WO2006096648A2 - Modele de souris - Google Patents

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WO2006096648A2
WO2006096648A2 PCT/US2006/007941 US2006007941W WO2006096648A2 WO 2006096648 A2 WO2006096648 A2 WO 2006096648A2 US 2006007941 W US2006007941 W US 2006007941W WO 2006096648 A2 WO2006096648 A2 WO 2006096648A2
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mutation
mice
kcnq3
seizure
kcnq2
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WO2006096648A3 (fr
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Nanda A. Singh
Mark F. Leppert
H. Steve White
Karen S. Wilcox
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University Of Utah Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • Chrna4 hemizygosity (such as that observed in the Sztl mouse) has not been well characterized.
  • Heterozygous knockout Chrna ⁇ 1' mice display no observable seizure phenotype (McCoIl et al., 2003) and display modestly increased sensitivity to the proconvulsant pentylenetetrazole (PTZ), kainic acid (KA), and bicuculline (BIC), and decreased sensitivity to 4-amino ⁇ yridine (4-AP) (Ross et al., 2000; Wong et al., 2002).
  • ARFGAP-1 is an ADP ribosylating factor that regulates the formation of coat protein 1 (COP-I) vesicles (Yang et al., 2000), but the effects of altered ARFGAP-1 function on neuroexcitability have not been examined.
  • the present invention relates to transgenic mice, particularly to knock-in mice, having mutations in the KCNQ2 gene or KCNQ3 gene.
  • the present invention also relates to targeting vectors intended for the generation of such mice.
  • the present invention relates to murine embryonic stem cells comprising said targeting vectors as well as to a screening method for the identification of compounds for the treatment of a human physiological condition, such as benign familial neonatal convulsions (BFNC), partial epilepsies, therapeutically resistant epilepsies, migraine, neuropathic pain, stroke, dementia and anxiety.
  • BFNC benign familial neonatal convulsions
  • partial epilepsies partial epilepsies
  • therapeutically resistant epilepsies migraine
  • neuropathic pain stroke
  • dementia dementia
  • the present invention provides a mouse with a mutation in one or both alleles of the endogenous KCNQ2 gene.
  • the mutation is a mutation found in a human BFNC family that has been introduced into the mouse's genome by homologous recombination.
  • the invention also provides cells derived from the mouse.
  • the present invention provides methods for screening candidate compounds to identify drugs useful in treating therapeutically resistant epilepsies.
  • the mice of the present invention are utilized as a model to screen for drugs that elevate seizure threshold and prevent seizures.
  • drugs are screened based on their effect on the electrophysiology of CAl pyramidal neurons isolated from the mice of the present invention.
  • Candidate substances so identified are useful for treating BFNC and are also be useful for treating seizures in individuals with other forms of generalized epilepsy and/or partial epilepsy.
  • the candidate substances are also useful for treating neuropathic pain, stroke, dementia, migraine and anxiety.
  • Figure IA shows wild-type construct of murine KCNQ2.
  • B BamHl restriction sites; 5'probe, position of probe used in Southern blots; green bars, locations of exons 2 through 7 of KCNQ2.
  • Figure IB shows targeting vector introduced into embryonic stem (ES) cells.
  • the ACN cassette is cloned into intron 5 of KCNQ2 and thymidine kinase (TK) vector at 3' end of targeting vector.
  • TK thymidine kinase
  • the neomycin (neo) gene driven by the mouse RNA polymerase II promoter (polll) confers positive selection and TK gene confers negative selection of ES cells.
  • the 5' arm is 5.4kB and the 3'arm is 4.6kB. *A306T mutation introduced into exon 6 of KCNQ2.
  • Figure 1C shows endogenous mutant construct following germline excision.
  • Figure ID shows southern blots of ES cell DNA to identify homologous recombination of ES cells.
  • ES cell DNA restricted by BamHI is electrophoresed on .055-1% agarose gel, transferred to nylon membrane and probed.
  • a 264bp probe located 5' of the targeting vector shows that 7.5kB is the wild-type allele and 6.9kB is the targeted allele verifying homologous recombination of the targeting vector.
  • the same southern blot is later probed with a 632bp neomycin probe to give the expected 2.85kB band demonstrating single copy insertion of the targeting vector. 28 ES cells out of a total 71 ES cells tested were correctly targeted and 12 were positive for the point mutation in exon 6.
  • Figure IE shows detection of the presence or absence of the A306T mutation in each experimental animal. Amplification of exon 6 using primers located in the flanking intronic sequence by PCR on mouse genomic DNA followed by single strand conformational polymorphism analysis (SSCP) of the PCR products on a 20% acrylamide gel electrophoresed at 4°C. +/+, homozygous wild-type, +/- heterozygous, -/-, homozygous mutant.
  • Figure IF shows presence or absence of a single loxP within intron 5 in each experimental animal.
  • Figure 2A shows wild-type construct of murine KCNQ3.
  • B BsaBI restriction sites; 5'probe, position of probe used in Southern blots; green bars, locations of exons 3 through 8 of KCNQ3.
  • FIG. 2B shows targeting vector introduced into embryonic stem (ES) cells.
  • the ACN cassette is cloned into intron 6 of KCNQ3 and thymidine kinase (TK) vector at 3' end of targeting vector.
  • TK thymidine kinase
  • the neomycin (neo) gene driven by the mouse RNA polymerase II promoter (polll) confers positive selection and TK gene confers negative selection of ES cells.
  • the 5 r arm is 8.2kB and the 3'arm is 1.9kB. *G310V mutation introduced into exon 5 of KCNQ3.
  • FIG. 2D shows southern blots of ES cell DNA to identify homologous recombination of ES cells.
  • ES cell DNA restricted by BsaBI is electrophoresed on 0.55-1% agarose gel, transferred to nylon membrane and probed.
  • a 600bp probe located 5' of the targeting vector shows that 10.3kB is the wild-type allele and 14.OkB is the targeted allele verifying homologous recombination of the targeting vector.
  • the same southern blot is later probed with a 632bp neomycin probe to give the expected 14.OkB band demonstrating single copy insertion of the targeting vector.
  • 27 ES cells out of a total 82 ES cells tested were correctly targeted and 14 were positive for the point mutation in exon 5.
  • Figure 2E shows Detection of the presence or absence of the G31 OV mutation in each experimental animal. Amplification of exon 5 using primers located in the flanking intronic sequence by PCR on mouse genomic DNA followed by single strand conformational polymorphism analysis (SSCP) of the PCR products on a 20% acrylamide gel electrophoresed at 4°C. +/+, homozygous wild-type, +/- heterozygous, -/-, homozygous mutant.
  • Figure 2F shows presence or absence of a single loxP within intron 6 in each experimental animal.
  • FIG. 3A Sample traces recorded from wild-type B6.129+/+ (WT; black), heterozygous mutant B6.129- Q2+/- (Het; darker grey), and homozygous mutant B6.129-Q2-/- (Horn; lighter grey) CAl neurons in response to the -20 to -60 mV step.
  • I K(M) amplitude is measured from the deactivation hump (10-20 msec after the hyperpolarizing step) to the steady-state level at the end of the trace.
  • Fig. 3A Sample traces recorded from wild-type B6.129+/+ (WT; black), heterozygous mutant B6.129- Q2+/- (Het; darker grey), and homozygous mutant B6.129-Q2-/- (Horn; lighter grey) CAl neurons in response to the -20 to -60 mV step.
  • I K(M) amplitude is measured from the deactivation hump (10-20 msec after the hyperpolarizing step) to the steady-state level
  • FIG. 3B I K(M) amplitude is decreased relative to wild-type in Horn CAl neurons (*, P ⁇ 0.001; all return steps).
  • Fig. 3C I K(M > density is also decreased in Horn CAl neurons (*, P ⁇ 0.005; all return steps).
  • Fig. 3D Peak amplitudes of sample traces are normalized to illustrate differences in deactivation kinetics ( ⁇ values).
  • Fig. 3E I K(M) deactication kinetics are faster in Het CAl neurons than WT at the -60 and -50 mV return step (
  • FIG. 4A Sample traces from WT, Het, and Horn CAl neurons illustrate the difference in spike firing patterns induced by the Kcnq2 A306T mutation.
  • a depolarizing current of +100 pA was injected for 800 msec to elicit spike trains. From the beginning to the end of the response, the frequency of the spike response decreases (adapts). Note the difference in the degree of adaptation in the Horn trace.
  • Fig. 4A Sample traces from WT, Het, and Horn CAl neurons illustrate the difference in spike firing patterns induced by the Kcnq2 A306T mutation.
  • a depolarizing current of +100 pA was injected for 800 msec to elicit spike trains. From the beginning to the end of the response, the frequency of the spike response decreases (adapts). Note the difference in the degree of adaptation in the Horn trace.
  • Fig. 4A Sample traces from WT, Het, and Horn CAl neurons illustrate the difference in spike firing patterns induced by the Kcnq2 A306T mutation
  • FIG. 4B Merspike interval number is plotted against the normalized frequency to depict population SFA in CAl neurons of WT, Het, and Horn mice. Het CAl neurons exhibit SFA similar to that of WT CAl neurons (n.s.). The SFA of Horn CAl neurons is significantly inhibited relative to WT (*, P ⁇ 0.0001).
  • Fig. 4C Sample traces in reponse to a stronger +140 pA depolarizing current injectionillustrate increased spike activity and in WT, Het, and Horn CAl neurons.
  • Fig. 4D In response to a stronger depolarizing current, Het CAl neurons now exhibit significantly decreased SFA relative to WT (*, P ⁇ 0.05).
  • FIG. 5A-5G show that processes involving repolarization following a single action potential are facilitated in C57/B16.129 CAl neurons carrying the Kcnq2 A306T mutation.
  • Fig. 5A Sample traces from WT (black), Het (darker grey), and Horn (lighter grey) CAl neurons illustrate several properties of single action potentials in response to a brief (5 msec) depolarizing current injection.
  • Fig. 5B Action potential 10-90% rise times are no different between WT, Het, and Horn.
  • Fig. 5C 90-10% decay time was not significantly altered in Het or Horn CAl neurons.
  • Fig. 5D The integrated area of the fast afterdepolarization (fADP) phase of the response was decreased in Horn CAl neurons only (*, P ⁇ 0.05).
  • Fig. 5E fADP deactivation kinetics were accelerated in both Het (f, P ⁇ 0.05) and Horn (*, P ⁇ 0.05) CAl neurons.
  • Fig. 5F A slow depolarizing current ramp was applied to determine the threshold for action potential generation (illustrated by the dotted line).
  • Fig. 5G Action potential threshold was determined to no different in Het or Horn CAl neurons.
  • FIG. 6A-6E show that I K(M) amplitude and density are decreased, and deactivation is accelerated in C57/B16.129 CAl neurons carrying the Kcnq3 G310V mutation.
  • Fig. 6A Sample traces recorded from wild-type B6.129+/+ (WT; black), heterozygous mutant B6.129- Q3+/- (Het; darker grey), and homozygous mutant B6.129-Q3-/- (Horn; lighter grey) CAl neurons in response to the -20 to -60 mV step.
  • I K(M > amplitude is measured from the deactivation hump (10-20 msec after the hyperpolarizing step) to the steady-state level at the end of the trace.
  • Fig 6B I K(M > amplitude is decreased relative to wild-type in Horn CAl neurons (*P ⁇ 0.005; all return steps).
  • Fig. 6C I K(M > density is also decreased in Horn CAl neurons (*P ⁇ 0.005; all return steps).
  • I R ( M) density is decreased in Het CAl neurons relative to WT (f , P ⁇ 0.05).
  • Fig. 6D Peak amplitudes of sample traces are normalized to illustrate differences in deactivation kinetics ( ⁇ values).
  • Fig. 6E I R(M) deactivation kinetics are faster in Het CAl neurons than WT only at the -60 mV return step (j, P ⁇ 0.01). In Horn CAl neurons, I K(M) deactivation is faster than WT only at the -70 mV step (*, P ⁇ 0.01).
  • FIGs 7A-7D show that Spike Frequency Adaptation (SFA) is inhibited in C57/B16.129 CAl neurons carrying the Kcnq3 G310V mutation.
  • Fig. 7A Sample traces from WT, Het, and Horn CAl neurons illustrate the difference in spike firing patterns induced by the Kcnq3 G310V mutation. A depolarizing current of +100 pA was injected for 800 msec to elicit spike trains. From the beginning to the end of the response, the frequency of the spike response decreases (adapts). Note the decreased adaptation in both the Het and Horn traces.
  • Fig. 7B Het CAl neurons exhibit less SFA than WT CAl neurons (j, P ⁇ 0.0001).
  • FIG. 7C Sample traces in reponse to a stronger +140 pA depolarizing current injection illustrate increased spike activity and in WT, Het, and Horn CAl neurons.
  • Fig. 7D Het and Horn CAl neurons exhibit decreased SFA relative to WT (f*, P ⁇ 0.0001). At this higher depolarizing current injection, SFA in Horn CAl neurons is significantly decreased relative to Het as well (J, P ⁇ 0.05).
  • Figures 8A-8G show that processes involving repolarization following a single action potential are facilitated in C57/B16.129 CAl neurons carrying the Kcnq3 G310V mutation. Fig.
  • FIG. 8A Sample traces from WT (black), Het (darker grey), and Horn (lighter grey) CAl neurons illustrate several properties of single action potentials in response to a brief (5 msec) depolarizing current injection.
  • Fig. 8B Action potential 10-90% rise times are no different between WT, Het, and Horn.
  • Fig. 8C 90-10% decay time was decreased relative to WT in both Het (t, P ⁇ 0.05) and Horn (*, P ⁇ 0.05) CAl neurons.
  • Fig. 8D The integrated area of the fast afterdepolarization (fADP) phase of the response was decreased in Horn CAl neurons only (*, P ⁇ 0.05).
  • Fig. 8ADP fast afterdepolarization
  • FIG. 8E fADP deactivation kinetics were accelerated in both Het (f , P ⁇ 0.05) and Horn (*, P ⁇ 0.05) CAl neurons.
  • Fig. 8F A slow depolarizing current ramp was applied to determine the threshold for action potential generation (illustrated by the dotted line).
  • Fig. 8 G Action potential threshold was determined to no different in Het or Horn CAl neurons.
  • Figures 9A-9E show that I ⁇ (M) amplitude and density are decreased, and deactivation is accelerated in FVB/N.129 CAl neurons carrying the Kcnq3 G310V mutation. Fig.
  • FIG. 9C I K(M) density is decreased in Horn CAl neurons at all return steps (*P ⁇ 0.05 for -70 mV step; P ⁇ 0.01 for -60, -50, -40 mV steps).
  • Fig. 9D Peak amplitudes of sample traces are normalized to illustrate differences in deactivation kinetics ( ⁇ values).
  • Fig. 9E I K(M) deactication kinetics are faster in Horn CAl neurons than WT at the -60 and -50 mV return step (*, P ⁇ 0.05). In Het CAl neurons, I K(M) deactivation is similar to WT.
  • FIG. 10A-10D show that Spike Frequency Adaptation (SFA) is inhibited in FVB/N.129 CAl neurons carrying the Kcnq3 G310V mutation.
  • Fig. 1OA Sample traces from WT, Het, and Horn CAl neurons illustrate the difference in spike firing patterns induced by the Kcnq3 G310V mutation. A depolarizing current of +100 pA was injected for 800 msec to elicit spike trains. From the beginning to the end of the response, the frequency of the spike response decreases (adapts). Note the difference in the degree of adaptation in the Horn trace.
  • Fig. 1OB Interspike interval number is plotted against the normalized frequency to depict population SFA in CAl neurons of WT, Het, and Horn mice.
  • Het CAl neurons exhibit SFA similar to that of WT CAl neurons (n.s.). The SFA of Horn CAl neurons is significantly inhibited relative to WT (*, P ⁇ 0.05).
  • Fig. 1OC Sample traces in reponse to a stronger +140 pA depolarizing current injection illustrate increased spike activity and in WT, Het, and Hom CAl neurons.
  • Fig. 10D In response to a stronger depolarizing current, Het CAl neurons still exhibit similar SFA to WT (n.s.). SFA is still significantly inhibited in Hom CAl neurons (*, P ⁇ 0.001).
  • Figures 1 IA-I ID show that processes involving repolarization following a single action potential are facilitated in FVB/N.129 CAl neurons carrying the Kcnq3 G310V mutation.
  • Fig. HA Sample traces from WT (black), Het (darker grey), and Hom (lighter grey) CAl neurons illustrate several properties of single action potentials in response to a brief (5 msec) depolarizing current injection.
  • Fig. HB Action potential 10-90% rise times are no different between WT, Het, and Hom.
  • Fig. 11C 90-10% decay time is not significantly affected in either Het or Hom CAl neurons.
  • Fig. HA Sample traces from WT (black), Het (darker grey), and Hom (lighter grey) CAl neurons illustrate several properties of single action potentials in response to a brief (5 msec) depolarizing current injection.
  • Fig. HB Action potential 10-90% rise times are no different between WT, Het, and Hom.
  • Fig. 11D The integrated area of the fast afterdepolarization (fADP) phase of the response was not significantly affected in Het or Hom CAl neurons.
  • Fig. 1 IE fADP deactivation kinetics show a trend toward significant slowing (P ⁇ 0.053) in Het CAl neurons.
  • Fig. HF A slow depolarizing current ramp was applied to determine the threshold for action potential generation (illustrated by the dotted line).
  • Fig. HG Action potential threshold was determined to no different in Het or Hom CAl neurons.
  • Figure 12 shows video monitoring of mice.
  • 17 N5F2 Q3-FVB mice were video-monitored to assess the seizure severity and frequency.
  • the X axis goes from 20 to 81 days of age. In each animal, monitoring was done 1-4 days before the first seizure and after the last seizure.
  • Black circles represent generalized tonic clonic seizures
  • gray circles respresent forelimb and hindlimb clonic seizures
  • white circles represent forelimb clonic seizures.
  • Figure 13 show upregulation of NPY in the mossy fibers of the dentate granule cells following seizures in N5F2 Q3-FVB homozygous mutant (-/-) mice compared to wild-type (+/+) mice.
  • Figure 14 shows reactive gliosis measured with an antibody to GFAP in the hippocampus of Q3-FVB homozygous mutant mice following multiple spontaneous recurrent seizures.
  • the present invention relates to transgenic mice, particularly to knock-in mice, having mutations in the KCNQ2 gene or KCNQ3 gene.
  • the present invention also relates to targeting vectors intended for the generation of such mice.
  • the present invention relates to murine embryonic stem cells and cryopreserved sperm comprising said targeting vectors as well as to a screening method for the identification of compounds for the treatment of a human physiological condition, such as benign familial neonatal convulsions (BFNC), partial epilepsies, therapeutically resistant epilepsies, migraine, neuropathic pain, stroke, dementia and anxiety.
  • BFNC benign familial neonatal convulsions
  • partial epilepsies partial epilepsies
  • therapeutically resistant epilepsies migraine
  • neuropathic pain stroke
  • dementia dementia
  • the present invention provides a mouse with a mutation in one or both alleles of the endogenous KCNQ2 gene.
  • the mutation in one or both of the endogenous KCNQ2 genes results in a mouse with a seizure-susceptible phenotype.
  • the mutation is a mutation found in human BFNC families that has been introduced into the mouse's genome by homologous recombination.
  • the invention also provides cells, including sperm derived from the mouse.
  • the present invention provides the knock-in mouse strain that carries a BFNC-causing missense point mutation - C57/B16 ⁇ 29-Kcnq2 A306 ⁇ (B6-Q2).
  • Wild- type, hemizygous, and homozygous N1F2 mice are viable. Spontaneous seizures have been observed that resemble the epileptic seizures of human BFNC.
  • electroconvulsive threshold testing was performed in heterozygous mutant and wild-type mice (Otto et al., 2004). The experiments presented herein were designed to test the hypothesis that mice expressing the exact Kcnq2 mutations that cause BFNC in humans will have reduced electroconvulsive seizure thresholds for forebrain, limbic, and hindbrain seizures.
  • mice heterozygous for the Kcnq2 A306T amino acid exchange mutation exhibits significantly decreased seizure thresholds in several electroconvulsive threshold (ECT) stimulus paradigms. These differences are dependent on the specific mutation expressed, seizure type elicited, background strain, as well as gender.
  • ECT electroconvulsive threshold
  • Studies conducted in Xenopus oocytes suggest that BFNC causing mutations decrease I K(M) amplitude, and some shift the voltage dependence of M channel activation toward the depolarized range (Schroeder et al., 1998; Singh et al., 2003).
  • mice expressing Kcnq2 point mutation that causes BFNC in humans will exhibit altered I ⁇ (M) function and single-cell neuroexcitability.
  • CAl neurons in mice homozygous for the Kcnq2 A306T mutation has significantly decreased I K(M) amplitude and spike frequency adaptation.
  • the present invention provides a mouse with a mutation in one or both alleles of the endogenous KCNQ3 gene.
  • the mutation in one or both of the endogenous KCNQ3 genes results in a mouse with a seizure-susceptible phenotype.
  • the mutation is a mutation found in human BFNC families that has been introduced into the mouse's genome by homologous recombination.
  • the invention also provides cells derived from the mouse.
  • C57/B ⁇ 6.l29-Kcnq3 G3WV (B6-Q3). Wild-type, hemizygous, and homozygous N2 mice are viable and born within the expected Mendelian proportions in all mutations and strains. Spontaneous seizures have been observed that resemble the epileptic seizures of human BFNC. The effects of this mutation on seizure susceptibility, electroconvulsive threshold testing was performed in heterozygous mutant and wild-type mice (Otto et al., 2004). The experiments presented herein were designed to test the hypothesis that mice expressing the exact Kcnq3 mutations that cause BFNC in humans will have reduced electroconvulsive seizure thresholds for forebrain, limbic, and hindbrain seizures.
  • FVB/N.129-iTcng3 G310V (FN-Q3). Wild-type, hemizygous, and homozygous N1F2 mice are viable. Spontaneous seizures have been observed that resemble the epileptic seizures of human BFNC. The effects of this mutation on seizure susceptibility, electroconvulsive threshold testing was performed in heterozygous mutant and wild-type mice (Otto et al., 2004). The experiments presented herein were designed to test the hypothesis that mice expressing the exact Kcnq3 mutations that cause BFNC in humans will have reduced electroconvulsive seizure thresholds for forebrain, limbic, and hindbrain seizures.
  • mice heterozygous for the KcnqS G310V amino acid exchange mutation exhibits significantly decreased seizure thresholds in several electroconvulsive threshold (ECT) stimulus paradigms. These differences are dependent on the specific mutation expressed, seizure type elicited, background strain, as well as gender.
  • ECT electroconvulsive threshold
  • Studies conducted in Xenopus oocytes suggest that BFNC causing mutations decrease I K(M) amplitude, and some shift the voltage dependence of M channel activation toward the depolarized range (Schroeder et al., 1998; Singh et al., 2003).
  • mice expressing Kcnq3 point mutation that causes BFNC in humans will exhibit altered I ⁇ (M) function and single-cell neuroexcitability.
  • CAl neurons in mice homozygous for the Kcnq3 G310V mutation has significantly decreased I K ⁇ M) amplitude and spike frequency adaptation.
  • mice of the present invention can be used in screening assays to determine whether a test agent (e.g., a drug, a chemical or a biologic) elevates seizure threshold or modulates the M-channel and thus can be used for treating or preventing seizures associated with BFNC or other neurological disorders where the M-channel function is perturbed.
  • a test agent e.g., a drug, a chemical or a biologic
  • compounds that modulate the M-channel can be used for treating pain, anxiety.
  • modulate refers to enhancement (e.g., an increase) or inhibition (e.g., a reduction) in the specified activity.
  • the ability of a test compound or agent to modulate the activity of the M-channel is tested in the mice of the present invention.
  • mice of the present invention are exposed to the test compound and the effect on the seizure phenotype or the electroconvulsion threshold is determined.
  • the ability of a test compound or agent to modulate the activity of the M-channel is tested in neurons isolated from the mice of the present invention.
  • CAl pyramidal neurons or brain slices are isolated from the mice of the present invention and are exposed to the test compound. The effect on the seizure pattern or the electrophysiology of the CAl pyramidal neurons is determined.
  • In vitro systems may also be utilized to screen for compounds that disrupt normal regulatory interactions.
  • a variety of test compounds can be evaluated in accordance with the present invention.
  • the compounds to be tested can be derived from libraries (i.e., are members of a library of compounds). While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin and Ellman, 1992; De Witt et al., 1993), peptoids (Zuckermann, 1994), oligocarbamates (Cho et al., 1993), and hydantoins (DeWitt et al., 1993).
  • the compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the ⁇ one-bead one-compound' library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997).
  • Other exemplary methods for the synthesis of molecular libraries can be found in the art, for example in Erb et al. (1994), Horwell et al. (1996) and Gallop et al. (1994).
  • Libraries of compounds may be presented in solution (e.g., Houghten et al., 1992), or on beads (Lam et al., 1991), chips (Fodor et al., 1993), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992) or on phage (Scott and Smith, 1990; Devlin et al., 1990; Cwirla et al., 1990; Felici et al., 1991).
  • the combinatorial polypeptides are produced from a cDNA library.
  • Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo.
  • Several approaches for use in rational drug design include analysis of three-dimensional structure, alanine scans, molecular modeling and use of anti-id antibodies. These techniques are well known to those skilled in the art.
  • Such techniques may include providing atomic coordinates defining a three-dimensional structure of a protein complex formed by said first polypeptide and said second polypeptide, and designing or selecting compounds capable of interfering with the interaction between a first polypeptide and a second polypeptide based on said atomic coordinates.
  • the substance may be further investigated. Furthermore, it may be manufactured and/or used in preparation, i.e., manufacture or formulation, or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • a substance identified as a modulator of polypeptide function may be peptide or non- peptide in nature. Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This approach might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process. Such techniques include those disclosed in U.S. Patent No. 6,080,576. [0062] A template molecule is then selected, onto which chemical groups that mimic the pharmacophore can be grafted.
  • a template molecule is then selected, onto which chemical groups that mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted thereon can be conveniently selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic is peptide-based
  • further stability can be achieved by cyclizing the peptide, increasing its rigidity.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent it is exhibited. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • any compounds which reverse any aspect of a given phenotype or expression of any gene in vivo and which modulates protein activity or binding with binding partner in vitro should be considered as candidates for further development or potential use in humans.
  • Dosages of test agents may be determined by deriving dose-response curves using methods well known in the art.
  • compositions containing an identified agent as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.). Typically, a therapeutically effective amount of an active ingredient is admixed with a pharmaceutically acceptable carrier. By a “therapeutically effective amount” or simply “effective amount” of an active compound is meant a sufficient amount of the compound to treat the desired condition at a reasonable benefit/risk ratio applicable to any medical treatment. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated.
  • Prescription of treatment e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington 's Pharmaceutical Sciences.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, parenteral, intramuscular, subcutaneous or intrathecal.
  • Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as com starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate,
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
  • antioxidants examples include, but are not limited to, water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, aloha-tocopherol and the like; and the metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like
  • oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (B
  • Exemplary methods for administering compounds will be apparent to the skilled artisan.
  • Certain methods suitable for administering compounds useful according to the present invention are set forth in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7th Ed. (1985).
  • the administration to the patient can be intermittent; or at a gradual, continuous, constant or controlled rate.
  • Administration can be to a warm-blooded animal (e.g. a mammal, such as a mouse, rat, cat, rabbit, dog, pig, cow or monkey); but advantageously is administered to a human being.
  • Administration occurs after general anesthesia is administered.
  • the frequency of administration normally is determined by an anesthesiologist, and typically varies from patient to patient.
  • the pharmaceutical compositions will generally contain from about 0.0001 to 99 wt. %, preferably about 0.001 to 50 wt. %, more preferably about 0.01 to 10 wt. % of the active ingredient by weight of the total composition.
  • the pharmaceutical compositions and medicaments can also contain other pharmaceutically active compounds.
  • other pharmaceutically active compounds include, but are not limited to, analgesic agents, cytokines and therapeutic agents in all of the major areas of clinical medicine.
  • the agents may be delivered in the form of drug cocktails.
  • a cocktail is a mixture of any one of the compounds useful with this invention with another drug or agent.
  • a common administration vehicle e.g., pill, tablet, implant, pump, injectable solution, etc.
  • a common administration vehicle e.g., pill, tablet, implant, pump, injectable solution, etc.
  • the individual drugs of the cocktail are each administered in therapeutically effective amounts.
  • a therapeutically effective amount will be determined by the parameters described above; but, in any event, is that amount which establishes a level of the drugs in the area of body where the drugs are required for a period of time which is effective in attaining the desired effects.
  • Transgenic Mice [0071] Vector Construction [0072] Wild-type clones of mouse KCNQ2 and KCNQ3 were isolated from a ⁇ phage library (Stratagene) and subcloned into pBSk.
  • Figure IA shows wild-type construct of murine KCNQ2.
  • Figure 2 A shows wild-type construct of murine KCNQ3. Point mutations, A306T mutation in exon 6 of KCNQ2 and G310V mutation in exon 5 of KCNQ3, were introduced into the respective constructs using PCR mutagenesis techniques with mutations contained in the primer.
  • the ACN (Bunting et al., 1999) cassette comprises the tACE/Cre/PolII/Neo gene was flanked with two loxP sites (Fig. IB and Fig. 2B).
  • the ACN cassette is cloned into an Xhol site in intron 5 of KCNQ2 and this construct is cloned into a thymidine kinase (TK) vector (Thomas and Capecchi, 1987).
  • TK thymidine kinase
  • the ACN cassette is cloned into a SnaBI site in intron 6 of KCNQ3 and this construct is cloned into TK.
  • Chimeric progeny were identified by coat color and a single male of each strain was mated to C57B1/6 or FVB/N females for the generation of Fl offspring.
  • Fl offspring were intercrossed to generate F2 experimental animals.
  • An additional 5 backcrosses were performed on the C57B1/6 or FVB/N backgrounds and these offspring were intercrossed to yield N5F2 experimental animals.
  • Genomic DNA obtained from tail biopsies of Fl and F2 animals were analyzed for
  • mice Male and female mice, varying in age between 180 and 320 days were used for all electroconvulsive threshold experiments. There were no significant differences in age between any of the groups. F2 generation mice of the following genotypes were used: wild-type C57/B16.129 (B6) and FVB/N.129 (FN); and heterozygous knock-in mutant C57/B16.129- Zc/jg2 A306T/+ (B6-Q2+/-), C57/Bl6 ⁇ 29-Kcnq2 Gmv ⁇ ' (B6-Q3+/-), and FVB/N. U9-Kcnq3 G3im+ (FN-Q3+/-).
  • Table 2 shows the CC 50 and CI 95 values in response to the minimal clonic seizure testing paradigm. Note that for this seizure type, the Kcnq2 A306T mutation in B6 mice, and Kcnq3 G310V mutation in B6 and FN mice, all resulted in a significant reduction in the seizure threshold of female mice. Conversely, none of the mutations expressed on any of the genetic background strains significantly affected the seizure threshold of male mice. The minimal clonic seizure was the only one in which this obvious gender-dependent susceptibility to Kcnql and Kcnq3 mutation was observed.
  • Table 3 summarizes the calculated CC 50 and CI95 values for the minimal THE seizure.
  • the seizure thresholds were decreased for all mutations, backgrounds, and genders. This is the only seizure phenotype in which heterozygous mutants all displayed decreased seizure susceptibility relative to littermate control WT mice.
  • KCNQ2 and KCNQ3 subunit proteins Differences in the expression of KCNQ2 and KCNQ3 subunit proteins, between brain regions and in discrete regions of the cell, might also contribute to these differences. For instance, while KCNQ2 and KCNQ3 are largely coexpressed throughout the brain, on a smaller scale, KCNQ2 is often expressed by itself in more discrete areas of the cell, specifically the axon initial segment and nodes of Ranvier (Devaux et al., 2004).
  • seizure thresholds might actually increase since interictal activity in the more vulnerable brain structures can offer short-term resistance to external stimulus-induced seizures, perhaps providing a preconditioning effect (Bortolotto et al., 1991; Rejdak et al., 2001).
  • EEG surface electroencephalogram
  • mice display lower seizure thresholds than males in general (Vernadakis and Woodbury, 1972; Frankel et al., 2001), and thus are considered to have increased seizure susceptibility, it is feasible that these mutations increase neuronal excitability such that a lower stimulus intensity is required to reach seizure- threshold and provoke seizure activity. [0096] It has been previously established that genetic background strain can considerably affect the seizure thresholds of mice (Ferraro et al., 1998; Frankel et al., 2001).
  • mice carrying the Kcnq3 G310V mutation on the B6 background exhibited a threshold no different from their WT littermates in partial psychomotor seizure testing, but this mutation did significantly reduce the threshold of male mice carrying the same mutation on the FN background.
  • the effect of the Kcnq3 G310V mutation appears to be dependent on the genetic background of the mouse.
  • Previous studies support this trend in background-specific changes in seizure activity. For instance, kainic acid treatment induces severe seizures in C57/B16 mice, but only slight seizure activity in 129/SvJ mice (McKhann et al., 2003). [0097] It should be noted that these studies were conducted in mice considerably older than what are typically used for ECT studies.
  • mice ranged from 180 to 320 days old, whereas most studies of this nature use 8-to-12 week old (56-to-84 day old) mice (Ferraro et al., 1998; Frankel et al., 2001). It should be noted, however, that the CC 5O values of these mice were retested for each seizure type as a final experiment, and were found to be consistent with those established initially. This indicates that seizure thresholds did not shift significantly throughout the study, even after repeated stimulations; nevertheless, these studies should be confirmed in 8- to- 12 week old mice to facilitate comparisons with the previous literature.
  • mice Male and female mice, 8-to-15 weeks of age were used for all electrophysiology experiments. F2 generation mice of the following genotypes were used: wild-type C57/B16.129 (B6+/+), heterozygous C57/B16.129-Z ⁇ z?2 A306T/+ (B6-Q2+/-) and homozygous C57/B16.129- Zc «#2 A306T/A306T (B6-Q2-/-) knock-in; wild-type C57/B16.129 (B6+/+), heterozygous C57/B16.129-Zc ⁇ 3 G310v/+ (B6-Q3+/-) and homozygous B6 ⁇ 29-Kcnq3 GnwiGmv (B6-Q3- ⁇ ) knock-in; and wild-type FVB/N.129 (FN+/+), heterozygous FVB/N.129-i_cng3 G310V/+ (FN- Q3+/
  • Brain slices were prepared from wild-type, heterozygous and homozygous Kcnq2
  • mice were anesthetized with Nembutal (25 mg/kg), decapitated, and their brains quickly removed and placed in oxygenated Ringer solution containing (in mM): 200 sucrose, 26 NaHCO 3 , 10 glucose, 3 KCl, 2 MgSO 4 , 2 CaCl 2 , 1.4 NaH 2 PO 4 . Brains were trimmed, mounted on a chuck, and 350 ⁇ m thick coronal slices were cut using a Vibratome sheer (Vibratome).
  • Electrophysiological Measurements [0104] Whole-cell perforated patch recordings were obtained from CAl pyramidal neurons in the acute brain slice preparation using a MultiClamp 700A amplifier (Axon Instruments). Signals in voltage-clamp and current-clamp modes were acquired at 10 KHz and 20 KHz, and filtered at 2 KHz and 10 KHz, respectively, for offline analysis using Clampfit 9. Glass capillaries (World Precision Instruments, Inc.) were pulled to 2.0-3.2 M ⁇ resistances using a micropipette electrode puller (Sutter Instrument Co.). Input and series resistance values of 80- 120 M ⁇ and ⁇ 15 M ⁇ , respectively, were used as selection criteria for accepting recordings.
  • Capacitance compensation and bridge balance functions were used for voltage-clamp and current-clamp experiments, respectively.
  • the external NaCl Ringer solution was supplemented with picrotoxin (50 ⁇ M) and NBQX (10 ⁇ M) to block GABA A receptor- and non-NMDA receptor-mediated responses, respectively.
  • I K(M > amplitude was measured as the relaxation current in response to a voltage step protocol from -20 mV to return potentials of -40 mV, -50 mV, -60 mV, and - 7OmV.
  • I K(M) density was calculated as I K(M) amplitude (pA) / whole-cell capacitance (pF) for each cell.
  • EXAMPLE 5 Effects of Kcnq2 and Kcnq3 Point Mutations on CAl Hippocampal Neurons [0109] As shown in the previous examples, characterization of B6.129 and FVBN.129 mice carrying the Kcnql A306T and Kcnq3 G310V knock-in mutations established a wide range of effects on seizure threshold. Although mice expressing these mutations should be genetically comparable to their wild-type littermates with the exception of a single amino acid switch, the changes in whole-animal seizure threshold detailed in the previous chapter do not specifically implicate alterations in I K ( M ) function at the single-cell level.
  • I ⁇ (M) in CAl neurons in brain slices prepared from mice heterozygous and homozygous for the Kcnql A306T and Kcnq3 G310V mutations, as well as their wild-type littermates, in an effort to characterize changes in I K(M) specifically.
  • I R(M ) amplitudes were no different than those measured in wild-type B6 slices (Fig. 3B).
  • I K ( M ) amplitude was significantly decreased at every return step tested. Since a decrease in current amplitude could be explained by a decrease in CAl neuron size, whole-cell capacitance, a relative approximation of cellular surface area, was used to convert amplitudes to current densities.
  • I K ( M) density was not significantly affected in B6-Q2+/- CAl neurons, but was decreased in B6-Q2-/- CAl neurons at all voltages (Fig. 3C).
  • I K(M > amplitude and density data at every hyperpolarizing return step are summarized in Table 4.
  • B6-Q2-/- value is significantly different from B6+/+ (P ⁇ 0.05).
  • I K(M > amplitude was significantly decreased at every return step tested.
  • I K(M) amplitude values were divided by membrane capacitance and converted to current density.
  • I K ( M) density was only significantly decreased in B6-Q3+/- CAl neurons at the -50 mV step, but was decreased in B6-Q3-/-CA1 neurons at all return steps (Fig. 6C).
  • the effects of Kcnq3 G310V mutation on I K(M) amplitude and density data at every hyperpolarizing return step are summarized in Table 7.
  • B6-Q3-/- value is significantly different from B6+/+ (P ⁇ 0.05).
  • the numbers of cells recorded from (n values) are identical for I K(M) amplitude and density values within the same genotype.
  • Spike frequency adaptation is also inhibited in the same neurons, which is consistent with a hypofunctional I K(M) , and provides evidence of increased neuronal excitability.
  • SFA Spike frequency adaptation
  • I K ⁇ M density, and deactivation kinetics are only significantly attenuated at the -50 mV and -60 mV steps, respectively.
  • SFA is inhibited in B6-Q3+/- neurons relative to B6+/+ at all depolarizing steps that produce action potential trains (data shown for +100 p A and +140 pA steps).
  • KCNQ2 protein is normally concentrated at the Nodes of Ranvier and the axon initial segment (AIS), the anatomical site of action potential initiation, and KCNQ3 is most often co-expressed with KCNQ2 (Devaux et al., 2004).
  • Kcnq2 A306T and Kcnq3 G310V mutations examined here produce such considerable and diverse changes in action potential characteristics.
  • Kcnq2-I- knock-out and homozygous Sztl-I- mice are perinatal lethal; both die of lung atelectasis (Watanabe et al, 2000; Yang et al., 2003). If the Kcnq2 A306T mutation precluded KCNQ2 protein insertion, mice homozygous for this mutation would likely die as well. To date, the only deaths observed in homozygous mutant knock-in mice have been postnatal as a result of lethal seizure events.
  • the M channel blocker linopirdine actually delays membrane repolarization in a recording paradigm similar to the one employed here (Yue and Yaari, 2004), an effect that is concordant with the role of I K ( M >
  • ADP area and ⁇ values are decreased in both B6-Q2+/- and B6-Q3+/- CAl neurons, and 90-10% action potential decay times are decreased in B6-Q3+/- CAl neurons.
  • Q3-FVBN Q3-B6, Q2-FVBN Q2-B6 mice
  • Q3-FVBN mice can have over 600 spontaneous recurrent seizures (SRS) before 82 days of age that begin with generalized tonic clonic seizures (grade 5, black circles) until approximately 40 days (?) which are followed by forelimb and hindlimb clonic seizures (grade 3, gray circles) until approximately 60 days, followed by forelimb clonic seizures (grade 2, white circles) ( Figure 12). This suggests that the seizure severity is reduced with increasing age.
  • SRS spontaneous recurrent seizures
  • mice During the first approx 50 days, animals will have periods of no seizures that can last for up to 5(?) days which are interrupted by at least one generalized tonic clonic seizure. All homozygous Q3-FVBN mice exhibit seizures, suggesting that the SRS phenotype is fully penetrant. Q3 mice with the same mutation on the B6 background exhibit spontaneous seizures in less than half of the mice that were video monitored, suggesting incomplete penetrance of seizures. [0154] KCNQ2 homozygous mutant mice on either B6 or FVBN genetic background exhibit at least one generalized tonic clonic seizure documented with video monitoring. KCNQ2-FVBN mice die as a result of seizures while KCNQ2-B6 mice survive at least one SRS. EXAMPLE 7
  • NPY neuropeptide Y
  • the upregulation of neuropeptide Y (NPY) in mossy fiber axons of dentate granule cells is a hallmark of seizure activity although the significance of this increased expression is unclear (reference: Scharfman & Gray, EXS. 2006;(95):193-211Plasticity of neuropeptide Y in the dentate gyrus after seizures, and its relevance to seizure-induced neurogenesis).
  • NPY upregulation was determined using a commercial antibody in N5F2 Q3-B6, Q3-FVB and Q2-B6 mouse brain slices following either single or multiple seizures.
  • NPY is upregulated in the mossy fibers of dentate granule cells of Q3-FVB (figure 13), Q3-B6 and Q2-B6 (data not shown) homozygous mutant mice following seizures. This upregulation is absent from wild-type mice. Of note is the absence of NPY staining in the inner molecular layer of the dentate gyrus indicating that no mossy fiber sprouting has occurred in these animals.
  • Reactive Gliosis Astrocytes play a significant role in glutamate uptake, potassium buffering and sequestration of reactive oxygen species during normal brain function. In CNS injury such as seizures, astrocytes in the hippocampus become activated, also termed reactive gliosis, but it is presently unclear whether this process contributes to CNS pathology or is a result of it. Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein in mature astrocytes and can be used as a marker of reactive gliosis.
  • GFAP Glial fibrillary acidic protein
  • mice that were genetically engineered to express the exact point mutations known to cause BFNC in humans were obtained from mice that were genetically engineered to express the exact point mutations known to cause BFNC in humans.
  • the Kcnql A306T mutation was expressed on the C57/B16.129 (B6) background
  • the Kcnq3 G310V mutation was expressed on both the C57/B16.129 and FVB/N.129 (FN) backgrounds.
  • Examples 2 and 3 summarized the alterations in seizure thresholds that result from knock-in point mutations in Kcnql and Kcnq3.
  • mice heterozygous for these mutations (expressed on both backgrounds) largely exhibited decreased seizure thresholds. This is the first report to show that mice carrying BFNC-causing mutations display reduced seizure thresholds.
  • Examples 4 and 5 are an electrophysiological characterization of CAl neurons in C57/B16.129-if ⁇ zg2 A305T , C57/B ⁇ 6 ⁇ 29-Kcnq3 Gmy , and FVB/N.129-J-cw#3 G310V mice.
  • I K(M > amplitude and density were attenuated, deactivation kinetics were accelerated, and SFA was significantly inhibited.
  • heterozygous mice however, a number of quite diverse effects were observed.
  • Table 13 summarizes the most salient observed differences in seizure threshold and electrophysiology parameters in the mouse models o ⁇ Kcnq2 and Kcnq3 mutations examined in the present study.
  • mice Due to limited availability of age-matched WT and heterozygous mutant mice, the ECT studies were conducted in mice that were much older (20-50 weeks) than those used for the electrophysiology studies (8-15 weeks). In addition, the electrophysiology studies were also only designed to test I K(M) function and other biophysical mechanisms that are affected by I K ( M > There are many other mechanisms that contribute to seizure threshold that were not examined in these studies. For instance, the tonic activity level of many neurotransmitter systems (i.e., glutamate, GABA, acetylcholine) might also be up- or down-regulated in response to the Kcnq mutations examined here.
  • neurotransmitter systems i.e., glutamate, GABA, acetylcholine
  • KCNQ2 residue 214 is contained within a different region of the M channel (the S4 voltage-sensing region).
  • results obtained in Xenopus oocytes often do not closely parallel those obtained in the native system (Dorr, 1993; Lewis et al., 1997; Sivilotti et al., 1997).
  • mice I K(M ) density and deactivation kinetics were significantly reduced in B6-Q3 +/" mice, producing a decrease in SFA.
  • I ⁇ (M) density was not affected, but deactivation kinetics were significantly reduced; CAl neurons of these mice did not display altered SFA.
  • BFNC-causing mutations only display about 80% penetrance in human families ⁇ 20% of family members carrying the same mutations are not afflicted with BFNC and do not display a higher incidence of adult onset epilepsy (Leppert et al., 1993). In mice, it appears that differences in B6.129 and FN.129 mouse genetic backgrounds may account for the differential sensitivity to M channel mutation.
  • the latent period between an initial insult and increased seizure susceptibility is a characteristic of many forms of epilepsy, specifically temporal lobe epilepsy (White, 2002).
  • a mutation in the Kcnq2IKcnq3 genes and an increased likelihood of developing epilepsy could be considered an initial insult and evidence of increased seizure susceptibility, respectively.
  • the Kcnq mutant knock-in mice could be used to conduct studies designed to observe the long- term consequences of genetic mutations on seizure susceptibility, or so-called "second hit” studies (Walker et al., 2002).
  • KCNQ2 is a nodal K+ channel. JNeurosci 24:1236-1244.
  • Singh, N.A. et al. (1998). A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat Genet 18:25-29.

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Abstract

La présente invention concerne des souris transgéniques, en particulier des souris knock-in (avec insertion d'un gène actif), qui présentent des mutations dans le gène KCNQ2 ou dans le gène KCNQ3, ainsi que le sperme de ces souris. Cette invention concerne également des vecteurs de ciblage conçus pour créer de telles souris. Elle concerne aussi des cellules souches embryonnaires de souris et du sperme cryoconservé comprenant lesdits vecteurs de ciblage, ainsi qu'un procédé de criblage permettant d'identifier des composés utilisés pour traiter des pathologies physiologiques humaines, telles que les convulsions néonatales familiales bénignes (BFNC), les épilepsies partielles, les épilepsies à résistance thérapeutique, les migraines, les douleurs neuropathiques, les accidents vasculaires cérébraux, la démence et l'anxiété.
PCT/US2006/007941 2005-03-07 2006-03-07 Modele de souris WO2006096648A2 (fr)

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EP2390348A1 (fr) * 2010-05-25 2011-11-30 Sanofi Procédés et utilisations liées à l'identification d'un composé impliqué dans la douleur et procédés pour le diagnostic de l'algésie
EP2390349A1 (fr) * 2010-05-25 2011-11-30 Sanofi Procédés et utilisations liées à l'identification d'un composé impliqué dans la douleur et procédés pour le diagnostic de l'algésie

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WO2011147851A1 (fr) * 2010-05-25 2011-12-01 Sanofi-Aventis Procédés et utilisations se rapportant à l'identification d'un composé impliqué dans la douleur ainsi que méthodes de diagnostic de l'algésie

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