WO2001032875A1 - Polypeptides et acides nucleiques de torsine c - Google Patents

Polypeptides et acides nucleiques de torsine c Download PDF

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Publication number
WO2001032875A1
WO2001032875A1 PCT/US2000/030355 US0030355W WO0132875A1 WO 2001032875 A1 WO2001032875 A1 WO 2001032875A1 US 0030355 W US0030355 W US 0030355W WO 0132875 A1 WO0132875 A1 WO 0132875A1
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Prior art keywords
nucleic acid
torc
torsin
protein
seq
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PCT/US2000/030355
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English (en)
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John P. Carulli
Alexander Lukashin
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Biogen, Inc.
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Priority to AU14611/01A priority Critical patent/AU1461101A/en
Publication of WO2001032875A1 publication Critical patent/WO2001032875A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention generally relates to nucleic acids and polypeptides.
  • the invention relates more particularly to nucleic acids encoding polypeptides related to torsin A and torsin B polypeptides, which are associated with peripheral neuropathies such as torsion dystonia.
  • the dystonias represent a class of movement disorders characterized by contracted, twisted postures in afflicted individuals.
  • the dystonia disorders are generally believed to arise from neuronal dysfunction, but not from neuronal degeneration er se.
  • the DYTl transcript is found at high levels in the substantia nigra pars compacta, hippocampus and cerebellum regions of the brain. The DYTl message appears to increase in abundance with stress prior to death.
  • the nucleic acid encodes a polypeptide which includes one or more amino acid sequences selected from the group consisting of GQHLA (SEQ ID NO: 19), LSLHGW (SEQ ID NO:7), GTGK (SEQ ID NO:8), LHFPH (SEQ ID NO:9); FDEMDK (SEQ ID NO:10), PFLPL (SEQ ID NO:l 1), TFFP (SEQ ID NO:12), and GCKTV (SEQ ID NO:13).
  • GQHLA SEQ ID NO: 19
  • LSLHGW SEQ ID NO:7
  • GTGK SEQ ID NO:8
  • LHFPH SEQ ID NO:9
  • FDEMDK SEQ ID NO:10
  • PFLPL SEQ ID NO:l 1
  • TFFP SEQ ID NO:12
  • GCKTV SEQ ID NO:13
  • the invention includes a substantially purified TorC polypeptide, e.g., any of the TorC polypeptides encoded by a torC nucleic acid.
  • the invention also includes a pharmaceutical composition that includes a TorC polypeptide and a pharmaceutically acceptable carrier or diluent.
  • kits comprising antibodies that bind to a polypeptide encoded by any of the nucleic acid molecules described above and a negative control antibody.
  • the present invention is also directed to methods of identifying a compound that binds to TorC polypeptide by contacting the TorC polypeptide with a compound and determining whether the compound binds to the TorC polypeptide.
  • the present invention is also directed to compounds that modulate TorC polypeptide activity identified by contacting a TorC polypeptide with the compound and determining whether the compound modifies activity of the TorC polypeptide, binds to the TorC polypeptide, or binds to a nucleic acid molecule encoding a TorC polypeptide.
  • a control sample is preferably taken from a matched individual, i.e., an individual of similar age, sex, or other general condition but who is not suspected of having a neurological condition.
  • the control sample may be taken from the subject at a time when the subject is not suspected of having a neurological disorder.
  • the TorC polypeptide is detected using a torC antibody.
  • the invention is also directed to methods of inducing an immune response in a mammal against a polypeptide encoded by any of the nucleic acid molecules described above.
  • the method includes administering to the mammal an amount of the polypeptide sufficient to induce the immune response.
  • the invention includes a method of diagnosing a neurological condition, e.g., a peripheral neuropathy such as torsion dystonia, in a subject.
  • the method includes providing a nucleic acid sample, e.g., RNA or DNA, or both, from the subject and measuring the amount of the torC nucleic acid in the subject nucleic acid sample.
  • the amount of torC nucleic acid sample in the subject nucleic acid is then compared to the amount of torC nucleic acid in a control sample.
  • An alteration in the amount of torC nucleic acid in the sample relative to the amount of torC in the control sample indicates the subject has a neurological condition.
  • the invention provides method of treating or preventing or delaying a neurological condition.
  • the method includes administering to a subject in which such treatment or prevention or delay is desired a torC nucleic acid, a TorC polypeptide, or a torC antibody in an amount sufficient to treat, prevent, or delay a neurological condition in the subject.
  • Torsin A and Torsin B are members of a family of secreted proteins that were initially discovered because the seminal member of the family, torsin A, is mutated in patients with an inherited form of torsion dystonia (Ozelius et al., Nature Genet. 17:40-48, 1997). Torsion dystonia is a peripheral neuropathy characterized by involuntary posturing of the trunk, neck, and occasionally the hands or feet.
  • This analysis identified a gene encoded in the "reverse" direction with respect to the orientation of the BAC clone AC007430.
  • the identified gene contained exons at positions 129092 - 128848 (SEQ ID NO:14), 129505 - 129378 (SEQ ID NO:15), 130374 - 130199 (SEQ ID NO:16), 131378 -131113 (SEQ ⁇ NO:17), and 132084 - 131934 (SEQ ID NO:18) in the BAC clone.
  • a sequence based on these assembled exons was used to search the GenBank EST database, and an EST by the accession number aa502940 was identified. The clone from which this EST was derived was sequenced.
  • the torsin C sequence in Table 1 is a composite of the sequence of this clone and sequences assembled from BAC AC007430.
  • Table 1 shows a human-derived DNA sequence encoding a TorC polypeptide, along with the amino acid sequence of an encoded polypeptide.
  • the polypeptide sequence has an amino terminal signal peptide at amino acids 1-20 according to the amino acid sequence shown in Table 1 and is indicated as an underlined sequence.
  • the amino terminal signal sequence in torC is shared with other members of the torsin gene family, and is typical of secreted proteins.
  • Torsin B DAIKPF DYYEQVDGVSYRKAIFIFLSNAGGD ITKTALDFWRAGRKREDIQ KDLEPV
  • the present invention discloses torC nucleic acids, isolated nucleic acids that encode TorC polypeptide or a portion thereof, TorC polypeptides, vectors containing these nucleic acids, host cells transformed with the torC nucleic acids, anti-TorC antibodies, and pharmaceutical compositions. Also disclosed are methods of making TorC polypeptides, as well as methods of screening, diagnosing, treating conditions using these compounds, and methods of screening compounds that modulate TorC polypeptide activity.
  • sequences and corresponding sequence identifier numbers discussed herein include the following:
  • nucleic acid molecules that encode TorC proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify t ⁇ rC-encoding nucleic acids (e.g., torC mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of torC nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • Probes refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt) or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA- like technologies.
  • an "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated torC nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3, or a complement of any of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • torC nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et ⁇ l, eds., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ⁇ l., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to torC nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having at least about 10 nt and as many as 50 nt, preferably about 15 nt to 30 nt. They may be chemically synthesized and may be used as probes.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:3.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:3, or a portion of this nucleotide sequence.
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO: 3 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:3, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:3, e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion oftorC.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
  • a "homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
  • homologous nucleotide sequences encode those sequences coding for isoforms of TorC polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for a TorC polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the nucleotide sequence encoding human torsin C protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO:4, as well as a polypeptide having torsin C activity.
  • a homologous amino acid sequence does not encode the amino acid sequence of a human torsin C polypeptide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3; or an anti-sense strand nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3; or of a naturally occurring mutant of SEQ ID NO:l or SEQ ID NO: 3.
  • Probes based on the human torC nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a
  • a polypeptide having a biologically active portion of torC refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically active portion oftorC” can be prepared by isolating a portion of SEQ ID NO:l or SEQ ID NO:3, that encodes a polypeptide having a torC biological activity (biological activities of the TorC proteins are described below), expressing the encoded portion of TorC protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion oftorC.
  • nucleic acid molecules encoding TorC proteins from other species and thus that have a nucleotide sequence that differs from the human sequence of SEQ ID NO:l or SEQ ID NO: 3, are intended to be within the scope of the invention.
  • a non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02%) BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l or SEQ ID NO:3 corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or 3, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • TorC proteins of the present invention can contain at least one domain that is a typically conserved region in TOR family members, i.e., Torsin A and Torsin B proteins, and torC homologs.
  • conserved domains include, e.g., GQHLA (SEQ ID NO: 19), LSLHGW (SEQ ID NO:7), GTGK (SEQ ID NO:8), LHFPH (SEQ ID NO:9); FDEMDK (SEQ ID NO: 10), PFLPL (SEQ ID NO:l 1), TFFP (SEQ ID NO:12), and GCKTV (SEQ ID NO:13).
  • GQHLA SEQ ID NO: 19
  • LSLHGW SEQ ID NO:7
  • GTGK SEQ ID NO:8
  • LHFPH SEQ ID NO:9
  • FDEMDK SEQ ID NO: 10
  • PFLPL SEQ ID NO:l 1
  • TFFP SEQ ID NO:12
  • GCKTV SEQ ID NO:13
  • the protein encoded by the nucleic acid molecule is at least about 60%) homologous to SEQ ID NO:2 or SEQ ID NO:4, more preferably at least about 70%, 80%o, 90%, 95%, 98%o, and most preferably at least about 99% homologous to SEQ ID NO:2 or SEQ ID NO:4.
  • Biologically active portions of a TorC protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the TorC protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 that include fewer amino acids than the full length TorC proteins, and exhibit at least one activity of a TorC protein.
  • biologically active portions comprise a domain or motif with at least one activity of the TorC protein.
  • a biologically active portion of a TorC protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential torC sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylafion signals). Such regulatory sequences are described, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • Suitable inducible non- fusion E. coli expression vectors include pTrc (Arnrann et al, (1988) Gene 69:301-315) and pET l id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • torC can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol Cell Biol 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • Neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477
  • pancreas-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166.
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to torC mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous torC gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the torC transgene to direct expression of TorC protein to particular cells.
  • the vector is designed such that, upon homologous recombination, the endogenous torC gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous torC gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous TorC protein).
  • the altered portion of the torC gene is flanked at its 5' and 3' ends by additional nucleic acid of the torC gene to allow for homologous recombination to occur between the exogenous torC gene carried by the vector and an endogenous torC gene in an embryonic stem cell.
  • the additional flanking torC nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • flanking DNA both at the 5' and 3' ends
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced torC gene has homologously recombined with the endogenous torC gene are selected (see e.g., Li et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras.
  • chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PL
  • Cre/loxP recombinase system of bacteriophage PL
  • FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 :1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a TorC protein or anti-torC antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a TorC protein or anti-torC antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by any of a number of routes, e.g., as described in U.S. Patent Nos. 5,703,055. Delivery can thus also include, e.g., intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or stereotactic injection (see e.g., Chen et al. (1994) PNAS 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • a TorC protein of the invention has the ability to bind ATP.
  • the isolated nucleic acid molecules of the invention can be used to express TorC protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect torC mRNA (e.g., in a biological sample) or a genetic lesion in a torC gene, and to modulate torC activity, as described further below.
  • TorC proteins can be used to screen drugs or compounds that modulate the torC activity or expression as well as to treat disorders characterized by insufficient or excessive production of TorC protein, e.g., peripheral neuropathies such as torsin dystonia, or production of TorC protein forms that have decreased or aberrant activity compared to torC wild type protein.
  • the anti-TorC antibodies of the invention can be used to detect and isolate TorC proteins and modulate torC activity.
  • This invention further pertains to novel agents identified by the above described screening assays and uses thereof for treatments as described herein.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to TorC proteins or have a stimulatory or inhibitory effect on, for example, torC expression or torC activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to TorC proteins or have a stimulatory or inhibitory effect on, for example, torC expression or torC activity.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a TorC protein or polypeptide or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des 12:145).
  • an assay is a cell-based assay in which a cell which expresses a membrane-bound form of TorC protein, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a TorC protein determined.
  • the cell for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the TorC protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the TorC protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane-bound form of TorC protein, or a biologically active portion thereof, on the cell surface with a known compound which binds torC to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a TorC protein, wherein determining the ability of the test compound to interact with a TorC protein comprises determining the ability of the test compound to preferentially bind to torC or a biologically active portion thereof as compared to the known compound.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of TorC protein, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the TorC protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity oftorC or a biologically active portion thereof can be accomplished, for example, by determining the ability of the TorC protein to bind to or interact with a torC target molecule.
  • a "target molecule” is a molecule with which a TorC protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a TorC protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • a torC target molecule can be a non-torC molecule or a TorC protein or polypeptide of the present invention.
  • a torC target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a membrane-bound torC molecule) through the cell membrane and into the cell.
  • the target for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with torC.
  • Determining the ability of the TorC protein to bind to or interact with a torC target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the TorC protein to bind to or interact with a torC target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e.
  • a reporter gene comprising a torC-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • a cellular response for example, cell survival, cellular differentiation, or cell proliferation.
  • an assay of the present invention is a cell-free assay comprising contacting a TorC protein or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the TorC protein or biologically active portion thereof. Binding of the test compound to the TorC protein can be determined either directly or indirectly as described above.
  • the assay comprises contacting the TorC protein or biologically active portion thereof with a known compound which binds torC to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a TorC protein, wherein determining the ability of the test compound to interact with a TorC protein comprises determining the ability of the test compound to preferentially bind to torC or biologically active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting TorC protein or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the TorC protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of torC can be accomplished, for example, by determining the ability of the TorC protein to bind to a torC target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of torC can be accomplished by determining the ability of the TorC protein further modulate a torC target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
  • the cell- free assay comprises contacting the TorC protein or biologically active portion thereof with a known compound which binds torC to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a TorC protein, wherein determining the ability of the test compound to interact with a TorC protein comprises determining the ability of the TorC protein to preferentially bind to or modulate the activity of a torC target molecule.
  • the cell-free assays of the present invention are amenable to use of both the soluble form or the membrane-bound form oftorC.
  • solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton ® X-100, Triton ® X-l 14, Thesit ® , Isotridecypoly(ethylene glycol ether) n , 3-(3-cholamidopropyl)dimethylamminiol-l-propane sulfonate (CHAPS), 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-l-propane sulfonate (CHAPSO), or N-dodecyl— N,N-dimethyl-3 -ammonio- 1 -propane sulfonate.
  • non-ionic detergents such as n-oc
  • binding of a test compound to torC, or interaction of torC with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.
  • GST-torC fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or TorC protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of torC binding or activity determined using standard techniques.
  • torC or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated torC or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with torC or target molecules can be derivatized to the wells of the plate, and unbound target or torC trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the torC or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the torC or target molecule.
  • modulators of torC expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of torC mRNA or protein in the cell is determined.
  • the level of expression of torC mRNA or protein in the presence of the candidate compound is compared to the level of expression oftorC mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator oftorC expression based on this comparison. For example, when expression of torC mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator oftorC mRNA or protein expression.
  • the candidate compound when expression of torC mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of torC mRNA or protein expression.
  • the level of torC mRNA or protein expression in the cells can be determined by methods described herein for detecting torC mRNA or protein.
  • the TorC proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • torC-binding proteins proteins that bind to or interact with torC
  • torC-binding proteins proteins that bind to or interact with torC
  • torC-binding proteins proteins that bind to or interact with torC
  • torC-binding proteins are also likely to be involved in the propagation of signals by the TorC proteins as, for example, upstream or downstream elements of the torC pathway.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for torC is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with torC.
  • a reporter gene e.g., LacZ
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein. Detection Assays
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • this sequence can be used to map the location of the gene on a chromosome.
  • This process is called chromosome mapping.
  • portions or fragments of the torC, sequences, described herein can be used to map the location of the torC genes, respectively, on a chromosome.
  • the mapping of the torC sequences to chromosomes is an important first step in conelating these sequences with genes associated with disease.
  • torC genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the torC sequences. Computer analysis of the torC, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual chromosomes of a given species. Only those hybrids containing the species-specific gene conesponding to the torC sequences will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the torC sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are prefened for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the torC gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the torC sequences of the present invention can also be used to identify individuals from minute biological samples.
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • the sequences of the present invention are useful as additional DNA markers for RFLP ("restriction fragment length polymorphisms," described in U.S. Pat. No. 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the torC sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of conesponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the torC sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
  • SNPs single nucleotide polymorphisms
  • RFLPs restriction fragment length polymorphisms
  • each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences of SEQ ID NO: 1 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining TorC protein and/or nucleic acid expression as well as torC activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with abenant torC expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with TorC protein, nucleic acid expression or activity. For example, mutations in a torC gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with TorC protein, nucleic acid expression or activity.
  • Another aspect of the invention provides methods for determining TorC protein, nucleic acid expression or torC activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (refened to herein as "pharmacogenomics").
  • Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.)
  • Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity oftorC in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of torC in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting TorC protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes TorC protein such that the presence of torC is detected in the biological sample.
  • a compound or an agent capable of detecting TorC protein or nucleic acid e.g., mRNA, genomic DNA
  • An agent for detecting torC mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to torC mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length torC nucleic acid, such as the nucleic acid of SEQ ID NO:l or SEQ ID NO:3, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to torC mRNA or genomic DNA.
  • a full-length torC nucleic acid such as the nucleic acid of SEQ ID NO:l or SEQ ID NO:3, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to torC mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • An agent for detecting TorC protein is an antibody capable of binding to TorC protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect torC mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection oftorC mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of TorC protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immuno fluorescence.
  • In vitro techniques for detection of torC genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of TorC protein include introducing into a subject a labeled anti-torC antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a prefened biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting TorC protein, mRNA, or genomic DNA, such that the presence of TorC protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of TorC protein, mRNA or genomic DNA in the control sample with the presence of TorC protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence oftorC in a biological sample can comprise: a labeled compound or agent capable of detecting TorC protein or mRNA in a biological sample; means for determining the amount of torC in the sample; and means for comparing the amount of torC in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect TorC protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant torC expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with TorC protein, nucleic acid expression or activity in, e.g., neurological conditions such as peripheral neuropathies, e.g., torsin dystonia.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant torC expression or activity in which a test sample is obtained from a subject and TorC protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of TorC protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant torC expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abenant torC expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • a subject can be effectively treated with an agent for a disorder, such as a neurological disorder, e.g. , a peripheral neuropathy such as torsion dystonia.
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with abenant torC expression or activity in which a test sample is obtained and TorC protein or nucleic acid is detected (e.g., wherein the presence of TorC protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant torC expression or activity.)
  • the methods of the invention can also be used to detect genetic lesions in a torC gene, thereby determining if a subject with the lesioned gene is at risk for, or suffers from, a neurological disorder, e.g., a peripheral neuropathy such as torsion dystonia.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a torC-protein, or the mis-expression of the torC gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (1) a deletion of one or more nucleotides from a torC gene; (2) an addition of one or more nucleotides to a torC gene; (3) a substitution of one or more nucleotides of a torC gene, (4) a chromosomal rearrangement of a torC gene; (5) an alteration in the level of a messenger RNA transcript of a torC gene, (6) abenant modification of a torC gene, such as of the methylation pattern of the genomic DNA, (7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a torC gene, (8) a non-wild type level of a torC-protein, (9) allelic loss of a torC gene, and (10) inappropriate post-translational modification of a torC-protein.
  • a prefened biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
  • detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS 91 :360-364), the latter of which can be particularly useful for detecting point mutations in the torC-gene (see Abravaya et al. (1995) Nucl Acids Res 23:675-682).
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a torC gene under conditions such that hybridization and amplification of the torC gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli et al, 1990, Proc Natl Acad Sci USA 87:1874-1878), transcriptional amplification system (Kwoh, et al, 1989, Proc Natl Acad Sci USA 86:1173-1177), Q-Beta Replicase (Lizardi et al, 1988, BioTechnology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a torC gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Pat. No. 5,493,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in torC can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al. (1996) Human Mutation 1: 244-255; Kozal et al. (1996) Nature Medicine 2: 753-759).
  • genetic mutations in torC can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al. above.
  • a first hybridization anay of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear anays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization anay that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation anay is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the torC gene and detect mutations by comparing the sequence of the sample torC with the conesponding wild-type (control) sequence.
  • sequencing reactions include those based on techniques developed by Maxim and Gilbert (1977) PNAS 74:560 or Sanger (1977) PNAS 74:5463.
  • any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve et al, (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publ. No. WO 94/16101; Cohen et al. (1996) Adv Chromatogr 36: 127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the torC gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type torC sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al (1988) Proc Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in torC cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on a torC sequence e.g., a wild-type torC sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Pat. No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in torC genes.
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control torC nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild- type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc Natl Acad Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a torC gene.
  • any cell type or tissue preferably peripheral blood leukocytes, in which torC is expressed may be utilized in the prognostic assays described herein.
  • any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
  • G6PD glucose-6-phosphate dehydrogenase
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations.
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity oftorC can be applied not only in basic drug screening, but also in clinical trials.
  • agents e.g., drugs, compounds
  • the effectiveness of an agent determined by a screening assay as described herein to increase torC gene expression, protein levels, or upregulate torC activity can be monitored in clinical trials of subjects exhibiting decreased torC gene expression, protein levels, or downregulated torC activity.
  • the effectiveness of an agent determined by a screening assay to decrease torC gene expression, protein levels, or downregulate torC activity can be monitored in clinical trials of subjects exhibiting increased torC gene expression, protein levels, or upregulated torC activity.
  • the expression or activity oftorC and, preferably, other genes that have been implicated in, for example, a neurological disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.

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Abstract

L'invention concerne des acides nucléiques codant pour les polypeptides de torsine C (torC) ainsi que des anticorps dirigés contre les polypeptides de TorC et des compositions pharmaceutiques les contenant. Les polypeptides de TorC présentent la même identité de séquence que les polypeptides de la Torsine A et de la Torsine B, lesquels ont été associés aux neuropathies périphériques, telles que la dystonie de torsion.
PCT/US2000/030355 1999-11-04 2000-11-03 Polypeptides et acides nucleiques de torsine c WO2001032875A1 (fr)

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WO2005095609A1 (fr) * 2004-04-02 2005-10-13 Japan Science And Technology Agency Nouveau peptide endogene cardio-inhibiteur/antihypertenseur physiologiquement actif

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1998057984A2 (fr) * 1997-06-19 1998-12-23 The General Hospital Corporation Torsine, genes de torsine et leurs utilisations

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Publication number Priority date Publication date Assignee Title
WO1998057984A2 (fr) * 1997-06-19 1998-12-23 The General Hospital Corporation Torsine, genes de torsine et leurs utilisations

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AUGOOD S ET AL: "Expression of the early-onset torsion dystonia gene (DYT1) in human brain", ANNALS OF NEUROLOGY,US,BOSTON, vol. 43, no. 5, 1 May 1998 (1998-05-01), pages 669 - 673, XP002086934, ISSN: 0364-5134 *
DATABASE EMBL SEQUENCE DATABASE 4 July 1997 (1997-07-04), NCI-CGAP: "National Cancer Institute, Cancer Genome Anatomy Project (CGAP); Tumor gene Index http://www.ncbi.nlm.nih.gov/ncicgap - unpublished", XP002159562 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005095609A1 (fr) * 2004-04-02 2005-10-13 Japan Science And Technology Agency Nouveau peptide endogene cardio-inhibiteur/antihypertenseur physiologiquement actif
US7829526B2 (en) 2004-04-02 2010-11-09 Japan Science And Technology Agency Cardioinhibitory/antihypertensive novel endogenous physiologically active peptide

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