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WO1996003043A1 - Kinase specifique du cerveau humain - Google Patents

Kinase specifique du cerveau humain Download PDF

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Publication number
WO1996003043A1
WO1996003043A1 PCT/US1995/009334 US9509334W WO9603043A1 WO 1996003043 A1 WO1996003043 A1 WO 1996003043A1 US 9509334 W US9509334 W US 9509334W WO 9603043 A1 WO9603043 A1 WO 9603043A1
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Prior art keywords
hbsk
nucleic acid
die
dna
sequence
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PCT/US1995/009334
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English (en)
Inventor
Renping Zhou
Clara Paulhiac
Original Assignee
Rutgers, The State University Of New Jersey
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Application filed by Rutgers, The State University Of New Jersey filed Critical Rutgers, The State University Of New Jersey
Priority to AU31441/95A priority Critical patent/AU3144195A/en
Publication of WO1996003043A1 publication Critical patent/WO1996003043A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention relates, in general, to human brain specific kinase, hBsk.
  • the present invention relates to nucleie acid molecules coding for hBsk; hBsk polypeptides; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antibodies having binding affinity specifically to hBsk; hybridomas containing the antibodies; nucleic acid probes for the detection of hBsk nucleic acid; a method of detecting hBsk nucleic acid or polypeptide in a sample; and kits containing nucleic acid probes or antibodies.
  • Neuronal degeneration has been shown to be involved in many neurological disorders (Price et al., in Selective Neuronal Death, Ciba Foundation Symposium 126, Wiley & Chichester, eds. (1987), pp. 30-48).
  • AD Alzheimer's Disease
  • brain structures including the locus coemleus and raphe complex of the brainstem, the basal forebrain cholinergic system, amygdala, hippocampus and neocortex (Coleman & Flood, Neurobiol. Aging 5:521-545 (1987)).
  • the pattern of cell loss in AD has similarities to that in the aging brain, the speed and amount of loss is far greater.
  • the most striking loss of neurons compared with the age-matched controls occurs in the hippocampal region, with a loss of up to 57% of the pyramidal cells (Coleman and Flood, Neurobiol. Aging 5:521-545 (1987)).
  • hippocampus is a key structure in the neurobiology of AD.
  • the extent of cell loss is most evident in the CA1 and subiculum, while areas CA3 and CA4 and granular cells of the dentate gy s are largely spared (Van Hoesen and Hyman, Progress in Brain Research 55:445-457 (1990)).
  • Neuronal degeneration in the hippocampus has long been known to be a site of pathological change in epileptic patients (Nadler, in The
  • CA3-CA4 damage is nearly always observed in pharmacologically intractable complex partial (limbic, temporal lobe, psychomotor) epileptic patients, while CA1 damage is also frequently observed (Nadler, in The Hippocampus-New Vistas, Chan-Palay V.,
  • Hippocampal defects are also suggested to be involved in schizophrenia (Bogerts et al., Arch. Gen. Psychiatry 42:184-191 (1985)).
  • Significant reductions in hippocampal volume were found in chronic schizophrenic patients, possibly due to degenerative shrinkages of unknown etiology (Bogerts et al., Arch. Gen. Psychiatry 42:1 '84-791 (1985); Bogerts et al , Biol. Psychiatry 55:236-246 (1993).
  • the reduced volume in hippocampus and other limbic system structures such as amygdala and parahippocampal gyms was associated with increased severity of psychopathology (Bogerts et al, Biol.
  • the hippocampus and its adjacent, anatomically related entorhinal, per ⁇ hinal, and parahippocampal cortices play an essential, although temporal, role for establishing long-term memory for facts and events (Squire and Zola-Morgan, Science 255:1380-1386 (1991)).
  • the widespread and reciprocal connections between hippocampal structures and neocortex may explain their degeneration in a variety of neurological diseases. Understanding the mechanism of neuronal survival in the hippocampus may help to develop effective treatments of neural degenerative diseases or disorders as well as neoplasms involving neuronal tissue.
  • Nerve growth factor has been a model trophic factor (Levi-Montalcini, Science 257: 1154-1162 (1987); Black et al , in Current Topics in Developmental Biology,
  • fibroblast growth factors are well known- mitogens (Gospodarowicz, in Current Topics in Developmental Biology, Vol. 24, Hilsen-Hamilton, M. ed., Academic Press Inc., San Diego (1990), pp. 57-93) that exhibit potent neurotrophic activity both in vivo (Anderson et al, Nature 552:360-361 (1988)) and on cultured neurons from many brain regions (Morrison et al, Proc. Natl. Acad. Sci. USA 55:7537-7541 (1986); Morrison, Neuroscience Res.
  • EGF Epidermal growth factor
  • BDNF neurotrophin-3
  • NT-3 neurotrophin-3
  • NT-3 was shown to support the growth of neurons from dorsal root ganghon, the neural placode-derived nodose ganghon, and the paravertebral chain sympathetic ganghon (Maisonpierre et al. , Science 247:1446-1451 (1990)).
  • the potential involvement of growth factors in neuronal regeneration aft ⁇ - injuries or in disease is demonstrated by the fact that brain injury causes a time dependent increase in neurotrophic activity at the lesion site (Nieto-Sampedro et al, Science 277:860-861 (1982).
  • intraventricular administration of NGF prevents retrograde degeneration of axotomized septal cholinergic neurons (Hefti J. Neurosci.
  • Growth/trophic factors function through their receptors which often possess intrinsic protein tyrosine kinase activity (Schlessinger & Ullrich, Neuron 9:383-931 (1992)).
  • the receptor protein-tyrosine kinases are composed of an extracellular domain, a membrane spanning domain and a catalytic domain (Schlessinger and Ullrich, Neuron 9:383-391 (1992). Binding of the growth/trophic factor to the extracellular domain activates the catalytic domain inside the cell and results in phosphorylation of substrates within the cell. Activation of the receptor is believed to mediate a variety of cellular processes including cell growth and differentiation.
  • tyrosine kinases are expressed during embryogenesis and are therefore believed to be important in the mechanisms underlying oncogenesis and cellular growth (Wilks, Advances in Cancer Research 60:43-13 (1993)). Increased or aberrant expressions of tyrosine kinase receptors has been associated with several human neoplasms, including glioblastomas, squamous carcinomas, breast and gastric cancers (Carpenter, Ann. Rev. Biochem. 56: 881-914 (1987); Muller et al., Cell 54:105-109 (1989); Kraus et al, Proc. Natl. Acad. Sci. USA 56 ⁇ 9193-9197 (1989)).
  • Alzheimer's epilepsy and schizophrenia are but a few of the diseases associated with degeneration of neurons in the hippocampus.
  • the factors needed for the regeneration and survival of neurons in the hippocampus and its associated limbic system are poorly characterized.
  • the identification of factors which promote the regeneration and survival of these neurons will be potentially useful in the treatment of the neoplasms, neurodegenerative diseases or disorders and brain injuries involving the limbic system.
  • the invention provides an isolated nucleic acid molecule coding for a polypeptide comprising an amino acid sequence corresponding to human brain specific kinase, hBsk.
  • the invention further provides a substantially pure polypeptide comprising an amino acid sequence corresponding to hBsk.
  • the invention also provides a nucleic acid probe for the specific detection of the presence of hBsk in a sample.
  • the invention further provides a method of detecting hBsk nucleic acid in a sample.
  • the invention also provides a kit for detecting the presence of hBsk nucleic acid in a sample.
  • the invention further provides a recombinant nucleic acid molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the above-described isolated nucleic acid molecule.
  • the invention also provides a recombinant nucleic acid molecule comprising a vector and the above-described isolated nucleic acid molecule.
  • the invention further provides a recombinant nucleic acid molecule comprising a sequence complimentary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide.
  • the invention also provides a cell that contains the above-described recombinant nucleic acid molecule.
  • the invention further provides a non-human organism that contains the above-described recombinant nucleic acid molecule.
  • the invention also provides an antibody having binding affinity specifically to a hBsk polypeptide.
  • the invention further provides a method of detecting a hBsk polypeptide in a sample.
  • the invention also provides a method of measuring the amount of hBsk in a sample.
  • the invention further provides a diagnostic kit comprising a first container means containing the above-described antibody, and a second container means containing a conjugate comprising a binding partner of said monoclonal antibody and a label.
  • the invention also provides a hybridoma which produces the above- described monoclonal antibody.
  • the invention further provides diagnostic methods for human disease, in particular neurodegenerative diseases, disorders, and injuries.
  • the invention also provides methods for therapeutic uses involving all or part of the nucleic acid sequence encoding hBsk and its corresponding protein.
  • the invention provides assays for the isolation of the ligand or ligands capable of activating the hBsk receptor and therapeutic uses for said ligand.
  • the invention also provides assays for the assessment and development of dmgs capable of activating the hBsk receptor and therapeutic uses for said dmgs.
  • FIGURE 1 Schematic of overlapping human Bsk cDNA Isolates.
  • Human Bsk cDNA clones are shown with their extent of overlap under a schematic of the mouse Bsk gene comprising: an extracellular domain (E), transmembrane domain (TM) and a kinase domain (K).
  • E extracellular domain
  • TM transmembrane domain
  • K kinase domain
  • the nucleotide numbers at the 3' and/or 5' ends of the isolates represent the corresponding nucleotide number within the mouse Bsk sequence (See, Zhou et al. (1994) J. Neuroscience Res. 37: 129- 143.
  • FIGURE 2A-2J Partial Nucleotide Sequence of a hBsk gene.
  • Figure 2A, 2C, 2E, 2G, and 21 describe a partial nucleotide sequence of
  • Figure 2B, 2D, 2F, 2H, and 2J describe a partial nucleotide sequence of Clones 6-1, 7-2, 8-1, 16-1, and 19-1, respectively, using a T7 primer.
  • rDNA recombinant DNA
  • isolated nucleic acid molecule refers to a polymer of nucleotides, and includes but should not be limited to DNA and RNA.
  • DNA Segment refers to a molecule comprising a linear stretch of nucleotides wherein the nucleotides are present in a sequence that may encode, through the genetic code, a molecule comprising a linear sequence of amino acid residues that is referred to as a protein, a protein fragment or a polypeptide.
  • a DNA sequence related to a single polypeptide chain or protein, and as used herein includes the 5' and 3' untranslated ends.
  • the polypeptide can be encoded by a full-length sequence or any portion of the coding sequence, so long as the functional activity of the protein is retained.
  • cDNA Complementary DNA
  • mRNA messenger RNA
  • Structural Gene A DNA sequence that is transcribed into mRNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • restriction endonuclease also restriction enzyme
  • a restriction endonuclease is an enzyme that has the capacity to recognize a specific base sequence (usually 4, 5, or 6 base pairs in length) in a DNA molecule, and to cleave the DNA molecule at every place where this sequence appears. For example, Ec ⁇ Sl recognizes the base sequence GAATTC/CTTAAG.
  • Restriction Fragment The DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome may be digested by a particular restriction endonuclease into a discrete set of restriction fragments.
  • the DNA fragments fractionated by agarose gel electrophoresis can be visualized directly by a staining procedure if the number of fragments included in the pattern is small.
  • the DNA fragments of genomes can be visualized successfully.
  • most genomes, including the human genome contain far too many DNA sequences to produce a simple pattem of restriction fragments.
  • the human genome is digested into approximately 1,000,000 different DNA fragments by -Ec ⁇ RI.
  • a methodology referred to as the Southern hybridization procedure can be applied.
  • Southern Transfer Procedure The purpose of the Southern transfer procedure (also referred to as blotting) is to physically transfer DNA fractionated by agarose gel electrophoresis onto a nitrocellulose filter paper or another appropriate surface or method, while retaining the relative positions of DNA fragments resulting from the fractionation procedure.
  • the methodology used to accomplish the transfer from agarose gel to nitrocellulose involves drawing the DNA from the gel into the nitrocellulose paper by capillary action.
  • Nucleic Acid Hybridization depends on the principle that two single-stranded nucleic acid molecules that have complementary base sequences will reform the me ⁇ nodynamically favored double-stranded structure if they are mixed under the proper conditions. The double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a nitrocellulose filter. In the Southern hybridization procedure, the latter situation occurs. As noted previously, the DNA of the individual to be tested is digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, converted to the single-stranded form, and transferred to nitrocellulose paper, making it available for reannealing to the hybridization probe.
  • Hybridization Probe To visualize a particular DNA sequence in the Southern hybridization procedure, a labeled DNA molecule or hybridization probe is reacted to the fractionated DNA bound to the nitrocellulose filter. The areas on the filter that carry DNA sequences complementary to the labeled DNA probe become labeled themselves as a consequence of the rea ⁇ ealing reaction. The areas of the filter that exhibit such labeling are visualized.
  • the hybridization probe is generally produced by molecular cloning of a specific DNA sequence. Oligonucleotide or Otigomer. A molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three.
  • oligonucleotide may be derived synthetically or by cloning.
  • Sequence Amplification A method for generating large amounts of a target sequence. In general, one or more amplification primers are annealed to a nucleic acid sequence. Using appropriate enzymes, sequences found adjacent to, or in between the primers are amplified.
  • Amplification Primer An oligonucleotide which is capable of annealing adjacent to a target sequence and serving as an initiation point for
  • DNA synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is initiated.
  • Vector A plasmid or phage DNA or other DNA sequence into which DNA may be inserted to be cloned.
  • the vector may replicate autonomously in a host cell, and may be further characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a dete ⁇ ninable fashion and into which DNA may be inserted.
  • the vector may further contain a marker suitable for use in die identification of cells transformed with the vector. Markers, for example, are tetracycline resistance or ampicillin resistance. The words "cloning vehicle" are sometimes used for "vector.”
  • Expression is the process by which a structural gene produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptide(s).
  • Expression Vector A vector or vehicle similar to a cloning vector but? which is capable of expressing a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
  • Expression control sequences will vary depending on whe ⁇ ier the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, te ⁇ nination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • a “functional derivative” of a sequence is a molecule that possesses a biological activity (either functional or stmctural) that is substantially similar to a biological activity of the protein or nucleic acid sequence.
  • a functional derivative of a protein may or may not contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional derivative” is intended to include e “fragments,” “segments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
  • a molecule is said to be a "chemical derivative" of anouier molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, and me like. The moieties may alternatively decrease the toxicity of die molecule, et ⁇ iiinate or attenuate any undesirable side effect of the molecule, and the like.
  • variants of a protein or nucleic acid are meant to refer to a molecule substantially similar in structure and biological activity to either the protein or nucleic acid. Thus, provided that two molecules possess a common activity and may substitute for each other, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary stmcture of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical.
  • a "substantially pure" protein or nucleic acid is a protein or nucleic acid preparation that is generally lacking in other cellular components.
  • Ligand refers to any protein or proteins that may interact with the hBsk receptor binding domain. Said ligand or ligands may be soluble or membrane bound. The ligand or ligands may be a naturally occurring protein, or synthetically or recombinantly produced. The ligand may also be a nonprotein molecule that acts as ligand when it interacts with the Bsk receptor binding domain. Interactions between the ligand and receptor binding domain include, but are not limited to, any covalent or non-covalent interactions.
  • the receptor binding domain is any region of the hBsk receptor molecule that interacts directly or indirectly with the hBsk ligand.
  • Neurodegenerative disease includes, but is not limited to, states in a mammal which can include chromosomal abnormalities, degenerative growth and developmental disorders, viral infections, bacterial infections, brain injuries, or neoplastic conditions. Examples of neurodegenerative diseases that can be diagnosed, assessed or treated by methods described in the present appUcation include, but are not limited to, Alzheimer's disease, epilepsy, schizophrenia. In a preferred embodiment diseases characterized by neurodegeneration in the limbic system are diagnosed, assessed or treated by methods disclosed in the present appUcation.
  • Dmgs include, but are not limited to proteins, peptides, degenerate peptides, agents purified form conditioned cell medium, organic molecules, inorganic molecules, antibodies or oUgonucleotides.
  • Other • candidate dmgs include analogs of the hBsk ligand or Ugands. The drug may be naturally occurring or synthetically or recombinantiy produced. One skilled in the art will understand that such dmgs may be developed by the assays described below.
  • the present invention relates to an isolated nucleic acid molecule coding for a polypeptide having an amino acid sequence corresponding to human brain specific kinase, hBsk.
  • e isolated nucleic acid molecule comprises the hBsk nucleotide sequence present in Clones 6-1 and 8-1 (See Figure 1) as deposited with the ATCC.
  • the isolated nucleic acid molecule encodes the hBsk amino acid sequence present in
  • Clones 6-1 and 8-1 (See Figure 1) as deposited witii the ATCC.
  • the hBsk sequence within Clones 6-1 and 8-1 is identified by its homology to the mouse bsk sequence (See Figure 1).
  • 6-1 and 8-1 may be used in the practice of the present invention.
  • nucleic acid molecules coding for polypeptides having amino acid sequences corresponding to hBsk are provided.
  • the nucleic acid molecule may be isolated from a biological sample containing human RNA or DNA.
  • the nucleic acid molecule may be isolated from a biological sample containing human RNA using the techniques of cDNA cloning and subtractive hybridization.
  • the nucleic acid molecule may also be isolated from a cDNA Ubrary using a homologous probe.
  • the nucleic acid molecule may be isolated from a biological sample containing human genomic DNA or from a genomic Ubrary. Suitable biological samples include, but are not limited to, blood, semen and tissue.
  • the method of obtaining the biological sample will vary depending upon the nature of me sample.
  • the isolated nucleic acid molecule is also intended to include alleUc variations, so long as the sequence is a functional derivative of the hBsk gene.
  • alleUc variations so long as the sequence is a functional derivative of the hBsk gene.
  • Isolated nucleic acid molecules of the present invention are also meant to include mose chemicaUy synthesized.
  • a nucleic acid molecule with me nucleotide sequence which codes for the expression product of a hBsk gene may be designed and, if necessary, divided into appropriate smaUer fragment. Then an oUgomer which corresponds to the nucleic acid molecule, or to each of me divided fragments, may be synthesized.
  • Such syn ⁇ ietic oUgonucleotides may be prepared, for example, by me triester method of Matteucci et al , J. Am. Chem. Soc. 705:3185-3191 (1981) or by using an automated DNA synthesizer.
  • An oUgonucleotide may be derived synthetically or by cloning. If necessary, the 5 '-ends of the oUgomers may be phosphorylated using T4 polynucleotide kinase. Kinasing of single strands prior to annealing or for labeling may be achieved using an excess of the enzyme. If kinasing is for the labeling of probe, the ATP may contain high specific activity radioisotopes. Then, the DNA oUgomer may be subjected to annealing and Ugation with T4 Ugase or the like.
  • title present invention relates to a substantially pure polypeptide having an amino acid sequence corresponding to hBsk.
  • the polypeptide has the amino acid sequence set forth in Clones 6-1 and 8-1 (See Figure 1).
  • the peptide is purified from human tissues or cells which naturally produce the peptide.
  • the above-described isolated nucleic acid fragments could be used to expressed the hBsk protein in any organism.
  • the samples of the present invention include cells, protein extracts or membrane extracts of cells, or biological fluids. The sample will vary based on the assay format, the detection method and title nature of the tissues, ceUs or extracts used as the sample.
  • the present invention relates to a nucleic acid probe for the specific detection of the presence of hBsk in a sample comprising the above-described nucleic acid molecules or at least 18 contiguous nucleotides thereof (preferably at least 20, 25, 30, 35, 40, or 50 thereof).
  • the probe is designed such that it does not have 100% homology with a similarly located mouse Bsk probe.
  • the nucleic acid probe may be used to probe an appropriate chromosomal or cDNA Ubrary by usual hybridization methods to obtain another nucleic acid molecule of the present invention.
  • a chromosomal may be used to probe an appropriate chromosomal or cDNA Ubrary by usual hybridization methods to obtain another nucleic acid molecule of the present invention.
  • DNA or cDNA Ubrary may be prepared from appropriate ceUs according to recognized methods in the art (cf. Molecular Cloning: A Laboratory
  • the synuiesized nucleic acid probes may be used as primers in a polymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, A Guide to
  • hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemuuminescence, and the like.
  • the nucleic acid probes of the present invention include RNA, as weU as DNA probes, such probes being generated using techniques known in the art.
  • a nucleic acid probe is immobilized on a soUd support.
  • soUd supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acryhc resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such soUd supports are well known in the art.
  • test samples suitable for nucleic acid probing methods of the . present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • the sample used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, ceUs or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are weU known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
  • the present invention relates to a method of detecting the presence of hBsk in a sample comprising a) contacting said sample with d e above-described nucleic acid probe, under conditions such that hybridization occurs, and b) detecting the presence of said probe bound to said nucleic acid molecule.
  • a method of detecting the presence of hBsk in a sample comprising a) contacting said sample with d e above-described nucleic acid probe, under conditions such that hybridization occurs, and b) detecting the presence of said probe bound to said nucleic acid molecule.
  • Samples to be tested include but should not be limited to RNA samples of human tissue.
  • hBsk has been found to be expressed in brain ceUs. Accordingly, hBsk probes may be used detect the presence of RNA from brain ceUs in a sample. Further, altered expression levels of hBsk RNA in an individual, as compared to normal levels, may indicate the presence of disease.
  • the present invention relates to a kit for detecting the presence of hBsk in a sample comprising at least one container means having disposed therein me above-described nucleic acid probe.
  • the kit further comprises other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe.
  • detection reagents include, but are not limited to radiolabeUed probes, enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin).
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and me agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, ampUfied product, or the like.
  • the present invention relates to a recombinant DNA molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecules.
  • the present invention relates to a recombinant DNA molecule comprising a vector and an above-described nucleic acid molecule.
  • the present invention relates to a nucleic acid molecule comprising a transcriptional control region functional in a ceU, a sequence complimentary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide, and a transcriptional termination region functional in said ceU.
  • the above-described molecules are isolated and/or purified DNA molecules.
  • the present invention relates to a ceU or non-human organism that contains an above-described nucleic acid molecule.
  • me peptide is purified from ceUs which have been altered to express the peptide.
  • a cell is said to be "altered to express a desired peptide" when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the ceU normaUy produces at low levels.
  • One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic ceUs.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
  • shaU in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as weU as the DNA sequences which, when transcribed into RNA, wiU signal synthesis initiation.
  • Such regions will normally include those 5 '-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and title like.
  • the non-coding region 3' to the sequence encoding an hBsk gene may be obtained by the above-described methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host ceU, then a 3' region functional in the host ceh may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of a hBsk gene sequence, or (3) interfere with title ability of the hBsk gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if die promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of the hBsk gene (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
  • Prokaryotic hosts are, generally, the most efficient and convenient for the production of recombinant proteins and, therefore, are preferred for title expression of the hBsk gene.
  • Prokaryotes most frequendy are represented by various strains of E. coli. However, other microbial strains may also be used, including other bacterial strains.
  • plasmid vectors that contain repUcation sites and control sequences derived from a species compatible witih the host may be used. Examples of suitable plasmid vectors may include pBR322, pUC118, ⁇ UC119 and me Uke; suitable phage or bacteriophage vectors may include ⁇ gtlO, ⁇ gtl 1 and the like; and suitable virus vectors may include pMAM-neo, pKRC and die Uke.
  • die selected vector of me present invention has the capacity to repUcate in title selected host ceU.
  • prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the peptide will not be glycosylated.
  • the prokaryotic host must be compatible with the rephcon and control sequences in the expression plasmid.
  • hBsk in a prokaryotic ceU, it is necessary to operably link the hBsk sequence to a functional prokaryotic promoter.
  • Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the j8-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pBR325, and the like.
  • inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage ⁇ (P L and P R ), the trp, recA, lacZ, lad, and gal promoters of E. coli, the ⁇ -amylase (Ulmanen et al, J. Bacteriol. 762: 176-182 (1985)) and the ⁇ -28-specific promoters of B.
  • subtilis (Gilman et al, Gene sequence 52:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward et al, Mol. Gen. Genet. 205:468-478 (1986)).
  • Prokaryotic promoters are reviewed by GUck
  • ceU ceU line
  • cell culture may be used interchangeably and all such designations include progeny.
  • trimformants or transformed ceUs
  • all progeny may not be precisely identical in DNA content, due to deUberate or inadvertent mutations.
  • mutant progeny have the same functionaUty as tiiat of me originaUy transformed ceU.
  • Host ceUs which may be used in the expression systems of the present invention are not strictiy limited, provided tiiat tiiey are suitable for use in the expression of the hBsk peptide of interest. Suitable hosts may often include eukaryotic ceUs.
  • Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian ceUs either in vivo, or in tissue culture.
  • Mammalian cells which may be useful as hosts include HeLa ceUs, cells of fibroblast origin such as VERO or CHO-K1 , or cells of lymphoid origin and their derivatives.
  • plant cells are also available as hosts, and control sequences compatible widi plant cells are available, such as the cauUfiower mosaic vims 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences.
  • Another preferred host is an insect cell, for example Drosophila larvae.
  • Drosophila alcohol dehydrogenase promoter can be used. Rubin, Science 240:1453-1459 (1988).
  • baculovirus vectors can be engineered to express large amounts of hBsk in insects ceUs (Jasny, Science 255:1653 (1987); Miller et al, In: Genetic Engineering (1986), Setlow, J.K., et al , eds., Plenum,
  • Different host ceUs have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage) of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • expression in a bacterial system can be used to produce an unglycosylated core protein product.
  • Expression in yeast will produce a glycosylated product.
  • Expression in mammalian ceUs can be used to ensure "native" glycosylation of the heterologous hBsk protein.
  • different vector/host expression systems may effect processing reactions such as proteolytic cleavages to different extents.
  • Any of a series of yeast gene sequence expression systems can be utilized which incorporate promoter and te ⁇ nination elements from the actively expressed gene sequences coding for glycolytic enzymes are produced in large quantities when yeast are grown in mediums rich in glucose.
  • Known glycolytic gene sequences can also provide very efficient transcriptional control signals.
  • Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications.
  • Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides).
  • leader sequences i.e., pre-peptides.
  • hBsk For a mammalian host, several possible vector systems are available for the expression of hBsk.
  • transcriptional and translational regulatory sequences may be employed, depending upon die nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovims, bovine papilloma vims, simian virus, or the Uke, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, and die like, may be employed.
  • Transcriptional initiation regulatory signals may be selected which aUow for repression or activation, so that expression of the gene sequences can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabohte) regulation.
  • eukaryotic regulatory regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include, for example, the promoter of the mouse metaUothionein I gene sequence (Hamer et al. , J. Mol Appl. Gen.
  • An hBsk nucleic acid molecule and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic ceU either as a non- repUcating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous rephcation, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced DNA sequence into the host chromosome.
  • a vector is employed which is capable of integrating the desired gene sequences into the host ceU chromosome.
  • CeUs which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which aUow for selection of host ceUs which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into die same ceU by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include sphce signals, as weU as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include tiiose described by Okayama, Molec. Cell. Biol. 5:280 (1983).
  • d e introduced nucleic acid molecule will be incorporated into a plasmid or viral vector capable of autonomous repUcation in the recipient host.
  • a plasmid or viral vector capable of autonomous repUcation in the recipient host.
  • Any of a wide variety of vectors may be employed for ti ⁇ is purpose. Factors of importance in selecting a particular plasmid or viral vector include: die ease with which recipient ceUs tiiat contain the vector may be recognized and selected from those recipient ceUs which do not contain the vector; die number of copies of the vector which are desired in a particular host; and whetitier it is desirable to be able to "shuttle" die vector between host ceUs of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of repUcation in
  • E. coli such as, for example, pBR322, ColEl, pSClOl, pACYC 184, T ⁇ VX.
  • plasmids are, for example, disclosed by Sambrook (cf. Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989)).
  • Bacillus plasrnids include pC194, pC221, pT127, and die Uke.
  • Such plasmids are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329).
  • Suitable Streptomyces plasmids include pJJlOl (KendaU et al, J. Bacteriol. 769:4177-4183 (1987)), and streptomyces bacteriophages such as ⁇ C31 (Chater et al, In: Sixth International Symposium on Actinomycetales Biology, Akademiai
  • Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-micron circle, and die like, or their derivatives.
  • Such plasmids are weU known in the art (Botstein et al, Miami Wntr. Symp. 79:265-274 (1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, Cell 25:203-204 (1982); BoUon et al, J. Clin. Hematol. Oncol. 70:39-48 (1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980)).
  • die DNA constmct(s) may be introduced into an appropriate host ceU by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and die like.
  • recipient ceUs are grown in a selective medium, which selects for the growtii of vector-containing ceUs.
  • Expression of the cloned gene molecule(s) results in the production of hBsk. This can take place in the transformed ceUs as such, or foUowing die induction of tiiese ceUs to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma ceUs or the like).
  • die present invention relates to an antibody having binding affinity specificaUy to a hBsk polypeptide as described above. Those which bind selectively to hBsk would be chosen for use in mediods which could include, but should not be limited to, die analysis of altered hBsk expression in tissue containing hBsk.
  • the hBsk proteins of the present invention can be used in a variety of procedures and methods, such as for die generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction.
  • the hBsk peptide of the present invention can be used to produce antibodies or hybridomas.
  • One skilled in the art will recognize that if an antibody is desired, such a peptide would be generated as described herein and used as an immunogen.
  • the antibodies of title present invention include monoclonal and polyclonal antibodies, as weU as fragments of these antibodies.
  • the invention further includes single chain antibodies. Antibody fragments which contain the idiotype of me molecule can be generated by known techniques.
  • such fragments include but are not limited to: die F(ab') 2 fragment; the Fab' fragments, Fab fragments, and Fv fragments.
  • antibodies to hBsk which are produced in humans, or are "humanized" (i.e. non-immunogenic in a human) by recombinant or other technology.
  • Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies) (Robinson, R.R. et al, International Patent PubUcation PCT/US86/02269; Akira, K.
  • CEA substitution Jones, P.T. et al, Nature 527:552-525 (1986); Verhoeyan et al, Science 259: 1534 (1988); Beidler, C.B. et al, J. Immunol. 747:4053-4060 (1988)).
  • die present invention relates to a hybridoma which produces die above-described monoclonal antibody.
  • a hybridoma is an immortalized ceU line which is capable of secreting a specific monoclonal antibody.
  • Any animal (mouse, rabbit, and the like) which is known to produce antibodies can be immunized widi die selected polypeptide.
  • Mediods for immunization are weU known in the art. Such methods include subcutaneous or interperitoneal injection of the polypeptide.
  • die amount of polypeptide used for immunization will vary based on die animal which is immunized, the antigenicity of the polypeptide and the site of injection.
  • the polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity.
  • Mediods of increasing the antigenicity of a polypeptide are weU known in the art. Such procedures include coupling the antigen widi a heterologous protein (such as globulin or /S-galactosidase) or through the inclusion of an adjuvant during immunization.
  • spleen ceUs from d e immunized animals are removed, fused widi myeloma ceUs, and aUowed to become monoclonal antibody producing hybridoma ceUs.
  • any one of a number of methods well known in the art can be used to identify the hybridoma ceU which produces an antibody widi die desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al, Exp. Cell Res. 775:109-124 (1988)).
  • Hybridomas secreting the desired antibodies are cloned and die class and subclass is determined using procedures known in the art (CampbeU, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, supra (1984)).
  • antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies widi the desired specificity using one of die above-described procedures.
  • Antibodies are detectably labeled.
  • Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and die like), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and die like), paramagnetic atoms, and die like. Procedures for accompUshing such labeling are weU-known in the art, for example, see (Sternberger et al., J. Histochem. Cytochem. 75:315 (1970); Bayer et al., Meth. Enzym.
  • the labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify ceUs or tissues which express a specific peptide.
  • soUd supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acryhc resins and such as polyacrylamide and latex beads.
  • plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acryhc resins and such as polyacrylamide and latex beads.
  • Techniques for coupling antibodies to such soUd supports are well known in the art (Weir et al. , "Handbook of Experimental Immunology” 4d ⁇ Ed., Blackwell Scientific PubUcations, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, N.Y.
  • the immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as weU as in immunochromotography .
  • Anti-peptide peptides can be generated in one of two fashions.
  • the anti-peptide peptides can be generated by replacing the basic amino acid residues found in the hBsk peptide sequence widi acidic residues, while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced widi aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine.
  • the present invention relates to a method of detecting a hBsk polypeptide in a sample, comprising: a) contacting the sample wid an above-described antibody, under conditions such that immunocomplexes form, and b) detecting the presence of said antibody bound to the polypeptide.
  • die methods comprise incubating a test sample widi one or more of die antibodies of die present invention and assaying whether die antibody binds to the test sample. Altered levels of hBsk in a sample as compared to normal levels may indicate a specific disease.
  • Incubation conditions depend on the format employed in the assay, die detection methods employed, and the type and nature of the antibody used in die assay.
  • One skilled in the art wiU recognize that any one of the commonly available immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays) can readily be adapted to employ the antibodies of die present invention. Examples of such assays can be found in Chard, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science PubUshers, Amsterdam, The Netherlands
  • the immunological assay test samples of the present invention include ceUs, protein or membrane extracts of ceUs, or biological fluids such as blood, serum, plasma, or urine.
  • the test sample used in the above- described method will vary based on the assay format, nature of the detection method and the tissues, ceUs or extracts used as the sample to be assayed.
  • Mediods for preparing protein extracts or membrane extracts of ceUs are weU known in the art and can be readily be adapted in order to obtain a sample which is capable widi d e system utilized.
  • a kit which contains aU the necessary reagents to carry out the previously described mediods of detection.
  • the kit may comprise: i) a first container means containing an above-described antibody, and u) second container means containing a conjugate comprising a binding partner of the antibody and a label.
  • die kit further comprises one or more other containers comprising one or more of die foUowing: wash reagents and reagents capable of detecting die presence of bound antibodies.
  • detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with die labeled antibody.
  • the compartmentalized kit may be as described above for nucleic acid probe kits.
  • the diagnostic and screening methods of the invention are especially useful for a patient suspected of being at risk for developing a disease associated with an altered expression level of hBsk based on family history, or a patient in which it is desired to diagnose an hBsk-related disease.
  • presymptomatic screening of an individual in need of such screening is now possible using DNA encoding die hBsk protein of the invention.
  • the screening method of the invention allows a presymptomatic diagnosis, including prenatal diagnosis, of die presence of a missing or aberrant hBsk gene in individuals, and thus an opinion concerning die likelihood tiiat such individual would develop or has developed an hBsk-associated disease. This is especiaUy valuable for the identification of carriers of altered or missing hBsk genes, for example, from individuals widi a family history of a hBsk-associated disease. Early diagnosis is also desired to maximize appropriate timely intervention.
  • a tissue sample would be taken from such individual, and screened for (1) the presence of die "normal" hBsk gene; (2) the presence of hBsk mRNA and/or (3) die presence of hBsk protein.
  • the normal human gene can be characterized based upon, for example, detection of restriction digestion patterns in "normal” versus the patient's DNA, including, RFLP analysis, using DNA probes prepared against the hBsk sequence (or a functional fragment thereof) taught in the invention.
  • hBsk mRNA can be characterized and compared to normal hBsk mRNA (a) levels and/or (b) size as found in a human population not at risk of developing hBsk- associated disease using similar probes.
  • hBsk protein can be (a) detected and/or (b) quantitated using a biological assay for hBsk activity or using an immunological assay and hBsk antibodies. When assaying hBsk protein, the omunological assay is preferred for its speed.
  • An (1) aberrant hBsk DNA size pattem, and/or (2) aberrant hBsk mRNA sizes or levels and/or (3) aberrant hBsk protein levels would indicate tiiat die patient is at risk for developing an hBsk-associated disease.
  • the screening and diagnostic methods of the invention do not require that die entire hBsk DNA coding sequence be used for the probe. Ratiher, it is only necessary to use a fragment or length of nucleic acid that is sufficient to detect die presence of the hBsk gene in a DNA preparation from a normal or affected individual, the absence of such gene, or an altered physical property of such gene (such as a change in electrophoretic migration pattem).
  • Prenatal diagnosis can be performed when desired, using any known method to obtain fetal cells, including amniocentesis, chorionic villous sampling (CVS), and fetoscopy.
  • Prenatal chromosome analysis can be used to determine if the portion of the chromosome possessing the normal hBsk gene is present in a heterozygous state.
  • functional hBsk DNA can be provided to the ceUs of such patient in a manner and amount tiiat permits die expression of the hBsk protein provided by such gene, for a time and in a quantity sufficient to treat such patient.
  • Many vector systems are known in the art to provide such deUvery to human patients in need of a gene or protein missing from the ceU.
  • retro virus systems can be used, especially modified retjpovims systems and especiaUy herpes simplex vims systems.
  • modified retjpovims systems and especiaUy herpes simplex vims systems are provided for, in, for example, the teachings of Breakefield, X.A. et al, The New Biologist 5:203-218 (1991); Huang, Q. et al, Experimental Neurology 775:303-316 (1992), WO93/03743 and
  • stem ceU populations for either neuronal or glial ceUs can be genetically engineered to express a functional hBsk receptor.
  • Such ceUs recombinantly expressing the hBsk receptor can be transplanted to die diseased or injured region of the mammal's limbic system (Neural Transplantation. A Practical Approach, Donnet & Djorklund, eds., Oxford University Press, New York, NY (1992)).
  • embryonic tissue or fetal neurons can be geneticaUy engineered to express functional hBsk receptor and transplanted to the diseased or injured region of the mammal's limibic system. The feasibility of transplanting fetal dopamine neurons into Parkinsonian patients has recendy been demonstrated. (LindvaU et al , Archives of Neurology 46:615-631 (1989)).
  • the extracellular domain of a receptor can be used as a probe to screen an expression cDNA Ubrary for die hBsk Ugand or Ugands.
  • placenta! alkaline phosphatase will be fused to die extraceUular domain of a receptor, and positive clones will be detected by the presence of alkaline phosphatase activity.
  • An alternative approach is to isolate the putative hBsk Ugand is to utilize die findings tiiat co-expression of a receptor and its Ugand in the same ceUs results in uncontroUed proliferation and maUgnant transformation (Klein et al , Cell 66:395-403 (1991); Gazit et al, Cell 59:89-97 (1984)).
  • a eukaryotic cDNA expression Ubrary can be transfected into ceUs expressing a receptor, and die presence of a Ugand will create an autocrine loop, resulting in a transformed phenotype.
  • hBsk receptor protein can be expressed in a ceU line or in Xenopus oocytes by the recombinant techniques described above and its Ugand stimulated activation of tyrosine kinase activity, as detected by an anti-phosphotryosine antibody (UBI, Happauge, New York) can be used to assay and purify the Ugand.
  • UBI anti-phosphotryosine antibody
  • cells expressing the recombinant hBsk receptor can be exposed to mammalian brain extract.
  • the brain extracts can be fractionated by chromatography and used to assay for die presence of the Ugand activity. Once an activity is identified in a particular fraction, it can be further purified by conventional biochemical techniques.
  • die hBsk extraceUular domain can be used to screen a random peptide Ubrary (Cull et al , Proc. Natl. Acad. Sci. USA 59:1865-1869 (1982); Lam et al , Nature 554:82-84 (1991)). Peptides isolated can be assayed for dieir Ugand activity in the above- described assays for drug screening.
  • the hBsk Ugand is expressed as a recombinant gene in a ceU, so tiiat die ceUs may be transplanted into a mammal, preferably a human in need of gene dierapy.
  • a genetic sequence which encodes for aU or part of the hBsk Ugand is inserted into a vector and introduced into a host ceU.
  • diseases tiiat may be suitable for gene dierapy include, but are not limited to, neurodegenerative diseases or disorders, Alzheimer's, schizophrenia, epilepsy, neoplasms and cancer.
  • the means by which the vector carrying the gene may be introduced into the ceU include but is not limited to, microinjection, electroporation, transduction, or transfection using DEAE-Dextran, Upofection, calcium phosphate or other procedures known to one skilled in the art (Molecular Cloning, A Laboratory Manual, Sambrook et al, eds., Cold Spring Harbor Press, Plainview, New York
  • the present invention relates to a method of administering hBsk to an animal (preferably, a mammal (specifically, a human)) in an amount sufficient to effect an altered level of hBsk in said animal.
  • the administered hBsk could specificaUy effect hBsk associated functions.
  • administration of hBsk could be used to alter hBsk levels in the brain.
  • hBsk can be administered parenteraUy by injection or by gradual perfusion over time. It can be administered intravenously, intraperitoneaUy, intramuscularly, or subcutaneously.
  • Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such_ as oUve oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcohoUc/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose and sodium chloride, lactated Ringer- s, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the Uke.
  • Preservatives and otiier additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and die like. See, generaUy, Remington's
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising hBsk in an amount sufficient to alter hBsk associated activity, and a pharmaceuticaUy acceptable diluent, carrier, or excipient.
  • concentrations and dosage unit sizes can be readily determined by one skilled in die art as described above (See, for example, Remington 's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980) and WO 91/19008).
  • a ⁇ ZAP Ubrary made from mRNA isolated from the brain of a
  • hBsk 18-week-old female human fetus was used to isolate hBsk.
  • hBsk was isolated using a 4.3 kb mouse Bsk probe labeled with [ 32 P] by random priming. 10 6 phage plaques were lifted onto nitroceUulose filters and prehybridized and hybridized under non-stringent conditions (5X SSPE,
  • Hybridization was performed at 42 °C for 16 hrs after prehybridization in the same buffer without the labeled probe for 2 hrs. After hybridization, the filters were washed under increasingly stringent conditions: 2X SSC, 0.5% SDS, 30 min, 50°C, 2 times; 0.1X SSC, 0.1 % SDS, 50°C, 30 min, 2 times; 0.1X SSC, 0.1 % SDS, 65 °C. Filters were exposed to X-ray films to assess the extent of signal loss after each wash. Positive plaques were picked and purified to homogeneity.
  • cDNA inserts in the purified positive clones were tiien excised from the phage vector into plasmid DNA using M13 helper phage. Partial sequence analysis (for general techniques See, Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, &
  • hBsk cDNA clones Thirty-four independent potential hBsk cDNA clones were isolated from mRNA purified from a human fetal brain using a mouse Bsk probe under stringent washing conditions. Partial sequence of these demonstrate tiiat five of these clones represent different parts of the hBs as shown schematicaUy in Figure 1.
  • Clones 6-1 and 8-1 contain an entire hBsk gene and were deposited
  • a clone containing die entire hBsk gene is constructed by 1) identifying a restriction site shared by Clones 6-1 and 8-1 which is only present in one location within these clones, 2) digesting die clones with die corresponding restriction enzyme and a restriction enzyme(s) which cuts at die 5' end of the 8-1 clone and die 3' end of the 6-1 clone, and 3) linking the fragments together widi, for example, Ugase. If convenient restriction enzyme sites are not present witiiin the clones, they may be inserted into the clones using site specific mutagenesis (cf. Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989).
  • Example 2 The Biological function of hBsk
  • M-CSFR extraceUular domain and title hBsk transmembrane and intraceUular domains (M-CSFR/Bsk).
  • An alternative construct, M- CSFR/hBsk 2 would contain M-CSFR extraceUular and transmembrane domains and hBsk intraceUular domain.
  • the chimeric receptor will be expressed under the LTR promoter in the pMEX expression vector (Oskam et al, Proc. Natl Acad. Sci. USA 55:2964-2968 (1988)) in NIH/3T3 cells to study whether it has a mitogenic effect and in PC 12 ceUs to study whether it induces differentiation.
  • the expression cassette pMEX-CR (CR for chimeric receptor) will be cotransfected widi pSV2Neo. Neo-resistant colonies will be grown up and tested for the expression of the chimeric receptor using immunoprecipitation or Western blot analysis (Ausubel et al. , in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (1987)).
  • ceU surface labeling will be performed.
  • the labeled ceUs wiU be lysed and immunoprecipitated widi either Bsk- or M-CSFR-specific antibodies. If the receptor is properly localized on d e ceU surface, a positive labeling of the chimeric product should result.
  • [ I25 I]-MCSF will be used to bind intact transfected or control ceUs.
  • the concentration of M-CSF needed to cause 50% inhibition of maximal [ 125 I]-MCSF binding to die ceUs wiU be determined (IC50). Scratchard analysis will be performed on die binding competition data (Scatchard, Ann. NY Acad. Sci. 57:660-672 (1949)) and the dissociation constant (Kd) will be calculated. CeUs transfected widi vector alone will be used as controls.
  • the chimeric receptor will be immunoprecipitated widi eitiier M-CSFR or hBsk-specific antibodies from M-CSF-stimulated ceUs for various times, using unstimulated ceUs as a control.
  • the precipitated receptor will be analyzed by die Western blot technique with anti-phosphotyrosine antibody to examine the activation of the receptor tyrosine kinase.
  • the biological effects of the stimulation of the chimeric receptor with M-CSF wiU be studied in NTH/3T3 cells by stimulation of DNA synthesis under low serum (calf serum at 0.3 %) conditions.
  • the effects of the chimeric receptor activation on neurite outgrowth in PC12 cells will be studied.
  • CeUs including, but not limited to, PC 12 cells, and NIH/3T3 ceUs, are grown to 80% confluence in 100-mm tissue culture dishes, washed twice in metitdonine/cysteine-free DMEM, (Dulbecco's Modified Eagle Media) starved in die same medium supplemented widi 5% dialyzed fetal calf serum for 30 min, and then incubated for 2, 4, or 8 hours in the same medium widi Translabel (Amersham, 70% ["S] cysteine. The labeled ceUs will be lysed and immunoprecipitated as described below.
  • metitdonine/cysteine-free DMEM Dulbecco's Modified Eagle Media
  • CeUs expressing the M-CSFR/hBsk chimeric receptor will be grown to 80% confluence on 175-cm 2 flasks. CeUs are washed twice widi PBS, removed gently widi a ceU scraper, and resuspended in PBS containing 6 U of lactoperoxidase, 20 U of glucose oxidase and 2mCi of [ ,25 rj. After 0, 10,; and 20 min, 140 ⁇ l of 1M glucose will be added. At 30 min, the reaction is stopped by three washes in PBS. The ceUs are then lysed and immunoprecipitated using appropriate antibodies.
  • CeUs will be washed in Wash Buffer (HEPES 50 mM, pH 7.4, NaCl 150 mM, glycerol 10%, EDTA 10 mM, NaF 100 mM, vanadate 2 mM, Na 4 P 2 0 7 10 mM, trypsin inhibitor 1000 U/ml, PMSF ImM, aprotinin ImM, leupeptin 10 ⁇ M); the ceUs are lysed for 30 min at 4°C in 200 ⁇ L of lysis buffer (Wash Buffer containing 1 % Triton X-100) and centrifuged for 30 min at 150,000 g in a Beckman TL-100 ultracentrifuge.
  • Wash Buffer HEP 10 mM, pH 7.4, NaCl 150 mM, glycerol 10%, EDTA 10 mM, NaF 100 mM, vanadate 2 mM, Na 4 P 2 0 7 10 mM, trypsin inhibitor 1000 U/ml,
  • the extracts are then cleared twice by 15 min incubation with protein A Sepharose (40 ⁇ l of 10% gel for 200 ⁇ l of ceU extracts). After a 5 min centrifugation, supernatants are mixed widi appropriate antibodies adsorbed on protein-A-Sepharose and incubated for 2 h at 4 °C widi agitation. The samples are then centrifuged for 30 sec and die peUets are washed 6 times (3 times widi wash buffer, 3 times with wash buffer supplemented with 500 mM NaCl, 0.1 % Triton X-100, 0.1 % SDS). The washed peUets are dien resuspended in SDS-PAGE buffer and subjected to SDS-PAGE analysis. Labeled proteins are visualized by autoradiography.
  • CeUs are grown in 100 mm culture dishes in DMEM to 80% confluence and tiien washed widi PBS and incubated widi 5 ml of 25 mM
  • KC1 1.2 mM MgS0 4 , 1 mM EDTA, 10 mM glucose, 15 mM sodium acetate, 1 % dialyzed BSA), and resuspended in 5 ml of Binding buffer to dete ⁇ nine die ceU number.
  • 400 ⁇ l of this ceU suspension is tiien incubated widi [ 125 rj-M-CSF (5 pM) and increasing concentrations of unlabeled M-CFS (from 0 to 10 *6 M) in a total volume of 500 ⁇ l for 90 min at 15 °C. After incubation, ceUs are washed with Binding Buffer.
  • Confluent ceU monolayers in 12-weU culture dishes will be grown to quiescence in medium containing 0.5 % fetal bovine serum for 24 hours (h).
  • DNA synthesis wiU be stimulated by adding various amount of M-CSF.
  • cells will be labeled for 4 h with 0.5 ⁇ Ci [methyl- 3 H] thymidine at 3 TBq/mmol, then washed tiiree times with ice cold PBS, incubated widi 1 ml of 10% trichloroacetic acid for 30 min, and washed twice widi die same solution at 4°C.
  • CeUs will be then solubilized in 0.5 ml of 0.2 N NaOH, 1 % SDS for 1 h at 37°C and die lysate will be. brought to neutral pH with Tris buffer. The incorporated radioactivity wiU be determined in a Uquid scintillation counter.
  • Ad-CR Recombinant Adenovirus
  • the recombinant adenovirus is constructed by in vivo homologous recombination between an adenoviral vector containing the chimeric receptor and an adenovirus deletion mutant Ad 1327 genomic DNA (Stratgord-Perricaudt et al. , J. Clin. Invest. 90:626-630 ( 1992)) in 293 ceUs which express adenoviral early genes (Graham et al , J. Gen. Virol. 56:59-72 (1977)). Briefly, 293 ceUs are cotransfected widi 5 ⁇ g of linearized plasmid pAd-CR and 5 ⁇ g of die Large Cla I fragment (2.6-100 mu) of Ad5 DNA.
  • plaques containing recombinant adenovimses are isolated and amplified in 293 ceUs, viral DNA is purified, and recombinant adenovims plaques containing die Bsk chimeric receptor are identified by restriction cleavage and Soutiiern analysis.
  • Hippocampi are dissected from El 8 rate embryos and coUected in F10 medium (Gibco).
  • the tissues are minced, rinsed twice widi F10 medium, and the ceUs are dissociated by gentle trituration and coUected by low speed centrifugation (500 rpm) for 30 sec.
  • the peUet is washed again in the same medium by resuspension and centrifugation.
  • the ceU peUets are resuspended in MEM supplemented widi 10% fetal calf serum, 2 mM glutamine, 25 U/ml PenicilUn and 25 ⁇ g/ml) and laminin (10 ⁇ g/ml) coated
  • CeUs are fixed widi 4% (v/v) paraformaldehyde for 4 hr at 4°C, permeabilized with 0.1 % (v/v) Triton X-100 in PBS for 15 min, and blocked with 10% FCS in PBS for 1 h. The ceUs are then incubated widi anti-neurofilament 20 antibody for 1 h at room temperature, washed twice widi PBS containing 10% FCS, and incubated widi die secondary antibody (horseradish peroxidase-conjugated) for 1 h.
  • the ceUs are incubated widi 0.2% (w/v) 0-phenylenediamine and 0.02% (v/v) H 2 0 2 in citrate buffer (50 mM) for 30 min.
  • the reaction is topped by adding an equal volume of 4.5 M
  • CeUs are rinsed twice widi PBS, fixed widi 4% paraformaldehyde for 30 min at room temperature, and blocked widi 10% FCS in PBS containing
  • CeUs are washed in die GABA uptake buffer containing 140 mM NaCl, 2.5 mM KC1, ImM KH 2 P0 4 , ImM N-i j HPO ⁇ 6mg/ml glucose, lMgCl 2 , 1 mM CaCl 2 , and 0.1 % BSA. FoUowing washing, ceUs are incubated in the GABA uptake buffer for 5 min at 37 °C. pHj-GABA is then added to a final concentration of 12 nM, and incubated at 37°C for 10 min. CeUs are kept on ice and washed tihree times with the uptake buffer. CeUs are then solubilized with 0.14 N NaOH for 2 h at room temperature, and pHJ-GABA in the extract is counted.
  • Uptake of GABA into non-neuronal ceUs is inhibited by die addition of 2 mM jS-alanine, whereas uptake specific for neurons is verified by inhibition with nipecotic acid at 1 mM. Specific neuronal GABA uptake is determined as GABA uptake that is blocked in die presence of 1 mM mpecotic acid.
  • the assay is performed by heating a portion of the supernatant at 65 °C for 10 min to inactivate background phosphatase activity and tiien measuring the optical density at 405 nm after incubation with 1 M diethanolamine (pH 9.8), 0.5 mM MgCl, 10 ⁇ M L-homoarginine (a phosphatase inhibitor), 0.5 mg/ml BSA, and 12 mM p-nitrophenyl phosphate.
  • 1 M diethanolamine pH 9.8
  • 0.5 mM MgCl 10 ⁇ M L-homoarginine (a phosphatase inhibitor)
  • 0.5 mg/ml BSA 0.5 mg/ml BSA
  • 12 mM p-nitrophenyl phosphate The highest alkaline phosphatase-expressing clone will be selected for the purification of AP-tag-hBsk fusion protein.
  • a brain expression cDNA Ubrary will be plated at a density of 50,000 pfu per 150 mm plate.
  • DupUcate filters will be lifted from me plates and rinsed in TBST. The filters are then blocked with TBST widi 10% goat serum, rinsed once in TBST, and incubated in TBST widi AP- tag-Bsk probe for 3 hours. The FUters are then washed in tiiree changes of TBST, 3 min each.
  • the positive clones will be detected by color formation when the filters are incubated wid alkaline phosphatase substrates 5-bromo-4-chloro-3-inodyl-phosphate (BCIP, 0.017 mg/ml) and nitrobluetetrazoUum (NBT, 0.33 mg/ml) in 100 mM Tris-HCl (pH 9.5),
  • This vector has been used successfuUy in the nervous system and no cytotoxicity was observed (LeGal LeSaUe et al , Science 259:988-990 (1993)).
  • long-term expression of genes was achieved widi this vector (LeGal LeSaUe et al. , Science 259:988-990 (1993)).
  • a vector plasmid, pAd-Cr, containing a chimeric receptor expressing cassette driven by M-MLV LTR promoter will be constructed.
  • the cassette will be bordered at the 5' end by the left end (map unit 0-1.3) of adenovims type 5 (Ad5) and at die 3' end by sequences from mu 9.4-17
  • hippocampal neuron culture will be estabUshed in polyornithine- and laminin-coated plastic dishes in MEM supplemented widi 10% fetal calf serum (FCS) and glutamine and infected widi Ad-CR or control virus. Later, the medium will be changed to a serum-free medium containing hormone supplements. M-CSF will be added at this time. After various times of treatment, ceUs will be stained widi antibody against neuron-specific enolase to identify neurons in the culture.
  • FCS fetal calf serum
  • the number of neurons in cultures infected witih vims containing M-CSFR/hBsk or with viral vector only will be compared to determine the effect of M-CSF stimulation on the survival of specific neurons.
  • ParaUel infected cultures will be studied for hBsk protein expression at various time points using Western blot or immunoprecipitation and for Ugand-dependent receptor (Kaplan et al, Nature 550:158-160 (1991); Klein et al. , Cell 65:189-197 (1991)).
  • To examine if hBsk has any effect on the neurite outgrowth of the hippocampal neurons changes in the level of neurofilament protein upon M-CSF treatment will be examined.
  • Hippocampal neurons wiU be infected with virus carrying M-CSF/hBsk and treated widi various concentrations of M-CSF (0.001-10 ng/ml) of M-CSF for 8 days and neurofilament protein levels will be measued by ELISA. Neurons infected widi vector alone wiU be used as controls. To delineate which neuronal population which respond to hBsk kinase activation, die effect of M-CSF treatment of neurons expressing the chimeric receptor on the number of GABAergic and calbindin-positive neurons wiU be studied. Infected neurons will be treated witih various concentrations of M-CSF (0.001-100 ng/ml).
  • ceUs After 8 days of treatment, ceUs will be stained widi anti-GABA receptor or anti-calbindin antibodies to study die effect of hBsk activation on the survival of various neuronal populations.
  • die changes of the Wgh-affinity uptake for GABA will be studied after various times of M-CSF treatment. pHJ-GABA binding by die neurons in cultures infected widi the vims expressing d e chimeric receptor or with control vims will be compared.
  • S-alanine wiU be used to inhibit die uptake of GABA into non-neuronal ceUs (Ip et al. , Neurosci. 77:3124-3134 (1991)).
  • An alternative vector system which is based on a herpes vims may also be used to dehver CSFR/hBsk into hippocampal neurons (Anderson et al, Human Gene Therapy 5:487-499 (1992); Fink et al , Human Gene Therapy 5:11-19 (1992)).
  • APtag-1 constructed by Flannagan & Leder, Cell 65:185-194
  • APtag-1 contains a set of restriction sites for the insertion of the region of die hBsk cDNA encoding die extraceUular domain. Downstream of the insertion sites is the fuU length sequence of SEAP, which will be fused to die upstream sequence.
  • the 5' end of die hBsk cDNA sequence wiU be inserted into APtag-1 , including sequences encoding die hBsk secretion signal peptide and die entire extraceUular domain, ending immediately before the first hydrophobic amino acid of die transmembrane region.
  • the resulting plasmid will merefore encode a fusion protein with die hBsk extraceUular domain joined to a SEAP.
  • a fusion protein will be expressed from a Moloney Murine Leukemia virus LTR promoter.
  • the fusion construct will be transfected into NIH/3T3 ceUs which have been shown to express high levels of an APtag-Kit fusion protein (Flannagan & Leder, Cell 65:185-194 (1990)).
  • the fusion construct will be contransfected with a selectable marker plasmid pSV2neo, and selected with G418 (400-800 ⁇ g/ml).
  • Neo-resistant colonies will be grown in 96-weU plates and screened for secretion of SEAP activity into the media (See above).
  • the fusion protein will be concentrated, purified and used as a probe to screen a cDNA expression Ubrary from mammalian brain, preferably mouse.
  • clones having background alkaline phosphatase activity (2) clones which bind non- specificaUy to die fusion protein; and (3) clones encoding die putative hBsk Ugand.
  • Background phosphatase clones will be positive without the added probe in die presence of alkaline phosphatase substrates.
  • extracts from bacteria expressing these clones will be used to stimulate die tyrosine kinase activity of hBsk in a hBsk expressing NIH/3T3 ceUs. Only the Ugand will be able to stimulate activation of hBsk tyrosine activity.
  • die receptor probe in NTH/3 T3 ceUs rather than bacteria to receive proper glycosylation of the hBsk extraceUular domain. It has however been demonstrated tiiat glycosylation of growth factors is often not necessary for their activity. For example, M-CSF (Metcalf, Blood 67:257-267 (1986) and NGF (available from Boehringer Mannheim) produced in bacteria are biologically active. Therefore, die glycosylated receptor probe should interact properly with its Ugand syntiiesized by E. coli in a phase plaque during die screening.
  • die probe can also be used in histological staining on mammalian brain section to localize expression of the Ugand. Determination of the loci of expression of the Bsk Ugand will aUow for biochemical purification of the Ugand from that tissue ceU source further for analysis.
  • hBsk Ugand An alternative approach to isolate the hBsk Ugand is to utilize a functional screening approach. FuU lengtii cDNA of hBsk will be cloned into an expression vector pMEX under a MMLV LTR promoter. The hBsk expression vector will be co-transfected into NTH/3T3 ceUs together with pSV2Hygro containing a hygromycin 3-phosphotransferase gene which confers hygromycin resistance (Gritz & Davies, Gene 25:179-188 (1983)). The transfected ceUs will be selected widi hygromycin B at a concentration of 350 ⁇ g/ml. The resistant clones will be grown in 12-weU plates and screened for hBsk expression widi anti-hBsk antibody by Western blot analysis.
  • the vector system developed by Miki et al , Gene 55:137-146 (1989), will be used to construct a directional eukaryotic cDNA Ubrary from mouse brain mRNA.
  • the vector has a MMLV LTR promoter for the expression of cDNA inserts and a SV40 early promoter-driven Neo gene as a selectable marker.
  • tiiis vector contains a pBR322 repUcation origin, and die cDNA inserts of interest can be obtained easily by Not I digestion of crude Lambda DNA preparations and Ugation foUowed by transfection of bacterial ceUs.
  • the cDNA Ubrary will be constructed as described in detail by Miki et al. Gene 83:131-146 (1989).
  • the cDNA Ubrary will be transfected into hBsk-expressing NIH/3T3 mouse embryo fibroblasts. Foci from transfected ceUs wUl be isolated and tested for Neo resistance to eliminate the background transformation in NIH/3T3 ceUs. Genomic DNA from each Neo-resistant transformant will be cleaved by Not I which will release the plasmid. Digested DNA will be Ugated under diluted conditions and used to transform competent bacteria. Plasmid DNA from each focus will be purified and transfected in NIH/3T3 ceUs with or witiiout hBsk expression.
  • ADDRESSEE STERNE, KESSLER, GOLDSTEIN & FOX
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CAAGCTGTCC ACGAATTTGC TAAGGAGATA GAAGCATCAT GTATCACCAT TGAGAGAGTT 180
  • MOLECULE TYPE DNA (genomic)
  • GCTCA 245 (2) INFORMATION FOR SEQ ID NO:6 :
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic) -by-
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention porte sur une kinase, la hBSK, spécifique du cerveau humain et en particulier sur des molécules d'acide nucléique codant pour la hBsk, des polypeptides de la hBsk, sur des molécules d'acide nucléique de recombinaison, des cellules contenant lesdites molécules d'acide nucléique de recombinaison, des anticorps présentant une affinité de liaison spécifique vis-à-vis de la hBsk, des hybridomes contenant lesdits anticorps, des sondes d'acide nucléique de détection de l'acide nucléique de la hBsk, une méthode de détection de l'acide nucléique ou du polypeptide de la hBsk, des matériels contenant lesdites sondes d'acide nucléique et lesdits anticorps. L'invention porte en outre sur des dosages biologiques utilisant la séquence de l'acide nucléique, la protéine réceptrice ou les anticorps objets de cette invention pour diagnostiquer, évaluer ou pronostiquer une maladie neurodégénérative chez un mammifère. Sont également inclus les utilisations thérapeutiques de la tyrosine kinase de type récepteur de la hBsk et le ligand du récepteur de la hBSK ainsi que ses utilisations thérapeutiques.
PCT/US1995/009334 1994-07-26 1995-07-26 Kinase specifique du cerveau humain WO1996003043A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7381410B2 (en) 2003-03-12 2008-06-03 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US7595044B2 (en) 1998-04-13 2009-09-29 California Institute Of Technology Artery-and vein-specific proteins and uses therefor
US7700297B2 (en) 1998-04-13 2010-04-20 California Institute Of Technology Artery- and vein-specific proteins and uses therefor
US7862816B2 (en) 2003-03-12 2011-01-04 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US7977463B2 (en) 2004-03-12 2011-07-12 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8975377B2 (en) 2007-08-13 2015-03-10 Vasgene Therapeutics, Inc Cancer treatment using humanized antibodies that bind to EphB4
US8981062B2 (en) 2004-03-12 2015-03-17 Vasgene Theapeutics, Inc Polypeptide compounds for inhibiting angiogenesis and tumor growth
US9533026B2 (en) 2004-09-23 2017-01-03 Vasgene Therapeutics, Inc Polypeptide compounds for inhibiting angiogenesis and tumor growth

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BRAIN RESEARCH AND MOLECULAR BRAIN RESEARCH, Volume 28, issued 1995, SCHULZ et al., "Isolation and Expression Analysis of tyro3, a Murine Growth Factor Receptor Tyrosine Kinase Preferentially Expressed in Adult Brain", pages 273-280. *
BRITISH JOURNAL OF CANCER, Volume 69, issued 1994, TUZI et al., "Eph, the Largest Known Family of Putative Growth Factor Receptors", pages 417-421. *
GENE, Volume 148, issued 1994, SIEVER et al., "Identification of a Complete Cek7 Receptor Protein Tyrosine Kinase Coding Sequence and cDNAs of Alternatively Spliced Transcripts", pages 219-226. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 269, Number 43, issued 28 October 1994, SHAO et al., "cDNA Cloning and Characterization of a Ligand for the Cek5 Receptor Protein Tyrosine Kinase", pages 26606-26609. *
JOURNAL OF NEUROSCIENCE RESEARCH, Volume 37, issued 1994, ZHOU et al., "Isolation and Characterization of Bsk, a Growth Factor Receptor-Like Tyrosine Kinase Associated With the Limbic System", pages 129-143. *
ONCOGENE, Volume 10, issued 1995, FOX et al., "cDNA Cloning and Tissue Distribution of Five Human EPH-Like Receptor Protein Tyrosine Kinases", pages 897-905. *
ONCOGENE, Volume 8, issued 1993, MAISONPIERRE et al., "Ehk-1 and Ehk-2: Two Novel Members of the Eph Receptor-Like Tyrosine Kinase Family With Distinctive Structures and Neuronal Expression", pages 3277-3288. *
ONCOGENE, Volume 8, issued 1993, SAJJADI et al., "Five Novel Avian Eph-Related Tyrosine Kinases are Differentially Expressed", pages 1807-1813. *
TRENDS IN BIOTECHNOLOGY, Volume 13, issued January 1995, MARTIN, "Gene Therapy and Pharmacological Treatment of Inherited Neurological Disorders", pages 28-35. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7595044B2 (en) 1998-04-13 2009-09-29 California Institute Of Technology Artery-and vein-specific proteins and uses therefor
US7700297B2 (en) 1998-04-13 2010-04-20 California Institute Of Technology Artery- and vein-specific proteins and uses therefor
US7741272B2 (en) 1998-04-13 2010-06-22 California Institute Of Technology Artery- and vein-specific proteins and uses therefor
US7939071B2 (en) 1998-04-13 2011-05-10 California Institute Of Technology Artery- and vein-specific proteins and uses therefor
US7381410B2 (en) 2003-03-12 2008-06-03 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US7862816B2 (en) 2003-03-12 2011-01-04 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8063183B2 (en) 2003-03-12 2011-11-22 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8273858B2 (en) 2003-03-12 2012-09-25 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US7977463B2 (en) 2004-03-12 2011-07-12 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8981062B2 (en) 2004-03-12 2015-03-17 Vasgene Theapeutics, Inc Polypeptide compounds for inhibiting angiogenesis and tumor growth
US9533026B2 (en) 2004-09-23 2017-01-03 Vasgene Therapeutics, Inc Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8975377B2 (en) 2007-08-13 2015-03-10 Vasgene Therapeutics, Inc Cancer treatment using humanized antibodies that bind to EphB4

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