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WO1997018822A9 - Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci - Google Patents

Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci

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Publication number
WO1997018822A9
WO1997018822A9 PCT/US1996/018675 US9618675W WO9718822A9 WO 1997018822 A9 WO1997018822 A9 WO 1997018822A9 US 9618675 W US9618675 W US 9618675W WO 9718822 A9 WO9718822 A9 WO 9718822A9
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WO
WIPO (PCT)
Prior art keywords
protein
deltex
nucleic acid
vertebrate
notch
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PCT/US1996/018675
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English (en)
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WO1997018822A1 (fr
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Publication date
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Priority to EP96942785A priority Critical patent/EP0869802A4/fr
Priority to AU11614/97A priority patent/AU728798B2/en
Priority to JP9519885A priority patent/JP2000502246A/ja
Publication of WO1997018822A1 publication Critical patent/WO1997018822A1/fr
Publication of WO1997018822A9 publication Critical patent/WO1997018822A9/fr

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Definitions

  • the present invention relates to vertebrate deltex genes and their encoded protein products, as well as derivatives and analogs thereof .
  • the invention further relates to production of vertebrate Deltex proteins, derivatives and antibodies.
  • Related therapeutic compositions and methods of therapy and diagnosis are also provided.
  • Notch locus plays a central role in regulative events influencing cell fate decisions in Drosophila in a very broad spectrum of developing tissues (reviewed in Artavanis-Tsakonas and Simpson, 1991 , Trends Genet. 7:403-408; and in Artavanis-Tsakonas et al. , 1991 , Ann.
  • Notch homologs have been isolated from a variety of vertebrate species and have been shown to be remarkably similar to their Drosophila counterpart in terms of structure, expression pattern and ligand binding properties (Rebay et al , 1991, Cell 67:687-699; Coffman et al., 1990, Science 249-1438-1441, Elhsen et al, 1991 , Cell 66-649-661, Weinmaster et al , 1991 , Development 113 199-205) Two human Notch homologs have been isolated (PCT Publication No WO 92/19737 dated November 12, 1992), termed hN and TAN-1 A human Notch (TAN-1) malfunction has been associated with a lymphatic cancer (Elhsen et al , 1991, Cell 66:649-661).
  • Notch encodes a large, structurally-complex transmembrane protein, consistent with an involvement in cell-cell communication (Wharton et al , 1985, Cell 43:567-581, Kidd et al , 1986, Mol Cell Biol 6 3094-3108)
  • Notch has an extracellular domain containing 36 tandem EGF-hke repeats and 3 Notch/linl2 repeats The intracellular domain bears several common structural motifs including 6 cdclO/SW16/ankyr ⁇ n repeats ("ANK" repeats) Lux et al., 1990, Nature 344:36-42, Breeden and Nasmyth, 1987, Nature 329:651-654, Michaely and Bennett, 1992, Trends Cell Biol.
  • ANK repeats are crucial for Notch-mediated signaling events. Both EGF-like repeats and ankyrin motifs are found in a variety of proteins known to interact with other protein molecules. Indeed, evidence has shown a direct interaction between Notch and the products of the Delta and Serrate loci, which also encode transmembrane proteins containing EGF- like repeats (Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699).
  • Drosophila Deltex has been demonstrated to play a critical role in development and other physiological processes, in particular, in the signaling pathway of Notch which is involved in cell fate (differentiation) determination.
  • Drosophila Deltex mediates the intracellular portion of the signal transduction cascade involved in Notch function (Diederich et al., 1994, Development 120:473-481).
  • Drosophila Deltex is localized within the cytoplasm, that it is a protein of unique sequence, that it displays homotypic interactions, and that it directly physically interacts with the Drosophila Notch intracellular ANK repeats.
  • Drosophila Deltex directly interacts with the ANK repeats of human Notch.
  • the ANK repeat motif is shared by many proteins and has been implicated in protein-protein interactions (Lux et al., 1990, Nature 344:36-42, Thompson et al., 1991 ,
  • deltex displays genetic interactions with Notch and Delta, both transmembrane proteins, and with mastermind, a nuclear localized protein (Smoller et al., 1990, Genes Dev 4: 1688-1700). This makes deltex the first identified cytoplasmic component of the Notch group of interacting loci.
  • the Drosophila Su(H) gene encodes a protein of 594 amino acids and binds to the promoters of several viral and cellular genes and interacts directly with a viral transactivator protein termed Epstein-Barr virus nuclear antigen 2 (EBNA2), which enables a virus to subvert the normal program of B cell differentiation (Sch Stammguth, F., et al, 1992. Cell 69. 1 199. Furukawa T , et al.. 1991 , J Biol. Chem.
  • EBNA2 Epstein-Barr virus nuclear antigen 2
  • the present invention relates to nucleotide sequences of vertebrate deltex genes, and ammo acid sequences of the encoded vertebrate Deltex proteins
  • the invention further relates to fragments and other derivatives, and analogs, of vertebrate Deltex proteins, as well as antibodies thereto Nucleic acids encoding such fragments or derivatives are also within the scope of the invention Production of the foregoing proteins and derivatives e g , by recombinant methods, is provided.
  • the invention relates to human deltex nucleic acids and proteins.
  • the invention relates to mammalian deltex nucleic acids and proteins.
  • the invention relates to vertebrate Deltex protein derivatives and analogs of the invention which are functionally active, or which comprise one or more domains of a vertebrate Deltex protein, including but not limited to the SH3- binding domains, ⁇ ng-H2-Z ⁇ nc fingers, domains which mediate binding to Notch or to a Notch dem atrve containing Notch cdclO/SW16/anky ⁇ n ("ANK' ) repeats, or am
  • the present invention also relates to therapeutic and diagnostic methods and compositions based on vertebrate Deltex proteins and nucleic acids.
  • the invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the invention .
  • Such therapeutic compounds include vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof, antibodies thereto, nucleic acids encoding the vertebrate Deltex proteins, analogs, or derivatives and vertebrate deltex antisense nucleic acids
  • a Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state.
  • a Therapeutic of the invention is administered to treat a nervous system disorder or to promote tissue regeneration and repair.
  • Therapeutics which antagonize, or inhibit, vertebrate Notch and/or Deltex function are administered for therapeutic effect.
  • Therapeutics which promote vertebrate Notch and/or Deltex function are administered for therapeutic effect.
  • disorders of cell fate in particular hyperproliferative (e.g., cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of vertebrate Notch and/or Deltex protein can be diagnosed by detecting such levels, as described more fully infra.
  • hyperproliferative e.g., cancer
  • hypoproliferative disorders involving aberrant or undesirable levels of expression or activity or localization of vertebrate Notch and/or Deltex protein can be diagnosed by detecting such levels, as described more fully infra.
  • a Therapeutic of the invention is a protein consisting of at least a fragment (termed herein "adhesive fragment") of vertebrate Deltex which mediates binding to a Notch protein or a fragment thereof.
  • the invention also provides methods of inactivating Notch function in a cell, methods of identifying a compound that inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein, and methods of expanding non-terminally differentiated cells.
  • Figure 1A-F Nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO:2) of Drosophila deltex cDNA.
  • FIG. 2A-C Composite nucleotide sequence (SEQ ID NO: 11) derived from the cDNA (nucleotide 1 to 2547), and deduced amino acid sequence (SEQ ID NO: 12) of the human deltex locus. The predicted amino acid sequence is depicted below the DNA sequence. The symbol: * designates the start of T05200 and $ the end of T05200. Core H and C residues in Ring-H2-zinc finger are shown by underlining. PCR primers hdx-1 to 4 (SEQ ID NO:26), (SEQ ID NO:27), (SEQ ID NO:28), and (SEQ ID NO:29), respectively, are indicated in bold. X and N represent amino acid residues and nucleotides, respectively, not yet determined.
  • FIG. 4A-B Amino acid sequence of Drosophila Deltex (SEQ ID NO: 2) and designated fragments implicated in protein-protein interactions. Fragments A-D
  • FIG. 1 Schematic diagram of Deltex fragments mediating Deltex-Deltex interactions.
  • the present invention relates to nucleotide sequences of vertebrate deltex genes, and amino acid sequences of their encoded Deltex proteins.
  • the invention further relates to fragments and other derivatives, and analogs, of vertebrate Deltex proteins.
  • Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is provided.
  • the invention relates to a human deltex gene and protein.
  • the invention relates to a mammalian deltex gene and protein.
  • the invention also relates to vertebrate Deltex protein derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) vertebrate Deltex protein.
  • Such functional activities include but are not limited to antigenicity [ability to bind (or compete with a vertebrate Deltex protein for binding) to an anti-vertebrate Deltex protein antibody], immunogenicity (ability to generate antibody which binds to a vertebrate Deltex protein), ability to bind (or compete with a vertebrate Deltex protein for binding) to Notch or a second Deltex protein or other proteins or fragments thereof, ability to bind (or compete with a vertebrate Deltex protein for binding) to a receptor or ligand for a vertebrate Deltex protein.
  • the invention further relates to fragments (and derivatives and analogs thereof) of a vertebrate Deltex protein which comprise one or more domains of a vertebrate Deltex protein (see infra), including but not limited to the SH3-binding domains, ring-H2- zinc fingers, domains which mediate binding to Notch (or a derivative thereof containing the Notch ANK repeats) or to a second Deltex molecule or fragment thereof, or any combination of the foregoing.
  • a vertebrate Deltex protein which comprise one or more domains of a vertebrate Deltex protein (see infra), including but not limited to the SH3-binding domains, ring-H2- zinc fingers, domains which mediate binding to Notch (or a derivative thereof containing the Notch ANK repeats) or to a second Deltex molecule or fragment thereof, or any combination of the foregoing.
  • Drosophila deltex as a probe were unsuccessful.
  • the present invention is based on the successful cloning of human deltex.
  • our results show a significant structural conservation of Deltex in humans, indicative of functional
  • the vertebrate deltex nucleic acid and amino acid sequences and antibodies thereto of the invention can be used for the detection and quantitation of vertebrate deltex mRNA and protein, to study expression thereof, to produce vertebrate Deltex proteins, fragments and other derivatives, and analogs thereof, in the study, assay, and manipulation of differentiation and other physiological processes, and are of therapeutic and diagnostic use, as described infra.
  • the agonists and antagonists of Deltex function can be used to alter the ability of a cell to differentiate.
  • the vertebrate deltex nucleic acids and antibodies can also be used to clone vertebrate deltex homologs of other species, as described infra. Such vertebrate deltex homologs are expected to exhibit significant homology to each other, and encode proteins which exhibit the ability to bind to a Notch protein.
  • the present invention also relates to therapeutic and diagnostic methods and compositions based on vertebrate Deltex proteins and nucleic acids.
  • the invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the invention.
  • therapeutic compounds include: vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the vertebrate Deltex proteins, analogs, or derivatives; and vertebrate deltex antisense nucleic acids.
  • a Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state.
  • a Therapeutic of the invention is administered to treat a nervous system disorder or to promote tissue regeneration and repair.
  • Therapeutics which antagonize, or inhibit, Notch and/or vertebrate Deltex function are administered for therapeutic effect.
  • Therapeutics which promote Notch and/or vertebrate Deltex function are administered for therapeutic effect.
  • disorders of cell fate in particular hyperproliferative (e.g., cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of Notch and/or vertebrate Deltex protein can be diagnosed by detecting such levels, as described more fully infra.
  • hyperproliferative e.g., cancer
  • hypoproliferative disorders involving aberrant or undesirable levels of expression or activity or localization of Notch and/or vertebrate Deltex protein can be diagnosed by detecting such levels, as described more fully infra.
  • a Therapeutic of the invention is a protein consisting of at least a fragment (termed herein "adhesive fragment") of vertebrate Deltex that mediates binding to a Notch protein, a second Deltex protein, or a fragment of Notch or Deltex.
  • the invention is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of human deltex, and the identification of regions of human Deltex which are predicted to bind to the ANK repeats of Notch, or which are predicted to bind to regions of human Deltex.
  • human deltex nucleic acids comprise the cDNA sequence shown in Figure 2A-C (SEQ ID NO: 11), or the coding region thereof (nucleotide numbers 504-2363), or nucleic acids encoding a human Deltex protein (e.g., having the sequence of
  • the invention provides nucleic acids consisting of at least 8 nucleotides
  • the nucleic acids consist of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides,
  • nucleic acids are provided which comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a vertebrate deltex gene.
  • the sequence is naturally occurring.
  • the invention provides nucleic acids comprising at least 110, 150, or 200 continuous nucleotides of the sequence of
  • the invention provides a nucleic acid comprising the first 25, 50, 100, 150, 200, or 230 amino acids of SEQ ID NO: 12.
  • vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encoding the amino terminal 180 amino acids (encoded, e.g. , by nucleotide numbers 504-1044 of SEQ ID: 11) of human deltex, or fragments thereof.
  • the vertebrate deltex nucleic acid has at least 50% identity over the corresponding nucleotide sequence of an identically sized human deltex. In another embodiment this identity is greater than 55 % .
  • the nucleotide sequence identity of the vertebrate deltex is greater than 60%. In a more preferred embodiment this identity is greater than 65 % .
  • the nucleotide sequence identity of the vertebrate deltex is greater than 70% over that of the corresponding nucleotide sequence of identically sized human deltex.
  • vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encoding the central region amino acids of human deltex (e.g., nucleotide numbers 1045-1821 of SEQ ID NO:11) or fragments thereof.
  • the nucleic acids encoding the central amino acids of the vertebrate Deltex protein has at least 50% nucleotide sequence identity with the corresponding human deltex sequence of identical size. In another embodiment this identity is greater than 55%. In a preferred embodiment, this nucleotide sequence identity is greater than 60%. In a more preferred embodiment this identity is greater than 65%.
  • the homology of the nucleic acids encoding the central region amino acids of the vertebrate deltex has a nucleotide sequence identity that is greater than
  • vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encoding the 180 carboxy terminal amino acids of human deltex (nucleotide numbers 1822-2366), or fragments thereof.
  • the nucleic acids encoding the carboxy terminal region of the vertebrate Deltex protein has at least 50% nucleotide sequence identity over the corresponding human deltex sequence of identical size. In another embodiment this identity is greater than 55%. In a preferred embodiment, this identity is greater than 60%.
  • this identity is greater than 65 % .
  • the identity of the nucleotides encoding the amino terminal amino acids of the vertebrate deltex is greater than 70% over that of the corresponding nucleotide sequence of identically sized human deltex.
  • a nucleic acid which is hybridizable to a vertebrate deltex nucleic acid e.g., having sequence SEQ ID NO: 11
  • a nucleic acid encoding a vertebrate deltex derivative under conditions of low stringency.
  • procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci.
  • Tris-HCl pH 7.4
  • 5 mM EDTA 5 mM EDTA
  • 0.1 % SDS Tris-HCl
  • the wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C.
  • Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 °C and reexposed to film.
  • Other conditions of low stringency which may be used are well known in the art (e.g. , as employed for cross-species hybridizations).
  • a nucleic acid which is hybridizable to a vertebrate deltex nucleic acid under conditions of high stringency is provided.
  • Washing of filters is done at 37°C for 1 h in a solution containing 2X SSC, 0.01 % PVP, 0.01 % Ficoll, and 0.01 % BSA. This is followed by a wash in 0.1X SSC at 50°C for 45 min before autoradiography.
  • Other conditions of high stringency which may be used are well known in the art.
  • Nucleic acids encoding derivatives (e.g., fragments) of vertebrate Deltex proteins (see Section 5.6), and vertebrate deltex antisense nucleic acids (see Section 5.11) are additionally provided.
  • a "nucleic acid encoding a fragment or portion of a vertebrate Deltex protein” shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the vertebrate Deltex protein and not the other contiguous portions of the vertebrate Deltex protein as a continuous sequence.
  • an expression library is obtained or is constructed by methods known in the art.
  • mRNA e.g. , human
  • cDNA is made and ligated into an expression vector (e.g. , a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced.
  • an expression vector e.g. , a bacteriophage derivative
  • Various screening assays can then be used to select for the expressed vertebrate Deltex product.
  • anti-human Deltex antibodies can be used to select the recombinant host cell expressing a cloned vertebrate deltex gene.
  • PCR is used to amplify the desired vertebrate deltex sequence in a genomic or cDNA library, prior to selection (see, by way of example Section 8, infra).
  • Oligonucleotide primers representing known vertebrate deltex sequences, preferably regions known to be conserved between Drosophila and human, can be used as primers in PCR.
  • the synthetic oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA), preferably a cDNA library, of potential interest.
  • PCR can be carried out, e.g. , by use of a Perkin-Elmer Cetus thermal cycler and
  • the DNA being amplified can include human mRNA or cDNA or genomic DNA.
  • a segment of a vertebrate deltex gene homolog After successful amplification of a segment of a vertebrate deltex gene homolog, that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra.
  • Deltex proteins may be identified in this fashion.
  • the PCR-amplified DNA can be inserted into an expression vector for expression cloning as described above.
  • oligonucleotide primers derived from the sequence of a vertebrate deltex gene of a different species can be isolated as set forth in Example 8, by screening with a probe, or priming for PCR with an oligonucleotide, containing deltex sequences encoding regions highly conserved between human and Drosophila.
  • the human Deltex amino acid stretches 414-419 (SEQ ID NO:30), 475-480 (SEQ ID NO:31), 504-511 (SEQ ID NO: 32), 531-539 (SEQ ID NO:33) and 557-564 (SEQ ID NO:34) are conserved in Drosophila Deltex amino acid stretches 549-555 (SEQ ID NO:35), 603-608 (SEQ ID NO:36), 632-639 (SEQ ID NO:37), 659-667 (SEQ ID NO:38) and 685- 692 (SEQ ID NO: 39), respectively.
  • a pair of oligonucleotides comprising sequences separated by a length in the range from 50-500 nucleotides is used as primers in PCR.
  • the invention envisions the use of nucleic acids encoding conserved regions of the Deltex protein in combination to isolate the Deltex encoding nucleic acids of other organisms, by use in PCR to amplify the desired sequence or less preferably, without PCR, as a probe in selection by virtue of direct colony hybridization (e.g. , Grunstein, M. and Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961).
  • the desired deltex gene can be isolated by a more gradual method of evolutionary walking via first isolating a deltex gene from a more closely related species, identifying the portions of deltex which are conserved cross-species, and then screening with a probe or priming for PCR with a nucleic acid containing the conserved sequence.
  • the evolutionary tree may first isolate a murine deltex gene using nucleic acids encoding human Deltex as a probe. A conserved portion of the murine deltex sequence is then used to screen or amplify deltex in an avian library; a conserved portion of the avian clone is used to screen an amphibian library, a conserved portion of the amphibian clone is used to screen a fish library, etc. If desired, the species to be selected in the next round of screening can be selected from among various species by hybridizing the deltex probe to a Southern blot containing genomic DNA from each species, and selecting a species to which hybridization occurs.
  • Any eukaryotic cell can potentially serve as the nucleic acid source for the molecular cloning of the vertebrate deltex gene.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g. , a DNA "library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired human cell (see, for example Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2d. Ed., Cold Spring
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • DNA fragments are generated, some of which will encode the desired gene.
  • the DNA may be cleaved at specific sites using various restriction enzymes.
  • DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication.
  • the lin a DNA agments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
  • identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, if an amount of a portion of a vertebrate deltex (of any species) gene or its specific RNA, or a fragment thereof e.g., the adhesive domain, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, R., 1977, Science 196, 180; Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961). Those DNA fragments with substantial homology to the probe will hybridize.
  • vertebrate Deltex has similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, binding to Notch, or antigenic properties as known for vertebrate Deltex.
  • the vertebrate Deltex protein may be identified by binding of labeled antibody to the putatively vertebrate Deltex synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay )-type procedure.
  • the vertebrate deltex gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified vertebrate deltex DNA of another species (e.g., human).
  • Immunoprecipitation analysis or functional assays e.g., ability to bind Notch
  • mRNA and, therefore, the complementary DNA fragments that contain the desired sequences e.g., specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against vertebrate Deltex protein.
  • radiolabelled vertebrate deltex cDNA can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. The radiolabelled mRNA or cDNA may then be used as a probe to identify the vertebrate deltex DNA fragments from among other genomic DNA fragments.
  • RNA for cDNA cloning of the vertebrate deltex gene can be isolated from cells which express vertebrate Deltex. Other methods are possible and within the scope of the invention.
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as PBR322, pUC, or Bluescript (Stratagene) plasmid derivatives.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • the cleaved vector and vertebrate deltex gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfecuon, infection, electroporauon, etc., so that many copies of the gene sequence are generated.
  • the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionizauon, can be done before insertion into the cloning vector.
  • transformauon of host cells with recombinant DNA molecules that incorporate the isolated vertebrate deltex gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene.
  • the gene may be obtamed in large quantities by growing transf ormants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
  • the vertebrate deltex sequences provided by the instant invention include those nucleotide sequences encodmg substanually the same ammo acid sequences as found in native vertebrate Deltex protein, and those encoded ammo acid sequences with functionally equivalent ammo acids, all as described m Section 5.6 infra for vertebrate Deltex derivatives.
  • the nucleic acid coding for a vertebrate Deltex protein or a functionally active fragment or other derivative thereof can be inserted mto an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • the necessary transc ⁇ ptional and translational signals can also be supplied by the native vertebrate deltex gene and/or its flanking regions.
  • host-vector systems may be utilized to express the protein- coding sequence. These include but are not limited to vertebrate cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g.
  • baculovirus baculovirus
  • microorgamsms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA
  • the expression elements of vectors vary m their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • a molecule comprising a portion of a vertebrate deltex gene which encodes a protein that binds to Notch or to a molecule comprising the Notch ANK repeats is expressed.
  • a molecule comprising a portion of a vertebrate deltex gene which encodes a protein that binds to a fragment of a Deltex protein is expressed.
  • mammalian deltex gene is expressed, or a sequence encoding a functionally active portion of mammalian Deltex.
  • the human deltex gene is expressed, or a sequence encoding a functionally active portion of human
  • a chimeric protein comprising a Notch-binding domain of a vertebrate Deltex protein is expressed.
  • a full-length vertebrate deltex cDNA is expressed, or a sequence encoding a functionally active portion of a vertebrate Deltex protein.
  • a fragment of a vertebrate Deltex protein comprising a domain of the protein, or other derivative, or analog of a vertebrate Deltex protein is expressed.
  • any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of a nucleic acid sequence encoding a vertebrate Deltex protein or peptide fragment may be regulated by a second nucleic acid sequence so that the vertebrate Deltex protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a vertebrate Deltex protein may be controlled by any promoter /enhancer element known in the art.
  • Promoters which may be used to control vertebrate deltex gene expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
  • tac (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25), ⁇ P L , or trc promoters; see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res.
  • promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring
  • Expression vectors containing vertebrate deltex gene inserts can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences.
  • first approach the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted vertebrate deltex gene.
  • second approach the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g.
  • recombinants containing the vertebrate deltex insert can be identified by the absence of the marker gene function.
  • recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the vertebrate deltex gene product in in vitro assay systems, e.g. , binding to
  • the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g. , lambda), and plasmid and cosmid DNA vectors, to name but a few.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered vertebrate Deltex protein may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g. , phosphorylation, cleavage) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • the vertebrate Deltex protein, fragment, analog, or derivative may be expressed as a fusion, or chimeric protein product (comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)).
  • a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
  • a chimeric product may be made by protein synthetic techniques, e.g. , by use of a peptide synthesizer. Both cDNA and genomic sequences can be cloned and expressed.
  • a vertebrate deltex cDNA sequence may be chromosomally integrated and expressed. Homologous recombination procedures known in the art may be used.
  • the invention provides amino acid sequences of vertebrate Deltex, preferably human Deltex, and fragments and derivatives thereof which comprise an antigenic determinant (i.e. , can be recognized by an antibody) or which are functionally active, as well as nucleic acid sequences encoding the foregoing.
  • "Functionally active" material as used herein refers to that material displaying one or more known functional activities associated with the fiill-length (wild-type) vertebrate Deltex protein product, e.g.
  • the invention provides fragments of a vertebrate
  • Deltex protein consisting of at least 6 amino acids, 10 amino acids, 50 amino acids, or of at least 75 amino acids.
  • the proteins comprise, or alternatively, consist essentially of; one or more of the SH3-binding domains (e.g. , SEQ ID NOS: 17-21 of Table
  • one or more ring-H2-zinc finger domains e.g., SEQ ID NO:25
  • a portion which binds to Notch e.g. , comprising the first approximately 230 amino acids of vertebrate
  • Deltex or any combination of the foregoing, of a vertebrate Deltex protein. Fragments, or proteins comprising fragments, lacking some or all of the foregoing regions of vertebrate
  • Deltex are also provided. Molecules comprising more than one copy of the foregoing regions are also provided. Nucleic acids encoding the foregoing are provided.
  • the gene product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive labelling of the product followed by analysis by gel electrophoresis, immunoassay, etc. Chemically synthesized proteins, derivatives, and analogs can be similarly analyzed.
  • a vertebrate Deltex protein Once a vertebrate Deltex protein is identified, it may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the functional properties may be evaluated using any suitable assay (see Section 5.7).
  • the amino acid sequence of a vertebrate Deltex protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant. Once the amino acid sequence is thus known, the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller et al., 1984, Nature 310:105- 111).
  • the structure of the vertebrate deltex genes and proteins can be analyzed by various methods known in the art.
  • the cloned DNA or cDNA corresponding to the vertebrate deltex gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975,
  • Southern hybridization with a vertebrate deltex-specific probe can allow the detection of the vertebrate deltex genes in DNA from various cell types.
  • Southern hybridization can be used to determine the genetic linkage of vertebrate deltex.
  • Northern hybridization analysis can be used to determine the expression of the vertebrate deltex genes.
  • Restriction endonuclease mapping can be used to roughly determine the genetic structure of the vertebrate deltex gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis. Alternatively, restriction maps can be deduced, once the nucleotide sequence is known.
  • DNA sequence analysis can be performed by any techniques known in the an, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger et al., 1977, Proc. Natl. Acad. Sci.
  • the amino acid sequence of a vertebrate Deltex protein can be derived by deduction from the DNA sequence, or alternatively, by direct sequencing of the protein, e.g., with an automated amino acid sequencer.
  • the amino acid sequence of a representative vertebrate Deltex protein comprises the sequence substantially as depicted in Figure 2A-C (SEQ ID NO: 12), and detailed in Section 6, infra.
  • the vertebrate Deltex protein sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824).
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of a vertebrate Deltex protein and the corresponding regions of the gene sequence which encode such regions. Hydrophilic regions are predicted to be immunogenic.
  • Manipulation, translation, and secondary structure prediction, as well as open reading frame prediction and plotting, can also be accomplished using computer software programs available in the art. Other methods of structural analysis can also be employed. These include but are not limited to X-ray crystallography (Engstom, 1974, Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick and Zoller (eds.), 1986, Computer Graphics and
  • a vertebrate Deltex protein may be used as an immunogen to generate antibodies which recognize such an immunogen.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • antibodies which specifically bind to vertebrate Deltex proteins are produced.
  • an antibody which binds to a vertebrate Deltex protein e.g. , mammalian, preferably human
  • such an antibody is produced by using as immunogen, regions least conserved between Drosophila melanogaster and the vertebrate Deltex protein.
  • antibodies to a particular domain of a vertebrate Deltex protein are produced.
  • an antibody is produced which binds to a fragment of vertebrate Deltex which binds to Notch; in another embodiment, an antibody binds to a molecule comprising the first 230 amino-terminal amino acids of vertebrate Deltex. In another embodiment the antibody binds to an amino-terminal fragment of vertebrate Deltex containing not more than the first 200 amino acids of vertebrate Deltex. In yet another embodiment, an antibody binds to a fragment of vertebrate Deltex which binds to a second Deltex molecule.
  • polyclonal antibodies to a vertebrate Deltex protein or derivative or analog may be obtained.
  • various host animals can be immunized by injection with a native vertebrate Deltex protein, or a synthetic version, or derivative (e.g. , fragment) thereof, including but not limited to rabbits, mice, rats, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, poly anions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • polyclonal or monoclonal antibodies are produced by use of a hydrophilic portion of a vertebrate Deltex peptide (e.g. , identified by the procedure of Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824)).
  • a hydrophilic portion of a vertebrate Deltex peptide e.g. , identified by the procedure of Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824).
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495- 497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies can be used.
  • monoclonal antibodies can be produced in germ-free animals (PCT Publication No.
  • human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96), or by other methods known in the art.
  • techniques developed for the production of "chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A.
  • Non-human antibodies can be humanized by the method of Winter (see U.S. Patent No. 5,225,539).
  • Antibody fragments and other derivatives which contain the idiotype (binding domain) of the molecule can be generated by known techniques.
  • fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • to select antibodies which recognize a specific domain of a vertebrate Deltex protein one may assay generated hybridomas for a product which binds to a vertebrate Deltex fragment containing such domain.
  • antibodies specific to a phosphorylated epitope of vertebrate Deltex are produced.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the protein sequences of the invention e.g. , for imaging these proteins, measuring levels thereof in appropriate physiological samples, etc.
  • Antibodies to vertebrate Deltex can be used to determine the intracellular distribution of Notch and/or vertebrate Deltex, in diagnostic methods such as described infra.
  • the antibodies also have use in immunoassays.
  • anti-vertebrate Deltex antibodies and fragments thereof containing the binding domain are Therapeutics.
  • the invention further provides vertebrate Deltex proteins, and derivatives
  • the vertebrate Deltex proteins are encoded by me vertebrate deltex nucleic acids described in Section 5.1 supra.
  • the proteins, derivatives, or analogs are of mouse or rat; agricultural stock such as cow, sheep, horse, goat, pig and the like; pets such as cats, dogs; or other domesticated mammals, or primate Deltex proteins.
  • Deltex are within the scope of the present invention.
  • the derivative or analog is functionally active, i.e. , capable of exhibiting one or more functional activities associated with a full-length, wild-type vertebrate Deltex protein.
  • vertebrate Deltex derivatives can be made by altering vertebrate deltex sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • nucleotide coding sequences Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a vertebrate deltex gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of vertebrate deltex genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • the vertebrate deltex genes are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • Deltex derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a vertebrate Deltex protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • proteins consisting of or comprising a fragment of a vertebrate Deltex protein consisting of at least 10 (continuous) amino acids of the vertebrate Deltex protein is provided.
  • the fragment consists of at least 20 or 50 amino acids of the vertebrate Deltex protem In specific embodiments, such fragments are not larger than 35, 100 or 200 ammo acids.
  • Derivatives or analogs of vertebrate Deltex m include but are not limited to those peptides which are substantially homologous to human Deltex or fragments thereof.
  • derivatives or analogs of vertebrate Deltex m include those pepudes which are substantially homologous to the amino terminal 180 ammo acids (1-
  • the amino terminal region of the vertebrate is amino terminal region of the vertebrate
  • Deltex protein has at least 30% identity over the -trnino terminal ammo acid sequence of idenucally sized human Deltex. In another embodiment this identity is greater than 35%. In a preferred embodiment, the amino terminal ammo acid identity of the vertebrate Deltex is greater than 45 % . In a more preferred embodiment this identity is greater than 55 % . In a most preferred embodiment, the homology of the amino terminal ammo acids of the vertebrate Deltex is greater than 65 % over the correspondmg human Deltex amino terminal ammo acid sequence of identical size.
  • derivatives or analogs of vertebrate Deltex include those peptides which are substantially homologous to the central region (ammo acids 181-441) of human Deltex, or fragments thereof.
  • the central region of the vertebrate Deltex protem has at least 30% identity with the correspondmg human Deltex sequence of identical size. In another embodiment this identity is greater than 35%.
  • the ammo acid identity of the central region of vertebrate Deltex and human Deltex is greater than 45% In a more preferred embodiment this identity is greater than 55 % .
  • the homology of the central ammo acids of the vertebrate Deltex to corresponding human Deltex amino acids of identical size is greater than 65%.
  • derivatives or analogs of vertebrate Deltex m include but are not limited to those peptides which are substantially homologous to the carboxy terminal ammo acids of human Deltex or fragments thereof.
  • the carboxy terminal region of the vertebrate Deltex protem (the carboxy terminal 180 ammo acids) has at least 45 % identity over the ammo acid sequence of identical size In another embodiment this identity is greater than 50%.
  • the ammo terminal amino acid identity of the vertebrate Deltex is greater than 55%. In a more preferred embodiment this identity is greater than 60%.
  • the homology of the amino terminal amino acids of the vertebrate Deltex is greater than 65% .
  • derivatives or analogs of vertebrate Deltex comprise regions conserved between Drosophila and human Deltex (see Section 8).
  • the vertebrate Deltex protein derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.
  • the cloned vertebrate deltex gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor
  • vertebrate Deltex-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction
  • mutagenesis Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem 253:6551), use of TAB ® linkers (Pharmacia), etc.
  • Manipulations of the vertebrate deltex sequence may also be made at the protein level. Included within the scope of the invention are vertebrate Deltex protein fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by acetylation, phosphorylation, carboxylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • phosphorylation or, alternatively, dephosphorylation is carried out, which can be to various extents, on the purified vertebrate Deltex protein or derivative thereof.
  • the phosphorylation state of the molecule may be important to its role in intracellular signal transduction of Notch function.
  • Phosphorylation can be carried out by reaction with an appropriate kinase (e.g., possibly cdc2 or CK H).
  • Dephosphorylation can be carried out by reaction with an appropriate phosphatase.
  • Deltex protein which comprises the desired domain, or which mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the vertebrate Deltex protein sequence.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid,
  • the vertebrate Deltex derivative is a chimeric, or fusion, protein comprising a vertebrate Deltex protein or fragment thereof (preferably consisting of at least a domain or motif of the vertebrate Deltex protein, or at least 10 amino acids of the vertebrate Deltex protein) joined at its amino or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein.
  • a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a vertebrate Deltex-coding sequence joined in-frame to a coding sequence for a different protein).
  • Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
  • a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • a specific embodiment relates to a chimeric protein comprising a fragment of a vertebrate Deltex protein which comprises a domain or motif of the vertebrate Deltex protein, e.g. , a portion which binds to a Notch protein or to a second Deltex protein, an SH-3 binding domain, a ring-H2-zinc finger domain, etc.
  • a chimeric nucleic acid can be constructed, encoding a fusion protein consisting of a vertebrate Deltex Notch-binding fragment joined to a non-Deltex protein.
  • a recombinant molecule can be constructed according to the invention, comprising coding portions of both a vertebrate deltex gene and another gene which is a member of the "Notch group.”
  • Another specific embodiment relates to a chimeric protein comprising a fragment of a vertebrate Deltex protein of at least six amino acids.
  • fusion proteins comprising human Deltex or various Notch fragments, are described in Section 7.
  • the invention provides vertebrate Deltex derivatives and analogs, in particular vertebrate Deltex fragments and derivatives of such fragments, that comprise or consist of one or more domains of the vertebrate Deltex protein, including but not limited to a region which binds to a Notch protein (or a molecule comprising the ANK repeats thereof), a region which binds to a second Deltex protein or portion thereof, an SH3-binding domain, or a ring-H2-zinc finger domain.
  • the vertebrate Deltex derivative may lack all or a portion of one or more of the foregoing domains.
  • the aforesaid domains consist of approximately the following amino-acid sequences (see Section 6.1.1 infra):
  • Ring-H2-zinc finger domain SEQ ID NO:25
  • binding fragments e.g., smaller than those set forth above, can be identified by routine methods, e.g., by construction of nucleic acids encoding such fragments and assays for binding (e.g. , via the interaction trap method described in Section
  • fragments comprising specific domains of vertebrate Deltex are those comprising domains in the respective vertebrate Deltex protein most homologous to the specific domain of the human Deltex protein.
  • Deltex as well as Deltex-Notch interactions were detected.
  • Deletion constructs encoding various fragments (described below) of Drosophila Deltex, Drosophila Notch and human Notch were expressed as fusion constructs (LexA or ACT fusions), and assayed.
  • FIG. 5 summarizes the Deltex-Deltex interactions we have detected.
  • Fragment A interacts with Fragment A (homotypic interactions).
  • Fragment B interacts with
  • Fragment B (homotypic interactions). Fragment C interacts with Fragment C (homotypic interactions). In addition, we detected interactions between fragments C and B. However, we can only detect the fragment C-B interaction if fragment C is tested as the "bait" (i.e., as the LexA fusion). If Fragment B is the bait, this interaction is not detected. All the other aforesaid interactions occur irrespective of which fragment is used as the bait. Fragment A consists of amino acids 1-303. Fragment B consists of amino acids 306-486. Fragment C consists of amino acids 514-737.
  • vertebrate Deltex regions are provided that are most homologous to Drosophila fragment A (SEQ ID NO: 13), fragment B (SEQ ID NO: 14), fragment C (SEQ ID NO: 15), and fragment D (SEQ ID NO: 16), shown in Figure 4A-B. Binding interactions between fragments are indicated by arrows in Figures 5 and 6. Such regions homologous to A-D are predicted also to display the binding interactions shown in
  • amino acids 1-237, 238-391, 392-620, and 1-175 of SEQ ID NO: 12 correspond to Drosophila fragments A-D, respectively.
  • Molecules comprising one or more of the foregoing regions are provided. Accordingly, by way of example, a molecule comprising amino acid numbers 1-237 of SEQ ID NO: 12 is predicted to bind the Notch ankyrin repeats.
  • inhibitors e.g. , peptide inhibitors of the foregoing protein interactions with Notch or with a second Deltex protein.
  • the nucleic acid sequences encoding Notch or vertebrate Deltex proteins or fragments thereof, for use in such assays, can be isolated from porcine, bovine, equine, feline, canine, as well as primate sources and any other mammals in which homologs of known genes can be identified.
  • the Notch protein or portion thereof comprising the ANK repeats which can be expressed and assayed for binding to Deltex or a Deltex derivative can be derived from any of the Notch homologs: human hN, human TAN-1, Xenopus, and Drosophila.
  • nucleotide coding sequences which encode substantially the same amino acid sequence as the aforesaid domains may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of the vertebrate deltex genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • vertebrate Deltex proteins, fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of the domains including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence ("conservative" changes).
  • the derivatives, analogs, and peptides of die invention can be produced by various methods known in the art.
  • the manipulations which result in their production can occur at the gene or protein level.
  • the nucleic acid sequence can be mutated in vitro or in vivo; and manipulations of the sequence may also be made at the protein level.
  • analogs and peptides can be chemically synthesized.
  • immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays,
  • immunoradiometric assays gel diffusion precipitm reactions, lmmunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • a method of identifying a molecule that inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein comprises the steps of contacting a Notch protein and a vertebrate Delta protein such that binding between the Notch protein and the Deltex protein can occur, in the presence of one or more molecules which are desired to be tested for the ability to inhibit or reduce binding between the Notch protein and the Deltex protein, and identifying the molecules that inhibit or reduce the binding of the Deltex protein to the Notch protein.
  • Any of various binding assays known in the art can be used to carry out such a method, including but not limited to yeast interaction trap assays, cell culture in vitro aggregation assays, and soluble binding assays using purified Notch and Deltex proteins.
  • a specific embodiment is as follows: Cultured cells are cotransfected with plasmid expression constructs that place Notch and deltex under distinct inducible promoters. Notch expression in these cells is first induced to ensure proper cell surface localization; Deltex expression is then induced.
  • This increased localization can be determined according to methods known in the art (e.g., immunofluorescent staining with antibody to Deltex).
  • the method can also be carried out using derivatives of Notch and Deltex that mediate binding to Deltex and to Notch, respectively.
  • the invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the invention.
  • therapeutic compounds include: vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof (e.g., as described hereinabove); antibodies thereto (as described hereinabove); nucleic acids encoding the vertebrate Deltex proteins, analogs, or derivatives (e.g., as described hereinabove); and vertebrate deltex antisense nucleic acids.
  • the Antagonist Therapeutics of the invention are those Therapeutics which antagonize, or inhibit, a vertebrate Deltex function and/or Notch function.
  • Antagonist Therapeutics are most preferably identified by use of known convenient in vitro assays, e.g. , based on their ability to inhibit binding of vertebrate Deltex to another protein (e.g. , a Notch protein), or inhibit any known Notch or vertebrate Deltex function as preferably assayed in vitro or in cell culture, although genetic assays (e.g., in
  • Drosophila or mouse may also be employed.
  • the Antagonist may also be employed.
  • Therapeutic is a protein or derivative thereof comprising a functionally active fragment such as a fragment of vertebrate Deltex which mediates binding to Notch, or an antibody thereto.
  • such an Antagonist Therapeutic is a nucleic acid capable of expressing a molecule comprising a fragment of vertebrate Deltex which binds to Notch, or a vertebrate deltex antisense nucleic acid (see Section 5.11 herein). It should be noted that preferably, suitable in vitro or in vivo assays, as described infra, should be utilized to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue, since the developmental history of the tissue may determine whether an Antagonist or Agonist Therapeutic is desired.
  • a nucleic acid containing a portion of a vertebrate deltex gene is used, as an Antagonist Therapeutic, to promote vertebrate deltex inactivation by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
  • the Agonist Therapeutics of the invention promote vertebrate Deltex function.
  • Such Agonist Therapeutics include but are not limited to proteins and derivatives comprising the portions of Notch that mediate binding to vertebrate Deltex, i.e. , the ANK repeats, and nucleic acids encoding the foregoing (which can be administered to express their encoded products in vivo).
  • Molecules which retain, or alternatively inhibit, a desired vertebrate Deltex property, e.g. , binding to Notch, binding to an intracellular ligand, can be used
  • a peptide e.g., in the range of 6-50 or 15-25 amino acids; and particularly of about 10, 15, 20 or 25 amino acids
  • an Antagonist Therapeutic is used to treat or prevent human or other malignancies associated with increased Notch expression (e.g., cervical cancer, colon cancer, breast cancer, squamous adenocarcinomas (see infra)).
  • Derivatives or analogs of vertebrate Deltex can be tested for the desired activity by procedures known in the art, including but not limited to the assays described in the examples infra.
  • molecules comprising Deltex fragments which bind to Notch ANK repeats can be obtained and selected by expressing deletion mutants of human Deltex (or of a nucleotide sequence of another species and assaying for binding of the expressed product to
  • peptide libraries can be screened to select a peptide with the desired activity; such screening can be carried out by assaying, e.g. , for binding to Notch or a molecule containing the Notch ANK repeats.
  • the Agonist and Antagonist Therapeutics of the invention have therapeutic utility for disorders of cell fate.
  • the Agonist Therapeutics are administered therapeutically (including prophylactically): (1) in diseases or disorders involving an absence or decreased (relative to normal, or desired) levels of Notch or vertebrate Deltex function, for example, in patients where Notch or vertebrate Deltex protein is lacking, genetically defective, biologically inactive or underactive, or underexpressed; and (2) in diseases or disorders wherein in vitro (or in vivo) assays (see infra) indicate the utility of vertebrate Deltex agonist administration.
  • Notch or vertebrate Deltex function can be readily detected, e.g., by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for protein levels, structure and/or activity of the expressed Notch or vertebrate Deltex protein.
  • a patient tissue sample e.g., from biopsy tissue
  • assaying it in vitro for protein levels, structure and/or activity of the expressed Notch or vertebrate Deltex protein.
  • Many methods standard in the an can be thus employed, including but not limited to immunoassays to detect and/or visualize Notch or vertebrate Deltex protein (e.g.
  • In vitro assays which can be used to determine whether administration of a specific Agonist Therapeutic or Antagonist Therapeutic is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a Therapeutic, and the effect of such Therapeutic upon the tissue sample is observed.
  • a sample of cells from such malignancy is plated out or grown in culture, and the cells are then exposed to a Therapeutic.
  • a Therapeutic which inhibits survival or growth of the malignant cells is selected for therapeutic use in vivo.
  • cell proliferation can be assayed by measuring 3 H-thymidine incorporation, by direct cell count, by detecting changes in transcriptional activity of known genes such as proto- oncogenes (e.g., fos, tnyc) or cell cycle markers; cell viability can be assessed by trypan blue staining, differentiation can be assessed visually based on changes in morphology, etc.
  • proto- oncogenes e.g., fos, tnyc
  • cell cycle markers e.g., cell cycle markers
  • cell viability can be assessed by trypan blue staining, differentiation can be assessed visually based on changes in morphology, etc.
  • the malignant cell cultures are separately exposed to (1) an Agonist Therapeutic, and (2) an Antagonist Therapeutic; the result of the assay can indicate which type of Therapeutic has therapeutic efficacy.
  • a Therapeutic is indicated for rse which exhibits the desired effect, inhibition or promotion of cell growth, upon a patient cell sample from tissue having or suspected of having a hyper- or hypoproliferative disorder, respectively.
  • hyper- or hypoproliferative disorders include but are not limited to those described in Sections
  • a Therapeutic is indicated for use in treating nerve injury or a nervous system degenerative disorder (see Section 5.8.2) which exhibits in vitro promotion of nerve regeneration/neurite extension from nerve cells of the affected patient type.
  • administering is also indicated in diseases or disorders determined or known to involve a Notch or Deltex dominant activated phenotype ("gain of function" mutations.)
  • Administration of an Agonist Therapeutic is indicated in diseases or disorders determined or known to involve a Notch or Deltex dominant negative phenotype ("loss of function" mutations).
  • the functions of various structural domains of the Notch protein have been investigated in vivo, by
  • Deltex binds to the Notch ANK repeat region.
  • in vitro assays can be carried out with representative cells of cell types involved in a patient's disorder, to determine if a
  • Therapeutic has a desired effect upon such cell types.
  • cells of a patient tissue sample suspected of being pre-neoplastic are similarly plated out or grown in vitro, and exposed to a Therapeutic.
  • Therapeutic which results in a cell phenotype that is more normal (i.e., less representative of a pre-neoplastic state, neoplastic state, malignant state, or transformed phenotype) is selected for therapeutic use.
  • Many assays standard in the art can be used to assess whether a pre-neoplastic state, neoplastic state, or a transformed or malignant phenotype, is present.
  • characteristics associated with a transformed phenotype include a more rounded cell morphology, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton surface protein, etc. (see Luria et al., 1978, General Virology, 3d Ed., John Wiley & Sons, New York pp. 436-446).
  • the in vitro assays described supra can be carried out using a cell line, rather than a cell sample derived from the specific patient to be treated, in which the cell line is derived from or displays characteristic(s) associated with the malignant, neoplastic or pre-neoplastic disorder desired to be treated or prevented, or is derived from the neural or other cell type upon which an effect is desired, according to the present invention.
  • an antagonist of Notch and/or Deltex function that can be used as an Antagonist Therapeutic is a molecule comprising a Deltex protein or portion thereof that mediates binding to Notch, covalently bound to a protease or proteoly tically active fragment thereof.
  • protease preferably is able to cleave a Notch protein.
  • the molecule is preferably a fusion protein (i.e., the covalent bond is a peptide bond).
  • the Deltex protein is preferably a vertebrate protein, most preferably human. Accordingly, the invention provides a method of targeting or inactivating proteins to which Deltex binds (e.g., Notch) in a cell.
  • the molecule comprising the Deltex protein or portion thereof and the protease sequences is produced through chemical or via molecular biological techniques.
  • This molecule e.g., fusion protein
  • This molecule is introduced into the cell by techniques known in the art (e.g., transfection of the cell with a nucleic acid encoding the molecule such that its expression occurs intracellularly).
  • the molecule can bind to Notch and/or other Deltex binding partners.
  • the protease portion of the molecule cleaves the protein to which the molecule is bound, thus inactivating it.
  • fusion protein containing domain I of human Deltex and the protease thermolysin when introduced into the cell would bind to and cleave Notch, thereby inactivating the Notch signaling pathway.
  • Molecules which would inactivate protein function e.g. , by binding thereto, can be used as an alternative to proteases.
  • the Antagonist Therapeutics are administered therapeutically (including prophylactically): (1) in diseases or disorders involving increased (relative to normal, or desired) levels of Notch or vertebrate Deltex function, for example, where the Notch or vertebrate Deltex protein is overexpressed or overactive; and (2) in diseases or disorders wherein in vitro (or in vivo) assays indicate the utility of vertebrate Deltex antagonist administration.
  • the increased levels of Notch or vertebrate Deltex function can be readily detected by methods such as those described above, by quantifying protein and/or RNA.
  • In vitro assays with cells of patient tissue sample or the appropriate cell line or cell type, to determine therapeutic utility can be carried out as described above.
  • Malignant and pre-neoplastic conditions which can be tested as described supra for efficacy of intervention with Antagonist or Agonist Therapeutics, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to those described below in Sections 5.8.1 and 5.9.1.
  • Malignancies and related disorders, cells of which type can be tested in vitro (and/or in vivo), and upon observing the appropriate assay result, treated according to the present invention include but are not limited to those listed in Table 1 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia):
  • malignancy or dysproliferative changes are treated or prevented in epithelial tissues such as those in the cervix, esophagus, and lung.
  • malignancies of the colon and cervix can exhibit increased expression of human Notch relative to such non-malignant tissue (see PCT Publication WO 94/07474 published April 14, 1994, incorporated by reference herein in its entirety).
  • malignancies of the colon or cervix are treated or prevented by administering an effective amount of an Antagonist Therapeutic of the invention.
  • the presence of increased Notch expression in colon, and cervical cancer suggests that many more cancerous and hyperproliferative conditions exhibit upregulated Notch.
  • various cancers e.g., breast cancer, squamous adenocarcinoma, seminoma, melanoma, and lung cancer, as well as other hyperproliferative disorders, can be treated or prevented by administration of an Antagonist Therapeutic.
  • Nervous system disorders involving cell types which can be tested as described supra for efficacy of intervention with Antagonist or Agonist Therapeutics, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
  • Nervous system lesions which may be treated in a patient (including human and non-human vertebrate patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems:
  • nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;
  • malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non- nervous system tissue;
  • degenerative lesions in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;
  • lesions associated with nutritional diseases or disorders in which a portion of the nervous system is destroyed or mjured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;
  • demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus- associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
  • Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons (see also Section 5.8). For example, and not by way of limitation. Therapeutics which elicit any of the following effects may be useful according to the invention:
  • a neuron-associated molecule in culture or in vivo, e.g. , choiine aceryltransferase or acetylcholinesterase with respect to motor neurons; or
  • motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
  • motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and
  • Hereditary Motorsensory Neuropathy (Char cot-Marie -Tooth Disease).
  • a Therapeutic of the invention is used for promotion of tissue regeneration and repair, including but not limited to treatment of benign dysproliferative disorders.
  • Specific embodiments are directed to treatment of cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), and baldness (a condition in which terminally differentiated hair follicles (a tissue rich in Notch) fail to function properly).
  • Deltex agonists and antagonists can also be used to manipulate the differentiation state of non-terminally differentiated (e.g., stem and progenitor) cells.
  • a stem cell can be exposed to such an agonist to inhibit its differentiation and achieve expansion of the stem cell population by incubation in vitro under growth conditions.
  • Such stem cells have use in transplantation for in vivo repopulation of their progeny cells and tissue regeneration.
  • a method for the expansion of a precursor cell comprises contacting the cell with an amount of a vertebrate (e.g. , human) Deltex protein or functionally active portion thereof effective to inhibit differentiation of the cell, and exposing the cell to cell growth conditions such that the cell proliferates.
  • the precursor cell can be but is not limited to a hematopoietic precursor cell, epithelial precursor cell, kidney precursor cell, neural precursor cell, skin precursor cell, osetoblast precursor cell, chondrocyte precursor cell, liver precursor cell, and muscle cell.
  • the Therapeutics of the invention can be administered to prevent progression to a neoplastic or malignant state, including but not limited to those disorders listed in Table 1. Such administration is indicated where the Therapeutic is shown in assays, as described supra, to have utility for treatment or prevention of such disorder.
  • Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp.
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometnal hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium.
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia
  • the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient can indicate the desirability of prophylactic/therapeutic administration of a Ther Chamberic of the invention
  • characteristics of a transformed phenotype m include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protem, etc (see also id , at pp 84-90 for characteristics associated with a transformed or malignant phenotype) .
  • leukoplakia a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are pre- neoplastic lesions indicative of the desirability of prophylactic intervention .
  • fibrocystic disease cystic hyperplasia, mammary dysplasia, particularly adenosis (benign epithelial hyperplasia) is indicative of the desirability of prophylactic intervention .
  • a patient which exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of a Therapeuti: a chromosomal translocation associated with a malignancy (e.g. , the
  • an Antagonist Therapeutic of the invention is administered to a human patient to prevent progression to breast, colon, or cervical cancer.
  • a Therapeutic of the invention can be administered to prevent a nervous system disorder described in Section 5.8.2, or other disorder (e.g. , liver cirrhosis, psoriasis, keloids, baldness) described in Section 5.8.3.
  • a nervous system disorder described in Section 5.8.2 or other disorder (e.g. , liver cirrhosis, psoriasis, keloids, baldness) described in Section 5.8.3.
  • the Therapeutics of the invention can be tested in vivo for the desired therapeutic or prophylactic activity.
  • such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
  • suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
  • any animal model system known in the art may be used.
  • the present invention provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding vertebrate
  • Antisense refers to a nucleic acid capable of hybridizing to a portion of a vertebrate deltex RNA (preferably mRNA) by virtue of some sequence complementarity. Such antisense nucleic acids have utility as Antagonist
  • Therapeutics of the invention can be used in the treatment or prevention of disorders as described supra in Section 5.8 and its subsections.
  • the antisense nucleic acids of the invention can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellular ly by transcription of exogenous, introduced sequences.
  • the vertebrate deltex antisense nucleic acids provided by the instant invention can be used for the treatment of tumors or other disorders, the cells of which tumor type or disorder can be demonstrated (in vitro or in vivo) to express a vertebrate deltex gene or a Notch gene. Such demonstration can be by detection of RNA or of protein.
  • the invention further provides pharmaceutical compositions comprising an effective amount of the vertebrate deltex antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra in Section 5.12.
  • a pharmaceutically acceptable carrier as described infra in Section 5.12.
  • Methods for treatment and prevention of disorders comprising administering the pharmaceutical compositions of the invention are also provided.
  • the invention is directed to methods for inhibiting the expression of a vertebrate deltex nucleic acid sequence in a prokaryotic or eukaryotic cell comprising providing the cell with an effective amount of a composition comprising an antisense vertebrate deltex nucleic acid of the invention.
  • Vertebrate deltex antisense nucleic acids and their uses are described in detail below.
  • the vertebrate deltex antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 oligonucleotides).
  • the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone.
  • the oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g. , Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO 88/09810, published December 15, 1988) or blood-brain barrier (see, e.g. , PCT Publication No. WO 89/10134, published April 25, 1988), hybridization-triggered cleavage agents (see, e.g.
  • a vertebrate deltex antisense oligonucleotide is provided, preferably of single-stranded DNA.
  • such an oligonucleotide comprises a sequence antisense to the sequence encoding an SH3- binding domain or a Notch-binding domain of vertebrate deltex or zinc finger domain, most preferably, of human deltex.
  • the oligonucleotide may be modified at any position on its structure with substituents generally known in the art.
  • the vertebrate deltex antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to
  • 5-fluorouracil 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
  • the oligonucleotide comprises at least one modified sugar moiety selected from the group including but not limited to arabinose,
  • the oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide may be conjugated to another molecule, e.g. , a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res.
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
  • the vertebrate deltex antisense oligonucleotide comprises catalytic RNA, or a ribozyme (see, e.g., PCT International Publication
  • the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987,
  • the vertebrate deltex antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
  • a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encodmg the vertebrate deltex antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells.
  • Expression of the sequence encoding the vertebrate deltex antisense RNA can be by any promoter known in the art to act in vertebrate, preferably human, cells. Such promoters can be inducible or constitutive.
  • Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc.
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a vertebrate deltex gene, preferably a human deltex gene.
  • a sequence complementary to at least a portion of an RNA means a sequence having sufficient complementarity to be able to hybridize with the
  • RNA forming a stable duplex; in the case of double-stranded vertebrate deltex antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of
  • the longer the hybridizing nucleic acid the more base mismatches with a vertebrate deltex RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • the vertebrate deltex antisense nucleic acids can be used to treat (or prevent) malignancies or other disorders, of a cell type which has been shown to express vertebrate deltex or Notch.
  • the malignancy is cervical, breast, or colon cancer, or squamous adenocarcinoma.
  • Malignant, neoplastic, and pre-neoplastic cells which can be tested for such expression include but are not limited to those described supra in
  • a single-stranded DNA antisense vertebrate deltex oligonucleotide is used.
  • Malignant (particularly, tumor) cell types which express vertebrate deltex or Notch RNA can be identified by various methods known in the art. Such methods include but are not limited to hybridization with a vertebrate deltex or Notch -specific nucleic acid
  • RNA from the cell type can be translated in vitro into Notch or vertebrate Deltex, immunoassay, etc.
  • primary tumor tissue from a patient can be assayed for Notch or vertebrate Deltex expression prior to treatment, e.g. , by
  • compositions of the invention comprising an effective amount of a vertebrate deltex antisense nucleic acid in a pharmaceutically acceptable carrier, can be administered to a patient having a malignancy which is of a type that expresses Notch or vertebrate deltex RNA or protein.
  • the amount of vertebrate deltex antisense nucleic acid which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Where possible, it is desirable to determine the antisense cytotoxicity of the tumor type to be treated in vitro, and then in useful animal model systems prior to testing and use in humans.
  • compositions comprising vertebrate deltex antisense nucleic acids are administered via liposomes, microparticles, or
  • microcapsules In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the vertebrate deltex antisense nucleic acids. In a specific embodiment, it may be desirable to utilize liposomes targeted via antibodies to specific identifiable tumor antigens (Leonetti et al., 1990, Proc. Natl. Acad. Sci. U.S.A.
  • the invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the invention.
  • the Therapeutic is substantially purified.
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
  • Therapeutic of the invention e.g., encapsulation in liposomes, microparticles,
  • microcapsules expression by recombinant cells, receptor mediated endocytosis (see, e.g. ,
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or
  • mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Ac_ministration can be systemic or local.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions of the invention may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • the Therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid. , pp. 317-327; see generally ibid.)
  • the Therapeutic can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres.,
  • a controlled release system can be placed in proximity of the therapeutic target, i.e. , the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • administration of a Therapeutic into a Notch- expressing cell is accomplished by linkage of the Therapeutic to a Delta (or other toporythmic) protein or portion thereof capable of mediating binding to Notch.
  • Linkage of the Therapeutic to a Delta (or other toporythmic) protein or portion thereof capable of mediating binding to Notch is accomplished by linkage of the Therapeutic to a Delta (or other toporythmic) protein or portion thereof capable of mediating binding to Notch.
  • the Therapeutic is delivered intracellularly (e.g., by expression from a nucleic acid vector, or by linkage to a Delta protein capable of binding to Notch followed by binding and internalization, or by receptor-mediated or diffusion mechanisms).
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g.
  • a gene gun Biolistic, Dupont
  • coating with lipids or cell-surface receptors or tran sfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al, 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868), etc.
  • a nucleic acid Therapeutic can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • Parkinson's disease Targeted intravenous; intrathecal
  • Alzheimer's disease Targeted intravenous; intrathecal
  • compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's
  • compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to mammals.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the Therapeutics of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be
  • Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical
  • compositions of the invention are optionally associated with such container(s) in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • Vertebrate Deltex proteins, analogues, derivatives, and subsequences thereof, vertebrate deltex nucleic acids (and sequences complementary thereto), anti-vertebrate Deltex antibodies have uses in diagnostics.
  • Such molecules can be used in assays, such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting vertebrate Deltex expression, or monitor the treatment thereof.
  • immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-vertebrate Deltex antibody under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.
  • such binding of antibody, in tissue sections, preferably in conjunction with binding of anti-Notch can be used to detect aberrant Notch and/or vertebrate Deltex localization or aberrant levels of Notch-vertebrate Deltex colocalization in a disease state.
  • antibody to vertebrate Deltex can be used to assay in a patient tissue or serum sample for the presence of vertebrate Deltex where an aberrant level of vertebrate Deltex is an indication of a diseased condition.
  • Aberrant levels of vertebrate Deltex binding ability in an endogenous Notch protein, or aberrant levels of binding ability to Notch (or other vertebrate Deltex ligand) in an endogenous vertebrate Deltex protein may be indicative of a disorder of cell fate (e.g., cancer, etc.)
  • aberrant levels is meant increased or decreased levels relative to that present, or a standard level representing that present, in an analogous sample from a subject not having the disorder.
  • immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked lmmunosorbent assay), "sandwich"
  • immunoassays immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Vertebrate deltex genes and related nucleic acid sequences and subsequences, including complementary sequences, and other toporythmic gene sequences, can also be used in hybridization assays.
  • Vertebrate deltex nucleic acid sequences, or subsequences thereof comprising about at least 8 nucleotides can be used as hybridization probes.
  • Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with aberrant changes in vertebrate Deltex expression and/or activity as described supra.
  • a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to vertebrate deltex DNA or RNA, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.
  • Human deltex was isolated through a combination of computer and biochemical screens. Initially, a human expressed sequence tag database was screened for homology against the amino acid sequence of Drosophila Deltex. The critical part of this search involved the assumption that stop codons in a particular reading frame of the database are the result of sequencing mistakes. Accordingly, stop codons were ignored and the open reading frame was extended in a different frame. The predicted amino acid sequence encoded by the hypothetical open reading frames were then compared with the protein product of the Drosophila deltex transcription unit.
  • a series of two 5' primers (hdx-1 (SEQ ID NO:26) and hdx-2 (SEQ ID NO:27)) and two 3' primers (hdx-3 (SEQ ID NO:28) and hdx-4 (SEQ ID NO:29 )) were synthesized based on the DNA sequence of gnl I dbest I 24254 T05200.
  • PCR reactions were performed using the four different primer combinations and a human fetal brain cDNA library (Invitrogene) as the template. The PCR product was sequenced and found to have the same DNA sequence as gnl I dbest I 24254 T05200.
  • the PCR product generated using the hdx-1 and hdx-4 primers was then labeled and used to screen another human fetal brain cDNA library.
  • the isolate was sequenced (SEQ ID NO: 11) and the predicted protein determined (SEQ ID NO: 12) ( Figure 2A-C). Not greater than 107 continuous nucleotides of SEQ ID NO: 11 were present in T05200. Applying standard techniques, the cDNA isolate obtained using the PCR product as probe was then labeled and itself used as a probe to screen a northern blot containing poly(A) + mRNA isolated from various human tissue samples.
  • This probe was observed to hybridize to a 5.4-kb RNA in heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas.
  • This probe was used to screen a zoo blot (a blot containing genomic EcoRI-digested
  • the predicted human Deltex product has 720 amino acids and an estimated molecular mass of approximately 80 kDa.
  • the 180 amino terminal residues of human Deltex have an approximate identity of 33 % with the corresponding amino acid residues of Drosophila Deltex and the nucleic acids encoding these amino acids have an approximate 52% identity.
  • the 180 carboxy terminal amino acids of human Deltex have an approximate 48% identity with the corresponding amino acid residues of Drosophila Deltex and the nucleic acids encoding these carboxy terminal amino acids have an approximate 49% identity.
  • Domain I corresponds to. the N-terminal 303 amino acids of Drosophila and the first 237 amino acids of human Deltex.
  • Drosophila we have demonstrated that the region of human Deltex corresponding to the first 175 amino acids of domain I is essential and sufficient to bind the Notch ANK repeats and overexpression of this domain can rescue loss-of-function phenotypes of Deltex. Since Drosophila Deltex can bind to human Notch-1 and 2, conservation of binding activity between human Deltex and Drosophila domain I is suggested.
  • Domain II of Drosophila Deltex contains a putative SH3 -binding site
  • SH2 and SH3 domains are conserved protein modules so named based on their homology to the oncogene Src (Src Homology). These motifs have been implicated in mediating protein-protein interactions in a number of signal transduction pathways (reviewed in Cell 71:359-362;Science 252:668-674; Trends
  • SH3-BD SH3-binding domain'
  • regions of the protein encoded by the Drosophila Son of sevenless (SOS) protein (SEQ ID NO:24), which may also contain SH3-BD, is shown.
  • the Son of sevenless encoded protein a putative guanine nucleotide exchange factor (GNEF)
  • GNEF putative guanine nucleotide exchange factor
  • GenBank FASTA server 85:2444-2448 available by the GenBank FASTA server through BITNET.
  • one protein segment is fused to the DNA-binding domain of the LexA protein, which in turn binds to the promoter of a LexAop-lacZ reporter construct without activating transcription. These constructs are referred as pEG.
  • a second foreign protein segment is fused to an acidic transcriptional activation domain that does not bind DNA on its own. These constructs are referred to as pJG.
  • Coexpression of these two proteins in yeast cells results in the functional reconstruction of an active LexA "hybrid" transcription factor if the foreign proteins physically interact with one another. Activity of the hybrid transcription factor is monitored by transcription of the ⁇ -galactosidase reporter gene.
  • fusion proteins from the pJG construct is induceu when yeast cells are cultured in the galactose media but not in the glucose media. Therefore, positive interaction should be observed only in galactose media.
  • the constructs examined using the yeast interaction were as follows: the pEGhDeltex construct contains the entire coding region of human Deltex;
  • pJGhNotch-1 encodes the ankyrin repeats region of human Notch- 1 from amino acids 1826-2147
  • pJGhNotch-2 encodes the ankyrin repeats region of human Notch- 2 from amino acids 1772-2084
  • pJGhNotch encodes the ankyrin repeats of
  • Deltex protein demonstrates a conserved structure in these two evolutionary distant species. Knowledge of the conserved regions of the protein allows one to design synthetic degenerate primers for use in hybridization and PCR reactions which enable the cloning of Deltex encoding nucleic acids in other organisms.
  • a murine deltex gene is obtained as follows: Standard techniques are utilized to synthesize a series of degenerate primers encoding amino acids 414-419 in Drosophila (SEQ ID NO: 30) and 549-555 in human (SEQ ID NO:35) in a 5' to 3' orientation. A second series of degenerate primers corresponding to the antisense strand of the nucleic acids encoding amino acids 475-480 in Drosophila (SEQ ID NO:31) and 603-608 in human (SEQ ID NO:36) is also synthesized. The two series of primers are added to a mixture containing mouse embryonic cDNA as template for the PCR amplification. PCR is carried out at a range of stringencies, according to methods commonly known, to allow for varying degrees of nucleotide similarity between the known deltex sequences and the mouse nucleic acid homolog being isolated.
  • mouse deltex gene is molecularly cloned and sequenced through techniques known in the art. This segment is used as a probe to isolate a complete cDNA and genomic clone. The complete nucleotide sequence of the mouse deltex homolog is determined by sequence analysis. 9. DEPOSIT OF MICROORGANISMS
  • Plasmid pBS hdx containing a cDNA inse ⁇ encoding a full-length human deltex as a EcoRI insert in Bluescript vector (Stratagene) was deposited by S. Leslie

Abstract

Séquences de nucléotides de gènes Deltex de vertébrés, et séquences d'acides aminés des protéines Deltex de vertébrés codées par ceux-ci. L'invention concerne également des fragments et d'autres dérivés et analogues des protéines Deltex de vertébrés, ainsi que les anticorps de ceux-ci. Les acides nucléiques codant pour ces fragments ou dérivés sont également compris dans l'invention. La production des protéines et dérivés cités, par exemple, par recombinaison, est décrite. Selon un mode de réalisation spécifique, l'invention porte sur les acides nucléiques et protéines Deltex chez l'homme. La présente invention porte également sur des procédés et des compositions thérapeutiques et diagnostiques à base de protéines, d'acides nucléiques et d'anticorps Deltex de vertébrés. L'invention concerne également des procédés d'inactivation d'une fonction Notch dans une cellule, des procédés d'identification d'un composé inhibant ou réduisant la liaison d'une protéine Deltex à une protéine Notch, et des procédés visant l'expansion de cellules à terminaisons non différenciées.
PCT/US1996/018675 1995-11-22 1996-11-22 Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci WO1997018822A1 (fr)

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EP96942785A EP0869802A4 (fr) 1995-11-22 1996-11-22 Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci
AU11614/97A AU728798B2 (en) 1995-11-22 1996-11-22 Vertebrate deltex proteins, nucleic acids, and antibodies, and related methods and compositions
JP9519885A JP2000502246A (ja) 1995-11-22 1996-11-22 脊椎動物Ｄｅｌｔｅｘタンパク質、核酸および抗体ならびに関連方法および組成物

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ES2334953T3 (es) 1995-06-28 2010-03-17 Imperial Cancer Research Technology Limited Secuencias de nucleotidos y proteinas de genes delta de vertebrados y metodos basados en los mismos.
US5780300A (en) * 1995-09-29 1998-07-14 Yale University Manipulation of non-terminally differentiated cells using the notch pathway
US6436650B1 (en) 1997-07-23 2002-08-20 Yale University Activated forms of notch and methods based thereon
US6692919B1 (en) 1997-07-23 2004-02-17 Yale University Activated forms of notch and methods based thereon
AU5524899A (en) * 1998-08-31 2000-03-21 Astrazeneca Uk Limited Human deltex-like gene zdx
GB9824045D0 (en) * 1998-11-03 1998-12-30 European Molecular Biology Lab Embl Regulator protein
GB9912132D0 (en) * 1999-05-26 1999-07-28 Univ Manchester Notch regulator-related gene and uses thereof
GB0019242D0 (en) * 2000-08-04 2000-09-27 Lorantis Ltd Assay

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US5750652A (en) * 1994-01-21 1998-05-12 Yale University Deltex proteins

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