US006060642A United States Patent (19) 11 Patent Number: 6,060,642 Tecott et al. (45) Date of Patent: May 9, 2000 54 SEROTONIN 5-HT6 RECEPTOR KNOCKOUT Roth, Bryan L., et al., “Binding of Typical and Atypical MOUSE Antipsychotic Agents 5-Hydroxytryptamine-6 and 5-Hydroxytryptamine–7 Receptors'." The Journal Of Phar 75 Inventors: Laurence H. Tecott, San Francisco; macology and Experimental Therapeutics (1994) vol. 268, Thomas J. Brennan, San Carlos, both No. (3):1403–1410. of Calif. Ruat, Martial, et al., “A Novel Rat Serotonin (5-HT) 73 Assignee: The Regents of the University of Receptor: Molecular Cloning, Localization And Stimulation Of cAMP Accumulation,” Biochemical and Biophysical California, Oakland, Calif. Communications Research Communications (May 28, 1993) 21 Appl. No.: 09/132,388 vol. 193, No. (1):268–276. Saudou, Frédéric, et al., “5-Hydroxytryptamine Receptor 22 Filed: Aug. 11, 1998 Subtypes. In Vertebrates And Inverterbrates,” Neurochem Int. (1994) vol. 25, No. (6):503–532. Related U.S. Application Data Sleight, A.J., et al., “Effects of Altered 5-HT Expression. In 60 Provisional application No. 60/055.817, Aug. 15, 1997. The Rat: Functional Studies Using Antisense Oligonucle 51 Int. Cl." ............................. C12N 5700; C12N 15/12; otides,” Behavioural Brain Research (1996) vol. AO1K 67/027; G01N 33/15; CO7H 21/04 73:245-248. 52 U.S. Cl. ................................... 800/3; 800/18; 800/21; Tecott, Laurence H., et al., “Behavioral Genetics: Genes And 800/9; 435/172.1; 435/1723; 435/325; Aggressiveness,” Current Biology (1996) vol. 6, No. 435/455; 536/23.5 (3):238–240. 58 Field of Search .................................... 800/3, 18, 21, Ward, Raymond P., et al., “Colocalization of Serotonin 800/9; 435/455, 172.1, 172.3, 325; 536/23.5 Receptor Subtypes 5-HT2A, 5-HT2C, and 5-HT6 With 56) References Cited Neuropeptides In Rat Striatum.” The Journal Of Compara tive Neurology (1996) vol. 370:405-414. U.S. PATENT DOCUMENTS Bradley et al., Bio/Technology, 10:534–539, 1992. 5,698,766 12/1997 Julius et al. ................................ 800/2 Saudou et al., Science, 265:1875-1878, 1994. OTHER PUBLICATIONS Kohen et al., GenBank Accession #L41146, 1996. Boss, F.G., et al., “Functional and Radioligand Binding Kohen et al., GenBank Accession #L41147, 1996. Characterization of Rat 5-HT Receptors Stably Expressed in HEK293 Cells,” Neuropharmacology (1997) vol. 36, No. Primary Examiner Deborah Crouch (4/5):713–720. ASSistant Examiner-Janet M. Kerr BourSon, Anne, et al., “Determination of the Role of the 5-HT Receptor in the Rat Brain: A Study Using Antisense Attorney, Agent, or Firm-Pamela J. Sherwood; Paula A. Oligonucleotides'.” The Journal of Pharmacology and Borden; Bozicevic, Field & Francis, LLP Experimental Therapeutics (1995) vol. 274, No. 57 ABSTRACT (1):173–180. Brennan, Thomas J., et al., “Sound-Induced Seizures. In The present invention features transgenic mice models for Serotonin 5-HT, Receptor Mutant Mice,” Nature Genetics gene function, wherein the transgenic mice are characterized (Aug. 1997) vol. 16:387–390. by having altered Serotonin 5-HT6 receptor gene function. Kohen, Ruth, et al., “Cloning, Characterization, and Chro The transgenic mice may be either homozygous or heterozy mosomal Localization of A Human 5-HT Serotonin Recep gous for a disruption in the endogenous 5-HT6 gene. Trans tor,” Journal of Neurochemistry (1996) vol. 66, No. genic mice homozygous for a disruption in the endogenous (1):47–56. 5-HT6 gene display a phenotype of increased anxiety behav Pierce, P.A., et al., “5-Hydroxytryptamine Receptor Sub ior including diminished investigation of foreign objects and type Messenger RNAS. In Rat Peripheral Sensory And Sym an elevation in Stretched attend postures. pathetic Ganglia: A Polymerase Chain Reaction Study,” Neuroscience (1996) vol. 70, No. (2):553–559. 3 Claims, 1 Drawing Sheet 6,060,642 1 2 SEROTONIN 5-HT6 RECEPTOR KNOCKOUT published rat Sequence contained a frame shift error. The MOUSE gene for the receptor maps to the human chromosome region 1p35-p36. Molecular cloning of the rat receptor is described This application claims the benefit of U.S. Provisional in Ruat et al. (1993) Biochem BiophyS Res Commun Application Ser. No. 60/055,817, filed Aug. 15, 1997. 193:268-276. This invention was made with Government support Boess et al. (1997) Neuropharmacology 36:713-720, under Grant no. DAO0282, awarded by the National Insti stably expressed the rat 5-HT6 receptor in HEK293 cells and tutes of Health. The Government has certain rights in this compared the affinity of a range of compounds in competi invention. tion binding experiments. The observed binding was LSD > omega-N-methyl-5-HT = bufote nine = 5 INTRODUCTION methoxy tryptamine > 5-HT22-methyl-5-HT =5- Background ben Zyl oxy trypt a mine = trypt a mine > 5 carboxamidotryptamined>5-HTQ. Receptor antagnosists The biogenic amine Serotonin (5-hydroxytryptamine; include methiothepin, clozapine, mianserin and ritanserin. 5-HT) is a brain neurotransmitter that has been strongly 15 The binding of typical and atypical antipsychotic agents to implicated in the pathophysiology and treatment of a wide 5-HT6 and 5-HT7 receptors is discussed in Roth et al. variety of neuropsychiatric disorders. It exerts its effects (1994) J Pharmacol Exp Ther 268: 1403–1410. Clozapine through a diverse family of Serotonin receptor Subtypes. Of and several related atypical antipsychotic agents (rilapine, the 14 different mammalian Serotonin receptors to have been olanzepine, tioSpirone, fluperlapine, clorotepine and cloned, all but one are members of the G-protein coupled Zotepine) had high affinities for 5-HT6 receptor. Several receptor Superfamily. Several of these, including the Sero dopamine-Selective antipsychotic agents (raclopride, rimca tonin 5-HT6 receptor, stimulate adenylyl cyclase via G zole and penfluridol) had essentially no affinity for either the coupling. 5-HT6 has a high affinity for several therapeuti 5-HT6 or 5-HT7 receptors. cally important antidepressant, antianxiety, hallucinogenic Ward and Dorsa (1996) J Comp Neurol 370:405-414 and antipsychotic drugs, particularly the atypical antipsy 25 describe the striatal distribution of the mRNAS of the chotics such as clozapine. The relevance of the 5-HT6 serotonin2A (5-HT2A), serotonin2C (5-HT2C), and seroto receptor to psychotherapeutics is indicated both through its niné (5-HT6) receptors in relation to enkephalin, Substance unique anatomical distribution and pharmacological prop P, and dynorphin expressing output neurons. Pierce et al. erties. (1996) Neuroscience 70:553–559 assayed for the presence Messenger RNA encoding the 5-HT6 receptor has been of messenger RNA for rat Serotonin receptor Subtypes in localized by in situ hybridization histochemistry to brain peripheral Sensory and Sympathetic ganglia was detected regions that regulate emotional responses, cognition and using the method of polymerase chain reaction. The pres motor function. This distribution pattern is generally ence of messenger RNA for 5-HT6 receptor was found matched by the localization of the 5-HT6 receptor protein within Superior cervical ganglia, but not lumbar Sympathetic identified by specific antibodies. This distribution is consis 35 ganglia. tent with the binding of psychoactive drugs. Therefore, The effects of administering 5-HT6 antisense oligonucle 5-HT6 receptors are implicated in the etiology of major otides to the rat brain are described in Sleight et al. (1996) psychiatric disorders and in the actions of psychiatric drugs. Behav Brain Res 73:245–248; and Bourson et al. (1995) J Antipsychotic medications are the treatment of choice for Pharmacol Exp Ther 274:173–180. A behavioral syndrome Schizoaffective disorders. Evidence to date Suggests that all 40 of yawning, Stretching and chewing was observed in treated of the antipsychotic drugs (except clozapine) are similarly ratS. effective in treating psychoses, with the differences being in milligram potency and Side effects. Sometimes patients view SUMMARY OF THE INVENTION the Side-effects of the antipsychotic drugs as being worse than their original psychosis. These include acute dystonic 45 Non-human transgenic animal models are provided for reactions, akathisia, Parkinsonism, in which akinesia is a serotonin 5-HT6 receptor (5-HT6) function, where the trans key feature, and tardive dyskinesia. Clozapine (Clozaril) has genic animal is characterized by having an altered 5-HT6 been proven to be more effective than all other antipsychotic gene. Alterations to the gene include deletions or other loSS drugs in treating certain disorders. It has reduced movement of function mutations, introduction of an exogenous gene Side-effects, but other Serious Side-effects limit its use. In 50 having a nucleotide Sequence with targeted or random mutations, introduction of an exogenous gene from another rare cases coma and death may result from the drug treat Species, or a combination thereof. The transgenic animals ment. may be either homozygous or heterozygous for the alter The importance of psychoactive drugs in present treat ation. The animals and cells derived therefrom are useful for ment of mental illness, and the presence of Serious and Screening biologically active agents that may modulate undesirable Side-effects with their use, makes the develop 55 ment of improved drugs of great interest. Animal models 5-HT6 receptor function. The screening methods are of useful in Screening assays provide a benefit by determining particular use for determining the Specificity and action of candidate agents that have improved specificity of action. antianxiety, antidepressant or antipsychotic drugs, particu Relevant Literature larly Serotoninergic drugs, or drugs that may interact with An Overview of 5-hydroxytryptamine receptor Subtypes 60 Serotonin receptors. The animals are useful as a model to in vertebrates and invertebrates may be found in Saudou and investigate the role of serotonin 5-HT6 receptors in normal Hen (1994) Neurochem Int 25:503–532. The cloning, brain function. characterization, and chromosomal localization of a human BRIEF DESCRIPTION OF THE DRAWINGS 5-HT6 serotonin receptor is described in Kohen et al. (1996) J Neurochem 66:47-56.