AU3469699A - Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders - Google Patents

Description

WO 99/51762 PCT/US99/07401 METHODS AND COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF NEUROPSYCHIATRIC DISORDERS 1. INTRODUCTION 5 The present invention relates to drug screening assays, and diagnostic and therapeutic methods for the treatment of neuropsychiatric disorders mediated by the expression of a mutant form of the pituitary adenylate cyclase activating polypeptide (PACAP) gene or by aberrant levels of PACAP 10 expression. The invention is based on Applicant's discovery that the PACAP gene is linked to the short arm of chromosome 18 in a region of the chromosome involved in mediating neuropsychiatric disorders such as bipolar-affective disorder (BAD). Thus, the invention relates to use of the PACAP gene 15 and/or gene products as markers for fine structure mapping of a region of human chromosome 18, including a region of the chromosome involved in mediating neuropsychiatric disorders. The invention also relates to methods for the identification of compounds that modulate the expression, synthesis and 20 activity of the PACAP protein/gene and to using compounds such as those identified as therapeutic agents in the treatment of a PACAP mediated disorder and/or a neuropsychiatric disorder, including, by way of example and not of limitation, bipolar affective disorder. The invention 25 also relates to methods for the diagnostic evaluation, genetic testing and prognosis of PACAP mediated disorders. 2. BACKGROUND OF THE INVENTION 2.1. NEUROPSYCHIATRIC DISORDERS 30 There are only a few psychiatric disorders in which clinical manifestations of the disorder can be correlated with demonstrable defects in the structure and/or function of the nervous system. Well-known examples of such disorders include Huntington's disease, which can be traced to a 35 mutation in a single gene and in which neurons in the striatum degenerate, and Parkinson's disease, in which dopaminergic neurons in the nigro-striatal pathway - 1- WO 99/51762 PCT/US99/07401 degenerate. The vast majority of psychiatric disorders, however, presumably involve subtle and/or undetectable changes, at the cellular and/or molecular levels, in nervous system structure and function. This lack of detectable 5 neurological defects distinguishes "neuropsychiatric" disorders, such as schizophrenia, attention deficit disorders, schizoaffective disorder, bipolar affective disorders, or unipolar affective disorder, from neurological disorders, in which anatomical or biochemical pathologies are 10 manifest. Hence, identification of the causative defects and the neuropathologies of neuropsychiatric disorders are needed in order to enable clinicians to evaluate and prescribe appropriate courses of treatment to cure or ameliorate the symptoms of these disorders. 15 One of the most prevalent and potentially devastating of neuropsychiatric disorders is bipolar affective disorder (BAD), also known as bipolar mood disorder (BP) or manic depressive illness, which ischaracterized by episodes of elevated mood (mania) and depression (Goodwin, et al., 1990, 20 Manic Depressive Illness, Oxford University Press, New York). The most severe and clinically distinctive forms of BAD are BP-I (severe bipolar affective (mood) disorder), which affects 2-3 million people in the United States, and SAD-M (schizoaffective disorder manic type). They are 25 characterized by at least one full episode of mania, with or without episodes of major depression (defined by lowered mood, or depression, with associated disturbances in rhythmic behaviors such as sleeping, eating, and sexual activity). BP-I often co-segregates in families with more etiologically 30 heterogeneous syndromes, such as with a unipolar affective disorder such as unipolar major depressive disorder (MDD), which is a more broadly defined phenotype (Freimer and Reus, 1992, in The Molecular and Genetic Basis of Neurological Disease, Rosenberg, et al., eds., Butterworths, New York, pp. 35 951-965; McInnes and Freimer, 1995, Curr. Opin. Genet. Develop. 5, 376-381). BP-I and SAD-M are severe mood disorders that are frequently difficult to distinguish from -2- WO 99/51762 PCT/US99/07401 one another on a cross-sectional basis, follow similar clinical courses, and segregate together in family studies (Rosenthal, et al., 1980, Arch. General Psychiat. 37, 804 810; Levinson and Levitt, 1987, Am. J. Psychiat. 144, 415 5 426; Goodwin, et al., 1990, Manic Depressive Illness, Oxford University Press, New York). Hence, methods for distinguishing neuropsychiatric disorders such as these are needed in order to effectively diagnose and treat afflicted individuals. 10 Currently, individuals are typically evaluated for BAD using the criteria set forth in the most current version of the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM). While many drugs have been used to treat individuals diagnosed with BAD, 15 including lithium salts, carbamazepine and valproic acid, none of the currently available drugs are adequate. For example, drug treatments are effective in only approximately 60-70% of individuals diagnosed with BP-I. Moreover, it is currently impossible to predict which drug treatments will be 20 effective in, for example, particular BP-I affected individuals. Commonly, upon diagnosis, affected individuals are prescribed one drug after another until one is found to be effective. Early prescription of an effective drug treatment, therefore, is critical for several reasons, 25 including the avoidance of extremely dangerous manic episodes and the risk of progressive deterioration if effective treatments are not found. The existence of a genetic component for BAD is strongly supported by segregation analyses and twin studies 30 (Bertelson, et al., 1977, Br. J. Psychiat. 130, 330-351; Freimer and Reus, 1992, in The.Molecular and Genetic Basis of Neurological Disease, Rosenberg, et al., eds., Butterworths, New York, pp. 951-965; Pauls, et al., 1992, Arch. Gen. Psychiat. 49, 703-708). Efforts to identify the chromosomal 35 location of genes that might be involved in BP-I, however, have yielded disappointing results in that reports of linkage between BP-I and markers on chromosomes X and 11 could not be - 3 - WO 99/51762 PCT/US99/07401 independently replicated nor confirmed in the re-analyses of the original pedigrees, indicating that with BAD linkage studies, even extremely high lod scores at a single locus, can be false positives (Baron, et al., 1987, Nature 326, 289 5 292; Egeland, et al., 1987, Nature 325, 783-787; Kelsoe, et al., 1989, Nature 342, 238-243; Baron, et al., 1993, Nature Genet. 3, 49-55). Recent investigations have suggested possible localization of BAD genes on chromosomes 18p and 21q, but in 10 both cases the proposed candidate region is not well defined and no unequivocal support exists for either location (Berrettini, et al., 1994, Proc. Natl. Acad. Sci. USA 91, 5918-5921; Murray, et al., 1994, Science 265, 2049-2054; Pauls, et al., 1995, Am. J. Hum. Genet. 57, 636-643; Maier, 15 et al., 1995, Psych. Res. 59, 7-15; Straub, et al., 1994, Nature Genet. 8, 291-296). Mapping genes for common diseases believed to be caused, by multiple genes, such as BAD, may be complicated by the typically imprecise definition of phenotypes, by etiologic 20 heterogeneity, and by uncertainty about the mode of genetic transmission of the disease trait. With neuropsychiatric disorders there is even greater ambiguity in distinguishing individuals who likely carry an affected genotype from those who are genetically unaffected. For example, one can define 25 an affected phenotype for BAD by including one or more of the broad grouping of diagnostic classifications that constitute the mood disorders: BP-I, SAD-M, MDD, and bipolar affective (mood) disorder with hypomania and major depression (BP-II). Thus, one of the greatest difficulties facing 30 psychiatric geneticists is uncertainty regarding the validity of phenotype designations, since clinical diagnoses are based solely on clinical observation and subjective reports. Also, with complex traits such as neuropsychiatric disorders, it is difficult to genetically map the trait-causing genes because: 35 (1) neuropsychiatric disorder phenotypes do not exhibit classic Mendelian recessive or dominant inheritance patterns attributable to a single genetic locus; (2) there may be - 4 - WO 99/51762 PCT/US99/07401 incomplete penetrance, i.e., individuals who inherit a predisposing allele may not manifest disease; (3) a phenocopy phenomenon may occur, i.e., individuals who do not inherit a predisposing allele may nevertheless develop disease due to 5 environmental or random causes; and (4) genetic heterogeneity may exist, in which case mutations in any one of several genes may result in identical phenotypes. Despite these difficulties, however, identification of the chromosomal location, sequence and function of genes and 10 gene products responsible for causing neuropsychiatric disorders such as bipolar affective disorders is of great importance for genetic counseling, diagnosis and treatment of individuals in affected families. 15 2.2. THE HUMAN PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE GENE (PACAP) PACAP is a bioactive polypeptide originally isolated from bovine hypothalamus by virtue of its ability to stimulate adenylate cyclase in anterior pituitary cell 20 cultures (Miyata, et al., 1989, Biochem. Biophys. Res. Commun. 164, 567-574). An amino-terminal domain of the PACAP polypeptide displays 68% identity to vasoactive intestinal polypeptide (VIP), and less similarity to growth hormone releasing hormone (GHRH), peptide histidine isoleucine amide 25 (PHI), secreting, and glucagon (Miyata, id.). The human PACAP gene (also known as ADCYAP) has been isolated and sequenced (GenBank* accession number X60435), and the structure of it's intron/exon borders determined, by comparison with a human PACAP cDNA (Hosoya, et al., 1992, Biochim. Biophys. Acta 30 1129, 199-206). Further, the gene has been localized to chromosome 18 on the basis of Southern blot hybridization with human-mouse hybrid somatic cell line DNA containing human chromosome 18 (Hosoya, id.). Still further, refinement to human chromosome 35 band 18p11 was achieved using a radioisotope in situ hybridization procedure (Hosoya, id.). Perez-Jurado and Francke (1993, Human Molecular Genetics 2, 827) have - 5 - WO 99/51762 PCT/US99/07401 described a dinucleotide repeat polymorphism in the 3' untranslated region of human PACAP. This marker, known as W3440, has been used in linkage disequilibrium analysis and was important for defining the candidate genetic interval 5 encoding human PACAP. Another STS marker known as ADCYAP1 is located in the 3' untranslated region of the human PACAP gene; this marker has been used to pinpoint the gene location precisely on the chromosome 18 physical map. Expression studies indicate that PACAP is synthesized in 10 retinal ganglion cells which terminate on VIP neurons in the suprachiasmatic nucleus (SCN), which is the location of the circadian clock. In addition, PACAP expression exhibits a circadian rhythm in rat SCN: low levels occur during light periods, high levels occur during dark periods, and stable 15 levels occur under continuously dark conditions (Fukuhara, et al., 1997, Neurosci. Lett. 229, 49-52). It has also been reported that PACAP induces the phosphorylation of CREB in the SCN during late subjective day and that melatonin inhibits this PACAP-induced phosphorylation (Kopp, et al., 20 1997, Neurosci. Lett. 227, 145-148). These findings suggest that PACAP expression levels and/or activity in the SCN may be changed by lighting conditions and that PACAP-containing neurons may play a role in the entrainment of circadian rhythms. 25 3. SUMMARY OF THE INVENTION It is an object of the present invention to identify genetic bases for neuropsychiatric disorders, provide methods of treating and diagnosing neuropsychiatric disorders, and 30 provide methods for identifying compounds for use as part of therapeutic and/or diagnostic methods. The invention further relates to methods for the treatment of PACAP mediated neuropsychiatric disorders, wherein such methods comprise administering a compound which 35 modulates the expression of a mammalian PACAP gene and/or the synthesis or activity of a mammalian PACAP gene product so symptoms of the disorder are ameliorated. -6- WO 99/51762 PCT/US99/07401 The invention further relates to methods for the treatment of mammalian PACAP mediated neuropsychiatric disorders resulting from PACAP gene mutations, wherein such methods comprise supplying the mammal with a nucleic acid 5 molecule encoding an unimpaired PACAP gene product such that an unimpaired PACAP gene product is expressed and symptoms of the disorder are ameliorated. The invention further relates to methods for the treatment of mammalian PACAP mediated neuropsychiatric 10 disorders resulting from PACAP gene mutations, wherein such methods comprise supplying the mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired PACAP gene product such that the cell expresses the unimpaired PACAP gene product and symptoms of the disorder are ameliorated. 15 In addition, the present invention is directed to methods that utilize the PACAP gene and/or gene product sequences for the diagnostic evaluation, genetic testing and prognosis of a PACAP mediated neuropsychiatric disorder. For example, the invention relates to methods for diagnosing 20 PACAP mediated neuropsychiatric disorders, wherein such methods comprise measuring PACAP gene expression in a patient sample, or detecting a PACAP mutation in the genome of the mammal suspected of exhibiting such a disorder. The invention still further relates to methods for 25 identifying compounds capable of modulating the expression of the mammalian PACAP gene and/or the synthesis or activity of the mammalian PACAP gene products, wherein such methods comprise contacting a compound to a cell that expresses an PACAP gene, measuring the level of PACAP gene expression, 30 gene product expression or gene product activity, and comparing this level to the level of PACAP gene expression, gene product expression or gene product activity produced by the cell in the absence of the compound, such that if the level obtained in the presence of the compound differs from 35 that obtained in its absence, a compound capable of modulating the expression of the mammalian PACAP gene and/or - 7 - WO 99/51762 PCT/US99/07401 the synthesis or activity of the mammalian PACAP gene products has been identified. The invention is based, in part, on the genetic and physical mapping of the PACAP gene to a specific portion of 5 human chromosome 18, and specifically to the short arm of human chromosome 18 between the telomere and D185481, described in the example presented below in Section 6. Thus, the invention also relates to the use of the PACAP gene and/or gene products as markers for fine structure mapping of 10 this region of human chromosome 18. The PACAP mediated neuropsychiatric disorders referred to herein include, but are not limited to, bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP I), bipolar affective (mood) disorder with hypomania and 15 major depression (BP-II). The term "PACAP mediated neuropsychiatric disorder" as used herein refers to a disorder involving an aberrant level of PACAP gene expression, gene product synthesis and/or gene product activity relative to levels found.in normal, 20 unaffected, unimpaired individuals, levels found in clinically normal individuals, and/or levels found in a population whose level represents a baseline, average PACAP level. 25 3.1. DEFINITIONS As used herein, the following terms shall have the abbreviations indicated. BAC, bacterial artificial chromosome(s) 30 BAD, bipolar affective disorder(s) BP, bipolar mood disorder BP-I, severe bipolar affective (mood) disorder BP-II, bipolar affective (mood) disorder with hypomania and major depression 35 bp, base pair(s) EST, expressed sequence tag lod, logarithm of odds -8- WO 99/51762 PCT/US99/07401 MDD, unipolar major depressive disorder ROS, reactive oxygen species RT-PCR, reverse transcriptase PCR SSCP, single-stranded conformational polymorphism 5 SAD-M, schizoaffective disorder manic type STS, short tag sequence YAC, yeast artificial chromosome 4. BRIEF DESCRIPTION OF THE FIGURES 10 FIG. 1. Sequence of the human PACAP gene. Amino acid sequences are indicated. FIG. 2A-2U. Genomic Sequence of the human PACAP gene. Exons are in bold and the 5' UTR is underlined. 15 5. DETAILED DESCRIPTION OF THE INVENTION This invention is based on the genetic and physical mapping of the PACAP gene to a specific, narrow portion of chromosome 18, also described in the Example presented below in Section 6. -20 The invention described in the subsections below encompasses screening methods (e.g., assays) for the identification of compounds which can be used to treat individuals suffering from a PACAP mediated neuropsychiatric disorder. The invention also encompasses agonists and 25 antagonists of the PACAP gene product, including small molecules, large molecules, and antibodies, as well as nucleotide sequences that can be used to inhibit PACAP gene expression (e.g., antisense and ribozyme molecules), and gene or regulatory sequence replacement constructs designed to 30 enhance PACAP gene expression (e.g., expression constructs that place the PACAP gene under the control of a strong promoter system). In particular, cellular and non-cellular assays are described that can be used to identify compounds that 35 interact with the PACAP gene product, e.g., modulate the activity of the PACAP and/or bind to the PACAP gene product. Such cell-based assays of the invention utilize cells, cell -9- WO 99/51762 PCT/US99/07401 lines, or engineered cells or cell lines that express the PACAP gene product. The invention also encompasses the use of cell-based assays or cell-lysate assays (e.g., in vitro transcription or 5 translation assays) to screen for compounds or compositions that modulate PACAP gene expression. To this end, constructs containing a reporter sequence linked to a regulatory element of the PACAP gene can be used in engineered cells, or in cell lysate extracts, to screen for compounds that modulate the 10 expression of the reporter gene product at the level of transcription. For example, such assays could be used to identify compounds that modulate the expression or activity of transcription factors involved in PACAP gene expression, or to test the activity of triple helix polynucleotides. 15 Alternatively, engineered cells or translation extracts can be used to screen for compounds (including antisense and ribozyme constructs) that modulate the translation of PACAP mRNA transcripts, and therefore, affect expression of the PACAP gene product. 20 The invention also encompasses PACAP gene products, polypeptides (including soluble PACAP polypeptides or peptides) and PACAP -fusion proteins for use in non-cell based screening assays, for use in generating antibodies, for diagnostics and therapeutics. Such peptides or polypeptides 25 can be fused to a heterologous protein, e.g., reporter, an Ig Fc region, etc., to yield a fusion protein. Such peptides, polypeptides and fusion proteins can be used in the non-cell based assays for screening compounds that interact with, e.g., modulate the activity of the PACAP gene product and/or 30 bind to the PACAP gene product. PACAP gene products can be used to treat PACAP mediated disorders. Such PACAP gene products include, but are not limited to, soluble derivatives such as peptides or polypeptides corresponding to one or more domains of the 35 PACAP gene product. Alternatively, antibodies to the PACAP protein or anti-idiotypic antibodies that mimic the PACAP gene product (including Fab fragments), antagonists or -10- WO 99/51762 PCT/US99/07401 agonists can be used to treat neuropsychiatric disorders involving PACAP. In yet another approach, nucleotide constructs encoding such PACAP gene products can be used to genetically engineer host cells to express such PACAP gene 5 products in vivo; these genetically engineered cells can function as "bioreactors" in the body delivering a continuous supply of PACAP gene product, PACAP peptides, soluble PACAP polypeptides. In addition, this invention presents methods for the 10 diagnostic evaluation and prognosis of PACAP mediated disorders. For example, nucleic acid molecules encoding PACAP can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of PACAP gene mutations, allelic variations and regulatory 15 defects in the PACAP gene. "Gene therapy" approaches for the modulation of PACAP gene expression and/or activity are within the scope of the invention. For example, nucleotide constructs encoding functional PACAP gene, mutant PACAP gene, as well as 20 antisense and ribozyme molecules can be used to modulate PACAP gene expression. The invention also encompasses pharmaceutical formulations and methods for treating disorders involving the PACAP gene. The present invention sets forth methods for 25 selecting an effective drug to administer to an individual having a PACAP mediated disorder. Such methods are based on the detection of genetic polymorphisms in the PACAP gene or variations in PACAP gene expression due to altered methylation, differential spinning, or post-transductional 30 modification of the PACAP gene product which can affect the safety and efficacy of a therapeutic agent. 5.1. THE PACAP GENE With respect to PACAP gene sequences as disclosed in 35 Figure 1, such sequences can, for example, be obtained readily by utilizing standard sequencing and bacterial artificial chromosome (BAC) technologies in connection with -11- WO 99/51762 PCT/US99/07401 BAC54 (Identification Reference EpHS996, ATCC Accession No. 98363). For example, sheared libraries can be made from BAC54. Fragments of a convenient size, e.g., in the size range of 5 approximately 1 kb, are cloned into a standard plasmid, and sequenced. Further PACAP sequences can then readily be identified by alignment of the BAC sequences with the PACAP sequences depicted in Figure 1. Alternatively, BAC subclones containing additional PACAP sequences can be identified by 10 identifying those subclones which hybridize to probes derived from the PACAP sequences depicted in Figure 1. With respect to the cloning of allelic variants of the human PACAP gene and homologues from other species (e.g., mouse), the isolated PACAP gene sequences disclosed herein 15 may be labeled and used to screen a cDNA library constructed from mRNA obtained from appropriate cells or tissues (e.g., brain tissues) derived from the organism (e.g., mouse) of interest. The hybridizationconditions used should be of a lower stringency when the cDNA library is derived from an 20 organism different from the type of organism from which the labeled sequence was derived. Alternatively, -the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions. 25 Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook, et al., 1989, Molecular Cloning, A 30 Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y.; and Ausubel, et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. Further, an PACAP gene allelic variant may be isolated 35 from, for example, human nucleic acid, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the PACAP gene -12- WO 99/51762 PCTIUS99/07401 product disclosed herein. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue known or suspected to express an PACAP gene allele (such as 5 human leukemia cell lines e.g., K562 BlA, H630 and H630-1, e.g., Dolnick, et al., 1996, Cancer Research 56, 1207-3260; Dolnick, et al., 1993, Nucleic Acids Res., 21, 1747-1752; Black, et al., 1996, Cancer Res. 56, 700-705). Preferably, the allelic variant will be isolated from an individual who 10 has a PACAP mediated neuropsychiatric disorder. The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a PACAP gene nucleic acid sequence. The PCR fragment may then be used to isolate a full length cDNA clone by a variety of 15 methods. For example, the amplified fragment may be labeled and used to screen a bacteriophage cDNA library. Alternatively, the labeled fragment may be used to isolate genomic clones via the screening of a genomic library. PCR technology may also be utilized to isolate full 20 length cDNA sequences. For example, RNA may be isolated, following standard.procedures, from an appropriate cellular or tissue source (i.-e., one known, or suspected, to express the PACAP gene, such as, for example, brain tissue samples obtained through biopsy or post-mortem). A reverse 25 transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the 30 hybrid may be digested with RNAse H, and second strand synthesis may then be primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of cloning strategies that may be used, see e.g., Sambrook et al., 1989, supra. 35 As mentioned above, the PACAP gene sequences may be used to isolate mutant PACAP gene alleles, preferably from a human subject. Such mutant alleles may be isolated from -13- WO 99/51762 PCT/US99/07401 individuals either known or proposed to have a genotype that contributes to the symptoms of an PACAP mediated disorder. Mutant alleles and mutant allele products may then be utilized in the therapeutic and diagnostic systems described 5 below. Additionally, such PACAP gene sequences can be used to detect PACAP gene regulatory (e.g., promoter) defects which can be associated with a PACAP mediated disorder. A cDNA of a mutant allelic variant of the PACAP gene may be isolated, for example, by using PCR, a technique that is 10 well known to those of skill in the art. In this case, the first cDNA strand may be synthesized by hybridizing an oligo dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual putatively carrying the mutant PACAP allele, and by extending the new 15 strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an oligonucleotide that .. hybridizes specifically to the 5' end of the normal gene. Using these two primers, the, product is then amplified via PCR, cloned into a suitable vector, and subjected.to DNA 20 sequence analysis through methods well known to those of -skill in the art. By comparing the DNA sequence of the mutant PACAP allele -to that of the normal PACAP allele,' the mutation(s) responsible for the loss or alteration of function of the mutant PACAP gene product can be ascertained. 25 Alternatively, a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry a mutant PACAP allele, or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express a mutant PACAP allele. An unimpaired PACAP gene or 30 any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant PACAP allele in such libraries. Clones containing the mutant PACAP gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill in 35 the art. Additionally, an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated - 14 - WO 99/51762 PCT/US99/07401 from a tissue known, or suspected, to express a mutant PACAP allele in an individual suspected of or known to carry such a mutant allele. In this manner, gene products made by the putatively mutant tissue may be expressed and screened using 5 standard antibody screening techniques in conjunction with antibodies raised against the normal PACAP gene product, as described, below, in Section 5.3. (For screening techniques, see, for example, Harlow and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring 10 Harbor, New York.) In cases where an PACAP mutation results in an expressed gene product with altered function (e.g., as a result of a missense or a frameshift mutation), a polyclonal set of anti PACAP gene product antibodies are likely to cross-react with 15 the mutant PACAP gene product. Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods well known to those of skill in the art. PACAP mutations can further be detected using PCR. 20 amplification techniques. Primers can routinely be designed to amplify overlapping regions of the whole PACAP sequence including the promoter region. In one embodiment, primers are designed to cover the exon-intron boundaries such that, coding regions can be scanned for mutations (see Figures 2A 25 2U). Several primers for analyzing various PACAP exons are provided in the Examples. Genomic DNA isolated from lymphocytes of normal and affected individuals is used as PCR template. PCR products from normal and affected individuals are compared, either by 30 single strand conformational polymorphism (SSCP) mutation detection techniques and/or by sequencing. The mutations responsible for the loss or alteration of function of the mutant PACAP gene product can then be ascertained. 35 5.2. PROTEIN PRODUCTS OF THE PACAP GENE PACAP gene products, or peptide fragments thereof, can be prepared for a variety of uses. For example, such gene - 15 - WO 99/51762 PCT/US99/07401 products, or peptide fragments thereof, can be used for the generation of antibodies, in diagnostic assays, or for the identification of other cellular or extracellular gene products involved in the regulation of PACAP mediated 5 disorders. The amino acid sequence depicted in Figure 1 represents a PACAP gene product. The PACAP gene product, sometimes referred to herein as a "PACAP protein," includes those gene products encoded by the PACAP gene sequences depicted in 10 Figure 1, as well as other human allelic variants of PACAP that can be identified by the methods herein described. In addition, PACAP gene products may include proteins that represent functionally equivalent gene products. Such an equivalent PACAP gene product may contain deletions, 15 including internal deletions, additions, including additions yielding fusion proteins, or substitutions of amino acid residues within and/or adjacent to the amino acid sequence encoded by the PACAP gene sequences described above, in Section 5.1, but that result in a "silent" change, in that 20 the change produces a functionally equivalent PACAP gene. product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar 25 (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include 30 arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, where alteration of function is desired, deletion or non-conservative alterations can be engineered to produce altered, including reduced PACAP gene products. Such 35 alterations can, for example, alter one or more of the biological functions of the PACAP gene product. Further, such alterations can be selected so as to generate PACAP gene - 16 - WO 99/51762 PCT/US99/07401 products that are better suited for expression, scale up, etc., in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges. 5 The PACAP gene products, peptide fragments thereof and fusion proteins thereof, may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the PACAP gene products, polypeptides, peptides, fusion peptides and fusion polypeptides of the 10 invention by expressing nucleic acid containing PACAP gene sequences are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing PACAP gene product coding sequences and appropriate transcriptional and translational 15 control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in-vivo genetic recombination. See, for example, the techniques described in Sambrook, -et al., 1989, supra, and 'Ausubel, et al., 1989, supra. Alternatively, RNA capable of 20 encoding PACAP gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, ed., IRL Press, Oxford. A variety of host-expression vector systems may be 25 utilized to express the PACAP gene product coding sequences of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells that may, when transformed or transfected with the 30 appropriate nucleotide coding sequences, exhibit the PACAP gene product of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing 35 PACAP gene product coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the PACAP gene product coding - 17 - WO 99/51762 PCT/US99/07401 sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the PACAP gene product coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., 5 cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing PACAP gene product coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs 10 containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In bacterial systems, a number of expression vectors may 15 be advantageously selected depending upon the use intended for the PACAP gene product being expressed. For example, when a large quantity of such a protein is to be produced for the generation of pharmaceutical compositions of PACAP gene product or for raising antibodies to PACAP gene.product, 20 for example, vectors that direct the expression of high levels of fusion. protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2, 1791), in which the PACAP gene product coding 25 sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13, 3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 264, 5503-5509); and the like. pGEX vectors may also 30 be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The 35 pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. - 18 - WO 99/51762 PCT/US99/07401 In an insect system, Autographa californica, nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The PACAP gene product coding sequence may be cloned 5 individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of PACAP gene product coding sequence will result in inactivation of the polyhedrin gene and 10 production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (e.g., see Smith, et al., 1983, J. Virol. 46, 15 584; Smith, U.S. Patent No. 4,215,051). In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an.expression vector, the PACAP gene product coding sequence of interest may be ligated to an 20 adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non essential region of the viral genome (e.g., region El or E3) 25 will result in a recombinant virus that is viable and capable of expressing PACAP gene product in infected hosts. (e.g., see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81, 3655-3659). Specific initiation signals may also be required for efficient translation of inserted PACAP gene product 30 coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire PACAP gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be 35 needed. However, in cases where only a portion of the PACAP gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation - 19 - WO 99/51762 PCT/US99/07401 codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons 5 can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153, 516-544). 10 In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be 15 important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be.chosen to ensure the correct modification and 20 processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, 25 VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the PACAP gene product may be engineered. Rather than using expression vectors that 30 contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the 35 foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant - 20 - WO 99/51762 PCT/US99/07401 plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method may advantageously be used to 5 engineer cell lines that express the PACAP gene product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the PACAP gene product. A number of selection systems may be used, including but 10 not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11, 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48, 2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22, 817) 15 genes can be employed in tk-, hgprt- or aprt~ cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77, 3567; O'Hare, et al., 1981, Proc. -20 Natl. Acad. Sci. USA 78, 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78, 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981,.J. Mol. Biol. 150, 1); and hygro, which confers resistance to 25 hygromycin (Santerre, et al., 1984, Gene 30, 147). Alternatively, any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht, et al. allows for the ready purification of non 30 denatured fusion proteins expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8972-8976). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an 35 amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2 -nitriloacetic acid-agarose columns and - 21 - WO 99/51762 PCT/US99/07401 histidine-tagged proteins are selectively eluted with imidazole-containing buffers. The PACAP gene products can also be expressed in transgenic animals. Animals of any species, including, but 5 not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate PACAP transgenic animals. The term "transgenic," as used herein, refers to animals expressing PACAP gene sequences 10 from a different species (e.g., mice expressing human PACAP gene sequences), as well as animals that have been genetically engineered to overexpress endogenous (i.e., same species) PACAP sequences or animals that have been genetically engineered to no longer express endogenous PACAP 15 gene sequences (i.e., "knock-out" animals), and their progeny. Any technique known in the art may be used to introduce a PACAP gene transgene into animals to produce the founder lines of transgenic animals. -Such techniques include,. but 2.0 are not limited to pronuclear microinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten, et al., 1985, Proc. Natl. Acad. Sci., USA 82, 6148-6152); gene targeting in embryonic stem cells (Thompson, et al., 1989, Cell 56, 313 25 321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol. 3, 1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57, 717-723) (For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115, 171-229) 30 Any technique known in the art may be used to produce transgenic animal clones containing a PACAP transgene, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal or adult cells induced to quiescence (Campbell, et al., 1996, Nature 380, 64-66; 35 Wilmut, et al., Nature 385, 810-813). The present invention provides for transgenic animals that carry a PACAP transgene in all their cells, as well as - 22 - WO 99/51762 PCT/US99/07401 animals that carry the transgene in some, but not all their cells, i.e., mosaic animals. The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be 5 selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko, et al., 1992, Proc. Natl. Acad. Sci. USA 89, 6232-6236). The regulatory sequences required for such a cell-type specific activation will depend upon the particular 10 cell type of interest, and will be apparent to those of skill in the art. When it is desired that the PACAP transgene be integrated into the chromosomal site of the endogenous PACAP gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some 15 nucleotide sequences homologous to the endogenous PACAP gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous PACAP gene. The transgene may also be selectively introduced. 20 into a particular cell type, thus inactivating the. endogenous PACAP gene in only that cell type, by following, for example, the teaching of Gu, et al. (Gu, et al., 1994, Science 265, 103-106). The regulatory sequences required for such a cell type specific inactivation will depend upon the particular 25 cell type of interest, and will be apparent to those of skill in the art. Once transgenic animals have been generated, the expression of the recombinant PACAP gene may be assayed utilizing standard techniques. Initial screening may be 30 accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression-of the transgene in the tissues of the transgenic animals may also be assessed using techniques that include but are not 35 limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR (reverse transcriptase PCR). Samples of PACAP gene - 23 - WO 99/51762 PCT/US99/07401 expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the PACAP transgene product. 5.3. ANTIBODIES TO PACAP GENE PRODUCTS 5 Described herein are methods for the production of antibodies capable of specifically recognizing one or more PACAP gene product epitopes or epitopes of conserved variants or peptide fragments of the PACAP gene products. Further, antibodies that specifically recognize mutant forms of PACAP, 10 are encompassed by the invention. Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced.by a Fab 15 expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of a PACAP gene product in an biological sample and may, therefore, be utilized as part of a diagnostic or prognostic 20 technique whereby patients may be tested for abnormal levels of PACAP gene products, and/or- for the presence of abnormal forms of such gene products. Such antibodies may-also be utilized in conjunction with, for example, compound screening schemes, as described, below, in Section 5.8, for the 25 evaluation of the effect of test compounds on PACAP gene product levels and/or activity. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described, below, in Section 5.9.2 to, for example, evaluate the normal and/or engineered PACAP 30 expressing cells prior to their introduction into the patient. Anti-PACAP gene product antibodies may additionally be used in methods for inhibiting abnormal PACAP gene product activity. Thus, such antibodies may, therefore, be utilized 35 as part of treatment methods for a PACAP mediated disorder. For the production of antibodies against a PACAP gene product, various host animals may be immunized by injection - 24 - WO 99/51762 PCT/US99/07401 with a PACAP gene product, or a portion thereof. Such host animals may include, but are not limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host 5 species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human 10 adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a PACAP gene product, or an 15 antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with PACAP gene product supplemented with-adjuvants as also described above. 20 Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of 25 Kohler and Milstein, (1975, Nature 256, 495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4, 72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80, 2026-2030), and the EBV hybridoma technique (Cole et al., 1985, Monoclonal Antibodies 30 And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo 35 makes this the presently preferred method of production. In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al., 1984, Proc. Natl. - 25 - WO 99/51762 PCT/US99/07401 Acad. Sci., 81, 6851-6855; Neuberger, et al., 1984, Nature 312, 604-608; Takeda, et al., 1985, Nature, 314, 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a 5 human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., 10 Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816397, which are incorporated herein by reference in their entirety.) In addition, techniques have been developed for the production of humanized antibodies. (See, e.g., Queen, U.S. 15 Patent No. 5,585,089, which is incorporated herein by reference in its entirety.) An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred.to as complementarily determining regions (CDRs). The extent of 20 the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Immunological Interest", Kabat, E. et al., U.-S.Department of Health and Human Services (1983). Briefly, humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non 25 human species and a framework region from a human immunoglobulin molecule. Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778; Bird, 1988, Science 242, 423-426; Huston, et al., 1988, Proc. Natl. 30 Acad. Sci. USA 85, 5879-5883; and Ward, et al., 1989, Nature 334, 544-546) can be adapted to produce single chain antibodies against PACAP gene products. Single chain. antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, 35 resulting in a single chain polypeptide. Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such - 26 - WO 99/51762 PCT/US99/07401 fragments include but are not limited to: the F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and the Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 5 fragments. Alternatively, Fab expression libraries may be constructed (Huse, et al., 1989, Science, 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. 10 5.4. USES OF PACAP GENE SEQUENCES GENE PRODUCTS. AND ANTIBODIES Described herein are various applications of PACAP gene sequences, PACAP gene products, including peptide fragments and fusion proteins thereof, and of antibodies directed 1 against PACAP gene products and peptide fragments thereof. Such applications include, for example, prognostic and diagnostic evaluation of a PACAP mediated disorder, and. the. identification of subjects with a predisposition to such disorders, as described, below, in Section 5.5. 20 Additionally, such applications include methods for the treatment of a PACAP mediated disorder as described below, in Section 5.9, and for the identification of compounds-that. modulate the expression of the PACAP gene and/or the synthesis or activity of the PACAP gene product, as described 25 below, in Section 5.8. Such compounds can include, for example, other cellular products that are involved in mood regulation and in PACAP mediated disorders. These compounds can be used, for example, in the amelioration of PACAP mediated disorders. 30 5.5. DIAGNOSIS OF ABNORMALITIES OF A PACAP MEDIATED DISORDER A variety of methods can be employed for the diagnostic and prognostic evaluation of PACAP mediated disorders and for the identification of subjects having a predisposition to such disorders. - 27 - WO 99/51762 PCT/US99/07401 Such methods may, for example, utilize reagents such as the PACAP gene nucleotide sequences described in Sections 5.1, and antibodies directed against PACAP gene products, including peptide fragments thereof, as described, above, in 5 Section 5.3. Specifically, such reagents may be used, for example, for: (1) the detection of the presence of PACAP gene mutations, or the detection of either over- or under expression of PACAP protein; 10 (2) the detection of over- or under-abundance of PACAP gene product; and (3) the detection of an aberrant level of PACAP gene product activity. PACAP gene nucleotide sequences can, for example, be 15 used to diagnose a PACAP mediated disorder using, for example, the techniques for PACAP mutation detection described above in Section 5.1. Mutations at a number of different genetic loci may lead to phenotypes related to neuropsychiatric disorders. 20 Ideally, the treatment of patients suffering from such neuropsychiatric disorder will be designed to target.the particular genetic loci containing the mutation mediating the disorder. Genetic polymorphisms have been linked to differences in drug effectiveness. Thus, identification of 25 alterations in the PACAP gene or protein can be utilized.to optimize therapeutic drug treatments. In an embodiment of the present invention, polymorphisms in the PACAP gene sequence, or variations in PACAP gene expression due to altered methylation, differential splicing, 30 or post-translational modification of the PACAP gene product, may be utilized to identify an individual having a disease or condition resulting from a PACAP mediated disorder and thus define the most effective and safest drug treatment. Assays such as those described herein may be used to identify such 35 polymorphisms or variations in PACAP gene expression activity. Once a polymorphism in the PACAP gene, or a variation in PACAP gene expression has been identified in an - 28 - WO 99/51762 PCTIUS99/07401 individual, an appropriate drug treatment can be prescribed to the individual. The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising 5 at least one specific PACAP gene nucleic acid or anti-PACAP gene product antibody reagent described herein, which may be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting abnormalities of a PACAP mediated disorder. 10 For the detection of PACAP gene mutations, any nucleated cell can be used as a starting source for genomic nucleic acid. For the detection of PACAP gene expression or PACAP gene products, any cell type or tissue in which the PACAP gene is expressed may be utilized. 15 Nucleic acid-based detection techniques are described, below, in Section 5.6. Peptide detection techniques are described, below, in Section 5.7. 5.6. DETECTION OF PACAP NUCLEIC ACID MOLECULES 20 A variety of methods can be employed to screen for the presence of PACAP gene-specific mutations and to detect and/or assay levels of PACAP nucleic acid sequences. Mutations within the PACAP gene can be detected by utilizing a number of techniques. Nucleic acid from any 25 nucleated cell can be used as the starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures that are well known to those of skill in the art. PACAP nucleic acid sequences may be used in 30 hybridization or amplification assays of biological samples to detect abnormalities involving PACAP gene structure, including point mutations, insertions, deletions, inversions, translocations and chromosomal rearrangements. Such-assays may include, but are not limited to, Southern analyses, 35 single-stranded conformational polymorphism analyses (SSCP), and PCR analyses. - 29 - WO 99/51762 PCT/US99/07401 Diagnostic methods for the detection of PACAP gene specific mutations can involve for example, contacting and incubating nucleic acids including recombinant DNA molecules, cloned genes or degenerate variants thereof, obtained from a 5 sample, e.g., derived from a patient sample or other appropriate cellular source, such as lymphocytes, with one or more labeled nucleic acid reagents including recombinant DNA molecules, cloned genes or degenerate variants thereof, as described in Section 5.1, under conditions favorable for the 10 specific annealing of these reagents to their complementary sequences within the PACAP gene. The diagnostic methods of the present invention further encompass contacting and incubating nucleic acids for the detection of single nucleotide mutations or polymorphisms of the PACAP gene. 15 Preferably, the lengths of these.nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all non annealed nucleic acids are removed from the nucleic acid: PACAP molecule hybrid. The presence of nucleic acids that have hybridized, if any such molecules.exist,. is then 20 detected. Using such a detection scheme, the nucleic acid from the cell type or tissue of interest can be immobilized, for example, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads. In this case, after incubation, non 25 annealed, labeled nucleic acid reagents of the type described in Section 5.1 are easily removed. Detection of the remaining, annealed, labeled PACAP nucleic acid reagents is accomplished using standard techniques well-known to those in the art. The PACAP gene sequences to which the nucleic acid 30 reagents have annealed can be compared to the annealing pattern expected from a normal PACAP gene sequence in order to determine whether a PACAP gene mutation is present. In a preferred embodiment, PACAP mutations or polymorphisms can be detected by using a microassay of PACAP 35 nucleic acid sequences immobilized to a substrate or "gene chip" (see, e.g. Cronin, et al., 1996, Human Mutation 7:244 255). - 30 - WO 99/51762 PCTIUS99/07401 Alternative diagnostic methods for the detection of PACAP gene specific nucleic acid molecules, in patient samples or other appropriate cell sources, may involve their amplification, e.g., by PCR (the experimental embodiment set 5 forth in Mullis, 1987, U.S. Patent No. 4,683,202), followed by the detection of the amplified molecules using techniques well known to those of skill in the art. The resulting amplified sequences can be compared to those that would be expected if the nucleic acid being amplified contained only 10 normal copies of the PACAP gene in order to determine whether a PACAP gene mutation exists. Additionally, well-known genotyping techniques can be performed to identify individuals carrying PACAP gene mutations. Such techniques include, for example, the use of 15 restriction fragment length polymorphisms (RFLPs), which involve sequence variations in one of the recognition sites for the specific restriction enzyme used. Additionally, improved methods for analyzing DNA polymorphisms, which can be utilized for the identification 20 of PACAP gene-specific mutations, have-been described that capitalize on the presence of variable numbers of short, tandemly repeated DNA sequences between the restriction enzyme sites. For example, Weber (U.S. Pat. No. 5,075,217) describes a DNA marker based on length polymorphisms in 25 blocks of (dC-dA)n-(dG-dT)n short tandem repeats. The average separation of (dC-dA)n-(dG-dT)n blocks is estimated to be 30,000-60,000 bp. Markers that are so closely spaced exhibit a high frequency co-inheritance, and are extremely useful in the identification of genetic.mutations, such as, 30 for example, mutations within the PACAP gene, and the diagnosis of diseases and disorders related to PACAP mutations. Also, Caskey et al. (U.S. Pat.No. 5,364,759) describe a DNA profiling assay for detecting short tri and tetra 35 nucleotide repeat sequences. The process includes extracting the DNA of interest, such as the PACAP gene, amplifying the - 31 - WO 99/51762 PCT/US99/07401 extracted DNA, and labelling the repeat sequences to form a genotypic map of the individual's DNA. The level of PACAP gene expression can also be assayed. For example, RNA from a cell type or tissue known, or 5 suspected, to express the PACAP gene, such as brain, may be isolated and tested utilizing hybridization or PCR techniques such as are described, above. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the 10 assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the PACAP gene. Such analyses may reveal both quantitative-and qualitative aspects of the expression pattern of the PACAP gene, including activation or 15 inactivation of PACAP gene expression. In one embodiment of such a detection scheme, a cDNA molecule is synthesized from an .RNA molecule of interest (e.g., by reverse transcription of the RNA molecule into -cDNA). A sequence within the cDNA is then used as the 20 template for a nucleic acid amplification reaction, such as a -PCR amplification reaction, or the like. The nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the 25 PACAP gene nucleic acid reagents described in Section 5.1. The preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides. For detection of the amplified product, the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. 30 Alternatively, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method. Additionally, it is possible to perform such PACAP gene 35 expression assays "in situ", i.e., directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid - 32 - WO 99/51762 PCTIUS99/07401 purification is necessary. Nucleic acid reagents such as those described in Section 5.1 may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G.J., 1992, "1PCR In Situ Hybridization: Protocols And 5 Applications", Raven Press, NY). Alternatively, if a sufficient quantity of the appropriate cells can be obtained, standard Northern analysis can be performed to determine the level of mRNA expression of the PACAP gene. 10 5.7. DETECTION OF PACAP GENE PRODUCTS Antibodies directed against unimpaired or mutant PACAP gene products or conserved variants or peptide fragments thereof, which are discussed, above, in Section 5.3, may also 15 be used as diagnostics and prognostics for a PACAP mediated disorder. Such methods may be used to detect abnormalities in the level of PACAP gene product synthesis or expression, or abnormalities in the structure, temporal expression, and/or physical location of PACAP gene product. The 20 antibodies and immunoassay methods described herein have, for example, important in vitro applications in assessing the efficacy of treatments for PACAP mediated disorders. Antibodies, or fragments of antibodies, such as those described below, may be used to screen potentially 25 therapeutic compounds in vitro to determine their effects on PACAP gene expression and PACAP gene product production. The compounds that have beneficial effects on a PACAP mediated disorder. In vitro immunoassays may also be used, for example, to 30 assess the efficacy of cell-based gene therapy for a PACAP mediated disorder. Antibodies directed against PACAP gene products may be used in vitro to determine, for example, the level of PACAP gene expression achieved in cells genetically engineered to produce PACAP gene product. In the case of 35 intracellular PACAP gene products, such an assessment is done, preferably, using cell lysates or extracts. Such analysis will allow for a determination of the number of - 33 - WO 99/51762 PCT/US99/07401 transformed cells necessary to achieve therapeutic efficacy in vivo, as well as optimization of the gene replacement protocol. The tissue or cell type to be analyzed will generally 5 include those that are known, or suspected, to express the PACAP gene. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The 10 isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the PACAP 15 gene. Preferred diagnostic methods for the detection of PACAP gene products, conserved variants or peptide fragments thereof, may involve, for example, immunoassays wherein the PACAP gene products or conserved variants or peptide 20 fragments are detected by their interaction with an anti PACAP gene product-specific antibody. For example, antibodies, or fragments of antibodies, such as those described, above, in Section 5.3, may be used to quantitatively or qualitatively detect the presence of 25 PACAP gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody (see below, this Section) coupled with light microscopic, flow cytometric, or fluorimetric detection. 30 Such techniques are especially preferred for PACAP gene products that are expressed on the cell surface. The antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron 35 microscopy, for in situ detection of PACAP gene products, conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological - 34 - WO 99/51762 PCT/US99/07401 specimen from a patient, and applying thereto a labeled antibody that binds to an PACAP polypeptide. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the 5 use of such a procedure, it is possible to determine not only the presence of the PACAP gene product, conserved variants or peptide fragments, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily recognize that any of a wide variety of 10 histological methods (such as staining procedures) can be modified in order to achieve in situ detection of a PACAP gene product. Immunoassays for PACAP gene products, conserved variants, or peptide fragments thereof will typically 15 comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells in the presence of a detectably labeled antibody capable ofT identifying PACAP gene product, conserved variants or peptide fragments thereof, and detecting the bound antibody by any of 20 a number of techniques well-known in the art. The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles or soluble proteins. The support may then be 25 washed with suitable buffers followed by treatment with the detectably labeled PACAP gene product specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support may then be detected by 30 conventional means. By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, 35 natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the - 35 - WO 99/51762 PCT/US99/07401 present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a 5 bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding 10 antibody or antigen, or will be able to ascertain the same by use of routine experimentation. One of the ways in which the PACAP gene product-specific antibody can be detectably labeled is by linking the same to an enzyme, such as for use in an enzyme immunoassay (EIA) 15 (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA) ", 1978, Diagnostic Horizons 2, 1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller, A. et al., 1978, J. Clin. Pathol. 31, 507-520; Butler, J.E., 1981, Meth. Enzymol. 73, 482-523; Maggio, E. (ed.), 1980, Enzyme 20 Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be 25 detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, a-glycerophosphate, 30 dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, #-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by 35 colorimetric methods that employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual - 36 - WO 99/51762 PCT/US99/07401 comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively 5 labeling the antibodies or antibody fragments, it is possible to detect PACAP gene products through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 10 1986). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled 15 antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and 20 fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as is 2 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as 25 diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by 30 detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. 35 Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in - 37 - WO 99/51762 PCT/US99/07401 which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for 5 purposes of labeling are luciferin, luciferase and aequorin. 5.8. SCREENING ASSAYS FOR COMPOUNDS THAT MODULATE PACAP GENE ACTIVITY The following assays are designed to identify compounds 10 that bind to a PACAP gene product, compounds that bind to intracellular proteins, or portions of proteins that interact with a PACAP gene product, compounds that interfere with the interaction of a PACAP gene product with intracellular proteins and compounds that modulate the activity of the 15 PACAP gene (i.e., modulate the level of PACAP gene expression and/or modulate the level of PACAP gene product activity). Assays may additionally be utilized that identify:compounds that bind to PACAP gene regulatory sequences (e.g., promoter sequences; see e.g., Platt, 1994, J. Biol. Chem. 269, 20 28558-28562), and that can modulate-the level of PACAP gene expression. Such compounds may include, but are not limited to, small organic molecules, such as ones that areable-to cross the blood-brain barrier, gain entry into an appropriate cell and affect expression of the PACAP gene or some other 25 gene involved in a PACAP regulatory pathway, or intracellular proteins. Methods for the identification of such intracellular proteins are described, below, in Section 5.8.2. Such intracellular proteins may be involved in the control and/or 30 regulation of mood. Further, among these compounds are compounds that affect the level of PACAP gene expression and/or PACAP gene product activity and that can be used in the therapeutic treatment of PACAP mediated disorders-as described, below, in Section 5.9. 35 Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to, Ig-tailed fusion peptides, and members of random - 38 - WO 99/51762 PCTIUS99/07401 peptide libraries; (see, e.g., Lam, et al., 1991, Nature 354, 82-84; Houghten, et al., 1991, Nature 354, 84-86), and combinatorial chemistry-derived molecular library made of D and/or L- configuration amino acids, phosphopeptides 5 (including, but not limited to members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et al., 1993, Cell 72, 767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain 10 antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules. Such compounds may further comprise compounds, in particular drugs or members of classes or families of drugs, 15 known to ameliorate or exacerbate the symptoms of a disorder. Such compounds include antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD), p-chlorophenylalanine,, p-propyldopacetamide dithiocarbamate derivatives e.g., FLA 63; anti-anxiety drugs, 20 e.g., diazepam; monoamine oxidase (MAO) inhibitors, e.g., iproniazid, clorgyline, phenelzine and isocarboxazid; biogenic amine uptake blockers, e.g., tricyclic antidepressants such as desipramine, imipramine and amitriptyline; serotonin reuptake inhibitors e.g., 25 fluoxetine; antipsychotic drugs such as phenothiazine derivatives (e.g., chlorpromazine (thorazine) and trifluopromazine)), butyrophenones (e.g., haloperidol (Haldol)), thioxanthene derivatives (e.g., chlorprothixene), and dibenzodiazepines (e.g., clozapine); benzodiazepines; 30 dopaminergic agonists and antagonists e.g., L-DOPA, cocaine, amphetamine, a-methyl-tyrosine, reserpine, tetrabenazine, benzotropine, pargyline; noradrenergic agonists and antagonists e.g., clonidine, phenoxybenzamine, phentolamine, tropolone. 35 Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the PACAP gene product and for - 39 - WO 99/51762 PCT/US99/07401 ameliorating PACAP mediated disorders. Assays for testing the effectiveness of compounds identified by, for example, techniques such as those described in Sections 5.8.1 - 5.8.3, are discussed, below, in Section 5.8.4. 5 5.8.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO THE PACAP GENE PRODUCT In vitro systems may be designed to identify compounds capable of binding the PACAP gene products of the invention. 10 Compounds identified may be useful, for example, in modulating the activity of unimpaired and/or mutant PACAP gene products, may be useful in elaborating the biological function of the PACAP gene product, may be utilized in screens for identifying compounds that disrupt normal PACAP 15 gene product interactions, or may in themselves disrupt such interactions. The principle of the assays used to identify compounds that bind to the PACAP gene product involves preparing a reaction mixture of the PACAP gene product and the test 20 compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay involves 25 anchoring a PACAP gene product or a test substance onto a solid support and detecting PACAP gene product/test compound complexes formed on the solid support at the end of the reaction. In one embodiment of such a method, the PACAP gene product may be anchored onto a solid support, and the test 30 compound, which is not anchored, may be labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized as the solid support. The anchored component may be immobilized by non-covalent or covalent attachments. Non 35 covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a - 40 - WO 99/51762 PCT/US99/07401 monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored. In order to conduct the assay, the non-immobilized 5 component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes 10 anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an 15 indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously non-immobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). 20 Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for PACAP gene product or the test compound to anchor any complexes formed in solution, and 25 a labeled antibody specific for the other component of the possible complex to detect anchored complexes. 5.8.2. ASSAYS FOR INTRACELLULAR PROTEINS THAT INTERACT WITH PACAP GENE PRODUCTS 30 Any method suitable for detecting protein-protein interactions may be employed for identifying PACAP gene product-protein interactions. Among the traditional methods that may be employed are co-immunoprecipitation, cross-linking and co-purification 35 through gradients or chromatographic columns. Utilizing procedures such as these allows for the identification of proteins, including intracellular proteins, that interact - 41 - WO 99/51762 PCT/US99/07401 with PACAP gene products. Once isolated, such a protein can be identified and can be used in conjunction with standard techniques, to identify proteins it interacts with. For example, at least a portion of the amino acid sequence of a 5 protein that interacts with the PACAP gene product can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, "Proteins: Structures and Molecular Principles," W.H. Freeman & Co., N.Y., pp.34-49). The amino 10 acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such proteins. Screening may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of 15 oligonucleotide mixtures and the screening are well-known. (See, e.g., Ausubel, supra, and 1990, "PCR Protocols: A Guide to Methods and Applications," Innis, et al., eds. Academic Press, Inc., New York). Additionally, methods may be employed that result in the 20 simultaneous identification of genes that encode a protein which interacts with an PACAP gene product. These methods include, for example, probing expression libraries with labeled PACAP gene product, using PACAP gene product in a manner similar to the well known technique of antibody 25 probing of Xgtl libraries. One method that detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described (Chien, et al., 1991, Proc. 30 Natl. Acad. Sci. USA, 88, 9578-9582) and is commercially available from Clontech (Palo Alto, CA). Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein 35 fused to the PACAP gene product and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been - 42 - WO 99/51762 PCT/US99/07401 recombined into this plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) 5 whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid cannot because it does not provide activation function and the activation domain hybrid cannot because it 10 cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product. 15 The two-hybrid system or related methodologies may be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, PACAP gene products may be used as the bait gene product. Total genomic or cDNA sequences 20 are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait PACAP gene product fused to the DNA-binding domain are co-transformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For 25 example, a bait PACAP gene sequence, such as the open reading frame of the PACAP gene, can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter 30 gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids. A cDNA library of the cell line from which proteins that interact with bait PACAP gene product are to be detected can be made using methods routinely practiced in the art. 35 According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the - 43 - WO 99/51762 PCT/US99/07401 transcriptional activation domain of GAL4. Such a library can be co-transformed along with the bait PACAP gene-GAL4 fusion plasmid into a yeast strain that contains a lacZ gene driven by a promoter that contains GAL4 activation sequence. 5 A cDNA encoded protein, fused to a GAL4 transcriptional activation domain that interacts with bait PACAP gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene. Colonies that express HIS3 can be detected by their growth on petri dishes containing semi 10 solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait PACAP gene product-interacting protein using techniques routinely practiced in the art. 15 5.8.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH PACAP GENE PRODUCT MACROMOLECULE INTERACTION The PACAP gene products may, in vivo, interact with one or more macromolecules, including intracellular macromolecules, such as proteins. Such macromolecules may 20 include, but are not limited to, nucleic acid molecules and those proteins identified via methods such as those described, above, in Sections 5.8.1 - 5.8.2. For purposes of this discussion, the macromolecules are referred to herein as "binding partners". Compounds that disrupt PACAP gene 25 product binding to a binding partner may be useful in regulating the activity of the PACAP gene product, especially mutant PACAP gene products. Such compounds may include, but are not limited to molecules such as peptides, and the like, as described, for example, in-Section 5.8.2 above. 30 The basic principle of an assay system used to identify compounds that interfere with the interaction between the PACAP gene product and a binding partner or partners involves preparing a reaction mixture containing the PACAP gene product and the binding partner under conditions and for a 35 time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the - 44 - WO 99/51762 PCT/US99/07401 presence and absence of the test compound. The test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of PACAP gene product and its binding partner. Control reaction mixtures 5 are incubated without the test compound or with a compound which is known not to block complex formation. The formation of any complexes between the PACAP gene product and the binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture 10 containing the test compound, indicates that the compound interferes with the interaction of the PACAP gene product and the binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal PACAP gene product may also be compared to complex formation 15 within reaction mixtures containing the test compound and a mutant PACAP gene product. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of, mutant but not normal PACAP gene product. 20 The assay for compounds that interfere with the interaction of the PACAP gene products and binding partners can be conducted in -a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the PACAP gene product or the binding partner onto a solid support and 25 detecting complexes formed on the solid support at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, 30 test compounds that interfere with the interaction between the PACAP gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction-in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously 35 with the PACAP gene product and interactive intracellular binding partner. Alternatively, test compounds that disrupt preformed complexes., e.g., compounds with higher binding - 45 - WO 99/51762 PCT/US99/07401 constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below. 5 In a heterogeneous assay system, either the PACAP gene product or the interactive binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may 10 be immobilized by non-covalent or covalent attachments. Non covalent attachment may be accomplished simply by coating the solid surface with a solution of the PACAP gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used 15 to anchor the species to the solid surface. The surfaces may be prepared in advance and stored. In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, 20 unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of 25 label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the 30 antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected. 35 Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and - 46 - WO 99/51762 PCTUS99/07401 complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, 5 depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex formation or that disrupt preformed complexes can be identified. In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed 10 complex of the PACAP gene product and the interactive binding partner is prepared in which either the PACAP gene product or its binding partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 by Rubenstein which utilizes this 15 approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt PACAP gene product/binding partner interaction can be 20 identified. In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the PACAP product and/or the binding partner (in cases where the binding partner is a 25 protein), in place of one or both of the full length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for 30 disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the 35 protein involved in interactive binding. Alternatively, one protein can be anchored to a solid surface using methods described in this Section above, and allowed to interact with - 47 - WO 99/51762 PCT/US99/07401 and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which 5 can be isolated and identified by amino acid sequencing. Also, once the gene coding for the segments is engineered to express peptide fragments of the protein, it can then be tested for binding activity and purified or synthesized. For example, and not by way of limitation, a PACAP gene 10 product can be anchored to a solid material as described, above, in this Section by making a GST-PACAP fusion protein and allowing it to bind to glutathione agarose beads. The binding partner can be labeled with a radioactive isotope, such as "S, and cleaved with a proteolytic enzyme such as 15 trypsin. Cleavage products can then be added to the anchored GST-PACAP fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known methods. 20 Peptides so identified can be produced synthetically or produced using recombinant DNA technology. 5.8.4. ASSAYS FOR IDENTIFICATION OF COMPOUNDS THAT AMELIORATE A PACAP MEDIATED DISORDER 25 Compounds, including but not limited to binding compounds identified via assay techniques such as those described, above, in Sections 5.8.1 - 5.8.4, can be tested for the ability to ameliorate symptoms of a PACAP mediated disorder. 30 It should be noted that the assays described herein can identify compounds that affect PACAP activity by either affecting PACAP gene expression or by affecting the level of PACAP gene product activity. For example, compounds-may be identified that are involved in another step in the pathway 35 in which the PACAP gene and/or PACAP gene product is involved and, by affecting this same pathway may modulate the effect of PACAP on the development of a PACAP mediated disorder. - 48 - WO 99/51762 PCTIUS99/07401 Such compounds can be used as part of a therapeutic method for the treatment of the disorder. Described below are cell-based and animal model-based assays for the identification of compounds exhibiting such an 5 ability to ameliorate symptoms of a PACAP mediated disorder. First, cell-based systems can be used to identify compounds that may act to ameliorate symptoms of a PACAP mediated disorder. Such cell systems can include, for example, recombinant or non-recombinant cell, such as cell 10 lines, that express the PACAP gene. In utilizing such cell systems, cells that express PACAP may be exposed to a compound suspected of exhibiting an ability to ameliorate symptoms of a PACAP mediated disorder, at a sufficient concentration and for a sufficient time to 15 elicit such an amelioration of such symptoms in the exposed cells. After exposure, the cells can be assayed to measure alterations in the expression of the PACAP gene, e.g., by assaying cell lysates for PACAP mRNA transcripts (e.g., by Northern analysis) or for PACAP gene products expressed by, 20 the cell; compounds that modulate expression of the PACAP gene are good candidates as therapeutics. In addition, animal-based systems or models for a PACAP mediated disorder, for example, transgenic mice containing a human or altered form of PACAP gene, may be used to identify 25 compounds capable of ameliorating symptoms of the disorder. Such animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies and interventions. For example, animal models may be exposed to a compound suspected of exhibiting an ability to ameliorate 30 symptoms, at a sufficient concentration and for a sufficient time to elicit such an amelioration of symptoms of a PACAP disorder. The response of the animals to the exposure may be monitored by assessing the reversal of the symptoms of the disorder. 35 With regard to intervention, any treatments that reverse any aspect of symptoms of a PACAP mediated disorder, should be considered as candidates for human therapeutic - 49 - WO 99/51762 PCT/US99/07401 intervention in such a disorder. Dosages of test agents may be determined by deriving dose-response curves, as discussed in Section 5.10.1, below. 5 5.9. COMPOUNDS AND METHODS FOR THE TREATMENT OF PACAP MEDIATED NEUROPSYCHIATRIC DISORDERS Described below are methods and compositions whereby a PACAP mediated disorder, may be treated. For example, such methods can comprise administering compounds which modulate 10 the expression of a mammalian PACAP gene and/or the synthesis or activity of a mammalian PACAP gene product so symptoms of the disorder are ameliorated. Alternatively, in those instances whereby the mammalia PACAP mediated disorders result from PACAP gene mutations, 15 such methods can comprise supplying the mammal with a nucleic acid molecule encoding an unimpaired PACAP gene product such that an unimpaired PACAP gene product is expressed and symptoms of the disorder are ameliorated. In another embodiment of methods for the treatment of 20 mammalia PACAP mediated disorders resulting from PACAP gene mutations, such methods can comprise supplying the mammal with a cell comprising a nucleic acid molecule that encodes an unimpaired PACAP gene product such that the cell expresses the unimpaired PACAP gene product and symptoms of the 25 disorder are ameliorated. In cases in which a loss of normal PACAP gene product function results in the development of a PACAP mediated disorder an increase in PACAP gene product activity would facilitate progress towards an asymptomatic state in 30 individuals exhibiting a deficient level of PACAP gene expression and/or PACAP gene product activity. Methods for enhancing the expression or synthesis of PACAP can include,. for example, methods such as those described below, in Section 5.9.2. 35 Alternatively, symptoms of PACAP mediated disorders, may be ameliorated by administering a compound that decreases the level of PACAP gene expression and/or PACAP gene product - 50 - WO 99/51762 PCT/US99/07401 activity. Methods for inhibiting or reducing the level of PACAP gene product synthesis or expression can include, for example, methods such as those described in Section 5.9.1. 5 5.9.1. INHIBITORY ANTISENSE, RIBOZYME AND TRIPLE HELIX APPROACHES In another embodiment, symptoms of PACAP mediated disorders may be ameliorated by decreasing the level of PACAP gene expression and/or PACAP gene product activity by using 10 PACAP gene sequences in conjunction with well-known antisense, gene "knock-out," ribozyme and/or triple helix methods to decrease the level of PACAP gene expression. Among the compounds that may exhibit the ability to modulate the activity, expression or synthesis of the PACAP gene, 15 including the ability to ameliorate the symptoms of a PACAP mediated disorder, are antisense, ribozyme, and triple helix molecules. Such molecules may be designed to reduce or inhibit either unimpaired, or if appropriate, mutant target gene activity. Techniques for the production and use of such 20 molecules are well known to those of skill in the art. Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense approaches involve the design of oligonucleotides that are complementary to a 25 target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarily, aJthough preferred, is not required. A sequence "complementary" to a portion of an RNA, as 30 referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to 35 hybridize will depend on both the degree of complementarily and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches - 51 - WO 99/51762 PCT/US99/07401 with an RNA it may contain and still form a stable duplex (or triplex, as the case-may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized 5 complex. In one embodiment, oligonucleotides complementary to non-coding regions of the PACAP gene could be used in an antisense approach to inhibit translation of endogenous PACAP mRNA. Antisense nucleic acids should be at least six 10 nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. 15 Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisensegene 20 inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense 25 oligonucleotide are compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more 30 than is necessary to prevent specific hybridization to the target sequence. - The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single stranded or double-stranded. The oligonucleotide can be 35 modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other - 52 - WO 99/51762 PCT/US99/07401 appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86, 6553-6556; Lemaitre, et 5 al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84, 648-652; PCT Publication No. W088/09810, published December 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134, published April 25, 1988), hybridization triggered cleavage agents (see, e.g., Krol et al., 1988, 10 BioTechniques 6, 958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5, 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc. 15 The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 20 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D galactosylqueosine, -inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 25 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, 30 queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil 5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. 35 The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including - 53 - WO 99/51762 PCT/US99/07401 but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose. In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected 5 from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof. In yet another embodiment, the antisense oligonucleotide 10 is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual -units, the strands run parallel to each other (Gautier, et al., 1987, Nucl. Acids Res. 15, 6625-6641). The oligonucleotide 15 is a 2'-0-methylribonucleotide (Inoue, et al., 1987, Nucl. Acids Res. 15, 6131-6148), or a chimeric RNA-DNA analogue (Inoue, et al., 1987, FEBS Lett. 215, 327-330). Oligonucleotides of theinvention may be synthesized by standard methods known in the art, e.g., by use of an 20 automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988, Nucl. Acids Res. 16, 3209), methylphosphonate oligonucleotides can be prepared by use of 25 controlled pore glass polymer supports (Sarin, et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85, 7448-7451), etc. While antisense nucleotides complementary to the target gene coding region sequence could be used, those complementary to the transcribed, untranslated region are 30 most preferred. Antisense molecules should be delivered to cells that express the target gene in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the 35 tissue site, or modified antisense molecules, designed to target the desired cells (eqg., antisense linked to peptides or antibodies that specifically bind receptors or antigens - 54 - WO 99/51762 PCT/US99/07401 expressed on the target cell surface) can be administered systemically. However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress 5 translation of endogenous mRNAs. Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will 10 result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation of the target gene mRNA. For example, a vector can be introduced e.g., such that it is taken up by a cell 15 and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methods standard in the art. 20 Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or 25 constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290, 304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22, 787-797), the herpes thymidine kinase promoter 30 (Wagner, et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., 1982, Nature 296, 39-42), etc. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced 35 directly into the tissue site. Alternatively, viral vectors can be used that selectively infect the desired tissue, in - 55 - WO 99/51762 PCTUS99/07401 which case administration may be accomplished by another route (e.g., systemically). Ribozyme molecules designed to catalytically cleave target gene mRNA transcripts can also be used to prevent 5 translation of target gene mRNA and, therefore, expression of target gene product. See, e.g., PCT International Publication W090/11364, published October 4, 1990; Sarver et al., 1990, Science 247, 1222-1225. Ribozymes are enzymatic RNA molecules capable of 10 catalyzing the specific cleavage of RNA. (For a review, see Rossi, 1994, Current Biology 4, 469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of 15 ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g.., U.S. Patent No. 5,093,246, which is incorporated herein by reference in its entirety. 20 While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the 25 target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. -The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk 30 Reference, VCH Publishers, New York, (see especially Figure 4, page 833) and in Haseloff and Gerlach, 1988, Nature, 334, 585-591, which is incorporated herein by reference in its entirety. Preferably the ribozyme is engineered so that the 35 cleavage recognition site is located near the 5' end of the target gene mRNA, i.e., to increase efficiency and minimize - 56 - WO 99/51762 PCT/US99/07401 the intracellular accumulation of non-functional mRNA transcripts. The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as 5 the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224, 574-578; Zaug and Cech, 1986, Science, 231, 470-475; Zaug, et al., 1986, Nature, 324, 429 10 433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47, 207-216). The Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. 15 The invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in the target gene. As in the antisense approach, the ribozymes can be composed of modified oligonuclectides (e.g.,.for improved.

20 stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo. A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce 25 sufficient quantities of the ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency. Endogenous target gene expression can also be reduced by 30 inactivating or "knocking out" the target gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al., 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al., 1989, Cell 5, 313-321; each of which is incorporated by reference 35 herein in its entirety). For example, a mutant, non functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target - 57 - WO 99/51762 PCT/US99/07401 gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA 5 construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene -(e.g., 10 see Thomas and Capecchi, 1987 and Thompson, 1989, supra). However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors. 15 Alternatively, endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i.e., the target gene promoter and/or enhancers) to form triple helical structures that prevent .transcription of the 20 target gene in target cells in the body. (See generally, Helene, 1991, Anticancer Drug Des., 6(6), 569-584; Helene, et al., 1992, Ann. N.Y.- Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays 14(12), 807-815). Nucleic acid molecules to be used in triplex helix 25 formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either 30 purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC* triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarily to a 35 purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-rich, for - 58 - WO 99/51762 PCT/US99/07401 example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting 5 in GGC triplets across the three strands in the triplex. Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 10 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex. In instances wherein the antisense, ribozyme, and/or 15 triple helix molecules described herein are utilized to inhibit mutant gene expression, it is possible that the technique may so efficiently reduce or inhibit the transcription (triple helix),and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles that. 20 the possibility may arise wherein the concentration of normal target gene product present may be lower than is necessary for a normal phenotype. In such cases, to ensure that substantially normal levels of target gene activity are maintained, therefore, nucleic acid molecules that encode and 25 express target gene polypeptides exhibiting normal target gene activity may, be introduced into cells via gene therapy methods such as those described, below, in Section 5.9.2 that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized. 30 Alternatively, in instances whereby the target gene encodes an extracellular protein, it may be preferable to co administer normal target gene protein in order to maintain the requisite level of target gene activity. Antisense RNA and DNA, ribozyme, and triple helix 35 molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above.. These include techniques for chemically - 59 - WO 99/51762 PCT/US99/07401 synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and 5 in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that 10 synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. 5.9.2. GENE REPLACEMENT THERAPY 15 PACAP gene nucleic acid sequences, described above in Section 5.1, can be utilized for the treatment of a PACAP mediated disorder. Such treatment can be in the form of gene replacement therapy. Specifically, one or more copies of a normal PACAP gene or a portion of the PACAP gene that directs 20 the production of a PACAP gene product exhibiting normal PACAP gene function, may be inserted into the appropriate cells within a patient, using vectors that include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that 25 introduce DNA into cells, such as liposomes. Because the PACAP gene is expressed in the brain, such gene replacement therapy techniques should be capable delivering PACAP gene sequences to these cell types within patients. Thus, in one embodiment, techniques that are well 30 known to those of skill in the art (see, e.g., PCT Publication No. W089/10134, published April 25, 1988) can be used to enable PACAP gene sequences to cross the blood-brain barrier readily and to deliver the sequences to cells in the brain. With respect to delivery that is capable of crossing 35 the blood-brain barrier, viral vectors such as, for example, those described above, are preferable. - 60 - WO 99/51762 PCTIUS99/07401 In another embodiment, techniques for delivery involve direct administration of such PACAP gene sequences to the site of the cells in which the PACAP gene sequences are to be expressed. 5 Additional methods that may be utilized to increase the overall level of PACAP gene expression and/or PACAP gene product activity include the introduction of appropriate PACAP-expressing cells, preferably autologous cells, into a patient at positions and in numbers that are sufficient to 10 ameliorate the symptoms of a PACAP mediated neuropsychiatric disorder. Such cells may be either recombinant or non recombinant. Among the cells that can be administered to increase the overall level of PACAP gene expression in a patient are 15 normal cells, preferably brain cells, that express the PACAP gene. Alternatively, cells, preferably autologous cells, can be engineered to express PACAP gene sequences, and may then, be introduced into a patient.in positions appropriate for the amelioration of the symptoms of a PACAP mediated 20 neuropsychiatric disorder. Alternately, cells that express an unimpaired PACAP gene and that are from a MHC matched individual can be utilized, and may include, for example, brain cells. The expression of the PACAP gene sequences is controlled by the appropriate gene regulatory sequences to 25 allow such expression in the necessary cell types. Such gene regulatory sequences are well known to the skilled artisan. Such cell-based gene therapy techniques are well known to those skilled in the art, see, e.g., Anderson, U.S. Patent No. 5,399,349. 30 When the cells to be administered are non-autologous cells, they can be administered using well known techniques that prevent a host immune response against the introduced cells from developing. For example, the cells may be introduced in an encapsulated form which, while allowing for 35 an exchange of components with the immediate extracellular environment, does not allow.the introduced cells to be recognized by the host immune system. - 61 - WO 99/51762 PCT/US99/07401 Additionally, compounds, such as those identified via techniques such as those described, above, in Section 5.8, that are capable of modulating PACAP gene product activity can be administered using standard techniques that are well 5 known to those of skill in the art. In instances in which the compounds to be administered are to involve an interaction with brain cells, the administration techniques should include well known ones that allow for a crossing of the blood-brain barrier. 10 5.10. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION The compounds that are determined to affect PACAP gene expression or gene product activity can be administered to a 15 patient at therapeutically effective doses to treat or ameliorate a PACAP mediated neuropsychiatric disorder. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of such a disorder. 20 5.10.1. EFFECTIVE DOSE Toxicity and therapeutic efficacy of such compoundscan-. be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the 25 LD50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50

/ED

50 . Compounds that exhibit large therapeutic 30 indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of. affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. 35 The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies - 62 - WO 99/51762 PCTIUS99/07401 preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any 5 compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test 10 compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. 15 5.10.2. FORMULATIONS AND USE Pharmaceutical compositions for use in accordance with the present invention may be, formulated in conventional manner using one or more physiologically acceptable carriers 20 or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration. 25 For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or 30 hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch ~ glycolate); or wetting agents (e.g., sodium lauryl sulphate). 35 The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they - 63 - WO 99/51762 PCT/US99/07401 may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending 5 agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or 10 sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound. 15 For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered 20 in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit 25 may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. 30 The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. The 35 compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or - 64 - WO 99/51762 PCT/US99/07401 dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated in rectal 5 compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. 10 Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an 15 acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage 20 forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. 25 6. EXAMPLE: LOCALIZATION OF THE PACAP GENE TO CHROMOSOME 18 In the Example presented in this Section, studies are described that, first, define an interval approximately 310 kb on the short arm of human chromosome 18 within which a 30 region associated with a neuropsychiatric disorder is located and, second, to the identification of the PACAP gene, as mapping within this region. 6.1. MATERIALS AND METHODS 35 6.1.1. LINKAGE DISEQUILIBRIUM Linkage disequilibrium (LD) studies were performed using DNA from a population sample of neuropsychiatric disorder - 65 - WO 99/51762 PCT/US99/07401 (BP-I) patients. The population sample and LD techniques were as described in Freimer et al., 1996, Nature Genetics 12, 436-441. The present LD study took advantage of the additional physical markers identified via the physical 5 mapping techniques described below. 6.1.2. YEAST ARTIFICIAL CHROMOSOME (YAC) MAPPING For physical mapping, yeast artificial chromosomes (YACs) containing human sequences were mapped to the region 10 being analyzed based on publicly available maps (Cohen et al., 1993, C.R. Acad. Sci. 316, 1484-1488). The YACs were then ordered and contig reconstructed by performing standard short tag sequence (STS)-content mapping with microsatellite markers and non-polymorphic STSs available from databases 15 that surround the genetically defined candidate region. 6.1.3. BACTERIAL ARTIFICIAL CHROMOSOME (BAC) MAPPING STSs from the short arm of human chromosome 18 were used to screen a human BAC library (Research Genetics, 20 Huntsville, AL). The ends of the BACs were cloned or directly sequenced. The end sequences were used to amplify the next overlapping BACs. From each BAC, additional microsatellites were identified. Specifically, random sheared libraries were prepared from overlapping BACs within 25 the defined genetic interval. BAC DNA was sheared with a nebulizer (CIS-US Inc., Bedford, MA). Fragments in the size range of 600 to 1,000 bp were utilized for the sublibrary production. Microsatellite sequences from the sublibraries were identified by corresponding microsatellite probes. 30 Sequences around such repeats were obtained to enable development of PCR primers for genomic DNA. 6.1.4. RADIATION HYBRID (RH) MAPPING Standard RH mapping techniques were applied to a 35 Stanford G3 RH mapping panel (Research Genetics, Huntsville, AL) to order all microsatellite markers and non-polymorphic STSs in the region being analyzed. - 66 - WO 99/51762 PCT/US99/07401 6.1.5. SAMPLE SEQUENCING Random sheared libraries were made from all the BACs within the defined genetic region. Approximately 9,000 subclones within the approximately 310 kb region were 5 sequenced with vector primers in order to achieve an 8-fold sequence coverage of the region. All sequences were processed through an automated sequence analysis pipeline that assessed quality, removed vector sequences and masked repetitive sequences. The resulting sequences were then 10 compared to public DNA and protein databases using BLAST algorithms (Altschul et al., 1990, J. Molec. Biol., 215, 403 410). 6.2. RESULTS 15 Genetic regions involved in bipolar affective disorder (BAD) human genes had previously been reported to map to portions of the long (18q) and short (18p) arms -of human chromosome 18, including a broad 18q genetic region of about 6-7 cM between markers D18S469 and D18S554 (U.S. Provisional 20 Applications Serial Nos. 60/014,498 and 60/023,438, filed on March 28, 1996 and August 23, 1996, respectively, the entire contents of each of -which are incorporated herein by reference; Freimer, et al.-, 1996, Neuropsychiat. Genet. 67, 254-263; Freimer, et al., 1996, Nature Genetics 12, 436-441), 25 the entire contents of each of which are incorporated herein by reference. Linkage Disequilibrium. Prior to attempting to identify gene sequences, studies were performed to further narrow the neuropsychiatric disorder region. Specifically, a linkage. 30 disequilibrium (LD) analysis was performed using population samples and techniques as described in Section 6.1, above, which took advantage of the additional physical markers identified via the physical mapping techniques described below. 35 High resolution physical mapping using YAC, BAC and RH techniques. In order to provide the precise order of genetic markers necessary for linkage and LD mapping, and to guide - 67 - WO 99/51762 PCT/US99/07401 new microsatellite marker development for finer mapping, a high resolution physical map of the 18q23 candidate region was developed using YAC, BAC and RH techniques. For such physical mapping, first, YACs were mapped to 5 the chromosome 18 region being analyzed. Using the mapped YAC contig as a framework, the region from publicly available markers D18S1161 and D18S554, which spans most of the D18S469-D18S554 region described above, was also mapped and contiged with BACs. Sublibraries from the contiged BACs were 10 constructed, from which microsatellite marker sequences were identified and sequenced. To ensure development of an accurate physical map, the radiation hybrid (RH) mapping technique was independently applied to the region being analyzed. RH was used to order 15 all microsatellite markers and non-polymorphic STSs in the region. Thus, the high resolution physical map ultimately constructed was obtained using data from RH mapping and STS content mapping. BAC clones within the newly identified 310 kb 20 neuropsychiatric disorder region were analyzed to identify specific genes within the region. A combination of sample sequencing, cDNA selection and transcription mapping analyses were combined to arrange sequences into tentative transcription units, that is, tentatively delineating the 25 coding sequences of genes within this genomic region of interest. One of the transcription units identified was termed PACAP. 30 The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various 35 modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying - 68 - WO 99/51762 PCT/US99/07401 drawings. Such modifications are intended to fall within the scope of the appended claims. All publications and patent applications mentioned in this specification are herein incorporated by reference to 5 the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. 10 15 20 25 30 35 - 69 -

Claims (17)

1. A method for determining whether a human subject has or is at risk for developing a PACAP mediated neuropsychiatric disorder comprising the step of detecting 5 the presence or absence of a genetic mutation in the PACAP gene (SEQ ID NO:1) of said subject, wherein said genetic mutation is selected from the group consisting of a nucleotide substitution, a nucleotide insertion and a nucleotide deletion and results in the production of a PACAP 10 protein having an amino acid sequence other than the wild type PACAP amino acid sequence and the presence of said genetic mutation identifies a subject that has or is at risk for developing a PACAP mediated neuropsychiatric disorder. 15

2. The method of Claim 1, further comprising the steps of: a) obtaining a sample comprising nucleic acid molecules from said subject;, b) amplifying nucleic acid molecules in said 20 sample that encode a PACAP protein using amplification primers that selectively anneal to and amplify nucleic acid molecules that encode said PACAP protein; and c) determining whether said genetic mutation is present. 25

3. The method of Claim 2, wherein said determination in step c) comprises sequencing said nucleic acid molecule that encodes said PACAP protein. 30

4. The method of Claim 1, wherein the nucleotide sequence of said nucleic acid molecule that encodes said PACAP protein is determined.

5. The method of Claim 1, further comprising the steps 35 of: a) obtaining a sample comprising nucleic acid molecules from said subject; - 70 - WO 99/51762 PCT/US99/07401 b) detecting the nucleic acid molecules in said sample that encode said PACAP protein using a nucleic acid probe that selectively hybridizes to nucleic acid molecules that encode said PACAP protein; and 5 c) determining whether said genetic mutation is present.

6. The method of Claim 1, further comprising the steps of: 10 a) obtaining a sample comprising nucleic acid molecules from said subject; b) detecting the nucleic acid molecules in said sample that encode said PACAP protein using a restriction endonuclease digestion of said nucleic acid molecules and a 15 nucleic acid probe that selectively hybridizes to nucleic acid molecules that encode said PACAP protein, or a fragment thereof; and c) determining whether said genetic mutation is present. 20

7. The method of Claim 1, wherein said genetic mutation is a base change.

8. The method of Claim 1, wherein said genetic 25 mutation is detected by determining whether an altered PACAP protein is produced in said subject.

9. The method of Claim 8, further comprising the steps of: 30 a) obtaining a sample comprising protein molecules from said subject; b) detecting the PACAP proteins in said sample using an antibody which binds to said PACAP protein; and c) determining whether said PACAP protein is 35 encoded by a nucleic acid molecule containing said genetic mutation. - 71 - WO 99/51762 PCT/US99/07401

10. A method for identifying a compound that can be used to treat a PACAP mediated neuropsychiatric disorder, comprising the steps of: a) contacting a test compound with a PACAP 5 protein; b) determining whether said test compound binds to said PACAP protein; and c) selecting a test compound that binds to said PACAP protein as being a compound that can be used to treat a 10 PACAP mediated neuropsychiatric disorder.

11. The method of Claim 10, wherein a PACAP having wild-type activity is used. 15

12. The method of Claim 10, wherein a PACAP having an altered activity is used.

13. A method for identifying compounds that can be used to treat a PACAP mediated neuropsychiatric disorder 20 comprising the steps of: a) incubating a cell that expresses a PACAP gene in the presence and -absence of a test compound; b) determining the activity of the PACAP gene product in the presence and absence of said test compound; 25 and c) selecting a test compound that alters the activity of said PACAP gene product as being a compound that can be used to treat a PACAP mediated neuropsychiatric disorder. 30

14. The method of Claim 13, wherein a PACAP having wild-type activity is used.

15. The method of Claim 13, wherein a PACAP having an 35 altered activity is used. - 72 - WO 99/51762 PCT/US99/07401

16. The method of Claim 1, wherein said PACAP mediated neuropsychiatric disorder is bi-polar affective disorder.

17. The method of Claim 1, wherein said PACAP mediated 5 neuropsychiatric disorder is schizoaffective disorder manic type. 10 15 20 25 30 35 - 73 -