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WO1999036527A1 - Gene de type ataxine 2 - Google Patents

Gene de type ataxine 2 Download PDF

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Publication number
WO1999036527A1
WO1999036527A1 PCT/CN1998/000009 CN9800009W WO9936527A1 WO 1999036527 A1 WO1999036527 A1 WO 1999036527A1 CN 9800009 W CN9800009 W CN 9800009W WO 9936527 A1 WO9936527 A1 WO 9936527A1
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Prior art keywords
polypeptide
ataxιn
gene
pro
seq
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PCT/CN1998/000009
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English (en)
Inventor
Jia-Hui Xia
Chun-Yu Liu
De-An Wang
Han-Xiang Deng
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Hunan Medical University
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Priority to PCT/CN1998/000009 priority Critical patent/WO1999036527A1/fr
Publication of WO1999036527A1 publication Critical patent/WO1999036527A1/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

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the ataxin-2 family, hereinafter referred to as ataxin-2 like gene. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.
  • Ataxin-2 is the gene responsible for spinocerebellar ataxia, type 2.
  • a partial cDNA sequence obtained by Pulst was found to be homologous to ataxin-2.
  • Ataxin-2 family has an established, proven history as therapeutic targets.
  • further members of the ataxin-2 family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, ataxia, cardiomyopathy, deafness, neurological disease, cancer, and AIDS.
  • the invention relates to ataxin-2 like gene polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such ataxin-2 like gene polypeptides and polynucleotides. Such uses include the treatment of ataxia, cardiomyopathy, deafness, neurological disease, cancer, and AIDS, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with ataxin-2 like gene imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate ataxin-2 like gene activity or levels.
  • Ataxin-2 like gene refers, among others, generally to a polypeptide having the ammo acid sequence set forth in SEQ ID NO:2 or an allelic variant thereof.
  • “Atax ⁇ n-2 like gene activity or atax ⁇ n-2 like gene polypep ⁇ de activity” or “biological activity of the atax ⁇ n-2 like gene or atax ⁇ n-2 like gene polypeptide” refers to the metabolic or physiologic function of said atax ⁇ n-2 like gene including similar acuvities or improved activities or these activities with decreased undesirable side-effects Also mcluded are antigemc and immunogenic activities of said atax ⁇ n-2 like gene
  • Tax ⁇ n-2 like gene refers to a polynucleotide having the nucleotide sequence set forth m SEQ ID NO 1 or allehc variants thereof and/or their complements
  • Antibodies as used herein mcludes polyclonal and monoclonal antibodies, chimenc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library
  • Isolated means altered “by the hand of man” from the natural state If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein
  • Polynucleotide generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides mclude, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hyb ⁇ d molecules comp ⁇ smg DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of smgle- and double-stranded regions
  • 'polynucleotide refers to t ⁇ ple-stranded regions comp ⁇ smg RNA or DNA or both RNA and DNA
  • polynucleotide also mcludes DNAs or RNAs containing one or more modified bases and DNAs or RNA
  • Polypeptide refers to any peptide or protein comp ⁇ smg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds, l e , peptide isosteres
  • Polypeptide refers to both short chains, commonly referred to as peptides, ohgopeptides or o gomers, and to longer chains, generally referred to as proteins
  • Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids
  • Polypeptides mclude ammo acid sequences modified either by natural processes, such as posttranslational processmg, or bv chemical modification techniques which are well known in the art Such modifications are well desc ⁇ bed m basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains
  • lodination methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation. sulfation, transfer-RNA mediated addition of a mo acids to proteins such as arginylation, and ubiquiunation
  • proteins such as arginylation, and ubiquiunation
  • PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES 2nd Ed , T E Cre ⁇ ghton, W H Freeman and Company, New York, 1993 and Wold, F , Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York. 1983.
  • Va ⁇ ant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical va ⁇ ant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the va ⁇ ant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • Identity is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques.
  • identity is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs.
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., JMolec Biol (1990) 215:403).
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ED NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2.
  • the polypeptide sequence having an amino acid sequence at least 95% identical to a reference amino acid sequence up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the present invention relates to ataxin-2 like gene polypeptides (or ataxin-2 like gene proteins).
  • the ataxin-2 like gene polypeptides include the polypeptide of SEQ ID NO:2; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO: 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly preferred.
  • Ataxin-2 like gene polypeptides are also included within ataxin-2 like gene polypeptides which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO:2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO:2. Furthermore, those with at least 97-99% are highly preferred.
  • ataxin-2 like gene polypeptides exhibit at least one biological activity of ataxin-2 like gene.
  • the ataxin-2 like gene polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Fragments of the ataxin-2 like gene polypeptides are also included in the invention. A fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned ataxin-2 like gene polypeptides.
  • fragments may be "free-standing,” or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of the ataxin-2 like gene polypeptide.
  • “about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
  • Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of ataxin-2 like gene polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet- foiming regions, turn andtum-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- forming regions, substrate binding region, and high antigenic index regions.
  • Other preferred fragments are biologically active fragments.
  • Biologically active fragments are those that mediate ataxin-2 like gene activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
  • all of these polypeptide fragments retain the biological activity of the ataxin-2 like gene, including antigenic activity.
  • Variants of the defined sequence and fragments also form part of the present invention.
  • Preferred variants are those that vary from the referents by conservative amino acid substitutions - i.e., those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
  • the ataxin-2 like gene polypeptides of the invention can be prepared in any suitable manner.
  • polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art. Polynucleotides of the Invention
  • Atax ⁇ n-2 like gene polynucleotides mclude isolated polynucleotides which encode the atax ⁇ n-2 like gene polypeptides and fragments, and polynucleotides closely related thereto More specifically, atax ⁇ n-2 like gene polynucleotides of the mvention mclude a polynucleotide comp ⁇ smg the nucleotide sequence contained in SEQ ID NO 1 encoding an atax ⁇ n-2 like gene polypeptide of SEQ ED NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 Atax ⁇ n-2 like gene polynucleotides further mclude a polynucleotide comp ⁇ smg a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the alax ⁇ n-2 like gene polypeptide of SEQ ID
  • the atax -2 like gene of the mvention is structurally related to other proteins of the atax ⁇ n-2 family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO 1) encoding human atax ⁇ n-2 like gene
  • SEQ ID NO 1 contains an open reading frame (nucleotide number 63 to 3215) encoding a polypeptide of 1051 ammo acids of SEQ ID NO 2.
  • the amino acid sequence of Table 2 (SEQ ID NO 2) has about 54 3% identity (using Bestfit of GCG) in 342 ammo acid residues with atax ⁇ n-2 (S M Pulst et al Nat Genet 14269-276, 1996)
  • the nucleotide sequence of Table 1 (SEQ ID NO 1) has about 62 7% identity (using Bestfit of GCG) in 1305 nucleotide residues with atax ⁇ n-2 (S M Pulst et al Nat Genet 14269-276, 1996)
  • atax ⁇ n-2 like gene polypeptides and polynucleotides of the present mvention are expected to have, inter a a, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art
  • One polynucleotide of the present mvention encoding the atax ⁇ n-2 like gene may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in cells of human adult brain using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252:1651-1656; Adams, M.D. et ai., Nature, (1992) 555:632-634; Adams, M.D., et ai, Nature (1995) 377 Supp.3-174).
  • Polynucleotides of the mvention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized usmg well known and commercially available techniques.
  • nucleotide sequence encoding ataxin-2 like gene polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 63 to 3215 of SEQ ID NO: 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al. , Proc Natl Acad Sci USA (1989) 86:821- 824, or is an HA tag.
  • the polynucleotide may also contain non-coding 5 ' and 3 ' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • polynucleotides encoding ataxin-2 like gene variants which comprise the amino acid sequence ataxin-2 like gene polypeptide of Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination.
  • the present invention further relates to polynucleotides that hybridize to the herein above- described sequences.
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences.
  • Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate fiill-length cDNAs and genomic clones encoding ataxin-2 like gene polypeptides, and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the ataxin-2 like gene.
  • hybridization techniques are known to those of skill in the art.
  • these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent.
  • the probes generally will comprise at least 15 nucleotides.
  • such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
  • a method comprises the steps of screening an appropriate library under stingent hybridization conditions with a labeled probe having the SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence
  • atax ⁇ n-2 like gene polynucleotides of the present invention further mclude a nucleotide sequence comprising a nucleotide sequence that hyb ⁇ dize under stringent condition to a nucleotide sequence having SEQ ED NO 1 or a fragment thereof
  • polypeptides comprising ammo acid sequences encoded by nucleotide sequences obtained by the above hyb ⁇ dization condition
  • Such hybndization techniques are well known to those of skill
  • the present mvention also relates to vectors which comprise a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engineered with vectors of the mvention and to the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs de ⁇ ved from the DNA constructs of the present mvention
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present mvention
  • Introduction of polynucleotides into host cells can be effected by methods desc ⁇ bed in many standard laboratory manuals, such as Davis et ai, BASIC METHODS M MOLECULAR BIOLOGY (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, NY ( 1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK. HEK 293 and Bowes melanoma cells, and plant cells
  • bacte ⁇ al cells such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK. HEK 293 and Bowes melanoma cells
  • a great va ⁇ ety of expression systems can be used Such systems mclude, among others, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retrovuuses, and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
  • the expression systems may contam control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used
  • approp ⁇ ate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the cells may be harvested p ⁇ or to use m the screenmg assay If the atax ⁇ n-2 like gene polypeptide is secreted mto the medium, the medium can be recovered m order to recover and pu ⁇ fy the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered
  • Atax ⁇ n-2 like gene polypeptides can be recovered and purified from recombinant cell cultures by well-known methods mcludmg ammonium sulfete or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for
  • This mvention also relates to the use of atax ⁇ n-2 like gene polynucleotides for use as diagnostic reagents Detection of a mutated form of the atax ⁇ n-2 like gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of atax ⁇ n-2 like gene Individuals carrying mutations the atax ⁇ n-2 like gene may be detected at the DNA level by a va ⁇ ety of techniques Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, u ⁇ ne, saliva, tissue biopsy or autopsy mate ⁇ al.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques pnor to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change m size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled ataxin-2 like gene nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures. DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing.
  • an array of oligonucleotide probes comp ⁇ smg atax ⁇ n-2 like gene nucleotide sequences or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Array technology methods are well known and have general applicability and can be used to address a va ⁇ ety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee etai., Science, Vol 274, pp 610-613 (1996)).
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to ataxia, cardiomyopathy, Deafness, neurological disease, cancer, and AIDS through detection of mutation in the ataxin-2 like gene by the methods described.
  • Ataxia, cardiomyopathy, Deafness, neurological disease, cancer, and AIDS can be diagnosed by methods comp ⁇ smg determining from a sample derived from a subject an abnormally decreased or increased level of atax ⁇ n-2 like gene polypeptide or atax ⁇ n-2 like gene mRNA Decreased or increased expression can be measured at the RNA level usmg any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR,
  • the present mvention relates to a diagonostic kit for a disease or suspectability to a disease, particularly ataxia, cardiomyopathy, deafness, neurological disease, cancer, and AIDS, which comp ⁇ ses:
  • any such kit, (a), (b), (c) or (d) may comp ⁇ se a substantial component
  • the nucleotide sequences of the present mvention are also valuable for chromosome identification
  • the sequence is specifically targeted to and can hyb ⁇ dize with a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention is an important first step m correlating those sequences with gene associated disease
  • genetic map data are found, for example, in V McKusick, Mendehan Inhe ⁇ tance in Man (available on line through Johns Hopkins University Welch Medical Library)
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinhe ⁇ tance of physically adjacent genes)
  • the differences m the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed m some or all of the affected individuals but
  • the human atax ⁇ n-2 like gene was mapped to chromosome 9cen where familial dilated cardiomyopathy, deafness, cartilage-hair hypoplasia, Melkersson-Rosenthal syndrome, and inclusion body myopathy, among others, were localized
  • polypeptides of the mvention or their fragments or analogs thereof, or cells expressing them can also be used as lmmunogens to produce antibodies immunospecific for the atax ⁇ n-2 like gene polypeptides
  • the teim "immunospecific" means that the antibodies have substantial!
  • the p ⁇ or art Antibodies generated against the atax ⁇ n-2 like gene polypeptides can be obtained by administering me polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples mclude the hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256495-497), the t ⁇ oma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV- hyb ⁇ doma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R Liss, Inc , 1985)
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to pu ⁇ fy the polypeptides bv affinity chromatography
  • Antibodies against atax ⁇ n-2 like gene polypeptides may also be employed to treat ataxia, cardiomyopathy, deafness, neurological disease, cancer, and AIDS, among others
  • Another aspect of the mvention relates to a method for mducmg an immunological response m a mammal which comp ⁇ ses inoculating the mammal with atax ⁇ n-2 like gene polypeptides, or fragments thereof, adequate to produce antibody and/or T cell immune response to protect said animal from ataxia, cardiomyopathy, deafness, neurological disease, cancer, and AIDS, among others
  • Yet another aspect of the mvention relates to a method of mducmg immunological response m a mammal which comp ⁇ ses delivering ataxm-2 like gene polypeptide via a vector directing expression of atax ⁇ n-2 like gene polynucleotide m vivo in order to mduce such an immunological response to produce antibody to protect said animal from diseases
  • a further aspect of the mvention relates to an lmmunological/vaccine formulation (composition) which, when introduced mto a mammalian host, mduces an immunological response m that mammal to an atax ⁇ n-2 like gene polypeptide wherein the composition comp ⁇ ses an atax ⁇ n-2 like gene polypeptide or atax ⁇ n-2 like gene
  • the vaccme formulation may further comp ⁇ se a suitable earner Since atax ⁇ n-2 like gene polypeptides may be broken down m the stomach, it is preferably administered parenterally (mcludmg subcutaneous, intramuscular, intravenous, lntradermal etc injection)
  • Formulations suitable for parenteral administration mclude aqueous and non-aqueous ste ⁇ le injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation mstonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • the ataxin-2 like gene polypeptides of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the ataxin-2 like gene polypeptide of the present invention.
  • polypeptides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
  • agonists or antagonists may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide of the present invention; or may be structural or functional mimetics of the polypeptide of the present invention. See Coligan et al, Current Protocols in Immunology l(2):Chapter 5 (1991).
  • Ataxin-2 like gene polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate ataxin-2 like gene polypeptides on the one hand and which can inhibit the function of ataxin-2 like gene polypeptides on the other hand.
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as ataxia, cardiomyopathy, deafness, neurological disease, cancer, and AIDS.
  • Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as ataxia, cardiomyopathy, deafness, neurological disease, cancer, and AIDS.
  • such screening procedures may involve using appropriate cells which express the ataxin-2 like gene polypeptide or respond to the ataxin-2 like gene polypeptide of the present invention.
  • Such cells include cells from mammals, yeast, Drosophila or E. coli.
  • Cells which express the ataxin-2 like gene polypeptide (or cell membrane containing the expressed polypeptide) or respond to the ataxin-2 like gene polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for ataxin-2 like gene activity.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the ataxin-2 like gene polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor Further, these assays may test whether the candidate compound results m a signal generated by activation of the atax ⁇ n-2 like gene polypeptide, usmg detection systems approp ⁇ ate to the cells bea ⁇ ng the atax ⁇ n-2 like gene polypeptide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • the assays may simply comprise the steps of mixing a candidate compound with a solution containing an atax ⁇ n-2 like gene polypeptide to form a mixture, measuring atax ⁇ n-2 like gene activity in the mixture, and comparing the atax ⁇ n-2 like gene activity of the mixture to a standard
  • the ataxm-2 like gene cDNA, protem and antibodies to the protem may also be used to configure assays for detectmg the effect of added compounds on the production of atax ⁇ n-2 like gene mRNA and protem m cells.
  • an ELISA may be constructed for measuring secreted or cell associated levels of atax ⁇ n-2 like gene protem usmg monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of atax -2 like gene (also called antagonist or agomst, respectively) from suitably manipulated cells or tissues.
  • the atax ⁇ n-2 like gene protem may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known m the art These mclude, but are not limited to, ligand binding and cross nking assays in which the atax ⁇ n-2 like gene is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or purification, and mcubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods mclude biophysical techniques such as surface plasmon resonance and spectroscopy In addition to bemg used for pu ⁇ fication and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of the atax ⁇ n-2 like gene which compete with the binding of the atax ⁇ n-2 like gene to its receptors, if any Standard methods for conducting screenm
  • Examples of potential atax ⁇ n-2 like gene polypeptide antagonists mclude antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the atax ⁇ n-2 like gene polypeptide, e g , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypetide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented
  • the present mvention relates to a screenmg kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for atax ⁇ n-2 like gene polypeptides, or compounds which decrease or enhance the production of atax ⁇ n-2 like gene polypeptides, which comp ⁇ ses
  • a recombinant cell expressmg an atax ⁇ n-2 like gene polypeptide, preferably that of SEQ ID NO 2,
  • a cell membrane expressmg an atax ⁇ n-2 like gene polypeptide, preferably that of SEQ ID NO 2, or
  • any such kit, (a), (b), (c) or (d) may comp ⁇ se a substantial component
  • This mvention provides methods of treating abnormal conditions such as ataxia, cardiomyopathy, deafness, neurological disease, cancer, and AIDS, related to both an excess of and insufficient amounts of, ataxm-2 like gene polypeptide activity
  • soluble forms of atax ⁇ n-2 like gene polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc m competition with endogenous atax ⁇ n-2 like gene polypeptides may be administered Typical embodiments of such competitors comp ⁇ se fragments of the ataxm-2 like gene polypeptide
  • expression of the gene encoding endogenous atax ⁇ n-2 like gene polypeptide can be inhibited usmg expression blockmg techmques Known such techmques mvolve the use of antisense sequences, either internally generated or separately administered See, for example, O'Connor, JNeurochem (1991) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression.
  • oligonucleotides which form t ⁇ ple helices with the gene can be supphed See, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et ai , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360 These ohgomers can be administered per se or the relevant oligomers can be expressed m vivo
  • atax ⁇ n-2 like gene and its activity several approaches are also available.
  • One approach comp ⁇ ses administering to a subject a therapeutically effective amount of a compound which activates the atax ⁇ n-2 like gene polypeptide, i e , an agonist as desc ⁇ bed above, in combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition
  • gene therapy may be employed to effect the endogenous production of atax ⁇ n-2 like gene by the relevant cells
  • Peptides such as the soluble form of atax ⁇ n-2 like gene polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical earner.
  • a suitable pharmaceutical earner Such formulations comp ⁇ se a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient.
  • earners mclude but are not limited to, saline, buffered saline, dextrose, water, glycerol.
  • the mvention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • systemic administration of the pharmaceutical compositions mclude injection, typically by intravenous injection
  • Other injection routes such as subcutaneous, intramuscular, or lntrape ⁇ toneal
  • Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents
  • oral administration may also be possible.
  • Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like
  • the dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in me range of 0.1 - 100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
  • Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • a polynucleotide such as a DNA or RNA
  • GTAGCTCCCC TGAGAACAGC CTGGATCCTT TTCCTCCCCG GATCTTAAAG GAGGAGCCCA 2040 AAGGAAAGGA GAAAGAGGTT GATGGTCTGT TGACTTCAGA GCCCATGGGG TCTCCCGTCT 2100
  • Gly Pro Gly Cys Pro lie Pro Leu Ala Ser Arg Ala Leu Gin Arg Gly

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Abstract

L'invention concerne des polypeptides et des polynucléotides de gène de type ataxine 2 et des procédés d'obtention de ces polypeptides par des techniques recombinantes. Font aussi l'objet de cette invention des procédés d'utilisation des polypeptides et des polynucléotides de gène de type ataxine 2 dans la conception de protocoles pour le traitement d'ataxie, de myocardiopathies, de surdité, d'affections neurologiques, de cancers et du SIDA, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000009 1998-01-19 1998-01-19 Gene de type ataxine 2 WO1999036527A1 (fr)

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PCT/CN1998/000009 WO1999036527A1 (fr) 1998-01-19 1998-01-19 Gene de type ataxine 2

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003024389A3 (fr) * 2001-07-30 2003-10-09 Immunex Corp Imx97018, polypeptide humain de type ataxine-1
CN109507436A (zh) * 2019-01-10 2019-03-22 南方医科大学南方医院 Atxn2l作为预测胃癌奥沙利铂原发性耐药的标志物的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991000572A1 (fr) * 1989-06-30 1991-01-10 Poqet Computer Corporation Blocage de bus sans utilisation de resistances d'appoint
WO1995001137A1 (fr) * 1993-06-29 1995-01-12 Voges Innovation Pty. Ltd. Distributeur

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991000572A1 (fr) * 1989-06-30 1991-01-10 Poqet Computer Corporation Blocage de bus sans utilisation de resistances d'appoint
WO1995001137A1 (fr) * 1993-06-29 1995-01-12 Voges Innovation Pty. Ltd. Distributeur

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003024389A3 (fr) * 2001-07-30 2003-10-09 Immunex Corp Imx97018, polypeptide humain de type ataxine-1
US6887687B2 (en) 2001-07-30 2005-05-03 Immunex Corporation Nucleic acids encoding human ataxin-1-like polypeptide IMX97018
US7183380B2 (en) 2001-07-30 2007-02-27 Immunex Corporation Human ataxin-1-like polypeptide IMX97018
US7704501B2 (en) 2001-07-30 2010-04-27 Immunex Corporation Antibodies binding to human ataxin-1-like polypeptide
CN109507436A (zh) * 2019-01-10 2019-03-22 南方医科大学南方医院 Atxn2l作为预测胃癌奥沙利铂原发性耐药的标志物的应用

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