the methods of this invention allow delivery of large molecules such as proteins in excess of 80 or 100 kDA across the blood-brain and blood-nerve barriers.
  • amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids.
  • exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • autonomic nervous system or "ANS” refers to neurons that are not under conscious control, comprising two antagonistic components, the sympathetic and parasympathetic nervous systems.
  • the autonomic nervous system regulates key functions including the activity of the cardiac muscle, smooth muscles (e.g., of the gut), and glands.
  • the autonomic nervous system has two divisions: 1. The sympathetic nervous system that accelerates the heart rate, constricts blood vessels, and raises blood pressure; and 2.
  • the parasympathetic nervous system slows the heart rate, increases intestinal and gland activity, and relaxes sphincter muscles.
  • blood-brain barrier refers to the selective mechanism opposing the passage of most ions and large-molecular weight compounds from the blood to brain tissue located in the lining of endothelial cells in the capillaries which comprise the blood-brain barrier.
  • BNB blood-nerve barrier
  • bloodstream refers to the flowing blood as it is encountered in the circulatory system. Something added to the bloodstream can be expected to become distributed to all parts of the body through which blood is flowing.
  • circululatory system refers to the system that moves blood throughout the body of a subject, which is comprised of the heart, arteries, capillaries, and veins.
  • central nervous system refers to the spinal cord and the brain, and includes the autonomic and somatic nervous systems.
  • central nervous system neuron refers to a neuron comprising the spinal cord and/or the brain.
  • neurons of the central nervous system include, but are not limited to, motoneurons, autonomic preganglionics, circumventricular organs, hypothalamic nuclei, and select brainstem reticular nuclei.
  • diagnostic molecule refers to the portion (either a polypeptide or other molecule) of the subject fusion molecules which allows the visualization of such fusion molecules in a diagnostic or imaging method.
  • portion can be a fluorophore, a radiolabel, a contrast agent, etc.
  • disorder of the central nervous system refers to a disturbance of function, structure, or both, of the central nervous system resulting from, for example, a genetic or embryonic failure in development or from exogenous factors such as poison, trauma, or disease of the CNS.
  • central nervous system disorders include, but are not limited to, the paroxysmal disorders (e.g., the epilepsies), autonomic nervous system dysfunction (e.g., arterial hypertension), movement disorders (e.g., hyperkinetic disorders, dyskinesias (resting tremor), basal ganglia hyperkinetic disorders (e.g., Huntington's chorea, hemiballismus), neuropsychiatric disorders (e.g., mania, psychosis obsessive compulsive disorder, and addiction), hypothalamic dysfunction (e.g., hyperlactemia), and neuropathic pain syndromes.
  • the paroxysmal disorders e.g., the epilepsies
  • autonomic nervous system dysfunction e.g., arterial hypertension
  • movement disorders e.g., hyperkinetic disorders, dyskinesias (resting tremor)
  • basal ganglia hyperkinetic disorders e.g., Huntington's chorea, hemiballismus
  • peripheral nervous system disorders refers to a disturbance of function, structure, or both, of the peripheral nervous system resulting from, for example, a genetic or embryonic failure in development or from exogenous factors such as poison, trauma, or disease of the peripheral nervous system.
  • peripheral nervous system disorders include, but are not limited to, acrodynia, Charcot-Marie-Tooth disease, diabetic neuropathies, nerve compression syndromes, neuralgias, neuromuscular junction diseases, and POEMS syndrome.
  • delivery polypeptide refers to the polypeptide portion of the subject fusion molecules which allow the fusion molecule to gain access to the CNS or otherwise be trans-synaptically transported.
  • exemplary delivery polypeptides can comprise all or a portion of wheat-germ agglutinin (and other related plant lectins like barley lectin), tetanus toxin (e.g., tetanus toxin C- fragment) and Thy-1.
  • enteric nervous system or "ENS” as used herein refers to the nervous system situated in the gastrointestinal tract. Two ganglionated neural plexuses in the gut wall which form one of the three major divisions of the autonomic nervous system.
  • the enteric nervous system innervates the gastrointestinal tract, the pancreas, and the gallbladder. It contains sensory neurons, interneurons, and motor neurons.
  • the circuitry can autonomously sense the tension and the chemical environment in the gut and regulate blood vessel tone, motility, secretions, and fluid transport.
  • the term "fenestrated capillary” refers to a type of capillary generally located in areas where there is substantial exchange between blood and tissues such as the choroid plexus and in a number of specialized areas such as the median eminence, neurohyophysis, area postrema, and pineal gland. Such areas are highly vascular areas and lack a BBB.
  • Fenestrated capillaries have fenestrations or pores (about 80-1 OOnm diameter) which are covered by diaphragms which are thinner than a plasma membrane.
  • Sinusoidal capillaries are irregular vessels with large diameters (30- 40nm). Certain sinusoid capillaries are also fenestrated and are thus included in the term "fenestrated capillary" as used herein.
  • Certain CNS neurons also have access to fenestrated capillaries either from the periphery (motoneurons, preganglionic autonomic neurons) or due to some privileged need to monitor blood (such as in circumventricular organs like the area postrema). Fenestrated capillaries also can be found in the vascular systems of the gut, muscle, and other end organs and organ systems.
  • isolated polypeptide refers to a polypeptide (e.g. a fusion molecule of the invention or a component thereof) which (1) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • a polypeptide e.g. a fusion molecule of the invention or a component thereof
  • isolated nucleic acid refers to a polynucleotide of genomic, cDNA, or synthetic origin or some combination there of, which (1) is not associated with the cell in which the "isolated nucleic acid” is found in nature, or (2) is operably linked to a polynucleotide to which it is not linked in nature.
  • mammals include humans, primates, bovines, porcines, canines, felines, and rodents (e.g., mice and rats).
  • neuroactive fusion molecule refers to fusion molecule which can exert a therapeutic or otherwise desirable result or effect upon delivery to a region of CNS or PNS tissue (e.g. a neuron, a glial cell, etc.) that is on the "brain" side of the blood-brain barrier.
  • a “fusion molecule” as used herein refers to a chimeric protein as that term is known in the art and can be constructed using methods known in the art, e.g.
  • the fusion molecule can also be a construct in which the therapeutic moiety is bonded to a polypeptide capable of trans- synaptically delivering the therapeutic moiety to sites where it can be active.
  • fusion proteins there are two different polypeptide sequences, and in certain cases, there can be more. The sequences can be linked in frame.
  • a fusion protein can include a domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it can be an "interspecies", "intergenic”, etc. fusion expressed by different kinds of organisms.
  • the fusion polypeptide can comprise one or more amino acid sequences linked to a first polypeptide.
  • the fusion sequences can be multiple copies of the same sequence, or alternatively, can be different amino acid sequences.
  • the fusion polypeptides can be fused to the N-terminus, the C-terminus, or the N- and C- terminus of the first polypeptide.
  • Exemplary fusion molecules include polypeptides comprising a therapeutic polypeptide or other therapeutic moiety and a delivery polypeptide, as described herein.
  • the term includes molecules in which the therapeutic portion and the delivery portion can be linked through means known to the art such as binding molecules, e.g., streptavidin-biotin linkages.
  • binding molecules e.g., streptavidin-biotin linkages.
  • streptavadin can be bonded to the delivery portion, and the biotin bonded to the therapeutic portion.
  • the neuroactive fusion molecules of this invention are capable of being taken up and communicated across the blood brain barrier and blood-nerve barrier by both afferent and efferent nerves.
  • the term "Secondary Targeting Portion” refers to a molecule or portion thereof, such as a binding moiety capable of selectively binding to a particular neuron receptor, to aid in targeting the fusion molecule to appropriate neurons.
  • a “Target Neuron” is a neuron intended to take up the neuroactive fusion molecule.
  • the target neuron can be the peripheral neuron which first contacts the fusion molecule, or any other neuron to which the fusion molecule is trans- synaptically transferred.
  • the "Ultimate Target Neuron” is the neuron on which the fusion molecule is intended to have its therapeutic effect.
  • Activating a target neuron means making sure the target neuron is in an active state and capable of taking up the neuroactive fusion molecule. Activation can be accomplished, for example, by placing the patient on a treadmill to activate nerves involved in walking, or otherwise stimulating the target neuron by means known to the art, including administration of neuron activating agents known to the art. Many neurons are active during a portion of each day. “Activating the target neuron” can also be accomplished by administering the neuroactive fusion molecule at a time such that it is available to be taken up by the target neuron at a time when the target neuron is naturally active.
  • non-naturally occurring refers to the fact that an object cannot be found in nature and has been intentionally modified or created by man.
  • a polypeptide or polynucleotide sequence that is a polypeptide produced by recombinant DNA techniques is "non-naturally occurring.”
  • nucleic acid refers to a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • a "patient,” “subject” or “host” to be treated by the subject method can mean either a human or non-human animal.
  • the term “peripheral nervous system” or “PNS” as used herein refers to the peripheral part of the nervous system external to the brain and spinal cord from their roots to their peripheral terminations.
  • the PNS includes, for example, the ganglia, both sensory and autonomic, and any plexuses through which the nerve fibers run. Nerves in the PNS connect the central nervous system (CNS) with sensory organs, other organs, muscles, circulatory system, and glands.
  • CNS central nervous system
  • peripheral neuron refers to a neuron comprising any component of the peripheral nervous system, for example, primary sensory neurons, which have their cell bodies in sensory ganglia.
  • non-intramuscular administration refers to the administration of a subject composition, neuroactive fusion molecule, therapeutic or other material other than (1) directly into the muscles or (2) directly into the central nervous system, wherein upon such administration such composition ultimately enters the patient's circulation and, thus, is subject to metabolism and other like processes.
  • exemplary administration methods that are examples of "non-intramuscular administration” include subcutaneous administration, intraperitoneal administration, oral administration, intravascular administration, and intraarterial administration.
  • polypeptide and the terms “protein” and “peptide” which are used interchangeably herein, refers to a polymer of amino acids.
  • exemplary polypeptides include gene products, naturally-occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.
  • polypeptide fragment when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy- terminus of the reference polypeptide, or alternatively both.
  • Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40 or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500 or more amino acids long.
  • a fragment can retain one or more of the biological activities of the reference polypeptide.
  • a fragment can comprise a druggable region, and optionally additional amino acids on one or both sides of the druggable region, which additional amino acids can number from 5, 10, 15, 20, 30, 40, 50, or up to 100 or more residues.
  • fragments can include a sub-fragment of a specific region, which sub-fragment retains a function of the region from which it is derived.
  • a fragment can have immunogenic properties.
  • the term "purified” refers to an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • a "purified fraction" is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all species present.
  • a purified composition will have one species that comprises more than about 80 percent of all species present in the composition, more than about 85%, 90%, 95%, 99% or more of all species present.
  • the object species can be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species.
  • a skilled artisan can purify a polypeptide of the invention using standard techniques for protein purification in light of the teachings herein.
  • recombinant protein or “recombinant polypeptide” refer to a polypeptide which is produced by recombinant DNA techniques.
  • An example of such techniques includes the case when DNA encoding the expressed protein is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the protein or polypeptide encoded by the DNA.
  • secondary targeting molecule refers to an optionally present portion of the subject fusion molecules that serves to target the fusion molecule, once it crosses the blood-brain barrier into peripheral neurons or subpopulations of central neurons.
  • phantom pain in amputees is produced by central circuitry signaling somatosensory information to body regions that no longer exist. In such cases it would be desirable to selectively destroy these circuits.
  • an exemplary fusion molecule could comprise a delivery polypeptide, for example tetanus toxin C-fragment, a therapeutic polypeptide that kills neurons when internalized, and a secondary targeting molecule which selectively targets the particular neuronal circuit.
  • Secondary Targeting Portion refers to a molecule or portion thereof, such as a binding moiety capable of selectively binding to a particular neuron receptor, to aid in targeting the fusion molecule to appropriate neurons.
  • therapeutic polypeptide refers to the polypeptide portion of the subject fusion molecules which acts as a therapeutic agent either as a fusion or after cleavage.
  • exemplary therapeutic polypeptides include, but are not limited to, neurotrophic factors, endocrine factors, growth factors, paracrine factors, hypothalamic releasing factors, neurotransmitter polypeptides, antibodies and antibody fragments which bind to neurotrophic factors, antibodies and antibody fragments which bind to neurotrophic factor receptors, polypeptide antagonists, agonists or antagonists for a receptor expressed by a CNS cell, and polypeptides involved in modifying intracellular processes including signal transduction cascades, trafficking, synaptic function, changes in gene expression and intracellular organelle function (e.g., lysosomal storage disease).
  • the "therapeutic portion" of a neuroactive fusion molecule can be a polypeptide or other therapeutic neuroactive molecule known to the art.
  • the term "therapeutic moiety” as used herein includes not only therapeutically active polypeptides, but also other chemical moieties known to the art to have a therapeutic effect on nervous function.
  • the term “therapeutically effective amount” refers to that amount of a neuroactive fusion molecule, drug or other molecule which is sufficient to effect treatment when administered to a subject in need of such treatment. The therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • vector refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked.
  • One type of vector which can be used in accord with the invention is an episome, i.e., a nucleic acid capable of extra- chromosomal replication.
  • Other vectors include those capable of autonomous replication and expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA molecules which, in their vector form are not bound to the chromosome.
  • plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • vector is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • molecules internalized by selective binding to membrane structures can re-enter the secretory vesicle in dendrites and result in trans-synaptic transfer.
  • Tetanus toxin normally enters the CNS from the systemic circulation with among the highest efficiency of any known protein.
  • the mechanism of entry begins with binding of the toxin at nerve endings in the periphery that have access to fenestrated capillaries.
  • the membrane binding site has been determined to be associated with specific gangliosides and immunoglobulins followed by activity- dependent intemalization and consequent fast retrograde axonal transport to cell bodies within the CNS.
  • the 47-kD C-terminus fragment of the heavy chain of tetanus toxin (C- fragment, "TTC") contains the binding site of the toxin, but is no longer toxic.
  • TTC retrograde transport following endocytotic uptake from terminals with access to fenestrated capillaries is similar to the distribution of endogenous plasma proteins, exogenous proteins, metals and tracer molecules injected intravenously.
  • binding sites for TTC are found on all neurons, they are highly concentrated in synaptosomal fractions as also observed with poliovirus.
  • the preferred affinity of theses substances for synaptic components can explain the greater accumulation in motoneurons compared to sensory neurons which are without synaptic terminals outside the BBB. Recently, it has been demonstrated that the uptake of TTC at the NMJ is activity dependent.
  • Yamamoto et al also observed a differential recruitment of serum albumin and other proteins in motoneurons.
  • This distribution of TTC accumulation in the mouse resembles the distribution of affected cell populations in amyotrophic lateral sclerosis (ALS) in humans.
  • ALS amyotrophic lateral sclerosis
  • the difference in accumulation of C-fragment between certain neuronal populations parallels differences in pathologic findings seen in ALS, where preganglionic neurons and ocular motoneurons are relatively spared. If transport of substances occurred in an activity-dependent manner this could in part explain the different intensities observed. For example, it is known that for clinical and pathological manifestations of both tetanus and paralytic poliomyelitis, affected muscles are those that are activated most.
  • the present invention provides methods and compositions for achieving retrograde therapeutic targeting through monosynaptic or polysynaptic pathways that overcome the limitations associated with intra-muscular injection.
  • systemic administration that is, peripheral administration by non-intramuscular means, of therapeutic fusion proteins that are specific to a targeted CNS circuit and that can then be delivered there in an activity-dependent manner.
  • Genetic expression profiles of neuronal classes and circuits can identify such circuits not previously known. Fortunately, in the postgenomic era, the availability of DNA microarrays and the technology of laser-capture microdissection (LCM) allow for high fidelity expression profiling of neural classes, both in control and disease states.
  • LCM laser-capture microdissection
  • TTC-fusion proteins can be delivered to CNS and PNS neurons via fenestrated capillaries and then trans-synaptically transferred to other neurons, including CNS neurons.
  • Activity-dependent trans-synaptic transfer permits selective neuronal targeting.
  • TTC-reporter fusion proteins e.g. ⁇ -gal, GFP
  • RNA of trans-synaptically labeled Renshaw cells can be captured with LCM for DNA microarray expression profiling to identify selective receptors for therapeutic and diagnostic targeting. Based on results from expression profiling, TTC can be fused to an identified neuroactive polypeptide that acts on Renshaw cells then delivered to measurably modify its function.
  • This approach permits therapeutic factors to be delivered to select CNS and other nervous system circuits (such as peripheral, enteric, autonomic) in an activity- dependent manner.
  • CNS and other nervous system disorders having hyperactive circuits are strong candidates for targeted drug delivery using the methods and compositions of the present invention.
  • drug delivery can target all neurons of the PNS and CNS. Therapeutic action in this case would be based on the selectivity of the therapeutic polypeptide.
  • the present invention provides methods for delivering a neuroactive fusion molecule across the blood-brain barrier or blood- nerve barrier comprising: (a) administering by non-intramuscular means to a subject a neuroactive fusion molecule comprising a therapeutic polypeptide and a delivery polypeptide, such that the bloodstream of said patient is capable of transporting said administered neuroactive fusion molecule to a fenestrated capillary of said patient; and (b) wherein, said administered fusion molecule is delivered across said fenestrated capillary into a neuron.
  • the neuron can be, for example, a central nervous system neuron, a peripheral neuron, an enteric nervous system neuron, or an autonomic nervous system neuron.
  • the methods can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from the peripheral neuron to at least one other neuron.
  • a method can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from the neuron to at least one other neuron, wherein the secondary targeting polypeptide causes said trans-synaptic transfer to selectively target at least one other neuron.
  • the administration of the neuroactive fusion molecule can be accomplished by a variety of non-intramuscular means.
  • the administering of the neuroactive fusion molecule can be intravenously.
  • Such intravenous administration can be intermittent or continuous.
  • the administering of the neuroactive fusion molecule can also be, for example, intraperitoneal, subcutaneously, or orally.
  • the present invention in another aspect, also provides neuroactive fusion molecules, as well as nucleic acids encoding such neuroactive fusion molecules, vectors, and host cells.
  • the neuroactive fusion molecule can comprise a therapeutic moiety and a delivery moiety.
  • the neuroactive fusion molecule can further comprise a secondary targeting moiety.
  • Such therapeutic fusion molecules can be specific to a targeted nerve circuit and can then be delivered there in an activity-dependent manner.
  • the subject neuroactive fusion molecule can be of any molecular weight as is resultant from its composition, e.g. the sum of the weights of the delivery moiety, the therapeutic moiety, and optionally the secondary targeting moiety.
  • a subject neuroactive fusion molecule can have a molecular weight of, for example, at least about 1 kDa, at least 10 kDa, at least about 20 kDa, at least about 30 kDa, at least about 40 kDa, at least about 50 kDa, at least about 60 kDa, at least about 70 kDa, at least about 80 kDa, at least about 90kDa, at least about 100 kDa, at least about 100 kDa, at least about 120 kDa, at least about 140 kDa, and so on, including molecules as large as 200kDa.
  • the present invention also provides nucleic acids encoding the subject neuroactive fusion molecules, and vectors and host cells for expressing the subject neuroactive fusion molecules.
  • the delivery moiety can be any polypeptide which is able to effect retrograde or anterograde (e.g., from primary sensory neuron connections to the CNS) delivery to a neuron.
  • the delivery moiety is a polypeptide can be selected from the group consisting of: wheat-germ agglutinin (and other related plant lectins like barley lectin), tetanus toxin (e.g., tetanus toxin C-fragment (TTC)), nerve growth factor, and Thy-1.
  • TTC tetanus toxin
  • TTC tetanus toxin
  • TTC-protein conjugates can also be transported trans-synaptically to pre-motor neurons within CNS circuits.
  • Trans-synaptic transfer of TTC is activity-dependent for selective targeting to active CNS circuits, thereby providing an opportunity for the preferential spread of therapeutic agents to select neuronal populations. Therefore, procedures that define the systemic delivery, selective targeting and fusion protein efficacy of TTC constructs should be viewed as a high priority area for translational research into CNS therapeutics.
  • complementary studies using TTC conjugated to reporter proteins e.g.
  • GFP allows activity-dependent identification of the same target population to profile its genetic expression patterns in health and disease. This profile can then identify critical factors to be fused to TTC to act as cell-specific therapeutic agents.
  • the therapeutic moiety of the neuroactive fusion molecule can be any polypeptide or other molecule that is able to exert a therapeutic effect.
  • the therapeutic moiety can be a neurotrophic factor, an endocrine factor, a growth factor, a paracrine factor, a hypothalamic releasing factor, a neurotransmitter polypeptide, an antibody or antibody fragment which binds to a neurotrophic factor, an antibody or antibody fragment which binds to a neurotrophic factor receptor, a polypeptide antagonist, an agonist or antagonist for a receptor expressed by a CNS cell, and a polypeptide involved in modifying intracellular processes including signal transduction cascades, trafficking, synaptic function, changes in gene expression and intracellular organelle function (e.g., lysosomal storage disease).
  • Other exemplary polypeptides or molecules that can comprise a therapeutic polypeptide of the invention include, but are not limited to:
  • Polypeptides or other molecules that act on receptors and other polypeptides including: acetylcholine receptors (muscarinic); acetylcholine receptors (nicotinic), acetylcholine synthesis and metabolism, adenosine receptors, a1-adrenoceptors, a2- adrenoceptors, b-adrenoceptors, biogenic amine transporters, cannabinoid receptors, dopamine receptors, dopamine, polypeptides involved in norepinephrine and epinephrine synthesis, polypeptides involved in dopamine and norepinephrine metabolism, excitatory amine acid transporters, GABAA receptors, GABAB receptors, GABAC receptors, GABA transporters, glutamate receptors (G Protein Family), glutamate receptors (ion channel family), polypeptides involved in glutamate/GABA synthesis and metabolism, glycine receptors, gly
  • Peptide receptors and polypeptides involved in peptide metabolism including, but not limited to, angiotensin receptors, bombesin receptors, bradykinin receptors, calcitonin gene-related peptide (and related peptides) receptors, chemokine receptors, cholecystokinin and gastrin receptors, corticotropin-releasing factor receptors, cytokine receptors, hematopoetin receptor family receptors, tumor necrosis receptor family receptors, interleukin-1/TIR receptor family receptors, endothelin receptors, galanin receptors, melanocortin receptors, neuropeptidases, neuropeptide Y receptors, neurotensin receptors, neurotrophin receptors, opioid receptors, orexin receptors, proteinase-activated receptors, somatostatin receptors, tachykinin receptors, vasoactive intestinal peptides (and related peptides) receptors, vasopressin
  • Ion Channels including, but not limited to: calcium channels, chloride channels, potassium channels, sigma receptors, sodium channels, and vanilloid receptors.
  • Peptides and other molecules affecting the following categories of disorders including but not limited to: neurological disorders, trauma of the head, neurotransmission, CNS infections, pain, CNS neoplasms, headache, neuro- ophthalmologic and cranial nerve disorders, function and dysfunction of the cerebral lobes, disorders of movement, stupor and coma, demyelinating diseases, delirium and dementia, seizure disorders, spinal cord disorders, sleep disorders, disorders of the peripheral nervous system, disorders of the autonomic nervous system, cerebrovascular disease, muscular disorders, attention deficit hyperactivity disorders, personality disorders, somatoform disorders, psychosexual disorders, anxiety disorders, schizophrenia and related disorders, dissociative disorders, psychiatric emergencies, mood disorders, drug use and dependence, suicidal behavior, and eating disorders, gastrointestinal disorders, gastroenteritis, esophageal disorders, antibiotic-associated colitis, inflammatory bowel diseases, gastritis and peptic ulcer disease, functional bowel disorders, pancreatitis, anorectal disorders, diarrhea and constipation.
  • Functional subpopulations of central neurons can be targeted by the identity of the therapeutic molecule delivered.
  • GDNF supports survival of dopaminergic substantia nigra neurons in vivo and in vitro and GDNF can be delivered to substantia nigra dopaminergic neurons via retrograde transport from its terminal fields in the striatum and act on its receptors RET and the GFRD receptor family.
  • Selective targeting of brain dopamine systems with TTC-GDNF could be used for diagnosis and therapeutic control of Parkinson's disease.
  • the neuroactive fusion molecule comprises a diagnostic molecule.
  • Such neuroactive fusion molecules also can comprise a therapeutic molecule or secondary target molecule.
  • the diagnostic molecules allow the neuroactive fusion molecule to be detected, such as for example, by fluorescence imaging such as fluoroscopy, position emission tomography (PET), microPET, computerized tomography (CT, CAT), magnetic resonance imaging (MRI), nuclear magnetic imaging (NMI), ultrasound, sonofluorescence, SPECT, optical imaging, endoscopy, microdialysis, autoradiography (x-ray), etc.
  • the diagnostic molecule is a molecule that is not a polypeptide, such as for example, a fluorophore or radiolabeled molecule.
  • the diagnostic molecule can be a polypeptide, such as for example, a fluorescent polypeptide such as green or yellow fluorescent protein, or a polypeptide carrying a radiolabel.
  • fluorophores include, but are not limited to: Fluorescein, Rhodamine, Texas Red, Cy2, Cy3, Cy5, VECTOR Red, ELF.TM. (Enzyme-Labeled Fluorescence), CyO, CyO.5, Cy1 , Cy1.5, Cy3, Cy3.5, Cy5, Cy7, FluorX, Calcein, Calcein-AM, CRYPTOFLUOR.TM.'S, Orange (42 kDa), Tangerine (35 kDa), Gold (31 kDa), Red (42 kDa), Crimson (40 kDa), BHMP, BHDMAP, Br-Oregon, Lucifer Yellow, Alexa dye family, N-[6-(7-nitrobenz-2-oxa-1 , 3-diazol-4-yl)amino]caproyl] (NBD), BODIPY.TM., boron dipyrromethene difluoride, Oregon Green, MITOTRACKER.TM.
  • the fluorophore is a fluorescent dye that spans the near-ultraviolet, visible and near-infrared spectrum, such as rhodamine or a fiuorescein-based dye such as FITC.
  • a diagnostic molecule of the invention is labeled with an isotopic label to facilitate its detection and or structural characterization using magnetic resonance or another applicable technique.
  • isotopic labels include radioisotopic labels such as, for example, potassium-40 (40K), carbon-14 (14C), tritium (3H), sulphur-35 (35S), phosphorus-32 (32P), technetium-99m (99mTc), thallium-201 (201TI), gallium-67 (67Ga), indium-111 (111 In), iodine-123 (1231), iodine-131 (1311), yttrium-90 (90Y), samarium-153 (153Sm), rhenium-186 (186Re), rhenium-188 (188Re), dysprosium-165 (165Dy) and holmium-166 (166Ho).
  • radioisotopic labels such as, for example, potassium-40 (40K), carbon-14 (14C), tritium (3H), sulphur-35 (35
  • the isotopic label can also be an atom with non zero nuclear spin, including, for example, hydrogen-1 (1 H), hydrogen-2 (2H), hydrogen-3 (3H), phosphorous-31 (31 P), sodium-23 (23Na), nitrogen-14 (14N), nitrogen-15 (15N), carbon-13 (13C) and fluorine-19 (19F).
  • the polypeptide is uniformly labeled with an isotopic label, for example, wherein at least 50%, 70%, 80%, 90%, 95%, or 98% of the possible labels in the polypeptide are labeled, e.g., wherein at least 50%, 70%, 80%, 90%, 95%, or 98% of the nitrogen atoms in the polypeptide are 15N, and/or wherein at least 50%, 70%, 80%, 90%, 95%, or 98% of the carbon atoms in the polypeptide are 13C, and/or wherein at least 50%, 70%, 80%, 90%, 95%, or 98% of the hydrogen atoms in the polypeptide are 2H.
  • an isotopic label for example, wherein at least 50%, 70%, 80%, 90%, 95%, or 98% of the possible labels in the polypeptide are labeled, e.g., wherein at least 50%, 70%, 80%, 90%, 95%, or 98% of the nitrogen atoms in the polypeptide are 15N, and/or where
  • the isotopic label is located in one or more specific locations within the molecule, for example, the label can be specifically incorporated into one or more of the leucine residues of a polypeptide.
  • the invention also encompasses embodiments wherein a single molecule comprises two, three or more different isotopic labels, for example, a polypeptide comprises both 15N and 13C labeling.
  • the diagnostic polypeptides of the invention are labeled with a fluorescent label to facilitate their detection, purification, or structural characterization.
  • a polypeptide of the invention is fused to a heterologous polypeptide sequence which produces a detectable fluorescent signal, such as, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED).
  • GFP green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • Renilla Reniformis green fluorescent protein GFPmut2, GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED).
  • a diagnostic molecule of the invention can be a contrast agent, i.e., a molecule used to target or highlight particular features or areas of interest during imaging.
  • the contrast agent comprising the diagnostic molecule can be linked to the neuroactive fusion molecule, which can carry it into the nervous system for imaging by one of any of the imaging modalities known to those of skill in the art, such as, for example, fluorescence imaging such as fluoroscopy, position emission tomography (PET), microPET, computerized tomography (CT, CAT), magnetic resonance imaging (MRI), nuclear magnetic imaging (NMI), ultrasound, sonofluorescence, SPECT, optical imaging, endoscopy, microdialysis, autoradiography (x-ray), etc.
  • fluorescence imaging such as fluoroscopy, position emission tomography (PET), microPET, computerized tomography (CT, CAT), magnetic resonance imaging (MRI), nuclear magnetic imaging (NMI), ultrasound, sonofluorescence, SPECT, optical imaging, endoscopy, microdi
  • each such imaging modality has its own set of contrast agents, known to those of skill in the art, that are suitable for use with a given imaging modality.
  • contrast agents known to those of skill in the art, that are suitable for use with a given imaging modality.
  • PET position emission tomography
  • CT computerized tomography
  • MRI magnetic resonance imaging
  • NMI nuclear magnetic imaging agents
  • fluoroscopy agents would include, but are not limited to, any of the aforementioned fluorophores or fluorescent proteins.
  • x-ray contrast agents include, but are not limited to, iodinated molecules, ionic contrast agents such as acetrizoic acid derivatives, diatrizoic acid derivatives, iothalamic acid derivatives, ioxithalamic acid derivatives, metrizoic acid derivatives, iodamide, lypophylic agents, aliphatic acid salts, iodipamide, and ioxaglic acid derivatives, as well as nonionic contrast agents such as metrizamide, iopamidol, iohexol, iopromide, iobitridol, iomeprol, iopentol, ioversol, ioxilan, and iodixanol.
  • ionic contrast agents such as acetrizoic acid derivatives, diatrizoic acid derivatives, iothalamic acid derivatives, ioxithalamic acid derivatives, metrizoic acid derivatives, i
  • MRI contrast agents include, but are not limited to gadolinium derivatives, manganese derivatives, and superparamagnetic iron oxide particles.
  • nuclear contrast agents include, but are not limited to, any of the aforementioned radiolabels.
  • Spacers such as for example, polypeptide spacers can be present, e.g., between the various components of the neuroactive fusion molecules.
  • the amino acid sequence of such spacers can be encoded by the same nucleic acid encoding the fusion molecule.
  • the length and composition of the spacer sequence can be chosen to achieve maximum flexibility between the connected domains and minimum steric hindrance to potential interactors or proteolytic agents of the domains.
  • the geometry of the spacer can be used to orient a molecule for optimal reaction with an interactor or agent.
  • a spacer with flexible geometry can allow the fusion molecules to conformationally adapt as they bind other compounds.
  • the nature of the spacer can be altered for other various purposes. For example, the charge or hydrophobicity of the spacer can be altered to promote the binding of a compound to the fusion molecule.
  • Spacer domains comprised of glycine residues generally result in protein folding conformations that allow for improved accessibility to the flanking domains. See Dan et, al. (1996), J. Biol. Chem. 271 :30717-30724; Borjigin, J and Nathans, J., (1994), J. Biol. Chem. 269:14715-147622.
  • the components of the fusion molecule can be cleavable, e.g., can be specifically cleaved by an agent or can self-cleave.
  • the cleavable domain is a protease cleavage site.
  • Such domains have unique polypeptide sequences that are recognized by a protease.
  • Exemplary proteases include Tobacco Etch Virus (TEV) protease, enterokinase, Factor Xa protease, thrombin, and kallikrein. See, e.g. Matsushima, et al. (1999) J. Biochem 125:847-51 ; Change, (1985), Eur. J. Biochem, 151 :217; Nagai, K, et al. (1984) Nature 308:810-812 for exemplary recognition sequences and cleavage conditions of selected proteases.
  • TSV Tobacco Etch Virus
  • neuroactive fusion molecules can be prepared by any method known in the art for the preparation of fusion molecules.
  • the neuroactive fusion molecule is comprised of a fusion polypeptide, it can be produced by expressing a genetic fusion of the desired polypeptide components in a host cell.
  • a neuroactive fusion molecule can be produced by chemically linking the polypeptides, or polypeptide and molecule, to be fused. Such methods are described in more detail below.
  • the present invention also provides nucleic acids encoding the above- described neuroactive fusion molecules.
  • Such nucleic acids can also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant polypeptides.
  • Techniques for making fusion genes are well known in the art. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
  • the present invention provides an isolated nucleic acid comprising a neuroactive fusion molecule of the invention.
  • the subject nucleic acid is provided in a vector comprising a nucleotide sequence encoding a neuroactive fusion molecule of the invention and operably linked to at least one regulatory sequence.
  • a vector comprising a nucleotide sequence encoding a neuroactive fusion molecule of the invention and operably linked to at least one regulatory sequence.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.
  • the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should be considered.
  • Such vectors can be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively transfecting cells either ex vivo or in vivo with genetic material encoding a chimeric polypeptide.
  • Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered alone with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers.
  • Nucleic acids can also be directly injected into cells. Alternatively, calcium phosphate precipitation can be carried out to facilitate entry of a nucleic acid into a cell.
  • the subject nucleic acids can be used to cause expression and over- expression of a fusion gene comprising a neuroactive fusion molecule of the invention in cells propagated in culture, e.g. to produce fusion proteins or polypeptides.
  • This invention also pertains to a host cell transfected with a recombinant gene in order to express a neuroactive fusion molecule of the invention.
  • the host cell can be any prokaryotic or eukaryotic cell.
  • a neuroactive fusion molecule of the invention can be expressed in bacterial cells, such as E. coli, insect cells (baculovirus), yeast, insect, plant, or mammalian cells. In those instances when the host cell is human, it can or may not be in a live subject.
  • Other suitable host cells are known to those skilled in the art.
  • the host cell can be supplemented with tRNA molecules not typically found in the host so as to optimize expression of the polypeptide. Other methods suitable for maximizing expression of the neuroactive fusion molecule of the invention will be known to those in the art.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • a neuroactive fusion molecule of the invention can be secreted and isolated from a mixture of cells and medium comprising the polypeptide. Alternatively, a neuroactive fusion molecule of the invention can be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a nucleotide sequence encoding all or part of a neuroactive fusion molecule of the invention can be used to produce a recombinant form of a protein via microbial or eukaryotic cellular processes.
  • Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, can be employed to prepare recombinant neuroactive fusion molecules of the invention by microbial means or tissue-culture technology in accord with the subject invention.
  • Expression vehicles for production of a recombinant protein include plasmids and other vectors.
  • suitable vectors for the expression of a fusion polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL- derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • the nucleic acid encoding a neuroactive fusion molecule of the invention is operably linked to a bacterial promoter, e.g., the anaerobic E. coli, NirB promoter or the E.
  • the bacterial promoter can be a constitutive promoter or an inducible promoter.
  • An exemplary inducible promoter is a promoter which is inducible by iron or in iron-limiting conditions.
  • iron-regulated promoters of FepA and TonB are known in the art and are described, e.g., in the following references: Headley, V. et al. (1997) Infection & Immunity 65:818; Ochsner, U.A. et al. (1995) Journal of Bacteriology 177:7194; Hunt, M.D. et al. (1994) Journal of Bacteriology 176:3944; Svinarich, D.M. and S. Palchaudhuri. (1992) Journal of Diarrhoeal Diseases Research 10:139; Prince, R.W. et al.
  • a plasmid for practicing the invention preferably comprises sequences required for appropriate transcription of the nucleic acid in bacteria, e.g., a transcription termination signal.
  • the vector can further comprise sequences encoding factors allowing for the selection of bacteria comprising the nucleic acid of interest, e.g., gene encoding a protein providing resistance to an antibiotic, sequences required for the amplification of the nucleic acid, e.g., a bacterial origin of replication.
  • a signal peptide sequence is added to the construct, such that the fusion polypeptide is secreted from cells.
  • signal peptides are well known in the art.
  • the powerful phage T5 promoter that is recognized by E. coli RNA polymerase is used together with a lac operator repression module to provide tightly regulated, high level expression or recombinant proteins in E. coli.
  • protein expression is blocked in the presence of high levels of lac repressor.
  • the DNA is operably linked to a first promoter and the bacterium further comprises a second DNA encoding a first polymerase which is capable of mediating transcription from the first promoter, wherein the DNA encoding the first polymerase is operably linked to a second promoter.
  • the second promoter is a bacterial promoter, such as those delineated above.
  • the polymerase is a bacteriophage polymerase, e.g., SP6, T3, or T7 polymerase and the first promoter is a bacteriophage promoter, e.g., an SP6, T3, or T7 promoter, respectively.
  • Plasmids comprising bacteriophage promoters and plasmids encoding bacteriophage polymerases can be obtained commercially, e.g., from Promega Corp. (Madison, Wis.) and InVitrogen (San Diego, Calif.), or can be obtained directly from the bacteriophage using standard recombinant DNA techniques (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, 1989). Bacteriophage polymerases and promoters are further described, e.g., in the following references: Sagawa, H. et al. (1996) Gene 168:37; Cheng, X. et al.
  • the bacterium further comprises a DNA encoding a second polymerase which is capable of mediating transcription from the second promoter, wherein the DNA encoding the second polymerase is operably linked to a third promoter.
  • the third promoter is a bacterial promoter.
  • more than two different polymerases and promoters could be introduced in a bacterium to obtain high levels of transcription.
  • the use of one or more polymerase for mediating transcription in the bacterium can provide a significant increase in the amount of polypeptide in the bacterium relative to a bacterium in which the DNA is directly under the control of a bacterial promoter.
  • the selection of the system to adopt will vary depending on the specific use of the invention, e.g., on the amount of protein that one desires to produce.
  • a nucleic acid encoding a neuroactive fusion molecule of the invention is introduced into a host cell, such as by transfection, and the host cell is cultured under conditions allowing expression of the neuroactive fusion molecule.
  • Methods of introducing nucleic acids into prokaryotic and eukaryotic cells are well known in the art. Suitable media for mammalian and prokaryotic host cell culture are well known in the art.
  • the nucleic acid encoding the subject fusion polypeptide is under the control of an inducible promoter, which is induced once the host cells comprising the nucleic acid have divided a certain number of times.
  • IPTG isopropyl beta-D-thiogalactopyranoside
  • the host cell can include a plasmid which expresses an internal T7 lysozyme, e.g., expressed from plasmid pLysSL (see Examples). Lysis of such host cells liberates the lysozyme which then degrades the bacterial membrane.
  • sequences that can be included in a vector for expression in bacterial or other prokaryotic cells include a synthetic ribosomal binding site; strong transcriptional terminators, e.g., to from phage lambda and t4 from the rrnB operon in E. coli, to prevent read through transcription and ensure stability of the expressed polypeptide; an origin of replication, e.g., ColE1 ; and beta-lactamase gene, conferring ampicillin resistance.
  • Other host cells include prokaryotic host cells. Even more preferred host cells are bacteria, e.g., E. coli. Other bacteria that can be used include Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp., Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., and Erysipel
  • YEP24, YIP5, YEP51 , YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al., (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83).
  • These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
  • drug resistance markers such as ampicillin can be used.
  • mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papilloma virus
  • pHEBo Epstein-Barr virus
  • the various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.
  • suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
  • baculovirus expression systems include pVL- derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWI), and pBlueBac-derived vectors (such as the ⁇ -gal comprising pBlueBac III).
  • in vitro translation systems are, generally, a translation system which is a cell-free extract comprising at least the minimum elements necessary for translation of an RNA molecule into a protein.
  • An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, elF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (elF4F).
  • CBP cap-binding protein
  • elF4F eukaryotic initiation factor 4F
  • in vitro translation systems examples include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, III.; and GIBCO/BRL, Grand Island, N.Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system can be used.
  • eukaryotic lysates such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla,
  • Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors.
  • An RNA nucleotide for in vitro translation can be produced using methods known in the art. In vitro transcription and translation can be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs.
  • a start codon AGT
  • methionine aminopeptidase MAP
  • a neuroactive fusion molecule of the invention can be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature, 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J., 6:307-311) can be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1994, EMBO J., 3:1671-1680; Broglie et al., 1984, Science, 224:838-843); or heat shock promoters, e.g., soybean hsp 17.5-E or hsp 17.3-B (Gurley et al., 1986, Mol.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the PGHS-2 sequence can be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • Successful insertion of the coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene).
  • the DNA encoding a neuroactive fusion molecule of the invention is cloned into the pBlueBaclll recombinant transfer vector (Invitrogen, San Diego, Calif.) downstream of the polyhedrin promoter and transfected into Sf9 insect cells (derived from Spodoptera frugiperda ovarian cells, available from Invitrogen, San Diego, Calif.) to generate recombinant virus.
  • Sf9 insect cells derived from Spodoptera frugiperda ovarian cells, available from Invitrogen, San Diego, Calif.
  • high-titer viral stocks are prepared that in turn would be used to infect Sf9 or High FiveTM (BTI-TN- 5B1-4 cells derived from Trichoplusia ni egg cell homogenates; available from
  • a neuroactive fusion molecule of the invention is prepared in transgenic animals, such that in certain embodiments, the polypeptide is secreted, e.g., in the milk of a female animal.
  • Viral vectors can also be used for efficient in vitro introduction of a nucleic acid into a cell. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid.
  • fusion polypeptides encoded by genetic material in the viral vector e.g., by a nucleic acid contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid.
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into mammals. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
  • a major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
  • the development of specialized cell lines (termed "packaging cells") which produce only replication- defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D.
  • recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one of the antisense E6AP constructs, rendering the retrovirus replication defective.
  • the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al.
  • retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include Crip, Cre, 2 and Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al.
  • retroviral vectors as a gene delivery system for nucleic acids encoding a neuroactive fusion molecule of the invention, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction of the genetic material, is that the target cells must be dividing. In general, this requirement will not be a hindrance to use of retroviral vectors. In fact, such limitation on infection can be beneficial in circumstances wherein the tissue (e.g., nontransformed cells) surrounding the target cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors.
  • tissue e.g., nontransformed cells
  • retroviral-based vectors by modifying the viral packaging proteins on the surface of the viral particle (see, for example, PCT publications W093/25234, WO94/06920, and W094/11524).
  • strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al.
  • Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating chimeric proteins (e.g., single-chain antibody/env chimeric proteins).
  • This technique while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.
  • retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the genetic material of the retroviral vector.
  • Another viral gene delivery system utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactive in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155).
  • adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
  • Recombinant adenoviruses can be advantageous in certain circumstances in that they are capable of infecting non-dividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482- 6486), hepatocytes (Herz and Gerard (1993) Proc.
  • virus particle is relatively stable and amenable to purification and concentration, and, as above, can be modified so as to affect the spectrum of infectivity.
  • introduced adenoviral DNA and foreign DNA contained therein is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).
  • Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic material (see, for example, Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466- 6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081 ; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51 :611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).
  • Other viral vector systems can be derived from herpes virus, vaccinia virus, and several RNA viruses.
  • non-viral methods can also be employed to cause expression of nucleic acids encoding a neuroactive fusion molecule of the invention, e.g. in a cell in vitro or in the tissue of an animal.
  • Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of genetic material by the targeted cell.
  • Exemplary gene delivery systems of this type include liposomal derived systems, polylysine conjugates, and artificial viral envelopes.
  • Cell lines of the above-described host cells can be established. Culturing techniques make it possible to obtain primary cultures which can be utilized directly as nontransformed lines, or can be transformed in order to obtain lines whose cells continue to proliferate, e.g. in an immortalized cell line. Such primary and immortalized cell cultures comprise the "cell lines" of the present invention.
  • the components of a neuroactive fusion molecule of the invention are produced separately and then linked, e.g. covalently linked, to each other.
  • polypeptides and/or molecules of interest are produced separately, purified, and mixed together under conditions under which they are able to be linked to each other.
  • Linkers also known as "linker molecules” or “cross- linkers” can be used to conjugate polypeptides.
  • Linkers include chemicals able to react with a defined chemical group of several, usually two, molecules and thus conjugate them. The majority of known cross-linkers react with amine, carboxyl, and sulfhydryl groups.
  • target chemical group is crucial if the group can be involved in the biological activity of the molecules to be conjugated.
  • maleimides which react with sulfhydryl groups, can inactivate Cys-comprising peptides or proteins that require the Cys to bind to a target.
  • Linkers can be homofunctional (comprising reactive groups of the same type), heterofunctional (comprising different reactive groups), or photoreactive (comprising groups that become reactive on illumination).
  • Linker molecules can be responsible for different properties of the conjugated compositions.
  • the length of the linker should be considered in light of molecular flexibility during the conjugation step, and the availability of the conjugated molecule for its target (cell surface molecules and the like.) Longer linkers can thus improve the biological activity of the compositions of the present invention, as well as the ease of preparation of them. Linkers that act in such capacity are also referred to as "spacers" herein.
  • the geometry of the linker can be used to orient a molecule for optimal reaction with a target.
  • a linker with flexible geometry can allow the cross- linked polypeptides to conformationally adapt as the bind other polypeptides.
  • the nature of the linker can be altered for other various purposes.
  • the aryl- structure of MBuS was found less immunogenic than the aromatic spacer of MBS.
  • the hydrophobicity and functionality of the linker molecules can be controlled by the physical properties of component molecules.
  • the hydrophobicity of a polymeric linker can be controlled by the order of monomeric units along the polymer, e.g. a block polymer in which there is a block of hydrophobic monomers interspersed with a block of hydrophilic monomers.
  • the invention provides methods of producing, identifying, and isolating a neuroactive fusion molecule of the invention.
  • a neuroactive fusion molecule of the invention can be isolated from cell culture medium, host cells, or reaction mixtures using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of a fusion.
  • the purified neuroactive fusion molecule of the invention is substantially free of other cellular material, e.g., proteins.
  • substantially pure or purified refers to preparations of molecules having less than about 20%) (by dry weight) contaminating cellular material, e.g., nucleic acids, proteins, and lipids, and preferably having less than about 5% contaminating material.
  • Preferred preparations of a neuroactive fusion molecule of the invention have less than about 2% contaminating material; even more preferably less than about 1 % contaminating material and most preferably less than about 0.5; 0.2; 0.1 ; 0.01 ; 0.001 % contaminating material.
  • purified preparations at least about 90%, about 95%, about 96%, 97%, 98%, 99%, 99.5%, or at least about 99.9%, 99.99%, or 99.999% purified neuroactive fusion molecule.
  • a preferred method for determining the amount of contaminating proteins in a preparation comprises subjecting the preparation to gel electrophoresis, e.g., polyacrylamide electrophoresis, in the presence of specific amounts of molecular markers, and staining the gel after the electrophoresis with a protein dye. A comparison of the intensity of the band of the subject polypeptide with the molecular markers indicates the purity of the subject polypeptide preparation.
  • Other methods for determining the amount of contaminating proteins include mass spectrometry, gel filtration and peptide sequencing according to methods known in the art.
  • a preferred method for determining the amount of contaminating cellular material in a polypeptide preparation comprises gel electrophoresis and silver staining of the gel.
  • Protein concentrations can be determined according to the following methods: Lowry-Folin-Ciocalteau reagent; UV absorption at 280 nm (aromatic band) or 205- 220nm (peptide band); dye binding (e.g., Coomassie Blue G-250); or bis-cinchonic acid (BCA; Pierce Chemicals (Rockford, IL)) reagent. All of these methods are described in, e.g., Robert K. Scopes, Protein Purification, Principles and Practice, Third Ed., Springer Verlag New York, 1993, and references cited therein. Briefly, the well-known Lowry method is a relatively sensitive method giving a good color with 0.1 mg/ml or protein or less.
  • the method using Coomassie Blue G-250 is very sensitive, fast and at least as accurate as the Lowry method.
  • the procedure consists in mixing a polypeptide sample with the reagent and measure the blue color at 595nm.
  • the preferred method for determining exact neuroactive fusion molecule amounts is by dry weight determination, since it provides a suitably accurate measurement.
  • the dry weight of a highly pure preparation of the molecule is determined, and this preparation is then used as a standard for determining the protein concentration of other preparations of neuroactive fusion molecules of the invention.
  • the percent recovery and degree of purity of a preparation of a neuroactive fusion molecule of the invention can be calculated from the total amount of fusion molecule recovered after purification and the amount and/or activity of the fusion molecule.
  • compositions comprising the neuroactive fusion molecules.
  • the pharmaceutical composition comprises an isolated, purified neuroactive fusion molecule.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the neuroactive fusion molecule comprises a therapeutic polypeptide and a delivery polypeptide.
  • the neuroactive fusion molecule further comprises a secondary targeting polypeptide.
  • the invention provides devices for administering the pharmaceutical compositions, for example, devices for intravenous, intraperitoneal, or subcutaneous injection.
  • compositions of the present invention can be administered by various means, depending on their intended use, as is well known in the art.
  • compositions of the present invention can be formulated as tablets, capsules, granules, powders or syrups.
  • formulations of the present invention can be administered parenterally as injections (intravenous, intraperitoneal or subcutaneous), drop infusion preparations or suppositories.
  • injections intravenous, intraperitoneal or subcutaneous
  • drop infusion preparations or suppositories for application by the ophthalmic mucous membrane route
  • compounds of the present invention can be formulated as eyedrops or eye ointments.
  • formulations can be prepared by conventional means, and, if desired, the compounds can be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent or a coating agent.
  • wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can be present in the formulated agents.
  • compositions can be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration.
  • the formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy.
  • the amount of agent that can be combined with a carrier material to produce a single dose vary depending upon the subject being treated, and the particular mode of administration.
  • Methods of preparing these formulations include the step of bringing into association agents of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association agents with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of a compound thereof as an active ingredient.
  • Compounds of the present invention can also be administered as a bolus, electuary, or paste.
  • the coordination complex thereof is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acety
  • a tablet can be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets can be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets can be made by molding in a suitable machine a mixture of the supplement or components thereof moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emuls
  • Suspensions in addition to compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations for rectal or vaginal administration can be presented as a suppository, which can be prepared by mixing a coordination complex of the present invention with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.
  • suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.
  • Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for transdermal administration of a supplement or component includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active component can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which can be required.
  • the complexes can include lipophilic and hydrophilic groups to achieve the desired water solubility and transport properties.
  • the ointments, pastes, creams and gels can contain, in addition to a supplement or components thereof, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a supplement or components thereof, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Compounds of the present invention can alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers can be used because they minimize exposing the agent to shear, which can result in degradation of the compound.
  • an aqueous aerosol is made by formulating an aqueous solution or suspension of the compound together with conventional pharmaceutically acceptable carriers and stabilizers.
  • the carriers and stabilizers vary with the requirements of the particular compound, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.
  • Aerosols generally are prepared from isotonic solutions.
  • compositions of this invention suitable for parenteral administration comprise one or more components of a supplement in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non- aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to use, which can contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and non-aqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • the present invention provides methods of treating a subject that a disorder of the nervous system comprising: (a) administering by non- intramuscular means to a subject a therapeutically effective amount of a neuroactive fusion molecule comprising a therapeutic polypeptide and a delivery polypeptide; (b) wherein said administered neuroactive fusion molecule crosses the blood-brain or blood-nerve barrier.
  • the disorder can be, in certain embodiments, a peripheral nervous system disorder and the neuron a peripheral neuron, or a central nervous system disorder and the neuron a central nervous system neuron.
  • the disorder can be an autonomic nervous system disorder and the neuron an autonomic nervous system neuron, or the disorder can be an enteric nervous system disorder and the neuron an enteric nervous system neuron.
  • the disorder is a central nervous system disorder, wherein the neuron is a peripheral neuron, and wherein the method further comprises trans-synaptically transferring the administered fusion molecule from the peripheral neuron to a central nervous system neuron.
  • the neuroactive fusion molecule in certain embodiments, can further comprise a secondary targeting polypeptide.
  • the method of treating can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from the neuron to at least one other neuron, wherein the secondary targeting polypeptide causes the trans-synaptic transfer to selectively target at least one other neuron.
  • administration can be, for example, peripheral, systemic, or local.
  • the neuroactive fusion molecules of the present invention can be delivered in these methods to select neuronal classes.
  • the epilepsies and other disorders of the CNS with aberrant hyperactive circuits e.g. arterial hypertension, hyperkinetic disorders, and obsessive-compulsive disorder
  • the neuroactive fusion molecules of the present invention can be internalized and trans-synaptically transferred in an activity-dependent manner allows for preferential targeting to these aberrant circuits.
  • hyperactive primary afferent signaling occurs in a plethora of chronic pain, which could be targeted by the methods of the present invention.
  • the hyperactive neural circuitry possesses no further physiological relevance.
  • phantom pain in amputees is produced by central circuitry signaling somatosensory information to body regions that no longer exist.
  • the delivery polypeptide could be fused to 2 proteins, the first used to selectively target a neuronal circuit and the second protein would be saporin, which kills neurons when internalized. In this manner hyperactive circuits can be selectively lesioned (e.g. thalamic pain syndromes).
  • disorders that can be treated by non-intramuscular administration of the neuroactive fusion molecules includes, but are not limited to, paroxysmal disorders (the epilepsies); neuropsychiatric disorder mania, psychosis (e.g. auditory hallucinations associated with excessive mesocortical DA activity), obsessive compulsive disorder (there is an actual defined circuitry), addiction (activation of reward circuitry for addiction), movement disorders such as the dyskinesias (resting tremor), basal ganglia hyperkinetic disorders (Huntington's chorea, hemiballismus), autonomic NS dysfunction arterial hypertension (following cervical spinal cord transection, secondary to cardiovascular disorders, essential hypertension, polyneuropathies), hypothalamic dysfunction, hyperlactemia, and neuropathic pain syndromes.
  • paroxysmal disorders the epilepsies
  • neuropsychiatric disorder mania e.g. auditory hallucinations associated with excessive mesocortical DA activity
  • any composition of the present invention will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the supplement. Any of the subject formulations can be administered in a single dose or in divided doses. Dosages for the compounds of the present invention can be readily determined by techniques known to those of skill in the art or as taught herein. Also, the present invention contemplates mixtures of more than one subject compound, as well as other therapeutic agents.
  • the dosage of the subject compositions will generally be in the range of about 0.01 ng to about 10 g per kg body weight, specifically in the range of about 1 ng to about 0.1 g per kg, and more specifically in the range of about 100 ng to about 10 mg per kg.
  • An effective dose or amount, and any possible affects on the timing of administration of the formulation can need to be identified for any particular compound of the present invention. This can be accomplished by routine experiment as described herein, using one or more groups of animals (preferably at least 5 animals per group), or in human trials if appropriate.
  • the effectiveness of any compound and method of treatment or prevention can be assessed by administering the supplement and assessing the effect of the administration by measuring one or more indices associated with the neoplasm of interest, and comparing the post- treatment values of these indices to the values of the same indices prior to treatment.
  • the precise time of administration and amount of any particular compound that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like.
  • the guidelines presented herein can be used to optimize the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing. While the subject is being treated, the health of the patient can be monitored by measuring one or more of the relevant indices at predetermined times during a 24-hour period.
  • Treatment can be optimized according to the results of such monitoring.
  • the patient can be periodically reevaluated to determine the extent of improvement by measuring the same parameters, the first such reevaluation typically occurring at the end of four weeks from the onset of therapy, and subsequent reevaluations occurring every four to eight weeks during therapy and then every three months thereafter.
  • Therapy can continue for several months or even years, with a minimum of one month being a typical length of therapy for humans. Adjustments to the amount(s) of agent administered and possibly to the time of administration can be made based on these reevaluations.
  • Treatment can be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage can be increased by small increments until the optimum therapeutic effect is attained.
  • the combined use of several compounds of the present invention, or alternatively other chemotherapeutic agents, can reduce the required dosage for any individual component because the onset and duration of effect of the different components can be complementary.
  • the different active agents can be delivered together or separately, and simultaneously or at different times within the day.
  • Toxicity and therapeutic efficacy of subject compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50. Compositions that exhibit large therapeutic indices are preferred. Although compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets the compounds to the desired site in order to reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of any supplement, or alternatively of any components therein lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • the present invention provides methods of diagnosing a disorder of the nervous system in a subject comprising: (a) administering by non- intramuscular means to a subject a neuroactive fusion molecule comprising a diagnostic molecule and a delivery polypeptide; and (b) wherein said administered neuroactive fusion molecule crosses the blood-brain barrier.
  • the diagnostic molecule can be a fluorophore, such as for example rhodamine.
  • the diagnostic molecule can be a polypeptide, such as for example, green fluorescent protein or yellow fluorescent protein.
  • such diagnostic methods can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from a peripheral neuron to at least one other neuron.
  • the neuroactive fusion molecule can further comprise a secondary targeting polypeptide.
  • the diagnostic method can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from a neuron to at least one other neuron, wherein the secondary targeting polypeptide causes the trans- synaptic transfer to selectively target at least one other neuron.
  • the neuroactive fusion molecules of the present invention can be delivered in these diagnostic methods to select neuronal classes.
  • the epilepsies and other disorders of the CNS with aberrant hyperactive circuits can be diagnosed by administration of the neuroactive fusion molecules of the present invention.
  • neuroactive fusion molecules of the present invention can be internalized and trans-synaptically transferred in an activity-dependent manner allows for preferential targeting to these aberrant circuits.
  • hyperactive primary afferent signaling occurs in a plethora of chronic pain, which could be identified by the methods of the present invention.
  • Other disorders that can be diagnosed by non-intramuscular administration of the neuroactive fusion molecules includes, but are not limited to, paroxysmal disorders (the epilepsies); neuropsychiatric disorder mania, psychosis (e.g.
  • auditory hallucinations associated with excessive mesocortical DA activity obsessive compulsive disorder (there is an actual defined circuitry), addiction (activation of reward circuitry for addiction), movement disorders such as the dyskinesias (resting tremor), basal ganglia hyperkinetic disorders (Huntington's chorea, hemiballismus), autonomic NS dysfunction arterial hypertension (following cervical spinal cord transection, secondary to cardiovascular disorders, essential hypertension, polyneuropathies), hypothalamic dysfunction, hyperlactemia, and neuropathic pain syndromes.
  • movement disorders such as the dyskinesias (resting tremor), basal ganglia hyperkinetic disorders (Huntington's chorea, hemiballismus), autonomic NS dysfunction arterial hypertension (following cervical spinal cord transection, secondary to cardiovascular disorders, essential hypertension, polyneuropathies), hypothalamic dysfunction, hyperlactemia, and neuropathic pain syndromes.
  • the presence and/or location of a neuroactive fusion molecule in a in a subject can be determined.
  • a biological sample can be taken from the subject, and prepared for visualization, e.g. by fluorescence.
  • the biological sample fluorescence can be visualized using appropriate magnification, excitation wavelengths and emission wavelengths.
  • the sample can be contacted with multiple neuroactive fusion molecules simultaneously.
  • the multiple neuroactive fusion molecules differ in their emission and/or excitation wavelengths.
  • Biological samples can include cells, tissue samples, lysates, or fluids from a living organism.
  • the cells are nerve cells, particularly neurons.
  • tissue samples are preferably sections of the peripheral or central nervous systems.
  • the detection of a neuroactive fusion molecule in a cell can include detection of the ligand in subcellular or extracellular compartments or organelles.
  • subcellular organelles and compartments include: Golgi networks and vesicles, pre-synaptic vesicles, lysosomes, vacuoles, nuclei, chromatin, mitochondria, chloroplasts, endoplasmic reticulum, coated vesicles (including clathrin coated vesicles), caveolae, periplasmic space and extracellular matrices.
  • the presence and/or location of a neuroactive fusion molecule in a subject can be determined directly in the subject.
  • a neuroactive fusion molecule can be administered to the subject, and the location visualized by, for example, any of the various imaging modalities known in the art, including but not limited to, fluorescence imaging such as fluoroscopy, position emission tomography (PET), microPET, computerized tomography (CT, CAT), magnetic resonance imaging (MRI), nuclear magnetic imaging (NMI), ultrasound, sonofluorescence, SPECT, optical imaging, endoscopy, microdialysis, autoradiography (x-ray), etc.
  • fluorescence imaging such as fluoroscopy, position emission tomography (PET), microPET, computerized tomography (CT, CAT), magnetic resonance imaging (MRI), nuclear magnetic imaging (NMI), ultrasound, sonofluorescence, SPECT, optical imaging, endoscopy, microdialysis, autoradiography (x-ray), etc.
  • the present invention provides methods of imaging a neuron in a subject comprising: (a) administering by non-intramuscular means to a subject a neuroactive fusion molecule comprising a diagnostic molecule and a delivery polypeptide; and (b) wherein said administered neuroactive fusion molecule crosses the blood-brain barrier.
  • the diagnostic molecule can be a contrast agent.
  • such diagnostic methods can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from a peripheral neuron to at least one other neuron.
  • the neuroactive fusion molecule can further comprise a secondary targeting polypeptide.
  • the imaging method can further comprise trans-synaptically transferring the administered neuroactive fusion molecule from a neuron to at least one other neuron, wherein the secondary targeting polypeptide causes the trans-synaptic transfer to selectively target at least one other neuron.
  • the diagnostic molecule of the neuroactive fusion molecule can be a contrast agent, i.e., a molecule used to target or highlight particular features or areas of interest during imaging.
  • the contrast agent comprising the diagnostic molecule can be linked to the neuroactive fusion molecule, which can carry it into the nervous system for imaging by one of any of the imaging modalities known to those of skill in the art.
  • imaging methods and compositions can be used, for example, to detect neurons, to map neurons and neuron connectivity, to localize neurons having a particular activity, etc. Kits
  • kits for treating and diagnosing disorders of the nervous system in a subject can also comprise one or more neuroactive fusion molecules of the present invention, or a pharmaceutical composition thereof.
  • Kit components can be packaged for either manual or partially or wholly automated practice of the foregoing methods.
  • this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use.
  • a kit can further comprise controls, reagents, buffers, and/or instructions for use.
  • Such kits can have a variety of uses, including, for example, imaging, diagnosis, therapy, and other applications.
  • TTC and WGA fluorescent conjugates can be delivered to motoneurons and then trans-synaptically transferred following subcutaneous, i.p. or i.v. injection.
  • Systemic injection of TTC has been observed to label all motoneurons, autonomic preganglionics, dorsal root ganglia, circumventricular nuclei and most likely select hypothalamic and brainstem reticular.
  • Systemic injection of WGA achieves similar results.
  • TTC- and WGA-fusions can be delivered to motoneurons and trans-synaptically transferred following subcutaneous, i.p. or i.v. injection.
  • WGA- or TTC- fluorescent conjugates reaches innumerably more CNS nerve cells than the conventional approach of intramuscular injection. Further, we have shown that WGA- or TTC-conjugates can be transported trans-synaptically in an activity dependent manner following its administration to the circulation. Thus, experimental paradigms or diseases that result in hyperactivity in select populations of CNS nerve cells should be preferentially targeted by WGA- or TTC- fusions.
  • Photographic evidence showed that, when delivered via the circulation, a WGA-rhodamine conjugate was transported to all CNS motoneurons, while intramuscular injection restricted labeling to motoneurons innervating the injected muscle only.
  • Intraperitoneal injection of WGA-rhodamine (0.2 mg) resulted in widespread motoneuron labeling on both sides of the spinal cord.
  • Intramuscular injection of WGA-FTIC (0.2 mg) in the ankle extensor muscle lateral gastrocnemious on one side of the animal restricted labeling to a limited number of motoneurons that project to it. It was observed that systemic delivery of WGA-rhodamine labeled all the motoneurons in this section including those also labeled selectively following intramuscular injection.
  • WGA-fluorescent conjugates were also transported trans-synaptically in an activity dependent manner.
  • Photographic evidence shows that WGA-conjugates were transported trans-synaptically (across synapses) in an activity-dependent manner following administration to the circulation.
  • a postnatal day 10 rat was injected intraperitoneally with 100 ⁇ l WGA-rhodamine (Vector labs) in 10mM HEPES buffer at 5mg/ml, and 50 ⁇ l of 1% Fluorogold in saline.
  • Fluorogold is known to be retrogradely transported to motoneuron cell bodies but not transported trans-synaptically. 24 hours later the cord was isolated and hemisected. One half of the cord had both L5 and L4 ventral roots electrically stimulated for 24 hrs. Stimulation intensity was 300 ⁇ A, 200 ⁇ s, and delivered in trains of 5 pulses at 200 Hz every 15 seconds (A1). The other half of the cord was not stimulated. In the un-stimulated cord no WGA-positive Fluorogold-negative cells were found, indicating that, in the absence of activity, WGA was not transported trans-synaptically. However, the stimulated side did contain such cells.
  • Fluorogold only labels motoneurons and cannot be transported trans-synaptically to other neurons that connect to motoneurons. Therefore, neurons which are labeled by a WGA-fluorescent conjugate but not Fluorogold must be interneurons that have been labeled trans- synaptically. Photographic evidence of systemic delivery of tracer molecules with subsequent cord isolation and sectioning both in fresh and fixed tissue showed that day following i.p. Fluorogold injection, ventral motoneurons, and sympathetic preganglionic neurons were retrogradely labeled and easily targeted for laser capture microdissection. Sympathetic cell clusters in the IML and in a medial cell group were found.
  • TTC and TTC fluorescent conjugates were also transported trans-synaptically following administration to the circulation.
  • 0.1-0.6 mg TTC or TTC- fluorescent conjugate was injected i.v., i.p., or subcutaneously into adult mice. Mice were sacrificed at 2 different time periods: 24 hours and 48 hours after systemic injection. At these time periods, the animals were anesthetized and transcardially- perfused with 1/3 w/v 0.9% NaCI, 0.1 %NaNO2, 1 unit/ml heparin, followed by equal w/v 4% paraformaldehyde, 0.1 M P03, ph 7.4.
  • TTC trans-synaptic transport was considerable.
  • TTC was injected subcutaneously in the nape at time 0 and at 24 hrs. Fluorogold was injected i.p. at time 0.
  • Fluorogold was injected i.p. at time 0.
  • the animal was sacrificed at 48 hours and processed for TTC.
  • the entire spinal cord neuropil was labeled consistent with its departure from neuronal cell bodies and retrograde transport to synaptic sites on dendrites. All motor nuclei in brainstem were also strongly labeled.
  • moderate labeling was seen in hippocampus neuropil, hypothalamic, habenular (a diencephalic structure implicated in limbic function) and cochlear nucleus (a sensory auditory relay nucleus) cell bodies.
  • TTC and TTC fusion proteins were shown to be transported with equal efficacy by co-injecting TTC and TTC- ⁇ -gal, wherein CNS tissue is examined for an identical distribution of immunolabeled TTC and ⁇ -gal. Photographic evidence showed capture of CNS TTC distribution 48 hours after subcutaneous injection at time 0 and 24 hours. The entire gray matter was labeled with TTC. A penumbra of weaker labeling more dorsally was from the TTC injected 48 hours earlier while the strong labeling in the motor column was due to the additional arrival of TTC from the second injection at 24 hours.
  • Fluorogold was found in the soma consistent with cellular transport into lysosomes while TTC, though present in the soma, was largely found in the neuropil in endosomes targeted for exocytosis. Fluorogold is found in all blood vessel capillaries coursing through the spinal cord (arrows). Photographic evidence showed TTC neuropil labeling in dorsal horn and intermediate grey matter respectively, and labeling in hippocampus, which is found preferentially in the neuropil. Neuronal somas were found to be preferentially labeled in the posterior hypothalamus, cochlear nucleus and medial habenula, due to their recent uptake from postsynaptic target sites.
  • TTC labeling was also shown in brainstem motor and reticular nuclei. TTC is found in a subpopulation of sensory neurons because TTC is taken up in an activity- dependent manner. TTC labeling is preferential in the larger-diameter non-pain encoding neurons. This is consistent with a lack of nociceptor activity in the animal subsequent to TTC injection.
  • Example 2 Labeling neurons in an activity-dependent manner to allow for future cell-specific manipulations including capture with LCM to identify unigue gene expression properties.
  • the procedure of Jankowska can be followed to selectively label, in an activity-dependent manner, Renshaw cells.
  • Renshaw cells are a population of intemeurons that mediate recurrent inhibition of ⁇ -motoneurons.
  • Several suction recording electrodes were attached to lumbar ventral roots of the isolated spinal cord maintained in artificial cerebrospinal fluid for electrical stimulation and recording.
  • TTC-GFP TTC-GFP fusion protein.
  • a TTC-lacZ or TTC-YFP/CFP/GFP fusion protein is systemically administered, and after 24-48 hours the spinal cord isolated and the ventral roots electrically stimulated for 24 hours in vitro. The tissue can then be processed to see if neurons are labeled in the location of known Renshaw cells.
  • Systemic delivery of Fluorogold is taken up and retrogradely transported by all neurons with access to fenestrated capillaries. However, Fluorogold not transported trans-synaptically. Therefore dual injections of FG as well as TTC fusion protein ensure that labeled Renshaw cells will not be mistaken for small ⁇ -motoneurons that are in an adjacent region in the motor nuclei.
  • the terminal experiment is undertaken 24-48 hours after systemic injection of TTC-reporter fusion protein.
  • the spinal cord was isolated and maintained in an oxygenated artificial cerebrospinal fluid (ACSF).
  • ACSF oxygenated artificial cerebrospinal fluid
  • the cord retained dorsal and ventral roots stimulation and recording activity.
  • Three suction electrodes were attached to lumbar ventral roots L3, L4, and L5 for electrical stimulation. These roots represent the vast majority of motor innervation of the hindlimb.
  • TTC-GFP fusion protein enters the CNS after delivery to the circulatory system via intraperitoneal injection and labels a subset of neurons.
  • a day 12 C57/BL6 mouse was injected with 300 ul (1.5mg/ml) TTC-EGFP and 50 ⁇ l 1%) Fluorogold intraperitoneally. Twenty four hours later 300 ⁇ l more of TTC- EGFP was injected. Twenty four hours later the 4.5 gram animal was perfused intraventricularly with 1.5 ml ice cold heparinized saline followed by 4.5 ml 4% paraformaldehyde, 0.1 M Phosphate buffered to pH 7.4. Tissue was isolated and postfixed in same fixative for 1 hour.
  • TTC-GFP spinal motoneurons, presumed interneurons, sympathetic preganglionics and dorsal root ganglia neurons were labeled. Only a subset of the motoneurons (Fluorogold positive) was TTC-GFP labeled. In addition, presumed TTC-GFP positive interneurons were labeled following trans-synaptic transport. Aundant TTC-GFP was found throughout the spinal cord, possibly in astrocytes or microglia. A low-power micrograph of TTC labeling in transverse section of spinal cord showed that the motor region of spinal cord was strongly labeled. Higher power magnification showed labeling of sympathetic preganglionic neurons and TTC labeling of peripheral sensory neurons in dorsal root ganglia.
  • TTC-GFP fusion protein ( ⁇ 80,000 molecular weight) entered the CNS after delivery to the circulatory system via intraperitoneal injection.
  • Fluorogold ( ⁇ 400 molecular weight) is known to be taken up by all motoneurons non-selectively by fluid-phase endocytosis from nerve terminals in the periphery.
  • TTC-GFP ( ⁇ 80,000 molecular weight) is taken up in an activity-dependent manner by selective receptor-mediated endocytosis. Only a subset of the motoneurons (Fluorogold ) were TTC-GFP labeled. In addition, presumed interneurons were labeled following trans-synaptic transport. There was also abundant TTC-GFP found throughout the spinal cord, possibly in astrocytes or microglia. In addition, autonomic sympathetic preganglionic and dorsal root ganglia sensory neurons were labeled.
  • Example 4 LCM and gene chip characterization to identify novel factors and changes in expression in disease Recurrent inhibitory interneurons (Renshaw cells) can be isolated using laser capture microdissection (LCM). Lumbar segments L2-L6 can be isolated from the rest of the cord, blocked and fresh-frozen onto cryostat chucks in appropriate orientation on dry ice. Tissue can be cryostat sectioned at 8 urn and thaw-mounted onto Teflon-sprayed microscope slides. All slides can be stored at -70 C prior to LCM. Prior to laser capture, slides are fixed in acetone at 4°C for 5 min, followed by dehydration in 70%, 95% and 100 % ethanol for 30 seconds each.
  • LCM laser capture microdissection
  • RNA can be reverse transcribed using a T7-oligodT primer.
  • Second strand synthesis can be performed using replacement synthesis, and the resulting cDNA can be linearly amplified by T7 RNA polymerase to create amplified RNA (aRNA).
  • aRNA amplified RNA
  • Biotin-Iabeled dCTP and dUTP can be included in the second in vitro transcription to create fluorescent probes that are fragmented and hybridized to an Affymetrix murine gene chip (U74 Av2; undertaken for a service fee at the microarray facility at Georgia Medical College), which contains approximately 6000 functionally known sequences. Analysis is performed to determine the presence (and relative abundance) or absence of the expression of a particular gene in the population of cells captured. Gene chip data obtained between sample populations can be compared to determine the gene expression differences. Both biological replicates (each neuronal population is obtained from 6 different animals) and also some technical replicates (either multiple chips for a given aRNA sample or another sample of the population from the same animal) are performed. Available software packages can be used to normalize data to correct for systematic biases in measured expression levels (e.g. introduced by differences in probe amounts, labeling efficiency, hybridization efficiency, and scanning) and to determine presence/absence of gene expression.
  • Affymetrix murine gene chip U74 Av2; undertaken
  • Expression profiling of identified CNS neurons was achieved by combining the technologies of fluorescent reporters, laser capture microdissection (LCM) and DNA microarrays.
  • LCM laser capture microdissection
  • Neurons were isolated from fresh frozen sections, 400 neurons per cell type in each animal, and extracted RNA was amplified, divided in half, and processed separately to produce 2 targets for 2-4 Affymetrix oligonucleotide arrays.
  • Fluorescently labeled cells were extracted using LCM and RNA was isolated from those cells. Low abundance transcripts were readily detected in our assay (e.g.
  • GABAA dopamine D2, 5HT2B, 1 D, 5A, and 4 receptors. Comparison of the expression of a given gene in each cell type before and after transection reinforces the idea that each cell type possesses a unique fingerprint and response to SCI (see Table 1).
  • somatostatin receptor 2 expression is highest in preganglionic sympathetics but after spinal cord injury it is down-regulated to levels observed in motoneurons.
  • Pair-wise comparisons of cell types in experimental animals show that the genes that change in MMN vs. LMN are non-overlapping, while only 3 and 7 genes overlap when comparing MMN and LMN to IML, respectively.
  • Unique transcription factors or transcription factor combinations have been identified for the specification of many neuron types in this region. The engrailed transcription factor is relatively specific for Renshaw cells.
  • the final step is to create a fusion protein of TTC with an important factor expressed in the target population, the Renshaw cell, identified by expression profiling that measurably modifies the properties of these cells.
  • the Renshaw cell identified by expression profiling that measurably modifies the properties of these cells.
  • that function of this population is selectively affected.
  • Both TTC-GFP and a TTC fusion protein hypothesized to modify cell function are co-injected.
  • the same experimental procedure for activity-dependent labeling, laser-capture, RNA amplification and microarray hybridization are employed. Control and therapeutic molecule treated populations are compared to determine whether expression changes are consistent with that expected form the application of the identified factor.
  • TTC- streptavidin TTC-SAV
  • TTC-SAV TTC- streptavidin
  • TTC tetanus toxin
  • TTC is atoxic. It has a potent and highly-selective affinity for neuronal membranes. It gains access to the CNS via activity-dependent intemalization and fast axonal transport from peripheral terminal fields. It traffics to somatodendritic regions and is exocytosed. It binds trans- synaptically and is internalized in an activity dependent manner in other central neurons whose circuitry is entirely within the CNS. This entire retrograde process is repeated through more circuits, reaching all CNS neurons.
  • TTC thelial growth factor
  • TTC-GFP distribution following systemic injection including: administration route, temporal profile, tissue distribution, immunogenicity, and binding site are detailed.
  • binding site several studies implicate an essential binding to the complex ganglioside GT1 b for trafficking.
  • Example 6 Dynamics of TTC-GFP distribution following systemic injection Administration and dosage Intravenous doses ranging from 0.1-0.6 mg total protein of TTC in adult mice have been found to produce qualitatively similar motoneuronal labeling. This equates to -3-17 mg/kg. These findings have been extended to include i.p. and subcutaneous doses in juvenile and adult mouse and neonatal rat. We have injected 15 mg/kg. Subcutaneous injection results in much greater central labeling.
  • i.p. injection the peritoneal cavity provides a large absorptive area for TTC to reach the circulation quickly.
  • entry is primarily by way of the portal vein so that first-pass hepatic losses can be considerable.
  • this is the least desirable injection method to use clinically due to risk of infection and adhesions.
  • intramuscular TTC injection restricts CNS entry to motoneurons innervating the muscle, subcutaneous injections are the most clinically relevant delivery method.
  • Adult mice are injected i.p. and subcutaneously with TTC-GFP at the following doses: 0.01 , 0.1 , 1 , 10 and 100 mg/kg. Four mice are used for each dose. Because i.p. injection results in consistent absorptive entry into the circulation, this administration route allows the most reliable inter-animal comparison.
  • i.p. injection is used for the experiments below to detect protein distribution in CNS and tissues immunohistochemically.
  • Tissue distribution We remove muscle (gastrocnemius), heart, kidney, liver, intestine, adrenal gland, spleen, pancreas, lung, bladder, large and small intestine, stomach, gall bladder, testes and skin. Following fixation tissue is cryostat sectioned and alternately processed on different slides for immunodetection of TTC and with hematoxylin and eosin for gross inspection of cell histology. Regarding TTC, particular attention is given to labeling in neural structures within these organs. All peripheral neurons are shown to be TTC + . The enteric nervous system contains over 100 million neurons and so is considered an important therapeutic target given the neural origin of many gastrointestinal diseases. Neurons and their processes are identified with neuron specific markers(e.g.
  • Optimal dose Extent of 'contaminating' binding in peripheral tissues, blood sample measures of electrolytes, evidence of CNS inflammation, and differences in CNS distribution are determined.
  • animals are anesthetized and prior to transcardial perfusion, blood is collected from the vena cava and serum stored at -20°C for chemistry measures (serum electrolytes and renal function indicators) by an autoanalyzer at Emory University Hospital.
  • TTC- ⁇ -gal, TTC-SAV, TTC-GFP, TTC-HRP and TTC-HRP-lgG fusion proteins reach central neurons following intramuscular or i.p. injection.
  • the largest of these protein conjugates, TTC-HRP-lgG has a combined molecular weight of ⁇ 275 kDa.
  • TTC-SAV we biotinylate large proteins to determine the approximate size constraint for systemic TTC-based delivery of molecules to the CNS. We biotinylate several of the proteins used as molecular weight standards.
  • EZ-Link® Sulfo-NHS-Biotinylation Kit from [Pierce] and the following molecular weight standards: thyroglobulin (670,000) myosin (200,000) y globulin (158,000) ⁇ - Galactosidase (116,000) Phosphorylase B (97,400) serum albumin (66,200) ovalbumin (45,000) and carbonic anhydrase (29,000).
  • Initial conjugations are used for the largest and smallest of these two (thyroglobulin and carbonic anhydrase) to quickly determine the need to re-adjust our protein weight standards to ranges outside the aforementioned molecular weights. Two animals are injected for each multi-protein complex and 24 hours later animals are examined for motoneuronal labeling.
  • Example 8 CNS entry, distribution and time dependence We examined time dependence and CNS distribution by injecting a dose of
  • TTC in all motoneuron populations, autonomic preganglionics, dorsal root ganglia, circumventricular nuclei, neurons in hypothalamus, brainstem reticular nuclei, hippocampus, cochlear nucleus and medial habenula.
  • TTC was injected subcutaneously and Fluorogold was injected i.p. 48 hours prior to sacrifice. The entire gray matter was labeled with TTC. Fluorogold was found in the soma consistent with cellular transport into lysosomes while TTC, though present in the soma, was largely found in the neurophil. Neural somata of the rostral hippocampi were labeled by Fluorogold as were all blood vessels coursing through the spinal cord.
  • TTC-GFP labeling was found in brainstem motor and reticular nuclei, cochlear nucleus and medial habenula. Hippocampus but not neocortex was labeled with TTC-GFP fusion protein. TTC-GFP labeling demonstrated that TTC-GFP was not found associated with blood vessels but rather in neurophil associated with neurons that label for Fluorogold. TTC-GFP labeling occurred in rostral hypothalamus and caudal hypothalamus.
  • TTC-SAV TTC-SAV
  • TTC-SAV-[molecular weight standards] Two time periods were explored - 24 and 48 hours.
  • CNS tissue was examined for immunolabeled GFP and SAV.
  • the distribution was compared to that observed when TTC alone was injected to show fusion proteins transported similarly to TTC In vivo with identical distribution.
  • a rat injected was i.p. with TTC-EGFP fusion protein 12 hours prior to sacrifice.
  • Immunostaining for TTC and GFP showed that the fusion protein co- localized both within neurons in the sensory ganglia and in spinal motoneurons. Labeled axons projected to labeled somata.
  • the motor region also illustrated similarity in staining pattern.
  • Example 9 Immunization Most people in the U.S. have been vaccinated for tetanus and so have neutralizing antibodies against the toxin that is also known to bind to TTC. Transport was shown to hypoglossal motoneurons after i.m. injection of TTC to hypoglossal nuclei of control and tetanus-immunized animals. No differences in TTC uptake into motoneurons in mice were found even though the titres of antibody (antitoxin) in mouse sera were very strong (averaging -20,000. The binding and intemalization of TTC into neurons is so rapid that most TTC is 'hidden' before an immune response can be mounted.
  • Example 10 Relating TTC binding site to the GT1 B ganglioside
  • TTC was injected i.p. and labeling was examined 24 and 48 hours later in 3 mice each.
  • GT1 b antibodies have been used very successfully to label neurons in vitro.
  • Cowley K.C., CINA O, Schmidt B.J., and Hochman S. (2002) The isolated rat spinal cord as an in vitro model to study the pharmacological control of spinal myoclonic- like activity.
  • Advances in Neurology ed. Fahn S. and Frucht S.
  • Lippincott Williams & Wilkins Lippincott Williams & Wilkins.
  • Glial cell line-derived neurotrophic factor a drug candidate for the treatment of Parkinson's disease. J. Neurol. 245, 35-42.
  • Lipid rafts act as specialized domains for tetanus toxin binding and intemalization into neurons. Mol. Biol. Cell 12, 2947-2960.
  • Tetanus toxin direct evidence for retrograde intraaxonal transport. Science 188, 945-947.
  • Tyrosine-1290 of tetanus neurotoxin plays a key role in its binding to gangliosides and functional binding to neurones.

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Abstract

La présente invention a trait à des procédés de traitement de troubles nerveux comprenant l'administration dans le système circulatoire d'un patient nécessitant un tel traitement, d'une molécule de fusion neuroactive comportant un groupe fonctionnel thérapeutique et un groupe fonctionnel apte au transport trans-synaptique du groupe fonctionnel thérapeutique vers un neurone sélectionné à travers la barrière hémato-encéphalique ou la barrière hémato-nerveuse. Des protéines thérapeutiques de grande dimension peuvent être transportées par les procédés de l'invention. Le groupe fonctionnel thérapeutique peut être ciblée vers des neurones dans le système nerveux central ainsi que dans le système nerveux périphérique, par exemple, vers des nerfs impliqués dans la sensation tels que des nerfs des systèmes de la douleur, le système nerveux entérique, le système nerveux autonome, et autres. Des dispositifs classiques tels que des comprimés, des moyens d'injection intraveineuse, des suppositoires, et d'autres moyens connus de l'art pour l'administration dans le système circulatoire peuvent être utilisés.
PCT/US2005/009714 2004-03-24 2005-03-24 Administration systemique d'agents therapeutiques au systeme nerveux central Ceased WO2005094497A2 (fr)

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US60/556,109 2004-03-24
US63909104P 2004-12-23 2004-12-23
US60/639,091 2004-12-23

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006070290A3 (fr) * 2004-06-23 2006-11-02 Ian A Ferguson Agents et procedes pour le diagnostic et le suivi precoces de la maladie d'alzheimer et d'autres troubles neurologiques
WO2008005889A3 (fr) * 2006-06-30 2008-12-31 Universtiy Of Texas M D Anders Procédés et agents d'imagerie basés sur le fragment c de la toxine tétanique, traitements d'ensembles de données pour la microscopie confocale
EP1985311A4 (fr) * 2006-01-24 2011-04-13 Univ Kagoshima Agent pour cibler un médicament sur un neurone cérébral
CN114163535A (zh) * 2021-11-09 2022-03-11 湖北科技学院 一种以ttc作为神经元细胞靶向基因的融合蛋白的诱导表达
WO2024026306A1 (fr) * 2022-07-25 2024-02-01 Wisconsin Alumni Research Foundation Conjugués ciblant la barrière hémato-nerveuse et procédés d'utilisation

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1341050C (fr) * 1988-11-04 2000-07-11 Martin E. Schwab Facteurs de regulations de la croissance des axones
US20030083299A1 (en) * 2000-11-04 2003-05-01 Ferguson Ian A. Non-invasive delivery of polypeptides through the blood-brain barrier

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006070290A3 (fr) * 2004-06-23 2006-11-02 Ian A Ferguson Agents et procedes pour le diagnostic et le suivi precoces de la maladie d'alzheimer et d'autres troubles neurologiques
GB2431348A (en) * 2004-06-23 2007-04-25 Ian A Ferguson Agents and methods for early diagnosis and monitoring of alzheimer's disease and other neurological disorders
GB2431348B (en) * 2004-06-23 2009-12-30 Ian A Ferguson Agents and methods for early diagnosis and monitoring of alzheimer's disease and other neurological disorders
AU2005321021B2 (en) * 2004-06-23 2011-11-24 Ian A. Ferguson Agents and methods for early diagnosis and monitoring of Alzheimer's disease and other neurological disorders
EP1985311A4 (fr) * 2006-01-24 2011-04-13 Univ Kagoshima Agent pour cibler un médicament sur un neurone cérébral
WO2008005889A3 (fr) * 2006-06-30 2008-12-31 Universtiy Of Texas M D Anders Procédés et agents d'imagerie basés sur le fragment c de la toxine tétanique, traitements d'ensembles de données pour la microscopie confocale
CN114163535A (zh) * 2021-11-09 2022-03-11 湖北科技学院 一种以ttc作为神经元细胞靶向基因的融合蛋白的诱导表达
WO2024026306A1 (fr) * 2022-07-25 2024-02-01 Wisconsin Alumni Research Foundation Conjugués ciblant la barrière hémato-nerveuse et procédés d'utilisation

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