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WO1996029433A1 - Proteine 'rest' et adn correspondant - Google Patents

Proteine 'rest' et adn correspondant Download PDF

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Publication number
WO1996029433A1
WO1996029433A1 PCT/US1996/003940 US9603940W WO9629433A1 WO 1996029433 A1 WO1996029433 A1 WO 1996029433A1 US 9603940 W US9603940 W US 9603940W WO 9629433 A1 WO9629433 A1 WO 9629433A1
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lys
glu
protein
rest
nucleic acid
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PCT/US1996/003940
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English (en)
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Gail Mandel
Jayhong Andrew Chong
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Research Foundation Of State University Of New York
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Priority to AU53693/96A priority Critical patent/AU5369396A/en
Publication of WO1996029433A1 publication Critical patent/WO1996029433A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to purified nucleic acids encoding REl -Silencing Transcription factors ("REST proteins”) and to purified proteins with REST activity.
  • REST proteins REl -Silencing Transcription factors
  • REST That protein herein referred to as "REST,” and the gene encoding it, is herein identified as having the amino acid sequence included in SEQ ID NO: 1.
  • SEQ ID NO: 10 The portion of the nucleic acid sequence included in SEQ ID NO: 1 that is an open reading frame for REST is identified as SEQ ID NO: 10.
  • the protein sequence for human REST and the nucleic acid sequence of the CDNA for human REST are shown in Figure 1.
  • One preferred embodiment of the present invention is a substantially pure nucleic acid comprising a nucleic acid encoding a protein having at least about 85 % homology to at least the DNA binding domain or the suppressor domain of an animal REST protein; the same substantially pure nucleic acid further comprising a nucleic acid encoding at least the DNA binding domain or the suppressor domain of an animal REST protein; the same substantially pure nucleic acid, wherein the REST protein is a mammalian REST protein; the same substantially pure nucleic acid, wherein the REST protein is a human REST protein; the same substantially pure nucleic acid, wherein the nucleic acid comprises SEQ ID NO:2; the same substantially pure nucleic acid, wherein the nucleic acid comprises SEQ ID NO: 10; the same substantially pure nucleic acid, further comprising a nucleic acid encoding both the DNA binding domain and the suppressor domain of an animal REST protein; the same substantially pure nucleic acid, wherein the REST protein is a mammalian R
  • Another preferred embodiment of the present invention is a substantially pure nucleic acid that hybridizes with an animal REST nucleic acid under stringent conditions; the same substantially pure nucleic acid, comprising the nucleic acid of SEQ ID NO:l.
  • a further preferred embodiment is a substantially pure nucleic acid comprising a nucleic acid encoding a protein that binds to a promoter having at least about 90% homology to nucleotides 6-28 of SEQ ID NO: 29 and acting to suppress the activity of a promoter having said promoter.
  • Yet another preferred embodiment is a substantially pure protein having at least about 85 % homology with at least the DNA binding domain or the suppressor domain of an animal REST protein; the same substantially pure protein, comprising at least the DNA binding domain or the suppressor domain of an animal REST protein; the same substantially pure protein, further comprising the protein of SEQ ID NO:2; the same substantially pure protein, further comprising both the DNA binding domain and the suppressor domain of an animal REST protein; the same substantially pure protein, further comprising the protein of SEQ ID NO: 10.
  • Yet another preferred embodiment is a transformed eukaryotic or prokaryotic cell comprising a nucleic acid encoding a protein having at least about 85 % homology to at least one of the DNA binding domain or the suppressor domain of an animal REST protein; the same transformed cell, further comprising a nucleic acid encoding at least the DNA binding domain or the suppressor domain of an animal REST protein; the same transformed cell, wherein the REST protein is a mammalian REST protein; the same transformed cell, wherein the REST protein is a human REST protein; the same transformed cell, wherein the nucleic acid comprises SEQ ID NO: 2.
  • the transformed cell expresses one of the inventive proteins described herein. - 3 -
  • Yet another preferred embodiment is a vector capable of reproducing in a eukaryotic or prokaryotic cell comprising a nucleic acid encoding a protein having at least about 85% homology to at least the DNA binding domain or the suppressor domain of an animal REST protein; the same vector capable of reproducing in a eukaryotic or prokaryotic cell, further comprising a nucleic acid encoding at least the DNA binding domain or the suppressor domain of an animal REST protein; the same vector capable of reproducing in a eukaryotic or prokaryotic cell, wherein the REST protein is a mammalian REST protein; the same vector capable of reproducing in a eukaryotic or prokaryotic cell, wherein the REST protein is a human REST protein; the same vector capable of reproducing in a eukaryotic or prokaryotic cell, wherein the nucleic acid comprises SEQ ID NO:2.
  • the inventive vector expresses, intracellularly or extracellularly, one of the inventive proteins described herein.
  • the inventive vector expresses, intracellularly or extracellularly, one of the inventive proteins described herein. 10
  • Yet another preferred embodiment is a method of preparing a protein having REST activity, wherein the protein has at least about 85% homology with at least the DNA binding domain or the suppressor domain of an animal REST protein, the method comprising:
  • Yet another preferred embodiment is a pharmaceutical composition for treating an animal having de-differentiated neural cells or neural cells exhibiting diminished activity
  • a pharmaceutical composition for treating an animal having de-differentiated neural cells or neural cells exhibiting diminished activity comprising an effective amount of a REST-interfering nucleic acid, wherein the REST-interfering nucleic acid cSfiiprises an antisense molecule directed against REST expression or an expression vector for expressing REST DNA binding activity but not REST silencer activity, and a pharmaceutically acceptable carrier; the same pharmaceutical composition, wherein the animal has brain cancer; the same pharmaceutical composition, wherein said animal has a demyelinating myasthenia gravis, muscular dystrophy, botulism, peripheral neuropathies, traumatic nerve injury, post stroke degeneration, post-traumatic spinal and neural degeneration, poliomyelitis or rabies.
  • Yet another preferred embodiment is a pharmaceutical composition for an animal having neural cells exhibiting excessive neural activity comprising an effective amount of an expression vector comprising a nucleic acid encoding a protein that inhibits the expression of neural proteins in non-neural tissues, and a pharmaceutically acceptable carrier; the same pharmaceutical composition, v ⁇ krein the animal has epilepsy, Lennox-Gastaut syndrome, spasticity, trauma-induced pain, schizophrenia, stroke or a neurodegenerative disease; the same pharmaceutical composition, wherein the animal has Alzheimer's, Parkinson's or Huntington's disease; the same pharmaceutical composition, wherein the animal has epilepsy; the same pharmaceutical composition, wherein the animal has a neurodegenerative disease.
  • Yet another preferred embodiment is an antibody that reacts specifically with the substantially pure protein having at least about 85% homology with at least the DNA binding domain or the suppressor domain of an animal REST protein, as recited above.
  • Figure 1 shows the protein encoded by the open reading frame of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO:l.
  • the DNA binding domain of REST is made up of eight zinc finger domains.
  • the portion of SEQ ID NO:l that encompasses the eight zinc finger domains of REST is identified as SEQ ID NO:2.
  • the underlined residues shown in Figure 1 are the zinc finger domains.
  • a search of the GenBank database found that the closest homology for this DNA binding domain is found with the Kruppel family of repressor proteins, particularly the GLI-Kriippel repressor protein. (For a review o ⁇ inc finger proteins, see Colman, Ann. Rev. Biochem.
  • REST has six repeat sequences having the following sequences:
  • a "REST nucleic acid” means the REST-encoding nucleic acid, whether RNA or DNA, synthetic or natural, found in a REST-expressing animal, or the complementary strand thereof.
  • "REST protein-encoding nucleic acid” or “nucleic acid encoding a REST protein” refers to any nucleic acid, whether native or synthetic, RNA, DNA, or cDNA, that encodes a REST protein.
  • codon usage preferences for the organism in v-Jflch such a nucleic acid is to be expressed are advantageously considered in designing a synthetic REST protein-encoding nucleic acid.
  • a “REST protein” is a REST homologous protein with the ability to bind an REl sequence and to repress the activity of a promoter containing an REl sequence.
  • An “animal REST protein” is a REST protein expressed by a member of the animal kingdom; a “human REST protein” is a REST protein expressed by a human.
  • Vectors encoding a protein with REl -binding activity but not suppressor activity are shown herein to reverse the transcriptional suppression caused by REST, apparently by competing for the REl promoter element through which REST functions. Accordingly, gene therapy with such vectors are used like the aforementioned and other antisense therapies known in the art to reduce REST's suppressor activity.
  • the vectors described in this paragraph and the antisense molecules dMkussed above are termed herein "REST-interfering nucleic acids.”
  • Probes for REST expression are used to measure the extent of a de-differentiation in biopsy tissue from tumors that are derived from neural tissue. Such probes are used to predict the extent of tissue transformation and the virulence of the tumor. Such probes include antibodies directed against REST or fragments thereof, nucleic acid probes that hybridize to REST mRNA under sflSngent conditions, and oligonucleotides that specifically prime a PCR amplification of REST mRNA.
  • neural stem cells for instance stem cells derived from embryos
  • diseases include Alzheimer's disease, PMkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (“Lou Gehrig's disease”) and demyelinating diseases such as multiple sclerosis.
  • Stem cells used in these therapies are induced to initiate differentiation to provide the needed replacement cells by treating them with REST antisense constructs or with vectors expressing the DNA-binding domain of REST but not the suppressor function of REST.
  • neural cells are transformed to express sufficient REST to down-regulate expression of the sodium channel.
  • up regulation of the expression of the neural voltage-dependent sodium channel is useful. This up regulation is done by antisense therapy based on REST nucleic acids to inhibit neural expression of REST or with gene therapy using a vector that expresses a protein that competes with REST for REl promoter sequences without suppressing the activity of the promoter.
  • the REST protein is also a useful target for drug screening efforts to identify drugs that interfere with its suppressor activity, either by inhibiting DNA binding or the negative effect of REST on transcription.
  • drug screening assays in one embodiment include cell-free transcription systems using the REST protein, cell-free transcription systems such as those described b
  • the screening methods also utilize in other embodiments expression studies conducted in cell culture, such as the chloramphenicol acetyl transferase (CAT) assay methods described herein below.
  • CAT chloramphenicol acetyl transferase
  • the suppression domain of REST is fused by recombinant methods to a DNA-binding domain of a positive transcription factor to create a protein that represses the activity of one or more promoters.
  • the suppressor domain is linked to pit- 1 » a transcription factor for the prolactin and growth hormone promoters (see Ingraham et al., Cell 55, 519-529, 1988), thereby creating a vector for gene therapeutics aimed at down regulating hyperactive pituitary production of growth hormone and/or prolactin.
  • Other examples of specific targets for this kind of therapy are the DNA-binding domains of steroid hormone or thyroid hormone receptors.
  • Fusion vectors expressing a DNA binding domain from a steroid hormone receptor and the REST suppressor domain are used in yet other embodiments to down regulate responsiveness to the steroid hormones in patients that overproduce the steroid or that have steroid hormone receptors that are too active.
  • the fusion protein in one embodiment includes the target DNA-binding element and substantially all of the REST protein.
  • the antibodies and nucleic acid probes of the present invention are also useful as histochemical reagents for marking the pathways of nerves that do not express the CNS-type sodium channel. Also, the staining of most non-neural tissue serves as a contrast agent to highlight neurons that do not express REST or express very low levels of REST. Thus, these histochemical agents are used to produce histochemical slides and preserved anatomy specimens useful for training students and physicians.
  • the first embodiment of the invention relates to a purified nucleic acid comprising a nucleic acid having at least 85% homology to at least the DNA binding domain or the suppressor domain of an animal REST protein.
  • a nucleic acid is referred to herein as a REST protein that binds the REl promoter element and/or suppresses the activity of the promoter for the CNS-type voltage- dependent sodium channel.
  • the encoded protein is preferably a REST protein of a mammalian aifinal, more preferably the human REST protein.
  • the encoded protein has the sequence of SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO: 10.
  • Another embodiment of the invention provides for one or more nucleic acids encoding a protein that binds to a promoter sequence having at least about 90% homology, preferably 95% homology, to nucleotides 6-28 the REl sequence (SEQ ID NO: 29) and acting to suppress the a20vity of a promoter containing that promoter sequence.
  • Yet another embodiment provides for a nucleic acid encoding a protein that inhibits the expression of neural proteins in non-neural tissues.
  • nucleic acid embodiments of the invention are preferably deoxyribonucleic acids, preferably double-stranded deoxyribonucleic acids, except that, for hybridization probes, single- stranded nucleic acids are preferred.
  • nucleic acids of the present invention also include rQ&nucleic acids.
  • the nucleic acids of the present invention are also referred to as polynucleotides or polynucleic acids.
  • the invention also relates to a mutated or deleted vSftion of a REST protein-encoding nucleic acid that encodes a protein that retains the ability to bind specifically to the REl promoter element and/or the ability to suppress an REl -responsive promoter when appropriately bound to the vicinity of the promoter.
  • the invention also relates to a nucleic acid encoding, in the proper order, at least 4 of the zinc finger domains of a REST protein, preferably at least 6 of the zinc finger domains, more preferably all of the zinc finger domains.
  • the zinc finger domains for human REST are identified in Figure 2.
  • the nucleic acid is SEQ ID NO:2.
  • Transcription suppressive proteins such as Kruppel, Kid-1, and ZNF2 generally have distinct suppressor domains which function so long as they are appropriately linked to DNA binding domains that suitably bring the suppressor domains into the vicinity of the target promoters. See, for instance, Licht et al., Nature 346, 76-79, 1990; Witzgall et al., Proc. Natl. Acad. Sci. USA 91 , 4514-4518, 1994.
  • Such a suppressor domain can readily be identified for the REST protein using deCfctional approaches and recombinant fusion protein approaches that are well known in the art.
  • the invention also is directed to a nucleic acid encoding a segment of the protein of a REST protein that is effective to repress the use of a promoter when attached to a protein that binds the promoter.
  • the encoded protein will be effective to repress the use of the promoter for the CNS-type voltage-dependent sodium channel gene.
  • the promoters discussed in reference to this embodiment are REl -responsive promoters.
  • the invention rSBtes to nucleic acid sequences that hybridize with such REST-encoding sequences under stringent conditions.
  • the nucleic acid of the present invention hybridizes with SEQ ID NO: 1 under stringent conditions.
  • the invention also relates to nucleic acids that hybridize with SEQ ID NO:2 under such stringent conditions.
  • “Stringent conditions” refers to conditions that allow for the hybridization of substantially r ⁇ rited nucleic acids, where relatedness is a function of the sequence of nucleotides in the respective nucleic acids. For instance, for a nucleic acid of 100 nucleotides, such conditions will generally allow hybridization thereto of a second nucleic acid having at least about 85% homology, preferably having at least about 90% homology. Such hybridization conditions are described by Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.
  • the invention further relates to REST proteins and to proteins having sufficient zinc finger domains to confer the ability to bind the REl promoter element.
  • the protein has at least 4 of the zinc finger domains REST, more preferably at least 6, yet more preferably at least 7. Still more preferably, the REl binding protein has all of the zinc finger domains.
  • the protein has the sequence of a contiguous stretch of at least about 252 amino acids of SEQ ID NO: l, more preferably, of a contiguous stretch of at least about 504 amino acids.
  • the embodiments of the present invention that relate to proteins also encompass analogs of REST proteins that retain one or both of the ability to bind the REl promoter element and to suppress the activity of a promoter to which the protein is bound.
  • These analogs preferably lack no more than about 360 amino acid residues of deleted sequence at the C-terminal or N-terminal ends, more preferably no more than about 180 amino acid rdfldues of deleted sequence.
  • the remaining sequence of the REST protein will preferably have no more than about 20 point mutations, preferably no more than about 10 point mutations, more preferably no more than about 5 point mutations.
  • the point mutations are preferably conservative point mutations.
  • the analogs will have at least about 85% homology, preferably at least about 90% homology, more preferably at least about 95% homology to a portion of an animal REST protein retaining one or both of REST's known activities, such as the proteins of SEQ ID NO: l or SEQ ID NO:2.
  • Antigens for eliciting the production of antibodies against the REST protein can be produced recombinantly by expressing all of or a part of the nucleic acid of a REST protein in a bacteria or a yeast or other eukaryotic cell line.
  • the recombinant protein is expressed as a fusion protein, with the non-REST portion of the protein serving either to facilitate purification or to enhance the immunogenicity of the fusion protein.
  • the non-REST portion comprises a protein for which there is a readily-available binding partner that is utilized for affinity purification of the fusion protein.
  • the antigen includes an "antigenic determinant," i.e., a minimum segment of amino acids sufficient to bind specifically with an anti-REST antibody.
  • an "antigenic determinant” i.e., a minimum segment of amino acids sufficient to bind specifically with an anti-REST antibody. 25 Rules for designing PCR primers are well known in the art, as reviewed by PCR Protocols, Cold Spring Harbor Press, 1991.
  • Degenerate primers i.e., preparations of primers that are heterogeneous at given sequence locations, are designed to amplify nucleic acid sequences that are highly related to, but not identical to, a REST protein.
  • degenerate primers in one embodiment, are designed from the human REST cDNA and used to amplify nucleic acid sequences for REST proteins from non-human species, as illustrated in the examples.
  • the method by which human REST cDNA was isolated which is described in detail in the examples, illustrates how readily REl-binding domains from REST proteins are identified.
  • a library was made of cDNA from a REST-expressing cell and inserted into a yeast expression vector for the GAL4 activation domain so that the library would express fusion proteins having one part derived from cDNA and another part that is the GAL4 activation domain.
  • Initial partial cDNA clones were identified by their ability to bind an REl element on the promoters for two reporter genes and activate expression of those genes by causing the fused GAL4 activation domain to act on the promoters. These initial clones were of portions of the REl binding domain of the human REST protein. The same methodology can be used to identify other sequences from other animal sources that are sufficient to bind the REl element.
  • nucleic acids having the sequence of SEQ ID NO:2, which encodes the zinc finger dtf ain of human REST are tested for the REl binding activity of the expressed protein.
  • One facile method of doing this is to sub-clone the constructs into the GAM vector discussed above. Successful constructs activate the two REl -containing reporter genes that were used in the initial cloning of human REST cDNA.
  • deletion mutants are constructed by subcloning restriction fragments ofta REST cDNA. The deletional constructs are cloned into expression vectors and tested for their ability to suppress the expression of a promoter that has a functional REl element.
  • a reporter construct having the promoter for the CNS-type voltage-dependent sodium channel linked to the gene for chloramphenicol acetyl transferase ("CAT") is used.
  • CAT chloramphenicol acetyl transferase
  • Functional constructs diminish the level of expression of CAT, an enzyme tfifi is readily measurable by well established techniques. See, for example, Gorman et al., Mol. Cell. Biol. 2, 1044-1051, 1982 and Young et al., DNA 4, 469-475, 1985.
  • the types of substitutions selected may be based on the analysis of the frequencies of amino 25 acid substitutions between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer- Verlag, 1978, pp. 14-16, on the analyses of structure- forming potentials developed by Chou and Fasman, Biochemistry 13, 211, 1974 or other such methods reviewed by Schulz et al. Principles in Protein Structure, Springer- Verlag, 1978, pp. 108-130, and on the analysis of hydrophobicity patterns in proteins developed by Kyte and 30 Doolittle, J. Mol. Biol. 157: 105-132, 1982. Numerous methods for determining percent homology are known in the art.
  • One preferred method is to use version 6.0 of the GAP computer program for making sequence comparisons.
  • the program is available from the University of Wisconsin Genetics Computer Group and utilizes the alignment method of Needleman and Wunsch, J. Mol. Biol. 48, 443, 1970, as revised by Smith and Waterman Adv. Appl. Math. 2, 482, 1981.
  • Nucleic acid molecules that bind to a REST-encoding nucleic acid under high stringency conditions are identified functionally, using methods outlined above, or by using the hybridization rules reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.
  • Antisera to REST are made by creating a REST antigen by linking a portion of the cDNA for human REST to a cDNA for glutathione s-transferase ("GST”) found on a commercial vector. The resulting vector expresses a fusion protein containing an antigenic portion of REST and GST that is readily purified from the expressing bacteria using a glutathione affinity column.
  • the purified antigenic fusion protein is used to immunize rabbits.
  • the same approach is used to make antigens based on other portions of the REST protein. Procedures for making antibodies and for identifying antigenic portions of proteins are well known. See, for instance, Harlow, Antibodies, Cold Spring Harbor Press, 1989.
  • the proteins of the invention are made, in one embodiment, using the identical approach as for generating REST antisera.
  • the cDNA specific for a given REST protein or analog thereof is linked using standard means to a cDNA for GST, found on a commercial vector, for example.
  • the fusion protein expressed by such a vector construct includes the REST protein or analog and GST, and can be treated as above for purification. Should the GST segment of the fusion protein interfere with function, it is removed by partial proteolytic digestion approaches that preferentially attack unstructured regions, such as the linkers between GST and the REST-derived protein.
  • the linkers are designed to lack structure, for instance using the rules for secondary structure-forming potential developed by Chou and Fasman, Biochemistry 13, 211 , 1974.
  • the linker is also designed to incorporate protease target amino acids, such as, for trypsin, arginine and lysine residues.
  • protease target amino acids such as, for trypsin, arginine and lysine residues.
  • standard synthetic approaches for making oligonucleotides are employed together with standard subcloning methodologies.
  • Other fusion partners other than GST can be used.
  • the REST proteins can be directly synthesized from nucleic acid (by the cellular machinery) without use of fusion partners.
  • nucleic acids having the sequence of SEQ ID NO: 10 are subcloned into an appropriate expression vector having an appropriate promoter and expressed in an appropriate organism.
  • REST lacks consensus glycosylation sites and, especially since it is not a membrane or exported protein, should lack glycosylations.
  • Antibodies against REST are employed to facilitate purification.
  • a protein or nucleic acid is "isolated” in accordance with the invention in that the molecular cloning of the nucleic acid of interest, for example, involves taking a human REST nucleic acid from a human cell, and isolating it from other human-derived nucleic acids. This isolated nucleic acid may then be inserted into a host cell, which may be yeast or bacteria, for example, or another human cell.
  • a protein or nucleic acid is "substantially pure” in accordance with the invention if it is predominantly free of other proteins or nucleic acids, respectively.
  • a macromolecule, such as a nucleic acid or a protein is predominantly free if it constitutes at least about 50% by weight of the given macromolecule in a composition.
  • the protein or nucleic acid of the present invention constitutes at least about 60% by weight of the total proteins or nucleic acids, respectively, that are present in a given composition thereof, more preferably about 80%, still more preferably about 90% , yet more preferably about 95% , and most preferably about 100%.
  • Such compositions are referred to herein as being proteins or nucleic acids that are 60% pure, 80% pure, 90% pure, 95% pure, or 100% pure, any of which are substantially pure.
  • One aspect of the present invention is directed to the use of "antisense" polynucleic acid to treat neural diseases, including de-differentiated neural tumor cells and diseases characterized by diminished neural activity. Such an approach is also used to trigger the differentiation of neural stem cells.
  • the approach involves the use of an antisense molecule designed to bind nascent mRNA (or "sense" strand) for a REST protein, thereby stopping or inhibiting the translation of the mRNA, or to bind to the REST gene to interfere with its transcription.
  • an antisense molecule is designed that binds the sense strand by the Watson-Crick base-pairing rules, forming a duplex structure analogous to the DNA double helix.
  • the phosphate backbone of die antisense molecules has been modified to remove the negative charge (see, for example, Agris et al., Biochemistry 25, 6268, 1986; Cazenave and Helene in Antisense Nucleic Acids and Proteins: Fundamentals and Applications, Mol and Van der Krol, eds., p. 47 et seq.. Marcel Dekker, New York, 1991) or the purine or pyrimidine bases have been modified (see, for example, Antisense Nucleic Acids and Proteins: Fundamentals and Applications, Mol and Van der Krol, eds., p. 47 et seq., Marcel Dekker, New York, 1991; Milligan et al.
  • the polynucleotide or nucleic acid compositions of the invention can be administered orally, topically, rectally, vaginally, by pulmonary route by use of an aerosol, or parenterally, i.e. intramuscularly, intraventricularly, subcutaneously, intraperitoneallly or intravenously.
  • the polynucleotide compositions are administered alone, or they are combined with a pharmaceutical ly-acceptable carrier or excipient according to standard pharmaceutical practice.
  • the polynucleotide compositions are used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like.
  • carriers that are used include lactose, sodium citrate and salts of phosphoric acid.
  • Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • aqueous suspensions are required for oral use, the polynucleotide compositions are combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
  • sterile solutions of the conjugate are usually prepared, and the pH of the solutions are suitably adjusted and buffered.
  • ointments or droppable liquids may be delivered by ocular delivery systems known to the art, such as applicators or eye droppers.
  • Such compositions include ucomimetics, such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives, such as sorbic acid or EDTA, and the usual quantities of diluents and/or carriers well known in the art.
  • diluents and/or carriers are selected so as to allow the formation of an aerosol.
  • the polynucleotide compositions are administered in an effective amount.
  • An effective amount is an amount effective to either (1) reduce the symptoms of the disease sought to be treated or (2) induce a pharmacological change relevant to treating or preventing the disease sought to be treated.
  • dosages are generally from about 1 ⁇ g to about 1 mg of nucleic acid per kg of body mass.
  • dosages are generally from about 1 ⁇ g to about 100 mg of nucleic acid per kg of body mass.
  • Antisense oligonucleotide dosages are generally from about 1 ⁇ g to about 100 mg of nucleic acid per kg of body mass.
  • the invention also encompasses the use of gene therapy approaches to insert a gene expressing an REl binding domain but not a suppressor domain into de-differentiated tumor cells or neural cells with diminished neural activity.
  • Gene therapy approaches for inserting a gene for a protein with REST activity into overactive neural cells are also within the invention.
  • gene therapy approaches for inserting a gene for a REST suppressor domain linked to a promoter binding element to suppress the activity of the promoter bound by the binding element are also within the invention.
  • medical workers prefer to incorporate, into one or more cell types of an organism, a DNA vector capable of directing the synthesis of a protein missing from the cell or useful to the cell or organism when expressed in greater amounts.
  • transfection The methods for introducing DNA to cause a cell to produce a new protein or a greater amount of a protein are called "transfection" methods. See, generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.
  • a number of the above-discussed methods of enhancing cell penetration by antisense nucleic acid are generally applicable methods of incorporating a variety of nucleic acids into cells.
  • Other general methods include calcium phosphate precipitation of nucleic acid and incubation with the target cells (Graham and Van der Eb, Virology, 52:456, 1983), co- incubation of nucleic acid, DEAE-dextran and cells (Sompayrac and Danna, Proc. Natl. Acad. Sci. , 12:7575, 1981), electroporation of cells in the presence of nucleic acid (Potter et al., Proc. Natl. Acad.
  • nucleic acid into virus coats to create transfection vehicles (Gitman et al., Proc. Natl. Acad. Sci. U.S.A., 82:7309-7313, 1985) and incubating cells with nucleic acid incorporated into liposomes (Wang and Huang, Proc. Natl. Acad. Sci., 84:7851-7855, 1987).
  • An approach in employing gene therapy is to incorporate the gene sought to be introduced into the cell into a virus, such as an adeno virus. See, for instance, Akli et al.. Nature Genetics 3, 224, 1993.
  • the stem cells that are useful in neural stem cell replacement therapy include human mesencephalic fetal brain cells, porcine fetal brain cells, human subventricular zone cells and glial progenitor cells, including O2A cells (which are progenitors for all glial cell types, including astrocytes and oligodendrocytes).
  • the invention also relates to methods of measuring a REST protein or mRNA from a tissue or staining a tissue for a REST protein or mRNA.
  • Useful methods of measuring mRNA include Southern blot analysis, dot blot analysis, nuclear transcription analysis, histochemical staining for mRNA and polymerase chain reaction amplification methods. See generally, Ausubel et al., Current Protocols in Molecular Biology, Wiley Press, 1993; PCR Protocols, Cold Spring Harbor Press, 1991; and Sambrook et al.. Molecular Cloning: A Laboratory Manual 2nd ed.. Cold Spring Harbor Press, 1989. For m situ nucleic acid hybridization techniques, see Baldino et al..
  • Methods of measuring protein in a tissue include enzyme- linked immunoassays ("ELISA"), immuno-diffusion assays, radio-immunoassays, immunoelectrophoresis, Western blot analyses and immunohistochemical staining techniques.
  • ELISA enzyme- linked immunoassays
  • PCR methods of amplifying nucleic acids utilize at least two primers.
  • One of these primers is capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction.
  • the other is capable of hybridizing the reciprocal sequence of the first strand (if die sequence to be amplified is single stranded, this sequence is initially hypothetical, but is synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards die site of hybridization for the first primer.
  • Conditions for conducting such amplifications are well known. See, for example, PCR Protocols, Cold Spring Harbor Press, 1991.
  • the samples that are amenable to assaying or staining for REST protein or nucleic acid include, without limitation, cells or tissues (including nerve tissues), protein extracts, nucleic acid extracts and biological fluids such as cerebral fluid, serum and plasma.
  • Preferred samples are nervous system-derived samples.
  • the agents to be screened include a great variety of chemicals including, but not limited to, biologically active molecules such as peptides, carbohydrates, alkaloids, aromatic compounds, polynucleotides and analogs thereof (particularly analogs that have been rendered more membrane permeable), DNA intercolating compounds and other pharmaceutical agents.
  • One cell-free assay comprises the steps of: providing a nuclear extract, providing a REST protein, providing the nucleotide triphosphates necessary for transcription, providing a promoter sequence that includes an element effective to bind to REST and thereby be inhibited, providing a candidate compound or a cocktail of candidate compounds, mixing the extract, protein, promoter, nucleotide triphosphates, and candidate compound(s), incubating the mixture to allow transcription to proceed, and determining the level of the resulting transcription from the promoter, relative increases in transcription reflecting an inhibition of either the binding of REST to the promoter element or the activity of the suppressor domain of REST.
  • the extract itself will generally provide sufficient amounts of die REST protein.
  • nucleotide triphosphates may also be found in the nuclear extract; however, generally, additional nucleotide triphosphates are added to reduce the variability of the assay.
  • the level of transcription is determined by primer extension as described by Bodner and Karin, Cell 50, 267-275, 1987.
  • One embodiment of the cellular assay comprises the steps of: providing a eukaryotic cell line that expresses the REST protein (either natively or through a stable or transient transfection), providing a suitable medium for maintaining the cell line, adding to the medium a candidate compound or a cocktail of candidate compounds, incubating the cells to allow transcription to proceed, and determining the level of transcription from a REST-responsive promoter.
  • RNA transcript is also measured by methods well known in the art, such as dot-blot hybridization or by Northern Blot analysis.
  • the REST protein has a negative influence on the activity of many promoters having an REl or an REl -like sequence (such as that of the promoter for SCG10). Direct cloning strategies for such negative factors are difficult since they require time consuming measurements of the loss of a property.
  • a HeLa cell cDNA library was created to express fusion proteins between cDNA-encoded polypeptides and the activation domain of the yeast GAL4 regulatory protein.
  • the library was designed to identify a clone encoding a fusion protein having an REl -binding domain and a GAM activation domain. Such a fusion protein acts as a positive transcription factor on appropriate REl -containing promoter.
  • a HeLa cell library was selected because HeLa cells do not express the type II voltage dependent sodium channel and express an REl -binding activity.
  • the cloning strategy employed yeast containing two reporter genes having REl regulatory sequences in or adjacent to their promoters.
  • One reporter gene was HIS3, which confers to yeast the ability to grow in media that lacks the amino acid histidine, functionally attached to the yeast GALl promoter.
  • the GALl promoter is normally inactive in the absence of a yeast activator protein such as GAM.
  • the other reporter gene was the bacterial lac z gene functionally coupled to the yeast CYC1 promoter.
  • the CYC1 promoter is normally inactive in the absence of a yeast activator protein such as GAM.
  • the HIS3 Construct Four copies of the 28 bp REl nucleic acid, SEQ ID NO:29, which had been synthesized by standard oligonucleotide synthesis methods, were cloned into a unique EcoRI site on yeast expression shuttle vector pTHl (described by Flick and Johnson, Mol. Cell. Biol. 10(9), 4757-4769, 1990). The EcoRI site is adjacent (and 5') to a yeast GALl promoter that is functionally linked to a HIS3 gene.
  • the shuttle vector also contained a marker gene that directed the expression of a gene that confers to yeast the ability to grow in the absence of the pyrimidine base uracil.
  • the reporter plasmids were linearized and introduced sequentially into a standard yeast strain (strain W303) by the LiAc method (Schiestl and Geitz, Curr. Gen. 16, 339-346, 1989). Transformants were selected by growth on plates lacking uracil (indicating the integration of pJAC12) and tryptophan (indicating the integration of pJAC13).
  • a HeLa cell cDNA library was constructed using the pGADGH plasmid containing the GAM activation domain (see Li and Herskowitz, Science 262: 1870-1874, 1993) functionally linked to a GAM promoter and having a polylinker site (including EcoRI and Xhol sites), located downstream of the activator domain sequence for inserting the cDNA.
  • the library plasmid contains a marker for the ability to grow in the absence of the amino acid leucine.
  • the library was linearized and introduced into the yeast reporter strain by the LiAc method.
  • the cells were plated in leucine minus and histidine minus agar plates to select colonies that are putatively transformed with a cDNA to express an fusion protein having an REl binding domain (derived from cDNA) and a GAM activation domain.
  • One hundred his + colonies were impressed onto filter paper and permeabilized by freeze-thawing.
  • the filter paper was layered onto another filter paper containing the ⁇ - galactosidase substrate 5-bromo-4-chloro-3-indoyl-b-D-galactoside (X-gal, available from Sigma Chemical Co., St. Louis).
  • the filter paper was incubated at room temperature and monitored for blue spots, which indicate /3-galactosidase positive colonies.
  • Plasmids containing the cDNA from these four colonies was isolated as described by Bartel et al., in Cellular Interactions in Development: A Practical Approach, D.A. Hartley, ed.. New York: Oxford University Press, 1994, pp 53-179, and amplified in bacteria.
  • the plasmids were introduced into the control yeast strain (wherein the reporter gene promoters contained mutant REl sequences). Three of the four plasmids failed to transform the control strain, indicating that the fusion proteins they encoded interacted specifically with the REl nucleic acid. These plasmids were designated p73, p90 and p613.
  • the three insert cDNAs were sequenced by the chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 998-1002, 1977) and found to include the sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, all of which encode overlapping portions of an apparent zinc-finger DNA-binding domain (nucleotides 216-1622, 636-1725 and 695-1622 of Fig. 1, respectively).
  • Example 2 Cloning of Two Overlapping Sequences Encoding REST SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 were used to probe another HeLa cell cDNA library that was cloned into the Lambda Zap II phage (Stratagene, Inc., San Diego, CA). Two phage isolates containing overlapping cDNAs of 3082 and 4408 bp were isolated (phages NH2 and NH7, respectively). These cDNAs are designated SEQ ID NO: 6 and SEQ ID NO:7 and encode nucleotides -175-1616 and 1472-5324 of Fig. 1, respectively.
  • REST cDNA From the overlap of these two cDNAs, most of the full length REST cDNA can be deduced.
  • SEQ ID NO: 1 The deduced amino acid sequence of REST is shown is Figure 1. Note that Lambda Zap II is readily convertible to the Bluescript plasmid using EcoRI as outlined by the supplier.
  • a 1.5 kilobase EcoRI-XhoI fragment of p73 comprising all of SEQ ID NO:3 was cloned in phase with the cDNA for glutathione s-transferase ("GST") in the commercial vector pGEX4T3 (Pharmacia, Uppsala, Sweden).
  • GST-REST fusion protein was produced in E.coli strain XL-1 blue (Stratagene, San Diego, CA) and purified on a glutathione-Sepharose column (Pharmacia, Uppsala, Sweden). The purified fusion protein was used to immunize two rabbits (Pocono Rabbit Farms, PA) to produce a polyclonal antibody preparation against REST.
  • RNA from HeLa cells, PC12 cells, L6 skeletal muscle cells and dorsal root ganglion was isolated as described by Toledo-Aral et al., Neuron, in press) and poly-A + -selected using a commercially available kit (Pharmacia, Inc., Uppsala, Sweden).
  • Messenger RNA (2-4 ⁇ g) was fractionated on denaturing gels and then electrophoretically transferred onto nylon paper for hybridization.
  • a DNA probe of human REST was generated by random primer labeling of the EcoRI - Xhol fragment of p73, which includes the nucleic acid of SEQ ID NO:3, to incorporate 32 P.
  • a rat REST cDNA (600 bp) was obtained by PCR (with an initial reverse-transcriptase step) of rat skeletal muscle mRNA using a degenerate primer modelled on the sequence of amino acids 146 to 153 (nucleotides 481 to 504) of the plus strand of SEQ ID NO: l and a degenerate primer modelled on the amino-acid-encoding sequence of amino acid residues 363 to 370 (nucleotides 1087 to 1110) of the minus strand of SEQ ID NO: l.
  • PCR-amplified cDNA was cloned into pGEM-7Z (Promega.Madison, WI), and workable amounts of the plasmid were grown in bacteria.
  • a rat REST riboprobe was manufactured by linearizing the plasmid with AccI and transcribing it with T7 polymerase in the presence of 32 P-UTP (Dupont, Wilmington, DE).
  • a riboprobe for the CNS-type sodium channel was made as described by D'Arcangelo et al., J. Cell Biol., 10(9), 4757-4769, 1993.
  • Hybridization and washing conditions used with the rat REST and sodium channel riboprobes were as described by Toledo- Aral et al., Neuron, in press; for the human REST DNA probe, the hybridization and washing solutions were the same as those used for the riboprobes, except that the blots were hybridized at 37°C and washed at 32°C.
  • HeLa cells none high levels rat L6 skeletal muscle cells none high levels rat PC 12 cells high level extremely low levels mouse dorsal root ganglia extremely low levels high levels
  • Example 5 Western Blot Analysis Western immunoblots of proteins derived from nuclear extracts were performed according to standard procedures, as described by Sambrook et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Harber Lab., Cold Spring Harbor, NY, 1989. Nuclear extracts were prepared by the single lysis method (Sambrook et al., 1989). Extracts were combined with an equal volume of 2X Laemmli sample buffer (Laemmli, Nature, 227, 680- 685, 1970) and boiled for 15 minutes. Samples were resolved by SDS-PAGE on 7.5% gels, transferred to nitrocellulose, and the nitrocellulose was blocked with 10% milk in TTBS (Sambrook et al., 1989).
  • 2X Laemmli sample buffer Laemmli, Nature, 227, 680- 685, 1970
  • Immunoblotting was performed using the enhanced chemiluminescence method using a commercial kit (Amersham, Burlington, MA).
  • the antibody to REST-GST was used at a 1:20 dilution after purification by FPLC on an alkyl Superose (a highly crosslinked agarose substituted with octyl groups) column (Pharmacia, Uppsala, Sweden).
  • Nuclear extracts were made from the PC 12 cell line derived from a neural pheochromocytoma, which expresses the CNS-type voltage-dependent sodium channel and does not express an REl binding activity, and from HeLa cells, which do not express the CNS-type voltage-dependent sodium channel and do express an REl binding activity.
  • the developmental pattern of expression of REST was analyzed by in situ hybridization in mouse embryos.
  • a 600 bp fragment of mouse REST cDNA (encompassing most of the zinc finger domain) was prepared from 8.5 day mouse embryos by the PCR method described in Example 4 for the preparation of rat REST cDNA.
  • the amplification product was cloned into a Bluescript vector (Stratagene, San Diego, CA) and partially sequenced using the Sequenase Kit (US Biochemicals, Cleveland, OH).
  • embryos were fixed overnight in paraformaldehyde, incubated in hydrogen peroxide to inactivate endogenous phosphatases, lightly proteinase K digested, refixed, and hybridized at 70°C in 1 ml of 50% formamide, 5 x SSC pH 4.5, 50 ⁇ g/ml yeast RNA, 1 % SDS, 50 ⁇ g/ml heparin, 0.1 % CHAPS, and 5mM EDTA containing 1 ⁇ g of probe.
  • the embryos were rinsed in a low wash solution (50% formamide, 5 x SSC, pH 4.5, 1 % SDS, 0.1 % CHAPS; 70°C), treated with RNAse A, rinsed with a high stringency wash solution (50% formamide, 2 x SSC, pH 4.5, 0.1 % CHAPS; 65°C), and incubated with an alkaline-phosphatase coupled rabbit anti-digoxin antisera (Boehringer Mannheim, Indianapolis, IN) The enzyme activity of the reporter was detected by a color reaction with 5-bromo-4- chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT), which resulted in the deposition of a water-insoluble purple precipitate. Embryos were rinsed, washed into 80% glycerol, and photographed intact and in slices. The in situ hybridization results for 9.5 day embryos indicated the presence of abundant
  • the NH2 vector containing the nucleic acid of SEQ ID NO: 6 was digested with Hind III and Hinc II; and the NH7 vector containing the nucleic acid of SEQ ID NO:7 was digested with Hinc II and Bgl II.
  • the excised inserts were subcloned into a Hind III and Bam HI digested pCMV I-amp (Invitrogen, Inc., San Diego) vector.
  • the Hinc II digestion cleaved the overlap region of NH2 and NH7 at nucleotide 1575, allowing for a contiguous insert of nucleotides -175 through 3656 to be isolated.
  • Example 9 Transfection Studies of REST Function Transient transfection of PC 12 cells with a plasmid containing the chloramphenicol acetyl transferase (CAT) gene attached to the REl -containing promoter for the CNS-type sodium channel results in the expression of CAT (the plasmid designated herein as "type II- CAT").
  • This plasmid has been described by Kraner et al., Neuron, 9, 37-44, 1992.
  • a control CAT vector driven by the strong rous sarcoma virus (RSV) promoter has been described by Kraner et al., 1992 and Gorman et al., Proc. Natl. Acad. Sci. USA 79, 6777-6781, 1982.
  • RSV rous sarcoma virus
  • REST-Express A fragment of the REST cDNA, encoding the entire REST protein, with HindlH and Bgll termini (including nucleotides -175 to 3656 of SEQ ID NO:l) was subcloned downstream of the CMV promoter in the commercial mammalian expression vector pCDNA 1-amp (InVitrogen, Inc., San Diego, CA) between the HindlH and BamHI sites to create the CMV-REST vector. The resulting expression vector was designated REST-Express.
  • Rat PC12 cells were transfected with 30 ⁇ g of REST-Express and 30 ⁇ g of either type II-CAT or RSV-CAT by electroporation (Kraner et al., 1992). Forty-eight hours after transfection the cells were harvested, centrifuged and lysed by freeze-thaw cycles. The supernatant was analyzed for CAT activity as previously described in Maue et al.. Neuron, 4, 223-231, 1990.
  • a cDNA encoding the Zn finger region of REST (including nucleotides 481 to 1236 of SEQ ID NO:l) was cloned independently into the pCDNAl-amp vector and was used as an interfering form of REST in transient transfection assays.
  • L6 muscle cells and PC 12 cells were transfected with 30 ⁇ g of the interfering REST vector along with 30 ⁇ g of type II- CAT plasmid by electroporation and treated as above.
  • ADDRESSEE Dechert Price & Rhoads
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE (A) ORGANISM: Human (H) CELL LINE: HeLa
  • IMMEDIATE SOURCE (A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • GGT GAA GGA AAT AAA GAA GCC CCT CTT CAG AAA GTA GGA GCA GAA 3024
  • GAG GCA GAT GAG AGC CTA CCT GGT CTT GCT GCT AAT ATC AAC GAA 3069 Glu Ala Asp Glu Ser Leu Pro Gly Leu Ala Ala Asn He Asn Glu
  • TYPE nucleic acid
  • C STRANDEDNESS: double
  • D TOPOLOGY: linear
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE (A) ORGANISM: Human (H) CELL LINE: HeLa
  • IMMEDIATE SOURCE (A) LIBRARY: CDNA (x) PUBLICATION INFORMATION:
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE (A) ORGANISM: Human (H) CELL LINE: HeLa
  • IMMEDIATE SOURCE (A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 415
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 805 Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu Glu Gly Asp 200 205 210
  • GGT ATA AAT TCA ACA GTT GAA GAA CCA GTT TCA CCA ATG CTT CCC 1454 Gly He Asn Ser Thr Val Glu Glu Pro Val Ser Pro Met Leu Pro 965 970 975
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 630 Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu Glu Gly Asp 200 205 210
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE (A) ORGANISM: Human (H) CELL LINE: HeLa
  • IMMEDIATE SOURCE
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • C JOURNAL: Cell
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • MOLECULE TYPE CDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE (A) ORGANISM: Human (H) CELL LINE: HeLa
  • IMMEDIATE SOURCE (A) LIBRARY: CDNA (x) PUBLICATION INFORMATION:
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • C JOURNAL: Cell
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE (A) ORGANISM: Human (H) CELL LINE: HeLa
  • IMMEDIATE SOURCE
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons
  • C JOURNAL: Cell
  • MOLECULE TYPE CDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE (A) ORGANISM: Human (H) CELL LINE: HeLa
  • IMMEDIATE SOURCE (A) LIBRARY: cDNA
  • PUBLICATION INFORMATION :
  • TITLE REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

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Abstract

La présente invention concerne un acide nucléique sensiblement pur codant pour une protéine inhibant l'expression de protéines neurales dans des tissus non neuraux. L'invention concerne également un acide nucléique sensiblement pur codant pour une protéine qui se fixe à une séquence promoteur présentant une homologie d'environ 90 % au moins avec les nucléotides 6-28 de la séquence RE1 et dont l'action tend à supprimer l'activité d'un promoteur comportant la séquence promoteur. L'invention concerne en outre un acide nucléique sensiblement pur codant une protéine homologue à environ 85 % au minimum avec l'un au moins des deux domaines suivants: domaine de liaison ADN ou domaine suppresseur du facteur de transcription silenceur de la RE1 d'un animal ou 'REST', pour 'RE1-Silencing Transcription factor'. L'invention concerne également des protéines codées de cette façon.
PCT/US1996/003940 1995-03-23 1996-03-22 Proteine 'rest' et adn correspondant WO1996029433A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996027665A3 (fr) * 1995-03-03 1997-04-24 California Inst Of Techn Proteines ayant les proprietes d'un facteur silenceur exerçant une action de repression sur les neurones
WO2003087289A2 (fr) * 2002-04-17 2003-10-23 Universita' Degli Studi Di Milano Procede de selection de composes utiles dans le traitement de la choree de huntington
JPWO2006030722A1 (ja) * 2004-09-15 2008-05-15 公立大学法人横浜市立大学 神経特異的遺伝子発現に必須なアミノ酸配列
WO2009147384A2 (fr) * 2008-06-07 2009-12-10 University College Cardiff Consultants Limited Peptide
US7943591B2 (en) 2007-05-11 2011-05-17 Adynxx, Inc. Gene expression and pain
CN102827947A (zh) * 2011-06-17 2012-12-19 东北制药集团辽宁生物医药有限公司 快速定量检测肝炎病毒核酸的试剂盒及检测方法
US9700624B2 (en) 2012-05-10 2017-07-11 Adynxx, Inc. Formulations for the delivery of active ingredients
US10287583B2 (en) 2014-08-15 2019-05-14 Adynxx, Inc. Oligonucleotide decoys for the treatment of pain

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HENRY A. ERLICH, "PCR Technology", Published 1992, by W.H. FREEMAN AND CO. (N.Y.), pages 7-16. *
SCIENCE, Volume 267, issued 03 March 1995, C.J. SCHOENHERR et al., "The Neuron-Restrictive Silencer Factor (NRSF): A Coordinate Repressor of Multiple Neuron-Specific Genes". *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996027665A3 (fr) * 1995-03-03 1997-04-24 California Inst Of Techn Proteines ayant les proprietes d'un facteur silenceur exerçant une action de repression sur les neurones
US5935811A (en) * 1995-03-03 1999-08-10 California Institute Of Technology Neuron-restrictive silencer factor nucleic acids
US6270990B1 (en) 1995-03-03 2001-08-07 California Institute Of Technology Neuron-restrictive silencer factor proteins
US6824774B2 (en) 1995-03-03 2004-11-30 California Institute Of Technology Antibodies that bind neuron restrictive silencer factor proteins
WO2003087289A2 (fr) * 2002-04-17 2003-10-23 Universita' Degli Studi Di Milano Procede de selection de composes utiles dans le traitement de la choree de huntington
WO2003087289A3 (fr) * 2002-04-17 2004-02-05 Univ Degli Studi Milano Procede de selection de composes utiles dans le traitement de la choree de huntington
JPWO2006030722A1 (ja) * 2004-09-15 2008-05-15 公立大学法人横浜市立大学 神経特異的遺伝子発現に必須なアミノ酸配列
US8093225B2 (en) 2007-05-11 2012-01-10 Adynxx, Inc. Gene expression and pain
US7943591B2 (en) 2007-05-11 2011-05-17 Adynxx, Inc. Gene expression and pain
US8741864B2 (en) 2007-05-11 2014-06-03 Adynxx, Inc Gene expression and pain
US9290762B2 (en) 2007-05-11 2016-03-22 Adynxx, Inc. Gene expression and pain
US10041069B2 (en) 2007-05-11 2018-08-07 Adynxx, Inc. Gene expression and pain
WO2009147384A3 (fr) * 2008-06-07 2010-01-28 University College Cardiff Consultants Limited Peptide
WO2009147384A2 (fr) * 2008-06-07 2009-12-10 University College Cardiff Consultants Limited Peptide
CN102827947A (zh) * 2011-06-17 2012-12-19 东北制药集团辽宁生物医药有限公司 快速定量检测肝炎病毒核酸的试剂盒及检测方法
CN102827947B (zh) * 2011-06-17 2014-09-24 东北制药集团辽宁生物医药有限公司 快速定量检测肝炎病毒核酸的试剂盒及检测方法
US9700624B2 (en) 2012-05-10 2017-07-11 Adynxx, Inc. Formulations for the delivery of active ingredients
US10434178B2 (en) 2012-05-10 2019-10-08 Adynxx Sub, Inc. Formulations for the delivery of active ingredients
US10287583B2 (en) 2014-08-15 2019-05-14 Adynxx, Inc. Oligonucleotide decoys for the treatment of pain
US10683502B2 (en) 2014-08-15 2020-06-16 Adynxx Sub, Inc. Oligonucleotide decoys for the treatment of pain

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