MXPA00009702A

MXPA00009702A - Methods and compositions for the diagnosis and treatment of neuropsychiatric disorders

Info

Publication number: MXPA00009702A
Application number: MXPA/A/2000/009702A
Authority: MX
Inventors: Hong Chen; Nelson B Freimer
Original assignee: Millennium Pharmaceuticals Inc; The Regents Of The University Of California
Priority date: 1998-04-03
Filing date: 2000-10-03
Publication date: 2001-12-13

Abstract

The present invention relates to the mammalian PACAP gene. The invention relates to methods for the identification of compounds that modulates the expression of PACAP and to using such compounds as therapeutics agents in the treatment of PACAP disorders. The invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of PACAP mediated disorders, and to methods and compositions for the treatment of these disorders. The invention also relates to use of the PACAP gene 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.

Description

METHODS AND COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF NEUROSIQUIÁTRIC ANOMALIES • INTRODUCTION The present invention relates to assays for detecting drugs, and to diagnostic and therapeutic methods for the treatment of neuropsychiatric disorders mediated by the expression of a mutant form of the adenylate activating polypeptide gene f) 10 cyclase of the pituitary { PACAP) or aberrant levels of PACAP expression. The invention is based on the 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 as an affective-bipolar disorder (BAD). Thus, the invention relates to the use of the PACAP gene and / or gene products as markers to map or determine the fine structure of a region of the 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 activity of the PACAP protein / gene and to the use of the compounds as those identified as therapeutic agents in the treatment of a disorder mediated by PACAP and / or a neuropsychiatric disorder, which includes, as an example and not as a limitation, bipolar affective disorder. The invention also relates to methods for diagnostic evaluation, genetic testing and prognosis of disorders mediated by PACAP. 2. BACKGROUND OF THE INVENTION 2.1. NEUROSIQUIATRIC DISORDERS There are only some psychiatric disorders in which the clinical manifestations of the disorders can be correlated with demonstrable defects in the structure and / or function of the nervous system. Well-known examples of these disorders include Huntington's disease, which can be traced to a mutation in a single gene, and in which neurons of the striatum are degenerated, and Parkinson's disease, in which the neurons degenerate dopaminergic in the nigro-striatal route. The vast majority of psychiatric anomalies, however, supposedly include subtle and / or undetectable changes at the cellular, muscular or both levels, in the structure and function of the nervous system. This lack of detectable neurological defects distinguishes "neuropsychiatric" disorders such as schizophrenia, attention deficit disorders, schizoaffective disorders, bipolar affective disorders or unipolar affective disorders, of neurological disorders in which anatomical or biological pathologies are manifested. Hence, the identification of the causal defects and neuropathologies of neuropsychiatric disorders are necessary to allow clinicians to evaluate and prescribe appropriate courses of treatment to cure or lessen the symptoms of these conditions. One of the most prevalent and potentially devastating disorders of neuropsychiatric disorders is bipolar affective disorder (BAD), also known as mood disorder, bipolar (BP) or manic depressive illness, which is characterized by episodes of elevated mood (mania) and depression (Goodwin, et al., 1990, Manic Depressive Illness, Oxford University Press, New York). The most severe and clinically distinct forms of BAD are BP-I (severe bipolar affective disorder (mood)), which affects 2 to 3 million people in the United States, and SAD-M (schizoaffective type manic disorder). These are characterized by at least one complete episode of mania, with or without major episodes of depression (defined by low mood or depression, with associated disturbances in rhythmic behaviors such as sleep, meals and sexual activity). BP-I often co-segregates in families with more etiologically heterogeneous syndromes, such as a unipolar affective disorder such as major depressive disorder, unipolar (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. 951-965; Mclnnes and Freimer, 1995, Curr. Opin. Genet Develop. 5,376-381). BP-I and SAD-M are serious mood disorders that are often difficult to distinguish between them with a cross-base, after similar clinical uses, and segregate in family studies.

(Rosenthal, et al., 1980, Arch. General Psychiat., 37, 804-810, Levinson and Levitt, 1987, Am. J. Psychiat., 144, 415-426; Goodwin, et al., 1990, Manic Depressive Illnes, Oxford University Press, New York). Hence, methods to distinguish neuropsychiatric disorders such as these are needed to effectively diagnose and treat affected individuals. At present, individuals are usually evaluated for ADB using the criteria established in the most current version of the Diagnostic and Statistical Manual of Mental Disorders (DSM) of the American Psychiatric Association. Although many medications have been used to treat individuals diagnosed with BAD, including lithium salts, carbamazepine, and valproic acid, none of the currently available medications are adequate. For example, drug treatments are effective only in approximately 60-70% of individuals diagnosed with BP-I. In addition, it is currently impossible to predict drug treatments that will be effective in, for example, individuals affected with particular BP-I. Usually, with the diagnosis, affected individuals are prescribed one medication after another until one is found that is effective. The early prescription of effective medication treatment is therefore crucial for a variety of reasons, including the prevention of extremely dangerous manic episodes and the risk of progressive deterioration if effective treatments are not found. The existence of a genetic component for ADB is strongly supported by the analysis of segregation and studies in twins (Bertelson, et al., 1977, Br. J. Psychiat, 130, 330-351, Freimer and Reus, 1992, in The Molecular and Genetic Basis of Neurological Disease, Rosenber, et al., eds., Butterworths, New York, pp. 951-956; Pauls, et al., 1992, Arch. Gen. Psychiat., 49, 703-708). Efforts to identify the chromosomal location of genes that could be involved in BP-1, however, have produced unsatisfactory results in that they report linkage between BP-I and markers on chromosomes X and 11 could not be replicated independently or confirmed in the reanalyses of the original pedigrees, indicating that linkage studies with BAD, even extremely high logs in a single locus, can be false positives (Barón, et al., 1987, Nature 326, 289-292; 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 research has suggested possible locations of the genes for BAD on chromosomes 18p and 21q, but in both cases the proposed candidate region is not well defined and there is no unequivocal support for its 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. , et al., 1995, Psych. Res. 59, 7-15; Straub, et al., 1994, Nature Genet., 8, 291-296). The determination or mapping of genes for common diseases considered to be caused by multiple genes, such as ADB, can be complicated by defining the phenotypes that are usually inaccurate, by etiological 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 probably carry an affected genotype from those who are genetically unaffected. For example, it is possible to define a phenotype affected by ADB including one or more of the broad groupings of the diagnostic classifications that constitute the mood disorders: BP-I, SAD-M, MDD, and bipolar affective disorder (condition of mood) with hypomania and major depression (BP-II). Thus, one of the biggest difficulties faced by psychiatric geneticists is the uncertainty related to the validity of phenotype designations, given that clinical diagnoses are based only on clinical observation and subjective reports. Likewise, with complex traits such as neuropsychiatric disorders, it is difficult to genetically map the genes that cause the trait because: (1) the phenotypes of the neuropsychiatric disorders do not present traditional recessive or dominant Mendelian inheritance patterns attributable to a single genetic locus; (2) there may be incomplete penetrance, that is, individuals who inherit a predisposing allele but do not manifest the disease; (3) a phenomenon of phenocopy can occur, that is, individuals who do not inherit a predisposing allele can nonetheless develop the disease due to environmental or random causes; and (4) there may be genetic heterogeneity, in which case mutations in any of the different genes can give rise to identical phenotypes. However, despite these difficulties, the identification of the chromosomal location, the sequence and function of the genes and 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. 2. 2 THE HUMAN PITUITARY CYLENASE ADENYLATE CYLPASA ACTIVATION GENIPEPTIDE (PACAP) PACAP is a bioactive polypeptide originally isolated from the bovine hypothalamus by virtue of its ability to stimulate adenylate cyclase in anterior pituitary cell cultures (Miyata, et al., 1989, Biochem Biophys, Res. Commun. 164, 567-574). An amino-terminal domain of the PACAP polypeptide shows 68% identity for the vasoactive intestinal polypeptide (VIP) and less similarity with growth hormone releasing hormone (GHRH), amide peptide histidine isoleucine (PHI), secretin, and glucagon ( Miyata, id.). The human PACAP gene (also known as ADCYAP1) has been isolated and sequenced (GenBank® accession number X60435), and the structure of its determined intron / exon limits, by comparison with a PACAP cDNA (Hosoya, et al., 1992, Biochim, Biophys, Acta 1129, 199-206). In addition, the gene has been located on chromosome 18 based on Southern blot hybridization with DNA from the human-mouse hybrid somatic cell line containing human chromosome 18 (Hosoya id.). Furthermore, refinement for the ldpll band of the human chromosome was achieved using a method of in situ hybridization with radioisotope (Hosoya, id.). Pérez-Jurado and Francke (1993, Human Molecular Genetics 2, 827) have described a repeat polymorphism of the dinocluotide in the 3 'untranslated region of human PACAP. This marker, known as W3440, has been used in the analysis of linkage disequilibrium and was important in defining the candidate genetic interval encoding human PACAP. Another marker, STS known as ADCYAP1 is located in the 3 'untranslated region of the human PACAP gene. This marker has been used to precisely identify the location of the gene in the physical map of chromosome 18. Expression studies indicate that PACAP is synthesized in the glandular cells of the retina that terminate in VIP neurons in the suprachiasmatic nucleus (SCN) , which is the location of the circadian clock. In addition, PACAP expression exhibits a circadian rhythm in the rat SCN: low levels occur during periods of light, high levels occur during periods of darkness, and stable levels occur under conditions of continuous darkness (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 the late subjective day and that melatonin inhibits this phosphorylation induced by PACAP (Kopp, et al., 1997, Neurosci Lett. 227, 145-148). These findings suggest that the levels of expression and / or activity of PACAP in the SCN can be changed by lighting conditions and that neurons containing PACAP may play a role in the entrainment [sic] of the circadian rhythm. 3. SUMMARY OF THE INVENTION An objective of the present invention is to identify genetic bases for neuropsychiatric disorders, provide methods for the treatment and diagnosis of neuropsychiatric disorders and provide methods for identifying compounds for use as part of the treatment methods and / or diagnosis. The invention furthermore relates to methods for the treatment of neuropsychiatric disorders mediated by PACAP, wherein these methods consist of administering a compound that modulates the expression of a mammalian PACAP gene, and / or the synthesis or activity of a gene product. PACAP of a mammal to reduce the symptoms of the disease. The invention furthermore relates to methods for the treatment of mammalian PACAP-mediated neuropsychiatric disorders resulting from PACAP gene mutations, wherein these methods consist of supplying the mammal with a nucleic acid molecule that codes for an undamaged PACAP gene product. , so that the non-damaged PACAP gene product is expressed and the symptoms of the disease are reduced. The invention also relates to methods for the treatment of neuropsychiatric disorders mediated by mammalian PACAP resulting from PACAP gene mutations., wherein the methods consist of supplying the mammal with a cell containing a nucleic acid molecule encoding a non-impaired PACAP gene product so that the cell expresses the non-impaired PACAP gene product and the symptoms of the condition are reduced. In addition, the present invention is directed to methods that use the PACAP gene and / or gene product sequences for diagnostic evaluation, gene testing and prognosis of a PACAP-mediated neuropsychiatric condition. For example, the invention relates to methods for the diagnosis of PACAP-mediated neuropsychiatric disorders, wherein these methods consist in measuring the expression of the PACAP gene in a patient sample, or detecting a PACAP mutation in the mammalian genome. which is suspected of having such an anomaly. The invention still further relates to methods for identifying compounds capable of modulating the expression of the mammalian PACAP gene and / or the synthesis or activity of mammalian PACAP gene products, wherein these methods consist in contacting a compound with a cell expressing a PACAP gene, measuring the level of expression of the PACAP gene, the expression of the gene product or activity of the gene product, and comparing this level with the level of PACAP gene expression, expression of the gene product or activity of the gene product produced by the cell in the absence of the compound, so that, if the level obtained in the presence of the compound differs from the obtained one in its absence, a compound capable of modulating the expression of the mammalian PACAP gene and / or the synthesis or activity of mammalian PACAP gene products. The invention is based, in part, on the genetic and physical determination of the PACAP gene to a specific portion of 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 the section. Thus, the invention relates to the use of the PACAP gene and / or gene products as markers for the determination of the fine structure of this region of human chromosome 18. The PACAP-mediated neuropsychiatric disorders herein include, but are not limited to, bipolar affective disorder, e.g., severe bipolar disorder (mood) (BP-I), bipolar affective disorder (mood) with hypomania. and major depression (BP-II). The term "PACAP-mediated neuropsychiatric disorder" when used in the present invention refers to a condition that involves an aberrant level of expression of the PACAP gene, the synthesis of the gene product and / or the activity of the gene product in relation to the levels found in normal, unaffected, undamaged individuals, levels found in clinically normal individuals and / or levels found in a population whose level represents a base, the average level of PACAP. 3. 1 DEFINITIONS When used herein, the following terms must have the abbreviations indicated.

BAC, bacterial artificial chromosome (s) BAD, bipolar affective disorder (s) BP, bipolar mood disorder BP-I, affective disorder (mood) bipolar, severe BP-II, affective disorder (mood) ) bipolar with hypomania and bp major depression, base pair (s) EST, lod expressed sequence tag, odd-negative MDD logarithm, major unipolar depressive disorder ROS, reactive oxygen species RT-PCR, with SSCP reverse transcriptase, conformational polymorphism of a single strand SAD-M, schizoaffective disorder type maniac STS, short label sequence YAC, artificial yeast chromosome 4. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Sequence of the human PACAP gene. The amino acid sequences are indicated. Figure 2A-2U. Genomic sequence of the human PACAP gene. The exons are in bold and underlined the 5 'UTR.

. DETAILED DESCRIPTION OF THE INVENTION This invention is based on the genetic and physical determination of the PACAP gene for a narrow, specific portion of chromosome 18, also described in the example presented later in Section 6. The invention described in the following subsections comprises detection methods (e.g., assays) for the identification of compounds that can be used to treat individuals suffering from a PACAP-mediated neuropsychiatric disorder. The invention also comprises the agonists and antagonists of the PACAP gene product, including small molecules, large molecules and antibodies, as well as nucleotide sequences that can be used to inhibit the expression of the PACAP gene (e.g., antisense molecules and ribozymes), and gene or regulatory sequence replacement constructs designed to improve the expression of the PACAP gene (for example, the expression constructs that place the PACAP gene under the control of a strong promoter system). In particular, cellular and non-cellular assays that can be used to identify compounds that interact with the PACAP gene product are described, for example, to modulate PACAP activity and / or binding to the PACAP gene product. These cell-based assays of the invention utilize cells, cell 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 (eg, transcription or in vitro translation assays) to explore compounds or compositions that modulate PACAP gene expression. For this purpose, constructs containing a gatekeeper sequence linked to a regulatory element of the PACAP gene can be used in manipulated cells, or in extracts of cell lysates, to detect compounds that modulate the expression of the reporter gene product at the level of the transcription, for example, these assays can be used to identify compounds that modulate the expression or activity of the transcription factors involved in the expression of the PACAP gene, or to test the activity of the triple helical polynucleotides. Otherwise, it is possible to use engineered cells or translation extracts to detect compounds (including antisense and ribozyme constructs) that modulate the translation of PACAP mRNA transcripts and, therefore, affect the expression of the PACAP gene product. The invention also encompasses the PACAP gene products, the polypeptides (including the soluble PACAP polypeptides or peptides) and the PACAP fusion proteins for use in non-cell-based detection assays, for use in the generation of antibodies, for diagnosis and therapeutic. These peptides or polypeptides can be fused to a heterologous protein, for example, reporter, an Ig Fc region, etc., to produce a fusion protein. These peptides, polypeptides and fusion proteins can be used in non-cell-based assays to detect compounds that interact with, for example, modulate the activities of the PACAP gene product and / or bind the PACAP product. The PACAP gene products can be used to treat disorders mediated by PACAP. These PACAP gene products include, but are not limited to, soluble derivatives such as peptides or polypeptides corresponding to one or more • 10 domains of the PACAP gene product. Otherwise, antibodies to the PACAP protein or anti-idiotypic antibodies that mimic the PACAP gene product (including Fab fragments) antagonists or agonists can be used to treat neuropsychiatric disorders that involve PACAP. In still another approach, the nucleotide constructs encoding these PACAP gene products can be used to genetically engineer host cells to express these PACAP gene products in vivo; these cells genetically The manipulated ones can function as "bioreactors" in the body providing a continuous supply of the PACAP gene product, PACAP peptides, soluble PACAP polypeptides. In addition, this invention presents the methods for the diagnostic and prognostic evaluation of the disorders mediated by PACAP. For example, the nucleic acid molecules encoding PACAP can be used as diagnostic hybridization probes or as primers for PCR analysis for diagnosis for the identification of PACAP gene mutations, allelic variations and regulatory defects in the PACAP gene. "Gene therapy" approaches for the modulation of the expression and / or activity of the PACAP gene are within the scope of the invention. For example, the nucleotide constructs encoding the functional PACAP gene, the mutant PACAP gene, as well as the antisense and ribozyme molecules can be used to modulate the expression of the PACAP gene. The invention also encompasses pharmaceutical formulations and methods for treating abnormalities involving the PACAP gene. The present invention establishes methods for selecting an effective medicament for administering to an individual with a disorder mediated by PACAP. These methods are based on the detection of genetic polymorphisms in the PACAP gene or variations in PACAP gene expression due to altered methylation, differential centrifugation or post transductional modification of the PACAP gene product that may affect the safety and efficacy of an agent. therapeutic. . 1. THE PACAP GENE With respect to the PACAP gene sequences as described in Figure 1, such sequences can, for example, be easily obtained using normal sequencing technologies and the bacterial artificial chromosome (BAC) in connection with BAC 54 ( identification reference EpHS996, ATCC accession number 98363). For example, shared libraries can be prepared from BAC54. Fragments of a suitable size, for example, in the size range of approximately 1 kb, are cloned into a standard plasmid, and the sequence is determined. Other PACAP sequences can then be easily identified by aligning the BAC sequences with the PACAP sequences depicted in Figure 1. Otherwise, it is possible to identify BAC subclones containing the additional PACAP sequences by identifying those clones that hybridize to probes from of the PACAP sequences depicted in Figure 1. With respect to the cloning of the allelic variants of the human PACAP gene and the homologs of other species (e.g., mouse), the isolated PACAP gene sequences described herein may be labeled and used to detect a cDNA library constructed from mRNA obtained from suitable cells or tissues (e.g., brain tissues) from the organism (e.g., mouse) of interest. The hybridization conditions used should be of a lower severity when the cDNA library comes from an organism 5 different from the type of organism from which the labeled sequence was obtained. Otherwise, it is possible to use the labeled fragment to detect a genomic library from the organism of interest, again, using the conditions adequate restrictives. Conditions with little restriction are well known to those skilled in the art, and will vary in a predictable manner depending on the specific organisms from which the library and the labeled sequences come from. For a guide regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A 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, NY 0 In addition, an allelic variant of the PACAP gene can be isolated from, for example, human nucleic acid, by performing PCR using two combinations of degenerate oligonucleotide primers designated based on the amino acid sequence within the PACAP 5 gene product described herein. The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from, for example, cell lines or human or non-human tissue of which they are known or suspected to express an allele of the PACAP gene (such as lines). of human leukemia cells for example K562 B1A, H630 and H630-1, for example, Dolnik, et al., 1996, Cancer Research 56, 1207-3260; Dolnik, 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 having a neuropsychiatric disorder mediated by PACAP. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a PACAP gene nucleic acid sequence. The PCR fragment can then be used to isolate a full-length cDNA clone by several methods, for example, the amplified fragment can be labeled and used to detect a bacteriophage cDNA library. Otherwise, the labeled fragment can be used to isolate genomic clones by detecting a genomic library. PCR technology can also be used to isolate full-length cDNA sequences. For example, it is possible to isolate the RNA, following normal procedures, from a suitable cell or tissue source (ie, one known or suspected to express the PACAP gene, such as, for example, samples). of brain tissue obtained from biopsy or post mortem). It is possible to perform a reverse transcription reaction on the RNA using an oligonucleotide primer specific for the 5 'end of the amplified fragment for the indication of the synthesis of the first strand. The resulting RNA / DNA hybrid can then be finished "tailed" with guanines using the reaction of the normal terminal transferase, the hybrid can be digested with RNAse H, and the synthesis of the second strand can then be initiated with the poly-C initiator. . Thus, the DNA sequences upstream of the amplified fragment can be easily isolated. For a review of the cloning strategies that may be used, see, for example, Sambrook, et al., 1989, supra. As already mentioned, the PACAP gene sequences can be used to isolate mutant alleles of the PACAP gene, preferably from a human individual. Such mutant alleles can be isolated from individuals known or proposed to have a genotype that contributes to the symptoms of a PACAP-mediated disorder. The mutant alleles and the products of the mutant allele can then be used in the therapeutic and diagnostic systems described below. In addition, these PACAP gene sequences can be used to detect regulatory defects (e.g., promoters) of PACAP that may be associated with a PACAP-mediated disorder. A cDNA from a mutant allelic variant of the PACAP gene can be isolated, for example, using PCR, a technique that is well known to those skilled in the art. In this case, the first strand of cDNA can be synthesized by hybridizing an oligo-dT oligonucleotide to the isolated mRNA of tissue known or suspected to be putatively expressed in an individual carrying the mutant PACAP allele. And extending the new strand with reverse transcriptase. The second strand of cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5 'end of the normal gene. Using these two initiators, the product is then amplified by PCR, cloned into convenient vector and subjected to DNA sequence analysis by methods well known to those skilled in the art. By comparing the DNA sequence of the PACAP mutant allele with that of a normal PACAP allele, it is possible to determine the mutation (s) responsible for the loss or alteration of the function of the mutant PACAP gene product.

Otherwise, it is possible to construct a genomic library using DNA obtained from an individual that is suspected or known to carry a PACAP mutant allele, or a cDNA library can be constructed using RNA from a tissue known or known to be Suspicion expresses a mutant allele of PACAP. Then, a non-damaged PACAP gene or any convenient fragment thereof can be labeled and used as a probe to identify the corresponding PACAP mutant allele 5 in such libraries. Clones containing the mutant PACAP gene sequences can then be purified and subjected to sequence analysis according to methods well known to those skilled in the art. In addition, it is possible to construct an expression library using the synthesized cDNA of, for example, RNA isolated from a tissue of which it is known or suspected to express a mutant PACAP allele in an individual of which it is suspected or known to carry such a mutant allele. In this way, gene products made by the putatively mutant tissue can be expressed and detected using normal antibody detection techniques together with antibodies raised against the normal PACAP gene product, as described below in section 0 5.3. For detection techniques see, for example, Harlow and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Soring Harbor Press, Cold Spring Harbor, New York). In cases where a PACAP mutation gives rise to a gene product expressed with altered function (eg, as a result of a codon with a wrong sense or a mutation by a change in reading frame), a polyclonal series of anti-viral antibodies PACAP gene product is likely to cross-react with the mutant PACAP gene product. Clones from the library detected by their reaction with these labeled antibodies can be purified and subjected to sequence analysis according to methods well known to those skilled in the art. In addition, PACAP mutations can be detected using PCR amplification techniques. The primers can be designed routinely to amplify overlapping regions of the complete PACAP sequence including the promoter region. In one embodiment, the primers are designed to cover the exon-intron boundaries so that the coding regions can be detected for mutations (see Figures 2A-2U). In the examples, several primers are provided to analyze different PACAP exons. Genomic DNA isolated from lymphocytes of normal and affected individuals is used as a template for PCR. The PCR products of normal and affected individuals are compared, by detection techniques of single-strand conformational polymorphism mutation (SSCP) and / or by sequencing. The mutations responsible for the loss or alteration of the function of the mutant PACAP gene product can then be determined. . 2 PRODUCTS OF PACAP GENE PROTEIN PACAP gene products, or peptide fragments thereof, can be prepared for various uses. For example, these gene 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 disorders mediated by PACAP. The amino acid sequence depicted in Figure 1 represents a product of the PACAP gene. The PACAP gene product, sometimes referred to herein as a "PACAP protein", includes those gene products encoded by the PACAP gene sequences depicted in Figure 1, as well as other human allelic variants of PACAP that can be identified by the methods described in the present. In addition, the PACAP gene products can include proteins that represent functionally equivalent gene products. Such an equivalent PACAP gene product may contain deletions, including internal deletions, additions, including additions that produce 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 giving origin to a "silent" change, in that the change produces a functionally equivalent PACAP gene product. The amino acid substitutions can be made based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the unfriendly nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; the neutral polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; the amino acids with positive charge (basic) include arginine, lysine and histidine; and the negatively charged amino acids (acids) include aspartic acid and glutamic acid. Otherwise, when an alteration of function is desired, the deletion or non-conservative alterations can be manipulated to produce altered, including reduced PACAP gene products. These alterations may, for example, alter one or more of the biological functions of the PACAP gene product. In addition, these alterations can be selected to generate PACAP gene products that are more convenient for the expression, on a higher scale, etc., in the chosen host cells. For example, the cysteine residues can be deleted or substituted with another amino acid residue to eliminate disulfide bridges. PACAP gene products, peptide fragments thereof and fusion proteins thereof can be produced by recombinant DNA technology using well known techniques. Thus, methods for preparing the PACAP gene products, polypeptides, peptides, fusion proteins and fusion polypeptides of the invention by expressing the nucleic acid containing the 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 the coding sequences of the PACAP gene product and the appropriate transcription and translation control signals. These methods include, for example, recombinant DNA techniques in vi tro, 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. Otherwise, the RNA capable of encoding the PACAP gene product sequences can be synthesized chemically using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, ed., IRL Press, Oxford. It is possible to use a variety of host-expression vector systems to express the coding sequences of the PACAP gene product of the invention. These host-expression systems [sic] represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells that can, when transformed or transfected with the coding sequences of the appropriate nucleotides, present the product of the invention. PACAP gene of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (eg E. coli, B. subtilis) transformed with bacteriophage recombinant DNA expression vectors, plasmid DNA or cosmid DNA containing the coding sequences of the PACAP gene product; yeast (eg, Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the coding sequences of the PACAP gene product; insect cell systems infected with recombinant virus expression vectors (for example, baculovirus) containing the coding sequences of the PACAP gene product; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, the Ti plasmid) containing the coding sequences of the PACAP gene product; or mammalian cell systems (eg, COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing the suitable promoters of the genome of mammalian cells (e.g. metallothionein promoter) or of mammalian virus (e.g. , the adenovirus late promoter, the 7.5K promoter of the vaccine virus). In bacterial systems, a number of expression vectors can be conveniently selected depending on the proposed use for the PACAP gene product being expressed. For example, when a large amount of this protein is to be produced, for the generation of pharmaceutical compositions of the PACAP gene product or for raising the antibodies to the PACAP gene product, for example, vectors that direct the expression of high levels of fusion protein products that are easily purified. These vectors include, but are not limited to, the pUR278 expression vector of E. coli (Ruther et al., 1983, EMBO J. 2, 1791), in which the coding sequence of the PACAP gene product can be individually linked. in the vector in the reading 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 similar. PGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, these fusion proteins are soluble and can be easily purified from cells used by absorption in glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin cleavage or dissociation sites or factor Xa protease so that the product of the chosen cloned gene can be released from the GST portion. In an insect system, Autographa californica, the nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence of the PACAP gene product can be cloned individually into the non-essential regions (eg the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg the polyhedrin promoter). Successful insertion of the coding sequence of the PACAP gene product will result in the inactivation of the polyhedrin gene and the production of non-occluded recombinant virus (i.e., a virus lacking the proteinaceous coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (See, for example, Smith et al., 1983, J. Virol. 46, 584; Smith, U.S. Patent No. 4,215,051). In mammalian host cells, it is possible to use different virus-based expression systems. In cases where adenovirus is used as the expression vector, the coding sequence of the PACAP gene product of interest may be linked to a complex for the control of transcription / translation of adenovirus, eg, the late promoter and the tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro recombination or in vivo. Insertion into a non-essential region of the viral genome (eg, the El or E3 region) will give rise to a recombinant virus that is viable and capable of expressing the gene product in infected hosts. (For example, see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81, 3655-3659). Specific initiation signals may also be necessary for efficient translation of the coding sequences of the inserted PACAP gene product. These signals include the ATG start codon and the adjacent sequences. In cases where a complete PACAP gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, additional translation control signals may not be necessary. However, in cases where only a portion of the coding sequence of the PACAP gene is inserted, exogenous translation control signals, including perhaps the ATG start codon, must be provided. In addition, 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 translation control signals and initiation codons can be from a variety of origins, both natural and synthetic. The efficacy of expression can be improved by the inclusion of suitable transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol, 153, 516-544). In addition, it is possible to choose a host cell strain that modulates the expression of the inserted sequences, or that modifies and processes the gene product in the specific way desired. These modifications (eg, glycosylation) and processing (eg, dissociation) of the protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. The appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. For this purpose, it is possible to use eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcription, glycosylation and phosphorylation of the gene product. These mammalian host cells include, but are not limited to: CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38. For the high-yield, long-term production of the recombinant proteins, stable expression is preferred. For example, cell lines that stably express the PACAP gene product can be manipulated. Instead of using expression vectors that contain viral replication origins, the host cells can be transformed with DNA controlled by suitable expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc. .), and a selectable marker. After the introduction of the foreign DNA, it is possible to allow the manipulated cells to grow for 1-2 days in an enriched medium, and then they are exchanged to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be formed and extended into cell lines. This method can be conveniently used to manipulate cell lines that express the PACAP gene product. These manipulated cell lines can be particularly useful in the detection and evaluation of compounds that affect the endogenous activity of the PACAP gene product. It is possible to use different selection systems, including, but not limited to: thymidine kinase genes (Wigler et al., 1977, Cell 11, 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc.

Natl. Acad. Sci. USA 48, 2026), and of adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22, 817) of the herpes simplex virus can be employed in tk ~, hgprt "or aprt" cells, respectively. It is also possible to use resistance to antimetabolites 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. Natl. Acad. Sci. USA 78, 1527); gpt that 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 hygromycin (Santerre et al., 1984, Gene 30, 147). Otherwise, any fusion protein can be easily purified using a specific antibody for the fusion protein that is being expressed. For example, a system described by Janknecht, et al., Allows rapid purification of non-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 recombinant vaccine plasmid so that the open reading frame of the gene is translationally fused to an amino terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2 + - nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with buffer solutions containing imidazole. The PACAP gene products can also be expressed in transgenic animals. Animals of any species, including but not limited to: mice, rats, rabbits, guinea pigs, pigs, micro-nerds, goats, sheep and non-human primates, eg, baboons, monkeys and chimpanzees can be used to generate PACAP transgenic animals . The term "transgenic", when used herein, refers to animals that express the PACAP gene sequences of a different species (eg, mice that express the sequences of the human PACAP gene), as well as animals that have been genetically manipulated to overexpress endogenous PACAP sequences, (ie, from the same species) or animals that have been genetically engineered to no longer express endogenous PACAP gene sequences (ie, "knock-out" animals) and their progeny. Any of the known techniques can be used to introduce a transgene of the PACAP gene in animals to produce the founder lines of the transgenic animals. These techniques include, but are not limited to pronuclear microinjection (Hoppe and Wagner, 1989, U.S. Patent No. 4,873,191); gene transfer mediated by retroviruses in germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82, 6148-6152); choice of the gene in primordial embryonic cells (Thompson et al., 1989, Cell 56, 313-321); embryo electroporation (Lo, 1983, Mol Cell Cell Biol. 3, 1803-1814); and wait-mediated gene transfer (Lavitrano et al., 1989, Cell 57, 717-723) (For a review of these techniques see Gordon, 1989, Transgenic Animáis, Intl. Rev. Cytol. 115, 171-229). Any of the known techniques can be used to produce clones of transgenic animals containing a PACAP transgene, for example, nuclear transfer in enucleated oocytes from nuclei from embryonic, fetal or cultured adult cells induced for rest or inactivity (Campbell et al. , 1996, Nature 380, 64-66; Wilmut, et al., Nature 385, 810-813). The present invention provides the transgenic animals that carry a PACAP transgene in all their cells, as well as the animals that carry the transgene in some cells., but not in all its cells, that is, mosaic animals. The transgene can be integrated in a single transgene or in concatamers, for example, in head-to-head cascades or head-to-tail cascades. The transgene can also be selectively introduced into and activated in a specific cell type following, for example, the teaching of Lasko et al. (Lasko et al., 1992, Proc. Natl. Acad. Sci. USA 89, 6232-6236). The regulatory sequences necessary for such specific activation of the cell type will depend on the particular cell type of interest, and will be apparent to those skilled in the art. When it is desired that the PACAP transgene be integrated into the chromosomal site of the endogenous PACAP gene, the choice or orientation of the gene is preferred. In summary, when such a technique is to be used, vectors containing some nucleotide sequences homologous to the endogenous PACAP gene are designed for the purpose of integration, through homologous recombination with chromosomal sequences, and disruption of function of the nucleotide sequence of the endogenous PACAP gene. The transgene can also be selectively introduced into a specific cell type, thereby inactivating the endogenous PACAP gene in only this cell type, following, for example, the teaching of Gu et al. (Gu et al., 1994, Science 265, 103-106). The regulatory sequences necessary for this specific inactivation of the cell type will depend on the type of cell of interest and will be apparent to those skilled in the art. Once the transgenic animals have been generated, the expression of the recombinant PACAP gene can be assayed using standard techniques. The initial detection can be carried out by means of Southern blot analysis or the PCR techniques to analyze the animal tissues to test if the integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals can also be assessed using techniques that include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, hybridization analysis in itself and RT -PCR (PCR with reverse transcriptase). Tissue samples expressing the PACAP gene can also be evaluated immunocytochemically using antibodies specific for the PACAP transgene product. . 3. ANTIBODIES FOR PACAP GEN PRODUCTS Production methods capable of specifically recognizing one or more epitopes of the PACAP gene product or epitopes of conserved variants or peptide fragments of the PACAP gene products are described herein. In addition, antibodies that specifically recognize the mutant forms of PACAP are included in the invention. These 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 expression library, anti-idiotypic (anti-ld) antibodies and epitope binding fragments of any of the foregoing. These antibodies can be used, for example, in the detection of a PACAP gene product in a biological sample and can, therefore, be used as part of a diagnostic or prognostic technique with which patients can be tested for abnormal levels of the PACAP gene products and / or for the presence of the abnormal forms of these gene products. These antibodies can also be used in conjunction with, for example, compound detection schemes, as described later in Section 5.8, for the evaluation of the effect of the test compounds on the levels and / or activity of the PACAP gene product. In addition, these antibodies can be used in conjunction with the gene therapy techniques described later in Section 5.9.2 to, for example, evaluate normal and / or manipulated cells expressing PACAP prior to their introduction into the patient. The anti-PACAP gene product antibodies can also be used in methods to inhibit the abnormal activity of the PACAP gene product. Thus, these antibodies can therefore be used as part of the treatment methods for a PACAP-mediated disorder. For the production of antibodies against a PACAP gene product, different host animals can be immunized by injection with a PACAP gene product, or a portion thereof. These host animals may include, but are not limited to: rabbits, mice and rats, to mention just a few. It is possible to use some adjuvants to increase the immune response, depending on the host species, which include but are not limited to Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, mollusc hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynejbacteriu_n parvum). Polyclonal antibodies are heterogeneous populations of antibody molecules from the sera of animals immunized with an antigen, such as a PACAP gene product, or an antigenic functional derivative thereof. For the production of the polyclonal antibodies, the host animals as described above can be immunized by injection with the PACAP gene product supplemented with adjuvants as also described above. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be obtained by any of the techniques that provide the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the Kohler and Milstein hybridoma technique, (1975, Nature 256-, 495-497; and U.S. Patent No. 4,376,110), the hybridoma technique of human B cells (Kosbor et al. 1983, Immunology Today 4, 72; Colé et al., 1983, Proc. Natl. Acad. Sci. USA 80. 2026-2030), and the EBV-hybridoma technique (Colé et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). These antibodies may be of any class of immunoglobulin including IgG, IgM, IgE, IgA, IgD and any of the subclasses thereof. The hybridoma that produces the mAb of this invention can be cultured in vi tro or in vivo. The production of high titers of mAbs in vivo makes this currently preferred production method. In addition, it is possible to use the techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. 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 suitable antigen specificity together with genes from a human antibody molecule with suitable biological activity. A chimeric antibody is a molecule in which different portions come from different animal species, such as those that have a variable region from a murine mAb and a human immunoglobulin constant region. (See, for example, Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816,397, which are incorporated herein by reference in their entireties.) In addition, they have been developed. techniques for the production of humanized antibodies. (See, for example, Queen, U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety). A variable region of light or heavy chain of immunoglobulin consists of a "framework" region interrupted by three hypervariable regions, known as complementarily determining regions (CDR). The extent of the framework region and the CDRs have been precisely defined (see "Sequences of Proteins of Immunological Interest", Kabat, E. et al., US Department of Health and Human Services (1983).) In summary, humanized antibodies are antibody molecules from non-human species having one or more CDRs of the non-human species and a lattice region of a human immunoglobulin molecule.Otherwise, the techniques described for the production of single-chain antibodies (U.S. Patent No. 4,946,778; Bird, 1988, Science 242, 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5879-5883; and Ward et al., 1989, Nature 334, 544-546) can are adapted to produce single-chain antibodies against the PACAP gene products Single-chain antibodies are formed by ligating the heavy and light chain fragments of the Fv region through an amino acid bridge, giving rise to a single-chain polypeptide Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, these fragments include, but are not limited to: F (ab ') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and Fab fragments that can be generated by reducing the disulphide bridges of the fragments F (ab ') 2. Otherwise, it is possible to construct Fab expression libraries (Huse et al., 1989, Science, 246, 1275-1281) to allow rapid and simple identification of the monoclonal Fab fragments with the desired specificity. . 4. USES OF THE PACAP GENE SEQUENCES, GENE PRODUCTS, AND ANTIBODIES Here different applications of the PACAP gene sequences, the PACAP gene products, including the peptide fragments and their fusion proteins are described, and of the antibodies directed against the PACAP gene products and the peptide fragments thereof. These applications include, for example, prognostic and diagnostic evaluation of a PACAP-mediated disorder, and identification of individuals predisposed to these conditions, as described later in Section 5.5. In addition, these applications include methods for the treatment of a PACAP-mediated abnormality 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 product. of the PACAP gene, as described later in Section 5.8.

These compounds may include, for example, other cellular products that are involved in the regulation of mood and PACAP-mediated disorders. These compounds can be used, for example, to alleviate disorders mediated by PACAP. . 5. DIAGNOSIS OF THE ANOMALIES OF A PACAP MEDIATED DISORDER It is possible to use different methods for the diagnosis and evaluation of the prognosis of the disorders mediated by PACAP and for the identification of individuals who are predisposed to these disorders. These methods may, for example, utilize reagents such as the nucleotide sequences of the PACAP gene described in Sections 5.1, and antibodies directed against the PACAP gene products, including peptide fragments thereof, as described above, in the Section 5.3. Specifically, these reagents can be used, for example, for: (1) detection of the presence of PACAP gene mutations, or detection of the envelope or sub-expression of the PACAP protein; (2) the detection of the envelope or sub-abundance of the PACAP gene product; and (3) the detection of an aberrant level of activity of the PACAP gene product. The nucleotide sequences of the PACAP gene can, for example, be used to diagnose a PACAP-mediated abnormality using, for example, the techniques for detecting the PACAP mutation described above in Section 5.1. Mutations in a number of different genetic loci can give rise to phenotypes related to neuropsychiatric disorders. In theory, the treatment of patients suffering from such neuropsychiatric disorder would be designed to choose the particular genetic loci containing the mutation that mediates the disorder. The genetic polymorphisms have been linked to the differences in the effectiveness of the drug. Thus, the identification of alterations in the PACAP gene or protein can be used to optimize therapeutic treatments with medications. In one embodiment of the present invention, polymorphisms in the PACAP gene sequence, or variations in PACAP gene expression due to altered methylation, differential splicing, or post translational modification of the PACAP gene product, can be used to identify an individual with a disease or condition that results from a condition mediated by PACAP and thus define the most effective and safe medication treatment. Assays such as those described herein can be used to identify these polymorphisms or variations in the activity of PACAP gene expression. Once a polymorphism in the PACAP gene, 5 or a variation in the expression of the PACAP gene have been identified in an individual, it is possible to prescribe an individual with the appropriate medication. The methods described herein can be performed, for example, using diagnostic kits • 10 prepackages containing at least one nucleic acid of the specific PACAP gene or a reagent of the antiproduct antibody of the PACAP gene described here, which can be conveniently used, for example in a clinical setting, to diagnose patients who have abnormalities of a PACAP-mediated condition. For the detection of PACAP gene mutations, it is possible to use any nucleated cell as the starting source for the genomic nucleic acid. For the detection of the PACAP gene expression or the PACAP gene products, it is possible to use any type of cell or tissue in which the PACAP gene is expressed. Nucleic acid-based detection techniques are described later in Section 5.6. Peptide detection techniques are described later in Section 5.7. . 6. DETECTION OF PACAP NUCLEIC ACID MOLECULES It is possible to employ various methods to detect the presence of specific mutations of the PACAP gene and to detect and / or assay the levels of PACAP nucleic acid sequences. Mutations within the PACAP gene can be detected using various techniques. The nucleic acid from any nucleated cell can be used as the starting point for these assay techniques, and can be isolated according to standard procedures for the preparation of nucleic acids that are well known to those skilled in the art. The nucleic acid sequences of PACAP can be used in hybridization or amplification assays of biological samples to detect abnormalities involving the structure of PACAP, including point mutations, insertions, deletions, inversions, translocations and chromosomal rearrangements. These assays may include, but are not limited to, Southern analysis, single-strand conformational polymorphism analysis (SSCP) and PCR analysis. Diagnostic methods for the detection of specific mutations of the PACAP gene may include, for example, contacting and incubating nucleic acids that include recombinant DNA molecules, cloned genes or degenerate variants thereof, obtained from a sample, by example, from a patient sample or other suitable cell 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 the favorable conditions for the specific tempering of these reagents for their complementary sequences within the PACAP gene. The diagnostic methods of the present invention further comprises contacting and incubating the nucleic acids for the detection of individual mutations or polymorphisms of the PACAP gene nucleotides. Preferably, the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all unhardened nucleic acids are removed from the nucleic acid: hybrid of the PACAP molecule. The presence of nucleic acids that have hybridized, if any of these molecules exist, is then detected, by using this detection scheme, the nucleic acid of the cell type or tissue of interest can be immobilized, for example, on a solid support such as it can be a membrane, or a plastic surface such as a microtiter plate or polystyrene beads. In this case, after incubation, the uncoupled nucleic acid reagents, labeled of the type described in Section 5.1, are easily separated. Detection of labeled, tempered, remanent PACAP nucleic acid reagents is achieved using standard techniques well known to those skilled in the art. The PACAP gene sequences for which the nucleic acid reagents have been annealed can be compared to the expected annealing pattern of a normal PACAP gene sequence to determine if a PACAP gene mutation is present. In a preferred embodiment, it is possible to detect PACAP mutations or polymorphisms using a microassay of PACAP nucleic acid sequences immobilized to a substrate or "piece of gene" (see, eg, Cronin, et al., 1996, Human Mutation 7: 244-255). Alternative diagnostic methods for the detection of specific nucleic acid molecules of the PACAP gene, in patient samples or other suitable cellular sources, may include their application, for example, by PCR (the experimental modality established in Mullis, 1987, Patent No. 4,683,202), followed by the detection of the amplified molecules using techniques well known to the experts. The resulting amplified sequences can be compared to those that would be expected if the nucleic acid being amplified had only the normal copies of the PACAP gene to determine if there is a mutation of the PACAP gene. further, Well-known genotype determination techniques can be performed to identify individuals carrying mutations of the PACAP gene. These techniques include, for example, the use of restriction fragment length polymorphisms (RFLP), which includes the sequence variations at one of the recognition sites for the specific restriction enzyme used. In addition, improved methods for analyzing DNA polymorphisms, which can be used for the identification of specific PACAP gene mutations, have been described which are performed in the presence of variable numbers of short cascaded DNA sequences between the sites of the restriction enzyme. For example, Weber (US Patent No. 5,075,217) describes a marker DNA based on polymorphisms of length in the blocks of short cascade repeats (dC-dA) n- (dG-dT) n. The average separation of the blocks (dC-dA) n- (dG-dT) n is estimated 30,000-60,000 bp. Markers that are so closely separated present a high frequency co-inheritance, and are extremely useful in the identification of genetic mutations, such as, for example, mutations within the PACAP gene, and the diagnosis of diseases and abnormalities related to the mutations of PACAP. Likewise, Caskey et al. (U.S. Patent No. 5,364,759) discloses an assay for the determination of the DNA profile to detect repeat sequences of short tri and tetranucleotides. The process includes extracting the DNA of interest, such as the PACAP gene, amplifying the extracted DNA and labeling the repeated sequences to form a genotypic map of the individual's DNA. The level of expression of the PACAP gene can be tested. For example, RNA of a cell type or tissue known or suspected to express the PACAP gene, such as brain, can be isolated and tested using hybridization or PCR techniques such as those already described. The isolated cells may be from the culture of cells or from a patient. The analysis taken from the culture may be a necessary step in the valuation of the cells to be used as part of the cell-based gene therapy technique or, otherwise, to test the effect of the compounds on the expression of the PACAP gene. These analyzes can reveal quantitative and qualitative aspects of the PACAP gene expression pattern, including the activation or inactivation of PACAP gene expression. In one embodiment, of this detection scheme, a cDNA molecule is synthesized from an RNA molecule of particular interest (for example, by reverse transcription of the RNA molecule in the cDNA). A sequence within the cDNA is then used as the template for the amplification reaction of the nucleic acid, 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 nucleic acid reagents of the PACAP gene described in Section 5.1. The preferred lengths of these nucleic acid reagents are at least 9-30 nucleotides. For the detection of the amplified product, nucleic acid amplification can be performed using radiolabeled or non-radioactive labeled nucleotides. Otherwise, it is possible to prepare enough amplified product so that the product can be visualized by normal staining with ethidium bromide or using any other convenient nucleic acid staining method. In addition, it is possible to perform these PACAP gene expression assays "in situ", that is, directly on tissue sections (fixed and / or frozen) of patient tissue obtained from biopsies or resections, so that purification is not necessary of the nucleic acid. Nucleic acid reagents as described in Section 5.1 can be used as probes and / or primers for such procedures in situ (see, for example, Nuovo, G. J. 1992, "PCR In Situ Hybridization: Protocols and Applications", Raven Press, NY). Otherwise, if it is possible to obtain a sufficient amount of the appropriate cells, it is possible to perform normal Northern analysis to determine the expression level of PACAP gene mRNA. . 7. DETECTION OF PACAP GENE PRODUCTS Antibodies directed against undamaged PACAP gene products or mutants or conserved variants or peptide fragments thereof, which were described in the above, in Section 5.3, can also be used as diagnostics and prognosis. for a disorder mediated by PACAP. These methods can be used to detect anomalies in the level of synthesis or expression of the PACAP gene product, or anomalies in the structure, temporal expression and / or physical location of the PACAP gene product. The antibodies and immunoassay methods described herein have, for example, important in vitro applications in the assessment of the efficacy of treatments for disorders mediated by PACAP. Antibodies, or fragments of antibodies such as those described below, can be used to detect potentially therapeutic compounds in vi tro to determine their effects on the expression of the PACAP gene and the production of the PACAP gene product. Compounds that have beneficial effects on a disorder mediated by PACAP. Immunoassays in vi tro can also be used, for example, to assess the efficacy of cell-based gene therapy for a PACAP-mediated disorder. Antibodies directed against the PACAP gene products can be used in vi tro to determine, for example, the level of expression of the PACAP gene obtained in genetically engineered cells to produce the PACAP gene product. In the case of intracellular PACAP gene products, this evaluation is preferably carried out using lysates or cell extracts. This analysis will allow a determination of the number of transformed cells necessary to obtain therapeutic efficacy in vivo, as well as the optimization of the gene replacement protocol. The tissue or cell type to be analyzed will generally include those that are known or suspected to express the PACAP gene. The methods of isolation of proteins 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 isolated cells may be from cell culture or from a patient. Analysis of the cells taken from culture may be a necessary step in the evaluation of the cells that are to be used as part of the cell-based gene therapy technique or, otherwise, to test the effect of the compounds in the expression of the PACAP gene. Preferred diagnostic methods for the detection of PACAP gene products, conserved variants or peptide fragments thereof may include, for example, immunoassays wherein PACAP gene products and conserved variants or peptide fragments are detected by their interaction with an antibody anti-product of the specific PACAP gene. For example, antibodies or antibody fragments, such as those described above in Section 5.3, can be used to quantitatively and qualitatively detect the presence of PACAP gene products with conserved variants or peptide fragments thereof. This can be achieved, for example, by immunofluorescence techniques employing a fluorescence-labeled antibody (see below in this section), coupled with light microscopy, flow cytometry or fluorometric detection. These techniques are especially preferred for PACAP gene products that are expressed on the cell surface. Antibodies (or fragments thereof) useful in the present invention can, moreover, be used histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of PACAP gene products, conserved variants or peptide fragments thereof. In situ detection can be carried out by separating a histological specimen from a patient, and applying thereto a labeled antibody that binds to a PACAP polypeptide. The antibody (or fragment) is preferably applied by superimposing the labeled antibody (or fragment) on a biological sample. By using such a method, it is possible to determine not only the presence of the PACAP gene product, the conserved variants or peptide fragments, but also its distribution in the examined tissue. In using the present invention, those skilled in the art will readily realize that any of a wide range of histological methods (such as staining procedures) can be modified to achieve in situ detection of a PACAP gene product. Immunoassays for PACAP gene products, conserved variants or peptide fragments thereof will normally consist of incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells or cell lysate in the presence of a labeled antibody in the form detectable capable of identifying the PACAP gene product, conserved variants or peptide fragments thereof, and detecting the bound antibody by any of the well-known techniques. The biological sample can be contacted and immobilized on a support or carrier in solid phase, such as nitrocellulose, which is capable of immobilizing cells, cellular particles or soluble proteins. The support can then be washed with suitable buffer solutions followed by treatment with the specific antibody of the detectably labeled PACAP gene product. The solid phase support can then be washed with the buffer solution a second time to remove unbound antibody. The amount of the bound mark on the solid support can then be detected by conventional means. By "carrier or carrier in solid phase" any support capable of binding an antigen or an antibody is proposed. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite. The nature of the carrier can be soluble to some degree or insoluble for the purposes of the present invention. The support material can have almost any possible structural configuration provided that the coupled molecule is capable of binding to an antigen or antibody. Thus, the configuration of the support can be spherical, as in a bead, or cylindrical, as in the internal surface of a test tube, or the external surface of a variant. Otherwise, the surface can be flat like a sheet, a test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers to bind antibody or antigen, or may find out the same by the use of routine experimentation. One of the methods in which the specific antibody of the PACAP gene product can be detectably labeled is by binding it to an enzyme, such as for use in an enzyme immunoassay (EIA) (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 Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa, E. et al., (Eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme that binds to the antibody will react with a suitable substrate, preferably a chromogenic substrate, in such form to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric or 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, α-glycerophosphate, dehydrogenase, triose phosphate isomerase, peroxidase radish, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection can be carried out by colorimetric methods that employ a chromogenic substrate for the enzyme. The detection can also be carried out by visual comparison of the degree of enzymatic reaction of a substrate compared to standards prepared in the same way. Detection can also be carried out using any of a number of other immunoassays. For example, by radiolabeling the antibodies and antibody fragments, it is possible to detect the PACAP gene products by 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 1986). The radioactive isotope can be detected by means such as the use of a gamma counter or scintillation counter or by autoradiography. It is also possible to label the antibody with a fluorescent compound. When the fluorescence-labeled antibody is exposed to light of the appropriate wavelength, its pree can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, haloficocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be bound to the antibody using metal chelating groups such as diethylenetriaminpentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The pree of the antibody with chemiluminescent label is then determined by detecting the pree of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ether, imidazole, acridinium salt and oxalate ester.

In the same way, it is possible to use a bioluminescent compound to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in 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 labeling purposes are luciferin, luciferaza and aequorin. . 8. DETECTION TESTS FOR COMPOUNDS THAT MODULATE PACAP GEN ACTIVITY The following assays are designed to identify compounds that bind to a PACAP gene product, compounds that bind to intracellular proteins, or portions of proteins that interact with a product of the PACAP gene, compounds that interfere with the interaction of a PACAP gene product with intracellular proteins and compounds that modulate PACAP gene activity (ie, they modulate the level of PACAP gene expression and / or modulate the level of the activity of the PACAP gene product). Tests may also be used [lacuna] which identify compounds that bind to the regulatory sequences of the PACAP gene (for example promoter sequences, see, for example, Platt, 1994, J. Biol. Chem. 269, 28558-28562) , and that can modulate the level of PACAP gene expression. These compounds may include, but are not limited to, small organic molecules such as those that can cross the blood-brain barrier, enter a suitable cell and affect the expression of the PACAP gene or some other genes involved in the PACAP regulatory pathway, or intracellular proteins. . Methods for the identification of these intracellular proteins are described, later in Section 5.8.2. These intracellular proteins may be involved in the control and / or regulation of mood. Further, among these compounds are those compounds that affect the level of PACAP gene expression and / or the activity of the PACAP gene product and that can be used in therapeutic treatment of PACAP-mediated disorders as described below in Section 5.9. The compounds may include, but are not limited to, peptides such as for example soluble peptides, including but not limited to, Ig-tail fusion peptides, and members of random peptide libraries; (see, for example, Lam et al., 1991, Nature 354, 82-84, Houghten, et al., 1991, Nature 354, 84-86) and combinatorial molecular library from chemical synthesis prepared from amino acids with D configuration and / or L, phosphopeptides, including, but not limited to, members of the library of random or partially degenerate, directed phosphopeptides; see, for example, Songyang, et al., 1993, Cell 72, 767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single-chain antibodies, and FAb, fragments of F (ab ') 2 and FAb expression libraries, and epitope-binding fragments thereof), and small organic or inorganic molecules. These compounds may further comprise the compounds, in particular drugs or members of classes or families of medicaments, known to lessen or exacerbate the symptoms of a disorder. These compounds include antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamine (LSD), p-chlorophenylalanine, p-propyldopacetamide, dithiocarbamate derivatives, for example FLA 63; antianxiety medications for example, diazepam; inhibitors of monoamine oxidase (MAO), for example, iproniazid, clorgyline, phenelzine and isocarboxazid; biogenic amine uptake blockers, for example, tricyclic antidepressants such as desipramine, imipramine and amitriptyline; serotonin reuptake inhibitors, for example fluoxetine; antipsychotic drugs such as phenothiazine derivatives (for example chlorpromazine (torazine) and trifluopromazine)), butyrophenones (for example haloperidol (Haldol)), thioxanthene derivatives (for example chlorprothixene), and dibenzodiazepines (for example, clozapine); benzodiazepines; dopamine agonists and antagonists, for example, L-DOPA, ***e, amphetamine, α-methyl tyrosine, reserpine, tetrabenazine, benztropine, pargiline; agonists and noradrenergic antagonists, for example, clonidine, phenoxybenzamine, phentolamine, tropolone. The compounds identified by the assays as those described herein may be useful, for example, in the elaboration of the biological function of the PACAP gene product and to alleviate the disorders mediated by PACAP. Tests to test the efficacy of the compounds identified, for example, by the techniques described in Sections 5.8.1 - 5.8.3, are described later in Section 5.8.4. . 8.1. IN VITRO DETECTION TESTS FOR THE COMPOUNDS THAT BIND TO THE PACAP GENE PRODUCT In vi tro systems may be designed to identify the compounds capable of binding to the PACAP gene products of the invention. The identified compounds can be useful, for example, in the modulation of the activity of the undamaged PACAP gene products and / or mutants, they can be useful in the elaboration of the biological function of the PACAP gene product, they can be used in detections to identify the compounds that break the normal interactions of the PACAP gene product, or can themselves disrupt these interactions. The principle of the assays used to identify the compounds that bind to the PACAP gene product includes preparing a reaction mixture of the PACAP gene product and the test compound under conditions and for a sufficient time to allow the two components to interact and interact. join, thus forming a complex that can be separated and / or detected in the reaction mixture. These tests can be performed in different ways. For example, a method for performing such a test includes anchoring a PACAP gene product or a test substance on a solid support and detecting the PACAP gene / test compound complexes formed on the solid support at the end of the reaction. In one embodiment of this method, the PACAP gene product can be anchored on a solid support, and the test compound, which is not anchored, can be labeled directly or indirectly. In practice, microtiter plates, such as the solid support, are conveniently used. The anchored component can be immobilized by non-covalent or covalent linkages. Non-covalent binding can be achieved simply by coating the solid surface with a solution of the protein and drying. Otherwise, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface. The surfaces can be prepared in advance and stored. To perform the test, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are separated (eg, by washing) under conditions such that any complex formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be achieved in different ways. Where the previously non-immobilized component is pre-marked, the detection of the immobilized mark on the surface indicates that the complexes were formed. When the previously immobilized component is not pre-marked, it is possible to use an indirect mark to detect the complexes anchored on the surface; for example, using a labeled antibody specific for the component not previously immobilized (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Otherwise, it is possible to perform a reaction in a liquid phase, the reaction products separated from the components that did not react and detect the complexes; for example, using an immobilized antibody specific for the PACAP gene product or the test compound to anchor any of the complexes formed in solution, and the labeled antibody specific for the other components of the complex possible to detect the anchored complexes. . 8.2. TESTS FOR INTRACELLULAR PROTEINS THAT INTERACT WITH THE PACAP GENE PRODUCTS Any convenient method to detect protein-protein interactions can be used to identify the interactions product of the PACAP-protein gene. Among the traditional methods that can be used is co-immunoprecipitation, cross-linking and co-purification by gradients or chromatographic columns. By using procedures such as these it is possible to identify proteins, including intracellular proteins, that interact with PACAP gene products. Once isolated, this protein can be identified and can be used in conjunction with normal techniques to identify proteins that interact with it. For example, at least a portion of the amino acid sequence of a protein that interacts with the PACAP gene product can be investigated using techniques well known to those skilled in the art, such as the Edman degradation technique (see, for example. , Creightnon, 1983, "Proteins: Structures and Molecular Principles," WH Freeman &; Co., N. Y., pp-34-94). The amino acid sequence obtained can be used as a guide for the generation of mixtures of oligonucleotides that can be used to detect gene sequences that encode these proteins. The detection can be carried out, for example, by normal hybridization or PCR techniques. The techniques for generation of oligonucleotide mixtures and detection are well known. (See, for example, Ausubel, supra, and 1990, "PCR Protocols; A Guide to Methods and Applications," Innis, et al., Academic Press, Inc., New York). In addition, it is possible to employ methods that lead to the simultaneous identification of the genes that code for a protein that interacts with a product of the PACAP gene. These methods include, for example, probing expression libraries with the labeled PACAP gene product, using the PACAP gene product in a manner similar to the well known antibody probing technique of the? Gtll libraries. A method that detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not as a limitation. A version of this system has been described (Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 9578-9582) and is available commercially in Clontech (Palo Alto, CA). In summary, using such a system, the plasmids encoding two hybrid proteins are constructed: one consists of the DNA-binding domain of a transcription-activating protein fused to the PACAP gene product, and the other consists of the PACAP domain. activating the transcription activator protein fused to an unknown protein that is encoded by a cDNA that has been recombined in this plasmid as part of a cDNA library. The fusion plasmid of the DNA binding domain and the cDNA library are transformed into a yeast Saccharomyces cerevisiae strain containing a reporter gene (eg, HBS or lacZ) whose regulatory region contains the activator binding site of the transcription. Any hybridized protein alone can not activate transcription of the reporter gene: the hybrid of the DNA binding domain can not because it does not provide activation function and the hybrid of the activation domain can not because it can not locate the activator binding sites. The interaction of the two hybrid proteins reconstitutes the functional activating protein and gives rise to the expression of the reporter gene, which is detected by an assay for the reporter gene product. The system of two hybrids or related methodologies can be used to detect libraries of the activation domain for proteins that interact with the product of the "bait" gene. As an example, and not as a limitation, PACAP gene products can be used as the product of the bait gene. The total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a PACAP gene product bait fused to the DNA binding domain are co-transformed into a yeast reporter strain, and the resulting transformants are detected for those expressing the reporter gene. For example, a sequence of the PACAP bait gene, such as the open reading frame of the PACAP gene, can be cloned into the vector so that it is translationally fused to the DNA encoding the DNA binding domain of the GAL4 protein. These colonies are purified and the plasmids library responsible for the expression of the reporter gene are isolated. The DNA sequencing is then used to identify the proteins encoded by the library plasmids. A cDNA library of the cell line from which the proteins that interact with the PACAP gene product bait are to be detected can be prepared using the methods practiced on a daily basis in the art. According to the particular system described in this, for example, it is possible to insert cDNA fragments into the vector so that they are fused translationally to the transcriptional activation domain of GAL4. Such a library can be co-transformed together with a PACAP bait-GAL4 gene fusion plasmid in a yeast strain containing a lacZ gene driven by a promoter that contains the GAL4 activation sequence. A protein encoded by cDNA, fused to a transcriptional activation domain of GAL4 that interacts with the PACAP gene product bait will reconstitute an active GAL4 protein and by this means will direct expression of the HIS3 gene. Colonies expressing HIS3 can be detected by their growth on petri dishes containing semi-solid medium based on agar lacking histidine. The cDNA can then be purified from these strains and used to produce and isolate the protein that interacts with the product of the PACAP bait gene using techniques that are practiced on a daily basis in the art. . 8.3. TESTING FOR COMPOUNDS THAT INTERFERE WITH THE INTERACTION OF MACACOMETRIC PACAP GENE PRODUCTS PACAP gene products can, in vivo, interact with one or more macromolecules, including intracellular macromolecules as proteins. These macromolecules may include, but are not limited to, nucleic acid molecules and these proteins may be identified by methods such as those described above, in Sections 5.8.1 -5.8.2. For purposes of this description, macromolecules are referred to herein as "binding counterparts". Compounds that effect disruption of the PACAP gene product that binds to a binding counterpart can be useful in regulating the activity of the PACAP gene product, especially the mutant PACAP gene products. These 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. The fundamental principle of a test system used to identify compounds that interfere with the interaction between the PACAP gene product and a counterpart or binding partner includes preparing a reaction mixture containing the PACAP gene product and the low binding counterpart. conditions and for a sufficient time to allow the two to interact and unite, thus forming a complex. To test a compound for its inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound may initially be included in the reaction mixture, or may be added at a time subsequent to the addition of the PACAP gene product and its binding counterpart. The control reaction mixtures are incubated without the test compound or without a compound known to not block the complex formation. The formation of any of the complexes between the PACAP gene product and the binding counterpart is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the PACAP gene product and the binding counterpart. In addition, complex formation within the reaction mixtures containing the test compound and the normal PACAP gene product can also be compared for complex formation within the reaction mixtures containing the test compound and a product of the mutant PACAP gene. This comparison may be important in those cases where it is desirable to identify the compounds that break the mutant but not normal PACAP gene product interactions. The assay for compounds that interfere with the interaction of the PACAP gene products and the binding counterparts can be performed in a heterogeneous or homogeneous format. The heterogeneous assays include anchoring the PACAP gene product and the binding counterpart on a solid support and detecting the complexes formed on the solid support at the end of the reaction. In the homogeneous tests, the complete reaction is carried out in the liquid phase. In any method, the order of addition of the reactants may vary to obtain different information about the compounds to be tested. For example, test compounds that interfere with the interaction between the PACAP gene products and the binding counterparts, eg, by competition, can be identified by performing the reaction in the presence of the test substance; that is, adding the test substance to the reaction mixture before or at the same time with the PACAP gene product and the interactive intracellular binding counterpart. Otherwise, test compounds that effect disruption of preformed complexes, for example, compounds with higher binding constants that displace one of the complex compounds, can be tested by adding the test compound to the reaction mixture after the complexes have been formed. The different formats are described shortly later. In a heterogeneous assay system, the PACAP gene product or the interactive binding partner is anchored on a solid surface, while the non-anchored species are labeled, in a direct or indirect manner. In practice, microtiter plates are conveniently used. The anchored species can be immobilized by non-covalent or covalent bonds. Non-covalent binding can be achieved simply by coating the solid surface with a solution of the PACAP gene product or the binding partner and drying. Otherwise, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored. To perform the assay, the counterpart of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, the non-reacting components are separated (eg, by washing) and any of the formed complexes will remain immobilized on the solid surface. The detection of the complexes anchored on the solid support can be carried out in different ways. When the non-immobilized species are pre-marked, the detection of the immobilized mark on the surface indicates that the complexes were formed. When non-immobilized species are not pre-marked, it is possible to use an indirect mark to detect complexes anchored on the surface; for example, using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly or indirectly labeled with a labeled anti-Ig antibody). Depending on the order of addition of the reaction components, the test compounds which inhibit the complex formation or which effect the disruption of the preformed complexes can be detected. Otherwise, the reaction can be carried out in a liquid phase in the presence or absence of the test compound, the reaction products are separated from the non-reacted components, and the complexes are detected; for example, using an immobilized antibody specific for one of the binding components to anchor any of the complexes formed in solution, and a labeled antibody specific for the counterpart to detect the anchored complexes. Again, depending on the order of addition of the reactants to the liquid phase, it is possible to identify the test compounds that inhibit the formation of the complex or that effect the disruption of the preformed complexes. In an alternative embodiment of the invention, it is possible to use a homogeneous assay. In this method, a preformed complex of the PACAP gene product and the interactive binding counterpart is prepared in which the PACAP gene product or its binding counterpart is labeled, but the signal generated by the tag is extinguished by complex formation (see, for example, U.S. Patent No. 4,109,496 to Rubenstein, which uses this method for immunoassays). The addition of a test substance that competes with and displaces one of the species of the preformed complex will give rise to the generation of a signal on the background. In this way, the test substances that effect the disruption of the interaction product of the PACAP gene / binding counterpart can be identified. In another embodiment of the invention, these same techniques can be employed using peptide fragments corresponding to the binding domains of the PACAP product and / or the binding counterpart (in cases where the binding partner is a protein), rather than a or both full-length proteins. Any of the different methods practiced routinely 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 detection for disruption of binding in a co-immunoprecipitation assay. The compensatory mutations in the gene encoding the second species in the complex can then be selected. The analysis of the sequences of the genes encoding the respective proteins will reveal the mutations corresponding to the region of the protein involved in the interactive binding. Otherwise, a protein can be anchored to a solid surface using the methods described in the previous section, and it is allowed to interact with and bind to its labeled binding counterpart, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a labeled short peptide containing the binding domain can remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the segments is manipulated to express the peptide fragments of the protein, it can then be tested for binding activity and purified or synthesized. As an example, and not as limitation, a PACAP gene product is anchored to a solid material as described, in the foregoing, in this section by making a GST-PACAP fusion protein and allowing binding to glutathione agarose beads. The counterpart of binding may be labeled with a radioactive isotope, such as 35S, and dissociated with a proteolytic enzyme, such as trypsin. The dissociation products can then be added to the anchored GST-PACAP fusion protein and binding is allowed. After washing the unbound peptides, the bound, labeled material representing the binding domain of the binding part, can be eluted, purified and analyzed for the amino acid sequence by well-known methods. The peptides thus identified can be produced in synthetic form or produced using recombinant DNA technology. . 8.4. TESTING FOR IDENTIFICATION OF COMPOUNDS AMINATING A PACAP MEASURED DISORDER Compounds including, but not limited to, the binding compounds identified by the assay techniques as described above in Sections 5.8.1-5.8. 4, can be tested for the ability to lessen the symptoms of a PACAP-mediated disorder. It should be noted that the assays described he can identify compounds that affect the activity of PACAP by affecting the expression of the PACAP gene or by affecting the level of activity of the PACAP gene product. For example, the compounds can be identified that are involved in another step in the path in which the PACAP gene and / or the PACAP gene product is involved, and affecting this same route can modulate the effect of PACAP on the development of a disorder mediated by PACAP. These compounds can be used as part of a therapeutic method for the treatment of the disorder. Below, cell-based assays based on animal models are described for the identification of compounds that exhibit such ability to lessen the symptoms of a PACAP-mediated disorder. First, cell-based systems can be used to identify compounds that can act to lessen the symptoms of a PACAP-mediated disorder. These cellular systems may include, for example, recombinant or non-recombinant cells such as cell lines that express the PACAP gene. In the use of these cell systems, cells expressing PACAP can be exposed to a compound which is suspected of having an ability to lessen the symptoms of a PACAP-mediated disorder, at a sufficient concentration and for a sufficient time to produce such a improvement of these symptoms in exposed cells. After exposure, the cells can be assayed to measure alterations in PACAP gene expression, for example, by assaying cell lysates for transcripts of PACAP mRNA (for example, by Northern analysis) or for PACAP gene products expressed by the cell; compounds that modulate the expression of the PACAP gene are good candidates as therapeutic. In addition, animal-based systems or models for a PACAP-mediated disorder, for example, transgenic mice that contain a human or altered form of the PACAP gene, can be used to identify compounds capable of ameliorating the symptoms of the disorder. These animal models can be used as test substrates for the identification of drugs, pharmaceutical compounds, therapies and interventions. For example, animal models can be exposed to a compound suspected of exhibiting the ability to lessen symptoms, at a sufficient concentration and for a sufficient time to produce such an improvement in the symptoms of a PACAP disorder. The response of the animals to the exposure can be monitored by assessing the inversion of the symptoms of the disorder. With respect to the intervention, any of the treatments that reverse any aspect of the symptoms of a PACAP-mediated disorder should be considered as a candidate for human therapeutic intervention in this condition. The doses of the test agents can be determined by producing dose-response curves, as described in Section 5.10.1, below. . 9. COMPOUNDS AND METHODS FOR THE TREATMENT OF DISORDERS MEDIATED NEUROSIQUIÁTRICOS BY PACAP The methods and compositions by means of which a mediated disorder can be treated are described below.

PACAP. For example, these methods may consist of administering compounds that modulate the expression of a mammalian PACAP gene and / or the synthesis or activity of a mammalian PACAP gene product to ameliorate the symptoms of the disorder. Otherwise, in cases where disorders mediated by mammalian PACAP result from mutations of the PACAP gene, these methods may comprise supplying the mammal with a nucleic acid molecule that codes for a non-impaired PACAP gene product so that a non-impaired PACAP gene product is expressed and the symptoms of the disorder are ameliorated. In another embodiment of the methods for the treatment of mammalian PACAP-mediated disorders resulting from PACAP gene mutations, these methods may involve supplying the mammal with a cell containing a nucleic acid molecule encoding a PACAP gene product. not deteriorated so that the cell expresses the product of the PACAP gene not deteriorated and the symptoms of the disorder are reduced. In cases in which a loss of the normal PACAP product function of origin leads to the development of a PACAP-mediated disorder, an increase in the activity of the PACAP gene product will facilitate progress toward an asymptomatic state in individuals with a poor level of expression of the PACAP gene and / or activity of the PACAP gene product. Methods for improving the expression or synthesis of PACAP may include, for example, methods such as those described below, in Section 5.9.2. Otherwise, the symptoms of PACAP-mediated disorders can be alleviated by administering a compound that decreases the level of expression of the PACAP gene and / or the activity of the PACAP gene product. Methods for inhibiting or reducing the level of synthesis or expression of the PACAP gene product may include, for example, methods such as those described in section 5.9.1. . 9.1 METHODS OF ANTI-AGING INHIBITION, RIBOZIMA AND TRIPLE HELIX In another embodiment the symptoms of PACAP-mediated disorders can be reduced by decreasing the level of PACAP gene expression and / or the activity of the PACAP gene product using PACAP gene sequences together with the PACAP gene sequences. antisense methods, gene "knockout", ribozyme and / or triple helix well known 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, including the ability to lessen the symptoms of a PACAP-mediated disorder, are the antisense, ribozyme and triple helix molecules. These molecules can be designed to reduce or inhibit the activity of the chosen gene not damaged, or if mutant is suitable. Techniques for the production and use of these molecules are well known to those skilled in the art. The RNA and antisense DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense methods include the design of oligonucleotides that are complementary to an mRNA of the chosen gene. The antisense oligonucleotides will bind to the mRNA transcripts for the chosen complementary gene and avoid translation. It is not required although absolutely complementary is preferred. A sequence "complementary" to a portion of an RNA, as mentioned herein, means a sequence that has sufficient complementarity to be able to hybridize with the RNA to form a stable duplex; in the case of double-stranded antisense nucleic acid, a single strand of the duplex DNA can thus be tested, or triplex formation can be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. In general, the greater the hybridizing nucleic acid, the more uncoupling of bases with an RNA can contain and still form a stable duplex or (triplex, as the case may be). One of ordinary skill in the art can ascertain a degree of tolerable decoupling by using standard procedures to determine the melting point of the hybridized complex. In one embodiment, the oligonucleotides complementary to the non-coding regions of the PACAP gene could be used in an antisense approach to inhibit the translation of mRNA for endogenous PACAP. The antisense nucleic acids should be at least 6 nucleotides in length, and preferably are oligonucleotides in the range 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. Regardless of the choice of the target sequence, it is preferred that in vitro studies are first performed to quantify the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies use controls that distinguish between antisense inhibition and non-specific biological effects of the oligonucleotides. It is also preferred that these studies compare levels of the target protein or RNA with those of an RNA or internal control protein. In addition, it is expected that the results obtained using the antisense oligonucleotide will be compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide be of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differ from the antisense sequence no more than is necessary to avoid specific hybridization to the chosen sequence. The oligonucleotides may be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified in the portion of the base, the sugar portion or the phosphate backbone, for example, to improve the stability of the molecule. Hybridization, etc. The oligonucleotide may include other adjoining groups such as peptides (e.g., to choose host cellular receptors in vivo), or agents that facilitate transport across the cell membrane (see, e.g., Letsinger, et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6553-6556; Lemaitre, et al., 1987, Proc. Natl. Acad. Sci. USA 84, 648-652; PCT Publication No. WO 88/09810, published December 15, 1988) or the blood-brain barrier (see, for example, PCT publication No. WO 89/10134, published April 25, 1988), activity dissociation agents by hybridization (see, for example, Krol et al., 1988, BioTechniques 6, 958-976) or intercalation agents (see, eg, Zon, 1988, Pharm. Res. 5, 539- 549). For this purpose, the oligonucleotide can be conjugated to another molecule, for example, a peptide, a crosslinking agent activated hybridization, a transport agent, an agent for dissociation activated by hybridization, etc. The antisense oligonucleotide may consist of at least one modified base portion selected from the group including, but not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-6-isopentenyl adenenin, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil , 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybtoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, uracil-5-oxyacetic acid (v), 5-methyl-2-t iouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diamino purine. The antisense oligonucleotide may also comprise at least one modified sugar portion selected from the group including, but not limited to: arabinose, 2-fluoroarabinose, xylulose and hexose. In still another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorothioothioate, a phosphoramidothioate, phosphoramidite, phosphoniamidate, a methylphosphonate, an alkyl phosphothioester and a formacetal or analogs thereof. In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to normal β units, the strands run parallel to each other (Gautier, et al., 1987, Nucí Acids, Res. 15, 6625-6641) . The oligonucleotide is a 2'-O-methylribonucleotide (Inoue, et al., 1987, Nucí Acids, Res. 15, 6131-6148), a chimeric RNA-DNA analog (Inouye, et al., 1987, FEBS Lett. 215, 327-330). The oligonucleotides of the invention can be synthesized by methods well known in the art, for example, by the use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.). As examples, the phosphorothioate oligonucleotides can be synthesized by the method of Stein et al., (1988, Nucí Acids Res. 16, 3209), the methylphosphonate oligonucleotides can be prepared by the use of controlled pore glass polymer supports ( Sarin, et al., 1988, Proc. Natl. Acad. Sci. USA 85, 7448-7451), et cetera. Although it is possible to use complementary antisense nucleotides for the sequence of the coding region of the chosen gene, those complementary to the translated, transcribed region are more preferred. Antisense molecules must be delivered to the cells expressing the chosen gene in vivo. Various methods have been developed to deliver antisense DNA or RNA to cells; for example, antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to select cells (e.g., antisense linked to peptides or antibodies that bind specifically to receptors or antigens expressed on the chosen cell surface) can be administered systemically. However, it is often difficult to obtain sufficient intracellular antisense concentrations to suppress the translation of the endogenous mRNAs. Therefore, a preferred method uses a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong poly III or pol II promoter. The use of this construct to transfect selected cells in the patient will result in the transcription of sufficient amounts of the single-stranded RNAs that will form base pairs complementary to the transcripts of the endogenous chosen gene and by this means will prevent translation of the mRNA of the chosen gene. For example, it is possible to introduce a vector, for example, so that it is captured by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or integrate into the chromosome, as long as it can be transcribed to produce the desired antisense RNA. These vectors can be constructed by the methods of recombinant DNA technology normal in the art. The vectors can be plasmids, viral or others known in the art, used for replication and expression in mammalian cells. The expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act on mammalian, preferably human, cells. These promoters can be inducible or constitutive. Such promoters include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nautre 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 (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78, 1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296, 39-42), et cetera. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the construction of recombinant DNA that can be introduced directly into the tissue site. Otherwise, it is possible to use viral vectors that selectively infect the desired tissue, in which case the administration can be carried out in another way (for example, systemically). Ribozyme molecules designed to catalytically dissociate the mRNA transcripts of the chosen gene can also be chosen to prevent translation of the mRNA of the chosen gene and, thus, the expression of the chosen gene product. See, for example, the PCT international publication WO 90/11364, published on October 4, 1990; Sarver et al., Science 247, 1222-1225. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific dissociation of RNA. (for a review, see Rossi, 1994, Current Biology 4, 469-471). The mechanism of action of the ribozyme includes specific hybridization of the sequence of the ribozyme molecule to the chosen, complementary RNA, followed by an event of endonucleolytic dissociation. The composition of the ribozyme molecules must include one or more complementary sequences for the mRNA of the chosen gene, and must include the well-known catalytic sequence responsible for the dissociation of the mRNA. For this sequence, see, for example, U.S. Patent No. 5,093,246, which is incorporated herein by reference in its entirety. Although it is possible to use ribozymes that dissociate or unfold the mRNA in site specific recognition sequences to destroy the chosen gene mRNA, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes dissociate the mRNA at locations dictated by flanking regions that form base pairs complementary to the chosen mRNA. The only requirement is that the chosen mRNA has the following sequence of two bases: 5'-UG-3X The construction and production of the hammerhead ribozymes is well known in the art and is described in more detail in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, (see especially Figure 4, page 833) and Haseloff and Gerlach, 1988, Nature, 334, 585-591, which is incorporated herein by reference in its integrity Preferably, the ribozyme is manipulated so that the dissociation recognition site is located near the 5 'end of the mRNA of the chosen gene, ie to increase efficiency and minimize intracellular accumulation of non-transcribed mRNA transcripts. functional The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-like ribozymes") such as those that occur naturally in Tetrahymena thermofila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech et al (Zaug et al., 1984, Science, 224, 574-578; Zaug and Cech, 1986, Science, 231, 470-475; Zaug et al., 1986, Nature, 324, 429-433; of International Patent published No. WO 88/04300 by University Patents Inc., Been and Cech, 1986, Cell, 47, 207-216). The Cech-type ribozymes have an active site of eight base pairs that hybridizes to a chosen RNA sequence where after the dissociation of the chosen RNA takes place. The invention comprises those Cech-like ribozymes that choose sequences from the active site of eight base pairs that are present in the chosen gene. As in the antisense method, the ribozymes may be composed of modified oligonucleotides (eg, to improve stability, orientation, etc.) and must be delivered to the cells expressing the chosen gene in vivo. A preferred delivery method includes using a DNA construct "encoding" the ribozyme under the control of a strong, constitutive pol III or pol II promoter, so that the transfected cells produce sufficient quantities of the ribozyme to destroy the messages of the gene endogenously chosen and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficacy. Expression of the endogenous chosen gene can also be reduced by inactivating or "knocking out" the chosen gene or its promoter using chosen homologous recombination (eg, see, Smityies 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 herein by reference in its completeness). for example, a non-functional, mutant chosen gene (or a completely unrelated DNA sequence) flanked by DNA homologs for the endogenous chosen gene (the coding regions or the regulatory regions of the chosen gene) can be used with or without a selectable marker and / or a negative selectable marker, to transfected cells expressing the chosen gene in vivo. The insertion of the DNA construct, through the chosen homologous recombination, gives rise to the inactivation of the chosen gene. These methods are particularly suitable in the agricultural field where modifications for ES cells (primordial embryos) can be used to generate animal progeny with an inactive chosen gene (for example, see, Thomas and Capecchi, 1987 and Thompson, 1989, supra). However, this approach can be adapted for use in humans provided that the recombinant DNA constructs are directly administered or targeted to the required site in vivo using suitable viral vectors. Otherwise, the expression of the endogenous chosen gene can be reduced by orienting the deoxyribonucleotide sequences complementary to the chosen gene regulatory region (ie, the promoter and / or enhancers of the chosen gene) to form triple helical structures that prevent transcription of the chosen gene in selected cells in the body, (see, in general, Helene, 1991, Anticancer Drugs Des., 6 (6), 569-584; Helene, et al., 1992, Ann, NY Acad. Sci., 660 , 27-36, and Maher, 1992, Biossays 14 (12), 807-815). The nucleic acid molecules that are to be used in the formation of the triple helix for the inhibition of transcription must be single-strand and composed of deoxynucleotides. The composition of the bases of these oligonucleotides should be designed to promote the formation of the triple helix through the rules of base pairing of Hoogsteen, which generally requires dimensional extensions of purines or pyrimidines that are present in a strand of a duplex. The nucleotide sequences can be based on pyrimidines.

Which will give rise to triplets TAT and C + through the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide bases in addition to a single-strand rich purine region of the duplex in a parallel orientation in this strand. In addition, the nucleic acid molecules can be chosen to be rich in purine, for example, containing an extension of residues G. These molecules will form a triple helix with a DNA duplex that is rich in GC parts, in which most of the purine residues are located in a single strand of the chosen duplex, giving rise to triplets GGC through the strands in the triplex.

Otherwise, the potential sequences that can be chosen for the formation of the triple helix can be increased by creating a nucleic acid molecule called "switchback". The switchback molecules are synthesized in an alternating mode 5 '-3'-, 3' -5 ', so that they pair their bases with the first strand of one duplex and then the other, eliminating the need for a resizable extension of purines or pyrimidines that are present in a strand of a duplex. In cases where the antisense ribozyme and / or triple helix molecules described herein are used to inhibit the expression of the mutant gene, it is possible that the technique can efficiently reduce or inhibit transcription (triple helix) and / or translation (antisense, ribozyme ) of the mRNA produced by normal alleles of the chosen gene that may arise where the concentration of the chosen normal gene product present may be lower than that necessary for a normal phenotype. In these cases, to ensure that the substantially normal levels of the activity of the chosen gene are maintained, therefore, the nucleic acid molecules that encode and express polypeptides of the chosen gene having normal activity of the chosen gene can be introduced into the cells by the gene therapy methods such as those described later in section 5.9.2 that do not contain sequences susceptible to any of the antisense, ribozyme or triple helix treatments being used. Otherwise, in cases whereby the chosen gene encodes an extracellular protein, it may be preferable to co-administer the protein of the chosen gene. The RNA and antisense, ribozyme and triple helix DNA molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules already described. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as for example chemical synthesis of phosphoramidite in solid phase. Otherwise, the RNA molecules can be generated by in vitro and in vivo transcription of the DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate the promoters of the suitable RNA polymerase, such as the T7 or SP6 polymerase promoters. Otherwise, the antisense cDNA constructs are synthesized antisense RNA constitutively or inducibly, - depending on the promoter used, they can be stably inserted into cell lines. . 9.2. GENE REPLACEMENT THERAPY PACAP gene nucleic acid sequences, already described in Section 5.1, can be used for the treatment of a condition mediated by PACAP. This 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 the production of a PACAP gene product that has normal PACAP gene function, can be grafted into the appropriate cells within a patient, using vectors that they include, but are not limited to: adenovirus, adeno-associated virus and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.

Because the PACAP gene is expressed in the brain, these gene replacement therapy techniques must be able to deliver PACAP gene sequences to these cell types within patients. Thus, in one embodiment, techniques well known to the experts (see, for example, PCT publication No. WO 89/10134, published April 25, 1988) can be used to allow PACAP gene sequences to cross the blood-brain barrier easily and supply the sequences to the cells in the brain. With respect to delivery that is capable of crossing the blood-brain barrier, viral vectors such as, for example, those described above, are preferable. In another embodiment, techniques for delivery include the direct administration of these PACAP gene sequences to the site of the cells in which the PACAP sequences are to be expressed. Additional methods that can be used to increase the total level of PACAP gene expression and / or the activity of the PACAP gene product include the introduction of suitable cells expressing PACAP, preferably autologous cells, in a patient at the positions and in the numbers that are sufficient to reduce the symptoms of a neuropsychiatric disorder mediated by PACAP. these cells can be recombinant or non-recombinant. Among the cells that can be administered to increase the total level of PACAP gene expression in a patient are normal cells, preferably brain cells, that express the PACAP gene. Otherwise, the cells, preferably autologous cells, can be manipulated to express the PACAP gene sequences, and then they can be introduced into a patient in the appropriate positions to lessen the symptoms of a PACAP-mediated neuropsychiatric disorder. Otherwise, cells expressing an undamaged PACAP gene from a coupled MHC individual [sic] may be used, and may include, for example, brain cells. The expression of the PACAP gene sequences is controlled by the appropriate gene regulatory sequences to allow such expression in the necessary cell types. These regulatory sequences of the gene are well known to the experts. These cell-based gene therapy techniques are well known to those skilled in the art, see, for example, Anderson, U.S. Patent No. 5,399,349. When the cells to be administered are non-autologous cells, they can be administered using well-known techniques that prevent the development of a host immune response against the introduced cells. For example, the cells can be introduced in an encapsulated form which, while allowing an exchange of the components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system. In addition, composed as those identified by techniques such as those already described, in section 5.8, which are capable of modulating the activity of the PACAP gene product, they can be administered using normal techniques well known to the experts. In cases where the compounds to be administered will involve an interaction with the brain cells, the administration techniques should include the well-known ones that allow crossing the blood-brain barrier. . 10. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION Compounds that are determined to affect PACAP gene expression or gene product activity can be administered to a patient in effective doses for therapeutic use to treat or ameliorate a neuropsychiatric disorder mediated by PACAP. A therapeutically effective dose refers to that amount of the compound sufficient to cause improvement of the symptoms of this disorder. . 10.1 EFFECTIVE DOSE The toxicity and therapeutic efficacy of these compounds can be determined by normal pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the Therapeutic Index can be expressed as the LD50 / ED50 ratio. Compounds having large therapeutic indices are preferred. Although it is possible to use compounds that have toxic side effects, attention should be paid to the design of a delivery system that directs such compounds to the site of the affected tissue to minimize the potential damage to uninfected cells [sic], to reduce by this means the collateral effects. The data obtained from cell culture assays and animal studies can be used to formulate a range of doses for human use. The dose of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from assays in cell cultures. It is possible to formulate a dose in animal models to obtain a concentration range in circulating plasma that includes the IC 50 (i.e., the concentration of test compound that obtains a semi-maximum inhibition of symptoms) as determined in cell cultures. Such information can be used to determine more precisely the useful dose in humans. Plasma concentrations can be measured, for example, by high-performance liquid chromatography. . 10.2 FORMULATIONS AND USE The pharmaceutical compositions used in accordance with the present invention can be formulated in a traditional manner using one or more acceptable carriers or excipients for physiological use. Thus, the compounds and their salts and solvates acceptable for physiological use can be formulated for administration by inhalation or insufflation (through the mouth or nose) or oral, buccal, parenteral or rectal administration. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with acceptable excipients, pharmaceutical use such as binding agents (eg, pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); filler materials (for example, lactose, microcrystalline cellulose or calcium acid phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets can 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 may be presented as an anhydrous powder for constitution with water or other suitable vehicle before use. These liquid preparations can be prepared by traditional means with additives acceptable for pharmaceutical use such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils) and preservatives (eg, methyl or propyl-p-hydroxybenzoates or sorbic acid) The preparations may also contain buffer salts, flavorings, dyes and sweetening agents, as appropriate The preparations for oral administration can be conveniently formulated to obtain controlled release of the active compound For buccal administration, the compositions can take the form of tablets or dragee formulations in a conventional manner. by inhalation, the compounds for use in accordance with the present invention, are conveniently supplied in the form of an aerosol spray preparation from pressurized containers or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane , dichlorotetrafluoroethane, carbon dioxide or another convenient gas. In the case of pressurized aerosol, the unit dose can be determined by providing a valve to supply a dosed amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mixture of the compound and a convenient powder base such as lactose or starch.

The compounds can be formulated for parenteral administration by injection, for example, by injection of a bolus or continuous intravenous route. Formulations for injection may be presented in unit dosage forms, for example, in ampules or in multi-dose containers, with added preservatives. The compositions may take such forms as suspensions. Solutions or emulsions in oily or aqueous vehicles and may contain formulation agents such as suspending, stabilizing and / or dispersing agents. Otherwise, the active ingredient may be in the form of powder for constitution with convenient vehicle, for example, sterile, pyrogen-free water, before use. The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, for example, containing bases for traditional suppositories such as cocoa butter or other glycerides. In addition to the formulations described above, the compounds can also be formulated as a depot preparation. These long-acting formulations can be administered by implantation (e.g., 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 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 container or dosing device that may contain one or more unit dosage forms containing the active ingredient. The package can, for example, consist of a metal or plastic sheet, such as a blister pack. The package or dosing device may be accompanied by instructions for administration. 6. EXAMPLE: LOCATION OF THE PACAP GENE IN CHROMOSOME 18 In the example presented in this section, we describe the studies that, first, define a range of approximately 310 kb in the short arm of human chromosome 18 within which a region associated with a neuropsychiatric disorder is located, and secondly , for the identification of the PACAP gene, as determined within the region. 6. 1. MATERIALS AND METHODS 6.1.1. UNBALANCE OF THE LIABILITY Linkage disequilibrium studies were conducted (LD) using DNA from a population sample of patients with neuropsychiatric disorder (BP-I). The sample of the population and the 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 by the physical mapping techniques described below. 6. 1.2. MAPPING OF THE ARTIFICIAL YEAR CHROMOSOMA (YAC) For the physical mapping, the yeast artificial chromosomes (YAC) containing the human sequences were mapped for the region being analyzed based on the maps available to the public (Cohen et al., 1993, CR Acad.

Sci. 316, 1484-1488). The YACs were then ordered and the contigs reconstructed by mapping the content of the normal short tag sequence (STS) with micro satellite and non-polymorphic STS markers available from the databases surrounding the genetically defined candidate region. 6. 1.3. MAPPING OF BACTERIAL ARTIFICIAL CHROMOSOMA (BAC) The STSs of the short arm of human chromosome 18 were used to detect a human BAC library (Research Genetics, Huntsville, AL). The ends of the BACs were cloned or directly sequenced. The extreme sequences were used to amplify the following superimposed BACs. From each BAC, additional micro satellites were identified, specifically, randomly shared libraries were prepared from BAC in superposition within the defined genetic range. The BAC DNA was shared with a nebulizer (CIS-US Inc. Bedford, MA). The fragments in the size range of 600 to 1,000 bp were used for the production of the sub-library. The microsatellite sequences of the sub-libraries were identified by corresponding micro-satellite probes. The sequences around these repeats were obtained to allow the development of primers for PCR for genomic DNA. 6. 1.4. HYBRID MAPPING BY RADIATION (RH) Standard RH mapping techniques were applied for an RH Standford G3 mapping panel (Research Genetics, Hunstville, AL) to order all micro satellite markers and non-polymorphic STS in the region under analysis. 6. 1.5 SEQUENCE OF THE SAMPLE The randomly shared libraries were prepared from all the BACs within the defined genetic region. Approximately 9000 subclones within the region of approximately 310 kb were sequenced with vector primers to achieve a sequence of eight times the coverage of the region. All the sequences were processed through a pipeline for an automated sequence analysis that assessed the quality, separated the vector sequences and masked the repeated sequences. The resulting sequences were then compared in public databases of DNA and protein using BLAST algorithms (Altschul et al., 1990, J. Molec. Biol., 215, 403-410). 6. 2 RESULTS Genetic regions involved in human genes for bipolar affective disorder ( have previously been reported on maps in portions of the long (18 q) and short (18 p) arms of human chromosome 18, including a broad genetic region of 18q (approximately 6-7 cM between markers D18S469 and D18S554 (Provisional Requests Series Nos. 60 / 014,498 and 60 / 023,438, filed on March 28, 1996 and August 23, 19996, respectively, the complete contents of each of which are incorporated herein by reference; Freimer et al., 1996, Neuoropsychiat. Genet 67, 254-263; Freimer et al., 19996, Nature Genetics 12, 436-441), the total contents of each of which is incorporated herein by reference. Linkage disequilibrium Before attempting to identify the gene sequences, studies were conducted to narrow the region of the neuropsychiatric disorder. Specifically, a linkage disequilibrium (LD) analysis was performed using samples from a population and techniques such as those described in section 6.1 above, taking advantage of the additional physical markers identified by the physical mapping techniques described below. High resolution physical mapping using the techniques of YAC BAC, and RH. To provide the precise order of the genetic markers necessary for linkage and LD mapping, and to guide a new development of microsatellite marker for a finer mapping, a high-resolution physical map of the candidate region 18q23 was developed using the YAC techniques , BAC and RH. For this physical mapping, YACs were first mapped to the region of chromosome 18 that was analyzed. Using the YAC contig mapped as a lattice, the region of the markers available to the public D18S161 and D18S554, which covers most of the D18S469-D18S554 region described above, was also mapped and assembled with the BACs. The sub-libraries of the contiguous BACs were constructed, from which the micro-satellite marker sequences were identified and sequenced.

To guarantee the development of a precise physical map, the radiation hybrid (RH) mapping technique was independently applied to the region being analyzed. The RH was used for all the micro-satellite markers and the non-polymorphic STCs in the region. Thus, the finally constructed high-resolution physical map was obtained using the data from the RH mapping and the STS content mapping. The BAC clones within the region of the newly identified 310 kb neuropsychiatric disorder were analyzed to identify specific genes within the region. A combination of sample sequencing, cDNA selection and transcription mapping analysis were combined to arrange the sequences into tentative transcription units, i.e., tentatively delineating the coding sequences of the genes within this genomic region of interest. One of the identified transcription units was called PACAP. The present invention should not be limited in scope by the specific embodiments described herein, which are proposed as only illustrations of the individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the aforementioned description and the accompanying drawings. These modifications are proposed to be within the scope of the appended claims. All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent one of which individual publications or patent applications is specified and individually indicated as incorporated by reference.

Claims

1. A method for determining whether a human individual has or is at risk of developing a PACAP-mediated neuropsychiatric disorder, which consists of the step of detecting the presence or absence of a genetic mutation in the PACAP gene (SEQ ID NO: 1) of the individual , wherein the genetic mutation is selected from the group consisting of: a substitution of nucleotides, an insertion of nucleotides and a deletion of nucleotides and gives rise to the production of a PACAP protein having a sequence of amino acids different from the amino sequence PACAP wild type acids and the presence of the genetic mutation identify an individual who has or is at risk of developing a neuropsychiatric disorder mediated by PACAP.

2. The method of claim 1, further comprising the steps of: a) obtaining a sample containing nucleic acid molecules from the individual; b) amplifying the nucleic acid molecules in the sample that encode the PACAP protein using primers for amplification that selectively tune and amplify the nucleic acid molecules encoding the PACAP protein; and c) determine if the genetic mutation is present.

3. The method of claim 2, wherein the determination in step c) is to sequence the nucleic acid molecule encoding the PACAP protein.

4. The method of claim 1, wherein the nucleotide sequence of the nucleic acid molecule encoding the PACAP protein is determined.

5. The method of claim 1, further comprising the steps of: a) obtaining a sample containing nucleic acid molecules from the individual; b) detecting the nucleic acid molecules in the sample that encode the PACAP protein using a nucleic acid probe that selectively hybridizes to the nucleic acid molecules encoding the PACAP protein; and c) determine if the genetic mutation is present.

The method of claim 1, further comprising the steps of: a) obtaining a mixture containing the nucleic acid molecules of the individual; b) detect in the sample the nucleic acid molecules encoding the PACAP protein using a restriction endonuclease digestion of the nucleic acid molecules and a nucleic acid probe that selectively hybridizes to the nucleic acid molecules encoding the PACAP protein, or a fragment of it; and c) determine if the genetic mutation is present.

The method of claim 1, wherein the genetic mutation is a base change.

The method of claim 1, wherein the genetic mutation is detected by determining whether an altered PACAP protein is produced in the individual.

The method of claim 8, further comprising the steps of: a) obtaining from the individual a sample containing protein molecules; b) detect PACAP proteins in the sample using an antibody that binds to the PACAP protein; and c) determining whether the PACAP protein is encoded by a nucleic acid molecule containing the genetic mutation.

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

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

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 neuropsychiatric disorders mediated by PACAP comprises the steps of: a) incubating a cell expressing 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 the test compound; and c) selecting a compound that alters the activity of the PACAP gene product being a compound that can be used to treat a neuropsychiatric disorder mediated by PACAP.

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 altered activity is used.

16. The method of claim 1, wherein the PACAP-mediated neuropsychiatric disorder is a bipolar affective disorder.

17. The method of claim 1, wherein the neuropsychiatric disorder mediated by PACAP is a manic type schizoaffective disorder.