EP1117782A2 - Canaux ioniques, en particulier recepteur de type recepteur de vanilloide (vr-l) - Google Patents

Canaux ioniques, en particulier recepteur de type recepteur de vanilloide (vr-l)

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Publication number
EP1117782A2
EP1117782A2 EP99949193A EP99949193A EP1117782A2 EP 1117782 A2 EP1117782 A2 EP 1117782A2 EP 99949193 A EP99949193 A EP 99949193A EP 99949193 A EP99949193 A EP 99949193A EP 1117782 A2 EP1117782 A2 EP 1117782A2
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EP
European Patent Office
Prior art keywords
nucleic acid
cell
sequence
protein
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP99949193A
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German (de)
English (en)
Inventor
Reynaldo Garcia
John Nicholas Uni. College London WOOD
Steven Pfizer Limited ENGLAND
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Ionix Pharmaceuticals Ltd
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University College London
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Publication of EP1117782A2 publication Critical patent/EP1117782A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to ion channels , particularly those which are responsive , inter alia, to noxious heat , and nucleic acid encoding the same . It further relates to methods and materials for generating and using these .
  • H + -gated channel that is additionally activated by noxious heat and hot peppers is the 'VRl' channel (or 'vanilloid receptor-1 1 ) which was cloned from rat by Caterina et al . (1997) Nature, 389: 816-824. This may play a role in pain pathways .
  • Ion channels are of interest generally, inter alia, for their utility in investigating and manipulating the neurological and physiological processes which they mediate in vivo .
  • novel channels can be used to screen for agonists or antagonists which may exert desirable physiological effects (e.g. analgesics which affect nociception) .
  • proteins or nucleic acids based on the channels themselves may be used directly to manipulate these processes.
  • the present inventors have cloned a novel ion channel from cultured Jurkat cells (a human leukemic T lymphocytes cell line) .
  • the protein functions as a non-selective ion channel and has been shown to be heat-sensing.
  • the ion channel has structural similarities to VR1 sharing 47% identity therewith (based on the number of identical amino acids divided by the total number of amino acids in the region being compared) .
  • the ion channel forming the basis of the present invention has been designated VR-Like or VR-L herein.
  • VR-L resembles members of the "trp" family of proteins in terms of topological organization Trp proteins are 6-transmembrane monomers first identified in Drosophila transient receptor potential (i.e. trp) mutants which shown deficits in photoreception.
  • trp Drosophila transient receptor potential
  • Both VR1 and VR-L have the characteristic N-terminal ankyrin repeats and considerable sequence similarity is also apparent in, but is limited to, the sixth transmembrane domain, its flanking sequences and the loop between transmembrane domains 5 and 6 believed to form part of the presumptive pore region.
  • trp genes provide the molecular basis for the phenomenon known as capacitative calcium entry which is loosely defined as the influx of Ca 2+ from the extracellular space following inositol 1, 4, 5 -triphosphate-induced mobilization of internal stores. It is known that trps play a critical role in phototransduction in Drosophila, and evidence that bradykinin, a hyperalgesic mediator, can gate trp-3 via activation of Gq heterotrimeric G-proteins has been obtained in a heterologous expression system. Studies by the Bargmann group in C. Elegans (Colbert et al . 1997, J Neurosci 17(21:8259-8269; Bargmann et al .
  • VRl itself is a capsaicin-gated channel. Such channels, present in sensory neurons, are believed to mediate the pain caused by acids and possibly other inflammatory mediators (e.g. bradykinin, prostaglandin E2 and 5-HT) which accompany tissue damage and ischaemic conditions. Heterologous expression of VRl induces a capsaicin-sensitive cation channel which is transiently activated by rapid extracellular acidification or capsaicin. The biophysical and pharmacological properties of the VRl channel closely match one of the capsaicin-gated cation channels described in sensory neurons (Helliwell et al , 1998 Neurosci Lett 10;250 (3) .177-180) .
  • VR-L is expressed in sensory neurons and is likely to play a role in mediating the pain and inflammation accompanying tissue damage (nociception) .
  • Other endogenous ligands may also activate the receptor in vivo, either directly, or through G-protein coupled receptors by analogy with other TRP channels above.
  • this new channel is, inter alia, an analgesic or anti-inflammatory drug target.
  • VR-L appears not to be capsaicin- sensitive under the conditions used in the experiments below. Additionally, it appears to have a different distribution to VRl which, for instance, is not present in Jurkat cells. The structural and functional information about VR-L made available by the present inventors suggest that it may have additional utility to VRl.
  • Biro et al (1998) Blood 91: 1332-1340 reported that certain cells of the immune system (mast cell lines) take up calcium in response to capsaicin and RTX (a plant toxin which has similar actions to capsaicin). Biro et al . also showed, in contrast to sensory neurons, capsaicin does not kill mast cells, or even induce their degranulation. However the putative receptor responsible for the demonstrated activity was not characterised.
  • VR-L in subsets of immune system cells suggests an important role as a receptor involved in the regulation of immune responses.
  • the channel also has utility in screening for agents that modulate immune responses .
  • Materials based on VR-L may be used in gene therapy both of disorders associated with sensory neurons (e.g. pain) and leucocytes (e.g. autoimmune disorders, leukemia).
  • disorders associated with sensory neurons e.g. pain
  • leucocytes e.g. autoimmune disorders, leukemia
  • the nucleic acid sequence and encoded protein of VR-L is shown within Fig 3A.
  • the nucleic acid sequence is referred to as Seq ID No 1, while the encoded protein is Seq ID No 2.
  • a non-selective cation channel protein which is activatable by noxious heat.
  • Channel proteins of the present invention may be .obtainable from cells of the immune system, preferably human cells.
  • the channel proteins are not capsaicin sensitive under the conditions described in the Examples below.
  • the channel may also be non-activatable by low pH i.e. not acid sensitive, by which is meant that the cation permeability of the channel is not significantly increased by low pH (increased acidity, or reduced alkalinity) e.g. pH of 4-5, particularly pH 4.6.
  • Proteins, or polypeptides, of the present invention may be provided in recombinantly produced, isolated, enriched or cell-free form. They may be present in cells heterologously, which is to say that they do not naturally occur there, but have been introduced through human intervention.
  • non-selective is meant that the activated channel is permeable to more than one cation, preferably at least Na + , K + , Ca 2+ , preferably having a broadly comparable permeability to each.
  • the protein will have one or more of the electrophysiological and pharmacological characteristics of the VR-L protein described herein.
  • the protein comprises amino acid sequence Seq ID No 2.
  • a VR-L variant protein having ion channel activity and comprising an amino acid sequence having at least about 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% sequence identity with Seq ID No 2.
  • the VRl channel of the prior art shares about 47% identity (56% similarity) with Seq ID No 2.
  • Ion channel activity may be tested as described above, using appropriate stimuli if required.
  • Similarity may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J " . Mol . Biol . 215: 403-10, which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman.
  • Parameters are preferably set, using the default matrix, as follows :
  • Gapopen (penalty for the first residue in a gap) : -12 for proteins / -16 for DNA
  • Gapext (penalty for additional residues in a gap) : -2 for proteins / -4 for DNA
  • KTUP word length 2 for proteins / 6 for DNA.
  • Homology may be over the full-length of the relevant sequence shown herein, or may be over a part of it, preferably over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 300, 400, 500, 600, 700 or more amino acids or compared with Seq ID No 2.
  • a variant polypeptide in accordance with the present invention may include within the sequence shown in Seq ID No 2, a single amino acid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes.
  • a variant polypeptide may include additional amino acids at the C- terminus and/or N-terminus. Alternatively it may represent an active (as an ion channel) fragment of the protein e.g. a pore forming fragment .
  • Proteins or polypeptides of the present invention may be prepared by the expression of nucleic acids encoding therefor in appropriate host cells, as described in more detail hereinafter.
  • nucleic encoding a protein of the invention as described above .
  • Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid). Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.
  • Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term “isolated” encompasses all of these possibilities.
  • the nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e.g. using an automated synthesiser.
  • nucleic acid encoding VR-L e.g. a nucleic acid encoding Seq ID No 2 (which nucleic acid sequence may be that set out within Seq ID No 1) .
  • sequence degeneratively equivalent to that sequence there is disclosed.
  • nucleic acid which encodes a VR-L variant as described above.
  • this will share homology or identity with the VR-L encoding sequence in similar terms as are described in relation to the variant proteins above, but wherein amino changes correspond to changes in codons or individual nucleotides.
  • Variants may include distinctive parts or fragment (however produced) corresponding to a portion of the sequence provided.
  • the fragments may encode particular functional parts of the polypeptide.
  • fragments may have utility in probing for, or amplifying, the sequence provided or closely related ones. Suitable lengths of fragment, and conditions, for such processes are discussed in more detail below.
  • nucleic acids which have been extended at the 3 ' or 5 ' terminus with respect any of these embodiments .
  • Preferred variant nucleic acids are those which, in addition to encoding VR-L variants, are capable of hybridizing with a poly- or oligonucleotide having a sequence which is complementary to a distinctive portion of SEQ ID No 1 under low stringency conditions, more preferably being capable of hybridizing with one or more of such poly- or oligonucleotides under high stringency conditions.
  • the screening is carried out at about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5 X SSC, or a temperature of about 50°C and a salt concentration of about 2 X SSPE.
  • T m 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42 °C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS , 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS.
  • suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.
  • Sequence variants which occur naturally may include alleles (which will include polymorphisms or mutations at one or more bases) or pseudoalleles . Also included within the scope of the present invention would be isogenes, or other homologous genes from other species sharing similarity with SEQ ID No 1.
  • a method of identifying and/or cloning a nucleic acid according to the present invention i.e. encoding an ion channel as described above
  • which method employs Seq ID No 1 or a distinctive fragment or region thereof.
  • 'distinctive' is meant based on a region which is not present in VRl or other trp proteins .
  • the sequence information in the region can be used directly to identify or clone variants, or be used to identify corresponding regions in a data-base (e.g. of expressed sequence tags, or sequence tagged sites), optionally from a different species.
  • VR-L homologues in both rat and mouse have been identified. Portions of the encoded amino acid sequences are shown in Fig 4.
  • a variant in accordance with the present invention is also obtainable by means of a method which includes :
  • nucleic acid e.g. from cells of the immune system, or from sensory neurons
  • nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and identifying said gene or homologue if present by its hybridisation with said nucleic acid molecule.
  • Probing may optionally be done by means of so-called “nucleic acid chips' (see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review) .
  • Nucleic acids of the invention may be amplified from template DNA from cells using a specific DNA amplification reaction with specific primers targeted to amplify the DNA required, e.g. of SEQ ID No 1, e.g. from genomic DNA, DRG cDNA, or mRNA templates, e.g. by using polymerase chain reaction or, from RNA, by using reverse transcription (RT) followed by polymerase chain reaction (PCR) (see “PCR protocols; A Guide to Methods and Applications", Eds. Innis et al , Academic Press, New York, 1990) .
  • SEQ ID No 1 e.g. from genomic DNA, DRG cDNA, or mRNA templates
  • RT reverse transcription
  • PCR polymerase chain reaction
  • a method involving use of PCR in obtaining nucleic acid according to the present invention may include:
  • nucleic acid e.g. from DRG, or other appropriate tissue or organ
  • clones or fragments identified in the search can be extended. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, mRNA preparation etc.) can be revisited to isolate missing portions e.g. using sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.
  • the original DNA source e.g. a clone library, mRNA preparation etc.
  • Distinctive fragments or oligonucleotides for use in probing or PCR may be selected prepared by those skilled in the art without burden in the light of the present disclosure. Typically they may be about 10 to 30 or fewer nucleotides in length (e.g. 18, 21 or 24). Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-24 nucleotides in length may be preferred. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100' s (e.g 500 or more) or even 1000 's of nucleotides in length.
  • oligonucleotides and their complements (but excluding any oligonucleotides consisting solely of regions disclosed per se in the form of EST clones, or other portions of undefined function) form one part of the present invention.
  • variants may be prepared by those skilled in the art on the basis of the sequences provided herein, for instance by site directed or random mutagenesis, or by direct synthesis.
  • the variant nucleic acid is generated either directly or indirectly (e.g. via one or more amplification or replication steps) from an original nucleic acid having all or part of the sequence shown in Seq ID No 1.
  • Changes to a sequence, to produce a derivative may be by way of one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.
  • Changes may be desirable for a number of reasons, including introducing or removing the following features: restriction endonuclease sequences; codon usage; other sites which are required for post translation modification; cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for post-translational modification.
  • Leader or other targeting sequences e.g. membrane or golgi locating sequences
  • All of these may assist in efficiently cloning and expressing an active polypeptide in recombinant form.
  • Other desirable mutation may be random (e.g. chemical) or site directed mutagenesis in order to alter the activity (e.g. specificity) or stability of the encoded polypeptide.
  • a vectorised DNA of SEQ ID No 1 is exposed to a mutagenic material such as hydroxylamine, or, in the case of SDM, a PCR reaction is carried out on that DNA using a mutagenic primer, whereby DNA is produced which encodes for a protein different in sequence to SEQ ID No 2 at a few predictable or predetermined sites respectively.
  • Particular target areas of the sequence are those which may determine any of the following: specificity of activation of the ion channel (e.g. whether or not a given stimulus, such as pH, elicits a response) or specificity of the ion channel (e.g. relative permeability to different cations).
  • specificity of activation of the ion channel e.g. whether or not a given stimulus, such as pH, elicits a response
  • specificity of the ion channel e.g. relative permeability to different cations.
  • amino acid sequence differs from SEQ ID No 2 only by conservative substitutions.
  • the expression ⁇ conservative substitutions' as used with respect to amino acids relates to the substitution of a given amino acid by an amino acid having physicochemical characteristics in the same class .
  • an amino acid in SEQ ID No 2 has a hydrophobic characterising group
  • a conservative substitution replaces it by another amino acid also having a hydrophobic characterising group; other such classes are those where the characterising group is hydrophilic, cationic, anionic or contains a thiol or thioether.
  • substitutions are well known to those of ordinary skill in the art (see e.g. US 5380712) and are only contemplated where the resultant protein has ion channel activity.
  • variants having non-conservative substitutions are also included. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide 's three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide.
  • the nucleic acids of the present invention are in the form of a recombinant and preferably replicable constructs, such as vectors.
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage or other vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) .
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e.g. mammalian, yeast or fungal cells) .
  • a vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome .
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial cell.
  • the vector may be a bi- functional expression vector which functions in multiple hosts. In the case of genomic VR-L DNA (isolatable as described above) this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell .
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) .
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention, such as the VR-L gene (see Seq ID NO 1) or a variant thereof .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • this aspect of the present invention provides a gene construct, preferably a replicable vector, comprising an inducible promoter operatively linked to a nucleotide sequence provided by the present invention, such as Seq ID No 1.
  • inducible as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on” or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.
  • vectors suitable for expression of mammalian DNA such as will occur to those skilled in the art, e.g. HSV or vaccinia vectors, or pcDNA3 shuttle vectors, e.g. as included within the lambda express system (Stratagene) , which are capable of expressing heterologous protein in both bacteria and in eucaryotic cells such as COS cells.
  • Suitable bacterial vectors will include lambda-Zap vectors such as the lambda-Zap-II vector available from Stratagene Cloning Systems.
  • Bacterial clones containing plasmids capable of gene expression can be obtained by excising pBluescript from the lambda-Zap-II construct in the presence of a filamentous helper phage also available from Stratagene. Typical protocols are provided in the examples below, in Stratagene kit inserts.
  • a further aspect of the present invention provides cells containing, or more preferably transformed with (or transfected with) the nucleic acids of the present invention.
  • Such cells are provided by transformation of a host cell, preferably a eucaryotic cell, e.g. a COS, CHO or HEK 293 cell or an oocyte, preferably a Xenopus oocyte, particularly COS cells, using DNA of the invention as incorporated by recombinant DNA techniques into a vector or as directly incorporated into the cells' genomic DNA e.g. by electroporation or other such DNA integrating technique.
  • a host cell preferably a eucaryotic cell, e.g. a COS, CHO or HEK 293 cell or an oocyte, preferably a Xenopus oocyte, particularly COS cells
  • Such cells may be capable of expressing, or having expressed, a VR-L protein as described above.
  • the cells may thus mimic, in some respects, the electrophysiological and pharmacological properties of native VR-L-expressing cells
  • RNA of the present invention it is also possible to produce cells bearing the receptor protein of the invention by direct injection of RNA of the present invention into the cells wherein it becomes translated.
  • down-regulation of expression of a target gene may be achieved using anti-sense technology.
  • a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene.
  • Rothstein et al 1987; Smith et al , (1988) Nature 334, 724-726; Zhang et al , (1992) The Plant Cell 4, 1575-1588, English et al . , (1996) The Plant Cell 8, 179-188.
  • Antisense technology is also reviewed in Bourque, (1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.
  • “Complementary to” means capable of base pairing with, whereby A base pairs with T (and U) ; G base pairs with C.
  • the "complement" of a reference nucleic acid consists of a sequence of nucleotides which are the counterpart of the entire nucleotide sequence of that reference nucleic acid.
  • An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression.
  • ribozymes e.g. hammerhead ribozymes, which can catalyse the site-specific cleavage of RNA, such as mRNA (see e.g. Jaeger (1997) “The new world of ribozymes” Curr Opin Struct Biol 7:324-335, or Gibson & Shillitoe (1997) “Ribozymes : their functions and strategies form their use” Mol Biotechnol 7: 242-251.)
  • RNA such as mRNA
  • Anti-sense or sense or ribozyme based regulation may be performed using vectors as described above, and may itself be regulated by employing an inducible promoter in an appropriate construct. For instance incorporation of this DNA into mammalian cells might be readily accomplished using vectors, e.g. such as HSV, vaccinia or adenovirus (see Principles of Gene Manipulation (1994) 5th Edit. Old and Primrose 5th Edition, Blackwell Scientific Publications) .
  • the complete sequence corresponding to the coding sequence need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of anti -sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes, such as a regulatory sequence .
  • the sequence employed may be about 500 nucleotides or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than about 500 nucleotides are preferable where possible, such as longer than about 600 nucleotides, than about 700 nucleotides, than about 800 nucleotides, than about 1000 nucleotides or more.
  • sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a variant of such a sequence.
  • the sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene. Effectively, the homology should be sufficient for the down-regulation of gene expression to take place .
  • One embodiment of this aspect of the invention employs DNA oligonucleotides, typically being of 10 to 30 bases long, conveniently about 20 bases long, optionally in degradation protected form, e.g. by being thiolated, and which conveniently have been chemically synthesized to be directed to hybridize with a part of the 5' coding region of the VR-L mRNA.
  • Annealing with the oligomeric DNA causes the mRNA to be degraded by activation of RNase H, or blocks the translation of the mRNA into protein.
  • the small size of such oligomers facilitates their direct access into target cells which express the present VR-L proteins.
  • An alternative embodiment produces antisense RNA in vivo by inserting a tissue specific inducible or constitutively active promoter, enhancer or locus control region or element upstream of the coding region, or part of the coding region, of DNA of SEQ ID No 1 in a construct which is then cloned into a vector.
  • a tissue specific inducible or constitutively active promoter, enhancer or locus control region or element upstream of the coding region, or part of the coding region, of DNA of SEQ ID No 1 for use in mammals in therapy such a vector should be capable of infecting but not killing target cells.
  • Convenient vectors for use in this embodiment which can target mammalian dorsal root ganglion cells are Herpes Simplex Virus (HSV) vaccinia or adenovirus derived vectors .
  • HSV Herpes Simplex Virus
  • Viral vectors for use in gene therapy are discussed by Vile (1997) Nature Biotechnology 15: 840-841.
  • the antisense downregulating DNA or RNA is provided in dorsal root ganglia cells it potentially inhibits the pain response by actually decreasing the number of VR-L channels on the surface of sensory cells.
  • the antisense downregulating DNA or RNA is provided in the immune system, it alters leucocyte function by actually decreasing the number of VR-L channels on the surface of neurons.
  • nucleotide sequence which is complementary to any of the coding-nucleic acids discussed in relation to earlier aspects of the invention forms one part of the present invention.
  • the invention further provides a method of influencing the electrophysiological and pharmacological properties of a cell, said method comprising the step of causing or allowing expression of a heterologous nucleic acid sequence as discussed above within the cell .
  • the present invention further provides the use of the nucleotide sequence of Seq ID No 1, or its complement, or a variant of either for down-regulation of gene expression, particularly down-regulation of expression of an ion channel - encoding gene, more preferably a cation channel, most preferably VR-L.
  • nucleic acid e.g. antisense DNA
  • nucleic acid of the present invention for use gene therapy, or for use in the preparation of medicaments for use in gene therapy.
  • an organism preferably a non-human mammal, comprising cells in which the activity of VR-L or a variant thereof have been altered, preferably impaired, by use of the methods and materials discussed above.
  • a rodent e.g. murine organism.
  • Methods of producing 'knock out' mammals in which specific gene activities have been impaired are now well known to those skilled in the art - see e.g. Boerrigter et al (1995) Nature 377: 657-659, or Gossen and Vijk (1993) Trends Genet 9: 27-31.
  • Such substances may, for instance, act as agonist, partial agonist or antagonist .
  • the protein or cell may be used in a method comprising exposing the protein (e.g which is associated with a membrane, for instance of a liposome) or cell surface to a solution of the substance such as to allow interaction between the substance and the VR-L (or variant VR-L) protein in the membrane and then measuring the electrophysiological response of the cell or membrane to this interaction. This measurement may optionally be compared with a reference figure.
  • exposing the protein e.g which is associated with a membrane, for instance of a liposome
  • a solution of the substance such as to allow interaction between the substance and the VR-L (or variant VR-L) protein in the membrane
  • This measurement may optionally be compared with a reference figure.
  • the response may be measured by use of a microelectrode technique accompanied by such measurement strategies as voltage clamping of the cell whereby activation of ion channels may be identified by inward or outward current flow as detected using the microelectrodes .
  • 2 Na, 86 Rb, 45 Ca radiolabeled cations or 14 C or 3 H guanidine may be used to assess such ion flux;
  • a sodium, calcium or potassium ion sensitive dye such as Fura-2, or indo
  • a potential sensitive dye may be used to monitor potential changes, e.g. such as in depolarization.
  • Agonists and partial agonists may be identified by their relative efficacy as compared with other known agonists and stimuli in activating the receptor or, in the case of partial agonists and antagonists, by their ability to block the activation caused by other known agonists.
  • Such substances will have potential as analgesics or other immuno- or neuro-modulatory agents, and as such form a further aspect of the present invention, optionally in the form medicamanents (e.g. compositions comprising a pharmaceutically acceptable carrier or filler) .
  • medicamanents e.g. compositions comprising a pharmaceutically acceptable carrier or filler
  • Purified VR-L protein or a variant thereof, e.g. produced recombinantly by expression from encoding nucleic acid therefor, may be used to raise antibodies employing techniques which are standard in the art .
  • Antibodies and other polypeptides comprising antigen-binding fragments of antibodies may be used (inte-r alia) as antagonists or inhibitors, or for use in identifying homologues .
  • Methods of producing antibodies include immunising a mammal with the protein or a fragment thereof.
  • Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest.
  • Antibodies may be polyclonal or monoclonal.
  • Antibodies may be modified in a number of ways. Indeed the term “antibody” should be construed as covering any specific binding substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of Chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023. It has been shown that fragments of a whole antibody can perform the function of binding antigens.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VI and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.
  • Fig 1 'virtual transcript' of VR-L based on overlapping human EST clones. Analogous positions in VRl from where primers were designed are indicated by numbers above the map. Primers and their orientations are indicated by arrowheads. Dashed vertical lines flank gaps not represented by any EST sequence. The 5 ' untranslated region (UTR) and the regions surrounding the stop codon were deduced from results of tentative human consensus sequence queries of the human genome index.
  • Fig 2 generation of full length sequence from PCR products by trans-PCR (Fig 2A) or utilisation of unique restriction site (Fig 2B) .
  • Fig 3A VR-L nucleotide and amino acid sequences.
  • Fig 3B amino acid sequence alignment of rat VRl (upper) and human VR-L (lower) .
  • Fig 4 multiple sequence alignment of rat VRl with mouse, rat and human (Jurkat cell) VR-L in the transmembrane 5/6 region.
  • Fig 5 whole cell voltage clamp recordings of COS-7 cells transfected with VR-L.
  • Fig 6 Table 1; EST clones showing homology to rat vanilloid receptor subtype 1 (VRl)
  • Fig 7 Table 2; primers used for cloning VR-L fragments.
  • a tblastn search of the Genbank dbEST identified at least 30 human and mouse entries (Fig 6, Table 1) showing considerable homology to different regions of the rat VRl amino acid sequence.
  • the overlapping human EST clones were assembled into a virtual transcript (Fig. 1) , albeit interrupted by gaps.
  • the contigs formed represent sequences which were broadly analogous to positions 86 to 763 of the rat VRl amino acid sequence . Since the EST clones were obtained from different cDNA libraries, and hence the contigs may comprise ESTs derived from different tissues, there was no way of knowing whether or not the 'transcript' was real.
  • RNA derived from Jurkat cells (a leukemic T cell line; and from where an EST clone was derived) was used as a template for reverse transcription. Sequencing of PCR products confirmed that the individual entries were one and the same transcript truncated at various points during reverse transcription.
  • HGI Human Gene Index
  • Fig. 3b shows a comparison of the human VR-L amino acid sequence with that of rat VRl.
  • VR-L-specific PCR primers based on a mouse EST clone (GenBank accession no. AA476107) were used to amplify VR-L from rat dorsal root ganglia cDNA from which VRl was isolated. Sequencing revealed a transcript similar to but distinct from VRl, confirming that there are at least two vanilloid receptor subtypes in the rat genome. An alignment of the rat VRl with the rat, mouse and human VR-L sequences in this region is shown in Fig. 4. RT-PCR and Subcloning Procedures
  • primer random hexamers or gene-specific oligonucleotides
  • the mixture was then incubated at 37°C for 2 min in 1 x buffer [50mM Tris-HCl (pH 8.3), 75 mM KC1 , 3mM MgCl , 10 mM dithiothreitol and 125 mM of each dNTP (dATP, dCTP, dGTP and dTTP) .
  • Superscript II reverse transcriptase 200 U was added to a final volume of 20 ⁇ l and the mixture was incubated for 60 minutes at 37°C. The reaction was stopped by a 5- minute incubation at 94°C.
  • reaction were carried out using 1 ⁇ l of first strand cDNA, 40 pmol of each primer, lx buffer [50 mM KC1, lOmM Tris-HCl pH9.0 , 0.1% Triton X-100 ], 1.5 mM MgCl2, 0.2mM dNTPs and 1U of Taq polymerase in a final volume of 20 ⁇ l .
  • the amplification program used included 1 cycle of 94 °C for 5 min, 53-57°C for 1 min, 72°C for 1-2 min; 35 cycles of 94°C for 1 min, 53-57°C for 45 sec, 72°C for 1-2 min; and a final extension at 72 °C for 5 min.
  • Gibco-BRL's eLONGase enzyme mix (a mixture of Taq polymerase and the proofreading Pyrococcus species GB-D thermostable DNA poly ⁇ nerases) was used in a 20 ⁇ l reaction including 1 ⁇ l first strand cDNA, 40 pmol of each primer, lx buffer [60 mM Tris-S0 4 (pH9.1), 18 mM (NH 4 ) 2 S0 4 with MgS0 4 between 1.5 to 1.9 mM] and 0.2 mM dNTPs .
  • Cycling conditions were: 1 cycle of 94°C for 3 min, 55-57°C for 1 min, 68°C for 45 sec to 2 mini 30-35 cycles of 94°C for 30 sec, 55-57°C for 30 sec, 68°C for 45 sec to 2 min; and a final extension at 68°C for 5 minutes.
  • PCR products were cloned into the vector pGem-T Easy (Promega) and sequenced using standard procedures. For blunt-ended PCR products generated by eLONGase, a 10 minute incubation with 1U Taq polymerase after the PCR reaction was necessary to add an "A" overhang necessary for TA-cloning into the pGem-T Easy vector.
  • the larger PCR products were spliced together to obtain the full-length VR-L clone, either by trans-PCR (splicing by overlap extension) or conventional ligation involving a unique restriction site (see Figs. 2a and 2b) .
  • the full-length clone was then transferred into a mammalian expression vector (pGW-1) and checked for correct orientation.
  • the resulting recombinant clone was subsequently used for transfection of COS-7 cells by electroporation.
  • Shuttle vectors e.g. pTracer-CMV or pgwl, containing VR-L were propagated and purified from bacterial cultures transformed with the recombinant plasmid. These vectors were used to express ion channels by transfecting COS cells. Cultured COS cells from a 100 mm petri dish (80- 90% confluent) were trypsinised and resuspended in 350 microlitres of ice cold HEBS buffer. 20-30 micrograms of plasmids of interest were dissolved with 150 microlitres of HEBS buffer, then mixed with the COS cell suspension in an electroporation cuvette and kept on ice to cool for 5 minutes.
  • the electroporator (Invitrogen) was set up at 250 microFarad, and charged for 3 minutes at 330V, 25mA, and 25W.
  • the cuvette was flicked to resuspend cells and electroporation effected.
  • COS cells were seeded in low density onto 35mm petri dishes and cultured with 2ml MEM/10% FCS at 37°C for 1-2 days.
  • Membrane currents were recorded using an Axopatch 200B amplifier. Currents were low-pass filtered at 5kHz (4- pole Bessel filter) , and digitized using a Digidata 1200 interface. Acquisition and analysis of currents was achieved using pClamp ⁇ software. Pipettes were filled with an intracellular solution containing (in mM) ; KCl 120, NaCl 8, MgCl2 3, HEPES 40, and BAPTA 10, at pH 7.35. Recordings were made at room temperature (18-22°C) .
  • the extracellular recording solution was composed of (in mM) ; NaCl 146; KCl 5; Glucose 10; MgCl 2 1, CaCl 2 0.01.
  • lOmM HEPES was used as the buffer, whilst for solutions of pH 6.5-4.0, MES at a concentration of lOmM was used.
  • VR-L forms a functional channel when transiently expressed in COS-7 cells. Despite the homology of VR-L to VRl, the channel does not appear to be gated by capsaicin or low pH.
  • Figure 5 shows responses from a typical cell. Application of 10 ⁇ M capsaicin to this cell, at a holding potential of -60mV, failed to evoke a change in membrane current
  • Example 4 Screenincr for ion channel modulating- agents
  • Compounds to be assessed as agonists, partial agonists or antagonists of the VR-L channels may be bath applied in the system above and inward current used as measurement of the activation or block of channels encoded by the transfected vectors.
  • VR-L permanently or transiently transfected cell lines expressing VR-L are used to measure increases in intracellular calcium in response to the stimuli discussed in Example 3 with or without the addition of compounds that may show agonist, partial agonist or antagonist activity.
  • Changes in intracellular calcium are measured using cells in multiwell plates using Ca-45 uptake, for instance using the method of Wood et al (1989) J Neurochem 53: 1203-1211, or activation of calcium sensitive dyes (e.g. Fura-II) as described in Zeilhofer et al (1996) J Neurophysiol 76(5): 2834-40 or Hansen et al (1998) Am J Physiol 274(6 Pt 1): C1552-62.
  • calcium sensitive dyes e.g. Fura-II

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Abstract

L'invention concerne des protéines de canal cationique isolées non sélectives qui: (a) peuvent être activées par une chaleur nocive; (b) peuvent provenir de lymphocytes T humains; (c) ne sont pas sensibles à la capsicine, notamment la protéine de VR-L (Seq. ID N° 2), outre ses variants (par exemple homologues ou fragments). L'invention concerne également des acides nucléiques codant pour ces protéines (par exemple Seq. ID N° 1) ou utiles pour l'exploration, l'amplification ou la régulation négative de ces protéines. L'invention concerne aussi des méthodes de production de ces protéines et acides nucléiques, des vecteurs, cellules hôtes et organismes les utilisant, ainsi que des anticorps. L'invention concerne enfin des méthodes permettant d'influencer les propriétés électrophysiologiques et/ou pharmacologiques d'une cellule (par exemple par voie thérapeutique) sur la base de la manipulation des protéines ou acides nucléiques et des méthodes de criblage de substances possédant une activité de modulation de canal ionique (pouvant être des analgésiques potentiels ou d'autres composés affectant la nociception, des agents immunomodulateurs ou des agents neuromodulateurs).
EP99949193A 1998-10-09 1999-10-08 Canaux ioniques, en particulier recepteur de type recepteur de vanilloide (vr-l) Withdrawn EP1117782A2 (fr)

Applications Claiming Priority (3)

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GB9822124 1998-10-09
GBGB9822124.5A GB9822124D0 (en) 1998-10-09 1998-10-09 Ion channels
PCT/GB1999/003348 WO2000022121A2 (fr) 1998-10-09 1999-10-08 Canaux ioniques, en particulier recepteur de type recepteur de vanilloide (vr-l)

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GB9827016D0 (en) * 1998-12-08 1999-02-03 Merck Sharp & Dohme Receptor protein
GB9907097D0 (en) 1999-03-26 1999-05-19 Novartis Ag Organic compounds
DE10043674A1 (de) * 2000-09-02 2002-03-21 Gruenenthal Gmbh Antisense Oligonukleotide
WO2002018407A2 (fr) * 2000-09-02 2002-03-07 Grünenthal GmbH Oligonucleotides antisens contre vr1
GB2377445A (en) * 2001-04-20 2003-01-15 Smithkline Beecham Plc Ion channel vanilloid receptor, VANILREP7
JP2002372530A (ja) * 2001-06-14 2002-12-26 Shiseido Co Ltd 皮膚刺激作用の検出方法
WO2004045638A1 (fr) * 2002-11-18 2004-06-03 Merck Sharp & Dohme Limited Ligands du recepteur vanilloide de type 2 pour traiter l'anxiete ou la depression

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DE69910538T2 (de) * 1998-01-22 2004-06-24 The Regents Of The University Of California, Oakland Nukleinsäuresequenzen, welche für capsaicin-rezeptoren kodieren
WO1999037765A1 (fr) * 1998-01-27 1999-07-29 Smithkline Beecham Plc Homologues du recepteur humain des vanilloides
EP0953638A1 (fr) * 1998-03-11 1999-11-03 Synthelabo Canal cationique d'origine humaine homologue au récepteur vanilloide

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See references of WO0022121A2 *

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WO2000022121A2 (fr) 2000-04-20

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