WO2003057825A2

WO2003057825A2 - Gene expression patterns in response to atypical and typical neuroleptic agents in the central nervous system

Info

Publication number: WO2003057825A2
Application number: PCT/US2002/036558
Authority: WO
Inventors: Elizabeth A. Thomas; Amy J. Warren; J. Gregor Sutcliffe
Original assignee: Digital Gene Technologies, Inc.
Priority date: 2001-11-13
Filing date: 2002-11-13
Publication date: 2003-07-17
Also published as: AU2002365190A8; AU2002365190A1; WO2003057825A3

Abstract

Polynucleotides, polypeptides, kits and methods are provided related to genes expressed in the central nervous system that are regulated by atypical and typical neuroleptics.

Description

GENE EXPRESSION PATTERNS IN RESPONSE TO ATYPICAL AND TYPICAL

NEUROLEPTIC AGENTS IN THE CENTRAL NERVOUS SYSTEM

RELATED APPLICATION

The present application claims priority to U.S. Provisional Patent Application No. 60/338,999, filed November 13, 2001, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Neuropsychiatric disorders, including schizophrenia, affective and behavioral disorders, are a heterogeneous group of devastating illnesses that can impair all aspects of a patient's life. Although positive symptoms, such as hallucinations and delusions are often emphasized, the negative symptoms of these disorders prevent patients from functioning in society, maintaining a job or exhibiting proper social behavior. Mental disorders, such as schizophrenia, represent a major public health problem that affects not only the patients and families, but imposes a costly impact on the health system and economy as well (Wasylenki, D. A., Can. J. Psych., 39:S35 (1994); Miller, D. D., Pharmacotherapy, 16: 2 (1996)). It has been estimated that approximately 30-50% of homeless Americans have some form of mental illness (Susser et al., Community Ment. Health J, 26: 391 (1990)). Genetic studies have implicated several susceptibility loci for schizophrenia on five distinct chromosomes; however, the etiology and pathophysiology of the disease have yet to be determined. Given the heterogeneity of the disease, it is not surprising that no single biological system or anatomical region has been proven to be pivotal to pathology. It is thought that dysfunction in multiple brain regions contributes to the overall manifestation of disease symptoms and numerous reports have identified abnormalities throughout the brain. Nevertheless, there is still no absolute consensus regarding which brain regions and neurochemical systems are most affected.

Given their apparent function in normal and diseased brain states, it is likely that midbrain dopamine neurons play an important role in the development of neuropathology. For example, many psychiatric disorders are associated with overactive dopaminergic activity in the meso-striatal dopamine system which refers to both the nigro-striatal dopamine pathway

NB1:580246.1 (neurons linking the substantia nigra to the striatum), and the meso-limbic dopamine pathway (neurons linking the ventral tegmental area to limbic regions, such as amygdala, olfactory tubercle and the nucleus accumbens, which is often considered a ventral extension of the striatum). Additionally, it is known that Parkinson's disease is caused by the degeneration of dopamine neurons of the nigro-striatal pathway.

In the general population, the risk for developing a psychiatric disorder is approximately 1% (Maier et al, Curr. Opin. in Psych., 11: 19 (1998); Kendler et al, Arch. Gen. Psych., 50: 9095 (1993)). However, this risk increases to 10% or 40% if one or both parents, respectively, have the disease. Concordance in monozygotic and dizygotic twins remains only as high 40- 50%) (Kendler et al., supra (1993)). While there is undoubtedly a genetic component to the transmission of psychiatric disorders, the lack of full concordance in dizygotic twins indicates that there are other environmental factors that contribute (Maier et al., supra (1998); Kendler et al., supra (1993)). A current challenge in genetic research on mental illnesses is the identification of mutations conferring susceptibility to, or genes associated with therapeutics for, such disorders. One approach addressing the latter is to identify genes whose expression is altered during the process of drug treatment. Considerable evidence demonstrates that the ameliorative effects of neuroleptic drugs, as well as their unwanted motor side effects, are the result of changes in gene expression.

Examples of neuroleptic drugs that are widely used in the long-term treatment of various psychiatric disorders, such as schizophrenia, include haloperidol and clozapine. The antipsychotic effects of neuroleptic drugs are generally attributed to blockade of D₂ receptors in the meso-limbic dopamine system (Metzler et al., Schizophrenia Bull, 2, 19-76 (1976)). The best evidence for this comes from the excellent correlation observed between the therapeutic potency of neuroleptics and their affinity for binding to the D receptor (Seeman et al., Curr. Opn. Neurol And Neurosurg., 6, 602-608 (1993); Creese et al., Science, 192, 481-483 (1976); Peroutka et al, Am. J. Psych., 137, 1518-1522 (1980); Deutch, et al., Schizophren. Res., 4, 121- 156 (1991); Seeman, P., Synapse 1, 133-152 (1987)). Although neuroleptic drags have affinity for other neurotransmitter receptors in the brain, such as muscarinic acetylcholine, 5-HT, alpha- adrenergic and histamine receptors, no correlation to clinical efficacy has been observed with these receptors (Peroutka et al, Am. J. Psych. (1980); Richelson et al, Eur. J. Phαrm., 103, 197- 204 (1984)).

NB 1:580246.1 Human brain imaging studies have demonstrated that dopamine receptors become blocked to a level of >70% after only a few hours of treatment with various neuroleptic drugs (Sedvall et al., Arch. Gen. Psych., 43: 995 (1986)). This blockade has been shown to lead to a compensatory increase in dopamine receptor number and supersensitivity of the unblocked receptors (Clow et al., Psychopharm., 69, 227-233 (1980); Rupniak et al, Life Sci., 32, 2289- 2311 (1983); Rogue et al, Eur. J. Pharm., 201, 165-169 (1991)). Furthermore, the short-term effects of dopamine antagonists on the brain are well known and include such effects as an increase in dopamine synthesis and catabolism, an increase in the firing rate of dopamine neurons resulting from the inhibition of pre-synaptic dopamine autoreceptors (Grace et al., J. Pharm. Exp. Ther., 238, 1092-1100 (1986), and a potentiation of cyclic AMP formation resulting from the blockade of post-synaptic dopamine receptors (Rupniak et al., Psychopharm., 84, 519- 521 (1984)).

In addition to their antipsychotic actions, neuroleptics can cause a series of mild to severe side effects. Some of these side-effects result from the non-specific nature of neuroleptic drugs, including hypotension and tachycardia, which results from alpha-adrenergic receptor blockade, and dry mouth and blurred vision, which results from the blockade of muscarinic acetylcholine receptors. The predominant and most undesirable effects that accompany neuroleptic treatment are the long-lasting motor deficits referred to as extrapyramidal side effects (Marsden et al., Psychol Med., 10, 55-72 (1980)). Extrapyramidal side effects are associated with the blockade of dopamine receptors in the dorsal striatum (Moore et al., Clin. Neuropharmacol, 12, 167-184 (1989) and include such motor deficits as dystonias (muscle spasms), akathisias (motor restlessness), Parkinson' s-like symptoms and Tardive Dyskinesia. Roughly 20% of patients taking antipsychotics demonstrate Parkinson' s-like symptoms, the blockade of dopamine D₂ receptors in the striatum being functionally equivalent to the degeneration of nigro-striatal dopamine neurons seen in Parkinson's Disease. Tardive Dyskinesia is a syndrome of abnormal involuntary movements that afflicts roughly 25% of patients on neuroleptic treatment (Jeste et al, Psychopharmacol, 106, 154-160 (1992); Casey, D. E., Schizo. Res., 35: S61 (1999)). The danger of this side effect is that it can be potentially irreversible, that is, patients can still have symptoms of Tardive Dyskinesia long after the antipsychotic has been discontinued. This implicates an epigenetic component to the effects of chronic neuroleptic treatment.

NB 1:580246.1 Interestingly, "typical" neuroleptics, such as haloperidol and fluphenazine, have a much higher propensity for causing extrapyramidal side effects than "atypical" neuroleptic drugs, such as clozapine, which rarely causes these types of effects. Although clozapine differs from haloperidol in its pharmalogical profile, the specific mechanism leading to the lack of motor side effects is unclear. Since clozapine has high affinity for other neurotransmitter receptors, such as muscarinic, adrenergic and serotonin receptors, it is possible that the antipsychotic actions of clozapine are partly due to blockade of these other receptors, which may restore proper balance of the dopamine input and output pathways of the basal ganglia.

Despite the immediate occupancy of dopamine receptors, neuroleptic drugs have a delayed onset of clinical action, which often can be up to several weeks. Further, as discussed above, neuroleptic drugs are characterized by their ability to cause late and long-lasting motor deficits (Marsden et al., Psychol. Med., 10: 55 (1980)). The distinct temporal discrepancy which exists between dopamine receptor occupancy and the onset of therapeutic and extrapyramidal side effects, suggests that additional molecular changes in the brain occur downstream from dopamine receptor blockade. In an attempt to identify the downstream molecular mechanisms, studies have focused on dopamine-receptor regulation of individual target genes in the striatum and nucleus accumbens.

For example, several studies have demonstrated that acute treatment with antipsychotic drugs causes induction of several immediate-early genes (Hughes et al., Pharmacol. Rev., 47: 133 (1995); Fibiger, H. C, J. Clin. Psych., 55: 33 (1994); Nguyen et al, Proc. Natl. Acad. Sci., 89, 4270-4274 (1992); MacGibbon et al., Mol. Brain. Res. 23, 21-32 (1994); Robertson et al., Neuro. Sci., 46, 315-328 (1992); Dragunow et al., Neuro. Sci, 37, 287-294 (1990); Miller J, Neurochem., 54, 1453-1455 (1990); Rogue et al, Brain Res. Bull, 29: 469 (1992)). Some immediate early gene proteins (IEGPs) act as transcription factors by binding to specific DNA sequences and regulating gene transcription. Thus, IEGPs can link receptor-mediated signalling effects to long-term changes in genomic activity. Recent studies have shown that haloperidol, a typical neuroleptic, induces the expression c-Fos in the rat striatum and nucleus accumbens, whereas, clozapine, an atypical neuroleptic, induces c-Fos in the nucleus accumbens only (Nguyen et al., Proc. Natl. Acad. Sci. (1992); MacGibbon et al., Mol. Brain Res. (1994); Robertson et al., Neurosci. (1992)). Haloperidol has also been shown to induce expression of other IEGPs, such as FosB, JunB, JunD and Krox24, in the striatum and nucleus accumbens

NB1:580246.1 (Rogue et al., Brain Res. Bull. 29, 469-472 (1992); Marsden et al., Psych. Med. (1980); Moore et al., Clin. Neuropharmacol. (1989)). hi contrast, clozapine has been shown to induce Krox24 and JunB in the nucleus accumbens only (Nguyen et al. (1992); MacGibbon et al. (1994)). These results suggest that clozapine's lower tendency to cause extrapyramidal side effects, compared to "typical" neuro leptics, may be associated with its failure to induce IEGPs in the striatum.

The appearance of immediate early genes after acute treatment with neuroleptics likely precedes a number of other molecular changes responsible for the delayed adaptive changes that occur with drug treatment in the striatum.

Chronic treatment with neuroleptic drugs has also been shown to cause changes in the expression of certain neuropeptides and neurotransmitter receptors. In distinct regions of the striatum, both neurotensin and enkephalin are upregulated after chronic (7 - 28 days) treatment with haloperidol, while levels of protachykinin mRNA are decreased (Merchant et al., J Pharm. Exp. Ther., 271, 460-471 (1994); Delfs et al, J Neurochem., 63, 777-780 (1994); Angulo et al, Neurosci. Lett. 113, 217-221 (1990)). In contrast, chronic clozapine treatment results in a decrease in enkephalin mRNA levels and only small changes in the expression of neurotensin and tachykinin (Merchant et al. (1994); Mercugliano et al, Neurosci. Lett., 136, 10-15 (1992); Angulo et al. (1990)). These differences suggest that neuropeptides may play a role in the motor deficits that result from treatment with typical neuroleptics.

Researchers have also demonstrated the regulation of genes associated with glutaminergic neuro transmission. For example, a decrease in mRNA expression of the glutamate transporter, GLT-1, was observed in the striatum after 30 days of haloperidol treatment, but not after clozapine exposure (Schneider et al., Neuroreport., 9, 133-136 (1998)). Similar treatment with haloperidol also resulted in an increase in the N-methyl-D-aspartate (NMD A) receptor subunits, NR1 and NR2, whereas clozapine treatment resulted in a lesser induction (Riva et al., Mol. Brain. Res. 50, 136-142 (1997)).

In addition, pathological and structural changes in the striatum have been observed after chronic drug treatment. Studies using experimental animals have detected a reduction in the size and number of striatal neurons and neuronal processes, as well as decreases in striatal neuronal density following chronic treatment with haloperidol (Christensen et al., Acta. Psych. Scand., 46, 14-23 (1910), Jeste et al., Psychopharm., 106, 154-160 (1992); Mahadik et al., Biol. Psych., 24, 199-217 (1988); Nielson et. al, Psychopharm., 59-85-89 (1978). These studies imply that

NB 1:580246.1 neuroleptics may have a neurotoxic effect on the striatum which could account for the ensuing neuroleptic-induced side effects.

Long-term changes in the expression of critical genes resulting from neuroleptic drug therapy may compensate for underlying genetically determined biochemical deficits, thereby restoring a state of normal mental activity, or alternatively, can cause detrimental or permanent consequences. Hence, genes that are regulated by drug treatment may provide information regarding pathways responsible for behavioral dysfunction. Although the above studies have examined the expression of a few individual target genes, there has been no comprehensive study of the effects of neuroleptics on gene expression over time in the striatum and nucleus accumbens, brain regions considered to be critically involved in the actions of neuroleptic drags. Thus, the number and identity of the genes which are differentially expressed following acute and chronic treatment with neuroleptics in these tissues remains unknown. Further, there has been no comprehensive examination of the differences between the striatal mRNA expression induced by typical neuroleptics and the expression induced by atypical neuroleptics.

Such a systematic characterization would allow the identification of genes that contribute to neuropathologies associated with neuropsychiatric disorders. This information can reveal pathways for the mechanism of actions of antipsychotic drags, as well as provide insight regarding the underlying basis of psychiatric dysfunction. Specifically, the identification of potentially harmful gene products is important to identify molecules that could be useful as diagnostic markers indicating neuropathology. Additionally, the identification of potentially harmful gene products is important to identify molecules that could be amenable to pharmaceutical intervention. A systematic characterization would also allow the identification of beneficial molecules that contribute to conditions of neuroprotection. Such identification of beneficial products could lead to the development of pharmaceutical agents useful in the treatment of neuropsychiatric disorders. Furthermore, the identification of harmful and beneficial products may lead to new lines of study towards the amelioration of symptoms associated with neuropsychiatric disorders.

Such a comparative study would also identify the genes that regulate the antipsychotic actions of neuroleptics versus those responsible for the unwanted side effects associated with these drags. This information would advance the development of an antipsychotic therapy that

NB 1:580246.1 would target specific actions of neuroleptic drugs or, alternatively, would selectively block proteins causing the motor side effects.

What is needed therefore, is a comprehensive examination of the differences between the striatal mRNA expression induced by typical neuroleptics and the expression induced by atypical neuroleptics. The Total Gene Expression Analysis (TOGA ) method, described in Sutcliffe et al., Proc. Natl. Acad. Sci. USA 97(5): 1976-81 (2000), International published application WO 00/26406, U.S. application serial no. 09/775,217, PCT/US02/02666, U.S. Patent No. 5,459,037, U.S. Patent No. 5,807,680, U.S. Patent No. 6,030,784, U.S. Patent No. 6,096,503, U.S. Patent No. 6,110,680, and 6,309,834, all of which are incorporated herein by reference, is a tool used to identify and analyze mRNA expression. The TOGA method is an improved method for the simultaneous sequence-specific identification of mRNAs in an mRNA population which allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA. The method can identify changes in expression of mRNA induced by typical neuroleptics and the expression induced by atypical neuroleptics.

MB1:580246.1 SUMMARY OF THE INVENTION

The present invention provides polynucleotides and the encoded polypeptides that are regulated by neuroleptic use. The present invention also provides different uses of these polynucleotides and polypeptides. The invention was made while performing studies using the PCR-based Total Gene Expression Analysis (TOGA method to analyze the expression patterns of thousands of genes and comparing the expression patterns among time courses following clozapine or haloperidol drug treatment. TOGA analysis identified several genes that were altered in their expression in response to clozapine and/or haloperidol administration in mouse brain. In particular, the TOGA system was used to examine how gene expression in the striatum and cortex is regulated by an atypical neuroleptic agent, such as clozapine, and a typical neuroleptic agent, such as haloperidol. These studies identified proteins and genes that are regulated by the treatment of atypical and typical drugs. Further, these studies identified several genes that are differentially regulated by typical and atypical drugs.

These studies identified proteins, genes and regions of genes that fit into three different classes that represent patterns of time-dependent changes in brain region-specific gene expression induced by chronic treatment with neuroletpic drugs. One class of sequences was identified that demonstrated a common regulation pattern for both neuroleptics, and thus are expected to account for some of the benefit (i.e., anti-psychotic activity) of these two types of drags. A second class of sequences demonstrated a regulation pattern unique to clozapine treatments, and thus would account for the clozapine-specific benefits and side-effects. Finally, a third class of sequences demonstrated a regulation pattern unique to haloperidol treatments, and thus would account for the haloperidol-specific (i.e., extrapyramidal) side-effects. In addition, brain region-specific patterns of response to clozapine were observed (i.e., striatum compared to cortex).

Thus, these studies were used to determine the genes specifically associated with antipsychotic activity versus those associated with extrapyramidal side effects, which information advances the development of improved antipsychotic therapies. Specifically, genes that demonstrate the same regulation pattern in response both atypical and typical neuroleptic treatments suggest pathways associated with the beneficial effects of these drugs in the treatment of neuropsychiatric disorders. In addition, genes uniquely regulated in response to only

NB1:580246.1 haloperidol, which has a higher propensity for causing extrapyramidal side effects, are likely to be associated with these types of effects. Knowledge of these sequences may explain the mechanisms through which the patients derive benefit, may explain the nature of the underlying pathology, and might provide new targets for designing therapeutics.

Further, the identified neuroleptic-regulated molecules are useful in therapeutic and diagnostic applications in the treatment of various neuropsychiatric disorders. Such molecules are also useful as probes as described by their size, partial nucleotide sequence and characteristic regulation pattern associated with neuroleptic administration.

The present invention associates previously known and novel polynucleotides and their encoded polypeptides to various neuropsychiatric disorders such that the polynucleotides and polypeptides may be useful for diagnosis and treament of such neuropsychiatric disorders. Another embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as a neuropsychiatric disorder, comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide of the invention or a polynucleotide of the invention. In one preferred embodiment, a method for preventing, treating, modulating or ameliorating schizophrenia is provided. In another preferred embodiment, a method for preventing, treating, modulating or ameliorating bipolar disorder, psychoses or Alzheimer's Disease is provided.

A further embodiment of the invention provides an isolated antibody that binds specifically to the isolated polypeptide of the invention. A preferred embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as a neuropsychiatric disorder, comprising administering to a mammalian subject a therapeutically effective amount of the antibody. In one preferred embodiment, a method for preventing, treating, modulating or ameliorating schizophrenia is provided. In another preferred embodiment, a method for preventing, treating, modulating or ameliorating bipolar disorders, psychoses or Alzheimer's Disease is provided.

An additional embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject. The method comprises determining the presence or absence of a mutation in a polynucleotide of the invention. A pathological condition or a susceptibility to a pathological condition, such as a neuropsychiatric disorder, is diagnosed based on the presence or absence of the mutation. In one

NB1:580246.1 preferred embodiment, a method for diagnosing schizophrenia is provided. In another preferred embodiment, a method for diagnosing bipolar disorders, psychoses or Alzheimer's Disease is provided.

Even another embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition, such as a neuropsychiatric disorder, in a subject. Especially preferred embodiments include methods of diagnosing schizoplirenia and bipolar disorders. The method comprises detecting an alteration in expression of a polypeptide encoded by the polynucleotide of the invention, wherein the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition. The alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression. In a preferred embodiment a first biological sample is obtained from a patient suspected of having a neuropsychiatric disorder, for example, schizophrenia or a bipolar disorder, and a second sample from a suitable comparable control source is obtained. The amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample. The amount of the polypeptide in the first and second samples is determined. A patient is diagnosed as having a neuropsychiatric disorder if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.

Where a polynucleotide of the invention is up-regulated, such as after chronic treatment with clozapine or haloperidol, the expression of the polynucleotide can be increased or the level of the intact polypeptide product can be increased in order to treat, prevent, ameliorate, or modulate the pathological condition. For example, increased expression of the SEQ ID NO:34 (NEU2_38) was observed after chronic treatment with clozapine, increased expression of the SEQ ID NO: 64 (NEU2_125) was observed after chronic treatment with haloperidol, and treatment with either neuroleptic lead to increased expression of SEQ ID NO:5 (NEU2_5). By increasing in vivo levels of such polynucleotides or polypeptide products, it may be possible to inhibit symptoms or reduce the severity of symptoms of schizophrenia or other neuropsychiatric disorders. This can be accomplished by, for example, administering a polynucleotide or polypeptide of the invention (or a set of polynucleotides and polypeptides including those of the invention) to the mammalian subject.

NB 1:580246.1 Where a polynucleotide of the invention is down-regulated, such as after chronic treatment with clozapine, the expression of the polynucleotide can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition, such as psychosis or other neuropsychiatric disorders. For example, decreased expression of the polynucleotide NEU2_29 (SEQ ID NO: 27) in striatum was observed after chronic administration of clozapine. In addition, the polynucleotide NEU2_6 (SEQ ID NO: 6) was down-regulated in cortex by chronic administration of clozapine. This activity may represent pathways common to the beneficial effects of clozapine treatment of psychosis or other neuropsychiatric disorders. By decreasing the in vivo levels of such polynucleotides or polypeptide products, it may be possible to inhibit symptoms or reduce the severity of symptoms of schizophrenia or other neuropsychiatric disorders. This can be accomplished by, for example, the use of antisense oligonucleotides, triple helix base pairing methodology or ribozymes. Alternatively, drugs or antibodies that bind to and inactivate the polypeptide product can be used.

Additionally, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides. One embodiment of the invention provides an isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NOs: 1-78, which is regulated by neuroleptic administration. Also provided is an isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to any one of these isolated nucleic acid molecules and an isolated nucleic acid molecule at least ten bases in length that is hybridizable to any one of these isolated nucleic acid molecules under stringent conditions. Any one of these isolated nucleic acid molecules can comprise sequential nucleotide deletions from either the 5 '-terminus or the 3 '-terminus. Further provided is a recombinant vector comprising any one of these isolated nucleic acid molecules and a recombinant host cell comprising any one of these isolated nucleic acid molecules. Also provided is the gene corresponding to the cDNA sequence of any one of these isolated nucleic acids.

Another embodiment of the invention provides an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NOs: 1-78. Also provided is an isolated nucleic acid molecule encoding any of these polypeptides, an isolated nucleic acid molecule encoding a fragment of any of these polypeptides, an isolated nucleic acid molecule

NB 1:580246.1 encoding a polypeptide epitope of any of these polypeptides, and an isolated nucleic acid encoding a species homologue of any of these polypeptides. Preferably, any one of these polypeptides has biological activity. Optionally, any one of the isolated polypeptides comprises sequential amino acid deletions from either the C-terminus or the N-terminus. Further provided is a recombinant host cell that expresses any one of these isolated polypeptides.

Yet another embodiment of the invention comprises an isolated antibody that binds specifically to an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO: 1-78. The isolated antibody can be a monoclonal antibody or a polyclonal antibody.

Another embodiment of the invention provides a method for identifying a binding partner to a polypeptide of the invention. A polypeptide of the invention is contacted with a binding partner and it is determined whether the binding partner affects an activity of the polypeptide.

Yet another embodiment of the invention is a method of identifying an activity of an expressed polypeptide in a biological assay. A polypeptide of the invention is expressed in a cell and isolated. The expressed polypeptide is tested for an activity in a biological assay and the activity of the expressed polypeptide is identified based on the test results.

Still another embodiment of the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in neuropsychiatric disorders, chosen from the group consisting of the DNA molecules shown in SEQ ID NOs: 1-78.

Even another embodiment of the invention provides a kit for detecting the presence of a polypeptide of the invention in a mammalian tissue sample. The kit comprises a first antibody which immunoreacts with a mammalian protein encoded by a gene corresponding to the polynucleotide of the invention or with a polypeptide encoded by the polynucleotide in an amount sufficient for at least one assay and suitable packaging material. The kit can further comprise a second antibody that binds to the first antibody. The second antibody can be labeled with enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, or bioluminescent compounds.

Another embodiment of the invention provides a kit for detecting the presence of genes or regions thereof encoding a protein comprising a polynucleotide of the invention, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay, and suitable packaging material.

NB1:580246.1 Yet another embodiment of the invention provides a method for detecting the presence of a nucleic acid encoding a protein in a mammalian tissue sample. A polynucleotide of the invention or fragment thereof having at least 10 contiguous bases is hybridized with the nucleic acid of the sample. The presence of the hybridization product is detected.

Additional embodiments of the invention provide a method for using a polynucleotide of the invention, a polypeptide of the invention, an antibody of the invention, or a gene of the invention or a region thereof for the manufacture of a medicament useful in the treatment of a neuropsychiatric disorder, such as, for example, psychoses, Alzheimer's disease, bipolar affective disorder or schizophrenia. An additional embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject. The method comprises determining the presence or absence of a mutation in a polynucleotide or gene of the invention or a region thereof. A pathological condition or a susceptibility to a pathological condition, such as neuropsychiatric disorder, such as, for example, psychoses, Alzheimer's disease, bipolar affective disorder or schizophrenia is diagnosed based on the presence or absence of the mutation.

Yet other embodiments of the invention involve assessing the stage of neuropsychiatric disorder, such as, for example, psychoses, Alzheimer's disease, bipolar affective disorder or schizophrenia by testing for regulation of at least one polynucleotide, polypeptide, antibody or gene of the invention or a region thereof. Further embodiments of the invention involve assessing the efficacy or toxicity of a therapeutic treatment for a neuropsychiatric disorder, such as, for example, psychoses, Alzheimer's disease, bipolar affective disorder or schizophrenia by testing for regulation of at least one polynucleotide, polypeptide, antibody or gene of the invention or a region thereof.

Another embodiment of the present invention provides a method of using a polynucleotide, polypeptide, antibody or gene of the invention or a region thereof for delivering to a patient in need thereof, genes, DNA vaccines, diagnostic reagents, peptides, proteins or macromolecules. Another embodiment of the invention provides a method of using a polypeptide or antibody of the invention to identify a binding partner to a polypeptide of the invention. In a preferred embodiment, a polypeptide of the invention is contacted with a binding partner and it is determined whether the binding partner affects an activity of the polypeptide.

NB1:580246.1 Additionally, the present invention provides novel polynucleotides, genes and their encoded polypeptides. One embodiment of the invention provides an isolated nucleic acid molecule comprising a polynucleotide listed in SEQ ID NO:4. Also provided is an isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to any one of the isolated nucleic acid molecules of the invention, an isolated nucleic acid molecule at least ten bases in length that is hybridizable to any one of the isolated nucleic acid molecules of the invention under stringent conditions, and an isolated nucleic acid molecule that is a homolog, ortholog, or paralog of any one of the isolated nucleic acid molecules of the invention. Any one of the isolated nucleic acid molecules of the invention can comprise sequential nucleotide deletions from either the 5 '-terminus or the 3 '-terminus. Also provided is the gene corresponding to the cDNA sequence of any one of the isolated nucleic acids of the invention, an isolated nucleic acid molecule hybridizable to such gene under stringent conditions, and an isolated nucleic acid molecule or gene that is a homolog, paralog or ortholog of such gene.

Another embodiment of the invention provides an isolated or purified polypeptide encoded by a polynucleotide listed in SEQ ID NO:4, a polynucleotide at least 95% identical to said polynucleotide or a gene corresponding to one of the foregoing polynucleotides and the complements and degenerate variants thereof. Also provided is an isolated or purified polypeptide 90% identical to one of the foregoing polypeptides, a fragment of one the foregoing polypeptides, and the homologs, paralogs, and orthologs of the foregoing polypeptides. Also provided is an isolated nucleic acid molecule or gene encoding any of the polypeptides or polypeptide fragments of the invention. Optionally, any one of the isolated polypeptides of the invention comprises sequential amino acid deletions from either the C-terminus or the N- terminus.

Yet another embodiment of the invention comprises an isolated antibody that binds specifically to an isolated polypeptide encoded by a polynucleotide listed in SEQ ID NO:4, a polynucleotide at least 95% identical to said polynucleotide or a gene corresponding to one of the foregoing polynucleotides and the complements and degenerate variants thereof. The isolated antibody can be a monoclonal antibody or a polyclonal antibody.

Still another embodiment of the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in a neuropsychiatric disorder, such as, for example, psychoses, Alzheimer's disease, bipolar affective disorder or schizophrenia, chosen

NB1:580246.1 from the group consisting of the DNA molecules shown in SEQ ID NO: 1-78, or their corresponding genes or regions thereof, or DNA molecules at least 95 > similar to one of the foregoing molecules.

Even another embodiment of the invention provides a kit for detecting the presence of a polypeptide of the invention in a mammalian tissue sample. In one embodiment, the kit comprises a first antibody that immunoreacts with a mammalian protein encoded by a gene corresponding to the polynucleotide of the invention or with a polypeptide encoded by the polynucleotide in an amount sufficient for at least one assay and suitable packaging material. The kit can further comprise a second antibody that binds to the first antibody. The second antibody can be labeled with enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, bioluminescent compounds, or with an organic moiety, such as biotin.

Another embodiment of the invention provides a kit for detecting the presence of genes encoding a protein comprising a polynucleotide of the invention, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay, and suitable packaging material.

An additional embodiment of the invention involves a method for identifying biomolecules associated with a neuropsychiatric disorder, such as, for example, psychoses, Alzheimer's disease, bipolar affective disorder or schizophrenia comprising the steps of: developing a cellular experiment specific for a neuropsychiatric disorder, such as, for example, psychoses, Alzheimer's disease, bipolar affective disorder or schizophrenia, harvesting the RNA from the cells used in the experiment, obtaining a gene expression profile, and using the gene expression profile for identifying biomolecules whose expression was altered during the experiment. The biomolecules identified may be polynucleotides, polypeptides or genes.

Another embodiment of the invention provides a method for assessing the efficacy a treatment for treating a neuropsychiatric disorder in a subject, wherein the neuropsychiatric disorder is selected form the group consisting of psychoses, Alzheimer's disease, bipolar affective disorder and schizophrenia, the method comprising the steps of comparing: (a) a level of expression of a polynucleotide selected from the group consisting of SEQ ID NOs:l-78, or a polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs:l-78, in a first sample obtained from the subject prior to providing at least a portion of the

NB1:580246.1 treatment to the subject; and (b) a level of expression of the same polynucleotide or polypeptide in a second sample following provision of the portion of the treatment, wherein a modulated level of expression of the polynucleotide or polypeptide in the second sample relative to the first sample, is an indication that the treatment is efficacious for treating the neuropsychiatric disorder. In a preferred embodiment, the treatment admimstered to the subject is clozapine treatment. In another preferred embodiment, the treatment administered to the subject is haloperidol treatment.

Another embodiment of the present invention provides a method for optimizing neuroleptic drug dosage in a patient afflicted with a neuropsychiatric disorder, such as, for example psychoses, Alzheimer's disease, bipolar affective disorder and schizophrenia. The method involves comparing a level of expression of a molecule of the present invention from a sample from the patient prior to providing the neuroleptic drug to the patient and comparing a level of expression of the same molecule of the present invention from a second sample taken from the patient after administration of the drug. These samples are then compared with a symptom of the neuropsychiatric disorder in the patient prior to administration of the treatment with the same symptom in the patient after administration of the drag . Finally, a correlation is made between the level of expression of the marker molecule of the present invention with the dosage of the neuroleptic drag and the symptom, wherein the lowest dosage of neuroleptic drug that modulates the level of expression of the marker molecule, as demonstrated by an amelioration of the symptom, is an indication that the dosage of the neuroleptic drag is optimized for the patient.

The foregoing merely summarizes certain aspects of the invention and is not intended, nor should it be construed as limiting the invention in any way.

NB1:580246.1 BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where:

Figure 1 is a graphical representation of the results of TOGA runs using a 5' PCR primer with parsing bases CTCG (SEQ ID NO:85) and the universal 3' PCR primer (SEQ ID NO: 82) showing PCR products produced from mRNA extracted from the striatum of mice treated with 7.5 mg/kg of clozapine for the following durations: no treatment (no clozapine) (Panel A), saline treatment (no clozapine) (Panel B), 45 minutes (Panel C), 12 days (Panel D), and 2 weeks (Panel E). Also shown is PCR products produced from mRNA extracted from the cortex of mice treated with clozapine for the following durations: saline (no clozapine) (Panel F), 5 days (Panel G), and 2 weeks (Panel H). The vertical index line indicates a PCR product of about 141 b.p. that is expressed to a greater level in the 12 and 14 day clozapine-treated striatum samples than in the untreated or saline treated striatum samples. Although not differentially regulated in cortex, this sequence was also highly expressed in cortex tissue. The horizontal axis represents the number of base pairs of the molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA analysis (which corresponds to the relative expression of the molecule of that address). The results of the TOGA ® runs have been normalized using the methods described in pending U.S. Patent Application Serial No.

09/318,699/U.S., and PCT Application Serial No. PCT/US00/14159, both entitled Methods and

System for Amplitude Normalization and Selection of Data Peaks (Dennis Grace, Jayson

Durham); and U.S. Patent 6,334,099, PCT Application Serial No. PCT/US00/14123, and pending U.S. Patent Application Serial Nos. 09/940,987/U.S., 09/940,581/U.S., 09/940,746/U.S., all entitled Methods for Normalization of Experimental Data (Dennis Grace, Jayson Durham), all of which are incorporated herein by reference. The vertical line drawn through the eight panels represents the DST molecule identified as NEU2_38 (SEQ TD NO:34).

Figure 2 presents a graphical example of the results obtained when a DST is verified by the Extended TOGA method using a primer generated from a cloned product (as described below). The PCR product corresponding to SEQ ID NO:34 (NEU2_38) was cloned and a 5'

PCR primer was built from the cloned DST (SEQ ID NO:86). The product obtained from PCR with this primer (SEQ ID NO:86) and the universal 3' PCR primer (SEQ ID NO:82) (as shown

NB1:580246.1 in the top panel, A) was compared to the length of the original PCR product that was produced in the TOGA^® reaction with mRNA extracted from the cortex of mice treated with 7.5 mg/kg of clozapine for 5 days using a 5' PCR primer with parsing bases CTCG (SEQ ID NO:85) and the universal 3' PCR primer (SEQ ID NO:82) (as shown in the middle panel, B). Again, for all panels, the number of base pairs is shown on the horizontal axis, and fluorescence intensity (which corresponds to relative expression) is found on the vertical axis. In the bottom panel (panel C), the traces from the top and middle panels are overlaid, demonstrating that the peak found using an extended primer from the cloned DST is the same number of base pairs as the original PCR product obtained through TOGA^® as NEU2_38 (SEQ ID NO:34). The bottom panel thus illustrates that NEU2_38 (SEQ ID NO:34) was the DST amplified in Extended

TOGA^®.

® Figure 3 is a graphical representation of the results of TOGA analysis, similar to Figure

1, using a 5' PCR primer with parsing bases AGCA (SEQ ID NO: 87) and the universal 3' primer (SEQ ID NO: 82), showing PCR products produced from mRNA extracted from the striatum of mice treated with 7.5 mg/kg of clozapine for the following durations: untreated (no clozapine) (Panel A), 45 minutes (Panel B), 12 days (Panel C), and 2 weeks (Panel D). The vertical index line indicates a PCR product of about 274 b.p. that is present in the untreated sample and is down-regulated within 45 minutes in the clozapine-treated sample, and remains down-regulated for 14 days in the presence of clozapine. The vertical line drawn through the five panels represents the DST molecule identified as NEU2_29 (SEQ ID NO:27).

® Figure 4 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases AGTT (SEQ ID NO: 88) and universal 3' primer (SEQ ID NO: 82), showing PCR products produced from mRNA extracted from the cortex of mice treated with 7.5 mg/kg of clozapine for the following durations: saline (no clozapine) (Panel A), 5 days (Panel

B), and 2 weeks (Panel C). The vertical index line indicates a PCR product of about 328 b.p. that is present in the saline treated cortex sample and decreases in expression over time in the clozapine-treated cortex samples. The vertical line drawn through the five panels represents the

DST molecule identified as NEU2_6 (SEQ ID NO:6).

® Figure 5 is a graphical representation of the results of TOGA runs using a 5' PCR primer with parsing bases TGTC (SEQ ID NO:89) and the universal 3' PCR primer (SEQ ID

NO: 82) showing PCR products produced from mRNA extracted from the striatum of mice

NB1:580246.1 treated with 4.0 mg/kg of haloperidol for the following durations: no treatment (no haloperidol) (Panel A), saline treatment (no haloperidol) (Panel B), 7 hour (Panel C), 10 days (Panel D), and 2 weeks (Panel E). The vertical index line indicates a PCR product of about 318 b.p. that is expressed to a greater level in the 10 and 14 day clozapine-treated striatum samples than in the untreated or saline treated striatum samples. The vertical line drawn through the five panels represents the DST molecule identified as NEU2_125 (SEQ ID NO:64).

Figure 6 is a graphical representation of the results of TOGA runs using a 5' PCR primer with parsing bases AGCA (SEQ ID NO:90) and the universal 3' PCR primer (SEQ ID NO: 82) showing PCR products produced from mRNA extracted from the striatum of mice treated with 7.5 mg/kg of clozapine for the following durations: no treatment (no clozapine) (Panel A), saline treatment (no clozapine) (Panel B), 45 minutes (Panel C), 12 days (Panel D), and 2 weeks (Panel E). The vertical index line indicates a PCR product of about 312 b.p. that is expressed to an increasingly greater level as increased duration of treatment with clozapine. This trend is summarized graphically in Panel F. Also shown is PCR products produced from 4.0 mg/kg of haloperidol for the following durations: no treatment (no haloperidol) (Panel G), saline treatment (no haloperidol) (Panel H), 7 hour (Panel I), 10 days (Panel J), and 2 weeks (Panel K). The vertical index line indicates this PCR product of about 312 b.p. is also expressed to an increasingly greater level with increased duration of treatment with haloperidol. This trend is summarized graphically in Panel L. The vertical line drawn through all these panels represents the DST molecule identified as NEU2_5 (SEQ ID NO:5).

NB1:580246.1 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention and the methods of obtaining and using the present invention will be described in detail after setting forth some preliminary definitions, which are provided to facilitate understanding of certain terms used in the present invention. Many of the techniques described herein are described in Dracopoli et al., Current Protocols in Human Genetics, John Wiley and Sons, New York (1999), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York (2000), both of which are incorporated herein by reference.

Definitions

"Isolated" refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state.

"Stringent hybridization conditions" refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5X SSC (5X SSC = 750 mM NaCl, 75 mM sodium citrate, 50 mM sodium phosphate pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at about 65°C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37°C in a solution comprising 6X SSPE (20X SSPE = 3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50°C with IX SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC). Note that variations in the above conditions may be accomplished tlirough the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO (5% w/v non-fat dried milk in phosphate buffered saline ("PBS"), heparin, denatured salmon sperm DNA, and other commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Of course, a polynucleotide which hybridizes only to polyA÷ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary

NB1:580246.1 stretch of T (or U) residues, would not be included in the definition of "polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

"Conservative amino acid substitution" refers to a substitution between similar amino acids that preserves an essential chemical characteristic of the original polypeptide.

"Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g., Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin and Griffin, Eds., Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov and Devereux, Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991)). While there exists a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo et al., SIAMJ Applied Math., 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in "Guide to Huge Computers," Martin J. Bishop, Ed., Academic Press, San Diego, (1994) and Carillo et al., (1988), Supra.

"EST" refers to an Expressed Sequence Tag, i.e. a short sequence of a gene made from cDNA, typically in the range of 200 to 500 base pairs. Since an EST corresponds to a specific region of a gene, it can be used as a tool to help identify unknown genes and map their position in the genome.

"DST" refers to a Digital Sequence Tag, i.e., a polynucleotide that is an expressed sequence tag of the 3' end of an mRNA.

Other terms used in the fields of biotechnology and molecular and cell biology as used herein will be as generally understood by one of ordinary skill in the applicable arts.

Treatment of Neurological and Psychiatric Disorders

Where a polynucleotide, polypeptide or gene of the invention or region thereof is upregulated, such as after chronic treatment with clozapine or haloperidol, the expression of the polynucleotide or gene or region thereof can be increased or the level of the intact polypeptide product can be increased in order to treat, prevent, ameliorate, or modulate the pathological condition. For example, increased expression of the SEQ ID NO:34 (NEU2_38) was observed

NB1:580246.1 after chronic treatment with clozapine, increased expression of the SEQ ID NO: 64 (NEU2_125) was observed after chronic treatment with haloperidol, and treatment with either neuroleptic lead to increased expression of SEQ ID NO:5 (NEU2_5). By increasing in vivo levels of such polynucleotides or polypeptide products, it may be possible to inhibit symptoms or reduce the severity of symptoms of schizophrenia or other neuropsychiatric disorders. This can be accomplished by, for example, administering a polynucleotide, polypeptide or gene of the invention or region thereof (or a set of polynucleotides, polypeptides, genes or regions thereof, including those of the invention) to the mammalian subject.

A polynucleotide or gene of the invention or region thereof can be administered to a mammalian subject alone or with other polynucleotides or genes by a recombinant expression vector comprising the polynucleotide or gene or region thereof. As used herein, a mammalian subject can be a human, baboon, chimpanzee, macaque, cow, horse, sheep, pig, dog, cat, rabbit, guinea pig, rat or mouse. Preferably, the recombinant vector comprises a polynucleotide shown in SEQ ID NOs: 1-78 inclusive or a polynucleotide which is at least 98% identical to a nucleic acid sequence shown in SEQ ID NOs: 1-78 inclusive or a gene corresponding to one of the foregoing polynucleotides or a region thereof. Also, preferably, the recombinant vector comprises a variant polynucleotide that is at least 80%, 90%), or 95% identical to a polynucleotide comprising at least one of SEQ ID NOs: 1-78 inclusive, a polynucleotide at least ten bases in length hybridizable to polynucleotide comprising at least one of SEQ ID NOs: 1-78 inclusive, a polynucleotide comprising at least one SEQ ID NOs: 1-78 inclusive with sequential nucleotide deletions from either the 5' terminus or the 3' terminus, or a species homolog of a polynucleotide comprising at least one of SEQ ID NOs: 1-78 inclusive or gene corresponding to any one of the foregoing polynucleotides of a region thereof.

The administration of a polynucleotide or gene of the invention, or region thereof or recombinant expression vector containing such polynucleotide, gene or region thereof to a mammalian subject can be used to express a polynucleotide in said subject for the treatment of neurological and psychiatric disorders, for example, schizophrenia. Expression of a polynucleotide or gene in target cells, including but not limited to brain cells, would effect greater production of the encoded polypeptide. In some cases, where the encoded polypeptide is a nuclear protein, the regulation of other genes may be secondarily up- or down-regulated. The administration of a polynucleotide or recombinant expression vector of the invention to a

NB1:580246.1 mammalian subject can be used to express a polynucleotide in the said subject for the treatment of, for example, psychosis or other neuropsychiatric disorders. Expression of a polynucleotide in target cells, including but not limited to the cells of the striatum and nucleus accumbens, would effect greater production of the encoded polypeptide. High expression of the polynucleotide would be advantageous since increased expression was observed after chronic treatment with clozapine or haloperidol.

There are available to one skilled in the art multiple viral and non- viral methods suitable for introduction of a nucleic acid molecule into a target cell, as described above. In addition, a naked polynucleotide, gene or region thereof can be administered to target cells. Polynucleotides and genes of the invention or regions thereof and recombinant expression vectors of the invention can be administered as a pharmaceutical composition (including, without limitation, genes delivered by vectors such as adeno-associated virus, liposomes, PLGA, canarypox virus, adenovirus, retroviruses including IL-1 and GM-CSF antagonists). Such a composition comprises an effective amount of a polynucleotide, gene or region thereof or recombinant expression vector, and a pharmaceutically acceptable formulation agent selected for suitability with the mode of administration. Suitable formulation materials preferably are non-toxic to recipients at the concentrations employed and can modify, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. See Remington 's Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990).

The pharmaceutically active compounds (i.e., a polynucleotide, gene or region thereof or a vector) can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals. Thus, the pharmaceutical composition comprising a polynucleotide, gene or region thereof or a recombinant expression vector may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).

The dosage regimen for treating a disease with a composition comprising a polynucleotide, gene or region thereof or expression vector is based on a variety of factors, including the type or severity of the neurological or psychiatric disorder, the age, weight, sex, medical condition of the patient, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using

NB 1:580246.1 standard methods. A typical dosage may range from about 0.1 mg/kg to about 100 mg/kg or more, depending on the factors mentioned above.

The frequency of dosing will depend upon the pharmacokinetic parameters of the polynucleotide, gene or region thereof or vector in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.

The cells of a mammalian subject may be transfected in vivo, ex vivo, or in vitro. Administration of a polynucleotide, gene or region thereof or a recombinant vector containing a polynucleotide, gene or region thereof to a target cell in vivo may be accomplished using any of a variety of techniques well known to those skilled in the art. For example, U.S. Patent No. 5,672,344 describes an in vivo viral-mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector. The above-described compositions of polynucleotides, genes and regions thereof and recombinant vectors can be transfected in vivo by oral, buccal, parenteral, rectal, or topical administration as well as by inhalation spray. The term "parenteral" as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.

While the nucleic acids and/or vectors of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors of the invention or other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

Another delivery system for polynucleotides or genes of the invention and regions thereof is a "non-viral" delivery system. Techniques that have been used or proposed for gene therapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO₄ precipitation, gene gun techniques, electroporation, lipofection, and colloidal dispersion (Mulligan, R., (1993) Science, 260 (5110):926-32). Any of these methods are widely available

NB1:580246.1 to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention may be accomplished using any of the available methods of transfection. Several such methodologies have been utilized by those skilled in the art with varying success. Id.

Where a polynucleotide, polypeptide or gene of the invention or region thereof is downregulated, such as after chronic treatment with clozapine, the expression of the polynucleotide or gene can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition, such as psychosis or other neuropsychiatric disorders. For example, decreased expression of the polynucleotide NEU2_29 (SEQ ID NO: 27) in striatum was observed after chronic administration of clozapine. In addition, the polynucleotide NEU2_6 (SEQ ID NO: 6) was down-regulated in cortex by chronic administration of clozapine. This activity may represent pathways common to the beneficial effects of clozapine treatment of psychosis or other neuropsychiatric disorders. By decreasing the in vivo levels of such polynucleotides or polypeptide products, it may be possible to inhibit symptoms or reduce the severity of symptoms of schizophrenia or other neuropsychiatric disorders. This can be accomplished by, for example, the use of antisense oligonucleotides, triple helix base pairing methodology or ribozymes. Alternatively, drags or antibodies that bind to and inactivate the polypeptide product can be used.

Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of gene products of the invention in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonotliioates, alkylphosphonates,

NB1:580246.1 phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, (1994) Meth. Mol. Biol, 20:1-8; Sonveaux, (1994) Meth. Mol. Biol, 26:1-72; Uhlmann et al., (1990) Chem. Rev., 90:543-583.

Modifications of gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of a gene of the invention. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred.

Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al., in Huber & Carr, Molecular and Immulogic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a polynucleotide. Antisense oligonucleotides that comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides that are precisely complementary to a polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent nucleotides, can provide sufficient targeting specificity for mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to a polynucleotide or gene of the invention or regions thereof. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3'

NB1-.580246.1 hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., (1992) Trends Biotechnol, 10:152-158; Uhlmann et al., (1990) Chem. Rev., 90:543-584; Uhlmann et al, (1987) Tetrahedron. Lett, 215:3539-3542.

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, (1987) Science, 236:1532-1539; Cech, (1990) Ann. Rev. Biochem., 59:543-568; Cech, (1992) Curr. Opin. Struct. Biol, 2:605-609; Couture & Stinchco b, (1996) Trends Genet., 12:510-515. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent No. 5,641,673). The mechanism of ribozyme action involves sequence- specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a polynucleotide or gene of the invention or a region thereof can be used to generate ribozymes that will specifically bind to mRNA transcribed from the polynucleotide. Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. (1988) Nature, 334:585-591). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, e.g., Gerlach et al., EP 321,201).

Specific ribozyme cleavage sites within a RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ JJD NOs: 1-78 inclusive, their complements and their corresponding genes and regions thereof provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing

NB1:580246.1 and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease polynucleotide expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al., U.S. Patent No. 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Polypeptides or antibodies to the polypeptides of the invention can also be used directly as therapeutics to prevent, treat, modulate, or ameliorate disease. The mammalian subject (preferably a human) can be given a recombinant or synthetic form of the polypeptide in one of many possible different formulations, including, but not limited to, subcutaneous, intravenous, intramuscular, mtraperitoneal, or intracranial injections of a solution of the polypeptide or antibody, or a suspension of a crystallized form of the polypeptide or antibody; topical creams or slow release cutaneous patch containing the polypeptide; encapsulated forms for oral or other gastrointestinal delivery of the polypeptide or antibody. In some cases, delivery of the polypeptide or antibody may be in the form of injection or transplantation of cells or tissues containing an expression vector such that a recombinant form of the polypeptide will be secreted by the cells or tissues, as described above for transfected cells.

The frequency and dosage of the administration of the polypeptides or antibodies will be determined by factors such as the biological activity of the pharmacological preparation, the persistence and clearance of the active protein, and the goals of treatment. In the case of antibody

NB1:580246.1 therapies, the frequency and dosage will also depend on the ability of the antibody to bind and neutralize the target molecules in the target tissues.

Diagnostic Tests

Pathological conditions or susceptibility to pathological conditions, such as psychosis or other neuropsychiatric disorders, can be diagnosed using methods of the invention. Testing for expression of a polynucleotide or gene of the invention or regions thereof or for the presence of the polynucleotide or gene product can correlate with the severity of the condition and can also indicate appropriate treatment. Furthermore, testing for regulation of a polynucleotide or gene of the invention or regions thereof or a panel of polynucleotides or genes of the invention or regions thereof can be used in drug development studies to assess the efficacy or toxicity of any experimental therapeutic. For example, the presence or absence of a mutation in a polynucleotide or gene of the invention or regions thereof can be determined through sequencing techniques known to those skilled in the art and a pathological condition or a susceptibility to a pathological condition can be diagnosed based on the presence or absence of the mutation. Further, an alteration in expression of a polypeptide encoded by a polynucleotide or gene of the invention can be detected, where the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition. The alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression, i.e. a modulation in expression.

The use of diagnostic tests is not limited to determining the presence of or susceptibility to disease. In many cases, the diagnostic test can be used to assess disease stage, especially in situations where such an objective lab test has no alternative reliable subjective test available. These tests can be used to follow the course of disease, help predict the future course of disease, or determine the possible reversal of the disease condition. For example, the level of expression of polynucleotides, genes, polypeptides of the invention or regions thereof may be indicative of disease stage or progression.

In drug development studies, these tests can be useful as efficacy markers, so that the ability of any new therapeutics to treat disease can be evaluated on the basis of these objective assays. The utility of these diagnostic tests will first be determined by developing statistical information correlating the specific lab test values with several clinical parameters so that the lab test values can be known to reliably predict certain clinical conditions.

NB1:580246.1 In many cases, the diagnostic lab tests based on the polynucleotides, genes, antibodies or polypeptides of the invention, i.e., gene expression profiles of polynucleotides or polypeptides encoded by the polynucleotides identified in SEQ ID NOs: 1-78, will be important markers of drug or disease toxicity. The markers of toxicity versus drag efficacy will be determined by studies correlating the effects of lαiown toxins or pathological conditions with specific alterations in gene regulation. Toxicity markers generated in this fashion will be useful to distinguish the various therapeutic versus deleterious effects on cells or tissues in the patient.

As an additional method of diagnosis, a first biological sample from a patient suspected of having a pathological condition, such as psychoses or other neuropsychiatric disorders, is obtained along with a second sample from a suitable comparable control source. A biological sample can comprise saliva, blood, cerebrospinal fluid, amniotic fluid, urine, feces, tissue, or the like. A suitable control source can be obtained from one or more mammalian subjects that do not have the pathological condition. For example, the average concentration and distribution of a polynucleotide, gene, or polypeptide of the invention or a region thereof can be determined from biological samples taken from a representative population of mammalian subjects, wherein the mammalian subjects are the same species as the subject from which the test sample was obtained. The amount of at least one polypeptide, gene, polynucleotide of the invention or region thereof is determined in the first and second sample. The amounts of the polypeptide in the first and second samples are compared. A patient is diagnosed as having the pathological condition if the amount of the polypeptide, gene, polynucleotide of the invention or a region thereof in the first sample is greater than or less than the amount of the polypeptide, gene, polynucleotide of the invention or a region thereof in the second sample. Preferably, the amount of polypeptide, gene, polynucleotide of the invention or a region thereof in the first sample falls in the range of samples taken from a representative group of patients with the pathological condition.

The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

The present invention also includes a diagnostic system, preferably in kit form, for assaying for the presence of the polypeptide of the present invention in a body sample, including,

NB 1:580246.1 but not limited to brain tissue, cell suspensions or tissue sections; or a body fluid sample, such as CSF, blood, plasma or serum, where it is desirable to detect the presence, and preferably the amount, of the polypeptide of this invention in the sample according to the diagnostic methods described herein.

In a related embodiment, the discovery of differential expression patterns for the molecules of the invention allows for screening of test compounds with an eye to modulating a particular expression pattern; for example, screening can be done for compounds that will convert an expression profile for a poor prognosis to a better prognosis. These methods can also be done on the protein basis; that is, protein expression levels of the molecules of the invention, such as, for example, polypeptides encoded by the polynucleotides identified in SEQ ID NOs: 1- 78, can be evaluated for diagnostic and prognostic purposes or to screen test compounds.

In addition, the invention provides methods of conducting high-throughput screening for test compounds capable of inhibiting activity of proteins encoded by the polynucleotides of the invention, i.e., SEQ ID NOs: 1-78. The method of high-throughput screening involves combining test compounds and the polypeptide and measuring an effect of the test compound on the encoded polypeptide. Functional assays such as cytosensor microphysiometer, calcium flux assays such as FLIPR (Molecular Devices Corp, Sunnyvale, CA), or the TUNEL assay may be employed to measure cellular activity.

The invention also provides a method of screening test compounds for inhibitors of psychosis or other neuropsychiatric disorders and the pharmaceutical compositions comprising the test compounds. The method for screening comprises obtaining samples from subjects afflicted with psychosis or other neuropsychiatric disorders, maintaining separate aliquots of the samples with a plurality of test compounds, and comparing expression of a molecules of the invention, i.e., SEQ ID NOs: 1-78, in each of the aliquots to determine whether any of the test compounds provides a substantially modulated level of expression relative to samples with other test compounds or to an untreated sample. In addition, methods of screening may be devised by combining a test compound with a protein and thereby determining the effect of the test compound on the polypeptide.

In a related embodiment, a nucleic acid molecule can be used as a probe (i.e., an oligonucleotide) to detect the presence of a polynucleotide of the present invention, a gene corresponding to a polynucleotide of the present invention or a region thereof, or a mRNA in a

NB1:580246.1 cell that is diagnostic for the presence or expression of a polypeptide of the present invention in the cell. The nucleic acid molecule probes can be of a variety of lengths from at least about 10 to about 5000 nucleotides long, although they will typically be about 20 to 500 nucleotides in length. The probe can be used to detect the polynucleotide, gene, gene region or mRNA through hybridization methods that are well known in the art.

In a related embodiment, detection of genes corresponding to the polynucleotides of the present invention can be conducted by primer extension reactions such as the polymerase chain reaction (PCR). To that end, PCR primers are utilized in pairs, as is well known, based on the nucleotide sequence of the gene to be detected. Preferably, the nucleotide sequence is a portion of the nucleotide sequence of a polynucleotide of the present invention. Particularly preferred PCR primers can be derived from any portion of a DNA sequence encoding a polypeptide of the present invention, but are preferentially from regions that are not conserved in other cellular proteins.

Preferred PCR primer pairs useful for detecting the genes corresponding to the polynucleotides of the present invention and expression of these genes are described below. Nucleotide primers from the corresponding region of the polypeptides of the present invention described herein are readily prepared and used as PCR primers for detection of the presence or expression of the corresponding gene in any of a variety of tissues.

In another embodiment, a diagnostic system, preferably in kit form, is contemplated for assaying for the presence of the polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention in a body fluid sample. Such diagnostic kit would be useful for monitoring the fate of a therapeutically admimstered polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention. The system includes, in an amount sufficient for at least one assay, a polypeptide of the present invention and/or a subject antibody as a separately packaged immunochemical reagent. Instructions for use of the packaged reagent(s) are also typically included.

A diagnostic system of the present invention preferably also includes a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule of the present invention.

NB1:580246.1 Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC), tetramethyhhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis et al., Eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference. Other suitable labeling agents are known to those skilled in the art.

In preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase or a vitamin, such as biotin, additional reagents are required to visualize the formation of the receptor-ligand complex. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor, such as diaminobenzidine. Such additional reagents for biotin include streptavidin. An additional reagent useful with glucose oxidase is 2,2'-amino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as ¹²⁴1, ¹²⁵1, ¹²⁸1, ¹³²I and ⁵¹Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is ¹²⁵I. Another group of useful labeling means are those elements such as ^πC, ¹⁸F, ¹⁵O and ¹³N which themselves emit positrons. The positrons so emitted produce gamma rays upon

NB 1:580246.1 encounters with electrons present in the animal's body. Also useful is a beta emitter, such ^mindium or ³H.

The linking of labels or labeling of polypeptides and proteins is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium (see, e.g., Galfre et al., Meth. Enzymol, 73:3-46 (1981)). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable (see, e.g., Aurameas, et al., Scand. J. Immunol, Vol. 8 Suppl. 7:7-23 (1978); Rodwell et al., Biotech., 3:889-894 (1984); and U.S. Patent No. 4,493,795).

The diagnostic systems can also include, preferably as a separate package, a specific binding agent. Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, such as, S. aureus protein A, and the like. Preferably the specific binding agent binds the reagent species when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent. In these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of the polypeptide of the present invention in a sample. A description of the ELISA technique is found in Sites et al., Basic and Clinical Immunology, 4th Ed., Chap. 22, Lange Medical Publications, Los Altos, CA (1982) and in U.S. Patent No. 3,654,090; U.S. Patent No. 3,850,752; and U.S. Patent No. 4,016,043, which are all incorporated herein by reference.

Thus, in some embodiments, a polypeptide of the present invention, an antibody or a monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from an aqueous medium, although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adsorption methods are described herein.

Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia

NB1:580246.1 Fine Chemicals (Piscataway, NJ), agarose, polystyrene beads of about 1 micron (μm) to about 5 millimeters (mm) in diameter available from several suppliers (e.g., Abbott Laboratories, Chicago, IL), polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs (sheets, strips or paddles) or tubes, plates or the wells of a microtiter plate, such as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent, or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.

Novel Polynucleotides

The present invention also is directed to a certain novel polynucleotide identified as SEQ ID NO: 4. Searches were performed against the GCG Nucleotide and EST databases and showed no significant homology between the polynucleotides identified as SEQ ID NO: 4.

Genes

The present invention also relates to the genes corresponding to SEQ ID NOs: 1-78, and the polypeptides encoded by the polynucleotides or genes or regions thereof of SEQ ID NOs:l- 78. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Homologs, Paralogs and Orthologs

Also provided in the present invention are homologs of the polynucleotides, polypeptides and genes of the invention and regions thereof, including paralogous genes and orthologous genes. Nucleic acid homologs may be isolated and identified using suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homolog. Studies of gene and protein evolution often involve the comparison of homologs, which are sequences that have common origins but may or may not have common activity.

NB1:580246.1 Sequences that share an arbitrary level of similarity determined by alignment of matching bases are called homologous.

There are many cases in which genes have duplicated, assumed somewhat different functions and been moved to other regions of the genome (e.g. alpha and beta globin). Such related genes in the same species are referred to as paralogs (e.g., Lundin, 1993, who refers to Fitch, 1976 for tins distinction). They must be distinguished from orthologs (homologous genes in different species, such as beta globin in human and mouse) if any sensible comparisons are to be made. These terms as relate to genes are formally defined as follows:

As used herein, "paralogous genes" are genes within the same species produced by gene duplication in the course of evolution. They may be arranged in clusters or distributed on different chromosomes, an arrangement which is usually conserved in a wide range of vertebrates.

As used herein, "orthologous genes" describes homologous genes in different species that are descended from the same gene in the nearest common ancestor. Orthologs tend to have similar function.

In reports of previous Human Gene Mapping Workshops, the Comparative Gene Mapping Committee recommended explicit criteria for establishing homology between genes mapped in different species, as well as urging inclusion of specific criteria in comparative gene mapping publications (O'Brien and Graves, 1991). The evidence for gene homology might also be recorded in The Comparative Animal Genome database (TCAGdb). Revised criteria for determining homology can include any of the following (the most stringent are asterixed):

Gene or other nucleotide sequence:

• similar nucleotide sequence*

• cross-hybridization to the same molecular probe*

• conserved map position*

Protein or polypeptide:

• similar amino acid sequence*

• similar subunit structure and formation of functional heteropolymer

• immunological cross-reaction

• similar expression profile

• similar subcellular location

• similar substrate specificity

• similar response to specific inhibitors

NB1:580246.1 Phenotype:

• similar mutant phenotype

• complementation of function*

Two new criteria have recently been added. Because of the accumulation of overwhelming evidence for linkage conservation among mammal and vertebrate species, conserved map position may now itself constitute an important criterion of homology, and is particularly valuable in distinguishing between members of a gene family. Complementation of function has also been added, because it is now possible to establish complementation of function by transfection across even the widest species barriers.

More recent studies have also demonstrated that some of these criteria are much more stringent than others. A strong basis for homology would be a demonstration of high DNA or amino acid sequence similarity, plus in addition to conservation of map position between flanking homologous markers. Less robust immunological and biochemical criteria for gene homology would need to be confirmed at least by gene position. The assumption of gene homology must be considered a working hypothesis, and may later be further confirmed when further scientific criteria are applied.

Preferred embodiments of the present invention include homologs, paralogs and orthologs of the polynucleotides, polypeptides and genes of the invention and regions thereof.

Polypeptides

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art. See, e.g., Curr. Prot. Mol. Bio., Chapter 16.

The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence that 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.

NB1:580246.1 The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified by the one-step method described in Smith & Johnson (Gene, 67:31-40, 1988). Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies of the invention raised against the secreted protein according to methods that are well known in the art.

Signal Sequences

Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein (Virus Res., 3:271-286 (1985)). The method of von Heinje uses the information from the residues surrounding the cleavage site, typically residues -13 to +2, where +1 indicates the amino tenninus of the secreted protein (Nucleic Acids Res., 14:4683-4690 (1986)). Therefore, from a deduced amino acid sequence, a signal sequence and mature sequence can be identified.

The deduced amino acid sequence of a secreted polypeptide can be analyzed by a computer program called Signal P (Nielsen et al., Protein Engineering, 10:1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated.

As one of ordinary skill in the art will appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence conesponding to the translations of SEQ ID NOs: 1-78 and their conesponding genes which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides and genes encoding such polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal

NB1:580246.1 sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. These polypeptides, and the polynucleotides and genes encoding such polypeptides, are contemplated by the present invention.

Polynucleotide, Polypeptide and Gene Variants

Polynucleotide, polypeptide and gene variants differ from the polynucleotides, polypeptides and genes of the present invention, but retain essential properties thereof. In general, variants have close similarity overall and are identical in many regions to the polynucleotide or polypeptide of the present invention.

Further embodiments of the present invention include polynucleotides having at least 80% identity, more preferably at least 90% identity, and most preferably at least 95%, 96%, 91%, 98%o or 99% identity to a sequence contained in SEQ ID NOs:l-78. Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the polynucleotides having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, polynucleotides at least ten bases in length hybridizable to polynucleotide comprising at least one of SEQ ID NOs: 1-78 inclusive, polynucleotides comprising at least one SEQ ID NOs: 1-78 inclusive with sequential nucleotide deletions from either the 5' tenninus or the 3' terminus, or a species homolog of polynucleotides comprising at least one of SEQ ID NOs: 1-78 inclusive will encode a polypeptide identical to an amino acid sequence contained in the translations of SEQ ID NOs: 1-78.

Further embodiments of the present invention include genes and regions thereof having at least 80%) identity, more preferably at 90% identity, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to genes conesponding to a sequence contained in SEQ ID NOs: 1-78 and regions thereof. Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the genes having at least 85%, 90%, 95%o, 96%, 97%, or 99% identity respectively to genes of the invention, genes hybridizable to genes of the invention, genes of the invention with sequential nucleotide deletions from either the 5' terminus or the 3' terminus, or a species homolog of genes of the invention will encode a polypeptide identical to an amino acid sequence contained in the translations of genes of the invention.

NB 1:580246.1 Further embodiments of the present invention also include polypeptides having at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence contained in translations of SEQ ID NOs:l-78 and their conesponding genes. Preferably, the above polypeptides should exhibit at least one biological activity of the protein. In a prefened embodiment, polypeptides of the present invention include polypeptides having at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%>, 97%, 98%, or 99% similarity to an amino acid sequence contained in translations of SEQ ID NOs: 1-78 and their conesponding genes. Methods for aligning polynucleotides, polypeptides, genes or regions thereof are codified in computer programs, including the GCG program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)), and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) which uses the local homology algorithm of Smith and Waterman (Adv. in App. Math., 2:482-489 (1981)).

When using any of the sequence alignment programs to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference polynucleotide or gene that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide or gene are allowed.

A prefened method for detennining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also refened to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Bratlag et al. (Comp. App. Biosci, 6:237-245 (1990)). The term "sequence" includes nucleotide and amino acid sequences. In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is presented in terms of percent identity. Prefened parameters used in a FASTDB search of a DNA sequence to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, and Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, and Window Size=500 or query sequence length in nucleotide bases, whichever is shorter. Prefened parameters employed to calculate percent

NB1:580246.1 identity and similarity of an amino acid alignment are: Matrix=PAM 150, k-tuple=2, Mismatch Penalty^ 1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequence length in amino acid residues, whichever is shorter.

For example, a polynucleotide having a nucleotide sequence of at least 95% "identity" to a sequence contained in SEQ ID NOs: 1-78 means that the polynucleotide is identical to a sequence contained in SEQ ID NOs: 1-78 or the cDNA except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the total length (not just within a given 100 nucleotide sfretch). In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to SEQ ID NOs: 1-78, up to 5% of the nucleotides in the sequence contained in SEQ ID NOs: 1-78 or the cDNA can be deleted, inserted, or substituted with other nucleotides. These changes may occur anywhere throughout the polynucleotide.

Similarly, a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference polypeptide, means that the amino acid sequence of the polypeptide is identical to the reference polypeptide except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the total length of the reference polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially prefened are polynucleotide variants containing alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are prefened. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also prefened. Polynucleotide variants can be produced for a variety of reasons. For instance, a polynucleotide variant may be produced to

NB1:580246.1 optimize codon expression for a particular host (i.e., codons in the human mRNA may be changed to those prefened by a bacterial host, such as E. coli). Variants may also arise by the process of ribosomal frameshifting, by translational read-through at naturally occurring stop codons, and by decoding of in-frame translational stop codons UGA through insertion of selanocysteine (See The RNA World, 2^nd edition, ed: Gesteland, R.F., Cech, T.R., & Atkins, J.F.; Cold Spring Harbor Laboratory Press, 1999).

The variants may be allelic variants. Naturally occurring variants are called "allelic variants," and refer to one of several alternate fonns of a gene occupying a given locus on a chromosome of an organism (Lewin, Εd., Genes II, John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. See, e.g., Curr. Prot. Mol. Bio., Chapter 8.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as decreased aggregation. As known, aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity (see, e.g., Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes, 36: 838-845 (1987); Cleland et al., Crit. Rev. Therap. Drug Carrier Sys., 10:307-377 (1993)). Similarly, interferon gamma exhibited up to ten times higher activity after deleting 8- 10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology, 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle et al. conducted extensive mutational analysis of human cytokine IL-la (J. Biol. Chem., 268:22105-22111 (1993)). These investigators used random mutagenesis to generate over 3,500 individual IL- 1 a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators concluded that "most of the molecule could be altered with little effect on either binding or biological activity." In fact, only 23 unique amino acid sequences, out of more than 3,500 amino acid

NB1:580246.1 sequences examined, produced a protein that differed significantly in activity from the wild-type sequence. Another experiment demonstrated that one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. Ron et al. reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues (J. Biol. Chem. 268: 2984-2988 (1993)).

Furthermore, even if deleting one or more amino acids from the N-tenninus or C- terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C- tenninus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise lαiown in the art.

Thus, the invention further includes polypeptide variants that show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, frameshifting, read- through translational variants, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make pheno typically silent amino acid substitutions is provided in Bowie et al., Science, 247:1306- 1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, the amino acid positions that have been conserved between species can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions in which substitutions have been tolerated by natural selection indicate positions which are not critical for protein function. Thus, positions tolerating amino acid substitution may be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site- directed mutagenesis or alanine-scanning mutagenesis (the introduction of single alanine

NB1:580246.1 mutations at every residue in the molecule) can be used (Cunningham et al, Science, 244: 1081- 1085 (1989)). The resulting mutant molecules can then be tested for biological activity.

According to Bowie et al., these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be pennissive at certain amino acid positions in the protein. For example, the most buried or interior (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface or exterior side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Nal, Leu and lie; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin; replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp; and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Besides conservative amino acid substitution, variants of the present invention include: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code; (ii) substitution with one or more of amino acid residues having a substituent group; (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (e.g., polyethylene glycol); (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, a leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

Polynucleotide and Polypeptide Fragments

In the present invention, a "polynucleotide fragment" and "region of a gene" refers to a short polynucleotide having a nucleic acid sequence contained in SEQ ID ΝOs:l-78. The short nucleotide fragments are preferably at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length. A fragment "at least 20 nt in length," for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in that shown in SEQ ID NOs:l-78. These nucleotide fragments are useful as diagnostic probes and primers as discussed

NB1:580246.1 herein. Of course, larger fragments (e.g., 50, 150, and greater than 150 nucleotides) are prefened.

Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments having a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, to the end of SEQ ID NOs:l-78. In this context "about" includes the particularly recited ranges, larger or smaller by several nucleotides (i.e., 5, 4, 3, 2, or 1 nt) at either terminus or at both termini. Preferably, these fragments encode a polypeptide that has biological activity.

In the present invention, a "polypeptide fragment" refers to a short amino acid sequence contained in the franslations of SEQ ID NOs: 1-78. Protein fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, or 60 amino acids in length. In this context "about" includes the particularly recited ranges, larger or smaller by several amino acids (5, 4, 3, 2, or 1) at either extreme or at both extremes.

In situations where a DST of the present invention is not a translatable polypeptide, i.e., where the DST is in whole or in part of the 3' untranslated region of its conesponding gene, the translation product or region of the translation product of the gene conesponding to the DST is intended to be encompassed by the terms "polypeptide" or "polypeptide fragment" as used herein.

Prefened polypeptide fragments include the secreted protein as well as the mature form. Further prefened polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids ranging from 1-30, can be deleted from the carboxy tenninus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are prefened. Similarly, polynucleotide fragments encoding these polypeptide fragments are also prefened.

NB1:580246.1 Also prefened are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix-forming regions, beta-sheet and beta-sheet-fonning regions, turn and turn-fonning regions, coil and coil- forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of the translations of SEQ ID NOs:l-78 and their conesponding genes falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotide fragments encoding these domains are also contemplated.

Other prefened fragments are biologically active fragments or the polynucleotide or gene encoding biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Epitopes And Antibodies Or Binding Partners To Them

Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA, 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).

In the present invention, immunogenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, which specifically bind the epitope. (See, e.g., Wilson et al., Cell, 37:767-778 (1984); Sutcliffe et al, Science, 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used to induce antibodies or to select binding partners according to methods well known in the art. (See, e.g., Sutcliffe et al., (1983) supra; Wilson et al., (1984) supra; Chow et al, Proc. Natl. Acad. Sci., USA, 82:910-914; and Bittle et al, J. Gen. Virol., 66:2347-2354 (1985)). A prefened immunogenic epitope includes the secreted protein. The immunogenic epitope may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse). Alternatively, the immunogenic epitope may be prescribed without a carrier, if the sequence is of sufficient length (at least about 25 amino acids). However, immunogenic epitopes comprising as few as 8 to 10

NB1:580246.1 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.)

As used herein, the term "antibody" (Ab) or "monoclonal antibody" (mAb) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med., 24:316-325, 1983). Thus, these fragments are prefened, as well as the products of a Fab or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and human and humanized antibodies.

The antibodies may be chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. See, e.g., Co et al., Nature, 351:501-2 (1991). In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al., Nature, 332:323, 1988, Liu et al, PNAS, 84:3439, 1987, Larrick et al, Bio/Technology, 7:934, 1989, and Winter and Harris, TIPS, 14:139, May, 1993, Zou et al, Science 262:1271-4, 1993, Zou et al., Curr. Biol, 4:1099-103, 1994, and Walls et al., Nucleic Acids Res., 21:2921-9, 1993.

One method for producing a human antibody comprises immunizing a non-human animal, such as a transgenic mouse, with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-78 or their conesponding genes, whereby antibodies directed against the polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-78 or their conesponding genes are generated in said animal. Procedures have been developed for generating human antibodies in non-human animals. The antibodies may be partially human, or preferably completely human. For example, mice have been prepared in which one or more endogenous immunoglobulin genes are inactivated by various means and human

NB1:580246.1 immunoglobulin genes are introduced into the mice to replace the inactivated mouse genes. Such transgenic mice may be genetically altered in a variety of ways. The genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some (preferably virtually all) antibodies produced by the animal upon immunization. Examples of techniques for production and use of such transgenic animals are described in U.S. Patent Nos. 5,814,318, 5,569,825, and 5,545,806, which are incoφorated by reference herein. Antibodies produced by immunizing transgenic animals with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-78 or their conesponding genes and methods of using such antibodies are provided herein.

Monoclonal antibodies may be produced by conventional procedures, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule. The spleen cells may be fused with myeloma cells to produce hybridomas by conventional procedures. Examples of such techniques are described in U.S. Patent No. 4,196,265, which is incorporated by reference herein.

A method for producing a hybridoma cell line comprises immunizing such a transgenic animal with an immunogen comprising at least seven contiguous amino acid residues of a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-78 or their conesponding genes; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-78 or their conesponding genes. Such hybridoma cell lines, and monoclonal antibodies produced therefrom, are encompassed by the present invention. Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques. Examples of such techniques are described in U.S. Patent No. 4,469,630 and U.S. Patent No. 4,361,549.

Antibodies are only one example of binding partners to epitopes or receptor molecules. Other examples include, but are not limited to, synthetic peptides, which can be selected as a binding partner to an epitope or receptor molecule. The peptide may be selected from a peptide library as described by Appel et al., Biotechniques, 13, 901-905; and Dooley et al., J. Biol. Chem. 273, 18848-18856, 1998.

NB 1:580246.1 Binding assays can select for those binding partners (antibody, synthetic peptide, or other molecule) with highest affinity for the epitope or receptor molecule, using methods known in the art. Such assays may be done by immobilizing the epitope or receptor on a solid support, allowing binding of the library of antibodies or other molecules, and washing away those molecules with little or no affinity. Those binding partners or antibodies with highest affinity for the epitope or receptor will remain bound to the solid support. Alternatively, anays of candidate binding partners may be immobilized, and a labeled soluble receptor molecule is allowed to interact with the anay, followed by washing unbound receptors. High affinity binding is detectable by the presence of bound label.

Antibodies or other binding partners may be employed in an in vitro procedure, or administered in vivo to inhibit biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-78 or their conesponding genes. Disorders caused or exacerbated (directly or indirectly) by the interaction of such polypeptides of the present invention with cell surface receptors thus may be treated. For example, chronic administration of neuroleptics can cause unwanted side effects. Administration of an antibody derived from the identified polynucleotides might block the signaling that causes these side effects. Alternatively, an antibody derived from the identified polynucleotides might selectively block proteins causing motor side effects. A therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective for reducing a biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-78 or their conesponding genes. For example, chronic administration of neuroleptics can cause unwanted side effects. Administration of an antibody derived from the translation sequence of identified polynucleotides might block the signaling that causes these side effects. Alternatively, an antibody derived from the translation sequence of identified polynucleotides might selectively block proteins causing motor side effects.

Generally, antibodies or binding partners to receptors or cell surface polypeptides also can be linked to moieties, such as, for example, drug-loaded particles, antigens, DNA vaccines, immune modulators, other peptides, proteins for specific binding, and the like to the cells for targeting and enhanced delivery of the drug-loaded particles, antigens, DNA vaccines, immune modulators, other peptides, proteins for specific binding, and the like. Exemplary vaccines that can be specifically targeted to particular cells include, but are not limited to, rotavirus, influenza,

NB1:580246.1 diptheria, tetanus, pertussis, Hepatitis A, B and C, as well as conjugate vaccines, including S. pneumonia. Similarly, exemplary drugs that may be specifically targeted to particular cells include, but are not limited to, insulin, LHRH, buserlein, vasopressin and recombinant interleukins, such as IL-2 and IL-12. Additionally, exemplary vectors, such as, for example, adeno-associated virus, canarypox virus, adenoviras, retrovirus, and other delivery vehicles, such as, for example, liposomes and PLGA may be used to specifically target therapeutic moieties, such as, for example, IL-1 antagonist, GM-CSF antagonists, and the like, to particular cells. As is apparent to one skilled in the art, numerous other vaccines, drugs, and vectors may be useful in targeting and delivering therapeutic agents to particular cells.

Also provided herein are conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs:l-78 or their conesponding genes. Examples of such agents are well lαiown, and include, but are not limited to diagnostic radionuclides, therapeutic radionuclides, and cytotoxic drags. See, e.g., Thrush et al., Annu.Rev. Immunol, 14:49-71, 1996. The conjugates may be useful in in vitro or in vivo procedures.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptides of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptides of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such

NB1:580246.1 regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides is a familiar and routine technique in the art.

In addition, polypeptides of the present invention, including fragments and, specifically, epitopes, can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP A 394,827; Traunecker et al., Nature, 331 :84-86, 1988). Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone (Fountoulakis et al., J. Biochem., 270:3958-3964 (1995)).

Similarly, EP A 0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (see, e.g., EP A 0 232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 (See, Bennett et al., J. Mol. Recognition 8:52-58 (1995); Johanson et al., J Biol. Chem., 270:9459- 9471,1995).

Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a peptide that facilitates purification of the fused polypeptide. In prefened embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available. As described in Gentz et al., for instance, hexa-histidine provides for convenient purification of the fusion protein (Proc. Natl. Acad. Sci. USA 86:821-824 (1989)). Another peptide tag useful for purification, the "HA" tag, conesponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37:767 (1984)). Other fusion proteins may

NB1:580246.1 use the ability of the polypeptides of the present invention to target the delivery of a biologically active peptide. This might include focused delivery of a toxin to tumor cells, or a growth factor to stem cells.

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention. See, e.g., Curr. Prot. Mol. Bio., Chapter 9.6.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the polynucleotide or gene of the present invention or regions thereof, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides, genes or regions thereof may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. See, e.g., Curr. Prot. Mol. Bio., Chapters 9.9, 16.15.

The polynucleotide or gene or gene region insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SN40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells;

ΝB1:580246.1 animal cells such as CHO, COS, 293, and Bowes melanoma cells, and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Vectors prefened for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, ρNH8A, PNH16A, PNH18A, ρNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, ρKK223-3, pKK233-3, ρDR540, ρRIT5 available from Pharmacia Biotech, Inc. Among prefened eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may, in fact, be expressed by a host cell lacking a recombinant vector.

A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.

Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N- terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-tenninal methionine is covalently linked.

NB1:580246.1 Polypeptides of the present invention, and preferably the secreted form, can also be recovered from products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.

Other Uses of the Polynucleotides of the Invention

Each of the polynucleotides and genes of the present invention and regions thereof identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides and genes of the present invention and regions thereof are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available. Each polynucleotide of the present invention can be used as a chromosome marker. Cunently, no specific diagnostic markers exist that can be used to prevent or delay psychotic episodes of schizophrenia. The polynucleotides of the present invention may be used as chromosome markers for diagnosis for schizophrenia.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NOs: 1-78 or their conesponding genes or regions thereof. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers may then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene conesponding to the SEQ ID NOs: 1-78 or their conesponding genes or regions thereof will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene-mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides, genes of the invention or regions thereof can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase

NB1:580246.1 chromosomal spread. Tins technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides of 2,000-4,000 bp are prefened. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides, genes of the invention or regions thereof can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Prefened polynucleotides conespond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross-hybridization during chromosomal mapping.

Once a polynucleotide, gene of the invention or region thereof has been mapped to a precise chromosomal location, the physical position of the polynucleotide, gene or region thereof can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library), Kruglyak et al. (Am. J. Hum. Genet, 56:1212-23, 1995); Curr. Prot Hum. Genet. Assuming one megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes. Thus, once coinheritance is established, differences in the polynucleotide and the conesponding gene or region thereof between affected and unaffected individuals can be examined.

The polynucleotides of SEQ ID NOs: 1-78 and their conesponding genes or regions thereof can be used for this analysis of individuals. As noted above, many psychiatric disorders have genetic etiology and using the polynucleotides of the present invenion in a diagnostic panel can facilitate in the diagnosis of patients or identify patients at risk.

First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations is ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the conesponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new

NB1:580246.1 polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using polynucleotides or genes of the present invention or regions thereof. Any of these alterations (altered expression, chromosomal reanangement, or mutation) can be used as a diagnostic or prognostic marker.

In addition to the foregoing, a polynucleotide or gene of the invention or regions thereof can be used to control gene expression tlirough triple helix fonnation or antisense DNA or RNA. Both methods rely on binding of the polynucleotide or gene or gene region to DNA or RNA. For these techniques, prefened polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (see, Lee et al., Nuc. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251:1360 (1991) for discussion of triple helix formation) or to the mRNA itself (see, Okano, J. Neurochem, 56:560 (1991); and Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988) for a discussion of antisense technique). Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease.

Other Uses of the Polypeptides and Antibodies of the Invention

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, et al., J. Cell. Biol, 101:976-985, 1985; Jalkanen et al., J. Cell. Biol, 105:3087-3096, 1987). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). See, e.g., Curr. Prot. Mol. Bio., Chapter 11. Suitable antibody assay labels are known in the ait and include enzyme labels, such as glucose oxidase; and radioisotopes, such as iodine (¹²⁵1, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),

NB1:580246.1 tritium (³H), indium (¹¹²In), and technetium (⁹⁹ Tc); fluorescent labels, such as fluorescein and rhodamine; and organic moieties, such as biotin.

In addition to assaying secreted protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, nuclear magnetic resonance (NMR), or electron spin resonance (ESR). For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment that has been labeled with an appropriate detectable imaging moiety such as a radioisotope (e.g., ¹³¹1, ¹¹²In, ^99mTc), a radio- opaque substance, or a material detectable by NMR, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, the quantity of radioactivity necessary for a human subject will normally range from about 5 to 20 millicuries of ⁹⁹ Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel and Rhodes, Eds., Masson Publishing h e. (1982)).

Thus, the invention provides a method of diagnosing a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder. Psychiatric disorders and treatment of psychiatric disorders with neuroleptics, including schizoplirenia, are associated with a dysregulation of neurotransmitter and/or neuropeptide levels that can result in the up- or down regulation of polynucleotides and polypeptides. These changes can be diagnosed or monitored by assaying changes in polypeptide levels in tissue or fluids such as CSF, blood, or in fecal samples.

NB1:580246.1 Moreover, polypeptides of the present invention can be used to treat disease. For example, schizophrenic patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin); to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B); to inhibit the activity of a polypeptide (e.g., an oncogene); to activate the activity of a polypeptide (e.g., by binding to a receptor); to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble tumor necrosis factor (TNF) receptors used in reducing inflammation); or to bring about a desired response (e.g., blood vessel growth).

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor). Polypeptides can be used as antigens to trigger immune responses. Local production of neurotransmitters and neuropeptides modulates many aspects of neuronal function. For example, in schizophrema overactive neurotransmitter activity is thought to be basis for the psychotic behavior. Administration of an antibody to an overproduced polypeptide can be used to modulate neuronal responses in psychiatric disorders such as schizophrenia.

A mammalian subject (preferably a human) can be given a recombinant or synthetic form of a polypeptide or antibody in one of many possible different formulations, preferably encapsulated and other forms for oral or other gastrointestinal delivery of the polypeptide or antibody. In some cases, delivery of the polypeptide or antibody may be in the form of injection or transplantation of cells or tissues containing an expression vector such that a recombinant form of the polypeptide will be secreted by the cells or tissues, as described above for transfected cells.

The frequency and dosage of the administration of the polypeptides or antibodies will be determined by factors such as the biological activity of the pharmacological preparation and the goals in the treatment of psychosis or other neuropsychiatric disorders. In the case of antibody deliveries, the frequency of dosage will also depend on the ability of the antibody to bind and neutralize the target molecules in the target tissues.

NB 1:580246.1 Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. See, e.g., Curr. Prot. Mol. Bio., Chapter 11.15. Moreover, the polypeptides of the present invention can be used to test the following biological activities.

Biological Activities

The polynucleotides, polypeptides and genes of the present invention and regions thereof can be used in assays to test for one or more biological activities. If these polynucleotides, polypeptides and genes or gene regions exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides, polypeptides, genes and gene regions can be used to prevent or treat the associated disease or pathological condition. Examples of the disease or pathological conditions that may be prevented or treated according to the methods described herein include, but are not limited to, neurological and psychiatric disorders.

Nervous System Activity

A polypeptide, polynucleotide or gene of the present invention or region thereof may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of neuroblasts, stem cells, or glial cells. Also, a polypeptide, polynucleotide or gene of the present invention may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inhibiting the mechanisms of synaptic transmission, synthesis, metabolism and inactivation of neural transmitters, neuromodulators and trophic factors, and by activating or inhibiting the expression and incorporation of enzymes, structural proteins, membrane channels, and receptors in neurons and glial cells, or altering neural membrane compositions.

The etiology of these deficiencies or disorders may be genetic, somatic (such as cancer or some autoimmune disorder), acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotide, polypeptide or gene of the present invention or region thereof can be used as a marker or detector of a particular nervous system disease or disorder. The disorder or disease can be any of Alzheimer's Disease, Pick's disease, Binswanger's disease, other senile dementia, Parkinson's disease, parkinsonism, obsessive compulsive disorders, epilepsy,

NB1:580246.1 encephaolopathy, ischemia, alcohol addiction, drug addiction, schizophrenia, amyofrophic lateral sclerosis, multiple sclerosis, depression, and bipolar manic-depressive disorder. Alternatively, the polypeptide, polynucleotide or gene of the present invention or region thereof can be used to study circadian variation, aging, or long-term potentiation, the latter affecting the hippocampus. Additionally, particularly with reference to mRNA species occurring in particular structures within the cenfral nervous system, the polypeptide, polynucleotide or gene of the present invention or region thereof can be used to study brain regions that are known to be involved in complex behaviors, such as learning and memory, emotion, drug addiction, glutamate neurotoxicity, feeding behavior, olfaction, viral infection, vision, and movement disorders.

Binding Activity

A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (i.e., an agonist), increase, inhibit (i.e., an antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic (see, e.g., Coligan et al., Current Protocols in Immunology 1(2), Chapter 5 (1991)). Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds or, at least, related to a fragment of the receptor capable of being bound by the polypeptide (e.g., an active site). In either case, the molecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Prefened cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

The assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the test compound results in a signal generated by binding to the polypeptide.

NB1:580246.1 Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a test compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. At present, diagnosis of schizophrenia is based on clinical assessment and not on any lab test. Thus, the availability of an objective laboratory diagnostic will be of great value in the diagnosis and assessment of patients through treatment regimens.

Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying a biological activity, and (c) determining if a biological activity of the polypeptide has been altered.

Other Activities

A polypeptide, polynucleotide or gene of the present invention or a region thereof may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, a polypeptide, polynucleotide, or gene of the present invention or region thereof may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

NB1:580246.1 A polypeptide, polynucleotide or gene of the present invention or a region thereof may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, the response to opiates and opioids, tolerance to opiates and opioids, withdrawal from opiates and opioids, reproductive capabilities (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

A polypeptide, polynucleotide or gene of the present invention or a region thereof may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors, or other nutritional components.

The following examples are intended to illustrate the invention, and are not to be construed as limiting the scope of the invention.

NB1:580246.1 EXAMPLE 1

Identification and Characterization of Polynucleotides Regulated by Typical and Atypical Neuroleptic Drugs

Male C57B1/6J mice (20-28 g) were housed in groups of four on a standard 12/12 hour light-dark cycle with ad libitum access to standard laboratory chow and tap water. For the experimental paradigms, mice were divided into groups of 25 and subjected to the following treatments:

Control groups: Mice received a single injection of sterile saline (0.1 ml volume), or no injection, and were sacrificed after 45 minutes.

Acute neuroleptic treatment: Mice received a single mtraperitoneal injection of the atypical neuroleptic clozapine (7.5 mg/kg). Animals were sacrificed after 45 minutes.

Chronic neuroleptic treatment: Mice received daily subcutaneous injections of clozapine (7.5 mg/kg) or haloperidol (4 mg/kg) for time periods of 5 days to 2 weeks.

All animals were sacrificed in their cages with CO₂ at the indicated times. Brains were rapidly removed and placed on ice. The striatum, including the nucleus accumbens, were dissected out and placed in ice-cold phosphate-buffered saline. The cytoplasmic RNA was isolated by phenokchloroform extraction of the homogenized tissue according to the method described in Schibler et al, J. Mol. Bio., 142, 93-116 (1980). Poly A enriched mRNA was prepared from cytoplasmic RNA using well-known methods of oligo dT chromatography.

Isolated RNA was then analyzed using a method of simultaneous sequence-specific

® identification of mRNAs known as TOGA (TOtal Gene expression Analysis) described below.

The TOGA^® Process

The isolated RNA from the cells and tissue samples was analyzed using a method of simultaneous sequence-specific identification of mRNAs using TOGA described in Sutcliffe, et al. Proc. Natl. Acad. Sci. USA, 97(5):1976-1981 (2000); International published application WO

026406; U.S. application serial no. 09/775,217, PCT/US 02/02666, U.S. Patent No. 5,459,037;

U.S. Patent No. 5,807,680; U.S. Patent No. 6,030,784; U.S. Patent No. 6,096,503, U.S. Patent

6,110,680, and U.S. Patent 6,309,834 hereby incorporated herein by reference. A final PCR step that used 256 5' PCR primers in separate reactions with a universal 3' PCR primer (SEQ ID

NB1:580246.1 NO:82) produced PCR products that conesponded to the 3' ends of RNAs in the starting mRNA population. The produced PCR products were then identified by: a) the initial 5' sequence comprising the sequence remainder of the recognition site of the restriction endonuclease used to cut and define the 5' end plus the sequence of the four parsing bases immediately 3 ' to the remainder of the recognition site, preferably the sequence of the entire fragment, and b) the length of the fragment. These two parameters, sequence and fragment length, were used to compare the obtained PCR products to a database of known polynucleotide sequences. Since the length of the obtained PCR products includes sequences encoding primer binding sites at the 5' and 3' ends of the insert, the sequence of the insert provided in the sequence listing is shorter than the fragment length that forms part of the digital address.

The method yields Digital Sequence Tags (DSTs), that is, polynucleotides that are expressed sequence tags of the 3' end of mRNAs. DSTs that fit the above criteria for the different experiments were selected for further study.

In general, double-stranded cDNA is generated from poly(A)-enriched cytoplasmic RNA extracted from the tissue samples of interest using an equimolar mixture or set of all 48 5'- biotinylated anchor primers to initiate reverse transcription. One such suitable set is G-A-A-T-T- C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-A-A-G-A-G-C-T-C-C-A-C-C-G-C-G-G-T- A-G-T-A-C-T-C-A-C-T-G-C-A-G-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 79), where V is A, C or G and N is A, C, G or T. One member of this mixture of 48 anchor primers initiates synthesis at a fixed position at the 3' end of all copies of each mRNA species in the sample, thereby defining a 3' endpoint for each species, resulting in biotinylated double- stranded cDNA.

Each biotinylated double-stranded cDNA sample was cleaved with the restriction endonuclease Mspl, which recognizes the sequence CCGG. The resulting fragments of cDNA conesponding to the 3' region of the starting mRNA were then isolated by capture of the biotinylated cDNA fragments on a streptavidin-coated substrate. Suitable streptavidin-coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads, and paramagnetic porous glass particles. A prefened streptavidin-coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Great Neck, NY).

The pool or library of captured cDNA products was modified by ligation of double- stranded polynucleotides at the 5' ends to contain sequences encoding a T3 RNA polymerase

NB1:580246.1 promoter and PCR primer binding sites. Suitable polynucleotides are A-A-T-T-C-G-G-T-A-C- C-A-A-T-T-A-A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A-C-C-T-C-G-A-G-G-T-C-G-A-C-G-G-T- A-T and C-G-A-T-A-C-C-G-T-C-G-A-C-C-T-C-G-A-G-G-T-C-C-C-T-T-T-A-G-T-G-A-G-G- G-T-T-A-A-T-T-G-G-T-A-C-C-G-A-A-T-T (SEQ ID NOS: 80 and 81, respectively). The modified cDNA library was subsequently used as a template for synthesis of cRNA (copy RNA) by incubation with T3 RNA polymerase.

At this stage, each of the cRNA preparations was processed in a three-step fashion. In step one, an aliquot of cRNA was used for synthesis of first-strand cDNA using the 5' RT primer (G-A-G-C-T-C-C-A-C-C-G-C-G-G-T, (SEQ ID NO:82). In step two, the cDNA product was used as a DNA template in four separate PCR reactions with each of the four 5' PCR primers of the form C-C-T-C-G-A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO:83), each paired with a "universal" 3' PCR primer G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO:82) to yield four sets of PCR reaction products ("Nl reaction products").

In step three, the product of each subpool was further divided into 64 subsubpools (2ng in 20μl) for the second PCR reaction. This PCR reaction comprised adding 100 ng of the fluoresceinated "universal" 3' PCR primer (SEQ ID NO: 82) conjugated to 6-FAM and 100 ng of the appropriate 5' PCR primer of the form C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 84), and using a program that included an annealing step at a temperature X slightly above the Tm of each 5' PCR primer to minimize artifactual misprinting and promote high fidelity copying. Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).

The products ("N4 reaction products") from the final polymerase chain reaction step for each of the tissue samples were resolved on a series of denaturing DNA sequencing gels using the automated ABI Prizm 377 sequencer. Data were collected using the GeneScan software package (ABI) and normalized for amplitude and migration. Complete execution of this series of reactions generated 64 product subpools for each of the four pools established by the 5' PCR primers of the first PCR reaction, for a total of 256 product subpools for the entire 5' PCR primer set of the second PCR reaction.

The mRNA samples extracted from the striatum and cortex of mice treated with 7.5 mg/kg of clozapine for the following durations: control (no clozapine), saline (no clozapine), 45 minutes, 7 hours, 5 days, 12 days, and 14 days were analyzed. In addition, the mRNA samples

NB 1:580246.1 extracted from the striatum of mice treated with 4.0 mg/kg of haloperidol for the following durations: saline (no haloperidol), 7 hours, 10 days, and 2 weeks were analyzed.

Clozapine, an atypical neuroleptic drug, displays a higher clinical efficacy than typical neuroleptics, such as haloperidol, and also has proven to be effective in refractory patients (Jann, M.W., Pharmacotherapy 11:179-195, 1991). In contrast to typical neuroleptics, which are dopamine D2 receptor antagonists, clozapine exhibits an expanded spectrum of affinity for other neurotransmitter receptors, including serotonin, histamine, muscarinic, and adrenergic recptors (Jann, M.W., Pharmacotherapy 11:179-195, 1991). Hence, it has been suggested that the superior clinical efficacy of clozapine is associated with antagonism at these other receptors. In addition, clozapine pharmacotherapy has a much lower incidence of extrapyramidal side-effects that frequently accompany treatment with typical neuroleptics, also suggesting a separate mechanism of action. Thus, after analysis of mRNAs significantly responsive after treatment to either drug, the mRNAs were compared and categorized into 3 different classes that represent patterns of time-dependent changes in striatal gene expression induced by chronic treatment with neuroleptic drags. The conespondence of each sequence with these three different classes is shown in Table 1. Class I sequences demonstrated a common regulation pattern for both neuroleptics, and thus would be expected to account for some of the benefit of these two types of drugs. Class II sequences demonstrated a regulation pattern unique to clozapine treatments, and thus would account for the clozapine-specific benefits and side-effects. Finally, Class III sequences demonstrated a regulation pattern unique to haloperidol treatments, and thus would account for the haloperidol-specific side-effects. An example of each class, as well as tissue specific regulation patterns (striatum vs. cortex), are described below. Knowledge of these may explain the mechanisms through which the patients derive benefit, may explain the nature of the underlying pathology, and might provide new targets for designing therapeutics.

Table 2A is a summary of the expression levels of the class I mRNAs determined from cDNA (haloperidol). Table 2B is a summary of the expression levels of the class I mRNAs determined from cDNA (clozapine). Table 3 is a summary of the expression levels of the class II mRNAs determined from cDNA (clozapine). Table 4 is a summary of the expression levels of the class III mRNAs determined from cDNA (haloperidol). These cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule, as well as the relative amount of the molecule produced at different

NB1:580246.1 time intervals after treatment. The 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl (CCGG) and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step. The digital address length of the fragment was determined by interpolation on a standard curve and, as such, may vary ± 1-2 b.p. from the actual length as determined by sequencing.

For example, the entry in Table 3 that describes a DNA molecule identified by the digital address Mspl CTCG 141, is further characterized as having a 5' terminus partial nucleotide sequence of CGGCTGG and a digital address length of 141 b.p. The DNA molecule identified as Mspl CTGG 141 is further described as being expressed at increasing levels after 2 weeks of treatment with clozapine (see Figure 1). Additionally, the DNA molecule identified as Mspl CTGG 141 is described by its nucleotide sequence, which conesponds with SEQ ID NO: 34.

Similarly, the other DNA molecules identified in Table 2B and 3 by their Mspl digital addresses are further characterized by: 1) the level of gene expression in the striatum of mice without clozapine treatment (control), 2) the level of gene expression in the striatum of mice with saline treatment (additional control), 3) the level of gene expression in the striatum of mice treated with clozapine for 45 minutes, 4) the level of gene expression in the striatum of mice treated with clozapine for 12 days, 5) the level of gene expression in the striatum of mice treated with clozapine for 2 weeks, 6) the level of gene expression in the cortex of mice treated with saline (control), 7) the level of gene expression in the cortex of mice treated with clozapine for 5 days, and 8) the level of gene expression in the cortex of mice treated with clozapine for 2 weeks. For all of the samples, the data was generated in duplicate (e.g., TOGA 1 and TOGA 2). In addition, other DNA molecules identified in Table 2A and 4 by their Mspl digital addresses are further characterized by: 1) the level of gene expression in the striatum of mice without haloperidol treatment (control), 2) the level of gene expression in the striatum of mice with saline treatment (additional control), 3) the level of gene expression in the striatum of mice treated with haloperidol for for 7 hours, 4) the level of gene expression in the striatum of mice treated with haloperidol for 10 days, and 5) the level of gene expression in the striatum of mice treated with haloperidol for 2 weeks.

Additionally, several of the DSTs were further characterized as shown in the Tables and their nucleotide sequences are provided as SEQ ID NOs: 1-78.

NB1:580246.1 The ligation of the sequence into a vector does not regenerate the Mspl site; the experimentally determined sequence reported herein has C-G-G as the first bases of the 5' end.

The data shown in Figure 1 were generated with a 5' -PCR primer (C-G-A-C-G-G-T-A-T- C-G-G-C-T-C-G; SEQ ID NO: 85) paired with the "universal" 3' primer (SEQ ID NO:82) labeled with 6-carboxyfluorescein (6FAM, ABI) at the 5' terminus. PCR reaction products were resolved by gel electrophoresis on 4.5% acrylamide gels and fluorescence data acquired on

ABI377 automated sequencers. Data were analyzed using GeneScan software (Perkin-Elmer).

® Figure 1 is a graphical representation of the results of TOGA runs using a 5' PCR primer with parsing bases CTCG (SEQ ID NO:85) and the universal 3' PCR primer (SEQ ID

NO: 82) showing PCR products produced from mRNA extracted from the striatum of mice treated with 7.5 mg/kg of clozapine for the following durations: no treatment (no clozapine)

(Panel A), saline treatment (no clozapine) (Panel B), 45 minutes (Panel C), 12 days (Panel D), and 2 weeks (Panel E). Also shown is PCR products produced from mRNA extracted from the cortex of mice treated with clozapine for the following durations: saline (no clozapine) (Panel

F), 5 days (Panel G), and 2 weeks (Panel H). The vertical index line indicates a PCR product of about 141 b.p. that is expressed to a greater level in the 12 and 14 day clozapine-treated striatum samples than in the untreated or saline treated striatum samples. Although not differentially regulated in cortex, this sequence was also highly expressed in cortex tissue. The horizontal axis represents the number of base pairs of the molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA ® analysis (which conesponds to the relative expression of the molecule of that address). The results of the TOGA runs have been normalized using the methods described in pending U.S. Patent Application Serial No. 09/318,699/U.S., and PCT Application Serial No. PCT/USOO/14159, both entitled Methods and System for Amplitude Normalization and Selection of Data Peaks (Dennis Grace, Jayson Durham); and U.S. Patent 6,334,099, PCT Application Serial No. PCT/US00/14123, and pending U.S. Patent Application Serial Nos. 09/940,987/U.S., 09/940,581/U.S., 09/940,746/U.S., all of which are incorporated herein by reference. The vertical line drawn through the eight panels represents the DST molecule identified as NEU2_38 (SEQ ID NO:34).

Some products, which were differentially represented, appeared to migrate in positions that suggest that the products were novel based on comparison to data extracted from GenBank.

NB1:580246.1 The sequences of such products were determined by cloning or candidate matches with existing databases.

Cloning of TOGA^® Generated PCR Products

In suitable cases, the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced. The database matches for each cloned DST sequence are listed in Table 5.

NEU2_38 (SEQ ID NO:34), the DNA molecule identifed by Mspl CTCG 141, was one such cloned product. In order to verify that the cloned product conesponds to the TOGA peak of

® interest, the extended TOGA assay was performed for each DST.

Verification Using the Extended TOGA Method

® In order to verify that the TOGA peak of interest conesponds to the identified DST, an

® extended TOGA assay was performed for each DST as described below. PCR primers

® ("Extended TOGA primers") were designed from sequence determined using one of two methods: (1) in suitable cases, the PCR product was isolated, cloned into a TOPO vector

(Invitrogen) and sequenced on both strands; (2) in other cases, the sequences listed for the TOGA ® PCR products were derived from candidate matches to sequences present in available

GenBank, EST, or proprietary databases.

® PCR was performed using the Extended TOGA primers and the Nl PCR reaction products as a substrate. Oligonucleotides were synthesized with the sequence G-A-T-C-G-A-A-

T-C extended at the 3' end with a partial Mspl site (C-G-G), and an additional 18 adjacent nucleotides from the determined sequence of the DST. For example, for the PCR product with the TOGA^® address CTCG 141 (NEU2_38; SEQ ID NO:34), the 5' PCR primer was G-A-T-C-

G-A-A-T-C-C-G-G-C-T-C-G-C-C-G-C-A-T-G-A-C-A-T-C-C-A (SEQ ID NO:86). This 5' PCR primer was paired with the fluorescence labeled universal 3 ' PCR primer (SEQ ID NO:82) in a

PCR reaction using the PCR Nl reaction product as substrate.

The length of the PCR product generated with the Extended TOGA" primer was compared to the length of the original PCR product that was produced in the TOGA" reaction.

The results for SEQ ID NO:34, for example, are shown in Figure 2. The length of the PCR

NB1:580246.1 product conesponding to SEQ ID NO:34 (NEU2_38) was cloned and a 5' PCR primer was built from the cloned DST (SEQ ID NO:86). The product obtained from PCR with this primer (SEQ ID NO.-86) and the universal 3' PCR primer (SEQ ID NO:82) (as shown in the top panel, A) was compared to the length of the original PCR product that was produced in the TOGA reaction with mRNA extracted from the cortex of mice treated with 7.5 mg/kg of clozapine for 5 days using a 5' PCR primer with parsing bases CTCG (SEQ ID NO: 85) and the universal 3 ' PCR primer (SEQ ID NO: 82) (as shown in the middle panel, B). Again, for all panels, the number of base pairs is shown on the horizontal axis, and fluorescence intensity (which conesponds to relative expression) is found on the vertical axis. In the bottom panel (panel C), the traces from the top and middle panels are overlaid, demonstrating that the peak found using an extended primer from the cloned DST is the same number of base pairs as the original PCR product obtained through TOGA^® as NEU2_38 (SEQ ID NO:34). The bottom panel thus illustrates that NEU2_38 (SEQ ID NO:34) was the DST amplified in Extended TOGA^®.

In other cases, the sequences listed for the TOGA PCR products were derived from candidate matches to sequences present in available Genbank, EST, or proprietary databases. Table 6 lists the candidate matches for each by accession number of the Genbank entry or by the accession numbers of a set of computer-assembled ESTs used to create a consensus sequence.

Extended TOGA primers were designed based on these sequences (as mentioned previously), and Extended TOGA^* was run to detennine if the database sequences were the DSTs amplified in TOGA^®.

Assignment of Identities to DSTs

Digital Sequence Tags (DSTs) can be easily associated with the gene encoding the full- length mRNA transcript including both 5' and 3' untranslated regions by methods known to those skilled in the art. For example, searches of the public databases of expressed sequences (e.g., GenBank) can identify cDNA sequences that overlap with the DST. Statistically significant sequence matches with greater than 95% nucleotide sequence matches across the overlap region can be used to generate a contiguous sequence ("contig") and serial searches with the accumulated contig sequence can be used to assemble extended sequence associated with the DST. In cases where the assembled contig includes an open reading frame (a nucleotide sequence encoding a continuous sequence of amino acids), the polypeptide encoded by the expressed mRNA can be predicted.

NB1:580246.1 In other cases, extended sequence can also be generated by making a probe containing the DST sequence. The probe would then be used to select cDNA clones by hybridization methods known in the art. These cDNA clones may be selected from libraries of cDNA clones developed from the original RNA sample, from other RNA samples, from fractionated mRNA samples, or from other widely available cDNA libraries, including those available from commercial sources. Sequences from the selected cDNA clones can be assembled into contigs in the same manner described for database sequences. The cDNA molecules can also be isolated directly from the mRNA by the rapid analysis of cDNA ends (RACE) and long range PCR. This method can be used to isolate the entire full-length cDNA or the intact 5' and 3' ends of the cDNA.

Methods for alignment of biological sequences for pairwise comparison are well known in the art. Local alignments between a query sequence and a subject sequence can be derived by using the algorithm of Smith (JMol Biol, 1981), by the homology alignment algorithm of Needleman (JMol Biol, 1970), or by the similarity search algorithm of Pearson (Proc Natl Acad Sci, 1988). A prefened method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also refened to as a sequence database, can be determined using the BLAST computer program based on the algorithm of Altschul and colleagues (Altschul, JMol Biol; 1990; Altschul, Nucleic Acids Res, 1997). The term "sequence" includes nucleotide and amino acid sequences. In a sequence alignment, the query sequence can be either protein or nucleic acid or any combination thereof. BLAST is a statistically driven search method that finds regions of similarity between a query and database sequences. These are called segment pairs, and consist of gapless alignments of any part of two sequences. Within these aligned regions, the sum of the scoring matrix values of their constituent symbol pairs is higher than a level expected to occur by chance alone. The scores obtained in a BLAST search can be interpreted by the experienced investigator to determine real relationships versus random similarities. The BLAST program supports four different search mechanisms:

• Nucleotide Query Searching a Nucleotide Database- Each database sequence is compared to the query in a separate nucleotide-nucleotide pairwise comparison.

• Protein Query Searching a Protein Database- Each database sequence is compared to the query in a separate protein-protein pairwise comparison.

NB 1:580246.1 • Nucleotide Query Searching a Protein Database- The query is translated, and each of the six products is compared to each database sequence in a separate protein- protein pairwise comparison.

• Protein Query Searching a Nucleotide Database- Each nucleotide database sequence is translated, and each of the six products is compared to the query in a separate protein-protein pairwise comparison.

By using the BLAST program to search for matches between a sequence of the present invention and sequences in GenBank and EST databases, identities were assigned whenever possible. A portion of these results is listed in Table 5.

DST Validation Using Real-Time Quantitative PCR

Validation of DSTs isolated by TOGA®⁾ was performed by using Real-Time Quantitative PCR using the ABI PRISM 7700 Sequence Detection System (PE Biosystems) that combines PCR, cycle-by-cycle fluorescence detection and analysis software for high-throughput quantitation of nucleic acid sequences. Reactions are characterized by the point in time when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. Relative quantitation of the amount of target in the sample is accomplished by measuring the cycle number at which a significant amount of product is produced. The entire process is performed by the integrated software of the 7700 system. Primers for Real-Time Quantitative PCR validation are selected by the integrated software package accompanying the ABI PRISM 7700. Standards for normalizing the quantitation of gene levels were chosen from a panel of 7 mouse and 6 human "housekeeping" genes. The normalization standard chosen for the striatum samples was HPRT (hypoxanthine phosphoribosyl transferase) and was based on the similarity of expression across all striatum sample templates. The results of this DST validation were compared to duplicate TOGA® runs, and the relative abundance of each DST validated in this manner is compared i Table 8. The relative abundance level of untreated striatum has a value of 1.00. The primers used in these studies are listed in Table 7.

EXAMPLE 2

NB1:580246.1 Characterization of Polynucleotides with Common and Unique Patterns of Regulation in Response to Atypical Compared to Typical Neuroleptics

Male C57B1/6J mice (20-28 g) were housed as previously described in Example 1. The same experimental paradigm used in Example 1 for clozapine and haloperidol treatment was used for the various analyses described below. Briefly, in the clozapine studies, the control group mice received a single injection of sterile saline (0.1 ml volume), or no injection, and were sacrificed after 45 minutes. The mice subjected to acute clozapine treatment were given a single mtraperitoneal injection of clozapine (7.5 mg/kg) and sacrificed after 45 minutes as described in Example 1. The mice subjected to chronic clozapine freatment received daily subcutaneous injections of clozapine (7.5 mg/kg) for 12 days or 2 weeks. The mice subjected to chronic haloperidol treatment received daily subcutaneous injection of haloperidol (4.0 mg/kg) for 7 hrs, 10 days, or 2 weeks. All animals were sacrificed in their cages with CO₂ at the indicated times. Brains were rapidly removed and placed on ice. For both the clozapine and haloperidol treated mice, the striatum, including the nucleus accumbens, were dissected out and placed in ice-cold phosphate-buffered saline. For clozapine treated mice, the cortex was also dissected out and placed in ice-cold phosphate-buffered saline. The mRNA was prepared according to the method described in the Example 1. After TOGA ® analysis, about 78 total neuroleptic responsive DSTs were found and their pattern of expression was compared between the two drugs. It was found that 25 genes demonstrated a common regulation pattern for both neuroleptics, of these 11 were downregulated and 14 were up-regulated. About 26 DSTs were found to be uniquely regulated in response to clozapine, and of these 17 were down-regulated and 9 were up-regulated. Finally, approximately 27 haloperidol specific DSTs were found, and interestingly, only 2 of these were down-regulated while 25 were up-regulated. It is interesting that haloperidol, the compound with the highest propensity for extrapyramidal side effects, exhibited such striking imbalance between the up and down-regulated responses. These overall trends may be suggestive of biological responses unique to haloperidol.

An example of a DST that was unique to clozapine treatment and not haloperidol is shown m Figure 1. The TOGA data shown in Figure 1 was generated with a 5 '-PCR primer (C- G-A-C-G-G-T-A-T-C-G-G-C-T-C-G; SEQ ID NO:85) paired with the "universal" 3' primer

NB1:580246.1 (SEQ ID NO:82) labeled with 6-carboxyfluorescein (6FAM, ABI) at the 5' terminus. PCR reaction products were resolved by gel electrophoresis, fluorescence data acquired on ABI377 automated sequencers, and data were analyzed using GeneScan software (Perkin-Elmer) as described in Example 1.

As shown in Table 1, the results of TOGA^® analysis indicate that NEU2_38 (SEQ ID NO:34) is expressed to a greater level in the 12 and 14 day clozapine-treated striatum samples than in the untreated or saline treated striatum samples. Although not differentially regulated in cortex, this sequence was also highly expressed in cortex tissue and up-regulated by 12 days of clozapine treatment. Thus, this DST demonstrated drag-specific regulation only in clozapine treated mice, as well as tissue-specific regulation only in striatum.

As shown in Table 5, the NEU2_38 clone (SEQ ID NO: 18) conesponds with an EST (similar to DJ-1 protein, clone MGC:7381 IMAGE:3487763, mRNA, complete eds; Accession number BC002187).

An additional example of a DST that was unique to clozapine treatment and not

® haloperidol is shown in Figure 3. Figure 3 is a graphical representation of the results of TOGA analysis, similar to Figure 1, using a 5' PCR primer with parsing bases AGC A (SEQ ID NO: 87) and the universal 3' primer (SEQ ID NO: 82), showing PCR products produced from mRNA extracted from the striatum of mice treated with 7.5 mg/kg of clozapine for the following durations: untreated (no clozapine) (Panel A), 45 minutes (Panel B), 12 days (Panel C), and 2 weeks (Panel D). The vertical index line indicates a PCR product of about 274 b.p. that is present in the untreated sample and is down-regulated within 45 minutes in the clozapine-treated sample, and remains down-regulated for 14 days in the presence of clozapine. The vertical line drawn tlirough the five panels represents the DST molecule identified as NEU2_29 (SEQ ID

NO:27).

As shown in Table 6, the NEU2_29 clone (SEQ ID NO:27) conesponds with GenBank sequence M64248.1, which was identified as mouse apolipoprotein A-JN (apoa-A). This gene belongs to a family of apolipoproteins, one of which has been previously associated with the mechanism of action of neuroleptic drags. Specifically, apolipoprotein D (apoD) has been associated with chronic administration of clozapine in mouse striatum/nucleus accumbens

(Thomas, E.A., et al., Clozapine increases apolipoprotein D expression in rodent brain: towards a mechanism for neuroleptic pharmacotherapy. J Νeurochem. 76:789-96, 2001) (described in

ΝB1:580246.1 pending PCT Application Serial No. PCT/US01/30695, entitled Gene Expression in the Central Nervous System Regulated by Neuroleptic Agents (Elizabeth Thomas, J. Gregor Sutcliffe, Thomas Pribyl, Brian Hilbush, Karl Hasel), incorporated by reference herein).

ApoD was initially identified as a constituent of plasma high-density lipoproteins (HDLs), which also contain phospholipids, cholesterol and fatty acids (McConathy et al., Fed. Eur. Biochem. Soc. Lett, 37: 178 (1973)). In the CNS of humans, as in other species (Provost et al., J. Lipid Res., 32: (1991); Seguin et al., Mol. Brain Res., 30: 242 (1995); Smith et al, J. Lipid Res., 31: 995 (1990)), apoD is expressed primarily in glial cells, pial cells, perivascular cells, and some neuronal populations (Navarro et al., Neurosci. Lett., 254: 17 (1995); Kahnan et al., enrol Res., 22: 330 (2000)). The physiological role for apoD within the CNS is not known, however, it has been shown to bind several hydrophobic ligands, including sterols and steroid hormones (Dilley et al., Breast Cane. Res. Treat, 16: 253 (1990); Lea, O. A., Steroids, 52: 337 (1988)) suggesting a role in extracellular lipid transport in the brain.

In addition to demonstrating increased apoD expression after chronic administration of clozapine in mouse striatum/nucleus accumbens (Thomas, E.A., et al., Clozapine increases apolipoprotein D expression in rodent brain: towards a mechanism for neuroleptic pharmacotherapy. J Neurochem. 76:789-96, 2001; PCT Application Serial No. PCT/US01/30695) an increase in apoD expression in the prefrontal cortex of schizophrenic and bipolar human subjects has been observed (Thomas, E.A., Dean, B., Pavey, G., Sutcliffe, J.G. Increased CNS levels of apolipoprotein D in schizophrenic and bipolar subjects: implications for the pathophysiology of psychiatric disorders. Proc Natl Acad Sci U S A. 98:4066-71, 2000). Further, increases in apoD mRNA expression were observed in brains of a mouse model for Alzheimer's disease (PDAPP transgenic mice) have been observed in the aged mice (Thomas, et al., manuscript in press; PCT Application Serial No. PCT/USOl/30695). These results implicate lipid homeostatic mechanisms in the pathological processes associated with Alzheimer's disease. Thus, apoD may be a region-specific marker for a neuropathological process that is initiated because of systemic lipid metabolism insufficiencies. The combined results suggest that apoD is a marker for neuropathology associated with psychiatric disorders and therefore can be used to target abnormalities in specific anatomical brain regions.

The finding that apoA, like apoD, is differentially expressed after clozapine treatment links apolipoproteins and the mechanism of action of neuroleptic drugs.

NB1:580246.1 An example of a DST that was unique to clozapine and not haloperidol treatment, and regulated specifically in cortex but not striatum is shown in Figure 4. Figure 4 is a graphical representation of the results of TOGA ^R analysis using a 5' PCR primer with parsing bases AGTT (SEQ ID NO: 88) and universal 3' primer (SEQ ID NO: 82), showing PCR products produced from mRNA extracted from the cortex of mice treated with 7.5 mg/kg of clozapine for the following durations: saline (no clozapine) (Panel A), 5 days (Panel B), and 2 weeks (Panel C). The vertical index line indicates a PCR product of about 328 b.p. that is present in the saline treated cortex sample and decreases in expression over time in the clozapine-treated cortex samples. The vertical line drawn through the five panels represents the DST molecule identified as NEU2_6 (SEQ ID NO:6). Thus, this DST demonstrated drug-specific regulation only in clozapine treated mice, as well as tissue-specific regulation only in cortex.

As shown in Table 5, the NEU2_6 clone (SEQ ID NO:6) conesponds with GenBank sequence D10011, which was identified as mouse glutamate receptor channel subunit gamma 2.

An example of a DST that was unique to haloperidol treatment is shown in Figure 5. Figure 5 is a graphical representation of the results of TOGA ® runs using a 5' PCR primer with parsing bases TGTC (SEQ ID NO:89) and the universal 3' PCR primer (SEQ ID NO:82) showing PCR products produced from mRNA extracted from the striatum of mice treated with

4.0 mg/kg of haloperidol for the following durations: no treatment (no haloperidol) (Panel A), saline treatment (no haloperidol) (Panel B), 7 hour (Panel C), 10 days (Panel D), and 2 weeks

(Panel E). The vertical index line indicates a PCR product of about 318 b.p. that is expressed to a greater level in the 10 and 14 day clozapine-treated striatum samples than in the untreated or saline treated striatum samples. The vertical line drawn tlirough the five panels represents the

DST molecule identified as NEU2_125 (SEQ ID NO:64). Thus, this DST was regulated only after response to haloperidol treatment, and thus may be associated with extrapyramidal side effects commonly observed in patients treated with this typical neuroleptic.

As shown in Table 5, the NEU2_125 clone (SEQ ID NO:64) conesponds with GenBank sequence AF108133, which was identified as mouse neuro-d4 gene.

An example of a DST showing the same regulation pattern after chronic treatment with both neuroleptics is shown in Figure 6. Figure 6 is a graphical representation of the results of TOGA^® runs using a 5' PCR primer with parsing bases AGCA (SEQ ID NO: 90) and the universal 3' PCR primer (SEQ RO NO: 82) showing PCR products produced from mRNA

NB 1:580246.1 extracted from the striatum of mice treated with 7.5 mg/kg of clozapine for the following durations: no treatment (no clozapine) (Panel A), saline treatment (no clozapine) (Panel B), 45 minutes (Panel C), 12 days (Panel D), and 2 weeks (Panel E). The vertical index line indicates a PCR product of about 312 b.p. that is expressed to an increasingly greater level as increased duration of treatment with clozapine. This trend is summarized graphically in Panel F. Also shown is PCR products produced from 4.0 mg/kg of haloperidol for the following durations: no treatment (no haloperidol) (Panel G), saline treatment (no haloperidol) (Panel H), 7 hour (Panel I), 10 days (Panel J), and 2 weeks (Panel K). The vertical index line indicates this PCR product of about 312 b.p. is also expressed to an increasingly greater level with increased duration of treatment with haloperidol. This trend is summarized graphically in Panel L. The vertical line drawn through the all these panels represents the DST molecule identified as NEU2_5 (SEQ ID NO:5). Thus, this DST represents an important class of neuroleptic-responsive genes that demonstrated a common regulation pattern for both an atypical (clozapine) and typical (haloperidol) neuroleptics. This common expression pattern suggests this DST is a strong candidate for an RNA encoding protein whose activity is involved in the benefit derived by patients from these two classes of neuroleptic drugs.

As shown in Table 5, the NEU2_5 clone (SEQ ID NO:5) conesponds with an EST sequence AK014368.

In summary, mRNAs significantly responsive after treatment to either neuroleptic fit into three different classes that represent patterns of time-dependent changes in striatal gene expression induced by chronic treatment with neuroleptic drugs. One class of sequences was identified that demonstrated a common regulation pattern for both neuroleptics, and thus would be expected to account for some of the benefit of these two types of drags. A second class of sequences demonstrated a regulation pattern unique to clozapine treatments, and thus would account for the clozapine-specific benefits and side-effects. Finally, a third class of sequences demonstrated a regulation pattern unique to haloperidol treatments, and thus would account for the haloperidol-specific side-effects. In addition, tissue specific patterns of response to clozapine were observed (i.e., striatum compared to cortex). Knowledge of these may explain the mechanisms through which the patients derive benefit, may explain the nature of the underlying pathology, and might provide new targets for designing therapeutics.

NB1:580246.1 The polynucleotides, polypeptides, kits and methods of the present invention may be embodied in other specific forms without department from the teachings or essential characteristics of the invention. The described embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are, therefore, to be embraced within.

NB1:580246.1

NB1:580246.1

NB1:580246.1

*Class I, common regulation pattern for both neuroleptics; Class II, regulation pattern unique to clozapine treatments; Class III, regulation pattern unique to haloperidol treatment

NB1:580246.1

NB 1:580246.1

NB1:580246.1

NB1:580246.1

90 Ul

NB1:580246.1

NB1:58026.1

NB1:580246.1

NB1:580₂6.1

NB1-.580246.1

NB1:580246.1

NB1:₅80246.1

NB1:S80₂6.1

NB1:580₂46.1

NB1:580246.1

NB1:58026.1

NB1:580246.1

NB1:580₂₄6.1

Claims

We claim:

1. An isolated nucleic acid molecule comprising a polynucleotide listed in SEQ ID NO:4, or a second isolated nucleic acid molecule at least 95% identical to said first isolated nucleic acid, wherem expression of the polynucleotide is modulated, relative to a control sample, in a patient afflicted with psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, or other neuropsychiatric disorders.

2. An isolated polypeptide encoded by:

(a) a first polynucleotide listed in SEQ ID NO:4; or

(b) a second polynucleotide at least 95% identical to said first polynucleotide; or

(c) a gene or region thereof corresponding to any of the foregoing polynucleotides or another gene at least 95% identical to said gene; or

(d) the complements and degenerate variants of any of the foregoing polynucleotides or genes, wherein expression of the polypeptide is modulated, relative to a control sample, in a patient afflicted with psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, or other neuropsychiatric disorders.

3. An isolated nucleic acid molecule at least ten bases in length that is hybridizable to the isolated nucleic acid molecule of claim 1 under stringent conditions.

4. An isolated nucleic acid molecule encoding a fragment of the polypeptide of claim 2.

5. The isolated nucleic acid of claim 4, wherein the nucleic acid encodes an epitope.

6. The polypeptide of claim 2, wherein the polypeptide has biological activity.

7. An isolated nucleic acid encoding a species homolog of the polypeptide of claim 2.

8. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the 5' end or the 3 'end.

9. An isolated fragment of any polypeptide of claim 2.

10. An isolated polypeptide that is a homolog, paralog or ortholog of any polypeptide of claim 2.

11. A recombinant vector comprising:

(a) the isolated nucleic acid molecule of claim 1 operably linked to a promoter;

(b) an origin of replication; and

(c) a selectable marker.

12. A recombinant host cell comprising the recombinant vector of claim 11.

13. A method for making a recombinant host cell comprising:

(a) introducing the recombinant vector of claim 11 into a host cell.

14. The isolated polypeptide of claim 2, wherein the isolated polypeptide comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

15. An isolated antibody that binds specifically to the isolated polypeptide of claim 2, wherein said isolated antibody is a polyclonal or monoclonal antibody.

16. A recombinant host cell that expresses the isolated polypeptide of claim 2.

17. An isolated polypeptide produced by the steps of:

(a) culturing the recombinant host cell of claim 12 under conditions such that said polypeptide is expressed; and

(b) isolating the polypeptide.

18. A method for preventing or treating psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, or other neuropsychiatric disorder, the method comprising administering to a mammalian subject a therapeutically effective amount of:

(a) a first polynucleotide selected from the group consisting of SEQ ID NOs: 1 -78;

(b) a second polynucleotide at least 95% identical to the first polynucleotide;

(c) a third polynucleotide at least ten bases in length that is hybridizable to the first polynucleotide under stringent conditions;

(d) a gene corresponding to any of the foregoing polynucleotides, another gene at least 95% identical to the gene, or a region of any of the foregoing genes;

(e) a first polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78;

(f) a second polypeptide encoded by a gene corresponding to any of the foregoing polynucleotides or another gene at least 95% identical to said gene;

(g) a third polypeptide at least 90% identical to one of the foregoing polypeptides; or

(h) a fragment of one of the foregoing polypeptides.

19. A method for preventing or treating psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, or other neuropsychiatric disorder, the method comprising administering to a mammalian subject a therapeutically effective amount of an antibody capable of specifically binding to:

(a) a first polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78;

(b) a second polypeptide encoded by a gene corresponding to any of the foregoing polynucleotides or another gene at least 95% identical to said gene;

(c) a third polypeptide at least 90% identical to one of the foregoing polypeptides; or

(d) a fragment of one of the foregoing polypeptides.

20. A method for diagnosing a neuropsychiatric disorder or the suscepuDii y ιυ the neuropsychiatric disorder in a subject, wherein said neuropsychiatric disorder is selected from the group consisting of psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, and other neuropsychiatric disorders, the method comprising:

(a) obtaining a biological sample from a subject suspected of having the neuropsychiatric disorder;

(b) determining in the biological sample, a presence of a mutation in a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78; and

(c) diagnosing the neuropsychiatric disorder or the susceptibility to the neuropsychiatric disorder based on the presence of said mutation.

21. A method for diagnosing a neuropsychiatric disorder or a susceptibility to the neuropsychiatric disorder, wherem the neuropsychiatric disorder is selected from the group consisting of psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, and other neuropsychiatric disorders, the method comprising the steps of:

(a) obtaining a first biological sample from a patient suspected of having the neuropsychiatric disorder;

(b) obtaining a second sample from a suitable comparable control source;

(c) determining in the first and second samples a level of expression of at least one of:

(i) a first polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78;

(ii) a second polypeptide encoded by a gene corresponding to any of the foregoing polynucleotides or another gene at least 95 % identical to said gene;

(iii) a third polypeptide at least 90% identical to one of the foregoing polypeptides;

(iv) a fragment of one of the foregoing polypeptides; or

(v) a polynucleotide selected from the group consisting of SEQ ID

NOs: 1-78; and

(d) comparing the level of expression of the at least one polypeptide or the polynucleotide in the first and second samples, wherein a patient is diagnosed as having the neuropsychiatric disorder or is susceptible to having the neuropsychiatric disorder if the level of expression of the at least one polypeptide or the polynucleotide in the first sample is substantially modulated relative to the level of expression of the at least one polypeptide or the polynucleotide in the second sample.

22. A method for identifying a binding partner to a polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78 comprising:

(a) contacting the polypeptide with a binding partner; and

(b) determining whether the binding partner effects an activity of the polypeptide.

23. A method for identifying an activity of an expressed polypeptide in a biological assay, the method comprising:

(a) expressing a polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78 in a cell;

(b) isolating the expressed polypeptide;

(c) testing the expressed polypeptide for an activity in a biological assay; and

(d) identifying the activity of the expressed polypeptide based on the test results.

24. A substantially pure isolated DNA molecule suitable for use as a probe for genes regulated by neuroleptics, chosen from the group consisting of the DNA molecules identified in SEQ ID NOs: 1-78, having a 5' partial nucleotide sequence and length as described by their digital address, and having a characteristic regulation pattern by neuroleptics.

25. A kit for detecting the presence of a polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78 in a mammalian tissue sample comprising a first antibody, wherein said first antibody immunoreacts with a mammalian protein encoded by a gene corresponding to a polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78 in an amount sufficient for at least one assay and suitable packaging material.

26. The kit of claim 25 further comprising a second antibody that binds to the first antibody.

27. The kit of claim 26, wherem the second antibody is labeled, wherein the label is selected from the group consisting of enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds.

28. A kit for detecting the presence of a gene encoding a protein compπsmg a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay, and suitable packaging material.

29. A method for detecting the presence of a nucleic acid encoding a protein in a mammalian tissue sample, comprising the steps of:

(a) hybridizing a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78 or fragment thereof having at least 10 contiguous bases, with the nucleic acid of the sample; and

(b) detecting the presence of the hybridization product.

30. A method of assessing the efficacy of a treatment for treating a neuropsychiatric disorder in a subject, wherein the neuropsychiatric disorder is selected form the group consisting of psychoses, Alzheimer's disease, bipolar affective disorder and schizophrenia, the method comprising the steps of comparing:

(a) a level of expression of a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78, or a polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78, in a first sample obtained from the subject prior to providing at least a portion of the treatment to the subject; and

(b) a level of expression of the same polynucleotide or polypeptide in a second sample following provision of the portion of the treatment, wherein a modulated level of expression of the polynucleotide or polypeptide in the second sample relative to the first sample, is an indication that the treatment is efficacious for treating the neuropsychiatric disorder.

31. The method of claim 30, wherem the treatment for treating a neuropsychiatric disorder in a subject comprises administering clozapine to the subject.

32. The method of claim 30, wherein the treatment for treating a neuropsychiatric disorder in a subject comprises administering haloperidol to the subject.

33. A method for optimizing neuroleptic drug dosage in a patient aimcteα wιτn a neuropsychiatric disorder, wherein the neuropsychiatric disorder is selected form the group consisting of psychoses, Alzheimer's disease, bipolar affective disorder and schizophrenia, the method comprising the steps of:

(a) comparing:

(i) a level of expression of a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78 or a polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78, in a first sample obtained from the patient prior to providing a dosage of neuroleptic drug; and

(ii) a level of expression of the same polynucleotide or polypeptide in a second sample obtained from the patient following provision of the dosage of neuroleptic drug, and

(b) comparing:

(i) a symptom of the neuropsychiatric disorder in the subject prior to providing the dosage of the neuroleptic drug; and

(ii) the symptom of the neuropsychiatric disorder in the subject following provision of the dosage of neuroleptic drug; and

(c) correlating the level of expression of the marker with the dosage of the neuroleptic drug and the symptom of the neuropsychiatric disorder, wherein the lowest dosage of neuroleptic drug that modulates the level of expression of the polynucleotide or polypeptide as demonstrated by amelioration of the symptom of the neuropsychiatric disorder is an indication that the dosage of neuroleptic drug is optimized for the patient.

34. A method for assessing the likelihood that administration of a neuroleptic drug to a patient in need thereof will result in extrapyridmal side effects, the method comprising the steps of;

(a) obtaining a sample from the patient; and

(b) comparing:

(i) a level of expression of a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78 or a polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1-78; and

(ii) a control level of expression of the same polynucleotide or polypeptide, wherem a substantially increased level of expression of the polynucleotide or polypeptide in the sample obtained from the patient, relative to the control level of expression of the polynucleotide or polypeptide, is an indication that administration of the neuroleptic drug to the patient will result in extrapyridmal side effects.

35. A method for manufacturing a medicament for the treatment of psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, or other neuropsychiatric disorder using at least one of:

(a) a first polynucleotide selected from the group consisting of SEQ ID NOs: 1-78;

(b) a second polynucleotide at least 95% identical to the first polynucleotide;

(d) a gene corresponding to any of the foregoing polynucleotides, another gene at least 95%) identical to the gene, or a region of any of the foregoing genes;

(h) a fragment of one of the foregoing polypeptides.

36. A method for manufacturing a medicament for the treatment ot psycnoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, or other neuropsychiatric disorder using an antibody that binds specifically to at least one of:

(d) a fragment of one of the foregoing polypeptides.

37. A method for assessing a stage of psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, or other neuropsychiatric disorder by testing for regulation of at least one of:

(b) a second polypeptide encoded by a gene corresponding to any of the foregoing polynucleotides or another gene at least 95%> identical to said gene;

(d) a fragment of one of the foregoing polypeptides.

38. A method for assessing the efficacy of a test compound for treating psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, or other neuropsychiatric disorder in a mammalian subject, the method comprising the step of comparing:

(a) a level of expression of a marker in a first sample obtained from the subject, wherein the first sample is exposed to the test compound and wherein the marker is selected from the group consisting of polynucleotides listed in SEQ ID NO: 1-78; polypeptides encoded by the polynucleotides listed in SEQ ID NO: 1-78; and fragments thereof; and

(b) a level of expression of the same marker in a second sample obtained from the subject, wherein the second sample is not exposed to the test compound, and wherein a substantially increased or decreased level of expression of the marker in the first sample, relative to the second sample, is an indication that the test compound is efficacious in treating psychoses, Alzheimer's disease, bipolar affective disorder, schizophrenia, or other neuropsychiatric disorder.