WO2006059069A2 - Canal ionique - Google Patents
Canal ionique Download PDFInfo
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- WO2006059069A2 WO2006059069A2 PCT/GB2005/004508 GB2005004508W WO2006059069A2 WO 2006059069 A2 WO2006059069 A2 WO 2006059069A2 GB 2005004508 W GB2005004508 W GB 2005004508W WO 2006059069 A2 WO2006059069 A2 WO 2006059069A2
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- kcnma3
- ion channel
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- 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
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
Definitions
- This invention relates to newly identified nucleic acids, polypeptides encoded by them and to their production and use. More particularly, the nucleic acids and polypeptides of the present invention relate to a KCNMA3 (Slo3) ion channel receptor, hereinafter referred to as KCNMA3 and its function. The invention also relates to inhibiting or activating the action of such nucleic acids and polypeptides.
- Potassium channels have evolved to play specialized roles in many inexcitable tissues. They are found in a wide variety of animal cells such as nervous, muscular, immune or epithelial tissues. The channels regulate these tissues by allowing the flow of potassium under certain circumstances. The outward flow of potassium ions upon opening the channel makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, eg. by calcium sensitivity, voltage- and ATP-sensitivity.
- an ion channel polypeptide comprising the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof.
- nucleic acid capable of encoding a polypeptide according to the first aspect of the invention.
- the nucleic acid comprises the nucleic acid sequence shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof.
- a polypeptide comprising a fragment of a polypeptide according to the first aspect of the invention.
- a fragment containing polypeptide comprises one or more regions which are homologous between SEQ ID No. 3 and SEQ ID No. 5, or which comprises one or more regions which are heterologous between SEQ ID No. 3 and SEQ ID No. 5.
- nucleic acid capable of encoding a polypeptide according to the third aspect of the invention.
- a vector comprising a nucleic acid according to the second or fourth aspect of the invention.
- the present invention in a sixth aspect, provides a host cell comprising a nucleic acid according to the second or fourth aspect of the invention, or vector according to the fifth aspect of the invention.
- a transgenic non-human animal comprising a nucleic acid according to the second or fourth aspect of the invention or a vector according to the fifth aspect of the invention.
- the transgenic non-human animal is a mouse.
- a polypeptide according to the first or third aspect of the invention in a method of identifying compound which is capable of interacting specifically with a KCNMA3 ion channel.
- a method for identifying an antagonist of a KCNMA3 ion channel comprising contacting a cell which expresses a KCNMA3 ion channel with a candidate compound capable of altering conductance or kinetics of the cell
- a method for identifying a compound capable of binding to a KCNMA3 ion channel polypeptide comprising contacting a KCNMA3 ion channel polypeptide with a candidate compound and determining whether the candidate compound binds to the ion channel polypeptide.
- a compound identified by a method according to any of the eighth to eleventh aspects of the invention is provided.
- a thirteenth aspect of the present invention we provide a compound capable of binding specifically to a polypeptide according to the first or third aspect of the invention.
- a fourteenth aspect of the present invention use of a polypeptide according to the first or third aspect of the invention, or part thereof; or a nucleic acid according to the second or fourth aspect of the invention, or part thereof, in a method for producing antibodies.
- an antibody capable of binding specifically to a polypeptide according to the first or third aspect of the invention, or part thereof; or a polypeptide encoded by a nucleic acid according to the second or fourth aspect of the invention, or part thereof.
- a pharmaceutical composition comprising any one or more of the following: a polypeptide according to the first or third aspect of the invention, or part thereof; a polypeptide encoded by a nucleic acid according to the second or fourth aspect of the invention, or part thereof; a vector according to the fifth aspect of the invention; a cell according to the sixth aspect of the invention; a compound according to the twelfth or thirteenth aspect of the invention; and an antibody according to the fifteenth aspect of the invention, together with a pharmaceutically acceptable carrier or diluent.
- a vaccine composition comprising any one or more of the following: a polypeptide according to the first or third aspect of the invention, or part thereof; a polypeptide encoded by a nucleic acid according to the second or fourth aspect of the invention, or part thereof; a vector according to the fifth aspect of the invention; a cell according to the sixth aspect of the invention; a compound according to the twelfth or thirteenth or aspect of the invention; and an antibody according to the fifteenth aspect of the invention.
- a diagnostic kit for a disease or susceptibility to a disease comprising any one or more of the following: a polypeptide according to the first or third aspect of the invention, or part thereof; a polypeptide encoded by a nucleic acid according to the second or fourth aspect of the invention, or part thereof; a vector according to the fifth aspect of the invention; a cell according to the sixth aspect of the invention; a compound according to the twelfth or thirteenth aspect of the invention; and an antibody according to the seventeenth aspect of the invention.
- a method of treating a patient suffering from a disease associated with enhanced activity of a KCNMA3 ion channel comprises administering to the patient an antagonist of a KCNMA3 ion channel.
- a method of treating a patient suffering from a disease associated with reduced activity of a KCNMA3 ion channel comprises administering to the patient an agonist of a KCNMA3 ion channel.
- the ion channel comprises a polypeptide having the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5.
- a method for treating and/or preventing a disease in a patient which comprises the step of administering any one or more of the following to the patient: a polypeptide according to the first or third aspect of the invention, or part thereof; a polypeptide encoded by a nucleic acid according to the second or fourth aspect of the invention, or part thereof; a vector according to the fifth aspect of the invention; a cell according to the sixth aspect of the invention; a compound according to the twelfth or thirteenth aspect of the invention; and an antibody according to the fifteenth aspect of the invention; a pharmaceutical composition according to the sixteenth aspect of the invention; and a vaccine according to the seventeenth aspect of the invention, to the subject.
- an agent comprising a polypeptide according to the first or third aspect of the invention, or part thereof; a polypeptide encoded by a nucleic acid according to the second or fourth aspect of the invention, or part thereof; a vector according to the fifth aspect of the invention; a cell according to the sixth aspect of the invention; a compound according to the twelfth or thirteenth aspect of the invention; and an antibody according to the fifteenth aspect of the invention, said agent for use in a method of treatment or prophylaxis of disease.
- a polypeptide according to the first or third aspect of the invention, or part thereof use of a polypeptide encoded by a nucleic acid according to the second or fourth aspect of the invention, or part thereof; a vector according to the fifth aspect of the invention; a cell according to the sixth aspect of the invention; a compound according to the twelfth or thirteenth aspect of the invention; and an antibody according to the fifteenth aspect of the invention, for the preparation of a pharmaceutical composition for the treatment or prophylaxis of a disease.
- non-human transgenic animal characterized in that the transgenic animal comprises an altered ion channel.
- the alteration is selected from the group consisting of: a deletion of ion channel, a mutation in ion channel resulting in loss of function, introduction of an exogenous gene having a nucleotide sequence with targeted or random mutations into ion channel, introduction of an exogenous gene from another species into ion channel, and a combination of any of these.
- a non-human transgenic animal having a functionally disrupted endogenous ion channel gene, in which the transgenic animal comprises in its genome and expresses a transgene encoding a heterologous ion channel protein.
- the present invention in a twenty-seventh aspect, provides a nucleic acid construct for functionally disrupting a KCNMA3 ion channel gene in a host cell, the nucleic acid construct comprising: (a) a non-homologous replacement portion; (b) a first homology region located upstream of the non-homologous replacement portion, the first homology region having a nucleotide sequence with substantial identity to a first ion channel gene sequence; and (c) a second homology region located downstream of the non-homologous replacement portion, the second homology region having a nucleotide sequence with substantial identity to a second ion channel gene sequence, the second ion channel gene sequence having a location downstream of the first ion channel gene sequence in a naturally occurring endogenous ion channel gene.
- a process for producing a KCNMA3 ion channel polypeptide comprising culturing a host cell according to the sixth aspect of the invention under conditions in which a nucleic acid encoding a KCNMA3 ion channel polypeptide is expressed.
- a method of detecting the presence of a nucleic acid according to the second or fourth aspect of the invention in a sample comprising contacting the sample with at least one nucleic acid probe which is specific for said nucleic acid and monitoring said sample for the presence of the nucleic acid.
- a method of detecting the presence of a polypeptide according to the first or third aspect of the invention in a sample comprising contacting the sample with an antibody according to the fifteenth aspect of the invention and monitoring said sample for the presence of the polypeptide.
- a method of diagnosis of a disease or syndrome caused by or associated with increased, decreased or otherwise abnormal expression of a KCNMA3 ion channel comprising the steps of: (a) detecting the level or pattern of expression of a KCNMA3 ion channel in an animal suffering or suspected to be suffering from such a disease; and (b) comparing the level or pattern of expression with that of a normal animal.
- KCNM A3 polypeptide comprising an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto, in a method of identifying a molecule suitable for the treatment or alleviation of an KCNMA3 associated disease.
- KCNMA3 polynucleotide comprising a nucleic acid sequence shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4 or a sequence having at least 90% sequence identity thereto, for the identification of a molecule suitable for the treatment or alleviation of an KCNMA3 associated disease.
- a method of identifying an agonist or antagonist of a KCNM A3 polypeptide comprising an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto, the method comprising administering a candidate molecule to an animal and determining whether the animal exhibits lower tendency to spontaneous alternate.
- a method for providing an indication useful in the diagnosis of or a determination of susceptibility to an KCNMA3 associated disease in an individual comprising detecting a change in the expression pattern or level of a KCNMA3 polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto in a sample from the individual.
- a method for providing an indication useful in the diagnosis of or a determination of susceptibility to an KCNMA3 associated disease in an individual comprising detecting a polymorphism in a KCNMA3 polynucleotide comprising a nucleic acid sequence shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4 or a sequence having at least 90% sequence identity thereto, in a sample from the individual.
- a non-human animal which displays lower tendency to spontaneous alternate when compared to a wild- type animal, the animal being a transgenic animal having a functionally disrupted endogenous KCNMA3 gene, in which the KCNMA3 gene comprises a nucleic acid sequence shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4 or a sequence having at least 90% sequence identity thereto.
- a 38 aspect of the present invention we provide use of such a non- human animal, or such an isolated cell or tissue thereof, in a method of identifying an agonist or antagonist of a KCNMA3 polypeptide for the treatment or alleviation of an KCNMA3 associated disease, the KCNMA3 polypeptide comprising an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto.
- a 40 th aspect of the present invention we provide use of an agonist or antagonist of a KCNMA3 polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto for the preparation of a pharmaceutical composition for the treatment of an KCNMA3 associated disease in an individual.
- Figure 1 is a diagram showing the results of analysis of the human ion channel polypeptide (SEQ ID NO: 3) using the HMM structural prediction software of pfam (http://www.sanger.ac.uk/Software/Pfam/search.shtml).
- Figure 2 is a diagram showing the structure of the targeting vector used to produce the knockout mouse.
- Figure 3 is a graph of the percentage spontaneous alternations of the KCNMA3 ion channel knockout mouse compared with the wildtype control mouse.
- Our invention relates in general to an ion channel, in particular, a pH- and voltage sensitve (but calcium-insensitive) potassium channel, which we refer to as KCNMA3, as well as homologues, variants or derivatives thereof.
- a pH- and voltage sensitve (but calcium-insensitive) potassium channel which we refer to as KCNMA3, as well as homologues, variants or derivatives thereof.
- KCNMA3 is structurally related to other proteins of the large conductance family, as shown by the results of sequencing the amplified cDNA products encoding human ion channel.
- the cDNA sequence of SEQ ID NO: 1 contains an open reading flame (SEQ ID NO: 2) encoding a polypeptide of 1150 amino acids shown in SEQ ID NO: 3.
- Human KCNMA3 is found to map to chromosome 8pl 1. Identities and Similarities to Ion channel
- KCNMA3 ion channel peptide is a voltage sensitive potassium channel (see Figure 1).
- the mouse homologue of the human ion channel has been cloned, and its nucleic acid sequence and amino acid sequence are shown as SEQ ID NO: 4 and SEQ ID NO: 5 respectively.
- the mouse ion channel cDNA of SEQ ID NO: 4 shows a high degree of identity with the human ion channel (SEQ ID NO: 2) sequence, while the amino acid sequence (SEQ ID NO: 5) of mouse ion channel shows a high degree of identity and similarity with human ion channel (SEQ ID NO: 3).
- Human and mouse ion channel are therefore members of a large family of a pH- and voltage sensitve (but calcium-insensitive) potassium channel.
- KCNMA3 cDNA Polymerase chain reaction (PCR) amplification of KCNMA3 cDNA detects expression of KCNMA3 to varying abundance in a number of tissues.
- KCNMA3 cDNA of SEQ ID NO: 1 to search the human EST data sources by BLASTN, identities are found in cDNA derived from libraries originating from the testis, hippocampus, and thymus. This indicates that KCNMA3 ion channel is expressed in these normal tissues.
- the KCNMA3 polypeptides, nucleic acids, probes, antibodies, expression vectors and ligands are useful for detection, diagnosis, treatment and other assays for diseases associated with over-, under- and abnormal expression of KCNMA3 in these and other tissues.
- KCNMA3 polypeptide is intended to refer to a polypeptide comprising the amino acid sequence shown in SEQ ID No. 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof.
- the polypeptide comprises or is a homologue, variant or derivative of the sequence shown in SEQ ID NO: 3.
- Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
- Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.
- Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
- Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-inking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-inks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
- the resultant amino acid sequence has ion channel activity, more preferably having at least the same activity of the ion channel shown as SEQ ID NO: 3 or SEQ ID NO: 5.
- the term "homologue” covers identity with respect to structure and/or function providing the resultant amino acid sequence has activity.
- sequence identity i.e. similarity
- sequence identity preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
- sequence identity i.e. similarity
- sequence identity preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity.
- receptor activity or “biological activity” of a receptor such as KCNMA3
- these terms are intended to refer to the metabolic or physiological function of the ion channel, including similar activities or improved activities or these activities with decreased undesirable side effects.
- antigenic and immunogenic activities of the ion channel are also included. Examples of ion channel activity, and methods of assaying and quantifying these activities, are known in the art, and are described in detail elsewhere in this document.
- a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
- an “insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring substance.
- substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
- negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
- KCNMA3 polypeptides may further comprise heterologous amino acid sequences, typically at the N-terminus or C-terminus, preferably the N-terminus.
- Heterologous sequences may include sequences that affect intra or extracellular protein targeting (such as leader sequences).
- Heterologous sequences may also include sequences that increase the immunogenicity of the polypeptide and/or which facilitate identification, extraction and/or purification of the polypeptides.
- Another heterologous sequence that is particularly preferred is a polyamino acid sequence such as polyhistidine which is preferably N-terminal.
- a polyhistidine sequence of at least 10 amino acids, preferably at least 17 amino acids but fewer than 50 amino acids is especially preferred.
- the KCNMA3 polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro- sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
- KCNMA3 polypeptides are advantageously made by recombinant means, using known techniques. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Polypeptides may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences, such as a thrombin cleavage site. Preferably the fusion protein will not hinder the function of the protein of interest sequence.
- KCNMA3 polypeptides may be in a substantially isolated form. This term is intended to refer to alteration by the hand of man from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
- a polynucleotide, nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide, nucleic acid or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.
- KCNMA3 protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
- a KCNMA3 polypeptide may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, for example, 95%, 98% or 99% of the protein in the preparation is a ion channel polypeptide.
- the present invention also relates to peptides comprising a portion of a KCNMA3 ion channel polypeptide according to the invention.
- peptides comprising a portion of a KCNMA3 ion channel polypeptide according to the invention.
- fragments of KCNMA3 and its homologues, variants or derivatives are included.
- the peptides of the present invention may be between 2 and 200 amino acids, preferably between 4 and 40 amino acids in length.
- the peptide may be derived from a KCNMA3 ion channel polypeptide as disclosed here, for example by digestion with a suitable enzyme, such as trypsin.
- the peptide, fragment, etc may be made by recombinant means, or synthesised synthetically,
- homologous regions regions of the amino acid sequence are conserved between different species
- heterologous regions regions of the amino acid sequence are conserved between different species
- the KCNMA3 polypeptides according to the invention may therefore comprise a sequence which corresponds to at least part of a homologous region.
- a homologous region shows a high degree of homology between at least two species.
- the homologous region may show at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identity at the amino acid level using the tests described above.
- Peptides which comprise a sequence which corresponds to a homologous region may be used in therapeutic strategies as explained in further detail below.
- the ion channel peptide may comprise a sequence which corresponds to at least part of a heterologous region.
- a heterologous region shows a low degree of homology between at least two species.
- This invention encompasses KCNMA3 polynucleotides, KCNMA3 nucleotides and KCNMA3 nucleic acids, methods of production, uses of these, etc, as described in further detail elsewhere in this document.
- KCNMA3 polynucleotide KCNMA3 nucleotide
- KCNMA3 nucleotide KCNMA3 nucleic acid
- the polynucleotide/nucleic acid comprises or is a homologue, variant or derivative of the nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, most preferably, SEQ ID NO: 2.
- KCNMA3 polynucleotides and nucleic acids comprise a nucleotide sequence capable of encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof.
- the ion channel polynucleotides and nucleic acids comprise a nucleotide sequence capable of encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, or a homologue, variant or derivative thereof.
- Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
- Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
- polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
- Modified bases include, for example, tritylated bases and unusual bases such as inosine.
- polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
- Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
- nucleotide sequence refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof).
- the nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin which may be double- stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
- nucleotide sequence may be prepared by use of recombinant DNA techniques (for example, recombinant DNA).
- nucleotide sequence means DNA.
- variants include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acids from or to the sequence of a KCNMA3 nucleotide sequence.
- references to "KCNMA3" and “ion channel” include references to such variants, homologues, derivatives and fragments of the KCNMA3 ion channel.
- the resultant nucleotide sequence encodes a polypeptide having KCNMA3 activity, preferably having at least the same activity of the ion channel shown as SEQ ID NO: 3 or SEQ ID NO: 5.
- the term "homologue” is intended to cover identity with respect to structure and/or function such that the resultant nucleotide sequence encodes a polypeptide which has ion channel activity.
- sequence identity i.e. similarity
- sequence identity preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
- KCNMA3 is useful for treating and diagnosing a range of diseases.
- human KCNMA3 ion channel maps to human chromosome 8pl 1.
- KCNMA3 may be used to treat or diagnose a disease which maps to this locus, chromosomal band, region, arm or the same chromosome.
- chromosome 8pl 1 Known diseases which have been determined as being linked to the same locus, chromosomal band, region, arm or chromosome as the chromosomal location of ion channel (i.e., chromosome 8pl 1) include the following (locations in brackets): Jackson- Weiss syndrome, (8pl 1.2-pl 1-1), Kallmann syndrome 2, (8pl 1.2-pll.l), Lipoid adrenal hyperplasia, (8pl l.2), Spherocytosis-2, (8pl l .2),
- KCNMA3 may be used to diagnose or treat, by any means as described in this document, Alzheimer's disease, ishaemic/vascular dementia, Pick's disease, diffuse Lewy body dementia, frontotemporal dementias, corticobasal degeneration, Huntington's disease, progressive supranuclear palsy, AIDS/HIV dementia, prion infections, encephalitis, neurosyphilis, vasculitis, progressive multifocal leukoencephalopathy.
- Partial epilepsies lesional, medial temporal lobe, non lesional neocortical and idiopathic (genetic) partial epilepsies, consisting simple partial seizures, complex partial seizures, secondarily generalised clonic tonic seizures, benign Rolandic and occipital seizures, generalised epilepsies including West's syndrome, Lennox Gastaut syndrome and progressive myoclonus epilepsy, consisting absence seizures, clonic tonix seizures, myoclonic seizures, tonic seizures, clonic seizures, atonic seizures and clonic-tonic-clonic seizures, recurrent status epilepticus, neonatal seizures and rare or isolated seizures. More preferably, KCNMA3 ion channel is used to diagnose or treat disease.
- transgenic knockout mice have symptoms that are charactistic of the following diseases and disorders: Alzheimer's disease, ishaemic/vascular dementia, Pick's disease, diffuse Lewy body dementia, frontotemporal dementias, corticobasal degeneration, Huntington's disease, progressive supranuclear palsy, AIDS/HIV dementia, prion infections, encephalitis, neurosyphilis, vasculitis, progressive multifocal leukoencephalopathy.
- KCNMA3 is used to diagnose or treat Alzheimer's disease, ishaemic/vascular dementia, Pick's disease, diffuse Lewy body dementia, frontotemporal dementias, corticobasal degeneration, Huntington's disease, progressive supranuclear palsy, AIDS/HIV dementia, prion infections, encephalitis, neurosyphilis, vasculitis, progressive multifocal leukoencephalopathy.
- Partial epilepsies lesional, medial temporal lobe, non lesional neocortical and idiopathic (genetic) partial epilepsies, consisting simple partial seizures, complex partial seizures, secondarily generalised clonic tonic seizures, benign Rolandic and occipital seizures, generalised epilepsies including West's syndrome, Lennox Gastaut syndrome and progressive myoclonus epilepsy, consisting absence seizures, clonic tonix seizures, myoclonic seizures, tonic seizures, clonic seizures, atonic seizures and clonic-tonic-clonic seizures, recurrent status epilepticus, neonatal seizures and rare or isolated seizures.
- KCNMA3 may be used to diagnose and/or treat any of these specific diseases using any of the methods and compositions described here.
- nucleic acids in particular, we specifically envisge the use of nucleic acids, vectors comprising KCNMA3 nucleic acids, polypeptides, including homologues, variants or derivatives thereof, pharmaceutical compositions, host cells, and transgenic animals comprising KCNMA3 nucleic acids and/or polypeptides, for the treatment or diagnosis of the specific diseases listed above.
- diagnostic kits for the detection of the specific diseases in an individual.
- Sequence identity with respect to any of the sequences presented here can be determined by a simple "eyeball” comparison (i.e. a strict comparison) of any one or more of the sequences with another sequence to see if that other sequence has, for example, at least 70% sequence identity to the sequence(s).
- Relative sequence identity can also be determined by commercially available computer programs that can calculate % identity between two or more sequences using any suitable algorithm for determining identity, using for example default parameters.
- a typical example of such a computer program is CLUSTAL.
- Other computer program methods to determine identify and similarity between the two sequences include but are not limited to the GCG program package (Devereux et al 1984 Nucleic Acids Research 12: 387) and FASTA (Atschul et al 1990 J Molec Biol 403-410).
- % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
- a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
- An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
- GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied. It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
- substantially identical when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more.
- the default threshold for EXPECT in BLAST searching is usually 10.
- BLAST Basic Local Alignment Search Tool
- blastp, blastn, blastx, tblastn, and tblastx these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (Karlin and Altschul 1990, Proc. Natl. Acad. Sci. USA 87:2264-68; Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-7; see http://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements.
- the BLAST programs are tailored for sequence similarity searching, for example to identify homologues to a query sequence. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994) Nature Genetics 6:119-129.
- blastp compares an amino acid query sequence against a protein sequence database
- blastn compares a nucleotide query sequence against a nucleotide sequence database
- blastx compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database
- tblastn compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands)
- tblastx compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
- BLAST uses the following search parameters:
- HISTOGRAM - Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual).
- DESCRIPTIONS Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page).
- EXPECT The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).
- CUTOFF - Cutoff score for reporting high-scoring segment pairs.
- the default value is calculated from the EXPECT value (see above).
- HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT.
- ALIGNMENTS Restricts database sequences to the number specified for which high-scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).
- MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). The valid alternative choices include: PAM40, PAMl 20, PAM250 and IDENTITY. No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.
- FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
- Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.
- NCBI-gi causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.
- sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.
- no gap penalties are used when determining sequence identity.
- the present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences presented herein, or any fragment or derivative thereof, or to the complement of any of the above.
- Hybridization means a "process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY) as well as the process of amplification as carried out in polymerase chain reaction technologies as described in Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plain view NY).
- Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, VoI 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
- Nucleotide sequences capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement will be generally at least 70%, preferably at least 75%, more preferably at least 85 or 90% and even more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
- Preferred nucleotide sequences will comprise regions homologous to SEQ ID NO: 1, 2 or 4, preferably at least 70%, 80% or 90% and more preferably at least 95% homologous to one of the sequences.
- the term "selectively hybridizable" means that the nucleotide sequence used as a probe is used under conditions where a target nucleotide sequence is found to hybridize to the probe at a level significantly above background.
- the background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened.
- background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA.
- the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P.
- nucleotide sequences that are capable of hybridizing to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.
- Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, VoI 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
- Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 1O 0 C to 20°C below Tm; and low stringency at about 20°C to 25 0 C below Tm.
- a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related nucleotide sequences.
- the present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof. Likewise, the present invention encompasses nucleotide sequences that are complementary to sequences that are capable of hybridising to the sequence of the present invention. These types of nucleotide sequences are examples of variant nucleotide sequences.
- stringent conditions eg. 65 0 C and 0. IxSSC ⁇ IxS
- the present invention also encompasses nucleotide sequences that are complementary to the sequences presented here, or any fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify and clone similar ion channel sequences in other organisms etc.
- the present invention thus enables the cloning of KCNMA3, its homologues and other structurally or functionally related genes from human and other species such as mouse, pig, sheep, etc to be accomplished.
- Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate partial or full-length cDNAs and genomic clones encoding KCNMA3 from appropriate libraries.
- Such probes may also be used to isolate cDNA and genomic clones of other genes (including genes encoding homologues and orthologues from species other than human) that have sequence similarity, preferably high sequence similarity, to the KCNMA3 gene.
- Hybridization screening, cloning and sequencing techniques are known to those of skill in the art and are described in, for example, Sambrook et al ⁇ supra).
- nucleotide sequences suitable for use as probes are 70% identical, preferably 80% identical, more preferably 90% identical, even more preferably 95% identical to that of the referent.
- the probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 150 and 500 nucleotides, more particularly about 300 nucleotides.
- to obtain a polynucleotide encoding a KCNMA3 polypeptide, including homologues and orthologues from species other than human comprises the steps of screening an appropriate library under stringent hybridization conditions with a labelled probe having the SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or a fragment thereof and isolating partial or full-length cDNA and genomic clones containing said polynucleotide sequence.
- stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42 degrees C.
- the cloned putative KCNMA3 ion channel polynucleotides may be verified by sequence analysis or functional assays.
- the conductance of Xenopus oocytes transfected as described may be detected as a means of gauging and quantifying KCNMA3 activity, useful for screening assays described below.
- Such a Xenopus oocyte electrophysiology assay is referred to for convenience as a "Functional Assay of KCNMA3 (Electrophysiology)".
- the putative KCNMA3 ion channel subunit or homologue may be assayed for activity in a "Functional Assay of KCNMA3 (Electrophysiology)" as follows.
- Capped RNA transcripts from linearized plasmid templates encoding the KCNMA3 cDNAs are synthesized in vitro with RNA polymerases in accordance with standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a microinjection apparatus.
- RNA encoding other ⁇ subunits of this channel may also be injected to form heteromeric channel complexes.
- Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response to agonist exposure. Recordings of the current are made in standard medium consisting of (in mM) NaCl 115, KCl 2.5, CaCl 2 1.8, NaOH-HEPES 10, pH7.2 at room temperature.
- the Xenopus system may also be used to screen known ligands and tissue/cell extracts for activating ligands, as described in further detail below.
- Alternative functional assays include patch clamp electrophysiology, Rb flux, fluorescence resonance energy transfer (FRET) analysis and FLIPR analysis, including the use of voltage sensitive dyes to investigate the membrane voltage of the cell.
- a FLIPR assay is described in Whiteaker et al. J Biomol Screen. 2001 Oct;6(5):305-1, while a FRET based assay is described in Falconer et al. J Biomol Screen. 2002 Oct;7(5):460-5.
- an assay which detects Rb flux as well as screens which detect change in Rb flux to identify agonists and antagonists of KCNMA3.
- Methods for measuring radiolabeled Rb flux are outlined in Rezazadeh et al J Biomol Screen. 2004 Oct;9(7):588-97 and a non-radiolabelled Rb flux assay in Assay Drug Dev Technol. 2004 Oct;2(5):525-34.
- % Rb efflux is measured in the assay.
- % Rb efflux is lowered by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more in the presence of an antagonist of KCNM A3.
- agonists of KCNMA3 increase the % Rb efflux of a suitably transfected cell.
- the % Rb efflux is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more in the presence of an agonist of KCNMA3.
- the KCNMA3 polypeptide may further be assayed for its kinetics, which include the activation, deactivation and inactivation.
- the activation time is the time taken for a full current to be established across a KCNMA3 containing channel under standard conditions, which the deactivation time is the time taken for a full current to zero under standard conditions.
- modulation, increase or decrease of KCNMA3 kinetics this should be taken to refer preferably to modulation, increase or decrease of KCNMA3 activation time, or KCNMA3 deactivation time, or both.
- the activation time constant is used as a measure of activation time
- the deactivation time constant is used as a measure of deactivation time.
- a typical activation time constant for KCNMA3 containing channels is 21ms.
- a typical potential for half- inactivation V 1 ⁇ i nact is -33mV, and the V ⁇ a inact may be assayed as a further or alternative kinetic parameter of inactivation.
- Modulators such as openers, agonists, blockers and antagonists of KCNMA3 containing channels are capable of changing, i.e., increasing or decreasing, the kinetics of the KCNMA3 containing channel, preferably any one or more of the activation time, the inactivation time, deactivation time, deactivation kinetics, potential for half-inactivation, etc.
- agonists and openers are molecules which are capable of decreasing the activation time and / or deactivation time (preferably the activation time and / or deactivation time constant), preferably by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, i.e., by decreasing the activation time to 20 ms, 18 ms, 16ms, or 15 ms or less, for example.
- antagonists or blockers of KCNMA3 are capable of increasing the activation time and / or deactivation time, preferably by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, i.e., by increasing the activation time to 22 ms, 25 ms or 27 ms or more, for example.
- the kinetics and specifically the activation time may be preferably measured using the "Functional Assay of KCNMA3 (Electrophysiology)", taking a time course of current and establishing the time taken for full current to be established.
- the inactivation time is measured using the "Functional Assay of KCNMA3 (Electrophysiology)", taking a time course of current and establishing the time taken for the full current to fall to zero.
- the deactivation kinetics which are a measure of the time the channel takes to deactivate after a repolarising pulse (eg to -4OmV) after a prepulse (eg. +50MV for 500ms), may be assayed.
- a repolarising pulse eg to -4OmV
- a prepulse eg. +50MV for 500ms
- a typical value for the deactivation kinetics of KCNMA3 containing channels is 44ms.
- KCNMA3 In order to design useful therapeutics for treating KCNMA3 associated diseases, it is useful to determine the expression profile of KCNMA3 (whether wild-type or a particular mutant).
- methods known in the art may be used to determine the organs, tissues and cell types (as well as the developmental stages) in which KCNMA3 is expressed.
- traditional or “electronic" Northerns may be conducted.
- Reverse-transcriptase PCR RT-PCR
- More sensitive methods for determining the expression profile of KCNMA3 include RNAse protection assays, as known in the art.
- Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound.
- Analogous computer techniques (“electronic Northerns") applying BLAST may be used to search for identical or related molecules in nucleotide databases such as GenBank or the LIFESEQ database (Incyte Pharmaceuticals). This type of analysis has advantages in that they may be faster than multiple membrane-based hybridizations.
- the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
- polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease, as explained in further detail elsewhere in this document.
- the invention includes a process for producing a KCNMA3 polypeptide.
- the method comprises in general culturing a host cell comprising a nucleic acid encoding KCNMA3 polypeptide, or a homologue, variant, or derivative thereof, under suitable conditions (i.e., conditions in which the KCNMA3 polypeptide is expressed).
- nucleotide sequences encoding KCNMA3 or homologues, variants, or derivatives thereof are inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
- a variety of expression vector/host systems may be utilized to contain and express sequences encoding KCNMA3. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
- the invention is not limited by the host cell employed.
- control elements are those non- translated regions of the vector (i.e., enhancers, promoters, and 5' and V untranslated regions) which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORTl plasmid (GIBCO/BRL), and the like, may be used.
- inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORTl plasmid (GIBCO/BRL), and the like, may be used.
- the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding ion channel, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
- Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
- plant viruses e.g., viral promoters or leader sequences
- a number of expression vectors may be selected depending upon the use intended for ion channel.
- vectors which direct high level expression of fusion proteins that are readily purified may be used.
- Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding KCNMA3 may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced, pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol.
- pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
- GST glutathione S-transferase
- fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
- Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
- yeast Saccharomyces cerevisae a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used.
- constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
- sequences encoding KCNMA3 may be driven by any of a number of promoters.
- viral promoters such as the 35S and 19S promoters of CaMV maybe used alone or in combination with the omega leader sequence from TMV.
- plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
- constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews. (See, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.).
- frugiperda cells or Trichoplusia larvae in which ion channel may be expressed In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding KCNMA3 may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing ion channel in infected host cells. (Logan, J.
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
- RSV Rous sarcoma virus
- the KCNMA3 receptors of the present invention are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells.
- HEK293 human embryonic kidney 293
- adherent dhfr CHO cells typically all 5' and 3' untranslated regions (UTRs) are removed from the receptor cDNA prior to insertion into a pCDN or pCDNA3 vector.
- the cells are transfected with individual receptor cDNAs by lipofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis.
- HEK293 or CHO cells transfected with the vector alone serve as negative controls.
- To isolate cell lines stably expressing the individual receptors about 24 clones are typically selected and analyzed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50% of the G418-resistant clones analyzed.
- HACs Human artificial chromosomes
- HACs may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
- HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
- Specific initiation signals may also be used to achieve more efficient translation of sequences encoding KCNMA3. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding KCNMA3 and its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular cell system used, such as those described in the literature. (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
- a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
- modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
- Post- translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding, and/or function.
- Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure the correct modification and processing of the foreign protein.
- ATCC American Type Culture Collection
- cell lines capable of stably expressing KCNMA3 can be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
- the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
- Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
- any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase genes (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase genes (Lowy, I. et al. (1980) Cell 22:817-23), which can be employed in tk " or apr " cells, respectively.
- antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
- npt confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. MoI. Biol. 150:1-14); and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
- marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
- sequence encoding ion channel is inserted within a marker gene sequence, transformed cells containing sequences encoding ion channel can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding KCNMA3 under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
- host cells which contain the nucleic acid sequence encoding KCNMA3 and express KCNMA3 may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
- polynucleotide sequences encoding KCNMA3 can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding KCNMA3.
- Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding KCNMA3 to detect transformants containing DNA or RNA encoding KCNMA3.
- a variety of protocols for detecting and measuring the expression of KCNMA3, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
- ELISAs enzyme-linked immunosorbent assays
- RIAs radioimmunoassays
- FACS fluorescence activated cell sorting
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on KCNMA3 is preferred, but a competitive binding assay may be employed.
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding KCNMA3 include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
- the sequences encoding KCNMA3, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
- RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
- T7, T3, or SP6 RNA polymerase
- Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host cells transformed with nucleotide sequences encoding KCNMA3 may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
- the protein produced by a transformed cell may be located in the cell membrane, secreted or contained intracellularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode KCNMA3 may be designed to contain signal sequences which direct secretion of KCNMA3 through a prokaryotic or eukaryotic cell membrane.
- Other constructions may be used to join sequences encoding KCNMA3 to nucleotide sequences encoding a polypeptide domain which will facilitate purification of soluble proteins.
- Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
- metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
- protein A domains that allow purification on immobilized immunoglobulin
- FLAGS extension/affinity purification system Immunex Corp., Seattle, Wash.
- cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif) between the purification domain and the KCNMA3 encoding sequence may be used to facilitate purification.
- One such expression vector provides for expression of a fusion protein containing KCNMA3 and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
- the histidine residues facilitate purification on immobilized metal ion affinity chromatography (IMIAC; described in Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281), while the enterokinase cleavage site provides a means for purifying KCNMA3 from the fusion protein.
- IMIAC immobilized metal ion affinity chromatography
- KCNMA3 may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 43 IA peptide synthesizer (Perkin Elmer). Various fragments of K.CNMA3 may be synthesized separately and then combined to produce the full length molecule.
- the KCNMA3 polypeptides, nucleic acids, probes, antibodies, expression vectors and ligands are useful as (and for the production of) biosensors.
- a biosensor is defined as being a unique combination of a receptor for molecular recognition, for example a selective layer with immobilized antibodies or receptors such as an ion channel, and a transducer for transmitting the values measured.
- a receptor for molecular recognition for example a selective layer with immobilized antibodies or receptors such as an ion channel
- a transducer for transmitting the values measured.
- One group of such biosensors will detect the change which is caused in the optical properties of a surface layer due to the interaction of the receptor with the surrounding medium.
- ellipso-metry and surface plasmon resonance may be mentioned especially ellipso-metry and surface plasmon resonance.
- Biosensors incorporating KCNMA3 may be used to detect the presence or level of KCNMA3 ligands, for example, nucleotides such as purines or purine analogues, or analogues of these ligands.
- nucleotides such as purines or purine analogues, or analogues of these ligands.
- the construction of such biosensors is well known in the art.
- KCNMA3 polypeptide of the present invention may be employed in a screening process for compounds which bind and which activate (agonists) or inhibit activation of (antagonists) of KCNMA3.
- KCNM A3 polypeptides may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell- free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).
- KCNMA3 polypeptides are responsible for many biological functions, including many pathology such as Alzheimer's disease, ishaemic/vascular dementia, Pick's disease, diffuse Lewy body dementia, frontotemporal dementias, corticobasal degeneration, Huntington's disease, progressive supranuclear palsy, AIDS/HIV dementia, prion infections, encephalitis, neurosyphilis, vasculitis, progressive multifocal leukoencephalopathy.
- Partial epilepsies lesional, medial temporal lobe, non lesional neocortical and idiopathic (genetic) partial epilepsies, consisting simple partial seizures, complex partial seizures, secondarily generalised clonic tonic seizures, benign Rolandic and occipital seizures, generalised epilepsies including West's syndrome, Lennox Gastaut syndrome and progressive myoclonus epilepsy, consisting absence seizures, clonic tonix seizures, myoclonic seizures, tonic seizures, clonic seizures, atonic seizures and clonic- tonic-clonic seizures, recurrent status epilepticus, neonatal seizures and rare or isolated seizures.
- Rational design of candidate compounds likely to be able to interact with KCNMA3 protein may be based upon structural studies of the molecular shapes of a polypeptide according to the invention.
- One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., X-ray crystallography or two-dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions.
- X-ray crystallography or two-dimensional NMR techniques.
- An alternative to rational design uses a screening procedure which involves in general producing appropriate cells which express the KCNMA3 receptor polypeptide of the present invention on the surface thereof.
- Such cells include cells from animals, yeast, Drosophila or E. coli.
- Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
- Xenopus oocytes may be injected with KCNMA3 mRNA or polypeptide, and currents induced by exposure to test compounds measured by use of voltage clamps measured, as described in further detail elsewhere.
- microphysiometric assays may be employed to assay KCNMA3 activity.
- Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell.
- the acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signalling process.
- the pH changes in the media surrounding the cell are very small but are detectable by, for example, the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, Calif).
- the CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing intracellular signaling pathway such as the G- protein coupled receptor of the present invention.
- the KCNMA3 ion channel may also be functionally screened (using ooyte electrophysiology, etc., functional screens) against tissue extracts to identify natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated, with the fractions being assayed as described here, until an activating ligand is isolated and identified.
- HEK 293 cells expressing KCNMA3 or recombinant KCNMA3 ion channel are loaded with fura 2 and in a single day more than 150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization.
- HEK 293 cells expressing KCNMA3 ion channel or recombinant KCNMA3 ion channel are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.
- Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as described in U.S. Pat. No. 5,482,835, incorporated by reference herein.
- Phage display is a protocol of molecular screening which utilises recombinant bacteriophage.
- the technology involves transforming bacteriophage with a gene that encodes one compound from the library of candidate compounds, such that each phage or phagemid expresses a particular candidate compound.
- the transformed bacteriophage (which preferably is tethered to a solid support) expresses the appropriate candidate compound and displays it on their phage coat.
- Specific candidate compounds which are capable of binding to a polypeptide or peptide are enriched by selection strategies based on affinity interaction.
- the successful candidate agents are then characterised.
- Phage display has advantages over standard affinity ligand screening technologies.
- the phage surface displays the candidate agent in a three dimensional configuration, more closely resembling its naturally occurring conformation. This allows for more specific and higher affinity binding for screening purposes.
- Another method of screening a library of compounds utilises eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing a library of compounds.
- Such cells either in viable or fixed form, can be used for standard binding-partner assays. See also Parce et al. (1989) Science 246:243-247; and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA 87;4007-4011, which describe sensitive methods to detect cellular responses.
- This separation step could typically involve a procedure such as adhesion to filters followed by washing, adhesion to plastic following by washing, or centrifugation of the cell membranes.
- the present invention therefore also provides a compound capable of binding specifically to a KCNMA3 ion channel of the present invention.
- compound refers to a chemical compound (naturally occurring or synthesised), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
- a biological macromolecule e.g., nucleic acid, protein, non-peptide, or organic molecule
- an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
- the compound is an antibody.
- the materials necessary for such screening to be conducted may be packaged into a screening kit.
- a screening kit is useful for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for KCNMA3 polypeptides or compounds which decrease or enhance the production of KCNMA3 polypeptides.
- the screening kit comprises: (a) a KCNMA3 ion channel polypeptide; (b) a recombinant cell expressing a KCNMA3 ion channel polypeptide; (c) a cell membrane expressing a KCNMA3 ion channel polypeptide; or (d) antibody to a KCNMA3 ion channel polypeptide.
- the screening kit may optionally comprise instructions for use. TRANSGENIC ANIMALS
- the present invention further encompasses transgenic animals capable of expressing natural or recombinant KCNMA3 ion channel, or a homologue, variant or derivative, at elevated or reduced levels compared to the normal expression level.
- transgenic animals ("ion channel knockouts") which do not express functional KCNMA3 ion channel as a result of one or more loss of function mutations, including a deletion, of the KCNMA3 ion channel gene.
- a transgenic animal is a non-human mammal, such as a pig, a sheep or a rodent.
- the transgenic animal is a mouse or a rat.
- Such transgenic animals as well as animals that wild-type for KCNMA3 may be used in screening procedures to identify agonists and/or antagonists of KCNMA3 ion channel, as well as to test for their efficacy as treatments for diseases in vivo.
- wild-type and transgenic animals that have been engineered to be deficient in the production of KCNMA3 ion channels may be used in assays to identify agonists and/or antagonists of KCNMA3.
- One assay is designed to evaluate a potential drug (a candidate ligand or compound) to determine if it produces a physiological response in the absence of KCNMA3. This may be accomplished by administering the drug to a transgenic animal as discussed above, and then assaying the animal for a particular response.
- preferred responses include one or more of the following: changes to disease resistance; altered inflammatory responses; altered tumour susceptability: a change in blood pressure; neovascularization; a change in eating behaviour; a change in body weight; a change in bone density; a change in body temperature; insulin secretion; gonadotropin secretion; nasal and bronchial secretion; vasoconstriction; loss reference and working memory; hyper- or hypoactivity constitutively or in response to novelty; anxiety; hyporeflexia or hyperreflexia; pain or stress responses.
- Tissues derived from wild-type and the KCNMA3 ion channel knockout animals may be used in assays to selective modulators (a candidate ligand or compound) of the KCNMA3 ion channel.
- a selective modulator would be seen to have an effect on wild- type but not knockout animal tissue.
- Such assays can be conducted by obtaining a first receptor preparation from the transgenic animal engineered to be deficient in KCNMA3 ion channel production and a second receptor preparation from a source known to bind any identified KCNMA3 ion channel ligands or compounds.
- the first and second receptor preparations will be similar in all respects except for the source from which they are obtained.
- the candidate ligand or compound will be examined at several different concentrations.
- Tissues derived from wild-type and transgenic animals may be used in assays directly or the tissues may be processed to isolate membranes or membrane proteins, which are themselves used in the assays.
- a preferred transgenic animal is the mouse.
- the ligand may be labeled using any means compatible with binding assays. This would include, without limitation, radioactive, enzymatic, fluorescent or chemiluminescent labeling (as well as other labelling techniques as described in further detail above).
- antagonists of KCNMA3 ion channel maybe identified by administering candidate compounds, etc, to wild type animals expressing functional KCNMA3 ion channel, and animals identified which exhibit any of the phenotypic characteristics associated with reduced or abolished expression of KCNMA3 ion channel function.
- Transgenic gene constructs can be introduced into the germ line of an animal to make a transgenic mammal. For example, one or several copies of the construct may be incorporated into the genome of a mammalian embryo by standard transgenic techniques.
- the transgene into the embryo can be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
- the KCNMA3 ion channel transgei ⁇ ⁇ be introduced into a mammal by microinjection of the construct into the pronuclei of the fertilized mammalian egg(s) to cause one or more copies of the construct to be retained in the cells of the developing mammal(s).
- the egg may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention.
- the progeny of the transgenically manipulated embryos can be tested for the presence of the construct by Southern blot analysis of the segment of tissue. If one or more copies of the exogenous cloned construct remains stably integrated into the genome of such transgenic embryos, it is possible to establish permanent transgenic mammal lines carrying the transgenically added construct.
- the litters of transgenically altered mammals can be assayed after birth for the incorporation of the construct into the genome of the offspring.
- this assay is accomplished by hybridizing a probe corresponding to the DNA sequence coding for the desired recombinant protein product or a segment thereof onto chromosomal material from the progeny.
- Those mammalian progeny found to contain at least one copy of the construct in their genome are grown to maturity.
- a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.
- the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes.
- the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism.
- a euploid zygote is preferred. If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
- the biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
- the number of copies of the trans gene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1 ,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. As regards the present invention, there will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences.
- exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.
- Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.
- Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
- DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene.
- the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
- Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
- suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.
- Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
- Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.
- the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
- the partner may be a parental line.
- in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
- the transgenic animals produced in accordance with the present invention will include exogenous genetic material.
- the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of a ion channel. Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
- the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
- transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
- ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448).
- Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction.
- Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
- Jaenisch, R. (1988) Science 240:1468-1474 For review see Jaenisch, R. (1988) Science 240:1468-1474.
- transgenic animals where the transgenic animal is characterized by having an altered KCNMA3 ion channel gene, preferably as described above, as models for KCNMA3 ion channel function.
- Alterations to the gene include deletions or other loss of function mutations, introduction of an exogenous gene having a nucleotide sequence with targeted or random mutations, introduction of an exogenous gene from another species, or a combination thereof.
- the transgenic animals may be either homozygous or heterozygous for the alteration.
- the animals and cells derived therefrom are useful for screening biologically active agents that may modulate KCNMA3 ion channel function. The screening methods are of particular use for determining the specificity and action of potential therapies for a number of diseases.
- the animals are useful as a model to investigate the role of ion channel in normal brain, heart, spleen and liver function.
- Another aspect pertains to a transgenic nonhuman animal having a functionally disrupted endogenous KCNMA3 ion channel gene but which also carries in its genome, and expresses, a transgene encoding a heterologous KCNMA3 ion channel protein (i.e., a ion channel from another species).
- the animal is a mouse and the heterologous KCNMA3 ion channel is a human KCNMA3 ion channel.
- An animal, or cell lines derived from such an animal, which has been reconstituted with human KCNMA3 ion channel, can be used to identify agents that inhibit human KCNMA3 ion channel in vivo and in vitro.
- a stimulus that induces signalling through human KCNMA3 ion channel can be administered to the animal, or cell line, in the presence and absence of an agent to be tested and the response in the animal, or cell line, can be measured.
- An agent that inhibits human KCNMA3 ion channel in vivo or in vitro can be identified based upon a decreased response in the presence of the agent compared to the response in the absence of the agent.
- the present invention also provides for a KCNMA3 ion channel deficient transgenic non-human animal (a "KCNMA3 ion channel knock-out").
- a KCNMA3 ion channel deficient transgenic non-human animal a "KCNMA3 ion channel knock-out"
- Such an animal is one which expresses lowered or no KCNMA3 ion channel activity, preferably as a result of an endogenous KCNMA3 ion channel genomic sequence being disrupted or deleted.
- such an animal expresses no activity.
- the animal expresses no activity of the KCNMA3 ion channel shown as SEQ ID NO: 3 or SEQ ID NO: 5.
- KCNMA3 ion channel knock-outs may be generated by various means known in the art, as described in further detail below.
- the present invention also pertains to a nucleic acid construct for functionally disrupting a KCNMA3 ion channel gene in a host cell.
- the nucleic acid construct comprises: a) a non-homologous replacement portion; b) a first homology region located upstream of the non-homologous replacement portion, the first homology region having a nucleotide sequence with substantial identity to a first KCNMA3 ion channel gene sequence; and c) a second homology region located downstream of the non-homologous replacement portion, the second homology region having a nucleotide sequence with substantial identity to a second KCNMA3 ion channel gene sequence, the second KCNMA3 ion channel gene sequence having a location downstream of the first KCNMA3 ion channel gene sequence in a naturally occurring endogenous KCNMA3 ion channel gene.
- Another aspect pertains to recombinant vectors into which the nucleic acid construct has been incorporated.
- Yet another aspect pertains to host cells into which the nucleic acid construct has been introduced to thereby allow homologous recombination between the nucleic acid construct and an endogenous KCNMA3 ion channel gene of the host cell, resulting in functional disruption of the endogenous KCNMA3 ion channel gene.
- the host cell can be a mammalian cell that normally expresses KCNMA3 ion channel from the liver, brain, spleen or heart, or a pluripotent cell, such as a mouse embryonic stem cell.
- an embryonic stem cell into which the nucleic acid construct has been introduced and homologously recombined with the endogenous KCNMA3 ion channel gene produces a transgenic nonhuman animal having cells that are descendant from the embryonic stem cell and thus carry the ion channel gene disruption in their genome.
- Animals that carry the KCNMA3 ion channel gene disruption in their germline can then be selected and bred to produce animals having the KCNMA3 ion channel gene disruption in all somatic and germ cells. Such mice can then be bred to homozygosity for the KCNMA3 ion channel gene disruption.
- the term "antibody”, unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
- Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
- the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
- antibodies with fully human variable regions (or their fragments), for example, as described in US Patent Nos. 5,545,807 and 6,075,181 may also be used.
- Neutralizing antibodies i.e., those which inhibit biological activity of the substance amino acid sequences, are especially preferred for diagnostics and therapeutics.
- Antibodies may be produced by standard techniques, such as by immunisation or by using a phage display library.
- a polypeptide or peptide of the present invention may be used to develop an antibody by known techniques.
- Such an antibody may be capable of binding specifically to the ion channel protein or homologue, fragment, etc.
- a selected mammal e.g., mouse, rabbit, goat, horse, etc.
- an immunogenic composition comprising a polypeptide or peptide of the present invention.
- various adjuvants may be used to increase immunological response.
- adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
- BCG Bacilli Calmette- Gueriri
- Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified the substance amino acid sequence is administered to immunologically compromised individuals for the purpose of stimulating systemic defence.
- Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope obtainable from a polypeptide of the present invention contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaff ⁇ nity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides amino acid sequences or fragments thereof haptenised to another amino acid sequence for use as immunogens in animals or humans.
- Monoclonal antibodies directed against epitopes obtainable from a polypeptide or peptide of the present invention can also be readily produced by one skilled in the art.
- the general methodology for making monoclonal antibodies by hybridomas is well known.
- Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
- Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
- Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).
- Antibodies both monoclonal and polyclonal, which are directed against epitopes obtainable from a polypeptide or peptide of the present invention are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy.
- Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
- Antiidiotype antibodies are immunoglobulins which carry an "internal image" of the substance and/or agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful in therapy.
- Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
- Antibody fragments which contain specific binding sites for the polypeptide or peptide may also be generated.
- fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
- Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1).
- Antibodies against ion channel polypeptides may also be employed to treat diseases.
- This invention also relates to the use of ion channel polynucleotides and polypeptides (as well as homologues, variants and derivatives thereof) for use in diagnosis as diagnostic reagents or in genetic analysis.
- Nucleic acids complementary to or capable of hybridising to ion channel nucleic acids (including homologues, variants and derivatives), as well as antibodies against KCNMA3 ion channel polypeptides are also useful in such assays.
- Detection of a mutated form of the KCNMA3 ion channel gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over- expression or altered expression of KCNMA3 ion channel.
- Individuals carrying mutations in the KCNMA3 ion channel gene may be detected at the DNA level by a variety of techniques.
- DNA may be isolated from a patient and the DNA polymorphism pattern of KCNMA3 ion channel determined. The identified pattern is compared to controls of patients known to be suffering from a disease associated with over-, under- or abnormal expression of KCNMA3 ion channel. Patients expressing a genetic polymorphism pattern associated with KCNMA3 ion channel associated disease may then be identified. Genetic analysis of the KCNMA3 ion channel gene may be conducted by any technique known in the art. For example, individuals may be screened by determining DNA sequence of a KCNMA3 ion channel allele, by RFLP or SNP analysis, etc.
- Patients may be identified as having a genetic predisposition for a disease associated with the over-, under-, or abnormal expression of KCNMA3 ion channel by detecting the presence of a DNA polymorphism in the gene sequence for KCNMA3 ion channel or any sequence controlling its expression.
- KCNMA3 ion channel associated diseases include cognitive disorders, for example Alzheimer's disease, Lewy body dementia, multiinfarct dementia, Pick's disease, frontotemporal dementias, progressive supranuclear palsy, epilepsy, autism, ADHD and status epilepticus.
- the present invention further discloses a kit for the identification of a patient's genetic polymorphism pattern associated with ion channel associated disease.
- the kit includes DNA sample collecting means and means for determining a genetic polymorphism pattern, which is then compared to control samples to determine a patient's susceptibility to KCNMA3 ion channel associated disease.
- Kits for diagnosis of a KCNMA3 ion channel associated disease comprising KCNMA3 ion channel polypeptide and/or an antibody against such a polypeptide (or fragment of it) are also provided.
- Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
- the DNA is obtained from blood cells obtained from a finger prick of the patient with the blood collected on absorbent paper.
- the blood will be collected on an AmpliCard.TM. (University of Sheffield, Department of Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, England SlO 2JF).
- the DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
- Oligonucleotide DNA primers that target the specific polymorphic DNA region within the genes of interest may be prepared so that in the PCR reaction amplification of the target sequences is achieved.
- RNA or cDNA may also be used as templates in similar fashion.
- the amplified DNA sequences from the template DNA may then be analyzed using restriction enzymes to determine the genetic polymorphisms present in the amplified sequences and thereby provide a genetic polymorphism profile of the patient. Restriction fragments lengths may be identified by gel analysis. Alternatively, or in conjunction, techniques such as SNP (single nucleotide polymorphisms) analysis may be employed.
- Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
- Point mutations can be identified by hybridizing amplified DNA to labeled ion channel nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, eg., Myers et al, Science (1985)230:1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method.
- an array of oligonucleotides probes comprising the KCNMA3 ion channel nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
- Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al., Science, VoI 274, pp 610-613 (1996)).
- Single strand conformation polymorphism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79).
- Single-stranded DNA fragments of sample and control KCNMA3 ion channel nucleic acids may 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 labelled or detected with labelled probes.
- RNA rather than DNA
- 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 diagnostic assays offer a process for diagnosing or determining a susceptibility to infections such as disease through detection of mutation in the KCNMA3 ion channel gene by the methods described.
- KCNMA3 ion channel polypeptides and nucleic acids may be detected in a sample.
- infections and diseases as listed above can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of the KCNMA3 ion channel polypeptide or KCNMA3 ion channel mRNA.
- the sample may comprise a cell or tissue sample from an organism suffering or suspected to be suffering from a disease associated with increased, reduced or otherwise abnormal KCNMA3 ion channel expression, including spatial or temporal changes in level or pattern of expression.
- the level or pattern of expression of KCNMA3 ion channel in an organism suffering from or suspected to be suffering from such a disease may be usefully compared with the level or pattern of expression in a normal organism as a means of diagnosis of disease.
- the invention includes a method of detecting the presence of a nucleic acid comprising a KCNMA3 ion channel nucleic acid in a sample, by contacting the sample with at least one nucleic acid probe which is specific for said nucleic acid and monitoring said sample for the presence of the nucleic acid.
- the nucleic acid probe may specifically bind to the KCNMA3 ion channel nucleic acid, or a portion of it, and binding between the two detected; the presence of the complex itself may also be detected.
- the invention encompasses a method of detecting the presence of a KCNMA3 ion channel polypeptide by contacting a cell sample with an antibody capable of binding the polypeptide and monitoring said sample for the presence of the polypeptide.
- Methods of detecting binding between two entities include FRET (fluorescence resonance energy transfer), surface plasmon resonance, etc.
- Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
- Assay techniques that can be used to determine levels of a protein, such as a KCNMA3 ion channel, in a sample derived from a host are well-known to those of skill in the art.
- Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
- the present invention relates to a diagnostic kit for a disease or susceptibility to a disease (including an infection), for example, disease.
- the diagnostic kit comprises a KCNMA3 ion channel polynucleotide or a fragment thereof; a complementary nucleotide sequence; a KCNMA3 ion channel polypeptide or a fragment thereof, or an antibody to a KCNMA3 ion channel polypeptide.
- nucleotide sequences of the present invention are also valuable for chromosome identification.
- the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. As described above, human ion channel is found to map to chromosome.
- mapping of relevant sequences to chromosomes is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian heritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
- the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
- This invention provides methods of treating an abnormal condition related to both an excess of or insufficient amount of KCNMA3 ion channel activity.
- One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the KCNMA3 ion channel, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
- soluble forms of KCNMA3 ion channel polypeptides still capable of binding the ligand in competition with endogenous KCNMA3 ion channel may be administered.
- Typical embodiments of such competitors comprise fragments of the KCNMA3 ion channel polypeptide.
- expression of the gene encoding endogenous KCNMA3 ion channel can be inhibited using expression blocking techniques.
- Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, JNeurochem (1991) 56:560 in Oligodeoxvnucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FIa. (1988).
- oligonucleotides which form triple helices with the gene can be supplied.
- KCNMA3 ion channel For treating abnormal conditions related to an under-expression of KCNMA3 ion channel and its activity, several approaches are also available.
- One approach comprises administering to a subject a therapeutically effective amount of a compound which activates ion channel, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition.
- gene therapy may be employed to effect the endogenous production of ion channel by the relevant cells in the subject.
- a polynucleotide may be engineered for expression in a replication defective retroviral vector, as discussed above.
- the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
- These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
- gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic- based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).
- Peptides such as the soluble form of KCNMA3 ion channel polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier.
- a suitable pharmaceutical carrier comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient.
- Such carriers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art.
- the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions.
- Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
- systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
- Other injection routes such as subcutaneous, intramuscular, or intraperitoneal, can be used.
- Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
- penetrants such as bile salts or fusidic acids or other detergents.
- oral administration may also be possible. Administration of these compounds may also be topical and/or localize, in the form of salves, pastes, gels and the like.
- the dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
- Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
- cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
- a polynucleotide such as a DNA or RNA
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising administering a therapeutically effective amount of the polypeptide, polynucleotide, peptide, vector or antibody of the present invention and optionally a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof).
- the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
- Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
- the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
- the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
- Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
- preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
- Antioxidants and suspending agents may be also used.
- the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
- the formulation may be designed to be delivered by both routes.
- the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
- compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.
- compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
- compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
- Another embodiment relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with the KCNMA3 ion channel polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from diseases among others.
- Yet another embodiment relates to a method of inducing immunological response in a mammal which comprises delivering a KCNMA3 ion channel polypeptide via a vector directing expression of a ion channel polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
- a further embodiment relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a KCNMA3 ion channel polypeptide wherein the composition comprises a KCNMA3 ion channel polypeptide or KCNMA3 ion channel gene.
- the vaccine formulation may further comprise a suitable carrier.
- the KCNMA3 ion channel polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
- parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
- the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
- the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
- Vaccines may be prepared from one or more polypeptides or peptides of the present invention.
- the preparation of vaccines which contain an immunogenic polypeptide(s) or peptide(s) as active ingredient(s), is known to one skilled in the art.
- such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
- the preparation may also be emulsified, or the protein encapsulated in liposomes.
- the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
- the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
- auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
- adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmurarnyl-L-alanyl-D-isoglutaminyl-L- alanine-2-(r-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835 A
- adjuvants and other agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
- adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan).
- adjuvants such as Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human use.
- the proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
- aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al 2 O 3 basis).
- the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 ⁇ g/ml, preferably 5 to 50 ⁇ g/ml, most preferably 15 ⁇ g/ml.
- the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4 0 C, or it may be freeze-dried. Lyophilisation permits long-term storage in a stabilised form.
- the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
- Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
- suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
- compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%.
- the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer
- Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S”, Eudragit "L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
- the polypeptides may be formulated into the vaccine as neutral or salt forms.
- Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine. ADMINISTRATION
- a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
- the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
- compositions of the present invention may be administered by direct injection.
- the composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
- each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
- the term "administered” includes delivery by viral or non-viral techniques.
- Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.
- Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
- the routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
- administered includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
- co-administered means that the site and time of administration of each of for example, the polypeptide of the present invention and an additional entity such as adjuvant are such that the necessary modulation of the immune system is achieved.
- the polypeptide and the adjuvant may be administered at the same moment in time and at the same site, there may be advantages in administering the polypeptide at a different time and to a different site from the adjuvant.
- the polypeptide and adjuvant may even be delivered in the same delivery vehicle - and the polypeptide and the antigen may be coupled and/or uncoupled and/or genetically coupled and/or uncoupled.
- the polypeptide, polynucleotide, peptide, nucleotide, antibody and optionally an adjuvant may be administered separately or co-administered to the host subject as a single dose or in multiple doses.
- the vaccine composition and pharmaceutical compositions of the present invention may be administered by a number of different routes such as injection (which includes parenteral, subcutaneous and intramuscular injection) intranasal, mucosal, oral, intra-vaginal, urethral or ocular administration.
- injection which includes parenteral, subcutaneous and intramuscular injection
- mucosal mucosal
- oral intra-vaginal
- urethral urethral
- the vaccines and pharmaceutical compositions of the present invention may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
- Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
- suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, may be 1% to 2%.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
- KCNMA3 polypeptide comprising an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto, in a method of identifying a molecule suitable for the treatment or alleviation of an KCNMA3 associated disease.
- Paragraph 3 A method according to Paragraph 1 or 2, in which the candidate molecule is exposed to a KCNMA3 polypeptide in order to determine if the candidate molecule is an agonist or antagonist thereof.
- Paragraph 4 A method according to Paragraph 1 , 2 or 3, in which candidate molecule is exposed to a cell expressing a KCNMA3 polypeptide.
- Paragraph 5 A method according to Paragraph 4, in which a change in intracellular cyclic AMP (cAMP) or calcium levels is detected.
- cAMP cyclic AMP
- Paragraph 6 A method according to Paragraph 4 or 5, in which a decrease in intracellular cyclic AMP levels is detected to identify an antagonist of KCNMA3 polypeptide.
- KCNM A3 polynucleotide comprising a nucleic acid sequence shown in SEQ ID No. 1 , SEQ ID No.2 or SEQ ID NO: 4 or a sequence having at least 90% sequence identity thereto, for the identification of a molecule suitable for the treatment or alleviation of an KCNMA3 associated disease.
- Paragraph 9 A method of identifying an agonist or antagonist of a KCNMA3 polypeptide comprising an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto, the method comprising administering a candidate molecule to an animal and determining whether the animal exhibits lower tendency to spontaneous alternate.
- Paragraph 10 A method according to Paragraph 9, in which the animal expresses functional KCNMA3 polypeptide.
- Paragraph 11 A method according to Paragraph 9 or 10, in which the animal is a wild type animal.
- Paragraph 12 A method according to Paragraph 9, 10 or 11, in which the animal is a rodent, preferably a mouse.
- Paragraph 13 A method according to any of Paragraph s 9 to 12, in which the determination is made using a Y-maze test.
- Paragraph 14 A method for providing an indication useful in the diagnosis of or a determination of susceptibility to an KCNMA3 associated disease in an individual, the method comprising detecting a change in the expression pattern or level of a KCNMA3 polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto in a sample from the individual.
- Paragraph 15 A method for providing an indication useful in the diagnosis of or a determination of susceptibility to an KCNMA3 associated disease in an individual, the method comprising detecting a polymorphism in a KCNMA3 polynucleotide comprising a nucleic acid sequence shown in SEQ ID No. 1 , SEQ ID No.2 or SEQ ID NO: 4 or a sequence having at least 90% sequence identity thereto, in a sample from the individual.
- Paragraph 16 A method according to any preceding Paragraph , in which the agonist or antagonist comprises an immunoglobulin, preferably an antibody preferably capable of binding specifically to a KCNMA3 polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto.
- an immunoglobulin preferably an antibody preferably capable of binding specifically to a KCNMA3 polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto.
- Paragraph 17 A non-human animal which displays lower tendency to spontaneous alternate when compared to a wild-type animal, the animal being a transgenic animal having a functionally disrupted endogenous KCNMA3 gene, in which the KCNMA3 gene comprises a nucleic acid sequence shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4 or a sequence having at least 90% sequence identity thereto.
- Paragraph 18 A non-human animal according to Paragraph 17, which has a deletion in a KCNMA3 gene or a portion thereof.
- Paragraph 19 A non-human animal according to Paragraph 17 or 18, in which the animal displays lower tendency to spontaneous alternate when measured in a Y-maze test.
- Paragraph 20 A non-human animal according to any of Paragraph s 17, 18 or 19 which is a rodent, preferably a mouse.
- Paragraph 21 A non-human animal according to Paragraph 20, which comprises a functionally disrupted KCNMA3 gene, preferably a deletion in a KCNMA3 gene, in which the KCNMA3 gene comprises a nucleic acid sequence shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4 or a sequence having at least 90% sequence identity thereto.
- Paragraph 22 An isolated cell or tissue from a non-human animal according to any of Paragraph s 17 to 21.
- Paragraph 23 Use of a non-human animal according to any of Paragraph s 17 to 21 , or an isolated cell or tissue thereof according to Paragraph 22, in a method of identifying an agonist or antagonist of a KCNMA3 polypeptide for the treatment or alleviation of an KCNMA3 associated disease, the KCNMA3 polypeptide comprising an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto.
- Paragraph 24 Use of a non-human animal according to any of Paragraph s 17 to 21, or an isolated cell or tissue thereof according to Paragraph 22 as a model for an KCNMA3 associated disease.
- Paragraph 25 Use of an agonist or antagonist of a KCNMA3 polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5 or a sequence having at least 90% sequence identity thereto for the preparation of a pharmaceutical composition for the treatment of an KCNMA3 associated disease in an individual.
- the KCNMA3 gene was identified bio-informatically using homology searches of genome databases. A 130kb gapped genomic contig was assembled from various databases. This contig provided sufficient flanking sequence information to enable the design of homologous arms to clone into the targeting vector.
- the murine KCNMA3 gene has 27 coding exons.
- the targeting strategy is designed to disrupt the gene at coding exon 3 by inserting a cassette containing multiple transcriptional termination signalsl.
- a 1.6kb 5' homologous arm and a 4.2kb 3' homologous arm flanking the region to be deleted are amplified by PCR and the fragments are cloned into the targeting vector.
- the 5' end of each oligonucleotide primer used to amplify the arms is synthesised to contain a different recognition site for a rare- cutting restriction enzyme, compatible with the cloning sites of the vector polylinkers and absent from the arms themselves.
- the primers are designed as listed in the primer table below, with 5' arm cloning sites of Notl/Spel and 3 'arm cloning sites of Ascl/Fsel (the structure of the targeting vector used, including the relevant restriction sites, is shown in Figure 2).
- primers specific to the KCNMA3 locus are designed for the following purposes: 5' and 3' probe primer pairs (5'prF/5'prR and 3'prF/3'prR) to amplify two short 150- 300bp fragments of non-repetitive genomic DNA external to and extending beyond each arm, to allow Southern analysis of the targeted locus, in isolated putative targeted clones; a mouse genotyping primer pair (hetF and hetR) which allows differentiation between wild-type, heterozygote and homozygous mice, when used in a multiplex PCR with a vector specific primer, in this case, Asc350; and lastly, a target screening primer (5'scr) which anneals upstream of the end of the 5' arm region, and which produces a target event specific 1.6kbkb amplimer when paired with a primer specific to the 5' end of the vector (TK5IBLM
- This amplimer can only be derived from template DNA from cells where the desired genomic alteration has occurred and allows the identification of correctly targeted cells from the background of clones containing randomly integrated copies of the vector.
- the location of these primers and the genomic structure of the regions of the KCNMA3 locus used in the targeting strategy is shown in SEQ ID NO: 19.
- KCNMA3 Primer Sequences musKCNMA3 5'prF AAGAGTACTGTTATGGGGGTCTCTGTG SEQ ID NO. 6 musKCNMA3 5'prR ACACGGACTTGAGCTAGTCATCACCAG SEQ ID NO.7 musKCNMA3 5'scr DR2 TCTGGTGATGACTAGCTCAAGTCCGTG SEQ ID NO.8 musKCNMA3 5'armF SEQ ID NO.9
- a targeting vector is prepared where the KCNMA3 region to be disrupted is replaced with non-homologous sequences composed of an endogenous gene expression reporter (a frame independent lacZ gene) upstream of a selection cassette composed of a promoted neomycin phosphotransferase (neo) gene arranged in the same orientation as the KCNMA3 gene.
- endogenous gene expression reporter a frame independent lacZ gene
- the transfected cells are cultured for 9 days in medium containing 200 ⁇ g/ml neomycin.
- Clones are picked into 96 well plates, replicated and expanded before being screened by PCR (using primers 5'scr and DR2, as described above) to identify clones in which homologous recombination has occurred between the endogenous KCNMA3 gene and the targeting construct. Positive clones can be identified at a rate of 1 to 5%.
- BspHI digested genomic DNA will give an 8.2kb wild-type band and a 13.7kb targeted band; and with the 3' probe, EcoRV cut genomic DNA will give a 10.9kb wild-type band and an 12.5kb targeted band.
- C57BL/6 female and male mice are mated and blastocysts are isolated at 3.5 days of gestation. 10-12 cells from a chosen clone are injected per blastocyst and 7-8 blastocysts are implanted in the uterus of a pseudopregnant Fl female. A litter of chimeric pups are born containing several high level (up to 100%) agouti males (the agouti coat colour indicates the contribution of cells descended from the targeted clone). These male chimeras are mated with female MFl and 129 mice, and germline transmission is determined by the agouti coat colour and by PCR genotyping respectively.
- PCR Genotyping is carried out on lysed tail clips, using the primers hetF and hetR with a third, vector specific primer (Asc350).
- This multiplex PCR allows amplification from the wild-type locus (if present) from primers hetF and hetR giving a 288bp band.
- the site for hetF is deleted in the knockout mice, so this amplification will fail from a targeted allele.
- the Asc350 primer will amplify a 418 bp band from the targeted locus, in combination with the hetR primer which anneals to a region just inside the 3 ' arm.
- this multiplex PCR reveals the genotype of the litters as follows: wild- type samples exhibit a single 288 bp band; heterozygous DNA samples yield two bands at 288 bp and 418bp; and the homozygous samples will show only the target specific 418 bp band.
- Aim To measure spontaneous alternation in mice. The Y-maze test exploits the natural exploratory behaviour in mice, they will spontaneously alternate between arms, entering the arm least frequently explored. Therefore, if there are three arms (A, B, C) they would enter A, then B, then C, then A, then B, then C.
- Apparatus Clear plastic Y-maze mounted on stand underneath camera.
- mice should be tested for each sex and for each genotype (wildtype control and knockout) on 129Ev/Sv background.
- Protocol 1. Set up Y-maze and video recording equipment so that the whole maze is within frame.
- mice Place the mice in the centre square, facing one of the open arm. Click on the Start Button to start recording. Each mouse will be tested once for 5 minutes; no retests can be carried out.
- the data is analysed and the number of spontaneous alternations calculated.
- the percentage of spontaneous alternations out of a possible alternations is expressed graphically.
- CGTCTCAGACTCTX ⁇ GGGTAGAAGACCTATGCTCCAAGTTATTAAATATTTAACGAGACCTAAACCCCACATCTCCTGTGT ⁇ TGAGCGTGTAAACCAGGT GCAGAGTCTK ⁇ GACTCCCATCTTCTGGATACGAGGTTCAATAATTTATAAATTGCTCTGGATTTGGGGTGTAGAGGACACAAACTCGCACATTTGCR ⁇
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