WO1996034619A1 - Methods of preventing neuron degeneration and promoting neuron regeneration - Google Patents

Methods of preventing neuron degeneration and promoting neuron regeneration Download PDF

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Publication number
WO1996034619A1
WO1996034619A1 PCT/US1996/005894 US9605894W WO9634619A1 WO 1996034619 A1 WO1996034619 A1 WO 1996034619A1 US 9605894 W US9605894 W US 9605894W WO 9634619 A1 WO9634619 A1 WO 9634619A1
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Prior art keywords
cart
neurons
disease
cells
cultures
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PCT/US1996/005894
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French (fr)
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Jean-Claude Marcel Louis
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Amgen Inc.
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Priority to AU56308/96A priority Critical patent/AU5630896A/en
Publication of WO1996034619A1 publication Critical patent/WO1996034619A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to uses of a novel protein called CART to decrease or prevent neuron degeneration and to promote neuron regeneration and restoration of function.
  • the invention concerns methods of treating various neurological conditions by administering CART.
  • a number of neurological disorders and diseases are caused at least in part by degeneration or death of particular classes of neurons.
  • Parkinson's disease is characterized by slowing of voluntary muscle movement, muscular rigidity, and tremor. Such symptoms are attributed at least in part to progressive degeneration of dopamine-producing neurons located in a specific region of the brain called the substantia nigra. Degeneration of these neurons (“dopaminergic neurons”) results in a decrease of dopamine levels in an adjacent region of the brain called the striatum.
  • the striatum contains neurons expressing receptors for dopamine; these neurons are involved in the control of motor activity.
  • ALS amyotrophic lateral sclerosis
  • progressive muscular atrophy progressive muscular atrophy
  • hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease) all result at least in part from a decay of motor neurons which are located in the ventral horn of the spinal cord.
  • the hippocampus a well defined structure that is part of the cerebral cortex of the brain, is important in the formation of long term memory. Destruction of the hippocampus, for example by ischemia, can result in an inability to form new memories. Degeneration of pyramidal CAl neurons, which are located in the CAl region of the hippocampus, is one characteristic of Alzheimer's disease. These same neurons are selectively vulnerable to ischemic and anoxic damage which occur in conditions such as stroke and head trauma. In addition, the CAl pyramidal hippocampal neurons as well as pyramidal neurons located in the CA3 region of the hippocampus, are selectively injured in epilepsy.
  • the striatum is the innervation region of the nerve terminals of dopaminergic-containing neurons from the substantia nigra.
  • the majority of striatal neurons utilize GABA (4-aminobutyric acid) as their neurotransmitter.
  • GABA 4-aminobutyric acid
  • the striatum is the major target of the progressive neurodegeneration that occurs in Huntington's disease, in which the major neuron loss is that of the striatal GABA-utiiizing neurons.
  • the serotonin-containing neurons are located in groups clustered around the midline of the hindbrain. These neurons are involved in the control of body temperature, mood, and sleep. Disorders of the serotonin-containing neuron system include, for example. depression, other mood disorders, and sleep disturbances.
  • Photoreceptor cells are a specialized subset of retina neurons, and are responsible for vision. Damage and/or death of photoreceptor cells can lead to blindness. Degeneration of the retina, such as by retinitis pigmentosa, age-related macular degeneration, and stationary night blindness, are all characterized by the progressive atrophy and loss of function of photoreceptor outer segments which are specialized structures containing the visual pigments that transform a light stimulus into electrical activity.
  • Neurotrophic factors are endogenous, soluble proteins that can regulate survival, growth, and/or morphological plasticity of neurons (see Fallon and Laughlin, Neurotrophic Factors, Academic Press, San Diego, CA [1993]) . In view of their ability to promote neuron regeneration and to prevent neuron death and degeneration, it has been postulated that neurotrophic factors might be useful in treating neurodegenerative conditions of the nervous system.
  • neurotrophic factors belong to several different protein superfamilies of polypeptide growth factors based on their amino acid sequence homology and/or their three-dimensional structure (MacDonald and Hendrikson, Cell, 73:421-424 [1993]).
  • One family of neurotrophic factors is the neurotrophin family. This family currently consists of NGF (nerve growth factor) , BDNF (brain derived neurotrophic factor), NT-3 (neurotrophin-3) , NT-4 (neurotrophin-4) , and NT-6 (neurotrophin-6) .
  • CNTF ciliary neurotrophic factor
  • LIF leukemia inhibitory factor
  • cytokine polypeptides that have neurotrophic activity. By virtue of their structural features and receptor components, these polypeptides are related to a family of hematopoietic cytokines that includes IL-6 (interleukin-6) , IL-11 (interleukin-11) , G-CSF (granulocyte-colony stimulating factor) , and oncostatin-M.
  • GDNF glial derived neurotrophic factor
  • TGF-beta transforming growth factor beta
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • TGF-alpha transforming growth factor-alpha
  • TGF-beta TGF-beta
  • IGF insulin like growth factor
  • CART cocaine and amphetamine regulated transcript
  • the gene appears to be alternatively spliced, resulting in the presence or absence of an in-frame 39 base insert within the putative coding region of rat CART mRNA.
  • predicted translation products are either 116 (CART-1) or 129 (CART-2) amino acids in length.
  • the human CART cDNA has been isolated from a human hypothalamic cDNA library and the human genomic sequence has been isolated as well (Genbank accession number for human cDNA: U16826; for human genomic sequence: U20325) . Based on the predicted amino acid sequence of both human and rat CART protein, CART appears to have a prototypic signal peptide, suggesting that the protein is secreted from the cells in which it is synthesized.
  • CART is not significantly homologous with any known neurotrophic factor at either the nucleotide sequence or amino acid sequence level.
  • the discoveries that comprise the present invention demonstrate that CART has unexpectedly been found to have neurotrophic activity for certain types of neurons. Prior to these discoveries, there was no suggestion or indication that CART might have such neurotrophic activity. Such findings suggest that CART may be the first identified member of a novel family of neurotrophic factors.
  • the present invention provides a method for promoting the survival, differentiation, or regeneration of neurons, comprising contacting the neurons with an effective amount of CART or a fragment thereof.
  • the neurons may be selected from the group consisting of: nigral dopaminergic neurons, hippocampal neurons, motor neurons, retinal neurons, and photoreceptor cells.
  • the method is conducted in vitro.
  • the method for promoting survival of neurons is conducted in vivo, optionally to treat a neurological disease or disorder affecting neurons selected from the group consisting of: nigral dopaminergic neurons, hippocampal neurons, motor neurons, retinal neurons, and photoreceptor cells.
  • the neurological disorder is selected from the group consisting of: substance addiction, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, ischemia, brain or spinal cord trauma, amyotrophic lateral sclerosis, Charcot- Marie-Tooth disease, dementia, retinitis pigmentosa, macular degeneration, retinal detachment, and retinal vascular disease.
  • a method for treating a neurological disorder or disease comprising administering a therapeutically effective amount of CART is provided.
  • the neurological disorder or disease is selected from the group consisting of: substance addiction, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, ischemia, brain or spinal cord trauma, amyotrophic lateral sclerosis, Charcot- Marie-Tooth disease, dementia, retinitis pigmentosa, macular degeneration, and retinal vascular disease.
  • the disease or disorder may comprise damage to the nervous system caused by trauma, infarction, surgery, infection, or malignancy, exposure to a toxin, or a nutritional deficiency.
  • the invention provides a composition comprising a therapeutically effective amount of CART in admixture with a pharmaceutically acceptable carrier.
  • Figure 1 depicts the cDNA sequence of rat CART (SEQ ID NO:l) .
  • Figure 2 depicts the predicted amino acid sequence of the rat CART protein based on the nucleotide sequence (SEQ ID NO:2) .
  • Figure 3 depicts the cDNA sequence of human CART (SEQ ID NO:3) .
  • Figure 4 depicts the full length nucleotide sequence of the human CART gene (SEQ ID NO:4) .
  • Figure 5 depicts the predicted amino acid sequence of the human CART protein based on the nucleotide sequence (SEQ ID NO:5) .
  • Figure 6 depicts the promotion of dopamine neuron survival by cart in cultures of embryonic day 15 (A) and post-natal day 6 (B) rat substantia nigra.
  • Cultures grown in DMEM/F12 with B27 medium supplement and 15% horse serum on polyornithine - and laminin-modified 96-well microplates were treated with rat recombinant CART-1 or CART-2 at 1 ng/ml (filled bars) , 10 ng/ml (open bars) and 100 ng/ml (hatched bars) and immunostained for tyrosine hydroxylase, a marker that identifies dopaminergic neurons in these cultures, after 6 days in vitro .
  • Figure 7 depicts the effect of CART on dopamine neurons in cultures of embryonic day 15 rat substantia nigra.
  • Cells were plated in DMEM/F12 with B27 medium supplement and 15% horse serum on polyornithine and laminin-modified culture surfaces at a density of about 25,000/6-mm well.
  • Dopamine neurons identified by tyrosine hydroxylase immunostaining are shown after 6 days in vitro in the presence of the following additions: (A) none; (B) rat recombinant CART-1, 100 pg/ml; (C) rat recombinant CART-2, 100 pg/ml.
  • Calibration bar 100 ⁇ m.
  • Figure 8 depicts the effect of CART on the morphological development of dopamine neurons in cultures of post-natal day 6 rat substantia nigra.
  • Cells were plated in DMEM/F12 with B27 medium supplement and 15% horse serum on polyornithine and laminin-modified culture surfaces at a density of about 40,000/6-mm well.
  • Dopamine neurons identified by tyrosine hydroxylase immunostaining are shown after 8 days in vitro in the presence of the following additions: (A,B) none; (C) rat recombinant CART- 1, 100 pg/ml.
  • Calibration bar 50 ⁇
  • Figure 9 depicts the stimulation of dopamine uptake by CART in cultures of embryonic day 15 rat substantia nigra.
  • Cultures grown in DMEM/F12 with B27 medium supplement and 15 percent horse serum on polyornithine and laminin- modified 96-well microplates were treated with rat recombinant CART-1 or CART-2.
  • cultures were processed for [3H]-dopamine uptake (50 nM; 1.5 million dpm/ml; 1 hour incubation at 37°C) .
  • the results are expressed as the percentages of the dopamine uptake values (in dpm/well) found in control cultures. Each data point is the mean of 2 wells from a representative experiment. Similar results were obtained in four independent experiments.
  • Figure 10 depicts the promotion of motor neuron survival by CART in cultures of embryonic day 15 rat spinal cord.
  • Cultures of metrizamide gradient-purified motor neurons (1000/6-mm well) treated with rat recombinant CART- 1 (ten-fold serial dilutions ranging from 10 ng/ml to 1 pg/ml) were fixed after 3 days and immunostained for neuronal-specific enolase.
  • Motor neuron survival was determined by counting the number of neuronal-specific enolase-positive neurons per 2.4 square mm fields. Each data point is the mean of 3 wells from a representative experiment. The standard deviation is indicated.
  • Figure 11 depicts the effect of CART on motor neurons in cultures of embryonic day 15 rat spinal cord.
  • Metrizamide gradient-purified motor neurons were plated in DMEM/F12 with B27 medium supplement and 15% horse serum on polyornithine and laminin-modified culture surfaces at a density of about 1000 neurons/6-mm well.
  • Motor neurons were identified by phase-contrast microscopy and are shown after 3 days in vitro in the presence of the following treatments: (A) none; (B) rat recombinant CART-1, 100 pg/ml; (C) rat recombinant CART-2, 100 pg/ml.
  • Calibration bar 50 ⁇ m.
  • Figure 12 depicts the promotion of hippocampal neuron survival by CART in cultures of post-natal day 4 rat hippocampus.
  • Cells were plated in DMEM/F12 with B27 medium supplement and 2.5% horse serum on polyornithine and laminin-modified 96-well microplates at a density of about 500/6-mm well.
  • A Changes in hippocampal neuron number over time in response to 100 pg/ml rat recombinant CART-1.
  • B Dose-dependence of CART-1 survival-promoting effect. The total number of neurons per well was determined by counting MAP-l/MAP-2 immunostained cells. Each value is the mean ⁇ s.d. of 3-4 cultures.
  • Figure 13 depicts the stimulation of neuritic development by CART in cultures of post-natal day 4 rat hippocampus. Cultures of hippocampal neurons were incubated for 12 days with or without 100 pg/ml rat recombinant CART-1 and then immunostained for MAP/l-MAP-2. All the neurons present in a 6-mm well were photographed and analyzed for neurite lengths and branching.
  • Mean neurite lengths were as follows: control (no CART) about 465 ⁇ m; CART-1 about 945 ⁇ m. The number of branching points per neuron was 1.9 (s.d. 2.1) for control; 6.1 (s.d. 5.2) for CART-1.
  • Figure 14 depicts the effect of CART on the morphological development of neurons in cultures of post ⁇ natal day 4 rat hippocampus.
  • Cells were plated in DMEM/F12 with B27 medium supplement and 2.5% horse serum on polyornithine and laminin-modified 96-well microplates at a density of about 500 cells/6-mm well.
  • Hippocampal neurons identified by immunostaining for MAP-1 and MAP-2 are shown after 12 days in vitro in the presence of the following additions: (A,B) none; (C,D) rat recombinant CART-1, 100 pg/ml and rat recombinant CART-2, 100 pg/ml.
  • Calibration bar 50 ⁇ m.
  • Figure 15 depicts the effect of CART on photoreceptor cells in cultures of post-natal day 5 mouse retina.
  • Cells were plated in DMEM/F12 with B27 medium supplement and 2.5% horse serum on polyornithine and laminin-modified 96-well microplates at a density of about 13,000 cells/6-mm well.
  • Photoreceptor cells identified by immunostaining for arrestin are shown after 6 days in vitro in the presence of the following additions: (A) none; (B) rat recombinant CART-1 , 100 pg/ml; (C) rat recombinant CART-2, 100 pg/ml.
  • Calibration bar 50 ⁇ m.
  • Figure 16 depicts the promotion of photoreceptor cell survival by CART in cultures of post-natal day 5 mouse retina. Cultures treated with rat recombinant CART-1 and CART-2 were fixed after 6 days and immunostained for arrestin. The number of arrestin-positive photoreceptor cells was determined in 2.4 sq.mm fields. The results are expressed as the percentages of photoreceptor cells found in untreated control cultures. Each value is the mean ⁇ s.d. of 3 cultures.
  • Figure 17 depicts the photoreceptor outer segment development and axonal growth in response to CART in cultures of embryonic day 17 chick retina.
  • Cells were plated in DMEM/F12 with B27 medium supplement and 2.5% horse serum on polyornithine and laminin-modified 96-well microplates at a density of about 10,000 cells/6-mm well.
  • Photoreceptor cells identified by phase-contrast morphology are shown after 7 days in vitro in the presence of (A) no treatment or (B-G) rat recombinant CART-1, 100 pg/ml.
  • CART-1 promotes the development of inner segments (IS; solid arrows) , outer segments (OS; open arrows) and of the axonal process (P) .
  • the lipid droplet (LD; arrowheads) marks the junction between inner and outer segments.
  • Calibration bar 25 ⁇ .
  • Figure 18 depicts the lack of effect of CART on the number of neurons and GABA uptake in cultures of embryonic day 15 striatum.
  • Cultures grown in DMEM/F12 with B27 medium supplement and 15 percent horse serum on polyornithine and laminin-modified 96-well microplates were treated with rat recombinant CART-1 or CART-2. After 6 days in vitro, the cultures were evaluated for immunostaining with MAP-1 or MAP-2, or for GABA uptake. The number of neurons (relative to control, untreated neurons) in striatal cultures treated with CART-1 or CART-2 is shown in (A) .
  • Each data point is the mean of 4 individual wells in which the number of MAP-positive cells was counted in 2.4 square mm fields. The standard deviation of each data point is indicated.
  • the effect of CART on GABA uptake relative to control (untreated neurons) is shown in (B) .
  • Each data point is the mean of duplicate cultures.
  • Figure 19 depicts the effect of CART on serotonin-containing neurons in cultures of embryonic day 15 rat hindbrain. Cultures were grown in DMEM/F12 with B27 medium supplement and 15 percent horse serum on polyornithine-and laminin-modified 96-well microplates, and were treated with rat recombinant CART-1 or CART-2 at the concentrations indicated.
  • This invention is based on the unexpected and surprising discovery that a CART polypeptide has been found to possess neurotrophic activity on certain types of neurons.
  • CART when used to describe a cDNA, gene or other nucleic acid sequence refers to such cDNA, gene or nucleic acid sequence (a) having any of the Genbank accession numbers U10071, U16826, or U20325; (b) having the nucleotide sequence as set forth in SEQ ID NO: 1, 3, or 4; (c) having a nucleic acid sequence which hybridizes under stringent conditions to the nucleic acid sequences of SEQ ID NO: 1, 3, or 4; (d) having a naturally occurring allelic variation of (a) or (b) ; and/or (e) nucleic acid variants of (a) -(d) produced as provided for herein.
  • stringent conditions refers to hybridization and washing under conditions that permit only binding of the probe to highly homologous sequences.
  • One stringent wash solution is 0.015 M NaCl, 0.005 M NaCitrate, and 0.1 percent SDS used at a temperature of 55°C-65°C.
  • Another stringent wash solution is 0.2 X SSC and 0.1 percent SDS used at a temperature of between 50°C-65°C.
  • oligonucleotide probes are used to screen cDNA or genomic libraries, the following stringent washing conditions may be used.
  • One protocol uses 6 X SSC with 0.05 percent sodium pyrophosphate at a temperature of 35°C-62°C, depending on the length of the oligonucleotide probe.
  • a second protocol utilizes tetramethylammonium chloride (TMAC) for washing oligonucleotide probes.
  • TMAC tetramethylammonium chloride
  • One stringent washing solution is 3 M TMAC, 50 mM Tris-HCl, pH 8.0, and 0.2 percent SDS. The washing temperature using this solution is a function of the length of the probe. For example, a 17 base pair probe is washed at about 45-50°C.
  • CART protein or “CART polypeptide” as used herein refers to any protein or polypeptide having the properties described herein for CART.
  • CART protein or CART polypeptide includes, an amino acid sequence encoded by any of items (a)-(e) above and peptide or polypeptide fragments derived therefrom, to the amino acid sequence set forth in SEQ ID NO:2 or 5, and/or to chemically modified derivatives as well as nucleic acid and or amino acid sequence variants thereof as provided for herein.
  • the phrase "differentiation of neurons” refers to the production axonal and/or dendritic processes ("neurites”) , and/or other morphological structures or features of neurons either in vitro or in vivo.
  • CART fragment refers to a peptide or polypeptide that is less than the full length amino acid sequence of naturally occurring CART protein but has substantially the same biological activity as CART polypeptide or CART protein described above. Such a fragment may be truncated at the amino terminus, the carboxy terminus, and/or internally, and may be chemically modified.
  • CART derivative or
  • CART variant refers to a CART polypeptide or CART protein that has 1) been chemically modified, as for example, by addition of polyethylene glycol or other compound, and/or 2) contains one or more nucleic acid or amino acid sequence substitutions, deletions, and/or insertions.
  • biologically active polypeptide and “biologically active fragment” refer to a peptide or polypeptide that 1) has CART activity (i.e., acts to support survival and/or differentiation of dopaminergic, hippocampal, motor, photoreceptor, and/or other neurons) , and/or 2) cross reacts with monoclonal or polyclonal antibodies raised against naturally occurring CART protein.
  • the terms “effective amount” and “therapeutically effective amount” refer to the amount of CART necessary to support the survival and/or differentiation of neurons as compared to neurons not treated with CART.
  • the CART polypeptides that are useful in practicing the present invention may be naturally occurring full length polypeptides, or truncated polypeptides or peptides (i.e, "fragments").
  • the polypeptides or fragments may be chemically modified, i . e . , glycosylated, phosphorylated, and/or linked to a polymer, as described below.
  • the polypeptides or fragments may be variants of the naturally occurring CART polypeptide (i.e., may contain one or more amino acid deletions, insertions, and/or substitutions as compared with naturally occurring CART) .
  • the full length CART polypeptide or fragment can be prepared using well known recombinant DNA technology methods such as those set forth in Sambrook et al . (Molecular Cloning: A Laboratoy Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989]) and/or Ausubel et a ⁇ . , eds, (Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY [1994]) .
  • a gene or cDNA encoding the CART protein or truncated version thereof may be obtained for example by screening a genomic or cDNA library, or by PCR amplification.
  • a gene encoding the CART polypeptide or fragment may be prepared by chemical synthesis using methods well known to the skilled artisan such as those described by Engels et al . (Angew. Chem. Intl. Ed., 28:716-734 [1989]). These methods include, inter alia, the phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis. A preferred method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry.
  • the DNA encoding the CART polypeptide will be several hundred nucleotides in length. Nucleic acids larger than about 100 nucleotides can be synthesized as several fragments using these methods.
  • nucleic acid and/or amino acid variants of naturally occurring CART may be produced using site directed mutagenesis or PCR amplification where the primer(s) have the desired point mutations (see Sambrook et al . , supra, and Ausubel et al . , supra, for descriptions of mutagenesis techniques) . Chemical synthesis using methods described by Engels et al . , supra, may also be used to prepare such variants.
  • Preferred nucleic acid variants are those containing nucleotide substitutions accounting for codon preference in the host cell that is to be used to produce CART.
  • Other preferred variants are those encoding conservative amino acid changes (e.g., wherein the charge or polarity of the naturally occurring amino acid side chain is not altered substantially by substitution with a different amino acid) as compared to wild type, and/or those designed to either generate a novel glycosylation and/or phosphorylation site(s) on CART, or those designed to delete an existing glycosylation and/or phosphorylation site(s) on CART.
  • the CART gene or cDNA can be inserted into an appropriate expression vector for expression in a host cell.
  • the vector is selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the CART gene and/or expression of the gene can occur) .
  • the CART polypeptide or fragment thereof may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells. Selection of the host cell will depend at least in part on whether the CART polypeptide or fragment thereof is to be glycosylated.
  • yeast, insect, or mammalian host cells are preferable; yeast cells will glycosylate the polypeptide, and insect and mammalian cells can glycosylate and/or phosphorylate the polypeptide as it naturally occurs on the CART polypeptide (i . e . , "native" glycosylation and/or phosphorylation) .
  • the vectors used in any of the host cells will contain 5' flanking sequence (also referred to as a "promoter") and other regulatory elements as well such as an enhancer (s), an origin of replication element, a transcriptional termination element, a complete intron sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • promoter an enhancer
  • an origin of replication element also referred to as a "promoter”
  • s an origin of replication element
  • a transcriptional termination element a complete intron sequence containing a donor and acceptor splice site
  • a signal peptide sequence a ribosome binding site element
  • polyadenylation sequence a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed
  • This tag will be expressed along with the protein, and can serve as an affinity tag for purification of the CART polypeptide from the host cell.
  • the tag can subsequently be removed from the purified CART polypeptide by various means such as using a selected peptidase for example.
  • the 5' flanking sequence may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of 5' flanking sequences from more than one source), synthetic, or it may be the native CART 5' flanking sequence.
  • the source of the 5' flanking sequence may be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the 5' flanking sequence is functional in, and can be activated by, the host cell machinery.
  • the 5' flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, 5' flanking sequences useful herein other than the CART 5' flanking sequence will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of the 5' flanking sequence may be known. Here, the 5' flanking sequence may be synthesized using the methods described above for nucleic acid synthesis or cloning.
  • the 5' flanking sequence may be obtained using PCR and/or by screening a genomic library with suitable oligonucleotide and/or 5' flanking sequence fragments from the same or another species.
  • a fragment of DNA containing a 5' flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion using one or more carefully selected enzymes to isolate the proper DNA fragment.
  • the desired fragment may be isolated by agarose gel purification, Qiagen® column or other methods known to the skilled artisan. Selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
  • the origin of replication element is typically a part of prokaryotic expression vectors purchased commercially, and aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for optimal expression of the CART polypeptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector.
  • the transcription termination element is typically located 3' to the end of the CART polypeptide coding sequence and serves to terminate transcription of the CART polypeptide. Usually, the transcription termination element in prokaryotic cells is a G-C rich fragment followed by a poly T sequence.
  • a selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium.
  • Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media.
  • Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.
  • the ribosome binding element commonly called the Shine-Dalgarno sequence (prokaryotes) or the Kozak sequence (eukaryotes) , is necessary for translation initiation of mRNA.
  • the element is typically located 3' to the promoter and 5' to the coding sequence of the CART polypeptide to be synthesized.
  • the Shine-Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content) .
  • Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth above and used in a prokaryotic vector.
  • a signal sequence may be used to direct the CART polypeptide out of the host cell where it is synthesized.
  • the signal sequence is positioned in the coding region of CART nucleic acid sequence, or directly at the 5' end of the CART coding region.
  • Many signal sequences have been identified, and any of them that are functional in the selected host cell may be used in conjunction with the CART gene. Therefore, the signal sequence may be homologous or heterologous to the CART polypeptide, and may be homologous or heterologous to the CART polypeptide. Additionally, the signal sequence may be chemically synthesized using methods set forth above.
  • transcription of the CART polypeptide is increased by the presence of one or more introns on the vector; this is particularly true for eukaryotic host cells, especially mammalian host cells.
  • the intron may be naturally occurring within the CART nucleic acid sequence, especially where the CART sequence used is a full length genomic sequence or a fragment thereof. Where the intron is not naturally occurring within the CART DNA sequence (as for most cDNAs) , the intro (s) may be obtained from another source.
  • the position of the intron with respect to the 5' flanking sequence and the CART coding sequence is important, as the intron must be transcribed to be effective. As such, where the CART nucleic acid sequence is a cDNA sequence, the preferred position for the intron is 3' to the transcription start site, and 5' to the polyA transcription termination sequence.
  • the intron will be located on one side or the other (i.e., 5' or 3') of the CART coding sequence such that it does not interrupt the this coding sequence.
  • Any intron from any source including any viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this invention, provided that it is compatible with the host cell (s) into which it is inserted.
  • synthetic introns are also included herein.
  • more than one intron may be used in the vector.
  • the elements set forth above are not already present in the vector to be used, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the elements are well known to the skilled artisan and are comparable to the methods set forth above (i.e., synthesis of the DNA, library screening, and the like) .
  • the final vectors used to practice this invention are typically constructed from a starting vectors such as a commercially available vector. Such vectors may or may not contain some of the elements to be included in the completed vector. If none of the desired elements are present in the starting vector, each element may be individually ligated into the vector by cutting the vector with the appropriate restriction endonuclease(s) such that the ends of the element to be ligated in and the ends of the vector are compatible for ligation. In some cases, it may be necessary to "blunt" the ends to be ligated together in order to obtain a satisfactory ligation. Blunting is accomplished by first filling in "sticky ends" using Klenow DNA polymerase or T4 DNA polymerase in the presence of all four nucleotides.
  • two or more of the elements to be inserted into the vector may first be ligated together (if they are to be positioned adjacent to each other) and then ligated into the vector.
  • vectors for practicing this invention are those which are compatible with bacterial, insect, and mammalian host cells.
  • Such vectors include, inter alia , pCRII (Invitrogen Company, San Diego, CA) , pBSII (Stratagene Company, LaJolla, CA) , and pETL (BlueBacII; Invitrogen) .
  • the completed vector may be inserted into a suitable host cell for amplification and/or CART polypeptide expression.
  • Host cells may be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast cell, an insect cell, or a vertebrate cell) .
  • the host cell when cultured under appropriate conditions, can synthesize CART protein which can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted) . After collection, the CART protein can be purified using methods such as molecular sieve chromatography, affinity chromatography, and the like.
  • the host cell will depend in part on whether the CART protein is to be glycosylated or phosphorylated (in which case eukaryotic host cells are preferred) , and the manner in which the host cell is able to "fold" the protein into its native tertiary structure (e.g., proper orientation of disulfide bridges, etc.) such that biologically active protein is prepared by the cell.
  • the CART may be "folded” after synthesis using appropriate chemical conditions as discussed below.
  • Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO) or 3T3 cells.
  • CHO Chinese hamster ovary cells
  • suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art.
  • Other suitable mammalian cell lines are the monkey COS-1 and COS-7 cell lines, and the CV-1 cell line.
  • Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable.
  • Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene.
  • suitable mammalian cell lines include but are not limited to, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.
  • host cells suitable for the present invention are bacterial cells.
  • E. coli e.g., HB101, DH5 ⁇ ,DH10, and MC1061
  • Various strains of B. subtilis are well-known as host cells in the field of biotechnology.
  • Pseudomonas spp . other Bacillus spp. , Streptomyces spp. , and the like may also be employed in this method.
  • yeast cells Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention (Miller et al . , Genetic Engineering 8: 277-298 [1986]) . Insertion (also referred to as
  • transformation or “transfection” of the vector into the selected host cell may be accomplished using such methods as calcium chloride, electroporation, microinjection, lipofection or the DEAE-dextran method.
  • the method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et al . , supra .
  • the host cells containing the vector i.e., transformed or transfected
  • the media will usually contain all nutrients necessary for the growth and survival of the cells. Suitable media for culturing E.
  • coli cells are for example, Luria Broth (LB) and/or Terrific Broth (TB) .
  • Suitable media for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which may be supplemented with serum and/or growth factors as required by the particular cell line being cultured.
  • a suitable medium for insect cultures is Grace's medium supplemented with yeastolate, lactalbumin hydrolysate, and/or fetal calf serum as necessary.
  • an antibiotic or other compound useful for selective growth of the transformed cells is added as a supplement to the media.
  • the compound to be used will be dictated by the selectable marker element present on the plasmid with which the host cell was transformed.
  • the selectable marker element is kanamycin resistance
  • the compound added to the culture medium will be kanamycin.
  • the amount of CART polypeptide produced in the host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, HPLC separation, immunoprecipitation, and/or activity assays such as DNA binding gel shift assays.
  • the CART polypeptide has been designed to be secreted from the host cells, the majority of polypeptide will likely be found in the cell culture medium. If however, the CART polypeptide is not secreted from the host cells, it will be present in the cytoplasm (for eukaryotic, gram positive bacteria, and insect host cells) or in the periplasm (for gram negative bacteria host cells) .
  • the host cells are typically first disrupted mechanically or osmotically to release the cytoplasmic contents into a buffered solution. CART polypeptide is then isolated from this solution.
  • CART polypeptide from solution can be accomplished using a variety of techniques. If the polypeptide has been synthesized such that it contains a tag such as Hexahistidine (CART/hexaHis) or other small peptide at either its carboxyl or amino terminus, it may essentially be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag or for the polypeptide directly (i . e . , a monoclonal antibody specifically recognizing CART) . For example, polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the Qiagen nickel columns) can be used for purification of CART/polyHis.
  • a tag such as Hexahistidine (CART/hexaHis) or other small peptide at either its carboxyl or amino terminus
  • affinity column where the column matrix has a high affinity for the tag or for the polypeptide directly (i . e
  • the contents of the periplasm or cytoplasm can be extracted from the host cell using any standard technique known to the skilled artisan.
  • the host cells can be lysed to release the contents of the periplasm by French press, homogenization, and/or sonication. The homogenate can then be centrifuged.
  • the inclusion bodies can often bind to the inner and/or outer cellular membranes and thus will be found primarily in the pellet material after centrifugation.
  • the pellet material can then be treated with a chaotropic agent such as guanidine or urea to release, break apart, and solubilize the inclusion bodies.
  • the CART polypeptide in its now soluble form can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If it is desired to isolate the CART polypeptide, isolation may be accomplished using standard methods such as those set forth below and in Marston et al . (Meth . Enz. , 182:264-275 [1990]) .
  • CART polypeptide inclusion bodies are not formed to a significant degree in the periplasm of the host cell, the CART polypeptide will be found primarily in the supernatant after centrifugation of the cell homogenate, and the CART polypeptide can be isolated from the supernatant using methods such as those set forth below.
  • purification can be accomplished using standard methods well known to the skilled artisan. Such methods include, without limitation, separation by electrophoresis followed by electroelution, various types of chromatography (immunoaffinity, molecular sieve, and/or ion exchange) , and/or high pressure liquid chromatography. In some cases, it may be preferable to use more than one of these methods for complete purification.
  • the CART polypeptides, fragments, and/or derivatives thereof may be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using methods known in the art such as those set forth by Merrifield et al . , (J. Am. Chem . Soc , 85:2149 [1964]), Houghten et al . (Proc Natl Acad. Sci . USA, 82:5132 [1985]), and Stewart and Young (Solid Phase Peptide Synthesis, Pierce Chem Co, Rockford, IL [1984]).
  • CART polypeptides or fragments may be oxidized using methods set forth in these references to form disulfide bridges.
  • the CART polypeptides or fragments may be employed as biologically active or immunological substitutes for natural, purified CART polypeptides in therapeutic and immunological processes.
  • CART-polymers Chemically modified CART compositions (i.e., "derivatives") where the CART polypeptide is linked to a polymer ("CART-polymers") are included within the scope of the present invention.
  • the polymer selected is typically water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer selected is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled as provided for in the present methods.
  • a preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see U.S. Patent 5,252,714).
  • the polymer may be branched or unbranched. Included within the scope of CART-polymers is a mixture of polymers. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable.
  • the water soluble polymer or mixture thereof may be selected from the group consisting of, for example, polyethylene glycol (PEG) , monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly- (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol.
  • PEG polyethylene glycol
  • monomethoxy-polyethylene glycol polyethylene glycol
  • dextran dextran
  • cellulose or other carbohydrate based polymers
  • poly- (N-vinyl pyrrolidone) polyethylene glycol propylene glycol homopolymers
  • a polypropylene oxide/ethylene oxide co-polymer polyoxyethylated polyols (e.g., glycerol)
  • Pegylation of CART may be carried out by any of the pegylation reactions known in the art, as described for example in the following references: Focus on Growth Factors 3 (2): 4-10 (1992); EP 0 154 316; and EP 0 401 384.
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer) as described below.
  • Pegylation by acylation generally involves reacting an active ester derivative of polyethylene glycol (PEG) with an CART protein. Any known or subsequently discovered reactive PEG molecule may be used to carry out the pegylation of CART.
  • a preferred activated PEG ester is PEG esterified to N-hydroxysuccinimide ("NHS") .
  • NHS N-hydroxysuccinimide
  • acylation is contemplated to include without limitation the following types of linkages between CART and a water soluble polymer such as PEG: amide, carbamate, urethane, and the like, as described in Bioconjugate Chem. 5: 133-140 (1994) .
  • Reaction conditions may be selected from any of those known in the pegylation art or those subsequently developed, provided that conditions such as temperature, solvent, and pH that would inactivate the CART species to be modified are avoided.
  • Pegylation by acylation usually results i n a poly-pegylated CART product, wherein the lysine ⁇ -amino groups are pegylated via an acyl linking group.
  • the connecting linkage will be an amide.
  • the resulting product will be at least about 95 percent mono, di- or tri- pegylated.
  • some species with higher degrees of pegylation up to the maximum number of lysine ⁇ -amino acid groups of CART plus one ⁇ -amino group at the amino terminus of CART will normally be formed in amounts depending on the specific reaction conditions used.
  • more purified pegylated species may be separated from the mixture, particularly unreacted species, by standard purification techniques, including, among others, dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel filtration chromatography and electrophoresis.
  • Pegylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with a protein such as CART in the presence of a reducing agent. Regardless of the degree of pegylation, the PEG groups are preferably attached to the protein via a -CH2-NH- group. With particular reference to the -CH2- group, this type of linkage is referred to herein as an "alkyl" linkage.
  • Derivatization via reductive alkylation to produce a monopegylated product exploits the differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in CART.
  • the reaction is performed at a pH (see below) which allows one to take advantage of the pK a differences between the ⁇ -amino groups of the lysine residues and that of the ⁇ -amino group of the N-terminal residue of the protein.
  • a pH see below
  • attachment of a water soluble polymer that contains a reactive group such as an aldehyde, to a protein is controlled: the conjugation with the polymer occurs predominantly at the N-terminus of the protein without significant modification of other reactive groups such as the lysine side chain amino groups.
  • the present invention provides for a substantially homogeneous preparation of CART- monopolymer protein conjugate molecules (meaning CART protein to which a polymer molecule has been attached substantially only (i.e., at least about 95%) in a single location on the CART protein. More specifically, if polyethylene glycol is used, the present invention also provides for pegylated CART protein lacking possibly antigenic linking groups, and having the polyethylene glycol molecule directly coupled to the CART protein.
  • a particularly preferred water-soluble polymer for use herein is polyethylene glycol, abbreviated PEG.
  • polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono- (C1-C10) alkoxy- or aryloxy-polyethylene glycol.
  • Methods for preparing pegylated CART will generally comprise the steps of (a) reacting an CART polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby CART becomes attached to one or more PEG groups, and (b) obtaining the reaction product (s) .
  • polyethylene glycol such as a reactive ester or aldehyde derivative of PEG
  • the optimal reaction conditions for the acylation reactions will be determined based on known parameters and the desired result. For example, the larger the ratio of PEG: protein, the greater the percentage of poly-pegylated product.
  • Reductive alkylation to produce a substantially homogeneous population of mono- polymer/CART protein conjugate molecule will generally comprise the steps of: (a) reacting a CART protein with a reactive PEG molecule under reductive alkylation conditions, at a pH suitable to permit selective modification of the ⁇ -amino group at the amino terminus of said CART protein; and (b) obtaining the reaction product (s) .
  • the reductive alkylation reaction conditions are those which permit the selective attachment of the water soluble polymer moiety to the N-terminus of CART.
  • Such reaction conditions generally provide for pKa differences between the lysine amino groups and the ⁇ -amino group at the N-terminus (the pK a being the pH at which 50% of the amino groups are protonated and 50% are not) .
  • the pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a larger excess of polymer to protein will be desired (i.e., the less reactive the N-terminal ⁇ -amino group, the more polymer needed to achieve optimal conditions) .
  • the pH will generally fall within the range of 3-9, preferably 3-6.
  • the molecular weight of the polymer is the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. Similarly, branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymer:protein ratio. In general, for the pegylation reactions contemplated herein, the preferred average molecular weight is about 2kDa to about lOOkDa (the term "about” indicating ⁇ lkDa) . The preferred average molecular weight is about 5kDa to about 50kDa, particularly preferably about 12kDa to about 25kDa.
  • the ratio of water-soluble polymer to CART protein will generally range from 1:1 to 100:1, preferably (for polypegylation) 1:1 to 20:1 and (for monopegylation) 1:1 to 5:1.
  • reductive alkylation will provide for selective attachment of the polymer to any CART protein having an ⁇ -amino group at the amino terminus, and provide for a substantially homogenous preparation of monopolymer/CART protein conjugate.
  • monopolymer/CART protein conjugate is used here to mean a composition comprised of a single polymer molecule attached to an CART protein molecule.
  • the monopolymer/CART protein conjugate preferably will have a polymer molecule located at the N-terminus, but not on lysine amino side groups.
  • the preparation will preferably be greater than 90% monopolymer/CART protein conjugate, and more preferably greater than 95% monopolymer CART protein conjugate, with the remainder of observable molecules being unreacted (i.e., protein lacking the polymer moiety) .
  • the examples below provide for a preparation which is at least about 90% monopolymer/ protein conjugate, and about 10% unreacted protein.
  • the monopolymer/protein conjugate has biological activity.
  • the reducing agent should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation.
  • Preferred reducing agents may be selected from the group consisting of sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane and pyridine borane.
  • a particularly preferred reducing agent is sodium cyanoborohydride.
  • reaction parameters such as solvent, reaction times, temperatures, etc., and means of purification of products, can be determined based on the published information relating to derivatization of proteins with water soluble polymers.
  • a mixture of polymer-CART protein conjugate molecules may be prepared by acylation and/or alkylation methods, as described above,and one may select the proportion of monopolymer/ protein conjugate to include in the mixture.
  • a mixture of various protein with various numbers of polymer molecules attached i.e., di-, tri-, tetra-, etc.
  • conditions which may be alleviated or modulated by administration of the present polymer/CART include those described herein for cart molecules in general.
  • the polymer/CART molecules disclosed herein may have additional activities, enhanced or reduced activities, or other characteristics, as compared to the non-derivatized molecules.
  • CART molecules that are not themselves active in the activity assays presented herein may be useful as modulators (e.g., inhibitors or stimulants) of the CART receptors in vitro or in vivo .
  • the CART polypeptides and fragments thereof, whether or not chemically modified, may be employed alone, or in combination with other pharmaceutical compositions such as, for example, neurotrophic factors, cytokines, interferons, interleukins, growth factors, antibiotics, anti-inflammatories, neurotransmitter receptor agonists or antagonists and/or antibodies, in the treatment of neurological conditions.
  • pharmaceutical compositions such as, for example, neurotrophic factors, cytokines, interferons, interleukins, growth factors, antibiotics, anti-inflammatories, neurotransmitter receptor agonists or antagonists and/or antibodies, in the treatment of neurological conditions.
  • the CART polypeptides and fragments thereof may be used to prepare antibodies generated by standard methods.
  • antibodies that react with the CART polypeptides, as well as reactive fragments of such antibodies are also contemplated as within the scope of the present invention.
  • the antibodies may be polyclonal, monoclonal, recombinant, chimeric, single- chain and/or bispecific, etc.
  • the antibody fragments may be any fragment that is reactive with the CART of the present invention, such as, F a b, Fab', etc.
  • hybridomas generated by presenting CART or a fragment thereof as an antigen to a selected mammal followed by fusing cells (e.g., spleen cells) of the animal with certain cancer cells to create immortalized cell lines by known techniques.
  • cells e.g., spleen cells
  • the methods employed to generate such cell lines and antibodies directed against all or portions of a human CART polypeptide of the present invention are also encompassed by this invention.
  • the antibodies may be used therapeutically, such as to inhibit binding of the CART to its receptor.
  • the antibodies may further be used for in vivo and in vitro diagnostic purposes, such as in labeled form to detect the presence of the CART in a body fluid.
  • compositions for treating various neurological disorders are within the scope of the present invention.
  • Such compositions may comprise a therapeutically effective amount of a CART polypeptide or fragment thereof (either of which may be chemically modified) in admixture with a pharmaceutically acceptable carrier.
  • the carrier material may be water for injection, preferably supplemented with other materials common in solutions for administration to mammals.
  • a CART therapeutic will be administered in the form of a composition comprising purified protein (which may be chemically modified) in conjunction with one or more physiologically acceptable carriers, excipients, or diluents.
  • Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers.
  • the product is formulated as a lyophilizate using appropriate excipients (e.g., sucrose).
  • excipients e.g., sucrose
  • Other standard carriers, diluents, and excipients may be included as desired.
  • Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0- 5.5, which may further include sorbitol or a suitable substitute therefor.
  • the CART compositions can be systemically administered parenterally. Alternatively, the compositions may be administered intravenously or subcutaneously. When systemically administered, the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • the preparation of such pharmaceutically acceptable protein solutions, with due regard to pH, isotonicity, stability and the like, is within the skill of the art.
  • Therapeutic formulations of CART compositions useful for practicing the present invention may be prepared for storage by mixing the selected composition having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington 's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, ed., Mack Publishing Company [1990]) in the form of a lyophilized cake or an aqueous solution.
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG) .
  • buffers such as phosphate, citrate, or other organic acids
  • antioxidants such as ascorbic acid
  • the CART composition to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Where the CART composition is lyophilized, sterilization using these methods may be conducted either prior to, or following, lyophilization and reconstitution.
  • the composition for parenteral administration ordinarily will be stored in lyophilized form or in solution.
  • Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • a sterile access port for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the route of administration of the composition is in accord with known methods, e . g. oral, injection or infusion by intravenous, intraperitoneal, intracerebral (intraparenchymal) , intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes, or by sustained release systems or implantation device which may optionally involve the use of a catheter.
  • the compositions may be administered continuously by infusion, bolus injection or by implantation device.
  • CART may be administered locally via implantation into the affected area of a membrane on to which CART polypeptide has been absorbed.
  • the device may be implanted into a cerebral ventricle or into brain parenchyma, and delivery of CART may be directly through the device via bolus or continuous administration, or via a catheter using continuous infusion.
  • CART polypeptide may be administered in a sustained release formulation or preparation.
  • sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e . g. films, or microcapsules.
  • Sustained release matrices include polyesters, hydrogels, polylactides (U.S.
  • Sustained- release compositions also may include liposomes, which can be prepared by any of several methods known in the art (e.g., DE 3,218,121; Epstein et al . , Proc . Natl . Acad. Sci . USA, 82: 3688-3692 [1985]; Hwang et al . , Proc . Natl . Acad. Sci . USA, 77: 4030-4034 [1980]; EP 52,322; EP 36,676; EP 88,046; EP 143,949).
  • liposomes can be prepared by any of several methods known in the art (e.g., DE 3,218,121; Epstein et al . , Proc . Natl . Acad. Sci . USA, 82: 3688-3692 [1985]; Hwang et al . , Proc . Natl . Acad. Sci . USA, 77: 4030-4034 [1980];
  • CART compositions in an ex vivo manner, i.e., to treat isolated neuronal tissues that may be subsequently implanted into a patient.
  • isolated dopaminergic neurons taken from fetal (or other tissue) may be treated with CART (either alone or in combination with other agents) prior to implanting of these neurons into the brain tissue of Parkinson's patients.
  • CART may be delivered through implanting into patients cells that have been genetically engineered (using methods described above) to express and secrete CART polypeptide.
  • Such cells may be human cells, and may be derived from the patient's own tissue or from another source, either human or non- human.
  • the cells may be immortalized.
  • the cells may be implanted into the brain or into other tissues of the nervous system, or into tissues that are innervated by the nervous system.
  • genomic DNA, cDNA, and/or synthetic DNA encoding cart or a fragment or variant thereof may be operably linked to a constitutive or inducible promoter that is active in the tissue into which the composition will be injected.
  • This CART DNA construct can be injected directly into brain or other neuronal tissue to be treated.
  • the CART DNA construct may be injected into muscle tissue where it can be taken up into the cells and expressed in the cells, provided that the CART DNA is operably linked to a promoter that is active in muscle tissue such as cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, or muscle creatine kinase promoter.
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • the DNA construct may include (in addition to the CART DNA and a promoter) , vector sequence obtained from vectors such as adenovirus vector, adeno-associated virus vector, a retroviral vector, and/or a herpes virus vector.
  • the vector/DNA construct may be admixed with a pharmaceutically acceptable carrier(s) for injection.
  • An effective amount of the CART composition (s) to be employed therapeutically will depend, for example, upon the therapeutic objectives such as the indication for which CART is being used, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.
  • a typical daily dosage may range from about 0.1 ⁇ g/kg to up to 100 mg/kg or more, depending on the factors mentioned above.
  • a clinician will administer the CART composition until a dosage is reached that achieves the desired effect.
  • the CART composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of CART) over time, or as a continuous infusion via implantation device or catheter.
  • the dosage will be between 0.01 ⁇ g/kg body weight (calculating the mass of the protein alone, without chemical modification) and 300 ⁇ g/kg (based on the same) .
  • the CART proteins, fragments and/or derivatives thereof may be utilized to treat diseases and disorders of the nervous system which may be associated with alterations in the pattern of CART expression or which may benefit from exposure to CART or anti-CART antibodies.
  • CART protein, and/or fragments or derivatives thereof may be used to treat patients in whom various cells of the nervous system have degenerated and/or have been damaged by trauma, surgery, stroke, ischemia, infection, metabolic disease, nutritional deficiency, malignancy, and/or toxic agents.
  • the CART protein or fragments thereof can be used to treat conditions in which damage caused by various events such as trauma, surgery, infarction, infection, malignancy, exposure to a toxin, or a nutritional deficiency, has occurred in hippocampal neurons, dopaminergic neurons, sensory neurons such as photoreceptor cells, and/or motor neurons.
  • CART protein and/or fragments or derivatives thereof can be used to treat congenital conditions or neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Parkinson-Plus syndromes (in which Parkinsonian symptoms result from degeneration of dopaminergic neurons) ; amyotrophic lateral sclerosis; epilepsy; emotional disorders and dementias; substance abuse or addiction (alcoholism, cocaine, heroin, amphetamine, or the like) ; ischemia resulting from stroke, head trauma, and/or chronic cerebral blood flow disorders; spinal cord diseases such as spinal muscular atrophy; amyotrophic lateral sclerosis (ALS) ; Charcot- Marie-Tooth syndrome; and/or dystrophies or degeneration of the neural retina such as retinitis pigmentosa (autosomal dominant, autosomal recessive, sex-linked) , fundus flaimaculatus (autosomal recessive) , senile macular degeneration, drug-induced retinopathies,
  • CART protein or peptide fragments or derivatives derived thereof can be used in conjunction with surgical implantation of tissue in the treatment of Alzheimer's disease and/or Parkinson's disease, or in other neurological diseases in which tissue implantation is indicated.
  • CART may be used to promote the survival and regeneration of dopaminergic or other neurons in a dose-dependent manner.
  • DMEM High glucose Dulbecco's Modified Eagle's Medium
  • F12 Ham's F12 medium
  • Leibovitz's L15 medium without sodium bicarbonate #41300-039
  • B27 medium supplement #17504-010
  • TRK527 about 94 Ci/mmol
  • Amersham Arlington Heights, IL
  • Optiphase Supermix scintillation cocktail from Wallac, Turku, Finland
  • white ViewPlate-96 microplates (#6005182) from Packard Instruments Corporation, Meriden, CT. All other reagents were obtained from Sigma Chemical Company, unless otherwise specified.
  • the basal medium was prepared as a 1:1 mixture of DMEM and F12 medium, and was supplemented with B27 medium supplement (GIBCO) added as a 50-fold concentrated stock solution.
  • B27 medium supplement (GIBCO) added as a 50-fold concentrated stock solution.
  • L-glutamine was added at a final concentration of about 2 mM, penicillin at about 100 IU/1, and streptomycin at about 100 mg/1.
  • Various additions were then made to this basal medium for the different types of neurons cultured. These additions wee as follows. For fetal rat substantia nigra, striatum, hindbrain, and spinal cord cultures, heat-inactivated horse serum was added to a final concentration of about 15 percent.
  • Purified rat recombinant CART-1 and CART-2 were prepared as 10 ⁇ g/ml solutions in D-PBS (phosphate buffered saline prepared with distilled water) containing 5 percent bovine serum albumin. The solutions were stored at 4°C in aliquots. Serial dilutions were prepared in 96-well microplates. Ten microliters of ten-fold concentrated CART solutions were added to cell cultures containing 90 ⁇ l of culture medium. Control cultures received 10 ⁇ l of D-PBS with 5 percent albumin. The treatments were initiated one hour after cells were seeded and, in some instances, repeated after 3 days.
  • D-PBS phosphate buffered saline prepared with distilled water
  • bovine serum albumin 5 percent bovine serum albumin
  • microtiter plate surfaces (the culture substratum) were modified by sequential coating with poly-L- ornithine followed by laminin as follows.
  • the plate surfaces were completely covered with a 0.1 mg/ml sterile solution of polyornithine in 0.1 M boric acid (pH 8.4) for at least 1 hour at room temperature, followed by a sterile wash with Super-Q water.
  • the water wash was then aspirated and a 1 ⁇ g/ml solution of mouse laminin (GIBCO) in PBS was added and incubated at 37°C for 2 hours.
  • Embryonic Rat Brain Cells were used as the source of substantia nigra, striatum, and hindbrain serotoninergic neurons. Timed-pregnant Sprague-Dawley rats at embryonic day 15 were used. A maximum of 36 embryos (about 3 litters) were processed per experiment. The pregnant rats were killed by exposure to CO 2 , their abdominal cavities opened with dissecting scissors and the fetuses were removed from the uteri.
  • Fetal brains were then dissected, cleaned of blood and meninges, and the desired regions (substantia nigra, striatum, and hindbrain) were dissected using well-defined anatomical landmarks (Altman and Bayer, Atlas of Prenatal Rat Brain Development, CRC Press, Boca Raton, FL [1995]).
  • the cells were collected in ice-cold D-PBS, and then transferred into 10 ml dissociation medium (120 units papain and 2000 units DNase in HBSS) .
  • the tissues were incubated for 45 minutes at about 37°C on a rotary platform shaker at about 200 rpm.
  • the cells were then dispersed by trituration through fire-polished Pasteur pipettes, sieved through a 20 ⁇ m Nitex mesh to discard undissociated tissue, and centrifuged for 5 minutes at 200 x g using an IEC clinical centrifuge.
  • the resulting cell pellet was resuspended into HBSS (Gibco/BRL, Grand Island, NY) containing ovalbumin and about 500 units DNAase, layered on top of a 4 percent ovalbumin solution (in HBSS) and centrifuged for about 10 minutes at 500 x g.
  • HBSS Gibco/BRL, Grand Island, NY
  • the final pellet was resuspended in complete culture medium (see above), adjusted to about 28,000 cells/ml, and seeded in 90 ⁇ l aliquots into the 6 mm-wells of 96-well microplates (Falcon) previously coated with polyornithine and laminin. Attachment of cells occurred rapidly, and the plating efficiency was about 75 percent.
  • Neonatal brains were used to establish cultures of hippocampal and substantia nigra neurons.
  • Sprague-Dawley post-natal 4-6 day rat pups were used to obtain neonatal brain cells. Each pup was held by its muzzle and sprayed with 70 percent ethanol from a spray bottle and then quickly decapitated. The cartilaginous skull was then exposed and incised starting at the foramen magnum. Each side of the skull was pulled away laterally with forceps to expose the brain surface. The whole brain was lifted from the base of the skull and placed in a small Petri dish with enough D-PBS to cover the brain. After the brains were collected, they were trimmed of residual meningeal tissue.
  • the regions containing the hippocampus and the substantia nigra were dissected with hypodermic needles from parasagittal brain slices, following published anatomical landmarks (Paxinos, T ⁇ rk, Tecott and Valentino, Atlas of the Developing Rat Brain, Academic Press, San Diego, CA [1991]). Dissociation procedures were the same as described for fetal brain cells (see above) . The final cell suspension was then preplated into 100-mm diameter plastic Petri dishes (about 1 million cells per dish) in DMEM/F12 containing 20 percent horse serum.
  • the unattached cells (representing about 75 percent of the initial cell population) were collected, pelleted by centrifugation, resuspended in complete culture medium and plated into 96- well microplates precoated with polyornithine and laminin.
  • the seeding densities were about 250 cells per well for hippocampal cultures and about 30,000 cells per well for substantia nigra.
  • the complete culture medium consisted of DMEM/F12 supplemented with B27, HEPES, insulin, transferrin, D-glucose and 2.5 percent horse serum (see above) .
  • the tissues were collected in ice-cold D-PBS and then transferred to dissociation medium (120 units papain and 2000 units DNAase in HBSS) .
  • Dissociation procedures were the same as for fetal brain cells (see above) , and the culture medium consisted of DMEM/F12 supplemented with B27, HEPES, insulin, transferrin, D-glucose and 2.5 percent horse serum (see above) .
  • Purification of Spinal Cord Motor Neurons Spinal cords were removed from 15-day old Sprague-Dawley rat embryos and freed of ganglia and adhering meninges. Only the lumbar segments of the cords were used.
  • Partially purified spinal cord motor neuron cultures (approximately 75 percent motor neurons) were prepared using methods described by Camu and Henderson (J. Neurosci . Meth . , 44:59-70 [1992]) with slight modifications as follows.
  • the spinal cord tissues were cut into small pieces (about 1 cubic mm) , subdivided into ventral and mediodorsal segments, and then incubated in 0.05 percent trypsin in D-PBS for about 17 minutes at 37°C, after which time a 10 percent volume of horse serum was added.
  • the spinal cords were then placed in dissociation medium consisting of Leibovitz L15 medium
  • tissue was made into a single cell suspension by gentle trituration through a fire- polished siliconized Pasteur pipette. The resulting cell suspension was then passed through a 20 ⁇ m nylon mesh and then subjected to a cell fractionation procedure using a metrizamide gradient.
  • the cell suspension (about 2 ml containing cells dissociated from 10-15 lumbar ventral cords) was applied to the top of 4 ml of 6.8 percent metrizamide previously placed in 15 ml sterile polystyrene tubes.
  • the tubes were centrifuged for about 20 minutes at 1650 rpm at room temperature in a bench-top Beckman centrifuge.
  • the large cells forming a sharp band at the interface of the metrizamide cushion (0-6.8 percent interface) were aspirated, washed in 4 volumes of dissociation medium, and layered on to 2 ml of 4% bovine serum albumin in L15 medium.
  • the cells were then centrifuged for about 10 minutes at 800 rpm and the resulting pellet was resuspended in about 1 ml of complete culture medium (DMEM/F12 + 15 percent horse serum; see above), adjusted to a concentration of about 17,000 cells per ml, and subsequently plated in 96-well microplates precoated with polyornithine and laminin at a density of about 1,500 cells/6-mm well in 90 ⁇ l of medium.
  • complete culture medium DMEM/F12 + 15 percent horse serum; see above
  • the cells were seeded into polyornithine and laminin coated 96 well microtiter plates at a density of about 12,500 cells/6-mm well (in 90 ⁇ l of complete culture medium consisting of DMEM/F12 supplemented with B27, HEPES, insulin, transferrin, D-glucose and 2.5 percent horse serum (see above). Under those conditions, retinal cultures consisted of more than about 80 percent (mouse) and about 60 percent (chick) photoreceptor cells.
  • the primary antibodies were then applied at a dilution of between 1:1000-1:4000 in the same buffer and incubated for 1 hour at 37°C on a rotary shaker. After three washes with D-PBS, the bound antibodies were detected using goat-anti-rabbit or horse-anti-mouse biotinylated IgG (Vectastain kit from Vector Laboratories, Burlingame, CA) at about a 1:500 dilution; these secondary antibodies were incubated with the cells for about 1 hour at 37°C.
  • the cells were then washed 3 times with D-PBS, and the secondary antibodies were detected with avidin-biotin-peroxidase complex diluted at 1:500, and the cells were incubated for about 45 minutes at 37°C. After three more washes with D- PBS, the cultures were reacted for 5-20 minutes in a solution of 0.1 M Tris-HCl, pH 7.4, containing 0.04 percent 3',3'- diaminobenzidine- (HC1) 4, 0.06 percent NiC12 and 0.02 percent hydrogen peroxide.
  • the specific markers used for particular cell types were as follows. Dopamine-producing neurons in the substantia nigra cultures were identified using a rabbit polyclonal antiserum to tyrosine hydroxylase (TH; Eugene Tech International) . Neurons in spinal cord, hippocampal, and striatal cultures were identified by staining with monoclonal antibodies to the markers MAP-1 and MAP-2 and neuronal- specific enolase. Photoreceptors cells in cultures of retina were identified by a polyclonal rabbit antibody directed to a synthetic peptide sequence of the rod-specific protein arrestin. The sequence is:
  • Neuronal cell cultures were fixed and processed for immunostaining as described above, and then examined with bright-field optics at 200X magnification. The number of stained neurons was counted in either the entire 6 mm-well (dopamine-containing neurons; serotonin-containing neurons; hippocampal neurons) or in one diametrical 0.4 X 6 mm strip, representing 10 percent of the total surface area of a 6 mm- well (motor neurons; photoreceptor cells; striatal neurons) . Viable neurons were characterized as having a regularly- shaped cell body, with a major axon-like process and several dendrite-like processes.
  • Neurons showing signs of degeneration such as having irregular, vacuolated perikarya or fragmented neurites, were excluded from the counts (most of the degenerating neurons, however, detached from the culture substratum) .
  • Cell numbers were expressed either as cells/6-mm well or as the fold-change relative to control cell density.
  • Morphometric analysis of neurite (i.e., both axons and dendrites) development was performed using 12-day-old cultures of rat hippocampus. Cultures containing about 100 neurons per 6-mm well were immunostained for MAP-1 and MAP-2 and examined with brightfield optics. Photographs of each individual neuron in a " 6-mm well of control and treated cultures were taken with an Optronics video-camera and enlarged to a final magnification of approximately 800-fold.
  • Neuritic size was determined by measuring the combined length of the neurites of each neuron with a stylus coupled to a SummaSketchll digitizing tablet (Summagraphics Corporation, Houston, TX) , utilizing a digitizing program (MacMeasure 1.9) and a Macintosh Centris 650 personal computer. Neuritic complexity was assessed by counting the number of primary neurites emerging from the soma and the number of branching points per neuron.
  • Dopamine uptake was determined in cultures of 15-day-old embryonic rat substantia nigra neurons that had been established in 96-well microplates. The cultures were washed with about 100 ⁇ l of pre-warmed uptake buffer which consists of a modified Krebs-Ringer solution, pH 7.4 containing about 120 mM NaCl, 4,7 mM KC1, 1.8 mM CaCl 2 , 1.2 mM MgS0 4 , 32 mM NaHP0 4 , 1.3 mM EDTA, and 5.6 mM D-glucose.
  • the uptake buffer also contained 1 mM ascorbic acid and 50 ⁇ M paragyline.
  • Tritiated dopamine 21 Ci/mmol
  • tritiated GABA 94 Ci/mmol
  • Non-specific dopamine uptake was determined by incubating the cultures with uptake buffer containing the dopamine uptake inhibitor GBR-12909 (1 ⁇ M) .
  • Non-specific uptake represented less than about 1 percent of total uptake.
  • the uptake assays were arrested by aspiration of the incubation medium followed by three rapid washes with about 120 ⁇ l of ice-cold uptake buffer.
  • the cells were then lysed by addition of 200 ⁇ l of Optiphase Supermix scintillation cocktail (Wallac) , and radioactivity was determined by scintillation spectrometry using a Wallac MicrobetaPlus 96- well microplate counter. The results are expressed either as dpm/6-mm well or as the fold-change relative to control cultures.
  • Cultures of embryonic day 15 (E15) and post-natal day 6 (P6) rat substantia nigra enriched in dopaminergic neurons were used to demonstrate the effect of CART on the survival of dopaminergic neurons.
  • the cultures were grown in polyornithine-laminin-coated 96-well microplates for up to 7 days alone or in the presence of 1, 10 or 100 ng/ml rat recombinant CART-1 or CART-2.
  • the culture medium consisted of DMEM/F12 supplemented with 15 percent heat-inactivated horse serum (E15 cultures) or 2.5 percent heat-inactivated horse serum, D-glucose, HEPES, insulin and transferrin (P6 cultures) .
  • TH tyrosine hydroxylase
  • both the E15 and P6 the cultures typically consisted of about 70 percent neurons as identified by neuronal specific enolase immunostaining (described above) and 30 percent non-neuronal cells (which had a flattened, phase-dark appearance) ; dopaminergic neurons represented about 5-15 percent of the neuron population.
  • Dopamine (DA) uptake measures the number and activity of high-affinity DA reuptake transporter sites and reflects the functional differentiation of dopaminergic neurons.
  • Enriched cultures of motor neurons about 75 percent pure were used to demonstrate the effect of CART on motor neuron survival.
  • the cultures were prepared from lumbar spinal cord tissue of 15-day-old rat embryos (the age at which motor neuron cell death occurs) , using the metrizamide density gradient cell separation method described above.
  • Purified motor neurons were removed from the 0-6.8 percent metrizamide interface and were seeded at a density of about 1,500 cells per 6-mm well (28 mm 2 of culture surface). More than 95 percent of the cells were phase bright and a similar percentage stained positively for neuronal-specific enolase (NSE) , a general neuronal cell marker. On average, approximately 900-1,100 of these cells (about 60-75 percent) were motor neurons as determined by the size of the cell bodies (at least about 35 ⁇ m in size) and by NSE staining.
  • NSE neuronal-specific enolase
  • metrizamide-purified spinal cord cultures have been previously reported to consist of 60- 80 percent of motor neurons, as identified by choline acetyltransferase (CAT) activity and immunostaining for CAT, low-affinity nerve growth factor receptor, L14 ( a motor neuron lectin) , and Islet-1 homeoprotein (Camu and Henderson, J. Neurosci . Meth . , 44:59-70 [1992]; Wong et al . , Eur. J. Neurosci . , 5:466-474 [1993]; Smith et al . , J. Neurosci . , 6:439-446 [1986]) .
  • CAT choline acetyltransferase
  • Hippocampal neurons were seeded in polyornithine-laminin- coated 96-well microplates at a density of about 250 cells per 6-mm well (28 mm 2 of culture surface) ; on average approximately 200 cells survived the culture procedure. Neuronal survival and development was assessed over a period of 12 days.
  • Figure 14 shows the typical morphologies of neurons from control (14A- B) and CART-treated cultures (14C-D) .
  • the population of hippocampal neurons supported by CART has morphological and immunohistochemical (expression of the glutamate receptors Glu-R2,-3 and -4; data not shown) properties characteristic of pyramidal neurons from the CAl and CA3 regions of the hippocampus.
  • Dissociated retinal cells were seeded into polyornithine-laminin-coated microplates at a density of about 12,500 per 6-mm well in DMEM/F12 supplemented with B27 medium supplement, 2.5 percent heat-inactivated horse serum, D-glucose, HEPES, insulin and transferrin. These cultures consisted of 75-90 percent photoreceptor cells, as identified by immunostaining for arrestin, a rod-specific antigen. The remaining cells were large multipolar and smaller unipolar NSE-positive neurons.
  • Photoreceptor cells appeared as rounded cells with a small cell body diameter, one or two neurites and, in some cases, a short vertical process that probably represents the connecting cilium ( Figure 15A-C) . At this level of resolution, there was no evidence of outer segment formation. In untreated cultures, the number of photoreceptors cells declined steadily over time to reach about 25 percent of the initial number after 6 days in culture. Treatment of the cultures with CART resulted in a significant increase in the number of viable photoreceptor cells after 6 days in culture (about 70 percent for CART-1 and 50 percent for CART-2) .
  • CART As can be seen in Figure 16, the effect of CART was maximal at about 100 ng/ml (CART-1) and 1 ng/ml (CART-2) , with the ED 50 of about 10 pg/ml (CART-1) and 40 pg/ml CART-2) .
  • CART also stimulated the extension of their axon- like process (Figure 15B-C) .
  • Striatal neuron cultures were prepared and cultured as described above, and were treated with varying concentrations of CART-1 or CART-2 as shown in Figure 18. After 6 days in culture, the number of neurons (18A) or GABA uptake (18B) was measured. As is apparent from Figures 18A and 18B, no significant increase in either neuron number or GABA uptake was apparent.
  • Serotonin-containing neurons from rat embryonic day 15 hindbrain were prepared and cultured as described above. After 8 days in culture, the cells were immunostained for serotonin. The results are shown in Figure 19. As is apparent, the number of serotonin-positive neurons in cultures treated with CART-1 or CART-2 was not significantly different from the number of these neurons in control (untreated) cultures. SEQUENCE LISTING
  • ATGTGTGCAA AGTATTCTTC TCTCGTTTTA TCCACCTGAC ACATTCTTGT GACCTTTCTG 660
  • Leu Gin lie Glu Ala Leu Gin Glu Val Leu Lys Lys Leu Lys Ser Lys 65 70 75 80
  • MOLECULE TYPE DNA (genomic)
  • CTACTCTGCC GTGGATGATG CCTCCCACGA GAAGGAGCTG GTCGGTATTC CCCTCGCTCT 300
  • Lys Ser Lys Arg Val Pro lie Tyr Glu Lys Lys Tyr Gly Gin Val Pro 65 70 75 80

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Abstract

Methods of reducing or preventing neuron degeneration and promoting neuron regeneration and restoration of function are disclosed. Also disclosed are methods of treating various neurological disorders.

Description

METHODS OF PREVENTING NEURON DEGENERATION AND PROMOTING
NEURON REGENERATION
BACKGROUND
Field of the Invention
This invention relates to uses of a novel protein called CART to decrease or prevent neuron degeneration and to promote neuron regeneration and restoration of function. In addition, the invention concerns methods of treating various neurological conditions by administering CART.
Description of Related Art
A number of neurological disorders and diseases are caused at least in part by degeneration or death of particular classes of neurons. For example, Parkinson's disease is characterized by slowing of voluntary muscle movement, muscular rigidity, and tremor. Such symptoms are attributed at least in part to progressive degeneration of dopamine-producing neurons located in a specific region of the brain called the substantia nigra. Degeneration of these neurons ("dopaminergic neurons") results in a decrease of dopamine levels in an adjacent region of the brain called the striatum. The striatum contains neurons expressing receptors for dopamine; these neurons are involved in the control of motor activity. The cause of the degeneration of dopaminergic neurons is unknown, but has been attributed to free radicals, excess iron content, environmental toxins, excitatory amino acid neurotoxicity, and possibly a deficiency of certain neurotrophic factors (Jenner, Neurology, Suppl. 3:S6-S12 [1995]; Adams and Victor, eds. Principles of Neurology, Chapter 42: Degerative Diseases of the Nervous System, McGraw Hill, NY [1993]).
Diseases such as amyotrophic lateral sclerosis (ALS) , progressive muscular atrophy, and hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease) all result at least in part from a decay of motor neurons which are located in the ventral horn of the spinal cord.
The hippocampus, a well defined structure that is part of the cerebral cortex of the brain, is important in the formation of long term memory. Destruction of the hippocampus, for example by ischemia, can result in an inability to form new memories. Degeneration of pyramidal CAl neurons, which are located in the CAl region of the hippocampus, is one characteristic of Alzheimer's disease. These same neurons are selectively vulnerable to ischemic and anoxic damage which occur in conditions such as stroke and head trauma. In addition, the CAl pyramidal hippocampal neurons as well as pyramidal neurons located in the CA3 region of the hippocampus, are selectively injured in epilepsy.
The striatum is the innervation region of the nerve terminals of dopaminergic-containing neurons from the substantia nigra. The majority of striatal neurons utilize GABA (4-aminobutyric acid) as their neurotransmitter. The striatum is the major target of the progressive neurodegeneration that occurs in Huntington's disease, in which the major neuron loss is that of the striatal GABA-utiiizing neurons.
The serotonin-containing neurons are located in groups clustered around the midline of the hindbrain. These neurons are involved in the control of body temperature, mood, and sleep. Disorders of the serotonin-containing neuron system include, for example. depression, other mood disorders, and sleep disturbances.
Photoreceptor cells are a specialized subset of retina neurons, and are responsible for vision. Injury and/or death of photoreceptor cells can lead to blindness. Degeneration of the retina, such as by retinitis pigmentosa, age-related macular degeneration, and stationary night blindness, are all characterized by the progressive atrophy and loss of function of photoreceptor outer segments which are specialized structures containing the visual pigments that transform a light stimulus into electrical activity.
While there are some therapies available to treat the symptoms and decrease the severity of such diseases ( e . g. , L-dopa to treat Parkinson's disease), there currently exists no effective treatment to prevent or reduce the degeneration of most of the above mentioned classes of affected neurons, or to promote their repair. Recently, several naturally occurring proteinaceous molecules have been identified based on their trophic activity on various neurons. These molecules are termed "neurotrophic factors". Neurotrophic factors are endogenous, soluble proteins that can regulate survival, growth, and/or morphological plasticity of neurons (see Fallon and Laughlin, Neurotrophic Factors, Academic Press, San Diego, CA [1993]) . In view of their ability to promote neuron regeneration and to prevent neuron death and degeneration, it has been postulated that neurotrophic factors might be useful in treating neurodegenerative conditions of the nervous system.
The known neurotrophic factors belong to several different protein superfamilies of polypeptide growth factors based on their amino acid sequence homology and/or their three-dimensional structure (MacDonald and Hendrikson, Cell, 73:421-424 [1993]). One family of neurotrophic factors is the neurotrophin family. This family currently consists of NGF (nerve growth factor) , BDNF (brain derived neurotrophic factor), NT-3 (neurotrophin-3) , NT-4 (neurotrophin-4) , and NT-6 (neurotrophin-6) .
CNTF (ciliary neurotrophic factor) and LIF (leukemia inhibitory factor) are cytokine polypeptides that have neurotrophic activity. By virtue of their structural features and receptor components, these polypeptides are related to a family of hematopoietic cytokines that includes IL-6 (interleukin-6) , IL-11 (interleukin-11) , G-CSF (granulocyte-colony stimulating factor) , and oncostatin-M. GDNF (glial derived neurotrophic factor) is a neurotrophic factor that belongs to the TGF-beta (transforming growth factor beta) superfamily. GDNF displays potent survival and differentiation-promoting actions for dopaminergic and motor neurons (Lin et al . , Science, 260:1130-1132 [1993]; Yan et al . , Nature, 373:341-344 [1995]).
Neurotrophic activity has been demonstrated for growth factors that are known for their mitogenic and/or differentiation-inducing activities on mesodermal and ectodermal tissues, such as FGF (fibroblast growth factor) , EGF (epidermal growth factor) TGF-alpha (transforming growth factor-alpha) , TGF-beta, and IGF (insulin like growth factor) .
Recently, a novel rat gene has been identified that is transcribed in neural and endocrine tissues in response to acute administration of psychomotor stimulants such as cocaine or amphetamine (Douglass et al . , J. Neurosci . , 15: 2471-2481 [1995]; Genbank accession number U10071) . The gene is termed CART (cocaine and amphetamine regulated transcript) . CART mRNA transcripts are induced by cocaine and amphetamine in brain striatum, and transcripts are also apparent (with or without induction) in hypothalamus, eye, thalamus, cortex, hippocampus, and hindbrain. The gene appears to be alternatively spliced, resulting in the presence or absence of an in-frame 39 base insert within the putative coding region of rat CART mRNA. As a result, predicted translation products are either 116 (CART-1) or 129 (CART-2) amino acids in length. The human CART cDNA has been isolated from a human hypothalamic cDNA library and the human genomic sequence has been isolated as well (Genbank accession number for human cDNA: U16826; for human genomic sequence: U20325) . Based on the predicted amino acid sequence of both human and rat CART protein, CART appears to have a prototypic signal peptide, suggesting that the protein is secreted from the cells in which it is synthesized. CART is not significantly homologous with any known neurotrophic factor at either the nucleotide sequence or amino acid sequence level. The discoveries that comprise the present invention demonstrate that CART has unexpectedly been found to have neurotrophic activity for certain types of neurons. Prior to these discoveries, there was no suggestion or indication that CART might have such neurotrophic activity. Such findings suggest that CART may be the first identified member of a novel family of neurotrophic factors.
In view of the fact that many nervous system disorders and diseases have no known cure, there is a need in the art to identify novel compounds for treating neurological conditions and diseases such as Parkinson's disease, amyotrophic lateral sclerosis (ALS) , Alzheimer's disease, stroke, and various degenerative disorders that affect vision.
Accordingly, it is an object of the present invention to provide methods of preventing neuron degeneration, and to provide methods of promoting neuron regeneration and restoring neural functions.
It is a further object of the invention to provide a method of treating certain neurological diseases.
These and other objects will be apparent to one of ordinary skill in the art from the present disclosure.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a method for promoting the survival, differentiation, or regeneration of neurons, comprising contacting the neurons with an effective amount of CART or a fragment thereof. The neurons may be selected from the group consisting of: nigral dopaminergic neurons, hippocampal neurons, motor neurons, retinal neurons, and photoreceptor cells. Optionally, the method is conducted in vitro.
In another embodiment, the method for promoting survival of neurons is conducted in vivo, optionally to treat a neurological disease or disorder affecting neurons selected from the group consisting of: nigral dopaminergic neurons, hippocampal neurons, motor neurons, retinal neurons, and photoreceptor cells.
In still another embodiment, the neurological disorder is selected from the group consisting of: substance addiction, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, ischemia, brain or spinal cord trauma, amyotrophic lateral sclerosis, Charcot- Marie-Tooth disease, dementia, retinitis pigmentosa, macular degeneration, retinal detachment, and retinal vascular disease. In yet another embodiment, a method for treating a neurological disorder or disease comprising administering a therapeutically effective amount of CART is provided. Optionally, the neurological disorder or disease is selected from the group consisting of: substance addiction, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, ischemia, brain or spinal cord trauma, amyotrophic lateral sclerosis, Charcot- Marie-Tooth disease, dementia, retinitis pigmentosa, macular degeneration, and retinal vascular disease. Additionally, the disease or disorder may comprise damage to the nervous system caused by trauma, infarction, surgery, infection, or malignancy, exposure to a toxin, or a nutritional deficiency. In one other embodiment, the invention provides a composition comprising a therapeutically effective amount of CART in admixture with a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the cDNA sequence of rat CART (SEQ ID NO:l) .
Figure 2 depicts the predicted amino acid sequence of the rat CART protein based on the nucleotide sequence (SEQ ID NO:2) .
Figure 3 depicts the cDNA sequence of human CART (SEQ ID NO:3) .
Figure 4 (A-C) depicts the full length nucleotide sequence of the human CART gene (SEQ ID NO:4) . Figure 5 depicts the predicted amino acid sequence of the human CART protein based on the nucleotide sequence (SEQ ID NO:5) .
Figure 6 (A-B) depicts the promotion of dopamine neuron survival by cart in cultures of embryonic day 15 (A) and post-natal day 6 (B) rat substantia nigra. Cultures grown in DMEM/F12 with B27 medium supplement and 15% horse serum on polyornithine - and laminin-modified 96-well microplates were treated with rat recombinant CART-1 or CART-2 at 1 ng/ml (filled bars) , 10 ng/ml (open bars) and 100 ng/ml (hatched bars) and immunostained for tyrosine hydroxylase, a marker that identifies dopaminergic neurons in these cultures, after 6 days in vitro . All the tyrosine-hydroxylase- positive neurons present in a 6-mm well were counted under brightfield optics. The results are expressed as the percentages of the number of tyrosine-hydroxylase- positive neurons found in control cultures. Each value in (A) is the mean of two different wells from a representative experiment. Similar results were obtained in three separate experiments. The values in (B) are the mean ± S.D. Of 5-6 wells from three independent experiments. Values are significantly different from control by Student's t-test at p<0.001.
Figure 7 (A-C) depicts the effect of CART on dopamine neurons in cultures of embryonic day 15 rat substantia nigra. Cells were plated in DMEM/F12 with B27 medium supplement and 15% horse serum on polyornithine and laminin-modified culture surfaces at a density of about 25,000/6-mm well. Dopamine neurons identified by tyrosine hydroxylase immunostaining are shown after 6 days in vitro in the presence of the following additions: (A) none; (B) rat recombinant CART-1, 100 pg/ml; (C) rat recombinant CART-2, 100 pg/ml. Calibration bar = 100 μm. Figure 8 (A-C) depicts the effect of CART on the morphological development of dopamine neurons in cultures of post-natal day 6 rat substantia nigra. Cells were plated in DMEM/F12 with B27 medium supplement and 15% horse serum on polyornithine and laminin-modified culture surfaces at a density of about 40,000/6-mm well. Dopamine neurons identified by tyrosine hydroxylase immunostaining are shown after 8 days in vitro in the presence of the following additions: (A,B) none; (C) rat recombinant CART- 1, 100 pg/ml. Calibration bar = 50 μ
Figure 9 depicts the stimulation of dopamine uptake by CART in cultures of embryonic day 15 rat substantia nigra. Cultures grown in DMEM/F12 with B27 medium supplement and 15 percent horse serum on polyornithine and laminin- modified 96-well microplates were treated with rat recombinant CART-1 or CART-2. After 3 days in vitro, cultures were processed for [3H]-dopamine uptake (50 nM; 1.5 million dpm/ml; 1 hour incubation at 37°C) . The results are expressed as the percentages of the dopamine uptake values (in dpm/well) found in control cultures. Each data point is the mean of 2 wells from a representative experiment. Similar results were obtained in four independent experiments.
Figure 10 depicts the promotion of motor neuron survival by CART in cultures of embryonic day 15 rat spinal cord. Cultures of metrizamide gradient-purified motor neurons (1000/6-mm well) treated with rat recombinant CART- 1 (ten-fold serial dilutions ranging from 10 ng/ml to 1 pg/ml) were fixed after 3 days and immunostained for neuronal-specific enolase. Motor neuron survival was determined by counting the number of neuronal-specific enolase-positive neurons per 2.4 square mm fields. Each data point is the mean of 3 wells from a representative experiment. The standard deviation is indicated.
Figure 11 (A-C) depicts the effect of CART on motor neurons in cultures of embryonic day 15 rat spinal cord. Metrizamide gradient-purified motor neurons were plated in DMEM/F12 with B27 medium supplement and 15% horse serum on polyornithine and laminin-modified culture surfaces at a density of about 1000 neurons/6-mm well. Motor neurons were identified by phase-contrast microscopy and are shown after 3 days in vitro in the presence of the following treatments: (A) none; (B) rat recombinant CART-1, 100 pg/ml; (C) rat recombinant CART-2, 100 pg/ml. Calibration bar = 50 μm.
Figure 12 (A-B) depicts the promotion of hippocampal neuron survival by CART in cultures of post-natal day 4 rat hippocampus. Cells were plated in DMEM/F12 with B27 medium supplement and 2.5% horse serum on polyornithine and laminin-modified 96-well microplates at a density of about 500/6-mm well. (A) Changes in hippocampal neuron number over time in response to 100 pg/ml rat recombinant CART-1. (B) Dose-dependence of CART-1 survival-promoting effect. The total number of neurons per well was determined by counting MAP-l/MAP-2 immunostained cells. Each value is the mean ± s.d. of 3-4 cultures.
Figure 13 (A-B) depicts the stimulation of neuritic development by CART in cultures of post-natal day 4 rat hippocampus. Cultures of hippocampal neurons were incubated for 12 days with or without 100 pg/ml rat recombinant CART-1 and then immunostained for MAP/l-MAP-2. All the neurons present in a 6-mm well were photographed and analyzed for neurite lengths and branching. (A) Total neurite length per neuron; (B) number of branching points per neuron. Each symbol represents one neuron. Mean neurite lengths were as follows: control (no CART) about 465 μm; CART-1 about 945 μm. The number of branching points per neuron was 1.9 (s.d. 2.1) for control; 6.1 (s.d. 5.2) for CART-1.
Figure 14 (A-D) depicts the effect of CART on the morphological development of neurons in cultures of post¬ natal day 4 rat hippocampus. Cells were plated in DMEM/F12 with B27 medium supplement and 2.5% horse serum on polyornithine and laminin-modified 96-well microplates at a density of about 500 cells/6-mm well. Hippocampal neurons identified by immunostaining for MAP-1 and MAP-2 are shown after 12 days in vitro in the presence of the following additions: (A,B) none; (C,D) rat recombinant CART-1, 100 pg/ml and rat recombinant CART-2, 100 pg/ml. Calibration bar = 50 μm.
Figure 15 (A-C) depicts the effect of CART on photoreceptor cells in cultures of post-natal day 5 mouse retina. Cells were plated in DMEM/F12 with B27 medium supplement and 2.5% horse serum on polyornithine and laminin-modified 96-well microplates at a density of about 13,000 cells/6-mm well. Photoreceptor cells identified by immunostaining for arrestin are shown after 6 days in vitro in the presence of the following additions: (A) none; (B) rat recombinant CART-1 , 100 pg/ml; (C) rat recombinant CART-2, 100 pg/ml. Calibration bar = 50 μm.
Figure 16 depicts the promotion of photoreceptor cell survival by CART in cultures of post-natal day 5 mouse retina. Cultures treated with rat recombinant CART-1 and CART-2 were fixed after 6 days and immunostained for arrestin. The number of arrestin-positive photoreceptor cells was determined in 2.4 sq.mm fields. The results are expressed as the percentages of photoreceptor cells found in untreated control cultures. Each value is the mean ± s.d. of 3 cultures.
Figure 17 (A-G) depicts the photoreceptor outer segment development and axonal growth in response to CART in cultures of embryonic day 17 chick retina. Cells were plated in DMEM/F12 with B27 medium supplement and 2.5% horse serum on polyornithine and laminin-modified 96-well microplates at a density of about 10,000 cells/6-mm well. Photoreceptor cells identified by phase-contrast morphology are shown after 7 days in vitro in the presence of (A) no treatment or (B-G) rat recombinant CART-1, 100 pg/ml. CART-1 promotes the development of inner segments (IS; solid arrows) , outer segments (OS; open arrows) and of the axonal process (P) . The lipid droplet (LD; arrowheads) marks the junction between inner and outer segments. Calibration bar = 25 μ .
Figure 18 (A-B) depicts the lack of effect of CART on the number of neurons and GABA uptake in cultures of embryonic day 15 striatum. Cultures grown in DMEM/F12 with B27 medium supplement and 15 percent horse serum on polyornithine and laminin-modified 96-well microplates were treated with rat recombinant CART-1 or CART-2. After 6 days in vitro, the cultures were evaluated for immunostaining with MAP-1 or MAP-2, or for GABA uptake. The number of neurons (relative to control, untreated neurons) in striatal cultures treated with CART-1 or CART-2 is shown in (A) . Each data point is the mean of 4 individual wells in which the number of MAP-positive cells was counted in 2.4 square mm fields. The standard deviation of each data point is indicated. The effect of CART on GABA uptake relative to control (untreated neurons) is shown in (B) . Each data point is the mean of duplicate cultures. Figure 19 depicts the effect of CART on serotonin-containing neurons in cultures of embryonic day 15 rat hindbrain. Cultures were grown in DMEM/F12 with B27 medium supplement and 15 percent horse serum on polyornithine-and laminin-modified 96-well microplates, and were treated with rat recombinant CART-1 or CART-2 at the concentrations indicated. After 8 days in culture, the cells were immunostained for serotonin. All of the serotonin-positive cells present in each 6-mm well of a microtiter plate were counted under brightfield optics. The results are expressed as percentages of the number of serotonin-positive neurons in control (no CART) cultures. Each data point is the mean of 2 wells.
DETAILED DESCRIPTION OF THE INVENTION
This invention is based on the unexpected and surprising discovery that a CART polypeptide has been found to possess neurotrophic activity on certain types of neurons.
As used herein, the term "CART" when used to describe a cDNA, gene or other nucleic acid sequence refers to such cDNA, gene or nucleic acid sequence (a) having any of the Genbank accession numbers U10071, U16826, or U20325; (b) having the nucleotide sequence as set forth in SEQ ID NO: 1, 3, or 4; (c) having a nucleic acid sequence which hybridizes under stringent conditions to the nucleic acid sequences of SEQ ID NO: 1, 3, or 4; (d) having a naturally occurring allelic variation of (a) or (b) ; and/or (e) nucleic acid variants of (a) -(d) produced as provided for herein. The term "stringent conditions" refers to hybridization and washing under conditions that permit only binding of the probe to highly homologous sequences. One stringent wash solution is 0.015 M NaCl, 0.005 M NaCitrate, and 0.1 percent SDS used at a temperature of 55°C-65°C. Another stringent wash solution is 0.2 X SSC and 0.1 percent SDS used at a temperature of between 50°C-65°C. Where oligonucleotide probes are used to screen cDNA or genomic libraries, the following stringent washing conditions may be used. One protocol uses 6 X SSC with 0.05 percent sodium pyrophosphate at a temperature of 35°C-62°C, depending on the length of the oligonucleotide probe. For example, 14 base pair probes are washed at 35-40°C, 17 base pair probes are washed at 45-50°C, 20 base pair probes are washed at 52-57°C, and 23 base pair probes are washed at 57-63°C. The temperature can be increased 2-3°C where the background non-specific binding appears high. A second protocol utilizes tetramethylammonium chloride (TMAC) for washing oligonucleotide probes. One stringent washing solution is 3 M TMAC, 50 mM Tris-HCl, pH 8.0, and 0.2 percent SDS. The washing temperature using this solution is a function of the length of the probe. For example, a 17 base pair probe is washed at about 45-50°C.
The term "CART protein" or "CART polypeptide" as used herein refers to any protein or polypeptide having the properties described herein for CART. By way of illustration, CART protein or CART polypeptide includes, an amino acid sequence encoded by any of items (a)-(e) above and peptide or polypeptide fragments derived therefrom, to the amino acid sequence set forth in SEQ ID NO:2 or 5, and/or to chemically modified derivatives as well as nucleic acid and or amino acid sequence variants thereof as provided for herein.
As used herein, the phrase "differentiation of neurons" refers to the production axonal and/or dendritic processes ("neurites") , and/or other morphological structures or features of neurons either in vitro or in vivo.
As used herein, the term "CART fragment" refers to a peptide or polypeptide that is less than the full length amino acid sequence of naturally occurring CART protein but has substantially the same biological activity as CART polypeptide or CART protein described above. Such a fragment may be truncated at the amino terminus, the carboxy terminus, and/or internally, and may be chemically modified. As used herein, the term "CART derivative" or
"CART variant" refers to a CART polypeptide or CART protein that has 1) been chemically modified, as for example, by addition of polyethylene glycol or other compound, and/or 2) contains one or more nucleic acid or amino acid sequence substitutions, deletions, and/or insertions. As used herein, the terms "biologically active polypeptide" and "biologically active fragment" refer to a peptide or polypeptide that 1) has CART activity (i.e., acts to support survival and/or differentiation of dopaminergic, hippocampal, motor, photoreceptor, and/or other neurons) , and/or 2) cross reacts with monoclonal or polyclonal antibodies raised against naturally occurring CART protein.
As used herein, the terms "effective amount" and "therapeutically effective amount" refer to the amount of CART necessary to support the survival and/or differentiation of neurons as compared to neurons not treated with CART.
The CART polypeptides that are useful in practicing the present invention may be naturally occurring full length polypeptides, or truncated polypeptides or peptides (i.e, "fragments"). The polypeptides or fragments may be chemically modified, i . e . , glycosylated, phosphorylated, and/or linked to a polymer, as described below. In addition, the polypeptides or fragments may be variants of the naturally occurring CART polypeptide (i.e., may contain one or more amino acid deletions, insertions, and/or substitutions as compared with naturally occurring CART) .
The full length CART polypeptide or fragment can be prepared using well known recombinant DNA technology methods such as those set forth in Sambrook et al . (Molecular Cloning: A Laboratoy Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989]) and/or Ausubel et aϊ. , eds, (Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY [1994]) . A gene or cDNA encoding the CART protein or truncated version thereof may be obtained for example by screening a genomic or cDNA library, or by PCR amplification. Alternatively, a gene encoding the CART polypeptide or fragment may be prepared by chemical synthesis using methods well known to the skilled artisan such as those described by Engels et al . (Angew. Chem. Intl. Ed., 28:716-734 [1989]). These methods include, inter alia, the phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis. A preferred method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry. Typically, the DNA encoding the CART polypeptide will be several hundred nucleotides in length. Nucleic acids larger than about 100 nucleotides can be synthesized as several fragments using these methods. The fragments can then be ligated together to form the full length CART polypeptide. In some cases, it may be desirable to prepare nucleic acid and/or amino acid variants of naturally occurring CART. Nucleic acid variants (wherein one or more nucleotides are designed to differ from the wild- type or naturally occurring CART) may be produced using site directed mutagenesis or PCR amplification where the primer(s) have the desired point mutations (see Sambrook et al . , supra, and Ausubel et al . , supra, for descriptions of mutagenesis techniques) . Chemical synthesis using methods described by Engels et al . , supra, may also be used to prepare such variants.
Other methods known to the skilled artisan may be used as well. Preferred nucleic acid variants are those containing nucleotide substitutions accounting for codon preference in the host cell that is to be used to produce CART. Other preferred variants are those encoding conservative amino acid changes (e.g., wherein the charge or polarity of the naturally occurring amino acid side chain is not altered substantially by substitution with a different amino acid) as compared to wild type, and/or those designed to either generate a novel glycosylation and/or phosphorylation site(s) on CART, or those designed to delete an existing glycosylation and/or phosphorylation site(s) on CART.
The CART gene or cDNA can be inserted into an appropriate expression vector for expression in a host cell. The vector is selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the CART gene and/or expression of the gene can occur) . The CART polypeptide or fragment thereof may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells. Selection of the host cell will depend at least in part on whether the CART polypeptide or fragment thereof is to be glycosylated. If so, yeast, insect, or mammalian host cells are preferable; yeast cells will glycosylate the polypeptide, and insect and mammalian cells can glycosylate and/or phosphorylate the polypeptide as it naturally occurs on the CART polypeptide (i . e . , "native" glycosylation and/or phosphorylation) .
Typically, the vectors used in any of the host cells will contain 5' flanking sequence (also referred to as a "promoter") and other regulatory elements as well such as an enhancer (s), an origin of replication element, a transcriptional termination element, a complete intron sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these elements is discussed below. Optionally, the vector may contain a "tag" sequence, i . e . , an oligonucleotide sequence located at the 5' or 3' end of the CART coding sequence that encodes polyHis (such as hexaHis) or another small immunogenic sequence. This tag will be expressed along with the protein, and can serve as an affinity tag for purification of the CART polypeptide from the host cell. Optionally, the tag can subsequently be removed from the purified CART polypeptide by various means such as using a selected peptidase for example.
The 5' flanking sequence may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of 5' flanking sequences from more than one source), synthetic, or it may be the native CART 5' flanking sequence. As such, the source of the 5' flanking sequence may be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the 5' flanking sequence is functional in, and can be activated by, the host cell machinery.
The 5' flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, 5' flanking sequences useful herein other than the CART 5' flanking sequence will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of the 5' flanking sequence may be known. Here, the 5' flanking sequence may be synthesized using the methods described above for nucleic acid synthesis or cloning. Where all or only a portion of the 5' flanking sequence is known, it may be obtained using PCR and/or by screening a genomic library with suitable oligonucleotide and/or 5' flanking sequence fragments from the same or another species. Where the 5' flanking sequence is not known, a fragment of DNA containing a 5' flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion using one or more carefully selected enzymes to isolate the proper DNA fragment.
After digestion, the desired fragment may be isolated by agarose gel purification, Qiagen® column or other methods known to the skilled artisan. Selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
The origin of replication element is typically a part of prokaryotic expression vectors purchased commercially, and aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for optimal expression of the CART polypeptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector. The transcription termination element is typically located 3' to the end of the CART polypeptide coding sequence and serves to terminate transcription of the CART polypeptide. Usually, the transcription termination element in prokaryotic cells is a G-C rich fragment followed by a poly T sequence. While the element is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described above. A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. The ribosome binding element, commonly called the Shine-Dalgarno sequence (prokaryotes) or the Kozak sequence (eukaryotes) , is necessary for translation initiation of mRNA. The element is typically located 3' to the promoter and 5' to the coding sequence of the CART polypeptide to be synthesized. The Shine-Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content) . Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth above and used in a prokaryotic vector.
In those cases where it is desirable for CART to be secreted from the host cell, a signal sequence may be used to direct the CART polypeptide out of the host cell where it is synthesized. Typically, the signal sequence is positioned in the coding region of CART nucleic acid sequence, or directly at the 5' end of the CART coding region. Many signal sequences have been identified, and any of them that are functional in the selected host cell may be used in conjunction with the CART gene. Therefore, the signal sequence may be homologous or heterologous to the CART polypeptide, and may be homologous or heterologous to the CART polypeptide. Additionally, the signal sequence may be chemically synthesized using methods set forth above. In many cases, transcription of the CART polypeptide is increased by the presence of one or more introns on the vector; this is particularly true for eukaryotic host cells, especially mammalian host cells. The intron may be naturally occurring within the CART nucleic acid sequence, especially where the CART sequence used is a full length genomic sequence or a fragment thereof. Where the intron is not naturally occurring within the CART DNA sequence (as for most cDNAs) , the intro (s) may be obtained from another source. The position of the intron with respect to the 5' flanking sequence and the CART coding sequence is important, as the intron must be transcribed to be effective. As such, where the CART nucleic acid sequence is a cDNA sequence, the preferred position for the intron is 3' to the transcription start site, and 5' to the polyA transcription termination sequence.
Preferably for CART cDNAs, the intron will be located on one side or the other (i.e., 5' or 3') of the CART coding sequence such that it does not interrupt the this coding sequence. Any intron from any source, including any viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this invention, provided that it is compatible with the host cell (s) into which it is inserted. Also included herein are synthetic introns. Optionally, more than one intron may be used in the vector.
Where one or more of the elements set forth above are not already present in the vector to be used, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the elements are well known to the skilled artisan and are comparable to the methods set forth above (i.e., synthesis of the DNA, library screening, and the like) .
The final vectors used to practice this invention are typically constructed from a starting vectors such as a commercially available vector. Such vectors may or may not contain some of the elements to be included in the completed vector. If none of the desired elements are present in the starting vector, each element may be individually ligated into the vector by cutting the vector with the appropriate restriction endonuclease(s) such that the ends of the element to be ligated in and the ends of the vector are compatible for ligation. In some cases, it may be necessary to "blunt" the ends to be ligated together in order to obtain a satisfactory ligation. Blunting is accomplished by first filling in "sticky ends" using Klenow DNA polymerase or T4 DNA polymerase in the presence of all four nucleotides. This procedure is well known in the art and is described for example in Sambrook et al ., supra . Alternatively, two or more of the elements to be inserted into the vector may first be ligated together (if they are to be positioned adjacent to each other) and then ligated into the vector.
One other method for constructing the vector to conduct all ligations of the various elements simultaneously in one reaction mixture. Here, many nonsense or nonfunctional vectors will be generated due to improper ligation or insertion of the elements, however the functional vector may be identified and selected by restriction endonuclease digestion. Preferred vectors for practicing this invention are those which are compatible with bacterial, insect, and mammalian host cells. Such vectors include, inter alia , pCRII (Invitrogen Company, San Diego, CA) , pBSII (Stratagene Company, LaJolla, CA) , and pETL (BlueBacII; Invitrogen) .
After the vector has been constructed and a CART nucleic acid has been inserted into the proper site of the vector, the completed vector may be inserted into a suitable host cell for amplification and/or CART polypeptide expression.
Host cells may be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast cell, an insect cell, or a vertebrate cell) . The host cell, when cultured under appropriate conditions, can synthesize CART protein which can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted) . After collection, the CART protein can be purified using methods such as molecular sieve chromatography, affinity chromatography, and the like.
Selection of the host cell will depend in part on whether the CART protein is to be glycosylated or phosphorylated (in which case eukaryotic host cells are preferred) , and the manner in which the host cell is able to "fold" the protein into its native tertiary structure (e.g., proper orientation of disulfide bridges, etc.) such that biologically active protein is prepared by the cell. However, where the host cell does not synthesize biologically active CART, the CART may be "folded" after synthesis using appropriate chemical conditions as discussed below.
Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO) or 3T3 cells. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. Other suitable mammalian cell lines, are the monkey COS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene. Other suitable mammalian cell lines include but are not limited to, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines. Similarly useful as host cells suitable for the present invention are bacterial cells. For example, the various strains of E. coli (e.g., HB101, DH5α,DH10, and MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis,
Pseudomonas spp . , other Bacillus spp. , Streptomyces spp. , and the like may also be employed in this method.
Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention (Miller et al . , Genetic Engineering 8: 277-298 [1986]) . Insertion (also referred to as
"transformation" or "transfection") of the vector into the selected host cell may be accomplished using such methods as calcium chloride, electroporation, microinjection, lipofection or the DEAE-dextran method. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et al . , supra . The host cells containing the vector (i.e., transformed or transfected) may be cultured using standard media well known to the skilled artisan. The media will usually contain all nutrients necessary for the growth and survival of the cells. Suitable media for culturing E. coli cells are for example, Luria Broth (LB) and/or Terrific Broth (TB) . Suitable media for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which may be supplemented with serum and/or growth factors as required by the particular cell line being cultured. A suitable medium for insect cultures is Grace's medium supplemented with yeastolate, lactalbumin hydrolysate, and/or fetal calf serum as necessary.
Typically, an antibiotic or other compound useful for selective growth of the transformed cells only is added as a supplement to the media. The compound to be used will be dictated by the selectable marker element present on the plasmid with which the host cell was transformed. For example, where the selectable marker element is kanamycin resistance, the compound added to the culture medium will be kanamycin. The amount of CART polypeptide produced in the host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, HPLC separation, immunoprecipitation, and/or activity assays such as DNA binding gel shift assays.
If the CART polypeptide has been designed to be secreted from the host cells, the majority of polypeptide will likely be found in the cell culture medium. If however, the CART polypeptide is not secreted from the host cells, it will be present in the cytoplasm (for eukaryotic, gram positive bacteria, and insect host cells) or in the periplasm (for gram negative bacteria host cells) .
For intracellular CART, the host cells are typically first disrupted mechanically or osmotically to release the cytoplasmic contents into a buffered solution. CART polypeptide is then isolated from this solution.
Purification of CART polypeptide from solution can be accomplished using a variety of techniques. If the polypeptide has been synthesized such that it contains a tag such as Hexahistidine (CART/hexaHis) or other small peptide at either its carboxyl or amino terminus, it may essentially be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag or for the polypeptide directly (i . e . , a monoclonal antibody specifically recognizing CART) . For example, polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the Qiagen nickel columns) can be used for purification of CART/polyHis. (See for example, Ausubel et al . , eds., Current Protocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, New York [1993]). Where the CART polypeptide has no tag and no antibodies are available, other well known procedures for purification can be used. Such procedures include, without limitation, ion exchange chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis in combination with gel elution, and preparative isoelectric focusing ("Isoprime" machine/technique, Hoefer Scientific) . In some cases, two or more of these techniques may be combined to achieve increased purity. Preferred methods for purification include polyHistidine tagging and ion exchange chromatography in combination with preparative isoelectric focusing.
If it is anticipated that the CART polypeptide will be found primarily in the periplasmic space of the bacteria or the cytoplasm of eukaryotic cells, the contents of the periplasm or cytoplasm, including inclusion bodies (e . g. , gram-negative bacteria) if the processed polypeptide has formed such complexes, can be extracted from the host cell using any standard technique known to the skilled artisan. For example, the host cells can be lysed to release the contents of the periplasm by French press, homogenization, and/or sonication. The homogenate can then be centrifuged. If the CART polypeptide has formed inclusion bodies in the periplasm, the inclusion bodies can often bind to the inner and/or outer cellular membranes and thus will be found primarily in the pellet material after centrifugation. The pellet material can then be treated with a chaotropic agent such as guanidine or urea to release, break apart, and solubilize the inclusion bodies. The CART polypeptide in its now soluble form can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If it is desired to isolate the CART polypeptide, isolation may be accomplished using standard methods such as those set forth below and in Marston et al . (Meth . Enz. , 182:264-275 [1990]) .
If CART polypeptide inclusion bodies are not formed to a significant degree in the periplasm of the host cell, the CART polypeptide will be found primarily in the supernatant after centrifugation of the cell homogenate, and the CART polypeptide can be isolated from the supernatant using methods such as those set forth below. In those situations where it is preferable to partially or completely isolate the CART polypeptide, purification can be accomplished using standard methods well known to the skilled artisan. Such methods include, without limitation, separation by electrophoresis followed by electroelution, various types of chromatography (immunoaffinity, molecular sieve, and/or ion exchange) , and/or high pressure liquid chromatography. In some cases, it may be preferable to use more than one of these methods for complete purification.
In addition to preparing and purifying CART polypeptide using recombinant DNA techniques, the CART polypeptides, fragments, and/or derivatives thereof, may be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using methods known in the art such as those set forth by Merrifield et al . , (J. Am. Chem . Soc , 85:2149 [1964]), Houghten et al . (Proc Natl Acad. Sci . USA, 82:5132 [1985]), and Stewart and Young (Solid Phase Peptide Synthesis, Pierce Chem Co, Rockford, IL [1984]). Chemically synthesized CART polypeptides or fragments may be oxidized using methods set forth in these references to form disulfide bridges. The CART polypeptides or fragments may be employed as biologically active or immunological substitutes for natural, purified CART polypeptides in therapeutic and immunological processes.
Chemically modified CART compositions (i.e., "derivatives") where the CART polypeptide is linked to a polymer ("CART-polymers") are included within the scope of the present invention. The polymer selected is typically water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer selected is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled as provided for in the present methods. A preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see U.S. Patent 5,252,714). The polymer may be branched or unbranched. Included within the scope of CART-polymers is a mixture of polymers. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. The water soluble polymer or mixture thereof may be selected from the group consisting of, for example, polyethylene glycol (PEG) , monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly- (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol. For the acylation reactions, the polymer(s) selected should have a single reactive ester group. For reductive alkylation, the polymer(s) selected should have a single reactive aldehyde group. The polymer may be of any molecular weight, and may be branched or unbranched.
Pegylation of CART may be carried out by any of the pegylation reactions known in the art, as described for example in the following references: Focus on Growth Factors 3 (2): 4-10 (1992); EP 0 154 316; and EP 0 401 384. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer) as described below. Pegylation by acylation generally involves reacting an active ester derivative of polyethylene glycol (PEG) with an CART protein. Any known or subsequently discovered reactive PEG molecule may be used to carry out the pegylation of CART. A preferred activated PEG ester is PEG esterified to N-hydroxysuccinimide ("NHS") . As used herein, "acylation" is contemplated to include without limitation the following types of linkages between CART and a water soluble polymer such as PEG: amide, carbamate, urethane, and the like, as described in Bioconjugate Chem. 5: 133-140 (1994) . Reaction conditions may be selected from any of those known in the pegylation art or those subsequently developed, provided that conditions such as temperature, solvent, and pH that would inactivate the CART species to be modified are avoided.
Pegylation by acylation usually results in a poly-pegylated CART product, wherein the lysine ε-amino groups are pegylated via an acyl linking group. Preferably, the connecting linkage will be an amide. Also preferably, the resulting product will be at least about 95 percent mono, di- or tri- pegylated. However, some species with higher degrees of pegylation (up to the maximum number of lysine ε-amino acid groups of CART plus one α-amino group at the amino terminus of CART) will normally be formed in amounts depending on the specific reaction conditions used. If desired, more purified pegylated species may be separated from the mixture, particularly unreacted species, by standard purification techniques, including, among others, dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel filtration chromatography and electrophoresis.
Pegylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with a protein such as CART in the presence of a reducing agent. Regardless of the degree of pegylation, the PEG groups are preferably attached to the protein via a -CH2-NH- group. With particular reference to the -CH2- group, this type of linkage is referred to herein as an "alkyl" linkage. Derivatization via reductive alkylation to produce a monopegylated product exploits the differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in CART. Typically, the reaction is performed at a pH (see below) which allows one to take advantage of the pKa differences between the ε-amino groups of the lysine residues and that of the α-amino group of the N-terminal residue of the protein. By such selective derivatization, attachment of a water soluble polymer that contains a reactive group such as an aldehyde, to a protein is controlled: the conjugation with the polymer occurs predominantly at the N-terminus of the protein without significant modification of other reactive groups such as the lysine side chain amino groups. The present invention provides for a substantially homogeneous preparation of CART- monopolymer protein conjugate molecules (meaning CART protein to which a polymer molecule has been attached substantially only (i.e., at least about 95%) in a single location on the CART protein. More specifically, if polyethylene glycol is used, the present invention also provides for pegylated CART protein lacking possibly antigenic linking groups, and having the polyethylene glycol molecule directly coupled to the CART protein. A particularly preferred water-soluble polymer for use herein is polyethylene glycol, abbreviated PEG. As used herein, polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono- (C1-C10) alkoxy- or aryloxy-polyethylene glycol.
In general, chemical derivatization may be performed under any suitable conditions used to react a biologically active substance with an activated polymer molecule. Methods for preparing pegylated CART will generally comprise the steps of (a) reacting an CART polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby CART becomes attached to one or more PEG groups, and (b) obtaining the reaction product (s) . In general, the optimal reaction conditions for the acylation reactions will be determined based on known parameters and the desired result. For example, the larger the ratio of PEG: protein, the greater the percentage of poly-pegylated product. Reductive alkylation to produce a substantially homogeneous population of mono- polymer/CART protein conjugate molecule will generally comprise the steps of: (a) reacting a CART protein with a reactive PEG molecule under reductive alkylation conditions, at a pH suitable to permit selective modification of the α-amino group at the amino terminus of said CART protein; and (b) obtaining the reaction product (s) .
For a substantially homogeneous population of mono-polymer/CART protein conjugate molecules, the reductive alkylation reaction conditions are those which permit the selective attachment of the water soluble polymer moiety to the N-terminus of CART. Such reaction conditions generally provide for pKa differences between the lysine amino groups and the α-amino group at the N-terminus (the pKa being the pH at which 50% of the amino groups are protonated and 50% are not) . The pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a larger excess of polymer to protein will be desired (i.e., the less reactive the N-terminal α-amino group, the more polymer needed to achieve optimal conditions) . If the pH is higher, the polymer:ρrotein ratio need not be as large (i.e., more reactive groups are available, so fewer polymer molecules are needed) . For purposes of the present invention, the pH will generally fall within the range of 3-9, preferably 3-6.
Another important consideration is the molecular weight of the polymer. In general, the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. Similarly, branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymer:protein ratio. In general, for the pegylation reactions contemplated herein, the preferred average molecular weight is about 2kDa to about lOOkDa (the term "about" indicating ± lkDa) . The preferred average molecular weight is about 5kDa to about 50kDa, particularly preferably about 12kDa to about 25kDa. The ratio of water-soluble polymer to CART protein will generally range from 1:1 to 100:1, preferably (for polypegylation) 1:1 to 20:1 and (for monopegylation) 1:1 to 5:1.
Using the conditions indicated above, reductive alkylation will provide for selective attachment of the polymer to any CART protein having an α-amino group at the amino terminus, and provide for a substantially homogenous preparation of monopolymer/CART protein conjugate. The term "monopolymer/CART protein conjugate" is used here to mean a composition comprised of a single polymer molecule attached to an CART protein molecule. The monopolymer/CART protein conjugate preferably will have a polymer molecule located at the N-terminus, but not on lysine amino side groups. The preparation will preferably be greater than 90% monopolymer/CART protein conjugate, and more preferably greater than 95% monopolymer CART protein conjugate, with the remainder of observable molecules being unreacted (i.e., protein lacking the polymer moiety) . The examples below provide for a preparation which is at least about 90% monopolymer/ protein conjugate, and about 10% unreacted protein. The monopolymer/protein conjugate has biological activity.
For the present reductive alkylation, the reducing agent should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation. Preferred reducing agents may be selected from the group consisting of sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane and pyridine borane. A particularly preferred reducing agent is sodium cyanoborohydride.
Other reaction parameters, such as solvent, reaction times, temperatures, etc., and means of purification of products, can be determined based on the published information relating to derivatization of proteins with water soluble polymers. A mixture of polymer-CART protein conjugate molecules may be prepared by acylation and/or alkylation methods, as described above,and one may select the proportion of monopolymer/ protein conjugate to include in the mixture. Thus, where desired, a mixture of various protein with various numbers of polymer molecules attached (i.e., di-, tri-, tetra-, etc.) may be prepared and combined with the monopolymer/CART protein conjugate material prepared using the present methods.
Generally, conditions which may be alleviated or modulated by administration of the present polymer/CART include those described herein for cart molecules in general. However, the polymer/CART molecules disclosed herein may have additional activities, enhanced or reduced activities, or other characteristics, as compared to the non-derivatized molecules.
CART molecules that are not themselves active in the activity assays presented herein may be useful as modulators (e.g., inhibitors or stimulants) of the CART receptors in vitro or in vivo .
The CART polypeptides and fragments thereof, whether or not chemically modified, may be employed alone, or in combination with other pharmaceutical compositions such as, for example, neurotrophic factors, cytokines, interferons, interleukins, growth factors, antibiotics, anti-inflammatories, neurotransmitter receptor agonists or antagonists and/or antibodies, in the treatment of neurological conditions.
The CART polypeptides and fragments thereof may be used to prepare antibodies generated by standard methods. Thus, antibodies that react with the CART polypeptides, as well as reactive fragments of such antibodies, are also contemplated as within the scope of the present invention. The antibodies may be polyclonal, monoclonal, recombinant, chimeric, single- chain and/or bispecific, etc. The antibody fragments may be any fragment that is reactive with the CART of the present invention, such as, Fab, Fab', etc. Also provided by this invention are the hybridomas generated by presenting CART or a fragment thereof as an antigen to a selected mammal, followed by fusing cells (e.g., spleen cells) of the animal with certain cancer cells to create immortalized cell lines by known techniques. The methods employed to generate such cell lines and antibodies directed against all or portions of a human CART polypeptide of the present invention are also encompassed by this invention.
The antibodies may be used therapeutically, such as to inhibit binding of the CART to its receptor. The antibodies may further be used for in vivo and in vitro diagnostic purposes, such as in labeled form to detect the presence of the CART in a body fluid.
Therapeutic Compositions and Administration
Therapeutic compositions for treating various neurological disorders are within the scope of the present invention. Such compositions may comprise a therapeutically effective amount of a CART polypeptide or fragment thereof (either of which may be chemically modified) in admixture with a pharmaceutically acceptable carrier. The carrier material may be water for injection, preferably supplemented with other materials common in solutions for administration to mammals. Typically, a CART therapeutic will be administered in the form of a composition comprising purified protein (which may be chemically modified) in conjunction with one or more physiologically acceptable carriers, excipients, or diluents. Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers. Preferably, the product is formulated as a lyophilizate using appropriate excipients (e.g., sucrose). Other standard carriers, diluents, and excipients may be included as desired. Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0- 5.5, which may further include sorbitol or a suitable substitute therefor.
The CART compositions can be systemically administered parenterally. Alternatively, the compositions may be administered intravenously or subcutaneously. When systemically administered, the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such pharmaceutically acceptable protein solutions, with due regard to pH, isotonicity, stability and the like, is within the skill of the art.
Therapeutic formulations of CART compositions useful for practicing the present invention may be prepared for storage by mixing the selected composition having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington 's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, ed., Mack Publishing Company [1990]) in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG) . The CART composition to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Where the CART composition is lyophilized, sterilization using these methods may be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration ordinarily will be stored in lyophilized form or in solution.
Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
The route of administration of the composition is in accord with known methods, e . g. oral, injection or infusion by intravenous, intraperitoneal, intracerebral (intraparenchymal) , intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes, or by sustained release systems or implantation device which may optionally involve the use of a catheter. Where desired, the compositions may be administered continuously by infusion, bolus injection or by implantation device. Alternatively or additionally, CART may be administered locally via implantation into the affected area of a membrane on to which CART polypeptide has been absorbed.
Where an implantation device is used, the device may be implanted into a cerebral ventricle or into brain parenchyma, and delivery of CART may be directly through the device via bolus or continuous administration, or via a catheter using continuous infusion. CART polypeptide may be administered in a sustained release formulation or preparation. Suitable examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e . g. films, or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-556 [1983]), poly (2-hydroxyethyl-methacrylate) (Langer et al . , J. Biomed. Mater. Res. , 15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105 [1982]), ethylene vinyl acetate (Langer et al . , supra) or poly- D(-)-3-hydroxybutyric acid (EP 133,988). Sustained- release compositions also may include liposomes, which can be prepared by any of several methods known in the art (e.g., DE 3,218,121; Epstein et al . , Proc . Natl . Acad. Sci . USA, 82: 3688-3692 [1985]; Hwang et al . , Proc . Natl . Acad. Sci . USA, 77: 4030-4034 [1980]; EP 52,322; EP 36,676; EP 88,046; EP 143,949). In some cases, it may be desirable to use CART compositions in an ex vivo manner, i.e., to treat isolated neuronal tissues that may be subsequently implanted into a patient. For example, isolated dopaminergic neurons taken from fetal (or other tissue) may be treated with CART (either alone or in combination with other agents) prior to implanting of these neurons into the brain tissue of Parkinson's patients.
In other cases, CART may be delivered through implanting into patients cells that have been genetically engineered (using methods described above) to express and secrete CART polypeptide. Such cells may be human cells, and may be derived from the patient's own tissue or from another source, either human or non- human. Optionally, the cells may be immortalized. The cells may be implanted into the brain or into other tissues of the nervous system, or into tissues that are innervated by the nervous system.
In certain situations, it may be desirable to use gene therapy methods for administration of CART to patients suffering from certain neurological disorders or diseases. Here, genomic DNA, cDNA, and/or synthetic DNA encoding cart or a fragment or variant thereof may be operably linked to a constitutive or inducible promoter that is active in the tissue into which the composition will be injected. This CART DNA construct can be injected directly into brain or other neuronal tissue to be treated. Alternatively, the CART DNA construct may be injected into muscle tissue where it can be taken up into the cells and expressed in the cells, provided that the CART DNA is operably linked to a promoter that is active in muscle tissue such as cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, or muscle creatine kinase promoter. Typically, the DNA construct may include (in addition to the CART DNA and a promoter) , vector sequence obtained from vectors such as adenovirus vector, adeno-associated virus vector, a retroviral vector, and/or a herpes virus vector. The vector/DNA construct may be admixed with a pharmaceutically acceptable carrier(s) for injection. An effective amount of the CART composition (s) to be employed therapeutically will depend, for example, upon the therapeutic objectives such as the indication for which CART is being used, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage may range from about 0.1 μg/kg to up to 100 mg/kg or more, depending on the factors mentioned above. Typically, a clinician will administer the CART composition until a dosage is reached that achieves the desired effect. The CART composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of CART) over time, or as a continuous infusion via implantation device or catheter.
As further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, the type of disorder under treatment, the age and general health of the recipient, will be able to ascertain proper dosing. Generally, the dosage will be between 0.01 μg/kg body weight (calculating the mass of the protein alone, without chemical modification) and 300 μg/kg (based on the same) .
The CART proteins, fragments and/or derivatives thereof may be utilized to treat diseases and disorders of the nervous system which may be associated with alterations in the pattern of CART expression or which may benefit from exposure to CART or anti-CART antibodies.
CART protein, and/or fragments or derivatives thereof, may be used to treat patients in whom various cells of the nervous system have degenerated and/or have been damaged by trauma, surgery, stroke, ischemia, infection, metabolic disease, nutritional deficiency, malignancy, and/or toxic agents. In particular, the CART protein or fragments thereof can be used to treat conditions in which damage caused by various events such as trauma, surgery, infarction, infection, malignancy, exposure to a toxin, or a nutritional deficiency, has occurred in hippocampal neurons, dopaminergic neurons, sensory neurons such as photoreceptor cells, and/or motor neurons. In other embodiments of the invention, CART protein and/or fragments or derivatives thereof can be used to treat congenital conditions or neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Parkinson-Plus syndromes (in which Parkinsonian symptoms result from degeneration of dopaminergic neurons) ; amyotrophic lateral sclerosis; epilepsy; emotional disorders and dementias; substance abuse or addiction (alcoholism, cocaine, heroin, amphetamine, or the like) ; ischemia resulting from stroke, head trauma, and/or chronic cerebral blood flow disorders; spinal cord diseases such as spinal muscular atrophy; amyotrophic lateral sclerosis (ALS) ; Charcot- Marie-Tooth syndrome; and/or dystrophies or degeneration of the neural retina such as retinitis pigmentosa (autosomal dominant, autosomal recessive, sex-linked) , fundus flaimaculatus (autosomal recessive) , senile macular degeneration, drug-induced retinopathies, progressive cone-rod degenerations, vascular retinopathies, stationary forms of night blindness, drug-induced retinopathies, photoic maculopathy, macular holes, and retinal detachment.
In addition, CART protein or peptide fragments or derivatives derived thereof can be used in conjunction with surgical implantation of tissue in the treatment of Alzheimer's disease and/or Parkinson's disease, or in other neurological diseases in which tissue implantation is indicated. CART may be used to promote the survival and regeneration of dopaminergic or other neurons in a dose-dependent manner.
The following examples are intended for illustration purposes only, and should not be construed as limiting the scope of the invention in any way. EXAMPLES
The materials used in the Examples were obtained as follows.
Cell Culture Media
High glucose Dulbecco's Modified Eagle's Medium (DMEM; #11965-092), Ham's F12 medium (F12; #11765-021), Leibovitz's L15 medium without sodium bicarbonate (#41300-039) ; B27 medium supplement (#17504-010) , penicillin/streptomycin
(#15070-014), L-glutamine (#25030-016), Dulbecco's phosphate- buffered saline (D-PBS; #14190-052), Hank's balanced salt solution with calcium and magnesium salts (HBSS; #24020-026), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acic (HEPES; #15630-015), mouse laminin (#23017-015), bovine serum albumin, fractionV (#110-18-017) all from GIBCO, Grand Island, NY; heat-inactivated horse serum from Hyclone, Logan, Utah; conalbumin (C-7786) , poly-L-ornithine hydrobromide (P- 3655), bovine insulin (1-5500), human transferrin (T-2252) , putrescine (P-6024) , progesterone (P-6149), sodium selenite (S-9133) , metrizamide (M-3383) all from Sigma Chemical Company, Saint-Louis, MO; papain, deoxyribonuclease I (DNAase) and ovalbumin (Papain dissociation system) from Worthington Biochemicals, Freehold, NJ; Falcon sterile 96- well microplates (#3072) , tissue culture plastic ware and polypropylene centrifuge tubes from Beckton-Dickinson, Oxnard, CA; Nunc Lab-Tek tissue culture chamber coverglasses (#136439) from Baxter, Irvine, CA; 20 μ (#460) nylon mesh from Tetko, Elmsford, NY; 4" dissecting forceps and 4" dissecting scissors from Roboz Surgical, Washington, DC.
Antibodies and Related Reaσents
Polyclonal rabbit anti-tyrosine hydroxylase (TE101) and polyclonal rabbit anti-serotonin (NT102) from Eugene Tech, Ridgefield Park, NJ; monoclonal anti-microtubule-associated protein-1 (MAP-1, M4278) from Sigma; monoclonal anti-MAP-2 (#1284-959) from Boehringer-Mannheim, Indianapolis, IN; polyclonal rabbit anti-neuronal-specific enolase (NSE, AB951) from Chemicon, Temecula, CA; biotinylated horse anti-mouse IgG, biotinylated goat anti-rabbit IgG and peroxidase- conjugated avidin/biotin complex (ABC Elite; kit PK-6100) from Vector Laboratories, Burlingame, CA; 3 ',3'- diaminobenzidine from Cappel Laboratories, West Chester, PA; Superblock blocking buffer in PBS (#37515) from Pierce, Rockford, IL; Triton X-100 (X100) , Nonidet P-40 (N6507) and hydrogen peroxide (30%, v/v; H1009) from Sigma; GBR-12909 dopamine uptake inhibitor (D-052) from RBI, Natick, MA; 3H- dopamine (NE-131; 21 Ci/mmol) from New England Nuclear, Boston, MA; [3H] -4-aminobutyric acid (GABA; catalog no. TRK527; about 94 Ci/mmol) from Amersham, Arlington Heights, IL; Optiphase Supermix scintillation cocktail from Wallac, Turku, Finland; white ViewPlate-96 microplates (#6005182) from Packard Instruments Corporation, Meriden, CT. All other reagents were obtained from Sigma Chemical Company, unless otherwise specified.
Preparation of Media
The basal medium was prepared as a 1:1 mixture of DMEM and F12 medium, and was supplemented with B27 medium supplement (GIBCO) added as a 50-fold concentrated stock solution. L-glutamine was added at a final concentration of about 2 mM, penicillin at about 100 IU/1, and streptomycin at about 100 mg/1. Various additions were then made to this basal medium for the different types of neurons cultured. These additions wee as follows. For fetal rat substantia nigra, striatum, hindbrain, and spinal cord cultures, heat-inactivated horse serum was added to a final concentration of about 15 percent.
For post-natal rat brain cultures, and mouse and chick retina cultures, heat-inactivated horse serum was added to a final concentration of about 2.5 percent, D-glucose was added to a final concentration of about 5 g/1, HEPES buffering agent was added to a final concentration of about 20 mM, bovine insulin was added to a final concentration of about 2.5 mg/ml, and human transferrin was added to a final concentration of about 0.1 mg/ml. After mixing, the pH was adjusted to about 7.3 and the media was kept at 4°C. The media were prepared fresh just before use in order to minimize inter-experimental variations. Plastic pipettes and containers were used throughout to minimize protein adsorption.
Preparation of CART
Purified rat recombinant CART-1 and CART-2 were prepared as 10 μg/ml solutions in D-PBS (phosphate buffered saline prepared with distilled water) containing 5 percent bovine serum albumin. The solutions were stored at 4°C in aliquots. Serial dilutions were prepared in 96-well microplates. Ten microliters of ten-fold concentrated CART solutions were added to cell cultures containing 90 μl of culture medium. Control cultures received 10 μl of D-PBS with 5 percent albumin. The treatments were initiated one hour after cells were seeded and, in some instances, repeated after 3 days.
Culture Substratum
To encourage optimal attachment of substratum neurons and neurite outgrowth, microtiter plate surfaces (the culture substratum) were modified by sequential coating with poly-L- ornithine followed by laminin as follows. The plate surfaces were completely covered with a 0.1 mg/ml sterile solution of polyornithine in 0.1 M boric acid (pH 8.4) for at least 1 hour at room temperature, followed by a sterile wash with Super-Q water. The water wash was then aspirated and a 1 μg/ml solution of mouse laminin (GIBCO) in PBS was added and incubated at 37°C for 2 hours. These procedures were conducted just before using the plates in order to ensure reproducibility of the results. Preparation of Embryonic Rat Brain Cells Embryonic rat brains were used as the source of substantia nigra, striatum, and hindbrain serotoninergic neurons. Timed-pregnant Sprague-Dawley rats at embryonic day 15 were used. A maximum of 36 embryos (about 3 litters) were processed per experiment. The pregnant rats were killed by exposure to CO2, their abdominal cavities opened with dissecting scissors and the fetuses were removed from the uteri. Fetal brains were then dissected, cleaned of blood and meninges, and the desired regions (substantia nigra, striatum, and hindbrain) were dissected using well-defined anatomical landmarks (Altman and Bayer, Atlas of Prenatal Rat Brain Development, CRC Press, Boca Raton, FL [1995]). The cells were collected in ice-cold D-PBS, and then transferred into 10 ml dissociation medium (120 units papain and 2000 units DNase in HBSS) . The tissues were incubated for 45 minutes at about 37°C on a rotary platform shaker at about 200 rpm. The cells were then dispersed by trituration through fire-polished Pasteur pipettes, sieved through a 20 μm Nitex mesh to discard undissociated tissue, and centrifuged for 5 minutes at 200 x g using an IEC clinical centrifuge. The resulting cell pellet was resuspended into HBSS (Gibco/BRL, Grand Island, NY) containing ovalbumin and about 500 units DNAase, layered on top of a 4 percent ovalbumin solution (in HBSS) and centrifuged for about 10 minutes at 500 x g. The final pellet was resuspended in complete culture medium (see above), adjusted to about 28,000 cells/ml, and seeded in 90 μl aliquots into the 6 mm-wells of 96-well microplates (Falcon) previously coated with polyornithine and laminin. Attachment of cells occurred rapidly, and the plating efficiency was about 75 percent.
Preparation of Dissociated Neonatal Brain Cells
Neonatal brains were used to establish cultures of hippocampal and substantia nigra neurons. Sprague-Dawley post-natal 4-6 day rat pups were used to obtain neonatal brain cells. Each pup was held by its muzzle and sprayed with 70 percent ethanol from a spray bottle and then quickly decapitated. The cartilaginous skull was then exposed and incised starting at the foramen magnum. Each side of the skull was pulled away laterally with forceps to expose the brain surface. The whole brain was lifted from the base of the skull and placed in a small Petri dish with enough D-PBS to cover the brain. After the brains were collected, they were trimmed of residual meningeal tissue. The regions containing the hippocampus and the substantia nigra were dissected with hypodermic needles from parasagittal brain slices, following published anatomical landmarks (Paxinos, Tδrk, Tecott and Valentino, Atlas of the Developing Rat Brain, Academic Press, San Diego, CA [1991]). Dissociation procedures were the same as described for fetal brain cells (see above) . The final cell suspension was then preplated into 100-mm diameter plastic Petri dishes (about 1 million cells per dish) in DMEM/F12 containing 20 percent horse serum. After about two hours of incubation, the unattached cells (representing about 75 percent of the initial cell population) were collected, pelleted by centrifugation, resuspended in complete culture medium and plated into 96- well microplates precoated with polyornithine and laminin. The seeding densities were about 250 cells per well for hippocampal cultures and about 30,000 cells per well for substantia nigra. The complete culture medium consisted of DMEM/F12 supplemented with B27, HEPES, insulin, transferrin, D-glucose and 2.5 percent horse serum (see above) . The tissues were collected in ice-cold D-PBS and then transferred to dissociation medium (120 units papain and 2000 units DNAase in HBSS) . Dissociation procedures were the same as for fetal brain cells (see above) , and the culture medium consisted of DMEM/F12 supplemented with B27, HEPES, insulin, transferrin, D-glucose and 2.5 percent horse serum (see above) . Purification of Spinal Cord Motor Neurons Spinal cords were removed from 15-day old Sprague-Dawley rat embryos and freed of ganglia and adhering meninges. Only the lumbar segments of the cords were used. Partially purified spinal cord motor neuron cultures (approximately 75 percent motor neurons) were prepared using methods described by Camu and Henderson (J. Neurosci . Meth . , 44:59-70 [1992]) with slight modifications as follows. The spinal cord tissues were cut into small pieces (about 1 cubic mm) , subdivided into ventral and mediodorsal segments, and then incubated in 0.05 percent trypsin in D-PBS for about 17 minutes at 37°C, after which time a 10 percent volume of horse serum was added. The spinal cords were then placed in dissociation medium consisting of Leibovitz L15 medium
(without sodium bicarbonate) supplemented with about 250 units/ml DNAase, about 100 μg/ml conalbumin, about 100 mg/ml bovine serum albumin, about 5 μg/ml bovine insulin, about 0.1 M putrescine, about 20 nM progesterone, about 30 nM sodium selenite, about 20 mM D-glucose, about 100 IU penicillin and about 100 μg/ml streptomycin. The tissue was made into a single cell suspension by gentle trituration through a fire- polished siliconized Pasteur pipette. The resulting cell suspension was then passed through a 20 μm nylon mesh and then subjected to a cell fractionation procedure using a metrizamide gradient. The cell suspension (about 2 ml containing cells dissociated from 10-15 lumbar ventral cords) was applied to the top of 4 ml of 6.8 percent metrizamide previously placed in 15 ml sterile polystyrene tubes. The tubes were centrifuged for about 20 minutes at 1650 rpm at room temperature in a bench-top Beckman centrifuge. The large cells forming a sharp band at the interface of the metrizamide cushion (0-6.8 percent interface) were aspirated, washed in 4 volumes of dissociation medium, and layered on to 2 ml of 4% bovine serum albumin in L15 medium. The cells were then centrifuged for about 10 minutes at 800 rpm and the resulting pellet was resuspended in about 1 ml of complete culture medium (DMEM/F12 + 15 percent horse serum; see above), adjusted to a concentration of about 17,000 cells per ml, and subsequently plated in 96-well microplates precoated with polyornithine and laminin at a density of about 1,500 cells/6-mm well in 90 μl of medium.
Preparation of Photoreceptor Cells
Seventeen-day-old White Leghorn chick embryos and 5-day- old C57B1/6 mouse pups were killed by decapitation and the eyes were dissected under sterile conditions into L15 medium (minus sodium bicarbonate) . The eyes were hemisected and the lens and vitreous were removed. The neural retinas were carefully removed and dissected free of the pigment epithelium, placed into L15, cut into small (about 1 square mm or less) fragments, incubated for about 45 minutes in papain/DNase dissociation medium, and dissociated into a single cell suspension, as described above. The cells were seeded into polyornithine and laminin coated 96 well microtiter plates at a density of about 12,500 cells/6-mm well (in 90 μl of complete culture medium consisting of DMEM/F12 supplemented with B27, HEPES, insulin, transferrin, D-glucose and 2.5 percent horse serum (see above). Under those conditions, retinal cultures consisted of more than about 80 percent (mouse) and about 60 percent (chick) photoreceptor cells.
Immunohistochemistry of Neurons
An indirect immunoperoxidase method described by Louis et al . (J. Pharmacol . Exp . Therap . , 262:1274-1283 [1992]; Science, 259:689-692 [1993]) was used with slight modifications as follows to characterize specific neuronal populations. Cultures of neurons were fixed for about 30 minutes at room temperature with 4 percent paraformaldehyde in D-PBS, pH 7.4, followed by three washes in D-PBS (200 μl per 6-mm well) . The fixed cultures were then incubated in Superblock blocking buffer (Pierce Chemical Company) in PBS containing 1 percent NP-40 to increase the penetration of the antibodies. The primary antibodies were then applied at a dilution of between 1:1000-1:4000 in the same buffer and incubated for 1 hour at 37°C on a rotary shaker. After three washes with D-PBS, the bound antibodies were detected using goat-anti-rabbit or horse-anti-mouse biotinylated IgG (Vectastain kit from Vector Laboratories, Burlingame, CA) at about a 1:500 dilution; these secondary antibodies were incubated with the cells for about 1 hour at 37°C. The cells were then washed 3 times with D-PBS, and the secondary antibodies were detected with avidin-biotin-peroxidase complex diluted at 1:500, and the cells were incubated for about 45 minutes at 37°C. After three more washes with D- PBS, the cultures were reacted for 5-20 minutes in a solution of 0.1 M Tris-HCl, pH 7.4, containing 0.04 percent 3',3'- diaminobenzidine- (HC1) 4, 0.06 percent NiC12 and 0.02 percent hydrogen peroxide.
The specific markers used for particular cell types were as follows. Dopamine-producing neurons in the substantia nigra cultures were identified using a rabbit polyclonal antiserum to tyrosine hydroxylase (TH; Eugene Tech International) . Neurons in spinal cord, hippocampal, and striatal cultures were identified by staining with monoclonal antibodies to the markers MAP-1 and MAP-2 and neuronal- specific enolase. Photoreceptors cells in cultures of retina were identified by a polyclonal rabbit antibody directed to a synthetic peptide sequence of the rod-specific protein arrestin. The sequence is:
Val-Phe-Glu-Glu-Phe-Ala-Arg-Gln-Asn-Leu-Lys-Cys (SEQ ID NO:6)
Determining Neuronal Survival
Neuronal cell cultures were fixed and processed for immunostaining as described above, and then examined with bright-field optics at 200X magnification. The number of stained neurons was counted in either the entire 6 mm-well (dopamine-containing neurons; serotonin-containing neurons; hippocampal neurons) or in one diametrical 0.4 X 6 mm strip, representing 10 percent of the total surface area of a 6 mm- well (motor neurons; photoreceptor cells; striatal neurons) . Viable neurons were characterized as having a regularly- shaped cell body, with a major axon-like process and several dendrite-like processes. Neurons showing signs of degeneration, such as having irregular, vacuolated perikarya or fragmented neurites, were excluded from the counts (most of the degenerating neurons, however, detached from the culture substratum) . Cell numbers were expressed either as cells/6-mm well or as the fold-change relative to control cell density.
Neurite Analysis
Morphometric analysis of neurite (i.e., both axons and dendrites) development was performed using 12-day-old cultures of rat hippocampus. Cultures containing about 100 neurons per 6-mm well were immunostained for MAP-1 and MAP-2 and examined with brightfield optics. Photographs of each individual neuron in a"6-mm well of control and treated cultures were taken with an Optronics video-camera and enlarged to a final magnification of approximately 800-fold. Neuritic size was determined by measuring the combined length of the neurites of each neuron with a stylus coupled to a SummaSketchll digitizing tablet (Summagraphics Corporation, Houston, TX) , utilizing a digitizing program (MacMeasure 1.9) and a Macintosh Centris 650 personal computer. Neuritic complexity was assessed by counting the number of primary neurites emerging from the soma and the number of branching points per neuron.
Dopamine and GABA Uptake Dopamine uptake was determined in cultures of 15-day-old embryonic rat substantia nigra neurons that had been established in 96-well microplates. The cultures were washed with about 100 μl of pre-warmed uptake buffer which consists of a modified Krebs-Ringer solution, pH 7.4 containing about 120 mM NaCl, 4,7 mM KC1, 1.8 mM CaCl2, 1.2 mM MgS04, 32 mM NaHP04, 1.3 mM EDTA, and 5.6 mM D-glucose. For dopamine uptake, the uptake buffer also contained 1 mM ascorbic acid and 50 μM paragyline. The cells were then preincubated at 37°C for about 10 minutes in uptake buffer. Tritiated dopamine (21 Ci/mmol) or tritiated GABA (94 Ci/mmol) was then added to substantia nigra or striatal cell cultures, respectively, at a concentration of about 50 nM in 75 μl of uptake buffer, and the cultures were incubated for about 60 minutes at 37°C. Non-specific dopamine uptake was determined by incubating the cultures with uptake buffer containing the dopamine uptake inhibitor GBR-12909 (1 μM) . Non-specific uptake represented less than about 1 percent of total uptake. The uptake assays were arrested by aspiration of the incubation medium followed by three rapid washes with about 120 μl of ice-cold uptake buffer. The cells were then lysed by addition of 200 μl of Optiphase Supermix scintillation cocktail (Wallac) , and radioactivity was determined by scintillation spectrometry using a Wallac MicrobetaPlus 96- well microplate counter. The results are expressed either as dpm/6-mm well or as the fold-change relative to control cultures.
EXAMPLE 1
Effect of CART on Substantia Niσra Dopaminergic Neurons
A. Dopaminergic Neuron Survival and Morphological Development
Cultures of embryonic day 15 (E15) and post-natal day 6 (P6) rat substantia nigra enriched in dopaminergic neurons were used to demonstrate the effect of CART on the survival of dopaminergic neurons. The cultures were grown in polyornithine-laminin-coated 96-well microplates for up to 7 days alone or in the presence of 1, 10 or 100 ng/ml rat recombinant CART-1 or CART-2. The culture medium consisted of DMEM/F12 supplemented with 15 percent heat-inactivated horse serum (E15 cultures) or 2.5 percent heat-inactivated horse serum, D-glucose, HEPES, insulin and transferrin (P6 cultures) . Additional CART (at the same concentration) was added to the culture medium on day 3. Immunostaining for tyrosine hydroxylase (TH) , the rate-limiting enzyme in dopamine biosynthesis, was used as a marker for dopaminergic neurons. Since noradrenergic neurons in the rhombencephalon also stain positive for TH, great care was taken to dissect an area restricted to the ventral tegmentum of the mesencephalon and to avoid the more caudal regions containing the noradrenergic cell bodies. After 6-7 days, both the E15 and P6 the cultures typically consisted of about 70 percent neurons as identified by neuronal specific enolase immunostaining (described above) and 30 percent non-neuronal cells (which had a flattened, phase-dark appearance) ; dopaminergic neurons represented about 5-15 percent of the neuron population.
Cultures of both E15 and P6 substantia nigra treated with either CART-1 or CART-2 contained about 40 to 80 percent more TH-immunoreactive neurons than untreated control cultures, suggesting that CART promotes the survival of dopaminergic neurons. Cultures of E15 substantia nigra treated with CART-1 or CART-2 showed an increase the number of TH-positive neurons in cultures after 3 days in vitro (Figure 6A) ; CART-1 was slightly more efficient (about 80 percent TH-postive neurons) than CART-2 (about 55 percent TH- positive neurons) . The saturating effect of CART was observed with concentrations as low as 1 ng/ml. The effect of CART on survival was still observed after 6 days in culture (Figure 7A-C) . CART also promoted the survival of dopaminergic neurons in cultures of P6 substantia nigra; after 7 days in culture, there were about 50 percent more TH- positive neurons in both CART-1 and CART-2 treated cultures as compared to controls (Figure 6B) . Figure 6A is representative of four independent experiments; Figure 6B is the average of three independent cultures.
Comparison of control and CART-treated cultures revealed pronounced effects of CART-1 and CART-2 on the morphological differentiation of dopaminergic neurons (Figures 7 and 8) . TH-immunoreactive neurons in CART-treated cultures possessed significantly more complex and extensive neuritic arborization, as well as a higher degree of neurite branching (Figure 7B-C for E15 cultures; Figure 8C for P6 cultures) and an overall larger soma size (Figure 8C) than TH-positive neurons in control cultures (Figure 7A; Figure 8A-B) .
B. Dopamine Uptake
Dopamine (DA) uptake measures the number and activity of high-affinity DA reuptake transporter sites and reflects the functional differentiation of dopaminergic neurons. DA uptake was measured in cultures of E15 rat substantia nigra after 3 days in vitro either with or without CART-1 or CART- 2. In these cultures, DA uptake had the pharmacological profile characteristic of dopaminergic neurons, i.e. it was nearly completely blocked (more than 98 percent) by 1 μM GBR- 12909, a DA transporter inhibitor specific for dopaminergic neurons (ID50 = 20 nM) . This indicates that DA uptake measurements do not reflect the presence of contaminating noradrenergic neurons, which can take up DA through norepinephrine transporters but are not sensitive to GBR- 12909 inhibition. CART stimulated DA uptake, with CART-1 being slightly more efficient than CART-2 (about 40 percent increase for CART-1 as compared to about a 30 percent increase for CART-2; Figure 9) . Saturating effects for both CART-1 and CART-2 were observed with 100 pg/ml. EXAMPLE 2
The Effect of CART on Spinal Cord Motor Neurons
Enriched cultures of motor neurons about 75 percent pure) were used to demonstrate the effect of CART on motor neuron survival. The cultures were prepared from lumbar spinal cord tissue of 15-day-old rat embryos (the age at which motor neuron cell death occurs) , using the metrizamide density gradient cell separation method described above.
Purified motor neurons were removed from the 0-6.8 percent metrizamide interface and were seeded at a density of about 1,500 cells per 6-mm well (28 mm2 of culture surface). More than 95 percent of the cells were phase bright and a similar percentage stained positively for neuronal-specific enolase (NSE) , a general neuronal cell marker. On average, approximately 900-1,100 of these cells (about 60-75 percent) were motor neurons as determined by the size of the cell bodies (at least about 35 μm in size) and by NSE staining. Consistent with these results, metrizamide-purified spinal cord cultures have been previously reported to consist of 60- 80 percent of motor neurons, as identified by choline acetyltransferase (CAT) activity and immunostaining for CAT, low-affinity nerve growth factor receptor, L14 ( a motor neuron lectin) , and Islet-1 homeoprotein (Camu and Henderson, J. Neurosci . Meth . , 44:59-70 [1992]; Wong et al . , Eur. J. Neurosci . , 5:466-474 [1993]; Smith et al . , J. Neurosci . , 6:439-446 [1986]) .
Cell viability was assessed after 3 days in culture by counting the number of large (greater than about 35 μm) NSE- positive neurons. At this time, about 65 percent of the original number of motor neurons in the control (untreated) cultures had already died (Figure 10) . Most of the surviving neurons in the control cultures displayed signs of degeneration, such as vacuolation, membrane blebbing and fragmentation of neurites (Figure 11A) . In CART-treated cultures, approximately 65 percent of the original population of large NSE-positive motor neurons remained healthy after 3 days in vitro (Figure 10) , and developed long and complex neurites (Figure 11B-C) . Maximal efficacy was observed with 100 pg/ml CART-1, with an ED50 of about 20 pg/ml (Figure 10) .
EXAMPLE 3
The Effect of CART on Hippocampal Neurons
Cultures of post-natal day 4 rat hippocampus cells consisting of more than 90 percent neurons were prepared as described previously (Louis et al . , Eur. J. Neurosci . , 5:1610-1621 [1993]) and used to demonstrate the effect of CART on hippocampal neuron survival and differentiation. Hippocampal neurons were seeded in polyornithine-laminin- coated 96-well microplates at a density of about 250 cells per 6-mm well (28 mm2 of culture surface) ; on average approximately 200 cells survived the culture procedure. Neuronal survival and development was assessed over a period of 12 days. Cultures were fixed in paraformaldehyde after 2 , 6 and 12 days, and were then immunostained for the specific neuronal markers MAP-1 and MAP-2. The survival in control cultures was excellent at 2 days. Thereafter, the number of neurons in the control cultures decreased with time in culture; after 6 days only about 55 percent of the cells survived, and after 12 days, only about 25 percent of the original population of neurons (about 200) remained (Figure 12A) . The addition of CART at days 0, 3, 6, and 9 to the culture medium significantly increased the survival of hippocampal neurons at both 6 and 12 days (Figure 12A) . About 60 percent of the initial population remained after 6 days, and about 50 percent of this population was still healthy after 12 days (this represents a two-fold increase over control cultures) . The effect of CART was maximal at about 10 pg/ml, with an ED50 of about 1 pg/ml (Figure 12B) . These results suggest that a subpopulation of hippocampal neurons depends on CART for survival. The morphometric analysis of all the neurons present in a control 6-mm well and a 6-mm well treated with 100 pg/ml CART-1 indicated that CART also enhanced the morphological differentiation of this subpopulation of neurons. CART induced an increase in the size of the cell bodies and an increase in the size and complexity of neuritic arborizations, namely the combined neurite length (Figure 13A) and the mean number of segments and branching points per neuron (Figure 13B) . Figure 14 shows the typical morphologies of neurons from control (14A- B) and CART-treated cultures (14C-D) . The population of hippocampal neurons supported by CART has morphological and immunohistochemical (expression of the glutamate receptors Glu-R2,-3 and -4; data not shown) properties characteristic of pyramidal neurons from the CAl and CA3 regions of the hippocampus.
EXAMPLE 4
The Effect of CART on Photoreceptor Cells
A. Photoreceptor Cell Survival and Axonal Growth
Cultures of post-natal day 6 mouse retinas were used to demonstrate the effect of CART on photoreceptor cell survival. Dissociated retinal cells were seeded into polyornithine-laminin-coated microplates at a density of about 12,500 per 6-mm well in DMEM/F12 supplemented with B27 medium supplement, 2.5 percent heat-inactivated horse serum, D-glucose, HEPES, insulin and transferrin. These cultures consisted of 75-90 percent photoreceptor cells, as identified by immunostaining for arrestin, a rod-specific antigen. The remaining cells were large multipolar and smaller unipolar NSE-positive neurons. Photoreceptor cells appeared as rounded cells with a small cell body diameter, one or two neurites and, in some cases, a short vertical process that probably represents the connecting cilium (Figure 15A-C) . At this level of resolution, there was no evidence of outer segment formation. In untreated cultures, the number of photoreceptors cells declined steadily over time to reach about 25 percent of the initial number after 6 days in culture. Treatment of the cultures with CART resulted in a significant increase in the number of viable photoreceptor cells after 6 days in culture (about 70 percent for CART-1 and 50 percent for CART-2) . As can be seen in Figure 16, the effect of CART was maximal at about 100 ng/ml (CART-1) and 1 ng/ml (CART-2) , with the ED50 of about 10 pg/ml (CART-1) and 40 pg/ml CART-2) . In addition to promoting photoreceptor cell survival, CART also stimulated the extension of their axon- like process (Figure 15B-C) .
B. Promotion of Outer Segment Development
The effect of CART on morphological development of photoreceptor cells was studied in cultures of embryonic day 17 chick retinas. The cultures were prepared using the same method as for mouse retinas. The resulting cultures contained about 60 percent photoreceptor cells and 40 percent large multipolar neurons. In control cultures after 6 days in vitro, the photoreceptor cells could be identified by oval cell bodies that were occupied almost exclusively by the nucleus, a short inner segment with a small lipid droplet, a single short, unbranched neurite emerging from a point opposite to the lipid droplet, and a short distal cilium (Figure 17A) . In CART-treated cultures, a large proportion of photoreceptor cells appeared as highly elongated, polarized, compartmentalized cells (Figure 17B-G) . These photoreceptor cells had an elongated inner segment that was in some cases connected to a tri-dimensional, phase-bright structure characteristic of an immature outer segment (Figure 17B and D-G) . Other photoreceptor cells developed a thick, long and branched neurite (Figure 17B-C) . The development of photoreceptor outer segments starts at embryonic day 15 in the chick embryo. Therefore, the effect of CART on outer segment development reflects the ability of this factor to promote the regeneration of outer segments damaged by the dissociation procedure.
EXAMPLE 5
The Effect of CART on Striatal Neurons
Striatal neuron cultures were prepared and cultured as described above, and were treated with varying concentrations of CART-1 or CART-2 as shown in Figure 18. After 6 days in culture, the number of neurons (18A) or GABA uptake (18B) was measured. As is apparent from Figures 18A and 18B, no significant increase in either neuron number or GABA uptake was apparent.
EXAMPLE 6
The Effect of CART on Serotonin Containing Neurons
Serotonin-containing neurons from rat embryonic day 15 hindbrain were prepared and cultured as described above. After 8 days in culture, the cells were immunostained for serotonin. The results are shown in Figure 19. As is apparent, the number of serotonin-positive neurons in cultures treated with CART-1 or CART-2 was not significantly different from the number of these neurons in control (untreated) cultures. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Amgen Inc.
(ii) TITLE OF INVENTION: Methods of Preventing Neuron Degeneration and Promoting Neuron Regeneration
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Amgen Inc.
(B) STREET: 1840 Dehavilland Drive
(C) CITY: Thousand Oaks
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 91320-1789
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 4-MAY-1995
(C) CLASSIFICATION:
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 840 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
AGCGAGGAAG TCCAGCACCA TGGAGAGCTC CCGCCTGCGG CTGCTACCCG TCCTGGGCGC 60
CGCCCTACTG CTGCTGCTAC CTTTGCTGGG TGCCGGTGCC CAGGAGGATG CCGAGCTGCA 120
GCCCCGAGCC CTGGACATCT ACTCTGCCGT GGATGATGCG TCCCATGAGA AGGAGCTGCC 180
AAGGCGGCAA CTTCGGGCTC CCGGCGCTGT GTTGCAGATT GAAGCGCTGC AGGAAGTCCT 240
GAAGAAGCTC AAGAGTAAAC GCATTCCGAT CTATGAGAAG AAGTACGGCC AAGTCCCCAT 300
GTGTGACGCT GGAGAGCAGT GCGCAGTGCG GAAAGGGGCC AGGATCGGGA AGCTGTGTGA 360 CTGTCCCCGA GGAACTTCTT GCAATTCTTT CCTCTTGAAG TGCTTGTGAA GGGGTGACAG 420
CCTCCTTCGG TTCCCATATT TCCTCTTTCC CCTAAAGGAG CGCTCTTTTG TCCCTGGAGC 480
CGCTTTAACA ACAATAAAGT TTGCGTTCCC CCCAGAGAGT GGATGGGCTC TTTCCCTGCT 540
GCTTCAAAAT AAAAGATTTG ATGTTATTGT GTGAAGGACA ATACCTTGAA TGGTGTTGGT 600
ATGTGTGCAA AGTATTCTTC TCTCGTTTTA TCCACCTGAC ACATTCTTGT GACCTTTCTG 660
GGAAGAAGAG GGACTTTCGC TTTAAAACTG TATTTTTGTA TGTGGCGGGT CACAATGAAG 720
ATTAGACCTA GTTAATTTTG GCAGATGACA TCATAACCCG GAAAACAAAT CACCCCAAAG 780
CAACACAAAT GGAAGCATGT GCAAATTACA CCCAATAAAG CATTTTTGAT AATTGCTCAC 840
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Glu Ser Ser Arg Leu Arg Leu Leu Pro Val Leu Gly Ala Ala Leu 1 5 10 15
Leu Leu Leu Leu Pro Leu Leu Gly Ala Gly Ala Gin Glu Asp Ala Glu 20 25 30
Leu Gin Pro Arg Ala Leu Asp lie Tyr Ser Ala Val Asp Asp Ala Ser 35 40 45
His Glu Lys Glu Leu Pro Arg Arg Gin Leu Arg Ala Pro Gly Ala Val 50 55 60
Leu Gin lie Glu Ala Leu Gin Glu Val Leu Lys Lys Leu Lys Ser Lys 65 70 75 80
Arg lie Pro lie Tyr Glu Lys Lys Tyr Gly Gin Val Pro Met Cys Asp 85 90 95
Ala Gly Glu Gin Cys Ala Val Arg Lys Gly Ala Arg lie Gly Lys Leu 100 105 110
Cys Asp Cys Pro Arg Gly Thr Ser Cys Asn Ser Phe Leu Leu Lys Cys 115 120 125
Leu (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 800 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AACGACGAGT TTCAGAACGA TGGAGAGCTC CCGCGTGAGG CTGCTGCCCC TCCTGGGCGC 60
CGCCCTGCTG CTGATGCTAC CTCTGTTGGG TACCCGTGCC CAGGAGGACG CCGAGCTCCA 120
GCCCCGAGCC CTGGACATCT ACTCTGCCGT GGATGATGCC TCCCACGAGA AGGAGCTGAT 180
CGAAGCGCTG CAAGAAGTCT TGAAGAAGCT CAAGAGTAAA CGTGTTCCCA TCTATGAGAA 240
GAAGTATGGC CAAGTCCCCA TGTGTGACGC CGGTGAGCAG TGTGCAGTGA GGAAAGGGGC 300
AAGGATCGGG AAGCTGTGTG ACTGTCCCCG AGGAACCTCC TGCAATTCCT TCCTCCTGAA 360
GTGCTTATGA AGGGGCGTCC ATTCTCCTCC ATACATCCCC ATCCCTCTAC TTTCCCCAGA 420
GGACCACACC TTCCTCCCTG GAGTTTGGCT TAAGCAACAG ATAAAGTTTT TATTTTCCTC 480
TGAAGGGAAA GGGCTCTTTT CCTGCTGTTT CAAAAATAAA AGAACACATT AGATGTTACT 540
GTGTGAAGAA TAATGCCTTG TATGGTGTTG ATACGTGTGT GAAGTATTCT TATTTTATTT 600
GTCTGACAAA CTCTTGTGTA CCTTTGTGTA AAGAAGGGAA GCTTTGTTTG AAAATTGTAT 660
TTTTGTATGT GGCATGGCAG AATGAAAATT AGATCTAGCT AATCTCGGTA GATGTCATTA 720
CAACCTGGAA AATAAATCAC CCTAAGTGAC ACAAATTGAA GCATGTACAA ATTATACATA 780
ATAAAGTGTT TTTAATAATT 800 (2) INFORMATION FOR SEQ ID NO:4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2483 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCCGGGCCCT CCTCCACCCC CCCTTCCTTC TTCGCCTCCT CCCTCTTTCC TGCACGGGGG 60 CTCGGGCTCA CTATAAAAGG TGGGAGCGCG TGGTGCCCCA GCAACGACGA GTTTCAGAAC 120
GATGGAGAGC TCCCGCGTGA GGCTGCTGCC CCTCCTGGGC GCCGCCCTGC TGCTGATGCT 180
ACCTCTGTTG GGTACCCGTG CCCAGGAGGA CGCCGAGCTC CAGCCCCGAG CCCTGGACAT 240
CTACTCTGCC GTGGATGATG CCTCCCACGA GAAGGAGCTG GTCGGTATTC CCCTCGCTCT 300
CGACCCCCTT GACGTGTCGC CTTGTCTCTT CTCTTGCACG CCTCCCTCCT TCCCCCCACC 360
CCCACTCCTA TTCCCAGAGT CAGGGCGCGG GGAGCTGAGC GCAACGCCCA GGCACCCACT 420
GCCATCCGAA GAGCGACTCG AGCTCACGGG CTCCTGGCAG TCTGTTGAGC GAATCCCTCA 480
TCCCGGCCCC TCTGAGCAAC AGGGGCCCCA GCGGCTCAGA GACCCGCGGT CAGTACCTGG 540
GACAGCGTCG CTAAGTTTCC ACCCCTCGAC CATTCCCTGT GTCCGCGGAG TCCCACCGCA 600
GAGTGCGTGT GGGTCCGGGG CTCCTTATAA CTAGGGCTGG AAGTGCGCAC CTGGGCTGGG 660
CTCGCAGCAA GGCGCAACTT CAGGCTCCGA AGCGGTGTGT TGCAGATCGA AGCGCTGCAA 720
GAAGTCTTGA AGAAGCTCAA GAGTAAACGT GTTCCCATCT ATGAGAAGAA GTATGGCCAA 780
GTCCCCATGG TAAGGTTTGT GGTCACTCCC TTCCCGTGTT TTTCCAAGAG AAAGTACACC 840
GCCTTGAATC GTACACACAG CTCCGTAGGA TGTGGCTAAA TAACTTAGGT AATGGGCTTG 900
CAGGATTCTG TGGGCTCCTT CTTCCTTCCC GGGTGAGGAA ATGGGAAAGC AGGAACAGGG 960
GTTGTAAGAA AGTGTAAGTC TATTGTTTGT TGCTCAGGAA AAAGGTCTGA TTTTTTTCCC 1020
TCTGAGAGGG CAAGAAAAGG AGCCAGGAAA TGTGATGCTC CCCTTCCCAC GCCCCCCAAC 1080
CCTCGCCACT TAAAGGTGGA AGAAACTAGG ATAAAACTAA TAATGTAAGT TTCTTTAAAA 1140
AATGTACTCT CACTGAGGTT ATAAGCACAA GGCTCCCTGT TTCAGATCTG ACTGTACGTC 1200
GACCTCTTGT GATGGTGATG GGGTCCAATT GCCCCTTTCA AGAGACAGAA ATTGCGTTGA 1260
CTGTGAGACT TGCCTGTTGG GAACCTGGGT TTGTTCATAC TCGATGACCA CACATTTTGT 1320
TGTTTCAGTG TGACGCCGGT GAGCAGTGTG CAGTGAGGAA AGGGGCAAGG ATCGGGAAGC 1380
TGTGTGACTG TCCCCGAGGA ACCTCCTGCA ATTCCTTCCT CCTGAAGTGC TTATGAAGGG 1440
GCGTCCATTC TCCTCCATAC ATCCCCATCC CTCTACTTTC CCCAGAGGAC CACACCTTCC 1500
TCCCTGGAGT TTGGCTTAAG CAACAGATAA AGTTTTTATT TTCCTCTGAA GGGAAAGGGC 1560
TCTTTTCCTG CTGTTTCAAA AATAAAAGAA CACATTAGAT GTTACTGTGT GAAGAATAAT 1620
GCCTTGTATG GTGTTGATAC GTGTGTGAAG TATTCTTATT TTATTTGTCT GACAAACTCT 1680
TGTGTACCTT TGTGTAAAGA AGGGAAGCTT TGTTTGAAAA TTGTATTTTT GTATGTGGCA 1740
TGGCAGAATG AAAATTAGAT CTAGCTAATC TCGGTAGATG TCATTACAAC CTGGAAAATA 1800 AATCACCCTA AGTGACACAA ATTGAAGCAT GTACAAATTA TACATAATAA AGTGTTTTTA 1860
ATAATTGCCC ATAGTGCACT GCTGTTTTCA TATAAGTAAT TTAAGTGGAA ATGGTGAGAT 1920
TAATCATGCT GTTGTTTTCA AAGAAAAATA TTTCAAAAAT AGCAGCCTAT TGGAAATGCA 1980
CTACGTCAGA GTTGATCGTA TAGAGTTGCA GCAGTTAGTA TACCTATTTC TTGATGCAGC 2040
GAGTGTGTGT GTATGTGTGT GTGTTAGTGT GTGTGTGTGT GTGTGTGTGA GAGAGAGAGA 2100
GAGAGAGAAA GAGAGAGATG AATGAGATGG AGATGGTTGG AGAAGAGGTT ATATAATTTT 2160
GTTTATTAAA ACCTTTAGCC AGACCCTTTA CTTTAAACAG TGAGACCAAT AAACTATAAA 2220
CAGTTTCATG TTTTAGTCAC ATTAAAAGCA ATTTGAAAAA TTAGAAATTT TGTTTTGACA 2280
ACTCCCTTAT TAGAAAATAT ACATTGATTT AAAGATATGG GCTGTTTAGG GTTGTTATTT 2340
GTCTAAAGAC TCCAAGGTTA TAAGACCCAT CCATCCCACA AGTAAATTCA CACTCTTGGA 2400
AAAATTCTCT ATTCCAGGAG AAAGAGTCAT TTCAGAAAAT AGTTTTGAGG GGAACAAATA 2460
AAAATTGGAG GAGGTGAGAA TTC 2483 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 116 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 :
Met Glu Ser Ser Arg Val Arg Leu Leu Pro Leu Leu Gly Ala Ala Leu 1 5 10 15
Leu Leu Met Leu Pro Leu Leu Gly Thr Arg Ala Gin Glu Asp Ala Glu 20 25 30
Leu Gin Pro Arg Ala Leu Asp lie Tyr Ser Ala Val Asp Asp Ala Ser 35 40 45
His Glu Lys Glu Leu lie Glu Ala Leu Gin Glu Val Leu Lys Lys Leu 50 55 60
Lys Ser Lys Arg Val Pro lie Tyr Glu Lys Lys Tyr Gly Gin Val Pro 65 70 75 80
Met Cys Asp Ala Gly Glu Gin Cys Ala Val Arg Lys Gly Ala Arg lie 85 90 95
Gly Lys Leu Cys Asp Cys Pro Arg Gly Thr Ser Cys Asn Ser Phe Leu 100 105 110 Leu Lys Cys Leu 115
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Val Phe Glu Glu Phe Ala Arg Gin Asn Leu Lys Cys 1 5 10

Claims

I CLAIM:
1. A method for promoting the survival, differentiation, or regeneration of neurons, comprising contacting the neurons with an effective amount of CART or a fragment thereof.
2. The method of claim 1, wherein the neurons are selected from a group consisting of: nigral dopaminergic neurons, hippocampal neurons, motor neurons, retinal neurons, and photoreceptor cells.
3. The method of claim 1 which is conducted in vitro.
4. The method of claim 1 which is conducted in vivo to treat a neurological disease or disorder affecting the neurons.
5. The method of claim 4 wherein the neurological disorder or disease is selected from the group consisting of: substance addiction, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, ischemia, brain or spinal cord trauma, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, dementia, retinitis pigmentosa, macular degeneration, retinal detachment, and retinal vascular disease.
6. A method for treating a neurological disorder or disease comprising administering a therapeutically effective amount of CART.
7. The method of claim 6 wherein the neurological disorder or disease is selected from the group consisting of: substance addiction, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, ischemia, brain or spinal cord trauma, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, spinal muscularatrophy, dementia, retinitis pigmentosa, macular degeneration, and retinal vascular disease.
8. The method of claim 6 wherein the disease or disorder comprises damage to the nervous system.
9. The method of claim 8 wherein the damage is caused by trauma, infarction, surgery, infection, or malignancy.
10. The method of claim 6 wherein the disease or disorder is caused by exposure to a toxin.
11. The method of claim 6 wherein the disease or disorder is caused by a nutritional deficiency.
12. A composition comprising a therapeutically effective amount of CART in admixture with a pharmaceutically acceptable carrier.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998042747A1 (en) * 1997-03-26 1998-10-01 Novo Nordisk A/S Polypeptide with appetite regulating activity
US6458927B1 (en) 1997-03-26 2002-10-01 Novo Nordisk A/S Polypeptide with appetite regulating activity
WO2000063370A2 (en) * 1999-04-15 2000-10-26 Warner-Lambert Company Nucleic acids coding for novel truncated cart proteins, corresponding truncated cart polypeptides and their use for therapeutic and diagnostic purposes
WO2000063370A3 (en) * 1999-04-15 2001-09-13 Warner Lambert Co Nucleic acids coding for novel truncated cart proteins, corresponding truncated cart polypeptides and their use for therapeutic and diagnostic purposes
WO2002057782A1 (en) * 2001-01-18 2002-07-25 Taisho Pharmaceutical Co., Ltd. Novel screening method

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AU5630896A (en) 1996-11-21
ZA963417B (en) 1997-01-08

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