WO2002096944A2 - Nouveaux canaux potassiques heterotetrameres et utilisations de ces derniers - Google Patents

Nouveaux canaux potassiques heterotetrameres et utilisations de ces derniers Download PDF

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WO2002096944A2
WO2002096944A2 PCT/EP2002/006082 EP0206082W WO02096944A2 WO 2002096944 A2 WO2002096944 A2 WO 2002096944A2 EP 0206082 W EP0206082 W EP 0206082W WO 02096944 A2 WO02096944 A2 WO 02096944A2
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potassium channel
heterotetrameric
subunit
voltage
subunits
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WO2002096944A3 (fr
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Dirk J. Snyders
Natacha Ottschytsch
Adam Raes
Diane Van Hoorick
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Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
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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
    • 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
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • New heterotetrameric potassium channels and uses thereof are described.
  • the present invention relates to new voltage-gated heterotetrameric potassium channels comprising Kv2.1 , Kv3.1 , Kv5.1 , Kv6.3, Kv10.1 or Kv11.1 potassium channel subunits and their direct or indirect uses for diagnosing, preventing and/or treating excitability disorders. Moreover, the present invention discloses the cDNA sequence of the Kv10.1 potassium channel subunit.
  • Voltage-gated potassium channels are transmembrane proteins consisting of four subunits, which tetramerize to form a central permeation pathway. Each subunit consists of six transmembrane domains (S1-S6) and a pore loop containing the GYG-motif, a signature sequence for potassium selectivity. The fourth transmembrane domain (S4) is positively charged and is the major part of the voltage sensor. Voltage-gated potassium channels serve a wide range of functions including regulation of the resting membrane potential and control of the shape, duration and frequency of action potentials [1]. At present, 26 genes have been described encoding for different Kv ⁇ -subunits divided into subfamilies by sequence similarities.
  • Kv family of potassium channels consists of nine subfamilies, Kv1 through Kv9, although Kv7 has only been described for Aplysia [11].
  • the members of the Kv1 through Kv4 subfamilies all show functional expression in a homotetrameric configuration.
  • the subunits of the Kv5, Kv6, Kv8 and Kv9 families cannot generate current by themselves, despite having the typical topology of voltage-gated potassium channels [2-5,8].
  • Kv6.1 fails to form homotetrameric channels, but it is able to form heterotetrameric channels with Kv2.1 ; expression of these heterotetramers revealed a current with clearly distinguishable properties [6].
  • the 'electrically silent' subunits have been shown to form heterotetrameric channels with the members of the Kv2 subfamily [4,5,8]. In a sense, these 'silent' subunits can be considered as regulatory subunits.
  • Patel and colleagues showed that the metabolic regulation of the Kv2.1/Kv9.3 heteromultimer might play an important role in hypoxic pulmonary artery vasoconstriction and in the possible development of pulmonary hypertension [5].
  • the present invention relates to the cloning and characterization of three voltage- gated potassium channel subunits, that were identified in the human genome: Kv6.3, Kv10.1 and Kv11.1. Although they have all the hallmarks of voltage-gated K + -channel subunits, they do not produce K + currents when expressed in mammalian cells. Yeast two-hybrid and co-immunoprecipitation experiments show that they do not form homotetrameric channels but that, surprisingly, they do form heterotetrameric channels with Kv2.1 , Kv3.1 and/or Kv5.1.
  • Fig. 2 Sequence alignment, phylogenetic tree and percent sequence identity of Kv6.3, Kv10.1 and Kv11.1.
  • A The amino acid sequence of Kv2.1 , Kv6.1 , Kv6.2, Kv6.3, Kv10.1 and Kv11.1 were aligned using MEGALIGN. For convenience, only the first 460 amino acids of Kv2.1 are shown. Gaps (indicated by dashes) were introduced in the sequence to maintain the alignment. conserveed amino acids are shaded in gray. The six putative transmembrane domains and the pore region are indicated by an overline.
  • B The proposed phylogenetic tree for the Kv family shows that Kv6.3, Kv10.1 and Kv11.1 are closer to the electrically silent channels than to the functional channels.
  • FIG. 3 Expression of Kv6.3, Kv10.1, Kv11.1 and Kv2.1 in human tissues.
  • a PCR was performed on a cDNA panel of human tissues with gene specific primers.
  • FIG. 4 Whole cell current recordings of Kv6.3, Kv10.1, Kv11.1 and the co- transfections with Kv2.1
  • Top panels show the typical recordings of A, not transfected Ltk " cells B, Kv6.3 C, Kv10.1 D, Kv11.1.
  • the holding potential was -80mV and cells were depolarized by 20mV increments from -80mV to +60mV, 500ms in duration, followed by a repolarizing puls at -25mV, 850ms in duration.
  • Bottom panels show typical recordings of A, Kv2.1 B, Kv2.1 + Kv6.3 C, Kv2.1 + Kv10.1 D, Kv2.1 + Kv11.1 in Ltk " cells.
  • the holding potential was -80mV and cells were depolarized by 10mV increments from -60mV to +70mV, 500ms in duration. Deactivating tails were recorded at -25mV or -35mV for 850ms.
  • Fig. 5 A. Activation curves. The activation curves of Kv2.1 , Kv2.1 + Kv6.3, Kv2.1 + Kv10.1 and Kv2.1 + Kv11.1 were obtained from the normalized tail amplitude recorded at a 1 sec test pulse of -25mV for Kv2.1 , Kv2.1 + Kv10.1 and Kv2.1 + Kv11.1 and of -50mV for Kv2.1 + Kv6.3 after prepulses ranging from -60mV to 70mV in 10mV steps, 500ms in duration. Experimental data were fitted with a Boltzmann function.
  • the inactivation curves of Kv2.1 , Kv2.1 + Kv6.3, Kv2.1 + Kv10.1 and Kv2.1 + Kv11.1 were obtained from the normalized peak currents recorded at a 250ms test pulse to 50mV as a function of the 5- sec prepulse ranging from -50mV to 10mV for Kv2.1 , Kv2.1 + Kv10.1 and Kv2.1 + Kv11.1 and from -80mV to -20mV for Kv2.1 + Kv6.3.
  • Experimental data were fitted with a Boltzmann function.
  • C Kinetics of activation and deactivation of Kv2.1 and Kv2.1 + Kv6.3.
  • Mean time constants SE of activation and deactivation are plotted on a linear scale as a function of a range of test potentials (- 100mV to 70mV).
  • test pulses ranging from -10mV to 70mV for Kv2.1 and -30mV to 70mV for Kv2.1 + Kv6.3 in 10mV steps, 500ms in duration, were applied.
  • time constants for deactivation a 200ms prepulse to 50mV followed by test pulses ranging from -20mV to -100mV in 10mV steps, 850ms in duration, were applied.
  • the experimental data were fitted with mono- or double exponential functions, as appropriate. D.
  • Kv2.1, Kv2.1 + Kv10.1 and Kv2.1 + Kv11.1 Mean time constants SE of activation are plotted on a linear scale as a function of a range of test potentials (- 10mV to 70mV).
  • the pulse protocol for Kv2.1 + Kv10.1 and Kv2.1 + Kv11.1 is the same as for Kv2.1 alone in figure 5.
  • Fig. 6 Interaction of Kv6.3, Kv10.1 and Kv11.1 with representative subunits of all Kv subfamilies, determined using the yeast two-hybrid method.
  • the intracellular aminoterminal segment that contains the subfamily specific NAB domain was used as bait and/or target.
  • Fig. 7 Alignment of the S6 segment of the Kv potassium channels. One member of each subfamily is represented. conserveed amino acids are shaded in gray. Sequence numbering of Kv2.1 is shown on top.
  • Fig. 8 Co-immunoprecipitation of Kv6.3GFP, Kv10.1GFP and Kv11.1GFP with Kv2.1c yc. Immunoprecipitation was done with anti-GFP antibodies, western blot was performed with anti-c-myc. Lanes 3, 4 and 5 show that Kv2.1c-myc was co- precipitated with Kv6.3GFP, Kv10.1GFP and Kv11.1GFP. GFP tagged Kv2.1 (lanel) and Kv1.5 (Iane2) were used as positive and negative controls, respectively.
  • Kv10.1 refers to a polypeptide that is a subunit or monomer of a voltage-gated potassium channel, a member of the Kv10 family, and a member of the Kv superfamily of potassium channel monomers.
  • the nucleic acid sequence of Kv6.3 is depicted in SEQ ID NO: 1, the protein sequence of Kv6.3 is depicted in SEQ ID NO: 2.
  • the nucleic acid sequence of Kv10.1 is depicted in SEQ ID NO: 3, the protein sequence of Kv10.1 is depicted in SEQ ID NO: 4.
  • the nucleic acid sequence of Kv11.1 is depicted in SEQ ID NO: 5, the protein sequence of Kv11.1 is depicted in SEQ ID NO: 6.
  • the nucleic acid sequence of Kv2.1 is depicted in SEQ ID NO: 7, the protein sequence of Kv2.1 is depicted in SEQ ID NO: 8.
  • the nucleic acid of Kv3.1 is depicted in SEQ ID NO: 9, the protein sequence of Kv3.1 is depicted in SEQ ID NO: 10.
  • the nucleic acid of Kv5.1 is depicted in SEQ ID NO: 11 , the protein sequence of Kv5.1 is depicted in SEQ ID NO: 12.
  • the term 'at least one' means that in a particular heterotetrameric channel any combination between two channel alfa subunits is possible. As an example a 3:1 , 2:2 and 1 :3 combination between two subunits is possible. However, a preferred combination is a 2:2 combination.
  • DNA sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome-binding site and possibly other sequences; Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
  • an "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • the monoclonal antibodies according to this embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
  • the monoclonal antibodies according to this embodiment of the invention may be human monoclonal antibodies.
  • Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice as described in PCT/EP 99/03605 or by using transgenic non-human animals capable of producing human antibodies as described in US patent 5,545,806.
  • PBL peripheral blood lymphocytes
  • SCID severe combined immune deficiency
  • a particular heterotetrameric channel employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • immobilize a particular heterotetrameric channel or its (their) target molecule(s) to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • Interaction (e.g., binding of) of a particular heterotetrameric channel to a target molecule can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • a particular heterotetrameric channel tagged can be adsorbed onto Ni-NTA microtiter plates, or a particular heterotetrameric channel -ProtA fusions adsorbed to IgG, which are then combined with the cell lysates (e.g., 35 S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the plates are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated.
  • Biotinylated particular heterotetrameric channel or fragments thereof 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 streptavid in-coated 96 well plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, 111.
  • streptavid in-coated 96 well plates Piereptavid in-coated 96 well plates
  • antibodies reactive with a particular heterotetrameric channel but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and a particular heterotetrameric channel trapped in the wells by antibody conjugation.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding a particular heterotetrameric channel specifically compete with a test compound for binding a particular heterotetrameric channel. In this manner, the antibodies can be used to detect the presence of any protein, which shares one or more antigenic determinants with a particular heterotetrameric channel.
  • Determining the functional effect refers to examining the effect of a compound that increases or decreases ion flux on a cell or cell membrane in terms of cell and cell membrane function.
  • the ion flux can be any ion that passes through a channel and analogues thereof, e.g., potassium, rubidium, and sodium.
  • the term refers to the functional effect of the compound on the channels described herein, e.g., changes in ion flux including radioisotopes, current amplitude, membrane potential, current flow, transcription, protein binding, phosphorylation, dephosphorylation, second messenger concentrations (cAMP, cGMP, Ca 2+ , IP 3 ) and other physiological effects such as hormone and neu retransmitter release, as well as changes in voltage and current.
  • Such functional effects can be measured by any means known to those skilled in the art, e.g., patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, and the like.
  • cells expressing endogenous channels described herein can be used in such assays.
  • samples or assays comprising said channels are treated with a potential activator or inhibitor and are compared to control samples without the inhibitor.
  • Control samples (untreated with inhibitors) are assigned a relative channel activity value of 100%.
  • Inhibition of channels comprising channels described herein is achieved when said channel activity value relative to the control is about 90%, preferably 50%, more preferably 25-0%.
  • Activation of channels comprising the channels described herein is achieved when the channel activity value relative to the control is 110%, more preferably 150%, most preferably at least 200-500% higher or 1000% or even higher.
  • the present invention provides the nucleic acids of the heterotetrameric channels described herein for the transfection of cells in vitro and in vivo. These nucleic acids can be inserted into any of a number of well-known vectors for the transfection of target cells and organisms as described below. The nucleic acids are transfected into cells, ex vivo or in vivo, through the interaction of the vector and the target cell. The nucleic acids for said channels, under the control of a promoter, then expresses a particular heterotetramer of the present invention, thereby mitigating the effects of absent, partial inactivation, or abnormal expression of the said heterotetrameric channels.
  • Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in, e.g., US Pat. No. 5,049,386, US Pat No. 4,946,787; and US Pat. No. 4,897,355 and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor- recognition lipofection of polynucleotides include those of Flegner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
  • the preparation of lipid: nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.
  • RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
  • Conventional viral based systems for the delivery of nucleic acids could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
  • Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
  • Lentiviral vectors are retroviral vector that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised on c/s-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum c/s-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al, J. Virol. 66:2731-2739 (1992); PCT/US94/05700).
  • adenoviral based systems are typically used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained.
  • Adeno-associated virus vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., U.S. Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka. Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Hermonat & Muzyczka, Proc. Natl. Acad. Sci.
  • All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system (Wagner et al, Lancet 351 :9117 1702-3 (1998). Replication-deficient recombinant adenoviral vectors (Ad) are predominantly used transient expression gene therapy, because they can be produced at high titer and they readily infect a number of different cell types.
  • Ad vectors are engineered such that a transgene replaced the Ad E1a, E1b, and E3 genes; subsequently the replication defector vector is propagated in human 293 cells that supply deleted gene function in trans.
  • Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney and muscle system tissues.
  • Conventional Ad vectors have a large carrying capacity.
  • An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al, Hum. Gene Ther. 7:1083-9 (1998)).
  • adenovirus vectors for gene transfer in clinical trials include Sterman et al, Hum. Gene Ther. 9:7 1083- 1089 (1998); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al, Gene Ther. 5:507-513 (1998)).
  • Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by producer cell line that packages a nucleic acid vector into a viral particle.
  • the vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed.
  • the missing viral functions are supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line is also infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
  • the gene therapy vector be delivered with a high degree of specificity to a particular tissue type.
  • a viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al, Proc. Natl. Acad. Sci. U.S.A.
  • Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
  • This principle can be extended to other pairs of virus expressing a ligand fusion protein and target cell expressing a receptor.
  • filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient).
  • a nucleic acid gene or cDNA
  • Various cell types suitable for ex vivo transfection are well known to those of skill n the art (see, e.g., Freshney et al, Culture of Animal Cells, A Manual of Basic Technique (3 rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).
  • stem cells are used in ex vivo procedures for cell transfection and gene therapy.
  • the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba et al, J. Exp. Med. 176: 1693-1702 (1992)).
  • cytokines such as GM-CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba et al, J. Exp. Med. 176: 1693-1702 (1992)).
  • Stem cells are isolated for transduction and differentiation using known methods.
  • stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells) (see Inaba et al, J. Exp. Med. 176:1693-1702 (1992)).
  • Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
  • therapeutic nucleic acids can be also administered directly to the organism for transduction of cells in vivo.
  • naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells.
  • Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells.
  • the nucleic acids are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • excitability disorders are disorders or diseases (genetic or acquired) which are caused by defects in ion channel currents, in the current invention caused by defects of potassium channel defects.
  • excitability disorders comprise arrhythmias (e.g. heart, muscle), long QT syndrome, hyperactivity disorders, epilepsia, mood disorders, mental disorders, behavioral disorders, anxiety disorders, ataxias, hypokalemic periodic paralysis, spasticity disorders, myotonia, paramyotonia.
  • the present invention also relates to a non-human transgenic animal harbouring a nucleic acid encoding for a Kv6.3, Kv10.1 and/or Kv11.1 potassium channel subunit and a non-human knock-out animal characterized in that the nucleic acid(s) encoding for the Kv6.3, Kv10.1 and/or Kv11.1 potassium channel subunit(s) can not be functionally expressed.
  • the present invention provides a transgenic non-human animal that carries in its somatic and germ cells at least one integrated copy of a human DNA sequence that encodes for a Kv6.3, Kv10.1 and/or Kv11.1 potassium subunit.
  • a human DNA sequence that encodes for a Kv6.3, Kv10.1 and/or Kv11.1 potassium subunit.
  • an entire human Kv6.3, Kv10.1 and/or Kv11.1 potassium subunit allele may be cloned and isolated, either in parts or as a whole, in a cloning vector (e.g. cosmid or yeast or human artificial chromosome).
  • Such mini-genes may contain a cDNA sequence encoding a Kv6.3, Kv10.1 and/or Kv11.1 potassium subunit polypeptide, preferably full-length, a combination of Kv6.3, Kv10.1 and/or Kv11.1 potassium subunit exons, or a combination thereof, linked to a downstream polyadenylation signal sequence and an upstream promoter (and preferably enhancer).
  • a mini-gene construct will, when introduced into an appropriate transgenic host (e.g., mouse or rat), express an encoded Kv6.3, Kv10.1 and/or Kv11.1 potassium subunit polypeptide or a fragment thereof.
  • Another approach to create transgenic animals is to target a mutation to the desired gene by homologous recombination in an embryonic stem (ES) cell line in vitro followed by microinjection of the modified ES cell line into a host blastocyst and subsequent incubation in a foster mother (see Frohman and Martin (1989) Cell 56:145).
  • ES embryonic stem
  • the technique of microinjection of the mutated gene, or a portion thereof, into a one-cell embryo followed by incubation in a foster mother can be used. Additional methods for producing transgenic animals are known in the art.
  • site-directed mutagenesis and/or gene conversion can be used to mutate a murine or other non-human Kv6.3, Kv10.1 and/or Kv11.1 potassium subunit gene/allele.
  • transgenic rats The procedure for generating transgenic rats is similar to that of mice (Hammer et al., Cell 63; 1099-112 (1990)). Thirty day-old female rats are given a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hours later each female placed with a proven male. At the same time, 40-80 day old females are placed in cages with vasectomized males. These will provide the foster mothers for embryo transfer. The next morning females are checked for vaginal plugs. Females who have mated with vasectomized males are held aside until the time of transfer. Donor females that have mated are sacrificed (CO 2 asphyxiation) and their oviducts removed, placed in
  • the ovarian bursa is torn, the embryos are picked up into the transfer pipet, and the tip of the transfer pipet is inserted into the infundibulum. Approximately 10-12 embryos are transferred into each rat oviduct through the infundibulum. The incision is then closed with sutures, and the foster mothers are housed singly.
  • Knock-out of the endogenous Kv6.3, Kv10.1 and/or Kv11.1 potassium subunit genes may be accomplished by the insertion of artificially modified fragments of the endogenous gene by homologous recombination.
  • the modifications include insertion of mutant stop codons, the deletion of DNA sequences, or the inclusion of recombination elements (lox p sites) recognized by enzymes such as Cre recombinase. Examples Materials and Methods
  • the channel sequences were obtained with a BLAST search of the high throughput genomic sequence (htgs) database (July 2000).
  • the coding sequences were cloned using PCR amplification from genomic DNA for Kv6.3, and from a brain library (Clontech) or a testis library (TaKaRa) for Kv10.1 and Kv11.1, respectively.
  • the following primers were used: 5 ' -AGCCATGACCTTCGGGCGCAG-3 ' and 5'- CAAGAATTGATTTGCAATGC-3 ' for Kv10.1 and 5 ' -AGCCATGCTCAAACAGAGTG- 3 ' and 5 ' -GAATCTACCAGCCACATGTC-3' for Kv11.1.
  • both coding exons were amplified.
  • the first coding exon was amplified using the forward primer 5'- CAGCAATGCCCATGCCTTCCAG-3 ' and the reverse primer 5 ' - AGCATTCGCCCTGGTCCTCCTCTGCCCTGA-3 ' , which contained the BsaMI site present at the start of the second exon.
  • the second exon was amplified with the primers 5'-CCGGAATGCTCTCGGAAG-3 ' and 5'-
  • the BsaMI restriction site was used to join the two coding exons.
  • Ltk ' -cells were cultured in DMEM supplemented with 10% horse serum and 1% penicillin/streptomycin under a 5% CO 2 atmosphere.
  • Ltk ' -cells were transiently transfected with cDNA using LipofectAMINE reagent (Life-Technologies, Inc.) as previously reported [12].
  • Each subunit was co- expressed with Kv2.1 (10:1 ratio). At this ratio, less than 0.01% of the channels will be wild type Kv2.1. 2-24 hours posttransfection the cells were trypsinized and used for analysis within 12 hours. -Whole cell current recording.
  • the cells were continuously perfused with a bath solution containing (in mM) NaCI 130, KCl 4, CaCI 2 1.8, MgCI 2 1 , HEPES 10, Glucose 10, adjusted to pH 7.35 with NaOH.
  • the pipettes were filled with intracellular solution containing (in mM) KCL 110, K 4 BAPTA 5, K 2 ATP 5, MgCI 2 1 , HEPES 10 and was adjusted to pH 7.2 using KOH. Junction potentials were zeroed with the filled pipette in the bath solution. After achieving a gigaohm seal, the whole cell configuration was achieved by mouth suction.
  • the access resistance varied from 4 to 9 M ⁇ . After compensation the series resistance was kept below 3 M ⁇ to ensure that voltage errors were ⁇ 5mV.
  • the MATCHMAKER Yeast Two-Hybrid System 3 (Clontech) was used to assay for protein-protein interactions.
  • Kv2.1 , Kv6.3, Kv10.1 and Kv11.1 were also cloned into the vector pGADT7.
  • Yeast strain AH109 was used for all experiments. AH109 cells were transformed with the plasmid constructs of interest (100ng of each) and plated on -Trp/-Leu/+XDGAL media to select for cells containing both vectors and to test for interaction. The degree of interaction was determined from the speed and intensity of the blue color development. -Confocal imaging.
  • Kv6.3, Kv10.1 and Kv11.1 were tagged with Green Fluorescent Protein (GFP) at their carboxyterminus using a mutagenesis PCR to eliminate the stop codon and to insert the GFP in frame with the channel cDNA.
  • GFP Green Fluorescent Protein
  • HEK293 cells were cultivated on coverslips in MEM supplemented with 10% fetal bovine serum, 1% non essential amino acids and 1% penicillin/streptomycin under a 5% CO 2 atmosphere. For co- transfections a 1 :10 ratio of channel DNA versus Kv2.1 was added.
  • the endoplasmatic reticulum (ER) was visualised with the DsRed ER localisation vector. This was constructed starting from the pDsRed vector (Clontech).
  • Kv6.3, Kv10.1 and Kv11.1 are subunits for human voltage gated potassium channels that fail to generate functional channels at the plasma membrane as shown by voltage clamp analysis and confocal microscopy. These subunits do not form homotetrameric channels, at least in part, because they fail to interact with themselves as demonstrated by the yeast two-hybrid approach. On the other hand, they each interacted with Kv2.1 , Kv3.1 and Kv5.1. Co-expression of Kv6.3 and Kv2.1 resulted in currents that differ significantly from typical Kv2.1 currents, while functional effects of Kv10.1 and Kv11.1 were less pronounced.

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Abstract

La présente invention concerne de nouveaux canaux potassiques hétérotétramères dépendant d'un potentiel d'action, comprenant des sous-unités de canaux potassiques Kv2.1, Kv3.1, Kv5.1, Kv6.3, Kv10.1 ou Kv11.1. L'invention traite de l'utilisation directe ou indirecte de ces derniers pour diagnostiquer, prévenir et/ou traiter les troubles d'excitabilité. De plus, la présente invention traite de la séquence de l'ADNc de la sous-unité du canal potassique Kv10.1.
PCT/EP2002/006082 2001-05-31 2002-05-31 Nouveaux canaux potassiques heterotetrameres et utilisations de ces derniers WO2002096944A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670335A (en) * 1993-08-06 1997-09-23 The Regents Of The University Of California Mammalian inward rectifier potasssium channel cDNAs, host cells expressing them, and screening assays using such cells
WO1998004521A1 (fr) * 1996-07-26 1998-02-05 Icagen, Inc. Inhibiteurs du canal de potassium

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670335A (en) * 1993-08-06 1997-09-23 The Regents Of The University Of California Mammalian inward rectifier potasssium channel cDNAs, host cells expressing them, and screening assays using such cells
WO1998004521A1 (fr) * 1996-07-26 1998-02-05 Icagen, Inc. Inhibiteurs du canal de potassium

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] Accession No. AC025750, 16 March 2000 (2000-03-16) "Homo sapiens chromosome UNK clone RP11-804P20" XP002181998 *
DATABASE EMBL [Online] Accession No. BF966122, 24 January 2001 (2001-01-24) "602286371f1 NIH_MGC_95 Homo sapiens cDNA clone IMAGE:4375480 5' mRNA sequence" XP002181999 *
DATABASE GENBANK [Online] 30 May 2001 (2001-05-30) retrieved from NCBI XP002223177 *
DWORAKOWSKA BEATA ET AL: "Ion channels-related diseases." ACTA BIOCHIMICA POLONICA, vol. 47, no. 3, 2000, pages 685-703, XP001022116 ISSN: 0001-527X *
PO S ET AL: "Heteromultimeric assembly of human potassium channels: Molecular basis of a transient outward current?" CIRCULATION RESEARCH, vol. 72, no. 6, 1993, pages 1326-1336, XP001029529 ISSN: 0009-7330 *
PONGS OLAF ET AL: "Functional and molecular aspects of voltage-gated K+ channel beta subunits." ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 868, 30 April 1999 (1999-04-30), pages 344-355, XP001119013 Conference on Molecular and Functional Diversity of Ion Channels and Receptors;New York, New York, USA; May 14-17, 1998, diversity of ion channels and receptors. April 30, 1999 New York Academy of Sciences 2 East 63rd Street, New York, New York 10021, USA ISBN: 1-57331-177-4 *
POST MARC A ET AL: "Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current." FEBS LETTERS, vol. 399, no. 1-2, 1996, pages 177-182, XP001022178 ISSN: 0014-5793 cited in the application *
SCHEFFER INGRID E ET AL: "Genetics of the epilepsies." CURRENT OPINION IN PEDIATRICS, vol. 12, no. 6, December 2000 (2000-12), pages 536-542, XP001033798 ISSN: 1040-8703 *

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