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WO2006013561A2 - Compositions et methodes de diagnostic et de traitement de trouble de stress post-traumatique - Google Patents

Compositions et methodes de diagnostic et de traitement de trouble de stress post-traumatique Download PDF

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Publication number
WO2006013561A2
WO2006013561A2 PCT/IL2005/000824 IL2005000824W WO2006013561A2 WO 2006013561 A2 WO2006013561 A2 WO 2006013561A2 IL 2005000824 W IL2005000824 W IL 2005000824W WO 2006013561 A2 WO2006013561 A2 WO 2006013561A2
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Prior art keywords
gene
ptsd
group
kit
expression
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PCT/IL2005/000824
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English (en)
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WO2006013561A3 (fr
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Ronnen Segman
Arieh Shalev
Tania Goltser
Nir Friedman
Noa Shefi
Naftali Kaminski
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Yissum Research Development Company Of The Hebrew University Of Jerusalem
Tel Hashomer Medical Research Iinfrastructure And Services Ltd.
Hadasit Medical Research Services And Development Ltd.
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Publication of WO2006013561A2 publication Critical patent/WO2006013561A2/fr
Publication of WO2006013561A3 publication Critical patent/WO2006013561A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to specific gene expression patterns in PBMC of acute and chronic PTSD affected individuals and, more particularly, to methods of predicting and diagnosing PTSD.
  • Post-traumatic stress disorder is a common mental disorder with a lifetime prevalence of 9-14 % (Breslau N. 2001. J. Clin. Psychiatry. 62 Suppl 17: 16-22; Kessler RC et al., 1995. Arch. Gen. Psychiatry 52: 1048-1060; Yehuda, R. 2002. N. Engl. J. Med. 346: 108-14).
  • PTSD results from a maladaptive response to life threatening events such as wars, natural disasters, domestic violence or sexual abuses; when such traumatic events cause psychological bruises beyond the usual corrective ability of the affected individual, PTSD develops.
  • PTSD is characterized by three specific groups of symptoms: intrusive behaviors (including flashbacks and intense physiologic distress), avoidance behaviors (including avoidance of reminders of the trauma and numbing of responsiveness), and hyperarousal (including insomnia and an exaggerated startle response).
  • intrusive behaviors including flashbacks and intense physiologic distress
  • avoidance behaviors including avoidance of reminders of the trauma and numbing of responsiveness
  • hyperarousal including insomnia and an exaggerated startle response.
  • Many trauma survivors exhibit PTSD symptoms at the early aftermath of traumatic events (e.g., 94 % of rape victims), with marked variability in terms of severity and persistence of each of the symptom clusters.
  • Twin studies have suggested that each of the clusters of symptoms possesses a distinct genetic basis and represent discrete biological dimensions (True WR, et al. 1993. Arch. Gen. Psychiatry, 50: 257-264).
  • PTSD While early symptoms are often transient, a significant number of survivors remain highly symptomatic, exhibiting the full persisting clinical disorder.
  • the diagnosis of acute PTSD is made if the intrusion, avoidance and hyperarousal cause significant clinical impairments which last for more than a month. On the other hand, if such symptoms last for a period of 2-30 days, a diagnosis of acute stress disorder is made.
  • Chronic PTSD is diagnosed if sufficiently severe symptoms in all 3 clusters are still apparent three or more months after trauma (DSM-IV, American Psychiatric Association).
  • PTSD are associated with activation of immune genes (Aloe L, et al., 1994. Proc Natl
  • PBMC peripheral blood monocyte cells
  • kits for determining predisposition of a subject to develop PTSD comprising at least 10 and no more than 574 polynucleotides wherein each of the polynucleotides is capable of specifically binding at least one specific polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-574.
  • an agent for the manufacture of a kit for determining predisposition to develop PTSD comprising at least 10 and no more than 574 polynucleotides wherein each of the polynucleotides is capable of specifically binding at least one specific polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-574.
  • kits for diagnosing PTSD in a subject comprising at least 10 and no more than 408 polynucleotides wherein each of the polynucleotides is capable of specifically binding at least one specific polynucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 10, 12, 17, 18, 20, 35, 62, 79, 81, 88, 93, 107, 128, 129, 139, 144, 154, 159, 164, 169, 170, 174, 185, 186, 194, 211, 214, 221, 236, 248, 252, 253, 255, 257, 260, 275, 277, 278, 283, 295, 296, 317, 320, 327, 331, 342, 344, 348, 352, 372, 376-378, 383, 389, 394, 395, 397, 408, 432, 437, 442, 452, 474, 475, 483, 504, 505, 523,
  • an agent for the manufacture of a kit for diagnosing PTSD comprising at least 10 and no more than 408 polynucleotide sequences wherein each of the polynucleotide sequences is at least 80 % identical to at least one specific polynucleotide selected from the group consisting of SEQ ID NOs: 7, 10, 12, 17, 18, 20, 35, 62, 79, 81, 88, 93, 107, 128, 129, 139, 144, 154, 159, 164, 169, 170, 174, 185, 186, 194, 211, 214, 221, 236, 248, 252, 253, 255, 257, 260, 275, 277, 278, 283, 295, 296, 317, 320, 327, 331, 342, 344, 348, 352, 372, 376-378, 383, 389, 394, 395, 397, 408, 432, 437, 442, 452, 474
  • a microarray comprising at least 10 and no more than 904 oligonucleotides wherein each of the oligonucleotides is capable of specifically binding at least one specific polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-904.
  • a method of diagnosing PTSD in a subject comprising obtaining a cell sample from the subject and detecting in the cell sample the level of expression of at least 10 and no more than 408 polynucleotide sequences wherein each of the polynucleotide sequences is at least 80 % identical to at least one specific polynucleotide selected from the group consisting of SEQ ID NOs: 7, 10, 12, 17, 18, 20, 35, 62, 79, 81, 88, 93, 107, 128, 129, 139, 144, 154, 159, 164, 169, 170, 174, 185, 186, 194, 211, 214, 221, 236, 248, 252, 253, 255, 257, 260, 275, 277, 278, 283, 295, 296, 317, 320, 327, 331, 342, 344, 348, 352, 372, 376-378, 383, 389, 394, 395, 397
  • a method of determining predisposition of a subject to develop PTSD comprising obtaining a cell sample from the subject and detecting in the cell sample the level of expression of at least 10 and no more than 574 polynucleotide sequences wherein each of the polynucleotide sequences is at least 80 % identical to at least one specific polynucleotide selected from the group consisting of SEQ ID NOs: 1-574.
  • a method of preventing PTSD in an individual predisposed to PTSD comprising regulating an expression level of at least one gene selected from the group consisting of SEQ ID NO: 1-574 thereby preventing PTSD in the individual.
  • an agent capable of regulating an expression level of at least one gene selected from the group consisting of SEQ ID NO: 1-574 for the manufacture of a medicament identified for preventing PTSD.
  • a method of treating PTSD in an individual suffering from PTSD comprising regulating an expression level of at least one gene selected from the group consisting of
  • each of the polynucleotides is selected from the group consisting of an oligonucleotide molecule, a cDNA molecule, a genomic molecule and an RNA molecule. According to still further features in the described preferred embodiments each of the polynucleotides is at least 10 and no more than 50 nucleic acids in length.
  • each of the polynucleotides is bound to a solid support.
  • the kit further comprising at least one reagent suitable for detecting hybridization of the polynucleotides and at least one RNA transcript.
  • the kit further comprising packaging materials packaging the at least one reagent and instructions of using the kit in determining predisposition of the subject to develop PTSD.
  • the kit further comprising packaging materials packaging the at least one reagent and instructions of using the kit in diagnosing PTSD in the subject.
  • each of the oligonucleotides is at least 10 and no more than 40 nucleic acids in length.
  • the cell sample is derived from a blood cell sample.
  • the subject has experienced a traumatic event.
  • the traumatic event is caused by a war, a natural disaster, a domestic violence and/or a sexual abuse.
  • detecting is effected at least one month following the traumatic event.
  • detecting is effected within 1-4 months following the traumatic event. According to still further features in the described preferred embodiments detecting is effected within 0.15-24 hours following the traumatic event.
  • detecting is effected within 45-200 minutes following the traumatic event.
  • regulating is upregulating an expression level and/or an activity of the at least one gene and/or a gene product thereof selected from the group consisting of SEQ ID NOs:2-5, 7-
  • upregulating is effected by at least one approach selected from the group consisting of: (a) expressing in cells of the individual an exogenous polynucleotide encoding the at least one gene; (b) increasing expression of the at least one gene in the individual; (c) increasing endogenous activity of the at least one gene product in the individual; (d) introducing an exogenous polypeptide including at least a functional portion of the at least one gene product to the individual; (e) administering cells expressing the at least one gene into the individual.
  • upregulating is effected by an agent selected from the group consisting of: (a) an exogenous polynucleotide encoding the at least one gene; (b) an agent capable of increasing expression of the at least one gene; (c) an agent capable of increasing endogenous activity of the at least one gene product; (d) an exogenous polypeptide including at least a functional portion of the at least one gene product; (e) cells expressing the at least one gene.
  • an agent selected from the group consisting of: (a) an exogenous polynucleotide encoding the at least one gene; (b) an agent capable of increasing expression of the at least one gene; (c) an agent capable of increasing endogenous activity of the at least one gene product; (d) an exogenous polypeptide including at least a functional portion of the at least one gene product; (e) cells expressing the at least one gene.
  • regulating is downregulating an expression level and/or an activity of the at least one gene and/or a gene product thereof selected from the group consisting of SEQ ID NOs: 1, 6, 14, 17, 22, 24, 25, 32, 33, 43, 54, 59, 61, 69, 70, 72, 74, 76, 88, 97, 105, 112, 114, 120, 121, 122, 125, 135, 136, 137, 140, 143, 150, 156, 158, 160, 163, 167, 180, 187, 191, 199, 205, 222, 235, 251, 265, 267, 304, 318, 319, 326, 327, 328, 339, 354, 356, 368, 369, 374, 375, 382, 385, 398, 401, 402, 406, 409, 412, 413, 414, 423, 424, 426, 439, 441, 469, 472, 474, 478, 480
  • downregulating is effected by an agent selected from the group consisting of: (a) a molecule which binds the at least one gene and/or a gene product thereof; (b) an enzyme which cleaves the at least one gene product; (c) an antisense polynucleotide capable of specifically hybridizing with at least part of an mRNA transcript encoded by the at least one gene; (d) a ribozyme which specifically cleaves at least part of an mRNA transcript encoded by the at least one gene; (e) a DNAzyme which specifically cleaves an mRNA transcript or DNA sequence of the at least one gene; (f) a small interfering RNA (siRNA) molecule which specifically cleaves at least part of a transcript encoded by the at least one gene; (g) a non-functional analogue of at least a catalytic or binding portion of the at least one gene product; (h) a molecule which prevents the at least one gene product activation
  • introducing is effected via systemic administration of the agent.
  • regulating is upregulating an expression level and/or an activity of the at least one gene and/or a gene product thereof selected from the group consisting of SEQ ID NOs:7, 10, 12, 18, 20, 35, 62, 79, 81, 93, 107, 128, 129, 139, 144, 154, 159, 164, 169, 170, 174, 185, 186, 194, 211, 214, 221, 236, 248, 252, 253, 255, 257, 260, 275, 277, 278, 283, 295, 296, 317, 320 ,331, 342, 344, 348, 352, 372, 376, 377, 378 ,383, 389, 394, 395, 397, 408, 432, 437, 442, 452, 475, 483, 504, 505, 523, 526, 531, 559, 561, 563, 565, 570, 575-581, 585- 591, 593, 5
  • regulating is downregulating an expression level and/or an activity of the at least one gene and/or a gene product thereof selected from the group consisting of SEQ ID NOs:17, 88, 327, 474, 530, 582-584, 592, 594-596, 603, 606, 613, 614, 618, 620, 621, 624-626, 629, 630, 634, 637, 638, 641, 642, 644-646, 649, 653-655, 663, 665, 669-671, 673, 678, 680, 681, 683-685, 688, 689, 691, 704, 705, 709-711, 716, 717, 720, 721, 728, 729, 732, 733, 735, 736, 741, 743, 745, 753, 756, 758, 764, 771, 774, 776, 779, 782, 786, 787, 789, 791, 792
  • the agent is formulated for systemic administration.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing methods of diagnosing and treating PTSD.
  • FIGs. la-h illustrate changes in expression profiles of genes following trauma.
  • Figures la-c show unsupervised hierarchical clustering of expression profiles of 4512 active genes in peripheral blood monocyte cells (PBMCs) drawn from trauma survivors.
  • Figure Ia all subjects;
  • Figure Ib one hour following a trauma;
  • Figure Ic four months following a trauma.
  • Columns correspond to samples and rows correspond to genes;
  • Control M4 subjects who did not meet any formal PTSD criterion at one and four months following a trauma.
  • Figure Id-f are overabundance plots depicting the number of genes differentiating PTSD from control samples.
  • Figure Id all subjects; Figure Ie - one hour following a trauma; Figure If - four months following a trauma.
  • Observed plot observed abundance of genes separating PTSD and control samples;
  • Expected by chance average number of separating genes, accepted under the null-hypothesis.
  • Figures lg-h are supervised expression profiles depicting the confidence of the classification algorithm along with the expression signature of most significant (p ⁇ 0.05) separating genes. Note the correct classification of 8 out of 9 samples drawn four months following a trauma ( Figure Ig), and 9 out of 11 samples drawn at the ER ( Figure Ih).
  • FIGs. 2a-l depict the correlation between the level of gene expression at four month following a trauma and the continuous Impact of Event Scale (IES) scores.
  • the expression level of various genes was determined in blood samples drawn at four months following a trauma, and was correlated with IES scores which were assessed four months following trauma exposure among all 24 survivors, regardless of the clinical designation above or bellow the threshold for formal PTSD diagnosis. Shown are the IES scores ( Figures 2a, d, g, and i), the level of gene expression ( Figures 2b, e, h, and k) and the Pearson correlation (p ⁇ 0.05) between the IES scores and the level of gene expression ( Figures 2c, f, i, and 1) of 18 available M4 samples exhibiting significant correlations.
  • FIGs. 3a-l depict the correlation between the level of gene expression as determined immediately following a trauma and the continuous Impact of Event Scale (IES) scores.
  • IES Event Scale
  • FIGs. 3a, d, g, and i Shown are the IES scores ( Figures 3a, d, g, and i), the level of gene expression ( Figures 3b, e, h, and k) and the Pearson correlation (p ⁇ 0.05) between the IES scores and the level of gene expression ( Figures 3c, f, i, and 1) of 15 available ER samples exhibiting significant correlations.
  • the significance of the number of correlated genes (p-value) and Pearson correlations were performed as described in Figures 2a-l. Note the significant correlation of the total IES scores (Figure 3c), avoidance (Figure 3f), intrusive memories (Figure 3i), and increased autonomic arousal (Figure 31) with the level of gene expression.
  • FIG. 4a-c illustrate functional groups among informative genes of transcripts differentiating consistent phenotype transcripts.
  • Figure 4a detailed expression profiles of several functional groups among acute PTSD (ER), chronic PTSD (M4), or subjects which did not meet any formal PTSD criterion immediately following a trauma (Control ER) or four months following a trauma (Control M4);
  • Figure 4b averaged over - or under - expression following trauma; Note the marked overall decreased expression of genes coding for transcription enhancers, regulators of protein biosynthesis, protein degradation and cell proliferation following trauma.
  • Figure 4c the frequency of gene ontology annotation among genes differentiating PTSD from control subjects (PTSD vs. Control) as compared with the total active genes on the chip (background). Note the significant increased representation (p ⁇ 0.0005) of genes involved in RNA metabolism or processing and nucleotide metabolism among the genes differentiating PTSD from control subjects.
  • FIGs. 5a-c illustrate the expression profile of genes co-expressed in neural and endocrine tissues following trauma.
  • Figure 5a detailed expression profiles of genes known to be co-expressed in neural and neuroenocrine tissues among subjects who had a definitive clinical diagnosis at both one and four months following a trauma;
  • Figure 5b - averaged over - or under - expression following trauma of genes known to be co- expressed in areas mediating stress reactivity;
  • Figure 5c the frequency of genes known to be co-expressed in areas mediating stress reactivity among the genes differentiating PTSD from control subjects (PTSD vs. Control) or the total active genes on the chip (background).
  • the present invention is of genes differentially expressed in PTSD affected subjects which can be used in determining predisposition to and/or diagnosing PTSD. Specifically, the present invention can be used in preventing and/or treating PTSD by regulating the expression level of the PTSD differentially expressed genes.
  • Post-traumatic stress disorder is a common mental disorder affecting individuals who experienced a life-threatening traumatic event. At the early aftermath of a traumatic event, many trauma survivors exhibit characteristic symptoms of intrusion, avoidance, and hyperarousal.
  • PTSD is usually treated with psychological interventions and psychotropic drugs aimed at reducing symptoms of anxiety and depression.
  • anti-depressant and anti anxiety drugs include serotonin reuptake inhibitors (e.g., fluoxetine, paroxetine, sertraline), atypical antidepressant drugs (e.g. trazodone, nefazodone) and anticonvulsant agents (e.g., gabapentin).
  • serotonin reuptake inhibitors e.g., fluoxetine, paroxetine, sertraline
  • atypical antidepressant drugs e.g. trazodone, nefazodone
  • anticonvulsant agents e.g., gabapentin
  • PTSD refers to post traumatic stress disorder, an anxiety disorder developing following serious traumatic events and characterized by symptoms of intrusion (i.e., re-experiencing or "flash-backs" of the trauma), avoidance (i.e., numbness, diminished emotions and lack of involvement with reality) and hyperarousal (i.e., being constantly threatened by the trauma, irritable or explosive, and having trouble in concentrating or remembering current information).
  • determining predisposition refers to determining susceptibility to develop a disorder.
  • a subject with a predisposition to develop a disorder is more likely to develop the disorder than a non-predisposed subject.
  • the term "subject” includes both young and old human beings of both sexes. Preferably, this term encompasses individuals who experienced a serious traumatic event and are therefore at risk of developing PTSD. Examples include, but are not limited to rape victims, subjects who experienced wars, terror attacks, motor vehicle accidents, natural disasters such as earthquakes, and the like. According to this aspect of the present invention the term “subject” also encompasses individuals who are at risk of being exposed to a traumatic event due to involvement in life-threatening assignments. Examples include, but are not limited to, soldiers, policemen and the like.
  • the method according to this aspect of the present invention is effected by detecting in a cell sample of the subject the level of expression of at least 10 and no more than 574 polynucleotide sequences, wherein each of the polynucleotide sequences is at least 80 % identical to a specific polynucleotide of the set of polynucleotides set forth by
  • the method is preferably effected by detecting the expression level of at least 10 and no more than 127 polynucleotide sequences corresponding to the set of polynucleotides including SEQ ID NOs:4, 7, 24, 27, 28, 29, 36, 37, 42, 46, 51, 53, 62, 64,
  • the phrase "at least 80 % identical" refers to homologous polynucleotide sequences which are at least 80 %, more preferably, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, more preferably at least 99 %, most preferably, 100 % identical to at least one of the polynucleotides of the present invention as determined using the BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters.
  • NCBI National Center of Biotechnology Information
  • the phrase "level of expression” refers to the degree of gene expression and/or gene product activity in a specific tissue or cell sample.
  • up-regulation or down-regulation of various genes can affect the level of the gene product (i.e., RNA and/or protein) in a specific tissue or cell sample.
  • detecting the level of expression of the polynucleotide sequences of the present invention is effected using RNA or protein molecules which are extracted from a cell sample of the subject.
  • RNA or protein molecules from cell samples (e.g., skin, blood, or bone marrow samples) are well known in the art.
  • RNA or protein molecules are preferably characterized for the expression and/or activity level of various RNA and/or protein molecules using methods known in the arts.
  • Northern Blot analysis This method involves the detection of a particular RNA in a mixture of RNAs.
  • An RNA sample is denatured by treatment with an agent (e.g., formaldehyde) that prevents hydrogen bonding between base pairs, ensuring that all the RNA molecules have an unfolded, linear conformation.
  • the individual RNA molecules are then separated according to size by gel electrophoresis and transferred to a nitrocellulose or a nylon-based membrane to which the denatured RNAs adhere.
  • the membrane is then exposed to labeled DNA probes.
  • Probes may be labeled using radio ⁇ isotopes or enzyme linked nucleotides. Detection may be using autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of particular RNA molecules and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the gel during electrophoresis.
  • RNA molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-
  • PCR amplification reaction is carried out in a PCR machine.
  • Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions ⁇ i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific
  • RNA molecules RNA molecules. It will be appreciated that a semi-quantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification product to known controls.
  • RNA in situ hybridization stain DNA or RNA probes are attached to the RNA molecules present in the cells.
  • the cells are first fixed to microscopic slides to preserve the cellular structure and to prevent the RNA molecules from being degraded and then are subjected to hybridization buffer containing the labeled probe.
  • the hybridization buffer includes reagents such as formamide and salts (e.g., sodium chloride and sodium citrate) which enable specific hybridization of the DNA or RNA probes with their target mRNA molecules in situ while avoiding non-specific binding of probe.
  • formamide and salts e.g., sodium chloride and sodium citrate
  • RT-PCR reverse transcriptase polymerase chain reaction
  • Pathol Res Pract. 1994, 190: 1017-25 the RT-PCR reaction is performed on fixed cells by incorporating labeled nucleotides to the PCR reaction.
  • the reaction is carried on using a specific in situ RT-PCR apparatus such as the laser-capture microdissection PixCell I LCM system available from Arcturus Engineering (Mountainview, CA).
  • Oligonucleotide microarray In this method oligonucleotide probes capable of specifically hybridizing with the polynucleotides of the present invention are attached to a solid surface (e.g., a glass wafer). Each oligonucleotide probe is of approximately 20- 25 nucleic acids in length.
  • a specific cell sample e.g., blood cells
  • RNA is extracted from the cell sample using methods known in the art (using e.g., a TRIZOL solution, Gibco BRL, USA).
  • Hybridization can take place using either labeled oligonucleotide probes (e.g., 5'- biotinylated probes) or labeled fragments of complementary DNA (cDNA) or RNA (cRNA).
  • labeled oligonucleotide probes e.g., 5'- biotinylated probes
  • cDNA complementary DNA
  • cRNA RNA
  • double stranded cDNA is prepared from the RNA using reverse transcriptase (RT) (e.g., Superscript II RT), DNA ligase and DNA polymerase I, all according to manufacturer's instructions (Invitrogen Life Technologies, Frederick, MD, USA).
  • RT reverse transcriptase
  • DNA ligase DNA polymerase I
  • the double stranded cDNA is subjected to an in vitro transcription reaction in the presence of biotinylated nucleotides using e.g., the BioArray High Yield RNA Transcript Labeling Kit (Enzo, Diagnostics, Affymetix Santa Clara CA).
  • the labeled cRNA can be fragmented by incubating the RNA in 40 mM Tris Acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate for 35 minutes at 94 °C.
  • the microarray is washed and the hybridization signal is scanned using a confocal laser fluorescence scanner which measures fluorescence intensity emitted by the labeled cRNA bound to the probe arrays.
  • a confocal laser fluorescence scanner which measures fluorescence intensity emitted by the labeled cRNA bound to the probe arrays.
  • each gene on the array is represented by a series of different oligonucleotide probes, of which, each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide.
  • Enzyme linked immunosorbent assay This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate.
  • Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody.
  • Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.
  • Western blot This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents.
  • Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
  • Radio-immunoassay In one version, this method involves precipitation of the desired protein (i.e., the substrate) with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labeled with I 125 ) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
  • a specific antibody and radiolabeled antibody binding protein e.g., protein A labeled with I 125
  • a labeled substrate and an unlabelled antibody binding protein are employed.
  • a sample containing an unknown amount of substrate is added in varying amounts.
  • the decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
  • Fluorescence activated cell sorting This method involves detection of a substrate in situ in cells by substrate specific antibodies.
  • the substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
  • Immunohistochemical analysis This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies.
  • the substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or Giemsa stain.
  • In situ activity assay According to this method, a chromogenic substrate is applied on the cells containing an active enzyme and the enzyme catalyzes a reaction in which the substrate is decomposed to produce a chromogenic product visible by a light or a fluorescent microscope.
  • In vitro activity assays In these methods the activity of a particular enzyme is measured in a protein mixture extracted from the cells. The activity can be measured in a spectrophotometer well using colorimetric methods or can be measured in a non- denaturing acrylamide gel (i.e., activity gel). Following electrophoresis the gel is soaked in a solution containing a substrate and colorimetric reagents. The resulting stained band corresponds to the enzymatic activity of the protein of interest. If well calibrated and within the linear range of response, the amount of enzyme present in the sample is proportional to the amount of color produced. An enzyme standard is generally employed to improve quantitative accuracy.
  • the cell sample used by the present invention can be any cell sample obtained from the subject or subject tissues. Examples include, but are not limited to blood cells, skin cells, bone marrow cells and the like. Methods of obtaining blood, bone marrow and/or epithelial cell samples from an individual are well known in the art.
  • the cell sample is a PBMC sample.
  • a sample can be obtained by drawing ten ml of blood by venipuncture (using EDTA as an anticoagulant) and separating the PBMCs from the blood sample using Histopaque solution gradient (Sigma-Aldrich, USA).
  • predisposition of a subject to develop PTSD can be detected at any time (e.g., prior to participation in a life-threatening assignment or following a traumatic event).
  • the predisposition of a subject to develop PTSD is detected in the cell sample of the subject shortly after a traumatic event, i.e., within 0.15-24 hours of a traumatic event, more preferably, within 0.3-22 hours, more preferably, within 0.5-18 hours, more preferably, within 0.5-10 hours, more preferably, within 0.5-5 hours, more preferably, within 45-200 minutes of a traumatic event.
  • the expression level of the polynucleotide sequences of the present invention can be correlated with the level of expression of PTSD affected individuals as shown in Table 2 and Figures Ia, b, e, and g, and Example 2 of the Examples section which follows and a differential expression of at least some or preferably all of the 574 genes described above is indicative of increased predisposition risk of the individual to develop PTSD.
  • the teachings of the present invention can be used to determine if an individual is predisposed to PTSD. Briefly, 45-200 minutes following a traumatic event a blood sample is obtained from the trauma survivor and PBMCs are isolated using, for example, the Histopaque solution gradient (Sigma-Aldrich, USA). Isolated PBMCs are immediately subjected to RNA extraction using e.g., a TRIZOL solution (Gibco BRL, USA), following which double stranded cDNA is prepared using e.g., Superscript II RT, DNA ligase and DNA polymerase I, all according to manufacturer's instructions (Invitrogen Life Technologies, Frederick, MD, USA).
  • a blood sample is obtained from the trauma survivor and PBMCs are isolated using, for example, the Histopaque solution gradient (Sigma-Aldrich, USA). Isolated PBMCs are immediately subjected to RNA extraction using e.g., a TRIZOL solution (Gib
  • labeled cRNA e.g., using biotin
  • cRNA is prepared in vitro from the double stranded cDNA [using e.g., the BioArray High Yield RNA Transcript Labeling Kit (Enzo)], and further fragmented as described above for 35 minutes at 94 0 C.
  • Hybridization is performed on an array of approximately 120-130 oligonucleotides, each of approximately 20-25 nucleic acids in length, which are capable of specifically hybridizing to the polynucleotides listed in Table 2 which exhibit a p-value of less than 0.047.
  • the arrays are washed to remove access of unbound cRNA probes and the hybridization signals are scanned using for example, the GeneArray scanner G2500A (Hewlett Packard).
  • the scanned images are analyzed using e.g., the Microarray Suite software No. 5 and the level of gene expression is determined in arbitrary units.
  • the observed expression pattern is then compared to the expression pattern of PTSD affected individuals as illustrated in Table 2 and a differential expression of at least some or preferably all of the 574 genes described above is indicative of increased predisposition risk of the individual to develop PTSD.
  • the reagents utilized by the method of determining predisposition to develop PTSD according to the present invention and which are described hereinabove can form a part of a kit.
  • kits for determining predisposition of a subject to develop PTSD includes at least 10 and no more than 574 polynucleotides, each of which can specifically bind at least one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-574.
  • the polynucleotides used by the kit of the present invention can be naturally occurring or synthetic polynucleotides such as oligonucleotides, RNA molecules, genomic DNA molecules, cDNA molecules and/or cRNA molecules.
  • the polynucleotides used by the kit of present invention are of at least 10 and no more than 50 nucleic acids in length, more preferably, at least 15 and no more than 45, more preferably, between 15-40, more preferably, between 20-35, more preferably, between
  • polynucleotides can be bound to a solid support e.g., a glass wafer in a specific order, i.e., in the form of a microarray.
  • oligonucleotides of an array can be synthesized directly on the solid support using well known prior art approaches (Seo TS, et al., 2004, Proc. Natl. Acad. Sci. USA, 101: 5488-93.).
  • the oligonucleotides are attached to the support in a location specific manner such that each specific oligonucleotide has a specific address on the support ⁇ i.e., an addressable location) which denotes the identity ⁇ i.e., the sequence) of that specific oligonucleotide.
  • the kit includes a microarray of 10-574 oligonucleotides, each of 20-25 nucleic acids in length, which correspond to the 574 polynucleotide sequences described hereinabove and which are listed in Table 2.
  • a kit includes 120-130 oligonucleotides corresponding to the polynucleotide sequences exhibiting a p-value of less than 0.047 as listed in Table 2 and described hereinabove.
  • Table 1 Gene expression level in M4 samples
  • Table 1 Differentially expressed genes in PTSD subjects at 4 months following a trauma are presented ( ⁇ Affymetrix ID NO.>_at_ ⁇ GeneBank Accession No.>) along with the normalized gene expression values in each study subject and the overall regulation of gene expression (Expres), i.e., upregulation (Up) or Downregulation (Down) in PTSD subjects as compared with control subjects. P- values represent the significance of the changes in gene expression between PTSD subjects and controls.
  • Table 2 Differentially expressed genes in PTSD subjects immediately following a trauma are presented ( ⁇ Affymetrix ID NO>_at_ ⁇ GeneBank Accession No.>) along with the normalized gene expression values in each study stubject and the overall regulation of gene expression (Expres), i.e., Upregulation (Up) or Downregulation (Down) in PTSD subjects as compared with control subjects. P-values represent the significance of the changes in gene expression between PTSD subjects and controls.
  • Table 3 Description of PTSD differentiating gene
  • Table 3 Differentially expressed genes in PTSD at the ER and four months following a trauma are presented ( ⁇ Affymetrix ID NO.>_at_ ⁇ GeneBank Accession No.>) along with the gene description.
  • the kit further includes at least one reagent as described hereinabove which is suitable for hybridization of the polynucleotides and at least one RNA transcript.
  • reagents include, but are not limited to formam ⁇ de, sodium chloride, and sodium citrate.
  • the kit further comprising packaging material packaging at least one reagent and a notification in or on the packaging material. Such a notification identifies the kit for use in determining predisposition to PTSD in the subject.
  • the kit also includes the appropriate instructions for use and labels indicating FDA approval for use in vitro.
  • the method and kit of determining predisposition to develop PTSD can be used to determine suitability of individuals who experienced a life threatening traumatic event and exhibit symptoms of acute stress disorder to an anti-PTSD treatment. This is of particular importance since current anti-PTSD treatment regimens include antidepressant, anxiolytic or antipsychotic agents which may often result in severe side effects such as sedation, tiredness, movement problems, weight gain, excessive salivation and dizziness. As a result, in some cases such a treatment is employed on individuals which are unlikely to develop chronic PTSD. On the other hand, in other cases, such a treatment is withheld from individuals which are at risk of developing chronic PTSD but are mis-diagnosed.
  • a method of diagnosing PTSD in a subject refers to determining the existence of a disease, which generally involves the evaluation of a patient's medical history, clinical symptoms and laboratory test results.
  • Methods of diagnosing PTSD are known in the art and include, for example, the Clinician Administered PTSD Scale (CAPS, Blake DD, et al. 1990; Behavior Therapist 13:187 -188), a structured clinical interview following the DSM IV diagnostic criteria for PTSD [Diagnostic and Statistical Manual of Mental Disorders - Fourth Edition (DSM-IV), The American Psychiatric Association, Washington D.C., 1994].
  • PTSD symptoms can be assessed using the Revised Impact of Events Scale (IES; Horowitz, M., et al., 1979; Psychosom. Med. 41: 209-218; Weiss DS and Marmar CR. The Impact of Event Scale-Revised. In Wilson JP, Keane TM Eds. Assessing Psychological Trauma and PTSD. New York, Guilford Press 1997:399- 411).
  • the diagnosis of PTSD is made if the symptoms of arousal, intrusion and avoidance which are described hereinabove last for more than one month. It will be appreciated that in cases where such symptoms last for less than a month, the diagnosis of acute stress disorder is made.
  • the method is effected by detecting in a cell sample of the subject the expression level of at least 10 and no more than 408 polynucleotide sequences, each of which being at least 80 % identical to at least one specific polynucleotide from the set of polynucleotides set forth by SEQ ID NOs: 7, 10, 12, 17, 18, 20, 35, 62, 79, 81, 88, 93, 107, 128, 129, 139, 144, 154, 159, 164, 169, 170, 174, 185, 186, 194, 211, 214, 221, 236, 248, 252, 253, 255, 257, 260, 275, 277, 278, 283, 295, 296, 317, 320, 327, 331, 342, 344, 348, 352, 372, 376-378, 383, 389, 394, 395, 397, 408, 432, 437, 442, 452, 474, 475, 483, 504, 505, 523, 5
  • the method is preferably effected by detecting the expression level of at least 10 and no more than 273 polynucleotides of a set including SEQ ID NOs: 17, 582, 583, 584, 595, 596, 606, 613, 614, 620 ,624, 626, 629, 634, 637, 638, 642, 644, 645, 646, 653, 655, 663, 669, 670, 671, 678, 680, 681, 684, 685, 688, 689, 710, 711, 717, 721, 728, 735, 736, 741, 743, 753, 756, 327, 771, 774, 779, 782, 786, 787, 789, 791, 792, 794, 795, 808, 811, 814,
  • detecting the level of expression is preferably performed within a time period of 1-4 months following a traumatic event, more preferably, 2-4 months, more preferably, 3-4 months, most preferably, 4 months following a traumatic event.
  • the method according to this aspect of the present invention can be used to diagnose PTSD in an individual which has experienced a traumatic event such as a motor car accident or act of terror. Briefly, four months following the traumatic event, a blood sample is drawn from the individual and the expression pattern of approximately 273 polynucleotide sequences which are listed in Table 1 and exhibit a p-value of less than
  • 0.016 is determined for RNA molecules obtained from PBMCs, essentially as described hereinabove. The observed expression pattern is then compared to the expression pattern of PTSD affected individuals as illustrated in Table 1. Differential expression of at least some and preferably all of the 408 gene transcripts described above is indicative of positive PTSD diagnosis in the individual.
  • Such a kit includes at least 10 and no more than 408 polynucleotides as described hereinabove.
  • preventing refers to avoiding the progression of chronic PTSD.
  • the phrase "an individual predisposed to PTSD” refers to any individual as described hereinabove which is likely to develop chronic PTSD. It will be appreciated that the phrase “an individual predisposed to PTSD” encompasses also an individual which is identified as predisposed to PTSD according to the teachings of the present invention.
  • the method is effected by regulating an expression level of at least one gene selected from the group consisting of SEQ ID NO: 1-574 thereby preventing PTSD in the individual.
  • regulating refers to upregulating (i.e., increasing) or downregulating (i.e., inhibiting or decreasing) of the expression and/or activity of at least one gene as mentioned hereinabove.
  • regulating is upregulating the expression level and/or an activity of at least one gene and/or gene product thereof selected from the group consisting of SEQ ID NOs: 2-5, 7-13, 15, 16, 18- 21, 23, 26-31, 34-42, 44-53, 55-58, 60, 62-68, 71, 73, 75, 77-87, 89-96, 98-104, 106-111, 113, 115-119, 123, 124, 126-134, 138, 139, 141, 142, 144-149, 151-155, 157, 159, 161, 162, 164-166, 168-179, 181-186, 188-190, 192-198, 200-204, 206-221, 223-234, 236- 250, 252-264, 266, 268-303, 305-317, 320-325, 329-338, 340-353, 355, 357-367, 370- 373, 376-381, 383, 384,
  • Upregulation of the downregulated gene of the present invention can be effected at the genomic level (i.e., activation of transcription via promoters, enhancers, regulatory elements), at the transcript level (i.e., correct splicing, polyadenylation, activation of translation) or at the protein level (i.e., post-translational modifications, interaction with substrates and the like).
  • An agent capable of upregulating expression level of the downregulated gene of the present invention may be an exogenous polynucleotide sequence designed and constructed to express at least a functional portion of the downregulated gene protein.
  • a polynucleotide sequence encoding the downregulated gene of the present invention [(e.g., GenBank Accession number AF065482 (SEQ ID NO:7) or AB020658 (SEQ ID NO: 12)] is preferably ligated into a nucleic acid construct suitable for mammalian cell expression.
  • a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
  • nucleic acid construct of the present invention can also utilize sequences which are homologous to the downregulated gene of the present invention, i.e., at least 80 % identical as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals —9.
  • Constitutive promoters suitable for use with the present invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).
  • Inducible promoters suitable for use with the present invention include for example the tetracycline-inducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804).
  • the nucleic acid construct (also referred to herein as an "expression vector") of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
  • a typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
  • Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
  • the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
  • the other upstream promoter elements determine the rate at which transcription is initiated.
  • Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 1983, which is incorporated herein by reference.
  • CMV cytomegalovirus
  • the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation of the downregulated gene of the present invention.
  • Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
  • Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40.
  • the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA.
  • a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
  • the vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
  • the expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • IRS internal ribosome entry site
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
  • viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the type of vector used by the present invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
  • Recombinant viral vectors are useful for in vivo expression of the downregulated gene of the present invention since they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • upregulation of the downregulated gene of the present invention can be also effected by administration of cells expressing the downregulated gene of the present invention into the individual.
  • Cells expressing the downregulated gene of the present invention can be any suitable cells, such as bone marrow stem cells, mesenchymal stem cells, lymphocyte cells, neural stem cells, and/or endocrine stem cells which are derived from the individuals and are transfected ex vivo with an expression vector containing the polynucleotide designed to express the downregulated gene of the present invention as described hereinabove.
  • Administration of the cells expressing the downregulated gene of the present invention can be effected using any suitable route such as intravenous, intra peritoneal, and intra-spinal.
  • Cells expressing the downregulated gene of the present invention can be derived from either autologous sources such as self bone marrow cells or from allogeneic sources such as bone marrow or other cells derived from non-autologous sources. Since non- autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non- autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells or tissues in immunoisolating, semipermeable membranes before transplantation. Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludagj H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).
  • microcapsules Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha- phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479- 83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. MoI Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha- cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.
  • microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 ⁇ m.
  • HEMA 2-hydroxyethyl methylacrylate
  • MAA methacrylic acid
  • MMA methyl methacrylate
  • Such microcapsules can be further encapsulated with additional 2-5 ⁇ m ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S.M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 200223: 849-56).
  • microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives.
  • microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
  • An agent capable of upregulating the downregulated gene of the present invention may also be any compound which is capable of increasing the transcription, translation and/or activity of an endogenous DNA, mRNA or protein encoded by the downregulated gene of the present invention and thus increasing endogenous activity of the downregulated gene of the present invention.
  • An agent capable of upregulating the downregulated gene of the present invention may also be an exogenous polypeptide including at least a functional portion (as described hereinabove) of the downregulated gene of the present invention.
  • regulating is downregulating an expression level and/or an activity of at least one gene and/or a gene product thereof selected from the group consisting of SEQ ID NOs: 1, 6, 14, 17, 22, 24, 25, 32, 33, 43, 54, 59, 61, 69, 70, 72, 74, 76, 88, 97, 105, 112, 114, 120, 121, 122, 125,
  • Downregulation of the upregulated gene of the present invention can be effected on the genomic and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., antisense, siRNA, Ribozyme, DNAzyme), or on the protein level using e.g., antagonists, enzymes that cleave the polypeptide and the like.
  • an agent capable of downregulating the upregulated gene of the present invention is an antibody or antibody fragment capable of specifically binding the upregulated gene of the present invention.
  • the antibody specifically binds at least one epitope of the upregulated gene of the present invention.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages.
  • These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage ⁇ of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage ⁇ of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (1972O].
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
  • the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et ah, Nature, 321:522-525 (1986); Riechmann et al, Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co- workers [Jones et al, Nature, 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al, J. MoI. Biol., 222:581 (1991)]. The techniques of Cole et a ⁇ . and Boerner et al.
  • human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • RNA interference is a two step process.
  • the first step which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP- dependent manner.
  • nt nucleotide
  • siRNA small interfering RNAs
  • RNA 19-21 bp duplexes (siRNA), each with 2-nucleotide 3' overhangs [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature 409:363-366 (2001)].
  • the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC.
  • the active RISC targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)].
  • each RISC contains a single siRNA and an RNase [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].
  • RNAi molecules suitable for use with the present invention can be effected as follows. First, the mRNA sequence of the upregulated gene of the present invention is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites.
  • UTRs untranslated regions
  • UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl, T. 2001, ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).
  • potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out. Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction.
  • Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • Another agent capable of downregulating the upregulated gene of the present invention is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the upregulated gene of the present invention. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F.
  • DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.
  • Downregulation of the upregulated gene of the present invention can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the upregulated gene of the present invention.
  • the first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells
  • the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
  • the prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Lucas J MoI Med 76: 75-6 (1998); Kronenwett et al. Blood 91: 852-62 (1998); Rajur et al.
  • antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et ah, Curr Opin MoI Ther 1:372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin MoI Ther 1:297-306 (1999)].
  • Another agent capable of downregulating the upregulated gene of the present invention is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding the upregulated gene of the present invention.
  • Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)].
  • the possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
  • ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al, Clin Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular
  • Endothelial Growth Factor receptor a key component in the angiogenesis pathway.
  • HEPTAZYME Hepatitis C Virus
  • Another agent capable of downregulating the upregulated gene of the present invention would be any molecule which binds to and/or cleaves the upregulated gene of the present invention. Such molecules can be antagonists or inhibitory peptides of the upregulated gene of the present invention.
  • a non-functional analogue of at least a catalytic or binding portion of the upregulated gene of the present invention can be also used as a suitable downregulating agent.
  • Another agent which can be used along with the present invention to downregulate the upregulated gene of the present invention is a molecule which prevents activation or substrate binding of the upregulated gene of the present invention.
  • upregulation or downregulation of specific genes is associated with the existence of chronic PTSD (see Example 2 of the Examples section which follows) downregulation or upregulation, respectively, thereof can be utilized to treat individuals suffering from chronic PTSD.
  • treating refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition in an individual suffering from, or diagnosed with, the disease, disorder or condition.
  • Those of skill in the art will be aware of various methodologies and assays which can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays which can be used to assess the reduction, remission or regression of a disease, disorder or condition.
  • the phrase “individual suffering from PTSD” refers to an individual which experienced a traumatic event as described hereinabove, and exhibits the full phenotype of PTSD as evaluated following at least one month of the trauma.
  • the phrase "individual suffering from PTSD” encompasses also individual who is diagnosed with PTSD according to the teachings of the present invention which are described hereinabove.
  • the method comprising regulating an expression level of at least one gene selected from the group consisting of SEQ ID NOs: 7, 10, 12, 17, 18, 20, 35, 62, 79, 81, 88, 93, 107, 128, 129, 139, 144, 154, 159, 164, 169, 170, 174, 185, 186, 194, 211, 214, 221, 236, 248, 252, 253, 255, 257, 260, 275, 277, 278, 283, 295, 296, 317, 320, 327, 331, 342, 344, 348, 352, 372, 376-378, 383, 389, 394, 395, 397, 408, 432, 437, 442, 452, 474, 475, 483, 504, 505, 523, 526, 530, 531, 559, 561, 563, 565, 570 and 575-904 thereby treating PTSD in the individual.
  • the term "regulating" refers to upregulating (as described above) the expression level and/or an activity of at least one gene and/or a gene product thereof selected from the group consisting of SEQ ID NOs: 7, 10, 12, 18, 20, 35, 62, 79, 81, 93, 107, 128, 129, 139, 144, 154, 159, 164, 169, 170, 174, 185, 186, 194, 211, 214, 221, 236, 248, 252, 253, 255, 257, 260, 275, 277, 278, 283, 295, 296, 317, 320, 331, 342, 344, 348, 352, 372, 376, 377, 378 ,383, 389, 394, 395, 397, 408, 432, 437, 442, 452, 475, 483, 504, 505, 523, 526, 531, 559, 561, 563, 565, 570, 575-581, 585-591
  • the term "regulating" is downregulating (as described above) the expression level and/or an activity of at least one gene and/or a gene product thereof selected from the group consisting of SEQ ID NOs:17, 88, 327, 474, 530, 582-584, 592, 594-596, 603, 606, 613, 614, 618, 620, 621, 624-626, 629, 630, 634, 637, 638, 641, 642, 644-646, 649, 653-655, 663, 665, 669-671, 673, 678, 680, 681, 683-685, 688, 689, 691, 704, 705, 709-711, 716, 717, 720, 721, 728, 729, 732, 733, 735, 736, 741, 743, 745, 753, 756, 758, 764, 771, 774, 776, 779, 782, 786, 787, 789
  • an expression vector e.g., a viral vector
  • a polynucleotide sequence encoding the downregulated gene of the present invention e.g., SEQ ID NO:26 which can be used in preventing PTSD and SEQ ID NO: 7 which can be used in treating PTSD
  • suitable promoter sequences to enable expression in brain cells is introduced into the individual via, e.g., intravenous administration.
  • Expression of such a vector in the brain is expected to upregulate the expression level and/or activity of the downregulated gene in such cells and thus to prevent and/or reduce the symptoms of PTSD (e.g., hallucinatory experiences).
  • Dosage of such an expression vector should be calibrated using cell culture experiments and animal models. Success of treatment is preferably evaluated by determining the individual mental status using for example the Clinician Administered PTSD Scale and the Revised Impact of Events Scale.
  • the prevention and/or treatment of PTSD can be also accomplished using for example, an siRNA molecule.
  • a suitable siRNA molecule which can be used to prevent or treat PTSD is the 5'- uguuccacaucgccugcuuca -3' (SEQ ID NO:905) or the 5'- caucgugauucuccagcucaa -3' (SEQ ID NO:906) which can specifically cleave the transcript of zyxin (SEQ ID NO:1) or ELA2, a neutrophil specific elastase 2 (SEQ ID NO: 17), respectively.
  • Such an siRNA molecule can be administered to the individual using intravenous administration.
  • dosage and duration of treatment can vary between individuals, depending on the individual general health status and severity of PTSD symptoms.
  • Each of the upregulating or downregulating agents described hereinabove or the expression vector encoding the downregulated gene of the present invention can be administered to the individual per se or as part of a pharmaceutical composition which also includes a physiologically acceptable carrier.
  • the purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.
  • a pharmaceutical composition refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the upregulating or downregulating agent or the expression vector encoding the downregulated gene of the present invention which are accountable for the biological effect.
  • pharmaceutically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • the pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (the upregulating or downregulating agent or the expression vector encoding the downregulated gene) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., avoidance, intrusion and arousal) or prevent the consistence full-PTSD phenotype in the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models [such as those described in Kaufer, 1998 (Supra)] to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active ingredient are sufficient to prevent and/or treat PTSD (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.
  • the term "about” refers to ⁇ 10 %.
  • Study subjects Study subjects were individuals who survived a trauma and were admitted to the emergency room immediately following a traumatic event (mean time between incident and arrival was 45 ⁇ 130 minutes). Trauma survivors were evaluated for the presence of acute and chronic PTSD at one and four months following a trauma event according to the DSM IV diagnostic criteria [Diagnostic and Statistical Manual of
  • inclusion criteria included subjects at the ages of 18-65, who met the DSM IV diagnostic criterion at one and four months following a trauma.
  • the control group included subjects who experience the trauma but did not meet any of the DSM IV diagnostic criterion following one month.
  • Exclusion criteria were the presence of a head, burn or serious physical injury, the presence or history of alcohol or illicit drugs abuse, psychiatric diagnoses other than depressive or anxiety disorders, or a medical or neurological illness that could confound the assessments.
  • PTSD evaluation - PTSD status at one and four months was determined using the Clinician Administered PTSD Scale (CAPS, Blake DD, et al. 1990; Behavior Therapist 13:187 -188), a structured clinical interview following the DSM IV diagnostic criteria for PTSD.
  • the co-occurrence of other mental disorders was ascertained by the Structured Clinical Interview for DSM IV Mental Disorders (SCID, Spitzer RL, et al., 1994; Biometric Research Department, New York State Psychiatric Institute). Trauma severity was assessed using a trauma severity scale (Shalev et al., 1998; American Journal of Psychiatry, 155:630-637) addressing the severity of the event in terms of threat to own life, and exposure to others dead or wounded or other undesirable sights.
  • CID Structured Clinical Interview for DSM IV Mental Disorders
  • Trauma severity was assessed using a trauma severity scale (Shalev et al., 1998; American Journal of Psychiatry, 155:630-637) addressing the severity of
  • PTSD symptom severity was assessed using the Revised Impact of Events Scale (IES; Horowitz, M., et al., 1979; Psychosom. Med. 41: 209-218; Weiss DS and Marmar CR. The Impact of Event Scale-Revised. In Wilson JP, Keane TM Eds. Assessing Psychological Trauma and PTSD. New York, Guilford Press 1997:399-411), by scoring three symptom clusters of PTSD: re-experiencing, avoidance and hyper arousal, or the total IES score summarizing all PTSD symptoms.
  • IES Revised Impact of Events Scale
  • Table 4 hereinbelow, out of the twenty-four trauma survivors, eight subjects exhibited persistent full diagnostic criteria at both one and four months after trauma (PTSD - consistent phenotype subjects) and six subjects met no formal clinical criterion for PTSD at any time (control - consistent phenotype subjects). Five subjects exhibited partial intermediate PTSD clinical criteria at one month and full criteria at four months following a trauma, and five other subjects exhibited partial intermediate PTSD clinical criteria at one month which were resolved at four month (i.e., partial phenotype subjects).
  • Table 4 Clinical diagnosis of PTSD following one (acute PTSD, Ml) or four months (chronic PTSD, M4) after trauma.
  • Y yes, full blown acute (one month) or chronic (four m diagnostic criteria for PTSD;
  • Pa partial, sub-threshold criteria for acute (one month) or chronic (four months) PTSD;
  • gene expression analysis was performed in blood samples taken immediately or four months following a trauma, and the expression pattern was correlated with the presence of acute or chronic PTSD, as follows.
  • Sample preparation and microarray hybridization Ten ml of blood were drawn by venipuncture from each subject at the ER and four months following exposure to trauma, using EDTA as anticoagulant. Blood samples were kept at room temperature for up to one hour until processing. Peripheral Blood Monocyte Cells (PMBCs) were separated using Histopaque solution gradient (Sigma-Aldrich, USA), immediately transferred to TRIZOL solution (Gibco BRL, USA) and total RNA was extracted according to the array manufacturer's instructions (Affymetrix, Santa Clara, CA).
  • PMBCs Peripheral Blood Monocyte Cells
  • RNA was purified using RNeasy Mini kit (Qiagen, Germany), precipitated at -20 °C overnight using 7.5 M ammonium acetate (0.4 times of the cRNA volume) (Sigma) and absolute ethanol (2.5 times of the total volume), and washed.
  • the expression value (average difference) for each gene was determined by calculating the average of differences of intensity (perfect match intensity minus mismatch intensity) between the probe pairs. Removal of the batch effect from the array was performed by calculating the average gene expression of each gene within each group and normalizing each expression measurement with the average expression of its group.
  • the expression analysis files created by GENECHIP 3.1 software were transferred to a database (Microsoft Access) and linked to Internet genome databases (e.g., NHLBI, Swiss Prot, and GeneCards).
  • Mean intensity for each sample was defined as the mean of average differences of individual samples from all subjects. Fold changes were determined by dividing the mean intensity of each sample by the mean intensity derived from all samples. All values below 20 were brought to 20 and all values above 10,000 were brought to 10,000. Genes were identified as active if exhibited at least one value between 50 to 7500, one present call by Microarray Analysis Suite 5.0, and exhibited more than 2-fold change from average value in at least one sample.
  • SPOTFIRE PRO 3.0 spotfire, Goteborg, Sweden
  • Clustering Un-supervised clustering was performed using DoublePCluster, an agglomerative hierarchical bi-clustering approach based on similarity in expression profiles. The similarity is measured with a generative probabilistic model identifying an optimal distribution model of the expression values in each of the clusters.
  • standard hierarchical clustering was performed using the GENE CLUSTER and TREEVIEW programs cluster softwares as well a newly developed cluster analysis algorithm (Eisen MB, et al., 1998; Proc. Natl. Acad. Sci. USA, 95: 14863-14868; Friedman N. PCluster: Probabilistic Agglomerative Clustering of Gene Expression Profile. Technical report 2003-80 School of Computer Science & Engineering, Hebrew University, 2003).
  • Differentially expressed signatures The significance of the number of differentially expressed signatures was determined using a randomized permutation test to compute the probability of selecting that many genes under the null hypothesis of random class assignment.
  • ScoreGenes analysis package http://compbio.cs.huii.ac.il/scoregenes/) by calculating for each p-value the accumulated number of genes that were scored with this p-value or better. The significance of the abundance plot was determined using a randomize permutation test with 1000 random reshufflings of labels.
  • Prediction of class assignment - Prediction of the label of a new sample was performed using the Naive Bayesian Classifier method (Duda RO, Hart PE. "Pattern Classification and Scene Analysis", John Wiley & Sons, New York, 1973).
  • the probability of gene expression value in each group and the probability of the observed pattern (for selected genes) are computed and the log-odds ratio between the two probabilities is calculated.
  • the sign of the log-odds defines the predicted class, and the magnitude represents the confidence in the prediction.
  • Leave-one-out cross validation (LOOCV) - The leave-one-out cross validation procedure [Ben-Dor, 2000 (Supra)] was used to evaluate the performance of the classifier on unseen samples.
  • PTSD symptoms include avoidance, re-experiencing, and hyper arousal, each manifesting with a variable intensity among survivors of trauma (Shalev AY., et al.,
  • Total IES score correlates with trauma severity but not the other personal and demographic variables - To identify factors affecting the overall PTSD symptoms, the
  • Table 7 Comparison by personal and demographic variables and pearson correlations with total month four Impact of Event Scale (IES) score among all trauma survivors.
  • NS not- significant using 2-tailed t test (p > 0.1).
  • transcripts co-expressed in neuroendocrine tissues Analysis of transcript expression in normal brain and endocrine tissues was performed by obtaining annotations from OMIM and UniGene and comparing with the U95 microarray transcripts using the Affymetrix netaffex query system. Each transcript was annotated with a tissue or process in which is expressed.
  • transcripts differentiating consistent phenotype groups of PTSD and controls were classified according to functional groups. As is shown in Figure 4a transcripts encoding for proteins which are involved in transcriptional activation, cell cycle and proliferation were downregulated among PTSD affected subjects. Noteworthy, similar results were obtained for transcripts differentiating spectral PTSD symptoms subjects from the controls (not shown). In addition, as is shown in Figure 4b, distinct expression signatures for transcripts involved in immune activation, signal transduction and apoptosis were observed.
  • PBMCs are known to be perturbed following acute psychological stress (Aloe L, et al., 1994. Proc. Natl. Acad. Sci. U. S. A. 91: 10440-4), in part through neuroendocrine and sympathetic modulation (McEwen BS. 1998. N. Engl. J. Med. 338: 171-9). Long-term alterations in sympathetic (Southwick SM., et al., 1999. Biol. Psychiatry. 46: 1192-204) and HPA reactivity (Yehuda, R. 2002. N. Engl. J. Med.
  • the current practice is based on grouping trauma survivors into those with or without clinical PTSD, by applying a severity threshold on the conglomerate score of the three PTSD symptom clusters (DSM-IV), with a consequent inherent loss of data (Andreasen NC. 1997. Science. 1997. 275: 1586-93; Radant A., et al., 2001. Psychiatry Res. 102: 203-15).
  • DSM-IV severity threshold on the conglomerate score of the three PTSD symptom clusters
  • the results presented in the present invention demonstrate that gene expression signatures in PBMCs contain information that is highly correlated with continuous symptom severity measures among all trauma survivors regardless of threshold clinical designation, and for each of the three key biological dimensions that compose PTSD.
  • initial PBMCs gene expression signatures are informative of later clinical course, and could have a significant potential for guiding early detection and thus early intervention among survivors of trauma.
  • gene expression patterns in cancer tissues are indicative of a patient's prognosis (van de Vijver MJ, et al. 2002. N. Engl. J. Med. 347: 1999-2009; Rosenwald A, et al., 2002. N. Engl. J. Med. 346: 1937-47)
  • these data suggest that such information exists in the much more accessible peripheral blood.
  • expression signatures among PBMCs in response to extreme psychological stress may reflect in part genomic predisposition to develop PTSD, beyond the putative participation of immune cells in this neuropsychiatric disorder.
  • Genes showing expression differences in lymphocytes from two patients with bipolar disorder have recently been shown to constitute promising candidates for search of causative genomic polymorphisms associated with risk for the disorder, suggesting that peripheral expression differences contain pathogenetically relevant information for the neuropsychiatric process (Kakiuchi C, et al., 2003. Nat. Genet. 35: 171-5).
  • Indirect support for this notion can be found in the increased proportion of genes co-expressed in brain and endocrine tissues, as well as specific genes related to neural transduction of stress among the informative transcripts observed in PBMCs.
  • results presented in the present invention demonstrate a general reduction in PBMCs' expression of transcription activators among psychologically affected trauma survivors in response to stress. This decrease may explain much of the differences in gene expression signatures observed between the PTSD and control subjects. It remains to be established if some of the robust differences among PBMCs in gene transcripts related to transcriptional activation, intracellular signaling pathways, cell cycle, and apoptosis, might be indicative of parallel changes occurring among cell populations more relevant to central stress reactivity. Genomic variation may drive related transcriptional reactivity among glial cells that share closer embryonal derivation to leukocytes or even among neuronal cells. Reduced hippocampal volumes have been described among PTSD patients (Gilbertson MW, et al. 2002. Nat.
  • Neurosci. 11: 1242-7 Altered neuroendocrine reactivity, signal transduction, and cellular proliferation and demise among neural and glial cells, have been implicated in hippocampal volume depletion [Kakiuchi, 2003 (Supra); Gilbertson, 2002 (Supra); Kim JJ, and Diamond DM. 2002. Nat. Rev. Neurosci. 6: 453-62], as well as in fear avoidance formation (Schafe GE, et al., 2001. Trends Neurosci. 24: 540-546) and memory consolidation (McEwen BS. 2001. Ann. N. Y. Acad. Sci.
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Abstract

La présente invention porte sur des gènes différentiellement exprimés chez des sujets atteints de trouble de stress post-traumatique, ces gènes pouvant être utilisés pour déterminer une prédisposition à un trouble de stress post-traumatique et/ou effectuer un diagnostic de ce trouble. Cette invention peut-être notamment utilisée dans la prévention et/ou le traitement de trouble de stress post-traumatique en régulant le taux d'expression des gènes différentiellement exprimés chez des sujets atteints de trouble de stress post-traumatique.
PCT/IL2005/000824 2004-08-02 2005-08-02 Compositions et methodes de diagnostic et de traitement de trouble de stress post-traumatique WO2006013561A2 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010029176A1 (fr) * 2008-09-12 2010-03-18 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Gènes associés à un trouble de stress post-traumatique (ptsd)
CN103068982A (zh) * 2010-07-14 2013-04-24 库尔纳公司 通过抑制盘状大同系物(dlg)的天然反义转录物而治疗dlg相关疾病
US8481507B2 (en) 2007-07-31 2013-07-09 The Board Of Regents, The University Of Texas System Micro-RNAs that control myosin expression and myofiber identity
US8629119B2 (en) 2009-02-04 2014-01-14 The Board Of Regents, The University Of Texas System Dual targeting of MIR-208 and MIR-499 in the treatment of cardiac disorders
WO2015027116A1 (fr) * 2013-08-21 2015-02-26 The Regents Of The University Of California Motifs de métabolites pour le diagnostic et la prédiction de troubles affectant le cerveau et le système nerveux
JP2017078720A (ja) * 2011-09-14 2017-04-27 ザ ヘンリー エム． ジャクソン ファウンデーション フォー ザ アドバンスメント オブ ミリタリー メディシン，インコーポレーテッド 心的外傷後ストレス障害（ｐｔｓｄ）のための診断用バイオマーカーを検出及び監視するため、並びに同障害の自殺型と非自殺型とを識別するためのプロセス及びキット
CN106701900A (zh) * 2015-11-16 2017-05-24 上海市东方医院 长链非编码rna herc2p3基因及其在胃癌中的用途
US9856532B2 (en) * 2012-09-07 2018-01-02 Institute For Systems Biology Markers and methods for detecting posttraumatic stress disorder (PTSD)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2414403A1 (fr) * 2000-07-06 2002-01-17 The Regents Of The University Of California Procede destine au diagnostic et au traitement de troubles mentaux
JP3694674B2 (ja) * 2001-02-28 2005-09-14 株式会社日立製作所 オリゴヌクレオチドアレイおよび検査方法

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8481507B2 (en) 2007-07-31 2013-07-09 The Board Of Regents, The University Of Texas System Micro-RNAs that control myosin expression and myofiber identity
US8962588B2 (en) 2007-07-31 2015-02-24 The Board Of Regents, The University Of Texas System Micro-RNAS that control myosin expression and myofiber identity
WO2010029176A1 (fr) * 2008-09-12 2010-03-18 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Gènes associés à un trouble de stress post-traumatique (ptsd)
US9243293B2 (en) 2008-09-12 2016-01-26 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Genes associated with posttraumatic-stress disorder (PTSD)
US8629119B2 (en) 2009-02-04 2014-01-14 The Board Of Regents, The University Of Texas System Dual targeting of MIR-208 and MIR-499 in the treatment of cardiac disorders
US8980860B2 (en) 2010-07-14 2015-03-17 Curna, Inc. Treatment of discs large homolog (DLG) related diseases by inhibition of natural antisense transcript to DLG
CN103068982A (zh) * 2010-07-14 2013-04-24 库尔纳公司 通过抑制盘状大同系物(dlg)的天然反义转录物而治疗dlg相关疾病
JP2017078720A (ja) * 2011-09-14 2017-04-27 ザ ヘンリー エム． ジャクソン ファウンデーション フォー ザ アドバンスメント オブ ミリタリー メディシン，インコーポレーテッド 心的外傷後ストレス障害（ｐｔｓｄ）のための診断用バイオマーカーを検出及び監視するため、並びに同障害の自殺型と非自殺型とを識別するためのプロセス及びキット
AU2012308305B2 (en) * 2011-09-14 2017-11-23 Banyan Biomarkers, Inc. Processes and kits to detect and monitor for diagnostic biomarkers for post traumatic stress disorder (PTSD) and to differentiate between suicidal and non-suicidal form of the disorder
US9856532B2 (en) * 2012-09-07 2018-01-02 Institute For Systems Biology Markers and methods for detecting posttraumatic stress disorder (PTSD)
WO2015027116A1 (fr) * 2013-08-21 2015-02-26 The Regents Of The University Of California Motifs de métabolites pour le diagnostic et la prédiction de troubles affectant le cerveau et le système nerveux
CN106701900A (zh) * 2015-11-16 2017-05-24 上海市东方医院 长链非编码rna herc2p3基因及其在胃癌中的用途
CN106701900B (zh) * 2015-11-16 2020-03-20 上海市东方医院 长链非编码rna herc2p3基因及其在胃癌中的用途

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