# Patent

## Title
Regulators of NFAT and/or store-operated calcium entry

## Abstract

Embodiments of the inventions relate to modulating NFAT activity, modulating store-operated Ca2+ entry into a cell and treating and/or preventing hyperactivity or inappropriate immune response by inhibiting the expression or activities of proteins involved in the calcineurin/NFAT axis.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/901,195 filed on Oct. 8, 2010, now U.S. Pat. No. 8,394,778, issued Mar. 12, 2013, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/249,709 filed Oct. 8, 2009, the contents of which are incorporated herein by reference in their entireties.


GOVERNMENT SUPPORT
This invention was made with Government support under contract Nos. A140127 and GM075256, awarded by the National Institutes of Health. The Government has certain rights in the invention.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 5, 2013, is named 20130207_SequenceListing-TextFile—033393—062756_C and is 60,175 bytes in size.
FIELD OF THE INVENTION
The invention relates to modulating nuclear factors of activated T-cell (NFAT) and/or store operated Ca2+ entry (SOCE) in cells, in particular T cells. The invention relates to the regulation of the activation of T cells and the modulation of immune responses.
BACKGROUND OF INVENTION
The calcium/calcineurin-dependent NFAT family is thought to have arisen following the recombination of an ancient precursor with a Rel domain about 500 million years ago, producing a new group of signaling and transcription factors (the NFAT genes) found only in the genomes of vertebrates. The family of NFAT transcription factor consists of five members NFAT1, NFAT2, NFAT3, NFAT4 and NFAT5. The NFAT proteins are activated by an increase in intracellular calcium levels, e.g., by means of store-operated calcium entry (SOCE). Calcium signaling is critical to NFAT activation because calmodulin, a well-known calcium sensor protein, activates the serine/threonine phosphatase calcineurin. Activated calcineurin rapidly dephosphorylates the serine rich region (SRR) and SP-repeats in the amino termini of NFAT proteins resulting in a conformational change that exposes a nuclear localization signal resulting in NFAT nuclear import. The activated NFAT proteins, in turn, induce transcription of cytokine genes which are required for an immune response.
Nuclear import of NFAT proteins is opposed by maintenance kinases in the cytoplasm and export kinases in the nucleus. Export kinases, such as PKA and GSK-3β, must be inactivated for NFAT nuclear retention. NFAT proteins have weak DNA binding capacity. Therefore, to effectively bind DNA NFAT proteins must cooperate with other nuclear resident transcription factors. This important feature of NFAT transcription factors enables integration and coincidence detection of calcium signals with other signaling pathways such as ras-MAPK or PKC. In fact, cell biological, genetic and biochemical evidence indicates that the circuitry of this pathway is well suited for intercalation with older pathways, such as MAP kinase, WNT and NOTCH. This recombination enabled Ca2+ signals to be redirected to a new transcriptional program, which provides part of the groundwork for vertebrate morphogenesis and organogenesis. Indeed, the calcineurin/NFAT axis is involved in numerous aspects of vertebrate morphogenesis: cell cycle regulation, cell differentiation, cell survival, angiogenesis, tumor cell invasion and metastasis, myogenesis, chondrocytes differentiation and the development of the cardiovascular system, the complex nervous system and the recombinational immune system. Consequently, deregulation of calcineurin/NFAT signaling and/or abnormal expression of its components have been associated with cell proliferation diseases such as cancer, autoimmune diseases, cardiovascular diseases, diabetes, and bone diseases to name a few. Discovery of modulators of Ca2+ influxes and/or the calcineurin/NFAT axis can provide therapeutic avenues for these diseases.
SUMMARY OF THE INVENTION
Embodiments of the invention are based on the discovery that several hundred genes in the human and mouse genomes whose gene products directly and/or indirectly modulate nuclear factors of activated T cell (NFAT) activation and/or modulate the store-operated Ca2+ entry (SOCE) into a cell. NFAT is a family of transcription factors that normally reside in the cytoplasm when inactive. When activated by dephosphorylation by calcineurin, the NFATs can translocate into the nucleus and “turn on” specific gene transcription. The inventors developed a cell-based reporter system for screening for modulators of (NFAT) and/or store-operated Ca2+ entry into a cell, with NFAT nuclear translocation as the readout for scoring a modulator. The cell-based reporter system comprises a mammalian cell co-expressing a NFAT-GFP, a STIM1-RFP, and a Orai1-FLAG. The markers: GFP, RFP and FLAG-tag facilitate the visual localization of the respectively expressed proteins within the cell compartments. Thapsigargin (TG), a tight-binding inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase, was used to deplete the Ca2+ in the endoplasmic reticulum and initiate SOCE, which in turn leads to NFAT dephosphorylation and NFAT nuclear translocation. The inventors used the cytoplasm-to-nuclear translocation of NFAT-GFP as their assay readout.
The inventors performed a large scale high-throughput siRNA screening of the human and mouse genome for genes that modulate NFAT nuclear translocation and/or SOCE. Genes that modulate the NFAT nuclear translocation and/or SOCE can either up-regulate (i.e. promote) or down-regulate (i.e. inhibit) NFAT nuclear translocation and/or SOCE. NFAT nuclear translocation and/or SOCE are necessary for the activation of T cells, the proliferation of activated T cells, and for maintaining the immune response involving T- and B-cells in the body. In addition, the NFAT translocation is associated with multiple signaling pathways such as the MAP kinase, WNT, and NOTCH signaling pathways. As such, NFATs directly and/or indirectly play important roles in cell proliferation and regeneration, cancer, angiogenesis, cardiovascular diseases, diabetes, neural regeneration, bone diseases and T cell adaptation to name a few. Therefore, identification of the modulator genes of NFAT nuclear translocation and/or SOCE can allow therapeutic regulation of the immune system, immune responses and other disease conditions associated with NFATs.
Inhibition of genes that up-regulate NFAT nuclear translocation and/or SOCE can help inhibit T-cell activation and immune response associated with hyperactivity or inappropriate activity of the immune system. Conversely, inhibition of genes that down-regulate NFAT nuclear translocation and/or SOCE can help increase T cells activation and immune responses associated with immune deficiency disease or conditions.
Secondary and tertiary screens of the hits from primary screens were conducted. Secondary and tertiary screens comprise Ca2+ influx as readout for scoring.
Accordingly, provided herein a method of modulating NFAT activity, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein and/or the expression of a gene identified in Table 1, 2, 3 or 4.
In one embodiment, provided herein is a method of modulating store-operated Ca2+ entry into a cell, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Table 1, 2, 3 or 4, and/or the expression of a gene identified in Table 1, 2, 3 or 4.
In one embodiment, provided herein is a method of treating and/or preventing hyperactivity or inappropriate immune responses in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Table 1, 2, 3 or 4, and/or the expression of a gene identified in Table 1, 2, 3 or 4, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). In some embodiment, the subject can be one who is at risk of developing hyperactivity or inappropriate immune response, for example, an organ transplant recipient.
In some aspects, the hyperactivity or inappropriate immune response in a subject is associated with acute and chronic immune diseases, e.g., allergic and atopic diseases, e.g., asthma, allergic rhinitis, allergic conjunctivitis and atopic dermatitis, and to autoimmune diseases, e.g., rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, autoimmune thyroiditis, hemolytic anemia and multiple sclerosis. Hyperactivity or inappropriate activity of the immune system is also involved in transplant graft rejections and graft-versus-host disease. Administering an agent that inhibits a gene identified in Table 1, 2, 3 or 4, can down-regulate NFAT activity and/or store-operated Ca2+ entry and thereby reduce chronic T cell activation.
In another embodiment, provided herein is a method of increasing immune response in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5, and/or the expression of a gene identified in Tables 1-5.
In an embodiment, provided herein is a method of treating a cell proliferation disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity of a protein expressed from a gene identified in Table 1, 2, 3 or 4 and/or the expression of a gene identified in Table 1, 2, 3 or 4, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). The cell proliferation disease or disorder is a neoplastic cell proliferation disorder and the neoplastic cell proliferation disorder is a therapy resistant cancer, a metastasis or malignant cancer.
In another embodiment, provided herein is a method of treating a cardiovascular disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Table 1, 2, 3 or 4 and/or the expression of a gene identified in Table 1, 2, 3 or 4, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). The cardiovascular disorders is cardiac hypertrophy, restenosis, atherosclerosis, or angiogenesis.
In another embodiment, provided herein is a method of treating an injury to the nervous system in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Table 1, 2, 3 or 4 and/or the expression of a gene identified in Table 1, 2, 3 or 4, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2).
In another embodiment, provided herein is a method of treating a bone disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Table 1, 2, 3 or 4 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2), wherein excessive osteoclast formation and activity is suppressed.
In another embodiment, provided herein is a method of treating diabetes in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Table 1-4 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2).
In another embodiment, provided herein is a method of treating an injury to the nervous system in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2).
In another embodiment, provided herein is a method of treating an angiogenic disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). The angiogenic disease or disorder is associated with VEGF-induced and IL-1 induced gene expression.
In some aspects, the angiogenesis disorder is selected from a group consisting of cancer, age-related macular degeneration, diabetic retinopathy; rheumatoid arthritis; Alzheimer's disease; obesity and endometriosis.
In another embodiment, the methods provided herein comprise a agent that is a nucleic acid inhibitor which inhibits gene expression.
In one aspect, the agent is a nucleic acid inhibitor. In some aspects, the nucleic acid is DNA, RNA, nucleic acid analogue, peptide nucleic acid (PNA), pseudo-complementary PNA (pcPNA), locked nucleic acid (LNA) or analogue thereof. In other aspects, the RNA is a small inhibitory RNA, siRNA, microRNA, shRNA, miRNA and analogues and homologues and variants thereof effective in gene silencing.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows schematic diagram of NFAT translocation and activation.
FIG. 2 shows the automated data acquisition by ImageXpress Micro
FIG. 3 shows the MetaXpress automated analysis of cell scoring and/or nuclear translocation of NFAT-GFP in thapsigargin treated cells.
FIG. 4 shows that combined STIM1 and Orai1 expression in Hela cells enhances nuclear translocation of NFAT-GFP.
FIG. 5 shows combined STIM1 and Orai1 expression in Hela cells enhances NFAT nuclear translocation.
FIG. 6 shows the reason for choosing to use NFAT translocation as an screening assay Stim2−/− T cells have a very slight defect in acutely measured store operated Ca2+ entry (SOCE) but a substantial defect in NFAT nuclear translocation and cytokine production.
FIG. 7 shows one of the flow diagram of the high-throughput screening protocol.
FIG. 8 shows a second flow diagram of the high-throughput screening protocol.
FIG. 9 shows the z score calculation for Hela cells transfected with STIM1 and Orai1.
FIG. 10 summarizes the steps for calculating Z scores.
FIG. 11 shows the graph of average Z scores of kinases screened in a sample well of the HTS.
FIG. 12 shows the graph of the average Z scores of genes screened in the sample wells of the HTS plate #50048. Note that the duplicate Z-scores for KCNN4 (from duplicate wells) showing the knockdown of potassium channel KCNN4.
FIG. 13 shows the distribution and classification of the identified genes/proteins that modulate NFAT and/or store operated Ca2+ entry (SOCE).
FIG. 14 shows the average Z score histogram of selected identified genes affecting NFAT.
FIG. 15 shows the average Z score histogram of selected identified genes affecting p65.
FIG. 16 shows the summary of the identified genes/proteins categorized into groups.
FIG. 17 shows the summary of the secondary screening protocol of the hits from the primary screen.
FIG. 18 shows the summary of the genes analyzed in the secondary screen.
FIG. 19 shows the instrumentation used in the tertiary screen.
FIG. 20 shows the methodology of the tertiary screen.
FIG. 21 shows eight reproducible traces of calcium fluxes in a tertiary screen in the presence (1.25 mM) or absence of lead (Pb).
FIG. 22 shows eight reproducible traces of calcium fluxes in a tertiary screen at two different temperatures, at 37° C. and at room temperature (RT) ( ̃25° C.).
FIG. 23 shows additional traces of calcium fluxes in a tertiary screen at room temperature (RT) ( ̃25° C.).
FIG. 24 shows additional traces of calcium fluxes in a tertiary screen.
FIG. 25 shows the summary of z scores obtained during the primary and secondary screen for a few select hits.
FIG. 26 shows additional traces of calcium fluxes of select hits in a tertiary screen at two different temperatures, at 37° C. and at room temperature (RT) ( ̃25° C.).
FIG. 27 shows the schematic diagram of mechanism of action of the potassium channel KCNN4 in relation to the intracellular Ca2+ concentration and the regulation of NFAT nuclear translocation and cytokine production by intracellular Ca2+ concentration.
FIG. 28 shows the effects of siRNA of STIM1, CanB1, and KCNN4 on NFAT nuclear translocation. The figure also shows that siRNA CanB1 has not effect on Ca2+ influx in contrast to the siRNA of STIM1 and KCNN4.
FIG. 29 shows that Stim1 is modified by thapsigargin treatment.
FIG. 30 shows that cross-linking enhances STIM1-Orai1 interaction.
FIG. 31 shows some traces of calcium fluxes of cells with siRNA to select hits.
FIG. 32 shows some traces of calcium fluxes of cells with siRNA to select hits.
FIG. 33 shows some traces of calcium fluxes of cells with siRNA to select hits.
FIG. 34 shows some traces of calcium fluxes of cells with siRNA to TROAP
FIG. 35 shows genes that enhances NFAT-GFP nuclear localization.
FIG. 36 is a summary of genes that affects NFAT-GFP nuclear localization, p65 translocation and Orai 1 cell surface localization.
FIG. 37 shows the summary of primary, secondary and tertiary screens.
FIGS. 38-57 show some traces of calcium fluxes in cells treated with siRNA to the respective genes: ACSBG1, ActB, ALCAM, ATN1, ATP6V0D1, C1ORF123, C20ORF96, C6ORF191, C8ORF42, CCDCl25, CCNB2, CNTN3, CPEB4, CPT2, DKFZP686A01247, DNAJC5G, ELMOD1, FAM108C1, FAS, FASTKD5, FLJ21986, FRMPD1, GGA3, GLT1D1, GOSR2, GPD1, GPD1L, GPR23, GSTM2, IL9, KCNIP2, KCCN4, KIAA0284, KRT35, KRTAP21-2, KPTAP5-8, L1TD1, LMAN1L, LMNB1, LOC338829, LOC388381, LYZL1, MGC34829, MRS2L, MYO9A, NAPA, NDUFA5, NIPA2, OSTM1, PASD1, PIK4CA, PILRA, PJA1, PRRT1, PRSS1, RAD9B, RNF185, RNPEPL1, RPGR, SEPT4/PNUTL2, SFXN5, SLC41A3, SPTLC2, STAM, STIM2, STIM1, ORA1, STXBP2, TMED10, TMEM110, TMEM142A, TNFSRF18, TRIM59, UBC, UEVLD, XKR5, ZNF289, ZNF706, ZZEF1 and JPH2.
FIGS. 58-70 show calcium fluxes traces of single cells treated with siRNA to the respective genes: ALCAM, ATP6V0D1, C1ORF123, C6ORF191, C7ORF58, CCNB2, CPT2, COPA, COPB1, COPZ1, DNAJC5G, FAS, FASTKD5, FBXO5, FRMPD1, GOSR2, GPR23, GSTM2-1, GSTM2-2, KIAA0284, KPTHAP5, KRTAP21-2, IL9, L1TD1, LOC338829, LYZL1, MICAL3, MYO9A-1, MYO9A-2, NDUFA5, PCOLN, PIK4CA, PILRA, PJA1, PRRT1, PRSS1, RAD9B, RNPEPL1, RPGR, SLC41A3, SPTLC2, STIM1, STIM2, STXBP2, SEPT4/PNUTL2, TRIM3, TRIM59, TMEM110, UHSKERB, USP13, XKR5, and ZNF289.



DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 18th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-14320499.2 911910-18-2); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology are found in Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987), Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.), Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.
It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages may mean±1%.
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “comprises” means “includes.” The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
All patents and other publications cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
Definitions of terms
The term “NFAT activation” refers to the nuclear translocation of NFAT from the cytoplasm to the nucleus. Nuclear factor of activated T-cells (NFAT) is a general name applied to a family of transcription factors shown to be important in immune response. Cytoplasmic NFAT proteins are phosphorylated. To enter the nucleus, NFAT has to be dephosphorylated. Activated serine/threonine phosphatase calcineurin rapidly dephosphorylates the serine rich region (SRR) and SP-repeats in the amino termini of NFAT proteins resulting in a conformational change that exposes a nuclear localization signal resulting in NFAT nuclear import. The term “NFAT activity” also means the nuclear translocation of NFAT from the cytoplasm to the nucleus.
As used herein, the term “pharmaceutical composition” refers to an active agent in combination with a pharmaceutically acceptable carrier of chemicals and compounds commonly used in the pharmaceutical industry. The term “pharmaceutically acceptable carriers” excludes tissue culture medium.
As used herein, the term “therapeutically effective amount” refers to that amount of active agent that can reduce the activity of a candidate protein by at least 5% or the expression of a gene identified in Tables 1-5 by at least 5%. The term also means a reduction of at least 5% in NFAT-GFP nuclear localization and/or SOCE and/or cytokine production in the cell-based assay as described herein or other methods that are known to one skilled in the art. The term also means providing “effective” treatment as that term is defined herein. An effective amount as used herein would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease.
As used herein, the term “treat’ or treatment” refers to reducing or alleviating at least one adverse effect or symptom associated with medical conditions that are associated with hyperactive or inappropriately active immune system. These include reducing the amount of cytokine production, suppression of T cell activation and proliferation, suppression of the immune system, and reducing inflammation.
As used herein, the terms “administering,” and “introducing” are used interchangeably and refer to the placement of the agents that inhibit gene identified in Tables 1-5 as disclosed herein into a subject by a method or route which results in at least partial localization of the agents at a desired site. The pharmaceutical compositions of the present invention can be administered by any appropriate route which results in an effective treatment in the subject.
The term “agent” refers to any entity which is normally not present or not present at the levels being administered, in the cell. Agents for use in the invention include, but are not limited to chemicals; small molecules; nucleic acid sequences; nucleic acid analogues; proteins; peptides; aptamers; antibodies; or fragments thereof. A nucleic acid sequence can be RNA or DNA, and can be single or double stranded, and can be selected from a group comprising; nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA) etc. Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc. A protein and/or peptide or fragment thereof can be any protein of interest, for example, but are not limited to: mutated proteins; therapeutic proteins and truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell. Proteins can also be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, midibodies, minibodies, triabodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof. Alternatively, the agent can be intracellular within the cell as a result of introduction of a nucleic acid sequence into the cell and its transcription resulting in the production of the nucleic acid and/or protein inhibitor of gene identified in Tables 1-5 within the cell. In some embodiments, the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments the agent is a small molecule having a chemical moiety. For example, chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
The term “inhibiting” as used herein as it pertains to the expression or activity of the protein or polypeptide of genes identified in Tables 1-5. The term does not necessarily mean complete inhibition of expression and/or activity. Rather, expression or activity of the protein, polypeptide or polynucleotide is inhibited to an extent, and/or for a time, sufficient to produce the desired effect, for example, reduced nuclear translocation of NFAT. In particular, inhibition of expression or activity of a gene from Tables 1-5 can be determined using an assay such as the bioassay for the protein encoded by the gene, for example, western blot analysis for the detection and quantification of expressed protein. Agents that inhibit the genes of Tables 1-5 are agents that inhibit the protein function and/or genes expression by at least 5%.
As used herein, “gene silencing” or “gene silenced” in reference to an activity of an RNAi molecule, for example a siRNA or miRNA refers to a decrease in the mRNA level in a cell for a target gene by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the miRNA or RNA interference molecule. In one preferred embodiment, the mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.
As used herein, the term “RNAi” refers to any type of interfering RNA, including but are not limited to, siRNAi, shRNAi, endogenous microRNA and artificial microRNA. For instance, it includes sequences previously identified as siRNA, regardless of the mechanism of down-stream processing of the RNA (i.e. although siRNAs are believed to have a specific method of in vivo processing resulting in the cleavage of mRNA, such sequences can be incorporated into the vectors in the context of the flanking sequences described herein). The term “RNAi” and “RNA interfering” with respect to an agent of the invention, are used interchangeably herein.
As used herein an “siRNA” refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is present or expressed in the same cell as the target gene, for example RANBP2. The double stranded RNA siRNA can be formed by the complementary strands. In one embodiment, a siRNA refers to a nucleic acid that can form a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 19-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).
As used herein “shRNA” or “small hairpin RNA” (also called stem loop) is a type of siRNA. In one embodiment, these shRNAs are composed of a short, e.g. about 19 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand can precede the nucleotide loop structure and the antisense strand can follow.
The terms “microRNA” or “miRNA” are used interchangeably herein are endogenous RNAs, some of which are known to regulate the expression of protein-coding genes at the posttranscriptional level. Endogenous microRNA are small RNAs naturally present in the genome which are capable of modulating the productive utilization of mRNA. The term artificial microRNA includes any type of RNA sequence, other than endogenous microRNA, which is capable of modulating the productive utilization of mRNA. MicroRNA sequences have been described in publications such as Lim, et al., Genes & Development, 17, p. 991-1008 (2003), Lim et al Science 299, 1540 (2003), Lee and Ambros Science, 294, 862 (2001), Lau et al., Science 294, 858-861 (2001), Lagos-Quintana et al, Current Biology, 12, 735-739 (2002), Lagos Quintana et al, Science 294, 853-857 (2001), and Lagos-Quintana et al, RNA, 9, 175-179 (2003), which are incorporated by reference. Multiple microRNAs can also be incorporated into a precursor molecule. Furthermore, miRNA-like stem-loops can be expressed in cells as a vehicle to deliver artificial miRNAs and short interfering RNAs (siRNAs) for the purpose of modulating the expression of endogenous genes through the miRNA and or RNAi pathways.
As used herein, “double stranded RNA” or “dsRNA” refers to RNA molecules that are comprised of two strands. Double-stranded molecules include those comprised of a single RNA molecule that doubles back on itself to form a two-stranded structure. For example, the stem loop structure of the progenitor molecules from which the single-stranded miRNA is derived, called the pre-miRNA (Bartel et al. 2004. Cell 116:281-297), comprises a dsRNA molecule.
As used herein, the term “complementary base pair” refers to A:T and G:C in DNA and A:U in RNA. Most DNA consists of sequences of nucleotide only four nitrogenous bases: base or base adenine (A), thymine (T), guanine (G), and cytosine (C). Together these bases form the genetic alphabet, and long ordered sequences of them contain, in coded form, much of the information present in genes. Most RNA also consists of sequences of only four bases. However, in RNA, thymine is replaced by uridine (U).
As used herein, the term “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one strand nucleic acid of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the template nucleic acid is DNA. In another aspect, the template is RNA. Suitable nucleic acid molecules are DNA, including genomic DNA, ribosomal DNA and cDNA. Other suitable nucleic acid molecules are RNA, including mRNA, rRNA and tRNA. The nucleic acid molecule can be naturally occurring, as in genomic DNA, or it may be synthetic, ie., prepared based up human action, or may be a combination of the two. The nucleic acid molecule can also have certain modification such as 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-β-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA), cholesterol addition, and phosphorothioate backbone as described in US Patent Application 20070213292; and certain ribonucleoside that are is linked between the 2′-oxygen and the 4′-carbon atoms with a methylene unit as described in U.S. Pat. No. 6,268,490, wherein both patent and patent application are incorporated hereby reference in their entirety.
The term “vector”, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. As used herein, a vector can be viral or non-viral.
As used herein, the term “expression vector” refers to a vector that has the ability to incorporate and express heterologous nucleic acid fragments in a cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
As used herein, the term “heterologous nucleic acid fragments” refers to nucleic acid sequences that are not naturally occurring in that cell. For example, when a human RANBP2 gene is inserted into the genome of a bacteria or virus, that human RANBP2 gene is heterologous to that recipient bacteria or virus because the bacteria and viral genome do not naturally have the human RANBP2 gene.
As used herein, the term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the shRNA for the human RANBP2 in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
The term “replication incompetent” as used herein means the viral vector cannot further replicate and package its genomes. For example, when the cells of a subject are infected with replication incompetent recombinant adeno-associated virus (rAAV) virions, the heterologous (also known as transgene) gene is expressed in the patient's cells, but, the rAAV is replication defective (e.g., lacks accessory genes that encode essential proteins from packaging the virus) and viral particles cannot be formed in the patient's cells.
The term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5′ untranslated (5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments. The term “gene” used herein can be a genomic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (e.g., introns, 5′- and 3′-untranslated sequences and regulatory sequences). The coding region of a gene can be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. A gene can also be an mRNA or cDNA corresponding to the coding regions (e.g. exons and miRNA) optionally comprising 5′- or 3′ untranslated sequences linked thereto. A gene can also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5′- or 3′-untranslated sequences linked thereto.
The term “subject” as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.
As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, for example the carrier does not decrease the impact of the agent on the treatment. In other words, a carrier is pharmaceutically inert.
As used herein, the term “modulate” means the regulation of the cellular activity of a protein. Modulation can mean up regulation of the cellular activity of the protein, whereby its activity is enhances and/or promoted. Modulation can also mean down regulation of the cellular activity of the protein, whereby its activity is reduced, blocked, and/or prevented.
As used herein, the term “a neoplastic cell proliferation disorder” refers to any disorder that is characterized by deregulated or unregulated cell proliferation that arises from a stem cell. A normal stem cell may be transformed into a cancer stem cell through disregulation of the proliferation and differentiation pathways controlling it. Examples include but are not limited to cancer and tumors formation.
As used herein, the term “tumor” refers to a mass of transformed cells that are characterized, at least in part, by containing angiogenic vasculature. The transformed cells are characterized by neoplastic uncontrolled cell multiplication which is rapid and continues even after the stimuli that initiated the new growth has ceased. The term “tumor” is used broadly to include the tumor parenchymal cells as well as the supporting stroma, including the angiogenic blood vessels that infiltrate the tumor parenchymal cell mass. Although a tumor generally is a malignant tumor, i.e., a cancer having the ability to metastasize (i.e. a metastatic tumor), a tumor also can be nonmalignant (i.e. non-metastatic tumor). Tumors are hallmarks of cancer, a neoplastic disease the natural course of which is fatal. Cancer cells exhibit the properties of invasion and metastasis and are highly anaplastic.
As used herein, the term “metastases” or “metastatic tumor” refers to a secondary tumor that grows separately elsewhere in the body from the primary tumor and has arisen from detached, transported cells, wherein the primary tumor is a solid tumor. The primary tumor, as used herein, refers to a tumor that originated in the location or organ in which it is present and did not metastasize to that location from another location. As used herein, a “malignant tumor” is one having the properties of invasion and metastasis and showing a high degree of anaplasia. Anaplasia is the reversion of cells to an immature or a less differentiated form, and it occurs in most malignant tumors.
The term “therapy resistant cancer” as used herein refers to a cancer present in a subject who is resistant to, or refractory to at least two different anti-cancer agents such as chemotherapy agents, which means, typically a subject has been treated with at least two different anti-cancer agents that did not provide effective treatment as that term is defined herein.
Embodiments of the invention are based on the discovery of several hundred genes in the human and mouse genomes whose gene products directly and/or indirectly modulate NFAT activation and/or modulate the store-operated Ca2+ entry (SOCE) into the cell. The inventors developed a cell-based reporter system for screening for modulators of nuclear factors of activated T cells (NFAT) and/or store-operated Ca2+ entry into a cell. The cell-based reporter system comprises a mammalian cell co-expressing a NFAT-GFP, a STIM1-RFP, and an Orai1-FLAG. The markers: GFP, RFP and FLAG-tag facilitate the visual localization of the respectively expressed proteins within the cell compartments. For example, whether NFAT is localized to the cytoplasm under non-Ca2+ depletion conditions (in the absence of thapsigargin (TG)) or has translocated to the nucleus upon treatment with TG, and whether STIM1/Orai1 are expressed and properly localized to the membranes. TG is a tight-binding inhibitor of a class of enzymes known by the acronym SERCA, which stands for sarco/endoplasmic reticulum Ca2+ ATPase. TG raises cytosolic calcium concentration by blocking the ability of the cell to pump calcium into the sarcoplasmic and endoplasmic reticula which causes these stores to become depleted. Store-depletion can secondarily activate plasma membrane calcium channels, triggering store-operated Ca2+ entry into a cell via plasma membrane channels. It was found that the co-expression of STIM1-RFP, and Orai1-FLAG in Hela cells enhanced SOCE in these cells upon TG treatment. The inventors used the cytoplasm-to-nuclear translocation of NFAT-GFP as their assay readout, counting the number of cells that have nuclear GFP fluorescence after TG treatment. For a population of these cells treated with TG, a mean number of cells will have NFAT-GFP nuclear localization after TG treatment for a fixed period of time, e.g. 10 minutes. This is the control population for the high-throughput screen. Within this population data, a standard deviation is also obtained. The data (number of cells having NFAT-GFP nuclear localization after TG treatment) is assumed to have a normal distribution. This data of this control population of cells are normalized to a standard normal distribution, which has a mean of 0 (the mean number of cell with nuclear NFAT-GAT) and standard deviation of 1.
To screen for modulators of NFAT and/or store-operated Ca2+ entry into a cell, the inventors performed a high-throughput siRNA screen of 23-mer siRNAs that target all human or mouse genes. For each gene, at least four different siRNAs were used. In such a cell-based assay, the inventors seek to discover genes that can modulate the cytoplasm-to-nuclear translocation of NFAT-GFP and/or store-operated Ca+ entry into a cell. The siRNAs to such a gene result in either a decrease or an increase in the nuclear GFP fluorescence after TG treatment. The decrease or increase is at least two fold of the standard deviation for the control population of cells treated with TG but conducted in the absence of any siRNA, i.e. at least an average Z score of −2.0 or +2.0. The number of standard deviations from the mean is called the Z-score and can be found by the formula:




  z
  =
  
    
      x
      -
      μ
    
    σ
  




where x is the mean number of cells having NFAT-GFP localization for the population of cells treated with siRNA, μ is mean number of cells having NFAT-GFP localization for the control population, and σ is the standard deviation for the control population of cells. The control population of cells is assayed in parallel with the siRNAs.

From this screen, the inventors uncovered  ̃500 genes that strongly modulate NFAT and/or store-operated Ca2+ entry into a cell, having an average Z-score of ≧|4| and  ̃650 genes that moderately/weakly at modulate NFAT and/or store-operated Ca2+ entry into a cell, having an average z-score of −4<Z<−2 or 2<Z<4. The designation |4| refers to the mathematical symbol for four absolute.
The screen identified known modulator of NFAT: calcineurin (CanB1 and CanAα) which are involved in the dephosphorylating NFAT which is necessary for nuclear translocation; known store-operated Ca2+ entry sensor proteins: Stim1 and Orai1; and KCNN4 (IKCa1, potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4). Calcineurin (CN) is a protein phosphatase also known as protein phosphatase 2B (PP2B). Protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform, also known as PPP3CA. The identification of known modulators of NFAT activity or store-operated Ca+ entry validates the accuracy and utility of the cell-based assay used by the inventors.
In addition to calcineurin, the siRNA screen identified KCNN4 (IKCa1, potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4) that is known to be indirectly involved in NFAT nuclear localization via SOCE. Several reports have demonstrated that Kv1.3 and IKCa1 K+ channels play crucial roles in T-cell activation, inflammation, progression of autoimmune diseases, and of other immunological disorders (Cahalan et al., 2001, Clin Immunol 21:235-252; Wulff et al., 2003, Curr. Opin. Drug Discov. Devel. 6:640-647; Chandy et al., 2004, Trends Pharmacolog Sci 25:280-289; Vicente et al., 2004, FEBS Lett 572:189-194). The use of Kv1.3 and IKCa1 K+ channel-blockers have been shown to ameliorate several types of disorders.
The high-throughput siRNA screen also identified several nuclear transport proteins: RAN (ras-related nuclear protein), RANBP2 (RAN binding protein 2), KPNB1 (karyopherin (importin) beta 1), CSE1L (chromosome segregation 1-like), and CRM1 (exportin 1, XPO1).
The entry and exit of large molecules from the cell nucleus is tightly controlled by the nuclear pore complexes (NPCs). Although small molecules can enter the nucleus without regulation, macromolecules such as RNA and proteins require association with karyopherins called importins to enter the nucleus and exportins to exit. The ability of both importins (KPNB1 and CSE1L) and exportins (CRM1) to transport their cargo is regulated by the small Ras related GTPase, RAN.
In some embodiments, the identified genes are SEQ. ID NOS:1-11 (Genbank Accession No. NM—000944; NM—021132.1; NM—006325; NM—006267.4; NM—002265.4, NM—001316; NM—003400.3; NM—003156.2, NM—020860.2, NM—032790.3, NM—002250.2).
Other examples of modulate genes identified in the cell-base assay as described herein include those that are involved in (1) Golgi-to-plasma membrane trafficking, (2) associated with mitochondria, (3) scaffold proteins (with PDZ domains, etc), (4) ubiquitin metabolism, (5) noncoding RNAs (possibility containing microRNAs), (6) RNA-binding proteins, and (7) potassium channels: KCNN4 (see Tables 1-5).
Accordingly, the invention provides a method of modulating NFAT activity, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein and/or the expression of a gene identified in Tables 1-5.
In one embodiment, provided herein is a method modulating store-operated Ca2+ entry into a cell, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5.
In one embodiment, provided herein is a method of treating and/or preventing hyperactivity or inappropriate immune responses in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). In some embodiment, the subject can be one who is at risk of developing hyperactivity or inappropriate immune response, for example, an organ transplant recipient.
In some aspects, the hyperactivity or inappropriate immune response in a subject is associated with acute and chronic immune diseases, e.g., allergic and atopic diseases, e.g., asthma, allergic rhinitis, allergic conjunctivitis and atopic dermatitis, and to autoimmune diseases, e.g., rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, autoimmune thyroiditis, hemolytic anemia and multiple sclerosis. Hyperactivity or inappropriate activity of the immune system is also involved in transplant graft rejections and graft-versus-host disease Administering an agent that inhibits a gene identified in Tables 1-5 can down-regulate NFAT activity and/or store-operated Ca2+ entry and thereby reduce chronic T cell activation.
In some embodiments, the genes identified in Tables 1-5 are involved in down-regulating NFAT activity and/or store-operated Ca+ entry. Agents that inhibit such genes can enhance NFAT activity and/or store-operated Ca2+ entry and thereby increase immune response. Accordingly, provided herein is method of increasing immune response in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5.
Subjects having immunodeficiency disorders can benefit from the method described herein of increasing immune response. Immunodeficiency disorders can include or result from but not limited to common variable immunodeficiency, selective antibody deficiency (such as IgA deficiency), transient hypogammaglobulinemia of infancy, X-linked agammaglobulinemia, chronic mucocutaneous candidiasis, DiGeorge anomaly, ataxia-telangiectasia, severe combined immunodeficiency disease, Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome, Chédiak-Higashi syndrome, chronic granulomatous disease, hyperimmunoglobulinemia E syndrome, leukocyte adhesion defects, leukocyte glucose-6-phosphate dehydrogenase deficiency, myeloperoxidase deficiency, complement component 1 (C1) inhibitor deficiency (hereditary angioedema), C3 deficiency, C6 deficiency, C7 deficiency, C8 deficiency, chemotherapy and radiation therapy, human immunodeficiency virus (HIV) infection, cancer, blood disorders (such as aplastic anemia, leukemia, and myelofibrosis), kidney failure, diabetes, liver disorders, and spleen disorders.
In some aspects, the subject is a mammal, for example, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus. The methods provided herein are applicable to any subject that comprises an immune system which comprises NFAT transcription activation factors and the need for sustained Ca2+ influx for NFAT activation.
In one embodiment, provided herein is a method of treating a cell proliferation disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). In some embodiment, the subject can be one who is at risk of developing a cell proliferation disease or disorder.
As used herein, cell proliferation disease or disorder is a neoplastic cell proliferation disorder, such as a therapy resistant cancer, a metastasis or malignant cancer. In one embodiment, the methods described herein are applied to subject who has or is at risk of having a metastasis or malignant cancer. The metastasis or malignant cancer can also be a recurring or relapsed cancer, after the subject has been treated with conventional cancer therapy such as radiation and/or chemotherapy. Accordingly, the neoplastic cell proliferation disorder is a therapy resistant cancer. Other cancers include but are not limited to solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, askocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Kaposi's sarcoma.
Cancers include, but are not limited to, bladder cancer; breast cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer including colorectal carcinomas; endometrial cancer; esophageal cancer; gastric cancer; head and neck cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia, multiple myeloma, AIDS associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease, liver cancer; lung cancer including small cell lung cancer and non-small cell lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; osteosarcomas; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, synovial sarcoma and osteosarcoma; skin cancer including melanomas, Kaposi's sarcoma, basocellular cancer, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; transitional cancer and renal cancer including adenocarcinoma and Wilm's tumor.
Cardiovascular disease is the major cause of death in industrialized nations. Targeted intervention in calcineurin, a calmodulin-dependent, calcium-activated phosphatase and its substrate, nuclear factor of activated T cells (NFAT), was demonstrated to be effective in the treatment of cardiovascular diseases. In one embodiment, provided herein is a method of treating a cardiovascular disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). In some embodiment, the subject can be one who is at risk of developing a cardiovascular disorder. Cardiovascular disorders including cardiac hypertrophy, restenosis, atherosclerosis, and angiogenesis.
Since there is a potential role for NFAT in axon re-growth and regeneration following axonal injury, modulating NFAT activity after such injury can promote axonal re-growth and regeneration. Accordingly, in one embodiment, provided herein is a method of treating an injury to the nervous system in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2).
Excessive osteoclast formation is characteristic of a variety of bone diseases such as rheumatoid arthritis. Hence a strategy for suppressing the excessive osteoclast formation can be novel therapeutic approach for the treatment of bone disease. Accordingly, in one embodiment, provided herein is a method of treating a bone disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). The method comprises suppressing the excessive osteoclast formation and activity.
In one embodiment, provided herein is a method of treating diabetes in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). In some embodiment, the subject can be one who is at risk of developing diabetes.
In one embodiment, provided herein is a method of treating an angiogenic disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). In some embodiment, the subject can be one who is at risk of developing an angiogenesis. In some embodiments, the angiogenic disease or disorder is related to VEGF-induced and IL-1 induced gene expression.
In one embodiment, provided herein is a method of promoting or inhibiting T cell anergy in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the activity a protein expressed from a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, wherein the gene is not calcineurin, calmodulin, Stim1, Stim2, Orai1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2).
In one embodiment, the agent that inhibits the activity of a protein expressed from a gene identified in Tables 1-5 and/or the expression of the gene identified in Tables 1-5 can be administered to the subject together with additional therapeutic agents, cancer therapy, immunosuppressant therapy, immunodeficiency therapy, steroid therapy, and psychotherapy.
In one embodiment, the agent that inhibits the activity of a protein expressed from a gene identified in Tables 1-5 and/or the expression of the gene identified in Tables 1-5 is a small molecule, nucleic acid, nucleic acid analogue, protein, antibody, peptide, aptamer or variants or fragments thereof. Such an agent can take the form of any entity which is normally not present or not present at the levels being administered to the cell or organism.
Other forms of inhibitors include a nucleic acid agent which is an RNAi agent such as a siRNA, shRNA, miRNA, dsRNA or ribozyme or variants thereof.
In one embodiment, the effects of the inhibitory agent such as an RNAi agent can be determined by measuring the Ca2+ fluxes in a treated cell using any method known in the art, e.g. Feske et al., (2006) Nature 441, 179-815 or a high-throughput assay as described below.
HTS Ca+ Assay—HeLa cells are transfected with 20 nM siRNA (siGenome SmartPools obtained from Dharmacon/ThermoFischer) in 96-well plates. The siRNA tested are against the gene expression of ACSBG1, ActB, ALCAM, ATN1, ATP6V0D1, C1ORF123, C20ORF96, C6ORF191, C8ORF42, CCDC125, CCNB2, CNTN3, CPEB4, CPT2, DKFZP686A01247, DNAJC5G, ELMOD1, FAM108C1, FAS, FASTKD5, FLJ21986, FRMPD1, GGA3, GLT1D1, GOSR2, GPD1, GPD1L, GPR23, GSTM2, IL9, KCNIP2, KCCN4, KIAA0284, KRT35, KRTAP21-2, KPTAP5-8, L1TD1, LMAN1L, LMNB1, LOC338829, LOC388381, LYZL1, MGC34829, MRS2L, MYO9A, NAPA, NDUFA5, NIPA2, OSTM1, PASD1, PIK4CA, PILRA, PJA1, PRRT1, PRSS1, RAD9B, RNF185, RNPEPL1, RPGR, SEPT4/PNUTL2, SFXN5, SLC41A3, SPTLC2, STAM, STIM2, STIM1, ORA1, STXBP2, TMED10, TMEM110, TMEM142A, TNFSRF18, TRIM59, UBC, UEVLD, XKR5, ZNF289, ZNF706, ZZEF1 and JPH2. The names of these genes are shown in Table 5.
After 72 hours, the cells are loaded with FURA2/AM and intra-cellular Ca2+ traces are measured on a Flexstation III kinetic fluorescent imager (Molecular Devices). Cells are then stimulated with 1 uM thapsigargin (TG) and 3 mM EGTA for 4 minutes, then 2 mM CaCl2 for an additional 6 minutes. Each gene-specific siRNA is analysed using 4 biological replicates, and positive hits are identified if at least 2 replicates showed a decrease in the second peak of Fura2 fluorescence greater or equal to 20% of the control. For each hit, one representative Fura2 trace is represented along with the corresponding trace from siControl or siSTIM1-treated cells. Examplary Ca2+ traces are shown in FIG. 38-57.
For single cell imaging of the calcium fluxes due to the treatment of the respective siRNA, the method of Feske et al., (2006) supra can be used and the method is briefly described below.
After the indicated siRNA treatment, i.e., treatment with siRNA against the gene expression of ACSBG1, ActB, ALCAM, ATN1, ATP6V0D1, C1ORF123, C20ORF96, C6ORF191, C8ORF42, CCDC125, CCNB2, CNTN3, CPEB4, CPT2, DKFZP686A01247, DNAJC5G, ELMOD1, FAM108C1, FAS, FASTKD5, FLJ21986, FRMPD1, GGA3, GLT1D1, GOSR2, GPD1, GPD1L, GPR23, GSTM2, IL9, KCNIP2, KCCN4, KIAA0284, KRT35, KRTAP21-2, KPTAP5-8, L1TD1, LMAN1L, LMNB1, LOC338829, LOC388381, LYZL1, MGC34829, MRS2L, MYO9A, NAPA, NDUFA5, NIPA2, OSTM1, PASD1, PIK4CA, PILRA, PJA1, PRRT1, PRSS1, RAD9B, RNF185, RNPEPL1, RPGR, SEPT4/PNUTL2, SFXN5, SLC41A3, SPTLC2, STAM, STIM2, STIM1, ORA1, STXBP2, TMED10, TMEM110, TMEM142A, TNFSRF18, TRIM59, UBC, UEVLD, XKR5, ZNF289, ZNF706, ZZEF1 and JPH2, the HeLa cells were loaded with the calcium indicator FURA-2, using the cell-permeant precursor FURA-2-AM. Coverslips are mounted in a flow chamber on the stage of a microscope for fluorescence imaging. Fluorescence emission is monitored at 510 nm, with alternating excitation at 340 nm and 380 nm. Initial perfusion is with calcium-free Ringer solution, then with calcium-free Ringer solution containing 1 micromolar thapsigargin to release calcium from ER stores (the first low peak in the graphs), and finally with ordinary Ringer solution that contains calcium and therefore supports calcium influx through store-operated channels (the second higher peak in the graphs). Fura-2 fluorescence data are then converted to cytoplasmic calcium concentrations as described in Feske et al (2006) Nature 441, 179-185. Cytoplasmic calcium concentration (nM) is plotted on the vertical axis, and time (s) on the horizontal axis.
Cells treated with control siRNA are also included in each experiment. The siRNAs that are effective in this assay produced differences in one or more of the following parameters: rate of rise of the signal due to store-operated calcium entry, its peak height, or its plateau. Examplary of single cell Ca2+ traces are shown in FIG. 58-70.
In some embodiments, it is possible that a siRNA may target more than one gene, especially when the genes are highly related in sequence. For example, the siRNA targeting SEPT4/PNUTL2 also targets SEPT5 gene expression, leading to reduced SEPT5 protein (see in FIG. 58).
Calcineurin/NFAT Axis in Vertebrates
Calcineurin is a calmodulin-dependent, calcium-activated protein phosphatase composed of catalytic and regulatory subunits. The serine/threonine-specific phosphatase functions within signal transduction pathways that regulate gene expression and biological responses in many developmentally important cell types. Calcineurin signaling was first defined in T lymphocytes as a regulator of nuclear factor of activated T cells (NFAT) transcription factor nuclear translocation and activation.
The NFAT transcription factor family consists of five members NFAT1, NFAT2, NFAT3, NFAT4 and NFAT5. NFAT1-4 are regulated by calcium signaling. All family members contain the rel DNA binding domain, however only NFAT1-4 contains the Ca2+ sensor/translocation domain. The activation process of the NFAT transcription factor family is tightly regulated by calcium-dependent phosphatase calcineurin. NFAT activation is dependent upon a rise in intracellular Ca2+, which activates the serine/threonine phosphatase, calcineurin. The increase in intracellular calcium levels can occur, e.g., by means of store-operated calcium entry (SOCE). Activated calcineurin rapidly dephosphorylates the serine rich region (SRR) and SP-repeats in the amino termini of NFAT proteins resulting in a conformational change that exposes a nuclear localization signal resulting in NFAT nuclear import.
Opposing this, the nuclear export of NFAT requires the sequential re-phosphorylation of this domain by several kinases including GSK-3β. Other post-translational modifications such as acetylation and sumoylation, as well as phosphorylation events distinct from those in the Ca2+/translocation domain, also modulate NFAT transcriptional activity.
As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signaling and many other cellular processes, in particular, for the calcium-release-activated calcium (CRAC) channels in T lymphocytes. The CRAC channels are essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation of the activated T cell. STIM1 and Orai 1 function as Ca2+ sensors of changes in the intracellular Ca2+ stores to activate CRAC channels.
NFAT functions as an integrator of multiple signaling pathways and achieves this through a combinatorial mechanism of transcriptional regulation. Other cellular signaling pathways including MAP kinase, WNT or NOTCH. NFAT, along with other transcription factors and co-activators, integrates signaling pathways by binding to chromatin in a highly specific and concerted fashion only upon receiving the appropriate signaling cues. The composition of the NFAT transcription complexes assembled at the promoter and enhancer elements of target genes is thus dependent upon both signaling and chromatin context, which determines when and where NFAT complexes activate or repress transcription. The NFAT family of transcription factors functions in combination with other transcription factors and co-activators to regulate genes central for many developmental systems. NFAT proteins have been found to be involved in numerous cellular processes, for example, cell cycle regulation, cell differentiation, cell survival, angiogenesis, tumor cell invasion and metastasis, myogenesis, chondrocyte differentiation and the development of the cardiovascular system, the complex nervous system, the recombinational immune system, and the cardiovascular system in a vertebrate (Graef I A et. al., Curr Opin Genet Dev. 2001, 11:505-12; Macian F., Nat Rev Immunol. 2005; 5:472-84; Schulz and Yutzey, Dev Biol. 2004, 266:1-16; Crabtree and Olson, Cell. 2002; 109(Suppl):567-79).
The development, activation, and maintenance of the immune system is dependent on several factors, of which Ca2+ influx and the activation of transcription factors are two of the most important factors. NFAT proteins are expressed in immune cells and play a key role in eliciting immune responses. Ca2+/calcineurin/NFAT signaling pathway is essential for lymphocyte activation, for short-term as well as long-term responses by immune-system cells, which include T and B cell proliferation and differentiation.
The activated NFAT proteins, in turn, induce transcription of cytokine genes which are required for an immune response. For example, NFAT1 and NFAT2 are much higher in memory and effector T cells than in naïve T cells, suggesting that they play an important function in memory T cells activation by way of IL-2 cytokine production in the memory T cells.
Calcineurin is indirectly responsible for activating the transcription of interleukin 2 (IL-2) that stimulates the growth and differentiation of T cell response. When an antigen presenting cell interacts with a T cell receptor on T cells, there is an increase in the cytoplasmic level of calcium, (Yamashita M., et. al., J Exp Med. 2000, 191: 1869-1880) which activates calcineurin, by binding a regulatory subunit and activating calmodulin binding. Calcineurin induces different transcription factors such as NFATs that are important in the transcription of IL-2 genes. Calcineurin dephosphorylates the cytoplasmic component of NFATs, transcription factors that can then go into the nucleus and turn on genes involved in IL-2 synthesis. IL-2 activates T-helper lymphocytes and induces the production of other cytokines. In this way, it governs the action of cytotoxic lymphocytes and NK cells. The amount of IL-2 being produced by the T-helper cells is believed to influence the extent of the immune response significantly. In immunosuppressive therapy, calcineurin is inhibited by cyclosporin, pimecrolimus (Elidel) and tacrolimus (FK506)—these drugs are known as calcineurin inhibitors.
Interleukin-21 (IL-21), a potent immunomodulatory four-alpha-helical-bundle type I cytokine, is produced by NKT and CD4(+) T cells and has pleiotropic effects on both innate and adaptive immune responses. These actions include positive effects such as enhanced proliferation of lymphoid cells, increased cytotoxicity of CD8(+) T cells and natural killer (NK) cells, and differentiation of B cells into plasma cells. Conversely, IL-21 also has direct inhibitory effects on the antigen-presenting function of dendritic cells and can be proapoptotic for B cells and NK cells. IL-21 is also produced by Th17 cells and is a critical regulator of Th17 development. The regulatory activity of IL-21 is modulated by the differentiation state of its target cells as well as by other cytokines or costimulatory molecules. IL-21 has potent antitumor activity but is also associated with the development of autoimmune disease. IL-21 transcription is dependent on a calcium signal and NFAT sites, and IL-21 requires Stat3 for its signaling. The key to harnessing the power of IL-21 will depend on better understanding its range of biological actions, its mechanism of action, and the molecular basis of regulation of expression of IL-21 and its receptor (Spolski and Leonard, Annu Rev Immunol. 2008, 26:57-79).
NFAT has also been shown to the crucial sensor of T cell receptor signaling in the interleukin (IL)-17 promoter and expression. IL-17 is a pro-inflammatory cytokine produced by T helper type 17 (Th17) cells, which have critical role in immunity to extracellular bacteria and the pathogenesis of several autoimmune disorders and asthma. There are two NFAT binding sites in the minimal promoter of IL-17. (Liu et. al., J Biol. Chem. 2004, 279:52762-71, Sundrud and Rao, Curr Opin Immunol. 2007, 9(3):287-93).
Central tolerance in the thymus is the primary mechanism for deleting autoreactive T cells. Despite this, escape of self-reactive T lymphocytes into the periphery reveals the threat of autoimmunity To compensate for its imperfection, the thymus also produces a naturally occurring subset of Foxp3+ CD4+ CD25+ regulatory T cells with suppressive function, capable of controlling autoreactive cells. Foxp3 (forkhead box P3), the lineage-specific marker for this subset of cells, is crucial to their thymic development and peripheral function. NFAT, in cooperation with Foxp 3, are crucial for the phenotype, development, maintenance, and function of these regulatory T cells, and the ultimately for maintaining immunological tolerance in an organism (Wu et. al, Cell. 2006, 126:375-87; Rudensky A Y, et. al., Cell. 2006, 126:253-6; Mays and Chen, Cell Res. 2007, 17:904-18; Oh-Hora M, et. al., 2008, Nat. Immunol. 2008, 9:432-43).
Inhibitory modulation of NFAT function can be a strategy for immunosuppressive therapy, a bottleneck of T cell receptor-dependent activation of T cells and for promoting T-cell anergy.
Recently report show that NFAT is involved in axonal growth and guidance during vertebrate development (Nguyen and Di Giovanni, Int J Dev Neurosci. 2008, 26: 141-145). The extension and organization of sensory axon projection and commissural axon growth are both dependent upon NFAT activity. Triple NFAT2/3/4 mutant mice demonstrate that the extension and organization of sensory axon projection and commissural axon growth are both dependent upon NFAT activity. Neurotrophin and L-type calcium channel signaling modulate intracellular calcium levels to regulate the nuclear import and transcriptional activity of NFAT by activating the phosphatase calcineurin. The rephosphorylation and subsequent export of NFAT from the nucleus is mediated by several kinases, including GSK-3 beta, which contribute to the fine tuning of NFAT transcriptional activity in neurons. Thus there is a potential role for NFAT in axon re-growth and regeneration following axonal injury.
The calcium/calcineurin/NFAT signaling is also involved in cardiovascular and skeletal muscle development in vertebrates Inhibition, mutation, or forced expression of calcineurin pathway genes result in defects or alterations in cardiomyocyte maturation, heart valve formation, vascular development, skeletal muscle differentiation and fiber-type switching, and cardiac and skeletal muscle hypertrophy (Schulz and Yutzey, Dev Biol. 2004, 266:1-16) Inhibition of calcineurin-NFAT is a negative regulator of cardiac myocyte (CM) hypertrophy (Fiedler et. al., Proc Natl Acad Sci USA. 2002, 99:11363-8). Since cardiovascular disease is the major cause of death in industrialized nations. Targeted intervention in calcineurin, a calmodulin-dependent, calcium-activated phosphatase and its substrate, nuclear factor of activated T cells (NFAT), can be effective in the treatment of cardiovascular diseases. Calcineurin/NFAT signaling pathway inhibition can be a therapeutic strategy in cardiovascular disorders including cardiac hypertrophy, restenosis, atherosclerosis, and angiogenesis.
Osteoclasts are multinucleated cells of monocyte/macrophage origin that degrade bone matrix. The differentiation of osteoclasts is dependent on a tumor necrosis factor (TNF) family cytokine, receptor activator of nuclear factor (NF)-kappaB ligand (RANKL), as well as macrophage colony-stimulating factor (M-CSF). Congenital lack of osteoclasts causes osteopetrosis. Among the essential molecules for osteoclastogenesis, including TNF receptor-associated factor (TRAF) 6, NF-kappaB, c-Fos and NFAT2. NFAT2 is activated by calcium signaling and binds to its own promoter, thus switching on an autoregulatory loop. C-Fos, as an activator protein (AP)-1 complex, is required for the autoamplification of NFAT2, enabling the robust induction of NFAT2. NFAT2 cooperates with other transcriptional partners to activate osteoclast-specific genes. Thus, NFAT2, the master transcription factor for osteoclast differentiation (Takayanagi, Ann. N.Y. Acad. Sci. 2007, 1116: 227-237). Excessive osteoclast formation characteristic of a variety of bone diseases. In rheumatoid arthritis, bone destruction is caused by the enhanced activity of osteoclasts. Suppressing the excessive osteoclast formation and/or the enhanced activity of osteoclasts by way of modulating the calcineurin/NFAT axis can be a strategy for the treatment and/or prevention of a variety of bone diseases.
Calcineurin/NFAT signaling axis is also important in the renal regulation of water homeostasis. A new member of the nuclear factor of activated T cells (NFAT) family has recently been discovered, NFAT 5, or Ton EBP. Ton EBP is the only known mammalian transcription factor that regulates gene expression in response to hypertonicity (Tyagi and Nandhakumar, Indian J Exp Biol. 2008, 46:89-93).
Deregulations of calcineurin/NFAT signaling and/or abnormal expression of its components have recently been reported in solid tumors of epithelial origin, lymphoma and lymphoid leukemia. Mouse models of human T-ALL/lymphoma shows that persistent activation of calcineurin/NFAT signaling is pro-oncogenic in vivo (Medyouf and Ghysdael, Cell Cycle. 2008, 7:297-303). Experimental evidence indicate the critical role of NFAT3 in some carcinogen-induced cell transformation and tumorigenicity (Lu and Huan, Curr Cancer Drug Targets. 2007, 7:343-53). There is an emerging role for Ca2+/calcineurin/NFAT signaling in cancerogenesis (Buchholz and Ellenrieder, Cell Cycle. 2007, 6(1):16-9). Modulation of NFAT can be suitable for the treatment of neoplastic cell proliferation diseases such as cancers.
Deregulation of calcineurin/NFAT signaling is also reported to be associated with defects in vertebrate development, since NFAT family of transcription factors are major regulators of vertebrate development. In human trisomy 21 or Down's syndrome, there is a human chromosome 21. Arron J R, et al. (Nature. 2006, 441:595-600) and Gwack Y, et al., (Nature, 2006, 441:646-50) report of two genes, DSCR1 and DYRK1A, that lie within the critical region of human chromosome 21 and the gene products act synergistically to inhibit the activation of NFATc transcription factors. The increase in expression of DSCR1 and DYRK1A can lead to a decrease in NFAT activation. In the mouse models of Down's syndrome, which are actually Dscr1- and Dyrk1a-overexpressing mice, these mice are found to be calcineurin- and NFAT-deficient. The reduced amount of NFAT can be associated with many of the features of Down's syndrome and also in many human diseases such as autoimmune disease and cancer as described herein.
Pancreatic beta-cells in the islet of Langerhans produce the hormone insulin, which maintains blood glucose homeostasis. Perturbations in beta-cell function may lead to impairment of insulin production and secretion and the onset of diabetes mellitus. Several essential beta-cell factors have been identified that are required for normal beta-cell function, including six genes that when mutated give rise to inherited forms of diabetes known as Maturity Onset Diabetes of the Young (MODY) (Heft, Bioessays. 2007, 29(10):1011-21). Mice with a beta-cell-specific deletion of the calcineurin phosphatase regulatory subunit, calcineurin b1 (Cnb1), develop age-dependent diabetes characterized by decreased beta-cell proliferation and mass, reduced pancreatic insulin content and hypoinsulinaemia. Moreover, beta-cells lacking Cnb1 have a reduced expression of established regulators of beta-cell proliferation. Conditional expression of active NFAT1 in Cnb1-deficient beta-cells rescues these defects and prevents diabetes. In normal adult beta-cells, conditional NFAT activation promotes the expression of cell-cycle regulators and increases beta-cell proliferation and mass, resulting in hyperinsulinaemia. Calcineurin/NFAT signaling regulates pancreatic beta-cell growth and function. Conditional NFAT activation also induces the expression of genes critical for beta-cell endocrine function, including all six genes mutated in hereditary forms of monogenic type 2 diabetes (Heit, Nature. 2006, 443(7109):345-9). Modulation of NFAT provides novel therapeutic approaches for the treatment of diabetes and for the prevention of diabetes for those at risk of developing diabetes.
There are evidences that the activation of calcineurin and NFAT and subsequently the PKC and the MEK/ERK MAPK pathways are induced by VEGF-A and IL-1 in endothelial cells. Gene activation via PLC-gamma provides VEGF with the potency to induce a wide spectrum of genes including many also upregulated by IL-1 (Schweighofer, Clin Hemorheol Microcirc. 2007, 37:57-62). Modulate calcineurin/NFAT can reduce VEGF-induced gene expression and reduced sprouting in undesired angiogenesis, such as in cancer, age-related macular degeneration, diabetic retinopathy; rheumatoid arthritis; Alzheimer's disease; obesity and endometriosis.
Nucleic Acid Inhibitors
In some embodiments, agents that inhibit the expression of a Dicer are nucleic acids. Nucleic acid inhibitors of a Dicer gene include, but not are limited to, RNA interference-inducing molecules (RNAi), for example, but not limited to, siRNA, dsRNA, stRNA, shRNA, an anti-sense oligonucleotide and modified versions thereof, where the RNA interference molecule silences the gene expression of the Dicer gene. In some embodiments, the nucleic acid inhibitor of a Dicer gene is an anti-sense oligonucleic acid, or a nucleic acid analogue, for example, but not limited to DNA, RNA, peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), or locked nucleic acid (LNA) and the like. In alternative embodiments, the nucleic acid is DNA or RNA, or nucleic acid analogues, for example, PNA, pcPNA and LNA. A nucleic acid can be single or double stranded, and can be selected from a group comprising nucleic acid encoding a protein of interest, oligonucleotides, PNA, etc. Such nucleic acid sequences include, for example, but not limited to, nucleic acid sequence encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc. Additional sequences can also be present.
RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) specifically suppresses the expression of a gene with its complementary sequence. Small interfering dsRNAs (siRNA) mediate post-transcriptional gene-silencing, and can be used to induce RNAi in mammalian cells. The dsRNA is processed intracellularly to release a short single stranded nucleic acid that can complementary base pair with the gene's primary transcript or mRNA. The resultant a double stranded RNA is susceptible to RNA degradation. Protein translation is thus prevent.
In some embodiments, single-stranded RNA (ssRNA), a form of RNA endogenously found in eukaryotic cells can be used to form an RNAi molecule. Cellular ssRNA molecules include messenger RNAs (and the progenitor pre-messenger RNAs), small nuclear RNAs, small nucleolar RNAs, transfer RNAs and ribosomal RNAs. Double-stranded RNA (dsRNA) induces a size-dependent immune response such that dsRNA larger than 30 bp activates the interferon response, while shorter dsRNAs feed into the cell's endogenous RNA interference machinery downstream of the Dicer enzyme.
Protein expression from the genes identified in Tables 1-5 can be reduced by inhibition of the expression of polypeptide (e.g., transcription, translation, post-translational processing) or by “gene silencing” methods commonly known by persons of ordinary skill in the art.
RNA interference (RNAi) provides a powerful approach for inhibiting the expression of selected target polypeptides. RNAi uses small interfering RNA (siRNA) duplexes that target the messenger RNA encoding the target polypeptide for selective degradation. siRNA-dependent post-transcriptional silencing of gene expression involves cutting the target messenger RNA molecule at a site guided by the siRNA.
RNA interference (RNAi) is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target gene results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76:9225), thereby inhibiting expression of the target gene. In one embodiment, the RNA is double stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs. siRNAs are incorporated into a protein complex (termed “RNA induced silencing complex,” or “RISC”) that recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target genes. As used herein, “inhibition of target gene expression” includes any decrease in expression or protein activity or level of the target gene or protein encoded by the target gene as compared to a situation wherein no RNA interference has been induced. The decrease can be of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target gene or the activity or level of the protein encoded by a target gene which has not been targeted by an RNA interfering agent.
“Short interfering RNA” (siRNA), also referred to herein as “small interfering RNA” is defined as an agent which functions to inhibit expression of a target gene, e.g., by RNAi. An siRNA can be chemically synthesized, can be produced by in vitro transcription, or can be produced within a host cell. In one embodiment, siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, 22, or 23 nucleotides in length, and can contain a 3′ and/or 5′ overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Preferably the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).
Double-stranded RNA (dsRNA) has been shown to trigger one of these posttranscriptional surveillance processes, in which gene silencing involves the degradation of single-stranded RNA (ssRNA) targets complementary to the dsRNA trigger (Fire A, 1999, Trends Genet. 15:358-363). RNA interference (RNAi) effects triggered by dsRNA have been demonstrated in a number of organisms including plants, protozoa, nematodes, and insects (Cogoni C. and Macino G, 2000, Curr Opin Genet Dev 10:638-643).
siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs). In one embodiment, these shRNAs are composed of a short (e.g., about 19 to about 25 nucleotide) antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand can precede the nucleotide loop structure and the antisense strand can follow. These shRNAs can be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA April; 9(4):493-501, incorporated by reference herein in its entirety).
The target gene or sequence of the RNA interfering agent can be a cellular gene or genomic sequence, e.g. that of calcineurin, Ran-GTPase, or Stim1 sequence. An siRNA can be substantially homologous to the target gene or genomic sequence, or a fragment thereof. As used in this context, the term “homologous” is defined as being substantially identical, sufficiently complementary, or similar to the target mRNA, or a fragment thereof, to effect RNA interference of the target. In addition to native RNA molecules, RNA suitable for inhibiting or interfering with the expression of a target sequence includes RNA derivatives and analogs. Preferably, the siRNA is identical to its target.
The siRNA preferably targets only one sequence. Each of the RNA interfering agents, such as siRNAs, can be screened for potential off-target effects by, for example, expression profiling. Such methods are known to one skilled in the art and are described, for example, in Jackson et al, Nature Biotechnology 6:635-637, 2003. In addition to expression profiling, one can also screen the potential target sequences for similar sequences in the sequence databases to identify potential sequences which can have off-target effects. For example, as few as 11 contiguous nucleotides of sequence identity are sufficient to direct silencing of non-targeted transcripts. Therefore, one can initially screen the proposed siRNAs to avoid potential off-target silencing using the sequence identity analysis by any known sequence comparison methods, such as BLAST.
siRNA molecules need not be limited to those molecules containing only RNA, but, for example, further encompasses chemically modified nucleotides and non-nucleotides, and also include molecules wherein a ribose sugar molecule is substituted for another sugar molecule or a molecule which performs a similar function. Moreover, a non-natural linkage between nucleotide residues can be used, such as a phosphorothioate linkage. For example, siRNA containing D-arabinofuranosyl structures in place of the naturally-occurring D-ribonucleosides found in RNA can be used in RNAi molecules according to the present invention (U.S. Pat. No. 5,177,196). Other examples include RNA molecules containing the o-linkage between the sugar and the heterocyclic base of the nucleoside, which confers nuclease resistance and tight complementary strand binding to the oligonucleotidesmolecules similar to the oligonucleotides containing 2′-O-methyl ribose, arabinose and particularly D-arabinose (U.S. Pat. No. 5,177,196).
The RNA strand can be derivatized with a reactive functional group of a reporter group, such as a fluorophore. Particularly useful derivatives are modified at a terminus or termini of an RNA strand, typically the 3′ terminus of the sense strand. For example, the 2′-hydroxyl at the 3′ terminus can be readily and selectively derivatized with a variety of groups.
Other useful RNA derivatives incorporate nucleotides having modified carbohydrate moieties, such as 2′O-alkylated residues or 2′-O-methyl ribosyl derivatives and 2′-O-fluoro ribosyl derivatives. The RNA bases can also be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence can be used. For example, halogenated bases, such as 5-bromouracil and 5-iodouracil can be incorporated. The bases can also be alkylated, for example, 7-methylguanosine can be incorporated in place of a guanosine residue. Non-natural bases that yield successful inhibition can also be incorporated.
The more preferred siRNA modifications include 2′-deoxy-2′-fluorouridine or locked nucleic acid (LNA) nucleotides and RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages. Such modifications are known to one skilled in the art and are described, for example, in Braasch et al., Biochemistry, 42: 7967-7975, 2003. Most of the useful modifications to the siRNA molecules can be introduced using chemistries established for antisense oligonucleotide technology. Preferably, the modifications involve minimal 2′-O-methyl modification, preferably excluding such modification. Modifications also preferably exclude modifications of the free 5′-hydroxyl groups of the siRNA.
Locked nucleic acids (LNAs), also known as bridged nucleic acids (BNAs), developed by Wengel and co-workers (Koshkin A. A., 1998, Tetrahedron, 54:3607-3630) and Imanishi and co-workers (Obika S., 1998, Tetrahedron Lett., 39:5401-5404). LNA bases are ribonucleotide analogs containing a methylene linkage between the 2′ oxygen and the 4′ carbon of the ribose ring. The constraint on the sugar moiety results in a locked 3′-endo conformation that preorganizes the base for hybridization and increases melting temperature (Tm) values as much as 10° C. per base (Wengel J., 1999, Acc. Chem. Res., 32:301-310; Braasch D. A. and Corey, D. R., 2001, Chem. Biol., 8:1-7). LNA bases can be incorporated into oligonucleotides using standard protocols for DNA synthesis. This commonality facilitates the rapid synthesis of chimeric oligonucleotides that contain both DNA and LNA bases and allows chimeric oligomers to be tailored for their binding affinity and ability to activate RNase H. Because oligomers that contain LNA bases have a native phosphate backbone they are readily soluble in water. Introduction of LNA bases also confers resistance to nucleases when incorporated at the 5′ and 3′ ends of oligomers (Crinelli R., et. al., 2002, Nucleic Acids Res., 30:2435-2443). The ability to use LNAs for in vivo applications is also favored by the finding that LNAs have demonstrated low toxicity when delivered intravenously to animals (Wahlestedt C., et. al., 2000, Proc. Natl. Acad. Sci. USA, 97: 5633-5638).
LNAs and LNA-DNA chimeras have been shown to be potent inhibitors of human telomerase and that a relatively short eight base LNA is a 1000-fold more potent agent than an analogous peptide nucleic acid (PNA) oligomer (Elayadi A. N., et. al., 2002, Biochemistry, 41: 9973-9981). LNAs and LNA-DNA chimeras have also been shown to be useful agents for antisense gene inhibition. Wengel and co-workers have used LNAs to inhibit gene expression in mice (Wahlestedt C., et. al., 2000, Proc. Natl. Acad. Sci. USA, 97:5633-5638), while Erdmann and colleagues have described the design of LNA-containing oligomers that recruit RNase H and have described the rules governing RNase H activation by LNA-DNA chimeras in cell-free systems (Kurreck J., et. al., 2002, Nucleic Acids Res., 30:1911-1918).
The syntheses of LNA-containing oligomers are known in the art, for examples, those described in U.S. Pat. Nos. 6,316,198, 6,670,461, 6,794,499, 6,977,295, 6,998,484, 7,053,195, and U.S Patent Publication No. US 2004/0014959, and all of which are hereby incorporated by reference in their entirety.
Another nucleic acid derivative envisioned in the methods described herein is phosphorodiamidate morpholino oligomer (PMO). PMOs are DNA mimics that inhibit expression of specific mRNA in eukaryotic cells (Arora, V., et. al., 2000, J. Pharmacol. Exp. Ther. 292:921-928; Qin, G., et. al., 2000, Antisense Nucleic Acid Drug Dev. 10:11-16; Summerton, J., et. al., 1997, Antisense Nucleic Acid Drug Dev. 7:63-70). They are synthesized by using the four natural bases, with a base sequence that is complementary (antisense) to a region of a specific mRNA. They are different than DNA in the chemical structure that links the bases together. Ribose has been replaced with a morpholine group, and the phosphodiester is replaced with a phosphorodiamidate. These alterations make the antisense molecule resistant to nucleases (Hudziak, R., et. al., 1996 Antisense Nucleic Acid Drug Dev. 6:267-272) and free of charges at physiological pH, yet it retains the molecular architecture required for binding specifically to a complementary strand of nucleic acid (Stein, D., et. al, 1997, Antisense Nucleic Acid Drug Dev. 7:151-157; Summerton, J., et. al., 1997, Antisense Nucleic Acid Drug Dev. 7:63-70; Summerton, J., and D. Weller., 1997, Antisense Nucleic Acid Drug Dev. 7:187-195).
The synthesis, structures, and binding characteristics of morpholine oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,127,866, 5,142,047, 5,166,315, 5,521,063, and 5,506,337, and all of which are hereby hereby incorporated by reference in their entirety. PMOs can be synthesized at AVI BioPharma (Corvallis, Oreg.) in accordance with known methods, as described, for example, in Summerton, J., and D. Weller U.S. Pat. No. 5,185,444; and Summerton, J., and D. Weller. 1997, Antisense Nucleic Acid Drug Dev. 7:187-195. For example, PMO against calcineurin or KCNN4 transcripts should containing between 12-40 nucleotide bases, and having a targeting sequence of at least 12 subunits complementary to the respective transcript. Methods of making and using PMO for the inhibition of gene expression in vivo are described in U.S. Patent Publication No. US 2003/0171335; US 2003/0224055; US 2005/0261249; US 2006/0148747; S 2007/0274957; US 2007/003776; and US 2007/0129323; and these are hereby incorporated by reference in their entirety.
siRNA and miRNA molecules having various “tails” covalently attached to either their 3′- or to their 5′-ends, or to both, are also known in the art and can be used to stabilize the siRNA and miRNA molecules delivered using the methods of the present invention. Generally speaking, intercalating groups, various kinds of reporter groups and lipophilic groups attached to the 3′ or 5′ ends of the RNA molecules are well known to one skilled in the art and are useful according to the methods of the present invention. Descriptions of syntheses of 3′-cholesterol or 3′-acridine modified oligonucleotides applicable to preparation of modified RNA molecules useful according to the present invention can be found, for example, in the articles: Gamper, H. B., Reed, M. W., Cox, T., Virosco, J. S., Adams, A. D., Gall, A., Scholler, J. K., and Meyer, R. B. (1993) Facile Preparation and Exonuclease Stability of 3′-Modified Oligodeoxynucleotides. Nucleic Acids Res. 21 145-150; and Reed, M. W., Adams, A. D., Nelson, J. S., and Meyer, R. B., Jr. (1991) Acridine and Cholesterol-Derivatized Solid Supports for Improved Synthesis of 3′-Modified Oligonucleotides. Bioconjugate Chem. 2 217-225 (1993).
Other siRNAs useful for targeting the genes identified in Tables 1-5 can be readily designed and tested. Accordingly, siRNAs useful for the methods described herein include siRNA molecules of about 15 to about 40 or about 15 to about 28 nucleotides in length, which are homologous to an gene identified in Tables 1-5. Preferably, the siRNA molecules targeting the gene identified in Tables 1-5 have a length of about 19 to about 25 nucleotides. More preferably, the siRNA molecules have a length of about 19, 20, 21, or 22 nucleotides. The siRNA molecules can also comprise a 3′ hydroxyl group. The siRNA molecules can be single-stranded or double stranded; such molecules can be blunt ended or comprise overhanging ends (e.g., 5′, 3′). In specific embodiments, the RNA molecule is double stranded and either blunt ended or comprises overhanging ends.
In one embodiment, at least one strand of the RNA molecule has a 3′ overhang from about 0 to about 6 nucleotides (e.g., pyrimidine nucleotides, purine nucleotides) in length. In other embodiments, the 3′ overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4 nucleotides in length. In one embodiment, the RNA molecule that targets the gene identified in Tables 1-5 is double stranded—one strand has a 3′ overhang and the other strand can be blunt-ended or have an overhang. In the embodiment in which the gene identified in Tables 1-5 targeting RNA molecule is double stranded and both strands comprise an overhang, the length of the overhangs can be the same or different for each strand. In a embodiment, the RNA comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides which are paired and which have overhangs of from about 1 to about 3, particularly about 2, nucleotides on both 3′ ends of the RNA. In one embodiment, the 3′ overhangs can be stabilized against degradation. In a preferred embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine 2 nucleotide 3′ overhangs by 2′-deoxythymidine is tolerated and does not affect the efficiency of RNAi. The absence of a 2′ hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium.
In some embodiments, assessment of the expression and/or knock down of gene identified in Tables 1-5 using gene specific siRNAs can be determined by methods that are well known in the art, such as western blot analysis or enzyme activity assays. Other methods can be readily prepared by those of skill in the art based on the known sequence of the target mRNA.
siRNA sequences are chosen to maximize the uptake of the antisense (guide) strand of the siRNA into RISC and thereby maximize the ability of RISC to target the mRNA of the gene identified in Tables 1-5 for degradation. This can be accomplished by scanning for sequences that have the lowest free energy of binding at the 5′-terminus of the antisense strand. The lower free energy leads to an enhancement of the unwinding of the 5′-end of the antisense strand of the siRNA duplex, thereby ensuring that the antisense strand will be taken up by RISC and direct the sequence-specific cleavage of the mRNA of the human gene identified in Tables 1-5.
In a preferred embodiment, the siRNA or modified siRNA is delivered in a pharmaceutically acceptable carrier. Additional carrier agents, such as liposomes, can be added to the pharmaceutically acceptable carrier.
In another embodiment, the siRNA is delivered by delivering a vector encoding small hairpin RNA (shRNA) in a pharmaceutically acceptable carrier to the cells in an organ of an individual. The shRNA is converted by the cells after transcription into a siRNA capable of targeting a specific gene identified in Tables 1-5. In one embodiment, the vector can be a plasmid, a cosmid, a phagmid, a hybrid thereof, or a virus. In one embodiment, the vector can be a regulatable vector, such as tetracycline inducible vector.
In one embodiment, the RNA interfering agents used in the methods described herein are taken up actively by cells in vivo following intravenous injection, e.g., hydrodynamic injection, without the use of a vector, illustrating efficient in vivo delivery of the RNA interfering agents, e.g., the siRNAs used in the methods of the invention.
Other strategies for delivery of the RNA interfering agents, e.g., the siRNAs or shRNAs used in the methods of the invention, can also be employed, such as, for example, delivery by a vector, e.g., a plasmid or viral vector, e.g., a lentiviral vector. Such vectors can be used as described, for example, in Xiao-Feng Qin et al. Proc. Natl. Acad. Sci. U.S.A., 100: 183-188. Other delivery methods include delivery of the RNA interfering agents, e.g., the siRNAs or shRNAs of the invention, using a basic peptide by conjugating or mixing the RNA interfering agent with a basic peptide, e.g., a fragment of a TAT peptide, mixing with cationic lipids or formulating into particles.
As noted, the dsRNA, such as siRNA or shRNA can be delivered using an inducible vector, such as a tetracycline inducible vector. Methods described, for example, in Wang et al. Proc. Natl. Acad. Sci. 100: 5103-5106, using pTet-On vectors (BD Biosciences Clontech, Palo Alto, Calif.) can be used. In some embodiments, a vector can be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion and foreign sequence and for the introduction into eukaryotic cells. The vector can be an expression vector capable of directing the transcription of the DNA sequence of the agonist or antagonist nucleic acid molecules into RNA. Viral expression vectors can be selected from a group comprising, for example, reteroviruses, lentiviruses, Epstein Barr virus-, bovine papilloma virus, adenovirus- and adeno-associated-based vectors or hybrid virus of any of the above. In one embodiment, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the antagonist nucleic acid molecule in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
RNA interference molecules and nucleic acid inhibitors useful in the methods as disclosed herein can be produced using any known techniques such as direct chemical synthesis, through processing of longer double stranded RNAs by exposure to recombinant Dicer protein or Drosophila embryo lysates, through an in vitro system derived from S2 cells, using phage RNA polymerase, RNA-dependant RNA polymerase, and DNA based vectors. Use of cell lysates or in vitro processing can further involve the subsequent isolation of the short, for example, about 21-23 nucleotide, siRNAs from the lysate, etc. Chemical synthesis usually proceeds by making two single stranded RNA-oligomers followed by the annealing of the two single stranded oligomers into a double stranded RNA. Other examples include methods disclosed in WO 99/32619 and WO 01/68836 that teach chemical and enzymatic synthesis of siRNA. Moreover, numerous commercial services are available for designing and manufacturing specific siRNAs (see, e.g., QIAGEN Inc., Valencia, Calif. and AMBION Inc., Austin, Tex.)
In some embodiments, an agent is protein or polypeptide or RNAi agent that inhibits the expression of genes identified in Tables 1-5 and/or activity of proteins encoded by gene identified in Tables 1-5. In such embodiments, cells can be modified (e.g., by homologous recombination) to provide increased expression of such an agent, for example, by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the natural inhibitor agent. For example, a protein or miRNA inhibitor of a gene identified in Tables 1-5 become expressed at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the desired nucleic acid encoding the agent. See, for example, PCT International Publication No. WO 94/12650 by Transkaryotic Therapies, Inc., PCT International Publication No. WO 92/20808 by Cell Genesys, Inc., and PCT International Publication No. WO 91/09955 by Applied Research Systems. Cells also can be engineered to express an endogenous gene comprising the agent under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene can be replaced by homologous recombination. Gene activation techniques are described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; PCT/US92/09627 (WO93/09222) by Selden et al.; and PCT/US90/06436 (WO91/06667) by Skoultchi et al. The agent can be prepared by culturing transformed host cells under culture conditions suitable to express the miRNA. The resulting expressed agent can then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of a peptide or nucleic acid agent inhibitor of the gene identified in Tables 1-5 can also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA Sepharose; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; immunoaffinity chromatography, or complementary cDNA affinity chromatography.
In one embodiment, the nucleic acid inhibitors of the genes identified in Tables 1-5 can be obtained synthetically, for example, by chemically synthesizing a nucleic acid by any method of synthesis known to the skilled artisan. The synthesized nucleic acid inhibitors of the gene identified in Tables 1-5 can then be purified by any method known in the art. Methods for chemical synthesis of nucleic acids include, but are not limited to, in vitro chemical synthesis using phosphotriester, phosphate or phosphoramidite chemistry and solid phase techniques, or via deoxynucleoside H-phosphonate intermediates (see U.S. Pat. No. 5,705,629 to Bhongle).
In some circumstances, for example, where increased nuclease stability is desired, nucleic acids having nucleic acid analogs and/or modified internucleoside linkages can be preferred. Nucleic acids containing modified internucleoside linkages can also be synthesized using reagents and methods that are well known in the art. For example, methods of synthesizing nucleic acids containing phosphonate phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate, formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate, dimethylene-sulfide (—CH2—S—CH2), dimethylene-sulfoxide (—CH2—SO—CH2), dimethylene-sulfone (—CH2—SO2—CH2), 2′-O-alkyl, and 2′-deoxy-2′-fluoro′phosphorothioate internucleoside linkages are well known in the art (see Uhlmann et al., 1990, Chem. Rev. 90:543-584; Schneider et al., 1990, Tetrahedron Lett. 31:335 and references cited therein). U.S. Pat. Nos. 5,614,617 and 5,223,618 to Cook, et al., U.S. Pat. No. 5,714,606 to Acevedo, et al, U.S. Pat. No. 5,378,825 to Cook, et al., U.S. Pat. Nos. 5,672,697 and 5,466,786 to Buhr, et al., U.S. Pat. No. 5,777,092 to Cook, et al., U.S. Pat. No. 5,602,240 to De Mesmacker, et al., U.S. Pat. No. 5,610,289 to Cook, et al. and U.S. Pat. No. 5,858,988 to Wang, also describe nucleic acid analogs for enhanced nuclease stability and cellular uptake.
The siRNA molecules of the present invention can be generated by annealing two complementary single-stranded RNA molecules together (one of which matches a portion of the target mRNA) (Fire et al., U.S. Pat. No. 6,506,559) or through the use of a single hairpin RNA molecule that folds back on itself to produce the requisite double-stranded portion (Yu et al. (2002) Proc. Natl. Acad. Sci. USA 99:6047-52). The siRNA molecules can also be chemically synthesized (Elbashir et al. (2001) Nature 411:494-98)
Synthetic siRNA molecules, including shRNA molecules, can be obtained using a number of techniques known to those of skill in the art. For example, the siRNA molecule can be chemically synthesized or recombinantly produced using methods known in the art, such as using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer (see, e.g., Elbashir, S. M. et al. (2001) Nature 411:494-498; Elbashir, S. M., W. Lendeckel and T. Tuschl (2001) Genes & Development 15:188-200; Harborth, J. et al. (2001) J. Cell Science 114:4557-4565; Masters, J. R. et al. (2001) Proc. Natl. Acad. Sci., USA 98:8012-8017; and Tuschl, T. et al. (1999) Genes & Development 13:3191-3197). Alternatively, several commercial RNA synthesis suppliers are available including, but are not limited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK). As such, siRNA molecules are not overly difficult to synthesize and are readily provided in a quality suitable for RNAi.
siRNA can also be produced by in vitro transcription using single-stranded DNA templates (Yu et al., supra). Alternatively, the siRNA molecules can be produced biologically, either transiently (Yu et al., supra; Sui et al. (2002) Proc. Natl. Acad. Sci. USA 99:5515-20) or stably (Paddison et al. (2002) Proc. Natl. Acad. Sci. USA 99:1443-48), using an expression vector(s) containing the sense and antisense siRNA sequences. siRNA can be designed into short hairpin RNA (shRNA) for plasmid- or vector-based approaches for supplying siRNAs to cells to produce stable gene identified in Tables 1-5 silencing. Examples of vectors for shRNA are #AM5779:—pSILENCERTM 4.1-CMV neo; #AM5777:—pSILENCERTM 4.1-CMV hygro; #AM5775:—pSILENCERTM 4.1-CMV puro; #AM7209:—pSILENCERTM 2.0-U6; #AM7210:—pSILENCERTM 3.0-H1; #AM5768: —pSILENCERTM 3.1-H1 puro; #AM5762:—pSILENCERTM 2.1-U6 puro; #AM5770:—pSILENCERTM 3.1-H1 neo; #AM5764:—pSILENCERTM 2.1-U6 neo; #AM5766:—pSILENCERTM 3.1-H1 hygro; #AM5760:—pSILENCERTM 2.1-U6 hygro; #AM7207:—pSILENCERTM 1.0-U6 (circular) from Ambion®.
Recently, reduction of levels of target mRNA in primary human cells, in an efficient and sequence-specific manner, was demonstrated using adenoviral vectors that express hairpin RNAs, which are further processed into siRNAs (Arts et al. (2003) Genome Res. 13:2325-32). In addition, dsRNAs can be expressed as stem loop structures encoded by plasmid vectors, retroviruses and lentiviruses (Paddison, P. J. et al. (2002) Genes Dev. 16:948-958; McManus, M. T. et al. (2002) RNA 8:842-850; Paul, C. P. et al. (2002) Nat. Biotechnol. 20:505-508; Miyagishi, M. et al. (2002) Nat. Biotechnol. 20:497-500; Sui, G. et al. (2002) Proc. Natl. Acad. Sci., USA 99:5515-5520; Brummelkamp, T. et al. (2002) Cancer Cell 2:243; Lee, N. S., et al. (2002) Nat. Biotechnol. 20:500-505; Yu, J. Y., et al. (2002) Proc. Natl. Acad. Sci., USA 99:6047-6052; Zeng, Y., et al. (2002) Mol. Cell. 9:1327-1333; Rubinson, D. A., et al. (2003) Nat. Genet. 33:401-406; Stewart, S. A., et al. (2003) RNA 9:493-501). These vectors generally have a polIII promoter upstream of the dsRNA and can express sense and antisense RNA strands separately and/or as a hairpin structures. Within cells, Dicer processes the short hairpin RNA (shRNA) into effective siRNA.
The targeted region of the siRNA molecule of the present invention can be selected from a given target gene sequence, e.g., the coding sequence of a gene identified in Tables 1-5, beginning from about 25 to 50 nucleotides, from about 50 to 75 nucleotides, or from about 75 to 100 nucleotides downstream of the start codon. Nucleotide sequences can contain 5′ or 3′ UTRs and regions nearby the start codon. One method of designing a siRNA molecule of the present invention involves identifying the 23 nucleotide sequence motif AA(N19)TT (SEQ ID NO: 12) (where N can be any nucleotide), and selecting hits with at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% G/C content. The “TT” portion of the sequence is optional. Alternatively, if no such sequence is found, the search can be extended using the motif NA(N21), where N can be any nucleotide. In this situation, the 3′ end of the sense siRNA can be converted to TT to allow for the generation of a symmetric duplex with respect to the sequence composition of the sense and antisense 3′ overhangs. The antisense siRNA molecule can then be synthesized as the complement to nucleotide positions 1 to 21 of the 23 nucleotide sequence motif. The use of symmetric 3′ TT overhangs can be advantageous to ensure that the small interfering ribonucleoprotein particles (siRNPs) are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs (Elbashir et al. (2001) supra and Elbashir et al. 2001 supra). Analysis of sequence databases, including but are not limited to the NCBI, BLAST, Derwent and GenSeq as well as commercially available oligosynthesis software such as Oligoengine®, can also be used to select siRNA sequences against EST libraries to ensure that only one gene is targeted.
Methods of predicting and selecting antisense oligonucleotides and siRNA are known in the art and are also found at the Website for GENSCRIPT, AMBION, DHARMACON, OLIGOENGINE, WADSWORTH, Whitehead Institute at the Massachusetts Institute of Technology and described in U.S. Pat. No. 6,060,248.
In some aspects, antisense nucleic acid technology can be used to inhibit the expression of gene identified in Tables 1-5. It is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene “off”. This is because mRNA has to be single stranded for it to be translated. This synthesized nucleic acid is termed an “anti-sense” oligonucleotide because its base sequence is complementary to the gene's messenger RNA (mRNA), which is called the “sense” sequence (so that a sense segment of mRNA “5′-AAGGUC-3′ ” would be blocked by the anti-sense mRNA segment “3′-UUCCAG-5′”).
Delivery of RNA Interfering Agents: Methods of delivering RNA interfering agents, e.g., an siRNA, or vectors containing an RNA interfering agent, to the target cells (e.g., cells of the brain or other desired target cells, for cells in the central and peripheral nervous systems), can include, for example (i) injection of a composition containing the RNA interfering agent, e.g., an siRNA, or (ii) directly contacting the cell, e.g., a cell of the brain, with a composition comprising an RNA interfering agent, e.g., an siRNA. In one embodiment, the RNA interfering agent can be targeted to the bone marrow where the lymphocytes expressing the genes identified in Tables 1-5 are made. In another embodiment, RNA interfering agents, e.g., an siRNA can be injected directly into any blood vessel, such as vein, artery, venule or arteriole, via, e.g., hydrodynamic injection or catheterization. In yet another embodiment, the RNA interfering agent can be injected or applied topically directly to the site of the skin ulcers.
Administration can be by a single injection or by two or more injections. The RNA interfering agent is delivered in a pharmaceutically acceptable carrier. One or more RNA interfering agents can be used simultaneously. The RNA interfering agents, e.g., the siRNAs targeting the mRNA of genes identified in Tables 1-5, can be delivered singly, or in combination with other RNA interfering agents, e.g., siRNAs, such as, for example siRNAs directed to other cellular genes. siRNAs targeting gene identified in Tables 1-5 can also be administered in combination with other pharmaceutical agents which are used to treat or prevent immunological diseases or disorders.
In one embodiment, specific cells are targeted with RNA interference, limiting potential side effects of RNA interference caused by non-specific targeting of RNA interference. The method can use, for example, a complex or a fusion molecule comprising a cell targeting moiety and an RNA interference binding moiety that is used to deliver RNA interference effectively into cells. For example, an antibody-protamine fusion protein when mixed with an siRNA, binds siRNA and selectively delivers the siRNA into cells expressing an antigen recognized by the antibody, resulting in silencing of gene expression only in those cells that express the antigen. The siRNA or RNA interference-inducing molecule binding moiety is a protein or a nucleic acid binding domain or fragment of a protein, and the binding moiety is fused to a portion of the targeting moiety. The location of the targeting moiety can be either in the carboxyl-terminal or amino-terminal end of the construct or in the middle of the fusion protein.
A viral-mediated delivery mechanism can also be employed to deliver siRNAs to cells in vitro and in vivo as described in Xia, H. et al. (2002) Nat Biotechnol 20(10):1006). Plasmid- or viral-mediated delivery mechanisms of shRNA can also be employed to deliver shRNAs to cells in vitro and in vivo as described in Rubinson, D. A., et al. ((2003) Nat. Genet. 33:401-406) and Stewart, S. A., et al. ((2003) RNA 9:493-501).
RNA interfering agents, for e.g., an siRNA, can also be introduced into cells via the vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid.
The dose of the particular RNA interfering agent will be in an amount necessary to effect RNA interference, e.g., post translational gene silencing (PTGS), of the particular target gene, thereby leading to inhibition of target gene expression or inhibition of activity or level of the protein encoded by the target gene.
It is also known that RNAi molecules do not have to match perfectly to their target sequence. Preferably, however, the 5′ and middle part of the antisense (guide) strand of the siRNA is perfectly complementary to the target nucleic acid sequence.
Accordingly, the RNAi molecules functioning as nucleic acid inhibitors of the genes identified in Tables 1-5 disclosed herein are, for example, but not limited to, unmodified and modified double stranded (ds) RNA molecules including short-temporal RNA (stRNA), small interfering RNA (siRNA), short-hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), (see, e.g. Baulcombe, Science 297:2002-2003, 2002). The dsRNA molecules, e.g. siRNA, also can contain 3′ overhangs, preferably 3′UU or 3′TT overhangs. In one embodiment, the siRNA molecules of the present invention do not include RNA molecules that comprise ssRNA greater than about 30-40 bases, about 40-50 bases, about 50 bases or more. In one embodiment, the siRNA molecules of the present invention are double stranded for more than about 25%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90% of their length. In some embodiments, a nucleic acid inhibitor of a gene identified in Tables 1-5 is any agent which binds to and inhibits the expression of mRNA of that gene identified in Tables 1-5, where the mRNA or a product of transcription of nucleic acid is encoded by SEQ. ID NOS:1-11 (Genbank Accession No. NM—000944; NM—021132.1; NM—006325; NM—006267.4; NM—002265.4, NM—001316; NM—003400.3; NM—003156.2, NM—020860.2, NM—032790.3, NM—002250.2).
In another embodiment, agents inhibiting the genes identified in Tables 1-5 are catalytic nucleic acid constructs, such as, for example ribozymes, which are capable of cleaving RNA transcripts and thereby preventing the production of wildtype protein. Ribozymes are targeted to and anneal with a particular sequence by virtue of two regions of sequence complementary to the target flanking the ribozyme catalytic site. After binding, the ribozyme cleaves the target in a site specific manner. The design and testing of ribozymes which specifically recognize and cleave sequences of the gene products described herein, for example, for the cleavage of the genes identified in Tables 1-5 or homologues or variants thereof can be achieved by techniques well known to those skilled in the art (for example Lleber and Strauss, (1995) Mol Cell Biol 15:540.551, the disclosure of which is incorporated herein by reference).
Pharmaceutical Compositions and Administration
In one embodiment, the invention provides a pharmaceutical composition comprising an agent that inhibits the activity of a protein encoded by a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5 and a pharmaceutically acceptable carrier. The agent can be a small molecule, nucleic acid, nucleic acid analogue, protein, antibody, peptide, aptamer or variants or fragments thereof. Other forms of inhibitors include a nucleic acid agent which is an RNAi agent such as a siRNA, shRNA, miRNA, dsRNA or ribozyme or variants thereof.
In one embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., 1990). In one embodiment, other ingredients can be added to pharmaceutical formulations, including antioxidants, e.g., ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; and sugar alcohols such as mannitol or sorbitol.
In a embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, to name a few.
Various delivery systems are known in the art and can be used to administer agent that inhibits the activity a candidate protein and/or the expression of a gene identified in Tables 1-5 of Tables 1-5, e.g., encapsulation in liposomes, microparticles, and microcapsules (see, e.g., Wu and Wu, J. Biol. Chem., 262:4429-4432 (1987)). The composition can be delivered in a vesicle, in particular a liposome (see, Langer, Science, 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler, eds. (Liss, New York 1989), pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see, generally, ibid.).
Pharmaceutical compositions can be administered by any known route. By way of example, the composition can be administered by a mucosal, pulmonary, topical, or other localized or systemic route (e.g., enteral and parenteral). The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection, infusion and other injection or infusion techniques, without limitation. The phrases “systemic administration,” “administered systemically”, “peripheral administration” and “administered peripherally” as used herein mean the administration of the agents as disclosed herein such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
Administration can be systemic or local. In addition, it can be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Omcana reservoir Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In one embodiment, the pharmaceutical formulation to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). The pH of the pharmaceutical formulation typically should be about from 6 to 8.
In one embodiment, the composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng., 14:201 (1987); Buchwald et al., Surgery, 88:507 (1980); Saudek et al., N. Engl. J. Med., 321:574 (1989)). In another embodiment, polymeric materials can be used (see, Medical Applications of Controlled Release, Langer and Wise, eds. (CRC Press, Boca Raton, Fla. 1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball, eds. (Wiley, New York 1984); Ranger and Peppas, Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983); see also Levy et al., Science, 228:190 (1985); During et al., Ann. Neurol., 25:35 1 (1989); Howard et al., J. Neurosurg., 7 1:105 (1989)). Other controlled release systems are discussed in the review by Langer (Science, 249:1527-1533 (1990)). For examples of sustained release compositions, see U.S. Pat. No. 3,773,919, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No. 1176565, U. Sidman et al., Biopolymers 22:547 (1983) and R. Langer et al., Chem. Tech. 12:98 (1982).
The precise dose to be employed in the formulation will also depend on the route of administration, and the severity of hyperactivity or inappropriate immune response, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. For gene therapy, viral vector should be in the range of 1×106 to 1014 viral vector particles per application per patient.
In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the route of administration, and the seriousness of the condition being treated and should be decided according to the judgment of the practitioner and each subject's circumstances in view of, e.g., published clinical studies. Suitable effective dosage amounts, however, range from about 10 micrograms to about 5 grams about every 4 hour, although they are typically about 500 mg or less per every 4 hours. In one embodiment the effective dosage is about 0.01 mg, 0.5 mg, about 1 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 g, about 1.2 g, about 1.4 g, about 1.6 g, about 1.8 g, about 2.0 g, about 2.2 g, about 2.4 g, about 2.6 g, about 2.8 g, about 3.0 g, about 3.2 g, about 3.4 g, about 3.6 g, about 3.8 g, about 4.0 g, about 4.2 g, about 4.4 g, about 4.6 g, about 4.8 g, or about 5.0 g, every 4 hours. Equivalent dosages may be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month, and about every two months. The effective dosage amounts described herein refer to total amounts administered. The compositions comprising agent that inhibits the activity of a protein encoded by a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5, including expression vectors and/or viral vectors are suitably administered to the patient at one time or over a series of treatments. For purposes herein, a “therapeutically effective amount” of a composition comprising an agent that inhibits the activity of a protein encoded by a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5 is an amount that is effective to reduce the amount of NFAT nuclear translocation, Ca+ influx and/or cytokine production by at least 20%, or reduce the symptom associated hyperactive or inappropriate immune response by at least 10%.
In an embodiment, the composition comprising an agent that inhibits the activity of a protein encoded by a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5 is administered in combination with immunosuppressive therapies including, but not limited to, azathioprine, infliximab, omalizumab, daclizumab, adalimumab, eculizumab, efalizumab, natalizumab, and omalizumab. In another embodiment, the composition comprising agent that inhibits the activity of a protein encoded by a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5 is administered in combination with immunosuppressive therapies and cyclophosphamide, chlorambucil, and/or rituximab.
Gene Therapy
In one embodiment, the agent that inhibits the activity of a protein encoded by a gene identified in Tables 1-5 and/or the expression of a gene identified in Tables 1-5 is administered to an individual by any one of several gene therapy techniques known to those of skill in the art. In general, gene therapy can be accomplished by either direct transformation of target cells within the mammalian subject (in vivo gene therapy) or transformation of cells in vitro and subsequent implantation of the transformed cells into the mammalian subject (ex vivo gene therapy). A viral vector carries an RNAi agent such as a shRNA or anti-sense oligonucleotide for a gene identified in Tables 1-5 under a tissue specific regulatory element is administered to an individual. The tissue specific regulatory element allows the expression of the RNAi agent in the target cells, for example, the lymph nodes.
The principles of gene therapy are disclosed by Oldham, R. K. (In: Principles of Biotherapy, Raven Press, N.Y., 1987), and similar texts. Disclosures of the methods and uses for gene therapy are provided by Boggs, S. S. (Int. J. Cell Clon. 8:80-96 (1990)); Karson, E. M. (Biol. Reprod. 42:39-49 (1990)); Ledley, F. D., In: Biotechnology, A Comprehensive Treatise, volume 7B, Gene Technology, VCH Publishers, Inc. NY, pp 399-458 (1989)), all of which references are incorporated herein by reference.
The nucleic acid encoding an RNAi agent such as shRNA can be introduced into the somatic cells of an animal (particularly mammals including humans) in gene therapy. Most preferably, viral or retroviral vectors are employed for as the transfer vehicle this purpose. The gene therapy virus can be in the form of an adenovirus, adeno-associated virus or lentivirus.
Retroviral vectors are a common mode of delivery and in this context are retroviruses from which all viral genes have been removed or altered so that no viral proteins are made in cells infected with the vector. Viral replication functions are provided by the use of retrovirus “packaging” cells that produce all of the viral proteins but that do not produce infectious virus.
Introduction of the retroviral vector DNA into packaging cells results in production of virions that carry vector RNA and can infect target cells, but such that no further virus spread occurs after infection. To distinguish this process from a natural virus infection where the virus continues to replicate and spread, the term transduction rather than infection is often used.
In one embodiment, the method of treating MN described herein provides a recombinant lentivirus for the delivery and expression of an RNAi agent in either dividing or non-dividing mammalian cells. The HIV-1 based lentivirus can effectively transduce a broader host range than the Moloney Leukemia Virus (MoMLV)-base retroviral systems. Preparation of the recombinant lentivirus can be achieved using the pLenti4/V5-DESTTM, pLenti6/V5-DESTTM or pLenti vectors together with ViraPowerTM Lentiviral Expression systems from Invitrogen.
Examples of use of lentiviral vectors for gene therapy for inherited disorders and various types of cancer, and these references are hereby incorporated by reference (Klein, C. and Baum, C. (2004). Hematol. J., 5, 103-111; Zufferey, R et al. (1997). Nat. Biotechnol., 15, 871-875; Morizono, K. et al. (2005). Nat. Med., 11, 346-352; Di Domenico, C. et. al. (2005), Hum. Gene Ther., 16, 81-90; Kim, E. Y., et al., (2004). Biochem. Biophys. Res. Comm., 318, 381-390).
Non-retroviral vectors also have been used in genetic therapy. One such alternative is the adenovirus (Rosenfeld, M. A., et al., Cell 68:143155 (1992); Jaffe, H. A. et al., Nature Genetics 1:372-378 (1992); Lemarchand, P. et al., Proc. Natl. Acad. Sci. USA 89:6482-6486 (1992)). Major advantages of adenovirus vectors are their potential to carry large segments of DNA (36 Kb genome), a very high titre (1011/ml), ability to infect non-replicating cells, and suitability for infecting tissues in situ, especially in the lung. The most striking use of this vector so far is to deliver a human cystic fibrosis transmembrane conductance regulator (CFTR) gene by intratracheal instillation to airway epithelium in cotton rats (Rosenfeld, M. A., et al., Cell 63:143-155 (1992)). Similarly, herpes viruses may also prove valuable for human gene therapy (Wolfe, J. H. et al., Nature Genetics 1:379-384 (1992)). Of course, any other suitable viral vector may be used for genetic therapy with the present invention.
U.S. Pat. No. 6,531,456 provides methods for the successful transfer of a gene into a solid tumor cell using recombinant AAV virions. Generally, the method described in U.S. Pat. No. 6,531,456 allows for the direct, in vivo injection of recombinant AAV virions into tumor cell masses, e.g., by intra-tumoral injection. The invention also provides for the simultaneous delivery of a second gene using the recombinant AAV virions, wherein the second gene is capable of providing an ancillary therapeutic effect when expressed within the transduced cell. U.S. Pat. No. 6,531,456 is hereby incorporated by reference in its entirety.
The viron used for gene therapy can be any viron known in the art including but not limited to those derived from adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus. Recombinant viruses provide a versatile system for gene expression studies and therapeutic applications.
The recombinant AAV virions described above, including the DNA of interest, can be produced using standard methodology, known to those of skill in the art. The methods generally involve the steps of (1) introducing an AAV vector into a host cell; (2) introducing an AAV helper construct into the host cell, where the helper construct includes AAV coding regions capable of being expressed in the host cell to complement AAV helper functions missing from the AAV vector; (3) introducing one or more helper viruses and/or accessory function vectors into the host cell, wherein the helper virus and/or accessory function vectors provide accessory functions capable of supporting efficient recombinant AAV (“rAAV”) virion production in the host cell; and (4) culturing the host cell to produce rAAV virions. The AAV vector, AAV helper construct and the helper virus or accessory function vector(s) can be introduced into the host cell either simultaneously or serially, using standard transfection techniques. Using rAAV vectors, genes can be delivered into a wide range of host cells including many different human and non-human cell lines or tissues. Because AAV is non-pathogenic and does not illicit an immune response, a multitude of pre-clinical studies have reported excellent safety profiles. rAAVs are capable of transducing a broad range of cell types and transduction is not dependent on active host cell division. High titers, >108 viral particle/ml, are easily obtained in the supernatant and 1011-1012 viral particle/ml with further concentration. The transgene is integrated into the host genome so expression is long term and stable.
A simplified system for generating recombinant adenoviruses is presented by He TC., et al. Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998. The gene of interest is first cloned into a shuttle vector, e.g. pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease Pme I, and subsequently cotransformed into E. coli. BJ5183 cells with an adenoviral backbone plasmid, e.g. pAdEasy-1 of Stratagene's AdEasyTM Adenoviral Vector System. Recombinant adenovirus vectors are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines, for example HEK 293 cells (E1-transformed human embryonic kidney cells) or 911 (E1-transformed human embryonic retinal cells) (Human Gene Therapy 7:215-222, 1996). Recombinant adenoviruses are generated within the HEK 293 cells.
The use of alternative AAV serotypes other than AAV-2 (Davidson et al (2000), Proc. Natl. Acad. Sci. USA 97(7)3428-32; Passini et al (2003), J. Virol. 77(12):7034-40) has demonstrated different cell tropisms and increased transduction capabilities. With respect to brain cancers, the development of novel injection techniques into the brain, specifically convection enhanced delivery (CED; Bobo et al (1994), Proc. Natl. Acad. Sci. USA 91(6):2076-80; Nguyen et al (2001), Neuroreport 12(9):1961-4), has significantly enhanced the ability to transduce large areas of the brain with an AAV vector.
Large scale preparation of AAV vectors is made by a three-plasmid cotransfection of a packaging cell line: AAV vector carrying a DNA coding sequence for an antisense oligonucleotide to hnRNPLL or an siRNA hnRNPLL nucleic acid molecule, AAV RC vector containing AAV rep and cap genes, and adenovirus helper plasmid pDF6, into 50×150 mm plates of subconfluent 293 cells. Cells are harvested three days after transfection, and viruses are released by three freeze-thaw cycles or by sonication.
AAV vectors are then purified by two different methods depending on the serotype of the vector. AAV2 vector is purified by the single-step gravity-flow column purification method based on its affinity for heparin (Auricchio, A., et. al., 2001, Human Gene therapy 12:71-6; Summerford, C. and R. Samulski, 1998, J. Virol. 72:1438-45; Summerford, C. and R. Samulski, 1999, Nat. Med. 5: 587-88). AAV2/1 and AAV2/5 vectors are currently purified by three sequential CsCl gradients.
Pharmaceutical compositions used in the methods described herein can be delivered systemically via in vivo gene therapy. A variety of methods have been developed to accomplish in vivo transformation including mechanical means (e.g, direct injection of nucleic acid into target cells or particle bombardment), recombinant viruses, liposomes, and receptor-mediated endocytosis (RME) (for reviews, see Chang et al. 1994 Gastroenterol. 106:1076-84; Morsy et al. 1993 JAMA 270:2338-45; and Ledley 1992 J. Pediatr. Gastroenterol. Nutr. 14:328-37).
Another gene transfer method for use in humans is the transfer of plasmid DNA in liposomes directly to human cells in situ (Nabel, E. G., et al., Science 249:1285-1288 (1990)). Plasmid DNA should be easy to certify for use in human gene therapy because, unlike retroviral vectors, it can be purified to homogeneity. In addition to liposome-mediated DNA transfer, several other physical DNA transfer methods, such as those targeting the DNA to receptors on cells by conjugating the plasmid DNA to proteins, have shown promise in human gene therapy (Wu, G. Y., et al., J. Biol. Chem. 266:14338-14342 (1991); Curiel, D. T., et al., Proc. Natl. Acad. Sci. USA, 88:8850-8854 (1991)).
For gene therapy viruses, the dosage ranges from 106 to 1014 particles per application. Alternatively the biolistic gene gun method of delivery may be used. The gene gun is a device for injecting cells with genetic information, originally designed for plant transformation. The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as biolistics. Another instrument that uses biolistics technology is the PDS-1000/He particle delivery system. The proteins, expression vector, and/or gene therapy virus can be coated on minute gold particles, and these coated particles are “shot” into biological tissues such as hemangiomas and melanoma under high pressure. An example of the gene gun-based method is described for DNA based vaccination of cattle by Loehr B. I. et al. J. Virol. 2000, 74:6077-86.
Materials and methods for the construction of the expression vectors NFAT-GFP and Stim1-RFP, and the transfection of expression vectors into Hela cells are well known to one skilled in the art and are also described in Okamura, et. al., Mol. Cell, 2000, 6:539-50; Aramburu, et. al., Science, 1999, 285:2129-33; Gwack Y, et. al., Nature, 2006, 441:646-50, Oh-hora et al, Nature immunology 2008, 9:432-43; US Patent Application Nos. US2007/0031814 and PCT/US2007/000280. These references are hereby incorporated by reference in their entirety.
More specifically, the expression vector Stim1-RFP was constructed by the following method. Full length murine Stim1 cDNA (Oh-hora et al, Nature immunology 2008, 9:432-43) was PCR-amplified and cloned into pDSRed-Monomer-N1 (Clontech) using the Xho1 and BamH1 sites.
The expression vector Orai-FLAG was constructed by the following method and by any molecular methods known to one skilled in the art. Full length human Orai1 cDNA (Feske et al, Nature 2006, 441:179-85) was PCR-amplified and cloned into pFLAG-CMV2 (Sigma) using the Not1 and Xho1 sites.
Hela cell line expressing NFAT1, Stim1, and Orai1: HeLa 13.10. A monoclonal population of HeLa NFAT1 (1-460)-GFP cells stably expressing the amino terminal signal responsive domain of NFAT1 fused to GFP (Gwack et al, Nature 2006, 441:646-50) were engineered to stably express full length Stim1-RFP and transiently transfected with full length Orai1-FLAG 1; efficiency of Orai1-FLAG expression was quantitated by anti-FLAG immunocytochemistry at 48 h post transfection (75%+6.7) and 96 h post transfection (42%+8). Cells were maintained at 37° C./10% CO2 in DMEM 10% bovine calf serum (BCS), penicillin/streptomycin, HEPES and β-mercaptoethanol/L-glutamine and 100 μg/mL Hygromycin B. Hygromycin B was removed 16 h before Orai1-FLAG transfection. All experiments were performed with cells kept at a passage number under 6.
HeLa 13.10 cells stably expressing NFAT1-GFP and Stim1-RFP and transiently expressing Orai1-FLAG were reverse transfected with 20 nM siRNA using Hiperfect Transfection Reagent (Qiagen) by robotic transfer of cells to 384-well plates (5000-6000 cells/well) pre-arrayed with siRNA corresponding to the annotated human genome (Dharmacon). 72 h post transfection with siRNA, cells were stimulated with thapsigargin (250 nM for 90 minutes at room temperature) to induce NFAT1-GFP nuclear translocation; cells were fixed with 3% paraformaldehyde, permeablized with 0.2% Triton-X 100, stained with the DNA intercalating dye DAPI and assessed for NFAT1-GFP nuclear translocation by fluorescent microscopy. Images were acquired using the ImageXpress Micro automated imaging system (Molecular Devices) using a 10× objective and analyzed using the Translocation Application module of MetaXpress software version 6.1 (Molecular Devices). Cytoplasmic to nuclear translocation was assessed by calculating a correlation of intensity between NFAT1-GFP fluorescence and DAPI staining: cells were scored as positive for nuclear NFAT1 when >60% of NFAT1-GFP fluorescence coincided with DAPI fluorescence. Each data point represents an average of at least 1200 individual cells per well and averaged for duplicate wells.
The references cited herein and throughout the specification are incorporated herein by reference in their entirety.
















TABLE 1







Stock_ID
Row
Type
Z score_A
Z score_B
Screen+
Gene Symbol
Entrez Gene ID
Accession #
Catalog #





PL-50049
L11
X
−3.09
−1.83
W
AB026190
27252
NM_014458
M-004893-00


PL-50049
J12
X
−3.90
−2.18
W
ABLIM2
84448
NM_032432
M-014892-00


PL-50047
A21
X
−3.03
−1.80
W
ACLY
47
NM_001096
M-004915-00


PL-50049
F16
X
−2.11
−1.83
W
ACY1L2
135293
XM_072402
M-024889-00


PL-50049
F02
X
−3.49
−2.11
W
ADCY4
196883
NM_139247
M-006800-00


PL-50001
G15
X
−3.55
−2.27
W
ADK
132
NM_001123
M-004733-02


PL-50004
C05
X
−2.16
−2.22
W
ADRA2B
151
NM_000682
M-005423-01


PL-50001
I23
X
−3.16
−2.17
W
AKAP11
11215
NM_016248
M-009277-01


PL-50049
D14
X
−1.58
−2.77
W
AKR1CL1
340811
XM_291723
M-029709-00


PL-50049
F08
X
−2.82
−1.74
W
ALS2CR13
150864
NM_173511
M-018538-00


PL-50016
D02
X
−2.44
−2.24
W
AMH
268.00
NM_000479
M-010991-00


PL-50079
J04
X
−2.46
−2.33
W
AMIGO2
347902
NM_181847
M-018701-00


PL-50058
J08
X
−2.91
−1.32
W
ANKFY1
51479
NM_016376
M-013161-00


PL-50001
M17
X
−1.97
−3.09
W
ANKK1
255239
NM_178510
M-004930-01


PL-50062
A20
X
−2.10
−1.94
W
ANKMY2
57037
NM_020319
M-013766-00


PL-50072
O18
X
−2.66
−2.06
W
AP1S3
130340
NM_178814
M-018537-00


PL-50051
D07
X
−0.16
−2.82
W
AP3B2
8120
NM_004644
M-021444-00


PL-50060
G04
X
−1.92
−2.66
W
APG16L
55054
NM_017974
M-021033-00


PL-50020
J11
X
−2.32
−3.58
W
APOBEC1
339
NM_001644
M-011573-00


PL-50047
K17
X
−2.04
−2.52
W
APXL
357
NM_001649
M-011577-00


PL-50052
N16
X
−2.02
−2.39
W
AQR
9716
NM_014691
M-022214-00


PL-50061
P09
X
−2.09
−2.98
W
ARHGAP15
55843
NM_018460
M-018019-00


PL-50060
D05
X
−1.31
−4.57
W
ARHGAP17
55114
NM_018054
M-008335-00


PL-50008
M17
X
−1.83
−2.56
W
ARHGDIA
396
NM_004309
M-016253-00


PL-50060
P12
X
−1.85
−2.81
W
ARL10C
55207
NM_018184
M-020294-00


PL-50067
A12
X
−2.08
−2.58
W
ARMC2
84071
NM_032131
M-018191-00


PL-50054
E09
X
−1.82
−2.26
W
ARPP-21
10777
NM_016300
M-016091-00


PL-50059
A19
X
−2.87
−2.80
W
ARS2
51593
NM_015908
M-019234-00


PL-50072
J21
X
−2.98
−2.50
W
ASB10
136371
NM_080871
M-007725-00


PL-50051
L06
X
−2.17
−1.94
W
ASMTL
8623
NM_004192
M-012663-00


PL-50089
G02
X
−3.39
−1.60
W
ASTL
431705
NM_001002036
M-032349-00


PL-50062
L11
X
−1.59
−2.02
W
ATP10D
57205
NM_020453
M-018004-00


PL-50047
O19
X
−3.42
−1.02
W
ATP2B4
493
NM_001684
M-006118-00


PL-50057
M21
X
−0.60
−3.79
W
ATP5S
27109
NM_015684
M-020544-00


PL-50064
G11
X
−2.18
−4.15
W
AZ2
64343
NM_022461
M-014092-00


PL-50062
E06
X
−1.54
−3.01
W
BBX
56987
NM_020235
M-015289-00


PL-50070
I20
X
−2.83
−2.20
W
BC002942
91289
NM_033200
M-015085-00


PL-50057
K14
X
−2.54
−2.67
W
BC-2
27243
NM_014453
M-020247-00


PL-50062
G05
X
−2.27
−1.90
W
BEXL1
56271
XM_043653
M-024780-00


PL-50047
K18
X
−1.40
−3.63
W
BFSP1
631
NM_001195
M-011218-00


PL-50017
A09
X
−2.42
−2.70
W
BMP15
9210
NM_005448
M-012018-01


PL-50075
G08
X
−2.90
−2.75
W
BMPER
168667
NM_133468
M-021489-00


PL-50008
A06
X
−2.04
−2.46
W
BRD8
10902
NM_006696
M-006377-00


PL-50063
P09
X
−3.57
−2.70
W
BRUNOL6
60677
NM_052840
M-015854-00


PL-50020
F10
X
−2.48
−1.85
W
BSCL2
26580
NM_032667
M-016749-00


PL-50053
H23
X
−2.62
−2.25
W
BTN3A3
10384
NM_006994
M-021359-00


PL-50061
A19
X
−3.13
−2.11
W
C10ORF59
55328
NM_018363
M-021211-00


PL-50070
J06
X
−2.62
−2.52
W
C10ORF94
93426
NM_130784
M-015298-00


PL-50062
K11
X
−2.68
−2.65
W
C11ORF17
56672
NM_020642
M-015631-00


PL-50063
H04
X
−1.74
−2.73
W
C13ORF10
64062
NM_022118
M-019088-00


PL-50069
D21
X
−1.91
−2.80
W
C14ORF126
112487
NM_080664
M-021299-00


PL-50075
F13
X
−2.34
−3.25
W
C14ORF147
171546
NM_138288
M-017156-00


PL-50070
A10
X
−2.77
−2.54
W
C14ORF43
91748
NM_194278
M-031938-00


PL-50070
B11
X
−2.37
−2.86
W
C14ORF73
91828
XM_040910
M-022006-00


PL-50071
G02
X
−2.58
−2.56
W
C14ORF8
122664
NM_173846
M-017754-00


PL-50053
G11
X
−3.12
−2.14
W
C14ORF92
9878
XM_375045
M-021236-00


PL-50064
P15
X
−2.47
−2.41
W
C16ORF23
79006
NM_024042
M-014274-00


PL-50004
G11
X
−2.61
−2.46
W
C17ORF35
8834
NM_003876
M-005440-01


PL-50080
M11
X
−2.47
−1.66
W
C18ORF34
374864
NM_198995
M-032008-00


PL-50060
K20
X
−2.53
−1.97
W
C19ORF24
55009
NM_017914
M-020936-00


PL-50020
B06
X
−2.95
−3.27
W
C21ORF107
54014
NM_018963
M-010963-00


PL-50059
K06
X
−2.50
−1.66
W
C21ORF45
54069
NM_018944
M-020789-00


PL-50053
K20
X
−1.98
−2.27
W
C21ORF6
10069
NM_016940
M-013856-00


PL-50069
P16
X
−2.84
−2.91
W
C21ORF84
114038
NM_153752
M-016161-00


PL-50051
L20
X
−1.99
−2.78
W
C4ORF8
8603
NM_003704
M-019541-00


PL-50075
K06
X
−2.03
−2.45
W
C5ORF11
167410
NM_153234
M-018373-00


PL-50061
G11
X
−2.72
−3.09
W
C6ORF110
55362
XM_371822
M-025105-00


PL-50069
D23
X
−2.08
−2.05
W
C6ORF51
112495
NM_138408
M-015508-00


PL-50072
F23
X
−2.27
−2.07
W
C6ORF57
135154
NM_145267
M-015985-00


PL-50065
B06
X
−2.19
−2.05
W
C6ORF59
79992
NM_024929



PL-50062
O17
X
−1.95
−2.24
W
C8ORF4
56892
NM_020130
M-015557-00


PL-50072
L10
X
−2.48
−2.75
W
C9ORF115
138428
XM_059972
M-026208-00


PL-50078
D16
X
−2.29
−1.86
W
C9ORF150
286343
NM_203403
M-031930-00


PL-50074
N18
X
−2.11
−3.35
W
C9ORF84
158401
NM_173521
M-018530-00


PL-50066
B20
X
−2.77
−1.61
W
CABLES2
81928
NM_031215
M-032282-00


PL-50020
A21
X
−4.27
−1.56
W
CACNA1A
773
NM_000068
M-006121-01


PL-50057
M05
X
−2.50
−1.67
W
CACNG4
27092
NM_014405
M-012519-00


PL-50051
L10
X
−2.82
−1.93
W
CADPS
8618
NM_003716
M-019218-00


PL-50006
M11
X
−2.76
−2.97
W
CARD12
58484
NM_021209
M-004396-00


PL-50021
J17
X
−2.30
−3.97
W
CAV3
859
NM_001234
M-011229-00


PL-50073
H05
X
−2.78
−2.55
W
CBLN2
147381
NM_182511



PL-50006
O05
X
−3.04
−1.72
W
CBX6
23466
NM_014292
M-009555-00


PL-50004
K15
X
−2.33
−3.05
W
CCR6
1235
NM_004367
M-005453-00


PL-50006
O19
X
−2.27
−2.03
W
CD151
977
NM_004357
M-003637-02


PL-50047
A18
X
−2.22
−2.55
W
CD1E
913
NM_030893
M-014647-00


PL-50017
I09
X
−2.00
−2.04
W
CD3G
917
NM_000073
M-011005-00


PL-50017
I15
X
−1.87
−3.86
W
CD5
921
NM_014207
M-007848-01


PL-50014
B06
X
−3.64
−2.09
W
CD74
972.00
NM_004355
M-012667-00


PL-50073
B10
X
−2.54
−1.73
W
CDC42EP5
148170
NM_145057



PL-50006
K08
X
−2.95
−2.84
W
CDH9
1007
NM_016279
M-013169-00


PL-50071
E05
X
−2.66
−3.17
W
CENTG1
116986
NM_014770
M-021010-00


PL-50047
B13
X
−3.12
−1.98
W
CFL2
1073
NM_021914
M-019078-00


PL-50067
N08
X
−2.59
−2.83
W
CHCHD5
84269
NM_032309
M-014849-00


PL-50015
G18
X
−2.23
−2.61
W
CHD4
1108.00
NM_001273
M-009774-00


PL-50053
L12
X
−2.50
−2.99
W
CHERP
10523
NM_006387
M-016176-00


PL-50015
G16
X
−2.93
−2.08
W
CHFR
55743.00
NM_018223
M-007018-01


PL-50004
M21
X
−2.16
−1.86
W
CHRM3
1131
NM_000740
M-005464-01


PL-50018
I11
X
−2.72
−2.11
W
CHRNA4
1137
NM_000744
M-006138-01


PL-50008
D23
X
−2.49
−2.10
W
CKN1
1161
NM_000082
M-011008-00


PL-50047
F05
X
−0.87
−3.50
W
CLCN4
1183
NM_001830
M-006152-00


PL-50057
M11
X
−0.66
−4.26
W
CLUL1
27098
NM_014410
M-017042-00


PL-50054
P09
X
−2.07
−2.02
W
CMRF-35H
11314
NM_007261
M-012778-00


PL-50012
P10
X
−2.55
−2.13
W
COMT
1312
NM_000754
M-009520-00


PL-50047
L05
X
−2.66
−1.83
W
COX8A
1351
NM_004074
M-011819-00


PL-50058
C06
X
−1.80
−2.20
W
CRBN
51185
NM_016302
M-021086-00


PL-50004
O11
X
−3.31
−1.98
W
CRHR2
1395
NM_001883
M-005470-01


PL-50018
I12
X
−2.54
−2.67
W
CRSP2
9282
NM_004229
M-011928-00


PL-50021
K20
X
−2.15
−2.49
W
CRSP3
9439
NM_004830
M-013220-00


PL-50018
I10
X
−2.43
−2.89
W
CRSP6
9440
NM_004268
M-006312-01


PL-50018
B18
X
−6.60
−1.80
W
CRYBA2
1412
NM_005209
M-012024-00


PL-50021
K12
X
−2.72
−1.44
W
CRYBB1
1414
NM_001887
M-011629-00


PL-50018
B16
X
−2.65
−2.48
W
CRYBB3
1417
NM_004076
M-006480-01


PL-50018
B12
X
−2.55
−3.76
W
CRYGC
1420
NM_020989
M-013142-00


PL-50010
J07
X
−2.43
−2.25
W
CSAD
51380
NM_015989
M-008314-00


PL-50021
I10
X
2.08
2.02
W
CST7
8530
NM_003650
M-017236-00


PL-50051
H11
X
−2.42
−2.76
W
CXORF12
8269
NM_003492
M-011428-00


PL-50047
P10
X
−3.46
−2.51
W
CYLC1
1538
XM_088636
M-024769-00


PL-50012
N08
X
−3.15
−2.69
W
CYP1A1
1543
NM_000499
M-004790-01


PL-50047
N12
X
−2.58
−3.29
W
CYP3A5
1577
NM_000777
M-009684-00


PL-50014
G17
X
−2.77
−2.17
W
D2S448
7837.00
XM_056455
M-022772-00


PL-50021
E20
X
−2.67
−1.85
W
DAAM1
23002
NM_014992
M-012925-00


PL-50017
O15
X
−2.15
−2.16
W
DBI
1622
NM_020548
M-006488-00


PL-50063
B23
X
−2.01
−1.97
W
DC2
58505
NM_021227
M-027193-00


PL-50053
G13
X
−3.37
−2.98
W
DDX46
9879
NM_014829
M-021234-00


PL-50047
H12
X
−3.31
−1.94
W
DHPS
1725
NM_001930
M-006670-00


PL-50070
K14
X
−2.81
−2.09
W
DKFZP434B1231
91156
NM_178275
M-018250-00


PL-50067
M17
X
−2.22
−2.32
W
DKFZP434I2117
83723
NM_031478
M-003996-00


PL-50078
A08
X
−3.07
−2.03
W
DKFZP686P0288
285190
NM_182588
M-018854-00


PL-50067
P23
X
−2.28
−3.62
W
DKFZP761B1514
84248
NM_032288
M-018604-00


PL-50047
H04
X
−0.84
−3.07
W
DLAT
1737
NM_001931
M-008490-00


PL-50012
J02
X
−2.78
−1.78
W
DNM1L
10059
NM_005690
M-012092-01


PL-50015
D06
X
−2.37
−2.28
W
DUSP16
80824.00
NM_030640
M-007890-00


PL-50015
D04
X
−3.96
−2.15
W
DUSP18
150290.00
NM_152511
M-007891-00


PL-50008
J19
X
−2.31
−2.93
W
DUX1
26584
NM_012146
M-019955-00


PL-50057
C13
X
−3.26
−2.30
W
DUX5
26581
NM_012149
M-019904-00


PL-50014
G08
X
−2.02
−2.27
W
DVL3
1857.00
NM_004423
M-004070-00


PL-50062
K04
X
−1.79
−2.87
W
E(Y)2
56943
NM_020189
M-018808-00


PL-50058
J09
X
−2.00
−2.33
W
E2IG2
51287
NM_016565
M-021148-00


PL-50068
O21
X
−2.47
−3.28
W
EBPL
84650
NM_032565
M-014920-00


PL-50063
O20
X
−2.25
−1.71
W
EPB41L5
57669
NM_020909
M-010729-00


PL-50017
H14
X
−2.63
−3.25
W
EREG
2069
NM_001432
M-011268-00


PL-50001
P14
X
−0.93
−3.04
W
ERK8
225689
NM_139021
M-004807-01


PL-50021
F20
X
−2.24
−3.12
W
ESRRBL1
55081
NM_018010
M-015367-00


PL-50064
D10
X
−3.04
−2.71
W
ET
79157
NM_024311
M-014329-00


PL-50089
H08
X
−2.63
−2.63
W
EVI5
7813
NM_005665
M-032510-00


PL-50067
E18
X
−2.76
−2.42
W
FAM14A
83982
NM_032036
M-014773-00


PL-50052
F08
X
−3.64
−2.59
W
FAM38A
9780
NM_014745
M-020870-00


PL-50015
K07
X
−2.52
−2.30
W
FBXL20
84961.00
NM_032875
M-015029-00


PL-50015
G02
X
−2.70
−3.21
W
FBXL3P
26223.00
NM_012159
M-012423-00


PL-50015
E04
X
−2.37
−1.84
W
FBXO22
26263.00
NM_012170
M-010812-01


PL-50055
L14
X
−3.13
−0.76
W
FBXO46
23403
XM_371179
M-023753-00


PL-50021
B02
X
−4.26
−2.10
W
FCGR3A
2214
NM_000569
M-016308-00


PL-50017
D20
X
−2.38
−3.61
W
FGF14
2259
NM_004115
M-011860-00


PL-50017
B16
X
−3.45
−2.97
W
FGF7
2252
NM_002009
M-011659-00


PL-50072
H13
X
−2.47
−3.34
W
FKBP1C
135521
XM_059776
M-028635-00


PL-50060
C12
X
−1.71
−2.56
W
FLJ10159
55084
NM_018013
M-021094-00


PL-50060
D19
X
−3.09
−1.32
W
FLJ10352
55125
NM_018069
M-032250-00


PL-50059
J05
X
−2.99
−1.80
W
FLJ10613
54552
NM_019067
M-015743-00


PL-50001
N08
X
−2.62
−2.33
W
FLJ10842
55750
NM_018238
M-007256-00


PL-50060
B14
X
−1.94
−2.52
W
FLJ11126
55308
NM_018332
M-015868-00


PL-50061
A09
X
−3.15
−1.98
W
FLJ11193
55322
NM_018356
M-021223-00


PL-50064
F11
X
−2.54
−2.12
W
FLJ12517
65094
NM_023007
M-014238-00


PL-50059
C04
X
−2.27
−2.61
W
FLJ20152
54463
NM_019000
M-016936-00


PL-50059
P23
X
−2.32
−4.10
W
FLJ20485
54517
NM_019042
M-015341-00


PL-50060
M09
X
−2.74
−2.48
W
FLJ20509
54956
NM_017851
M-020837-01


PL-50060
M21
X
−2.62
−1.68
W
FLJ20519
54964
NM_017860
M-016312-00


PL-50066
G23
X
−2.48
−2.37
W
FLJ20972
80098
NM_025030
M-014556-00


PL-50066
O12
X
−2.65
−3.61
W
FLJ22688
80199
NM_025129
M-016342-00


PL-50065
P09
X
−1.79
−2.78
W
FLJ23554
79864
NM_024806



PL-50074
C18
X
−2.54
−2.85
W
FLJ25286
153443
NM_152546
M-015445-00


PL-50072
P23
X
−2.72
−2.62
W
FLJ32356
144717
NM_144671
M-015976-00


PL-50073
N18
X
−1.95
−2.38
W
FLJ32421
148362
NM_144695



PL-50073
L02
X
−2.70
−2.68
W
FLJ32569
148811
NM_152491



PL-50076
B12
X
−2.68
−2.73
W
FLJ32682
220081
NM_182542
M-018973-00


PL-50073
B05
X
−2.02
−1.72
W
FLJ32734
146849
NM_144681



PL-50074
C09
X
−2.94
−2.73
W
FLJ33814
150275
NM_173510
M-018556-00


PL-50078
A05
X
−2.34
−2.88
W
FLJ34690
284034
NM_182567
M-018978-00


PL-50075
A23
X
−1.99
−2.64
W
FLJ35757
162333
NM_152598
M-007150-00


PL-50075
K07
X
−2.32
−2.31
W
FLJ35838
163479
NM_173532
M-018510-00


PL-50074
F04
X
−3.13
−1.97
W
FLJ35843
160762
NM_152591
M-016858-00


PL-50071
D02
X
−2.23
−3.04
W
FLJ35961
127294
NM_152372
M-017014-00


PL-50078
C15
X
−1.88
−3.07
W
FLJ36878
284114
NM_178518
M-018245-00


PL-50078
C14
X
−1.82
−4.10
W
FLJ38379
285097
NM_178530
M-017660-00


PL-50072
A13
X
−2.47
−1.83
W
FLJ38984
127703
NM_152374
M-016893-00


PL-50071
H12
X
−2.59
−2.77
W
FLJ39117
126638
XM_371312
M-027449-00


PL-50072
A04
X
−2.25
−2.87
W
FLJ39155
133584
NM_152403
M-019235-00


PL-50072
C21
X
−2.26
−2.24
W
FLJ40160
128209
NM_173484
M-018431-00


PL-50081
A23
X
−2.01
−1.94
W
FLJ42953
400892
NM_207474
M-032163-00


PL-50080
F18
X
−3.91
−2.00
W
FLJ42957
400077
NM_207436
M-032113-00


PL-50080
P17
X
−1.88
−2.66
W
FLJ43965
389206
NM_207406
M-032079-00


PL-50080
H12
X
−2.85
−2.57
W
FLJ45803
399948
NM_207429
M-032104-00


PL-50080
O14
X
−2.09
−2.75
W
FLJ46354
374977
NM_198547
M-027321-00


PL-50080
P11
X
−1.43
−2.78
W
FLJ46481
389197
NM_207405
M-032082-00


PL-50008
P08
X
−2.71
−3.35
W
FOXB1
27023
NM_012182
M-008906-00


PL-50008
L04
X
−4.33
−1.87
W
FOXP2
93986
NM_014491
M-010359-01


PL-50070
I11
X
−2.12
−2.27
W
FOXP4
116113
NM_138457
M-008255-00


PL-50048
G11
X
−2.21
−2.52
W
FSHPRH1
2491
NM_006733
M-005268-00


PL-50006
J23
X
−2.04
−2.38
W
FTH1
2495
NM_002032
M-019634-01


PL-50057
A13
X
−2.11
−2.61
W
FXC1
26515
NM_012192
M-018242-00


PL-50001
H18
X
−3.04
−1.84
W
FYN
2534
NM_002037
M-003140-03


PL-50018
M05
X
−2.18
−1.95
W
GABRB1
2560
NM_000812
M-006168-00


PL-50011
A05
X
−2.25
−3.32
W
GART
2618
NM_000819
M-008594-00


PL-50013
A23
X
−2.25
−2.53
W
GBP1
2633
NM_002053
M-005153-01


PL-50017
M08
X
−3.40
−1.34
W
GDNF
2668
NM_000514
M-011040-00


PL-50019
J09
X
−3.26
−2.11
W
GJB3
2707
NM_024009
M-019948-00


PL-50062
I16
X
−1.91
−2.17
W
GL004
56947
NM_020194
M-018261-00


PL-50001
F18
X
−2.66
−1.52
W
GMFG
9535
NM_004877
M-019878-01


PL-50004
A18
X
−2.43
−2.90
W
GNAQ
2776
NM_002072
M-008562-00


PL-50061
N08
X
−2.11
−2.21
W
GOLGA6
55889
NM_018652
M-013307-00


PL-50064
O08
X
−3.58
−2.42
W
GORASP1
64689
NM_031899
M-013510-00


PL-50048
O17
X
−2.13
−2.49
W
GOT1
2805
NM_002079
M-011673-00


PL-50057
K20
X
−2.15
−1.58
W
GPKOW
27238
NM_015698
M-015129-00


PL-50048
O16
X
−2.42
−1.85
W
GPM6B
2824
NM_005278
M-018825-00


PL-50004
D11
X
−2.86
−2.53
W
GPR101
83550
NM_054021
M-005526-01


PL-50015
N19
X
−3.90
−2.32
W
GPR114
221188.00
NM_153837
M-005535-01


PL-50004
P05
X
−2.10
−1.83
W
GPR50
9248
NM_004224
M-005578-00


PL-50004
P15
X
−3.01
−2.29
W
GPR56
9289
NM_005682
M-004552-00


PL-50004
P04
X
−2.32
−2.27
W
GPR73L1
128674
NM_144733
M-005594-01


PL-50016
I16
X
−2.60
−1.70
W
GRB7
2886.00
NM_005310
M-012701-00


PL-50048
O04
X
−2.74
−1.34
W
GRID1
2894
XM_043613
M-007917-00


PL-50016
I06
X
−2.41
−2.33
W
GRID2
2895.00
NM_001510
M-006188-00


PL-50004
J16
X
−2.36
−1.70
W
GRK7
131890
NM_139209
M-004628-00


PL-50052
F07
X
−2.09
−1.79
W
GTPBP1
9567
NM_004286
M-017321-00


PL-50006
P09
X
−2.87
−2.40
W
H2AFZ
3015
NM_002106
M-011683-01


PL-50052
F05
X
−2.54
−2.09
W
H6PD
9563
NM_004285
M-004692-00


PL-50022
G10
X
−2.20
−2.29
W
HBE1
3046
NM_005330
M-012069-00


PL-50053
J16
X
−2.96
−1.83
W
HBXIP
10542
NM_006402
M-012269-00


PL-50008
F14
X
−2.40
−2.43
W
HCFC1
3054
NM_005334
M-019953-00


PL-50020
G12
X
−2.47
−2.33
W
HD
3064
NM_002111
M-003737-00


PL-50013
C17
X
−2.51
−2.37
W
HDAC3
8841
NM_003883
M-003496-00


PL-50061
G13
X
−2.86
−3.19
W
HEMGN
55363
NM_018437
M-021355-00


PL-50073
B02
X
−2.57
−1.95
W
HERV-FRD
405754
NM_207582



PL-50048
K06
X
−2.94
−3.13
W
HIST1H1B
3009
NM_005322
M-012049-00


PL-50081
O18
X
−3.97
−1.63
W
HIST1H2AL
8332
NM_003511
M-011434-00


PL-50051
L09
X
−4.54
−1.37
W
HIST1H3B
8358
NM_003537
M-006475-00


PL-50051
L11
X
−3.15
−2.10
W
HIST1H4A
8359
NM_003538
M-011456-00


PL-50008
B12
X
−2.39
−2.71
W
HOXA7
3204
NM_006896
M-017573-00


PL-50048
A18
X
−0.65
−2.78
W
HOXB8
3218
NM_024016
M-017527-00


PL-50048
A14
X
−1.59
−2.94
W
HOXC8
3224
NM_022658
M-012995-00


PL-50060
O23
X
−3.65
−1.92
W
HRASLS2
54979
NM_017878
M-020862-00


PL-50056
O23
X
−2.46
−2.58
W
HS747E2A
25770
NM_015370
M-020284-00


PL-50062
P14
X
−2.63
−2.55
W
HSCARG
57407
NM_020677
M-020759-00


PL-50070
D04
X
−1.86
−2.59
W
HSPB9
94086
NM_033194
M-009005-00


PL-50004
F18
X
−3.01
−2.53
W
HTR1A
3350
NM_000524
M-005633-00


PL-50001
B04
X
−5.47
−2.28
W
HUNK
30811
NM_014586
M-004214-01


PL-50051
H06
X
−0.80
−3.81
W
HYAL2
8692
NM_003773
M-013689-00


PL-50048
D21
X
−1.83
−2.41
W
IDH3G
3421
NM_004135
M-009361-00


PL-50064
A05
X
−2.30
−1.52
W
IFRG15
64163
NM_022347
M-014044-00


PL-50070
L06
X
−1.41
−2.46
W
IGSF8
93185
NM_052868
M-015148-00


PL-50017
C20
X
−2.89
−2.23
W
IL10RB
3588
NM_000628
M-007926-01


PL-50017
C06
X
−2.44
−2.31
W
IL15RA
3601
NM_002189
M-007935-00


PL-50017
C02
X
−2.24
−3.02
W
IL17
3605
NM_002190
M-007937-00


PL-50069
F15
X
−2.57
−1.76
W
IL17F
112744
NM_052872
M-007942-00


PL-50017
B13
X
−2.58
−1.46
W
IL1F9
56300
NM_019618
M-007959-00


PL-50017
B23
X
−1.30
−2.70
W
IL1RL1
9173
NM_003856
M-007963-00


PL-50017
D11
X
−3.96
−2.20
W
IL22
50616
NM_020525
M-007972-00


PL-50004
D08
X
−2.78
−1.96
W
IL8RB
3579
NM_001557
M-005647-00


PL-50022
F07
X
−2.43
−2.63
W
INSIG1
3638
NM_005542
M-017880-01


PL-50006
H12
X
−3.18
−2.23
W
INSM1
3642
NM_002196
M-006535-00


PL-50061
O18
X
−1.70
−2.66
W
INTERSEX
55588
XM_290829
M-023854-00


PL-50009
G21
X
−2.37
−3.10
W
IRF7
3665
NM_001572
M-011810-01


PL-50065
A23
X
−3.60
−1.48
W
IRX1
79192
XM_380171



PL-50022
H13
X
−2.41
−3.95
W
ITSN2
50618
NM_006277
M-009841-00


PL-50057
B11
X
−2.63
−3.04
W
JM1
28952
NM_014008
M-020554-00


PL-50069
M05
X
−2.57
−2.29
W
JM11
90060
NM_033626
M-015130-00


PL-50068
P18
X
−2.41
−2.32
W
JUB
84962
NM_198086
M-021473-00


PL-50014
H15
X
−2.07
−1.96
W
KALRN
8997.00
NM_003947
M-010019-00


PL-50061
B06
X
−2.43
−2.45
W
KBTBD7
84078
NM_032138
M-015708-00


PL-50020
I17
X
−0.96
−3.17
W
KCNC4
3749
NM_004978
M-006223-01


PL-50016
A12
X
−2.71
−2.63
W
KCNH4
23415.00
NM_012285
M-006234-01


PL-50004
B20
X
−2.62
−1.67
W
KCNJ3
3760
NM_002239
M-006248-00


PL-50064
J17
X
−2.90
−2.72
W
KCTD14
65987
NM_023930
M-014252-00


PL-50002
E19
X
−2.70
−2.08
W
KDR
3791
NM_002253
M-003148-01


PL-50052
B04
X
−2.94
−2.64
W
KEAP1
9817
NM_012289
M-012453-00


PL-50013
E19
X
−2.38
−2.39
W
KIAA0217
23185
XM_040265
M-026388-00


PL-50052
B08
X
−2.95
−1.93
W
KIAA0542
9814
XM_038520
M-024567-00


PL-50055
A07
X
−2.32
−2.25
W
KIAA0980
22981
NM_025176
M-018162-00


PL-50055
N10
X
−2.87
−1.91
W
KIAA1068
23386
NM_015332
M-014018-00


PL-50062
L04
X
−2.17
−1.97
W
KIAA1189
57471
XM_371576
M-027986-00


PL-50062
L02
X
−3.14
−2.74
W
KIAA1194
57472
NM_015455
M-019101-00


PL-50002
G09
X
−3.22
−2.00
W
KIAA1361
57551
XM_290796
M-004846-01


PL-50063
O18
X
−2.09
−2.19
W
KIAA1549
57670
XM_371956
M-025462-00


PL-50063
O04
X
−2.92
−1.90
W
KIAA1573
57685
NM_020925
M-014178-00


PL-50075
D05
X
−1.73
−2.14
W
KIAA1987
170951
XM_375298
M-030681-00


PL-50015
P14
X
−2.15
−3.76
W
KIF11
3832.00
NM_004523
M-003317-01


PL-50048
J21
X
−3.22
−2.12
W
KIR2DL4
3805
NM_002255
M-018983-00


PL-50069
A02
X
−2.13
−2.22
W
KRTAP9-4
85280
NM_033191
M-013676-00


PL-50076
L06
X
−2.86
−2.22
W
LACE1
246269
NM_145315
M-008222-00


PL-50013
G09
X
−3.29
−2.26
W
LAP3
51056
NM_015907
M-005923-03


PL-50048
P09
X
−3.03
−1.97
W
LASP1
3927
NM_006148
M-010519-00


PL-50089
K04
X
−1.63
−2.44
W
LCN10
414332
NM_001001712
M-032327-00


PL-50064
A17
X
−2.69
−1.83
W
LEPRE1
64175
NM_022356
M-004271-00


PL-50060
P20
X
−0.80
−3.39
W
LGI2
55203
NM_018176
M-017097-00


PL-50002
I23
X
−2.41
−2.59
W
LIM
10611
NM_006457
M-006930-00


PL-50015
O09
X
−2.25
−2.78
W
LMO7
4008.00
NM_005358
M-019252-00


PL-50002
G21
X
−1.85
−2.25
W
LMTK3
114783
XM_055866
M-005338-01


PL-50076
N21
X
−2.84
−1.85
W
LNX2
222484
NM_153371
M-007164-00


PL-50069
P15
X
−2.49
−2.56
W
LOC113828
113828
NM_138435
M-015492-00


PL-50070
I05
X
−2.29
−3.17
W
LOC116068
116068
XM_371760
M-024521-00


PL-50071
O16
X
−2.62
−2.28
W
LOC120376
120376
XM_071712
M-026516-00


PL-50071
F07
X
−1.59
−3.04
W
LOC124402
124402
NM_145253
M-015987-00


PL-50071
N23
X
−2.21
−2.15
W
LOC125893
125893
XM_064856
M-023813-00


PL-50071
J04
X
−2.12
−2.12
W
LOC126520
126520
XM_059051
M-023896-00


PL-50072
B11
X
−2.71
−2.54
W
LOC134145
134145
NM_199133
M-024274-00


PL-50073
O11
X
−2.34
−2.31
W
LOC144097
144097
NM_138471



PL-50081
J11
X
−1.54
−2.58
W
LOC145414
0
XM_085138
M-021965-00


PL-50073
C20
X
−1.77
−2.26
W
LOC146443
146443
XM_378558



PL-50073
A14
X
−2.89
−2.56
W
LOC146713
146713
XM_378712



PL-50081
A20
X
−2.17
−2.43
W
LOC149643
0
XM_086616
M-021537-00


PL-50074
O11
X
−2.57
−2.20
W
LOC151484
151484
XM_379159
M-028022-00


PL-50082
F23
X
−2.31
−1.98
W
LOC152877
0
XM_094066
M-024144-00


PL-50074
E06
X
−2.75
−2.53
W
LOC153328
153328
NM_145282
M-007347-00


PL-50074
D11
X
−2.07
−1.79
W
LOC154222
154222
XM_379456
M-028649-00


PL-50083
K09
X
−2.24
−2.33
W
LOC154907
0
XM_088072
M-025440-00


PL-50081
C16
X
−2.08
−2.30
W
LOC155036
155036
XM_376722
M-028875-00


PL-50083
A21
X
−1.87
−2.14
W
LOC158796
0
XM_088677
M-025004-00


PL-50074
J08
X
−2.30
−2.90
W
LOC159090
159090
NM_145284
M-016085-00


PL-50075
C11
X
−2.52
−2.87
W
LOC162427
162427
NM_178126
M-018456-00


PL-50075
K13
X
−1.93
−4.79
W
LOC163590
163590
NM_145034
M-016470-00


PL-50082
A17
X
−2.23
−1.70
W
LOC200493
0
XM_115715
M-022826-00


PL-50076
E15
X
−2.05
−2.86
W
LOC202051
202051
XM_114430
M-024410-00


PL-50076
M23
X
−2.49
−2.58
W
LOC205251
205251
NM_174925
M-017831-00


PL-50081
N05
X
−3.34
−1.92
W
LOC254897
0
XM_170950
M-022066-00


PL-50077
F13
X
−2.72
−1.86
W
LOC283152
283152
XM_378314



PL-50077
D04
X
−2.21
−4.21
W
LOC283989
283989
NM_207346



PL-50078
A11
X
−2.08
−3.17
W
LOC284058
284058
NM_015443
M-031748-00


PL-50078
K13
X
−2.09
−2.15
W
LOC284361
284361
NM_175063
M-018434-00


PL-50078
M13
X
−1.80
−2.88
W
LOC284390
284390
XM_371138
M-031229-00


PL-50078
M04
X
−2.89
−1.65
W
LOC284661
284661
XM_378832
M-027567-00


PL-50078
K08
X
−2.34
−1.86
W
LOC284739
284739
NM_207349
M-031294-00


PL-50078
I02
X
−2.15
−2.35
W
LOC284825
284825
XM_375935
M-031369-00


PL-50078
A12
X
−1.94
−3.54
W
LOC285194
285194
XM_379207
M-028149-00


PL-50082
I14
X
−3.00
−2.75
W
LOC285248
0
XM_211816
M-023664-00


PL-50083
G10
X
−2.14
−2.08
W
LOC338734
0
XM_290547
M-026505-00


PL-50083
F05
X
−2.20
−2.58
W
LOC338756
0
XM_291989
M-026922-00


PL-50079
B09
X
−2.64
−1.81
W
LOC340109
340109
XM_379322
M-028502-00


PL-50083
I14
X
−2.28
−1.93
W
LOC340843
0
XM_291726
M-026400-00


PL-50083
J05
X
−2.17
−2.03
W
LOC341356
0
XM_292023
M-027076-00


PL-50082
P12
X
−5.82
−2.32
W
LOC345651
0
XM_293924
M-024480-00


PL-50080
M14
X
−4.93
−2.81
W
LOC375133
375133
NM_199345
M-032119-00


PL-50080
C02
X
−3.73
−1.73
W
LOC386597
386597
XM_379073
M-027974-00


PL-50086
B14
X
−3.37
−2.68
W
LOC387784
0
XM_373506
M-029865-00


PL-50086
H08
X
−1.81
−2.55
W
LOC387810
0
XM_373513
M-029805-00


PL-50087
K18
X
−1.48
−3.48
W
LOC387825
0
XM_370668



PL-50087
O04
X
−2.59
−2.12
W
LOC387845
0
XM_370684



PL-50087
G02
X
−4.14
−2.44
W
LOC387914
0
XM_370718



PL-50088
C11
X
−2.83
−2.18
W
LOC388298
0
XM_370992



PL-50088
K21
X
−2.71
−2.99
W
LOC388432
0
XM_371086



PL-50081
C12
X
−4.87
−2.48
W
LOC388585
0
XM_371215
M-007769-00


PL-50083
P04
X
−2.40
−3.20
W
LOC388697
0
XM_373868
M-027450-00


PL-50084
G20
X
−3.63
−2.37
W
LOC389000
0
XM_371534
M-027916-00


PL-50084
A14
X
−2.42
−1.82
W
LOC389067
0
XM_374021
M-028005-00


PL-50084
A10
X
−2.39
−2.59
W
LOC389070
0
XM_374022
M-028009-00


PL-50084
H11
X
−2.84
−2.27
W
LOC389102
0
XM_371623
M-028089-00


PL-50084
P13
X
−2.00
−2.17
W
LOC389153
0
XM_374053
M-028171-00


PL-50085
A15
X
−1.85
−2.10
W
LOC389273
0
XM_374115
M-028392-00


PL-50085
C02
X
−2.12
−3.30
W
LOC389370
0
XM_374162
M-028681-00


PL-50085
B05
X
−2.03
−1.53
W
LOC389386
0
XM_371818
M-028707-00


PL-50085
K08
X
−3.31
−2.67
W
LOC389416
0
XM_371837
M-028580-00


PL-50085
P10
X
−1.46
−2.24
W
LOC389541
0
XM_371939
M-028918-00


PL-50086
C08
X
−2.54
−2.06
W
LOC389727
0
XM_372092
M-029462-00


PL-50086
B05
X
−2.30
−2.18
W
LOC389753
0
XM_372112
M-029491-00


PL-50086
H18
X
−3.76
−1.94
W
LOC389950
0
XM_372307
M-029790-00


PL-50087
C02
X
−5.64
−2.10
W
LOC390377
0
XM_372486



PL-50083
P09
X
−1.83
−3.25
W
LOC391059
0
XM_372784
M-027419-00


PL-50084
G23
X
−2.56
−1.54
W
LOC391209
0
XM_372840
M-027739-00


PL-50085
B20
X
−3.51
−1.79
W
LOC392702
0
XM_374730
M-029081-00


PL-50085
B08
X
−3.38
−1.62
W
LOC392726
0
XM_374734
M-029100-00


PL-50086
C17
X
−1.42
−3.60
W
LOC392791
0
XM_374752
M-029140-00


PL-50086
L06
X
−2.71
−2.95
W
LOC399786
0
XM_378236
M-029755-00


PL-50087
A09
X
−2.48
−2.14
W
LOC399920
0
XM_378300



PL-50087
O14
X
−3.77
−2.10
W
LOC400092
0
XM_378398



PL-50088
G13
X
−2.71
−2.21
W
LOC400479
0
XM_375282



PL-50088
I19
X
−2.45
−2.29
W
LOC400619
0
XM_378703



PL-50083
P15
X
−1.94
−3.99
W
LOC400740
0
XM_378840
M-027426-00


PL-50084
B06
X
−1.60
−2.62
W
LOC401169
0
XM_379306
M-028377-00


PL-50085
A17
X
−2.43
−1.78
W
LOC401175
0
XM_379317
M-028393-00


PL-50085
E18
X
−2.43
−1.56
W
LOC401286
0
XM_376555
M-028648-00


PL-50085
J11
X
−3.03
−2.86
W
LOC401314
0
XM_376586
M-028821-00


PL-50085
J13
X
−2.58
−2.50
W
LOC401316
0
XM_376587
M-028825-00


PL-50085
J15
X
−1.74
−2.76
W
LOC401317
0
XM_379479
M-028826-00


PL-50085
J19
X
−2.77
−1.28
W
LOC401321
0
XM_379483
M-028830-00


PL-50086
A16
X
−2.87
−1.88
W
LOC401518
0
XM_379638
M-029475-00


PL-50086
J05
X
−2.03
−3.09
W
LOC401548
0
XM_376902
M-029584-00


PL-50086
J15
X
−2.49
−2.92
W
LOC401552
0
XM_379668
M-029594-00


PL-50084
L20
X
−3.16
−2.65
W
LOC402148
0
XM_377818
M-028220-00


PL-50085
J04
X
−2.32
−2.36
W
LOC402477
0
XM_379803
M-029005-00


PL-50085
D18
X
−3.23
−2.03
W
LOC402515
0
XM_380112
M-029067-00


PL-50085
D12
X
−1.79
−4.81
W
LOC402521
0
XM_379848
M-029073-00


PL-50085
B14
X
−1.49
−2.07
W
LOC402537
0
XM_380120
M-029089-00


PL-50089
O21
X
−3.27
−2.66
W
LOC402556
0
XM_379877
M-031795-00


PL-50090
A07
X
−2.47
−1.86
W
LOC402560
0
XM_380127
M-031802-00


PL-50086
A19
X
−2.56
−3.08
W
LOC402586
0
XM_380138
M-029126-00


PL-50086
A21
X
−2.25
−2.69
W
LOC402587
0
XM_380139
M-029127-00


PL-50086
E21
X
−1.65
−3.01
W
LOC402625
0
XM_379975
M-029163-00


PL-50085
F04
X
−2.78
−2.23
W
LOC402641
0
XM_379995
M-029057-00


PL-50081
M17
X
−3.94
−1.80
W
LOC404785
404785
NM_207513
M-032197-00


PL-50058
M23
X
−3.15
−0.63
W
LOC51054
51054
NM_015899
M-020941-00


PL-50057
A16
X
−2.13
−1.90
W
LOC51066
51066
NM_015931
M-020960-00


PL-50058
P07
X
−2.64
−3.28
W
LOC51333
51333
NM_016643
M-006990-00


PL-50059
K05
X
−2.62
−2.50
W
LOC51693
51693
NM_016209
M-021254-00


PL-50070
P05
X
−2.95
−2.76
W
LOC92689
92689
NM_138389
M-015552-00


PL-50069
B23
X
−2.30
−2.80
W
LOC96597
96597
XM_378655
M-023053-00


PL-50019
G07
X
−3.09
−2.93
W
LOR
4014
NM_000427
M-011077-00


PL-50048
P16
X
−3.33
−1.23
W
LPO
4025
NM_006151
M-023219-00


PL-50022
F18
X
−2.00
−2.26
W
LTBP3
4054
NM_021070
M-014144-00


PL-50016
D07
X
−2.46
−3.34
W
LU
4059.00
NM_005581
M-010608-00


PL-50017
P04
X
−1.87
−2.52
W
LY64
4064
NM_005582
M-020015-00


PL-50007
A15
X
−2.28
−2.10
W
M96
22823
NM_007358
M-012796-00


PL-50022
D16
X
−2.13
−1.89
W
MAD2L2
10459
NM_006341
M-003272-03


PL-50059
H23
X
−2.81
−2.75
W
MAGEL2
54551
NM_019066
M-013374-00


PL-50002
M10
X
−1.81
−2.45
W
MAPK13
5603
NM_002754
M-003591-02


PL-50007
C09
X
−1.94
−2.22
W
MAPRE2
10982
NM_014268
M-012501-00


PL-50049
B16
X
−2.58
−2.31
W
MASP1
5648
NM_001879
M-005937-00


PL-50013
I15
X
−2.56
−2.75
W
MBTPS2
51360
NM_015884
M-005940-01


PL-50005
C07
X
−2.90
−2.51
W
MC4R
4160
NM_005912
M-005660-00


PL-50016
D17
X
−2.17
−2.39
W
MCC
4163.00
NM_002387
M-010523-01


PL-50053
N17
X
−2.20
−2.76
W
MCRS1
10445
NM_006337
M-018557-00


PL-50022
B06
X
−2.01
−3.85
W
MDGA1
266727
NM_153487
M-016082-00


PL-50072
M14
X
−1.50
−3.11
W
MDH1B
130752
XM_059468
M-023122-00


PL-50009
O11
X
−2.86
−2.47
W
MEF2A
4205
NM_005587
M-009362-00


PL-50009
O13
X
−1.72
−2.04
W
MEF2B
4207
NM_005919
M-009342-00


PL-50068
C08
X
−2.94
−2.77
W
MGC13168
84821
NM_032735
M-014977-00


PL-50068
N14
X
−3.07
−2.59
W
MGC14126
84984
NM_032898
M-015039-00


PL-50070
P23
X
−2.16
−1.23
W
MGC16372
92749
NM_145038
M-016558-00


PL-50079
O04
X
−4.23
−2.82
W
MGC16597
339230
XM_375500
M-030964-00


PL-50070
K02
X
−2.66
−1.94
W
MGC17337
91283
NM_080655
M-015247-00


PL-50081
K21
X
−2.40
−2.20
W
MGC21394
404203
NM_205841
M-031857-00


PL-50074
M16
X
−2.23
−2.69
W
MGC23918
151903
NM_144716
M-015455-00


PL-50072
H08
X
−2.51
−3.29
W
MGC23937
139596
NM_145052
M-016141-00


PL-50005
C13
X
−2.91
−2.23
W
MGC26856
256710
NM_152779
M-016259-00


PL-50074
A12
X
−1.93
−2.28
W
MGC39633
153733
NM_152549
M-015448-00


PL-50072
L02
X
−2.46
−2.58
W
MGC41945
138724
NM_203299
M-031891-00


PL-50067
J04
X
−3.26
−2.46
W
MGC4238
84292
NM_032332
M-014860-00


PL-50073
D11
X
−2.10
−2.65
W
MGC45714
147007
NM_152464



PL-50076
D04
X
−1.77
−2.91
W
MGC50559
254013
NM_173802
M-018388-00


PL-50080
M06
X
−1.69
−2.85
W
MGC52000
375260
NM_198943
M-031853-00


PL-50063
A09
X
−3.04
−2.88
W
MICAL3
57553
XM_032997
M-024432-00


PL-50063
D21
X
−4.46
−2.44
W
MIG12
58526
NM_021242
M-015884-00


PL-50068
F16
X
−3.31
−2.68
W
MIRAB13
85377
NM_033386
M-015102-00


PL-50014
I19
X
−2.67
−3.05
W
MMP24
10893.00
NM_006690
M-005963-01


PL-50059
K17
X
−1.71
−2.84
W
MO25
51719
NM_016289
M-015407-00


PL-50054
O16
X
−2.84
−2.14
W
MORF4L1
10933
NM_006791
M-006379-00


PL-50002
E02
X
−2.67
−2.48
W
MRC2
9902
NM_006039
M-020064-00


PL-50059
E21
X
−1.55
−2.18
W
MRPL48
51642
NM_016055
M-017512-00


PL-50059
C10
X
−1.16
−3.52
W
MRPS21
54460
NM_018997
M-013388-00


PL-50007
A04
X
−3.23
−1.63
W
MT1A
4489
NM_005946
M-012724-00


PL-50071
C02
X
−2.23
−1.74
W
MTFMT
123263
NM_139242
M-009633-00


PL-50013
O21
X
−3.05
−2.44
W
MTMR9
66036
NM_015458
M-019244-01


PL-50010
O05
X
−3.21
−2.46
W
MTRF1L
54516
NM_019041
M-015386-00


PL-50007
K07
X
−1.21
−2.88
W
MYH1
4619
NM_005963
M-013486-00


PL-50057
N09
X
−2.73
−1.89
W
MYLIP
29116
NM_013262
M-006976-00


PL-50061
J06
X
−1.58
−2.08
W
MYO5C
55930
NM_018728
M-031960-00


PL-50051
B13
X
−3.48
−1.64
W
MYST3
7994
NM_006766
M-019849-00


PL-50049
E19
X
−1.80
−2.48
W
NAP1L4
4676
NM_005969
M-012183-00


PL-50051
D06
X
−0.40
−2.39
W
NAPG
8774
NM_003826
M-011529-00


PL-50011
C16
X
−2.28
−2.15
W
NCB5OR
51167
NM_016230
M-009347-00


PL-50049
G07
X
−3.23
−2.49
W
NCF4
4689
NM_000631
M-011128-00


PL-50023
B18
X
−2.59
−1.76
W
NCOA5
57727
NM_020967
M-013157-00


PL-50023
B10
X
−2.36
−4.21
W
NDEL1
81565
NM_030808
M-018571-00


PL-50049
K07
X
−2.82
−2.10
W
NDUFB9
4715
NM_005005
M-019899-00


PL-50049
K11
X
−2.38
−2.61
W
NDUFC1
4717
NM_002494
M-019601-00


PL-50013
B04
X
−2.95
−2.65
W
NDUFS1
4719
NM_005006
M-019069-00


PL-50049
K19
X
−1.20
−3.23
W
NDUFS6
4726
NM_004553
M-019817-00


PL-50054
K17
X
−2.42
−1.80
W
NET-5
10867
NM_006675
M-012293-00


PL-50072
O07
X
−2.53
−2.48
W
NEU4
129807
NM_080741
M-013263-00


PL-50009
I06
X
−3.19
−2.49
W
NFATC2
4773
NM_012340
M-003606-01


PL-50009
E20
X
−2.89
−1.53
W
NFKB2
4791
NM_002502
M-003918-00


PL-50052
G09
X
−2.89
−2.46
W
NFS1
9054
NM_021100
M-011564-00


PL-50049
M17
X
−3.01
−2.15
W
NHLH2
4808
NM_005599
M-020020-00


PL-50063
I16
X
−2.01
−2.62
W
NOPE
57722
NM_020962
M-014170-00


PL-50051
L08
X
−2.19
−2.30
W
NPFF
8620
NM_003717
M-011502-00


PL-50005
G09
X
−2.39
−2.05
W
NPY2R
4887
NM_000910
M-005673-01


PL-50005
G11
X
−1.74
−2.32
W
NPY5R
4889
NM_006174
M-005674-00


PL-50054
N13
X
−1.63
−2.71
W
NRM
11270
NM_007243
M-012779-00


PL-50007
O12
X
−2.70
−3.03
W
NUCB1
4924
NM_006184
M-015822-00


PL-50062
D13
X
−1.82
−2.48
W
NUP107
57122
NM_020401
M-020440-00


PL-50061
D21
X
−2.28
−1.73
W
NUP133
55746
NM_018230
M-013322-00


PL-50061
H02
X
−2.48
−3.27
W
NXF2
56001
NM_017809
M-010445-00


PL-50061
H06
X
−1.54
−3.06
W
NXF5
55998
NM_032946
M-013599-00


PL-50019
P20
X
−4.00
−2.02
W
NXT1
29107
NM_013248
M-017194-00


PL-50069
A23
X
−1.88
−2.00
W
NYD-SP28
85478
NM_033124
M-015072-00


PL-50023
G16
X
−2.24
−3.32
W
OFD1
8481
NM_003611
M-009300-01


PL-50005
G23
X
−1.96
−2.04
W
OPN3
23596
NM_014322
M-005681-01


PL-50089
P07
X
−1.47
−2.83
W
OR3A4
390756
NM_001005334
M-032453-00


PL-50089
L07
X
−1.44
−2.78
W
OR4A5
81318
NM_001005272
M-032433-00


PL-50089
J20
X
−2.50
−2.48
W
OR5B2
390190
NM_001005566
M-032494-00


PL-50089
L05
X
−1.83
−3.72
W
OR5M11
219487
NM_001005245
M-032432-00


PL-50089
P04
X
−3.78
−2.12
W
OR6C74
254783
NM_001005490
M-032472-00


PL-50056
E19
X
−2.02
−1.91
W
ORC3L
23595
NM_012381
M-003285-01


PL-50023
E16
X
−2.49
−4.24
W
OTOR
56914
NM_020157
M-017390-00


PL-50059
K11
X
−4.17
−2.07
W
PADI3
51702
NM_016233
M-021051-00


PL-50069
E06
X
−1.66
−2.78
W
PAGE-5
90737
NM_130467
M-017468-00


PL-50056
L02
X
−2.88
−1.42
W
PAI-RBP1
26135
NM_015640
M-020528-00


PL-50060
M10
X
−3.27
−1.51
W
PAK1IP1
55003
NM_017906
M-020912-00


PL-50076
L05
X
−2.26
−1.72
W
PAQR10
221938
NM_198403
M-008052-00


PL-50009
A18
X
−3.07
−2.42
W
PAWR
5074
NM_002583
M-004434-00


PL-50049
K02
X
−3.58
−2.13
W
PCBP1
5093
NM_006196
M-012243-01


PL-50062
B07
X
−3.19
−2.38
W
PCNP
57092
NM_020357
M-020263-00


PL-50007
C20
X
−2.34
−1.95
W
PCOLCE
5118
NM_002593
M-011747-00


PL-50023
B07
X
−2.40
−2.28
W
PDE6A
5145
NM_000440
M-007651-00


PL-50063
B04
X
−2.10
−2.62
W
PDF
64146
NM_022341
M-003851-00


PL-50011
F21
X
−2.31
−1.75
W
PDHA2
5161
NM_005390
M-023925-00


PL-50016
A19
X
−2.67
−2.94
W
PDP2
57546.00
NM_020786
M-022572-00


PL-50070
L02
X
−3.12
−2.13
W
PERLD1
93210
NM_033419
M-017912-00


PL-50061
E12
X
−2.26
−2.28
W
PEX26
55670
NM_017929
M-019128-00


PL-50002
J23
X
−2.09
−2.44
W
PFKFB3
5209
NM_004566
M-006763-00


PL-50073
N08
X
−2.07
−2.21
W
PHF13
148479
NM_153812



PL-50068
C23
X
−2.49
−2.19
W
PHYHIPL
84457
NM_032439
M-014894-00


PL-50078
A23
X
−2.16
−2.00
W
PIGW
284098
NM_178517
M-021480-00


PL-50002
P07
X
−1.58
−3.31
W
PIK3R3
8503
NM_003629
M-019546-00


PL-50002
P13
X
−1.95
−2.15
W
PIK4CB
5298
NM_002651
M-006777-02


PL-50058
H07
X
−2.39
−2.07
W
PIPOX
51268
NM_016518
M-010199-00


PL-50075
G16
X
−2.79
−2.75
W
PKD1L1
168507
NM_138295
M-017434-00


PL-50011
L13
X
−2.53
−2.13
W
PNLIP
5406
NM_000936
M-008973-00


PL-50011
L15
X
−2.42
−2.27
W
PNLIPRP1
5407
NM_006229
M-009145-00


PL-50014
G05
X
−3.24
−2.84
W
PRDX3
10935.00
NM_006793
M-010355-00


PL-50002
L20
X
−1.82
−2.35
W
PRKACA
5566
NM_002730
M-004649-00


PL-50018
J11
X
−2.05
−1.97
W
PROK1
84432
NM_032414
M-014883-00


PL-50057
A18
X
−1.87
−3.54
W
PROL5
26952
NM_012390
M-020197-00


PL-50002
K13
X
−2.45
−1.79
W
PRPS1L1
221823
NM_175886
M-006804-00


PL-50050
A09
X
−2.02
−3.81
W
PSG3
5671
NM_021016
M-014137-00


PL-50057
P15
X
−0.93
−2.95
W
PTD004
29789
NM_013341
M-015680-00


PL-50058
J11
X
−2.32
−2.24
W
PTX1
51290
NM_016570
M-021151-00


PL-50056
G19
X
−2.66
−3.04
W
RABGAP1
23637
NM_012197
M-012803-00


PL-50013
D11
X
−2.20
−1.93
W
RABL2A
11159
NM_007082
M-013620-00


PL-50013
F11
X
−2.65
−3.09
W
RANBP2
5903
NM_006267
M-004746-01


PL-50056
A09
X
−2.52
−1.67
W
RASD2
23551
NM_014310
M-009560-00


PL-50070
E02
X
−1.96
−2.22
W
RASL10B
91608
NM_033315
M-008344-00


PL-50059
E06
X
−2.00
−2.83
W
RBM27
54439
XM_291128
M-024337-00


PL-50007
J19
X
−2.21
−2.60
W
RBM5
10181
NM_005778
M-009220-01


PL-50011
P10
X
−2.94
−2.47
W
RCE1
9986
NM_005133
M-006025-00


PL-50073
O02
X
−2.85
−1.93
W
RDH12
145226
NM_152443



PL-50011
P08
X
−2.92
−1.42
W
RDH5
5959
NM_002905
M-008220-01


PL-50057
P18
X
−1.73
−2.54
W
REPIN1
29803
NM_013400
M-006978-00


PL-50054
C09
X
−2.31
−2.46
W
RFPL3
10738
NM_006604
M-006934-00


PL-50054
P10
X
−1.95
−2.06
W
RNF13
11342
NM_007282
M-006944-00


PL-50018
D17
X
−2.24
−2.52
W
RORB
6096
NM_006914
M-003441-01


PL-50080
K20
X
−1.57
−2.35
W
RP26
375298
NM_201548
M-027336-00


PL-50074
D21
X
−2.03
−2.89
W
RPIB9
154661
NM_138290
M-015403-00


PL-50050
O09
X
−3.26
−1.86
W
RPL3L
6123
NM_005061
M-012009-00


PL-50003
C19
X
−2.44
−1.89
W
RPS6KA2
6196
NM_021135
M-004663-01


PL-50005
C20
X
−2.04
−3.11
W
RRH
10692
NM_006583
M-005723-01


PL-50050
E08
X
−1.85
−4.78
W
SAA2
6289
NM_030754
M-016279-00


PL-50005
C18
X
−2.99
−2.50
W
SALPR
51289
NM_016568
M-004774-00


PL-50050
C18
X
−2.18
−2.16
W
SATB1
6304
NM_002971
M-011771-00


PL-50007
P16
X
−2.75
−1.65
W
SCA7
6314
NM_000333
M-011106-00


PL-50016
N15
X
−2.88
−1.93
W
SCG3
29106.00
NM_013243
M-013710-00


PL-50007
N20
X
−2.05
−2.03
W
SEC22L1
9554
NM_004892
M-011963-00


PL-50064
N02
X
−2.81
−2.48
W
SECISBP2
79048
NM_024077
M-015634-00


PL-50011
L20
X
−2.73
−2.88
W
SENP1
29843
NM_014554
M-006357-00


PL-50062
P17
X
−1.38
−2.19
W
SENP7
57337
NM_020654
M-006035-00


PL-50071
C04
X
−2.84
−1.66
W
SENP8
123228
NM_145204
M-004071-00


PL-50055
B08
X
−1.54
−2.91
W
SEZ6L
23544
NM_021115
M-008081-00


PL-50063
G20
X
−3.18
−2.00
W
SF4
57794
NM_172231
M-017511-00


PL-50007
L12
X
−2.30
−2.29
W
SFRS7
6432
NM_006276
M-015909-00


PL-50062
G16
X
−1.99
−3.46
W
SHD
56961
NM_020209
M-023905-00


PL-50016
P12
X
−2.04
−2.07
W
SLAMF6
114836.00
NM_052931
M-013423-01


PL-50062
C18
X
−2.38
−1.44
W
SLC12A9
56996
NM_020246
M-007390-00


PL-50049
M08
X
−3.20
−1.97
W
SLC22A1LS
5003
NM_007105
M-019642-00


PL-50064
J04
X
−2.51
−2.24
W
SLC25A23
79085
NM_024103
M-007360-00


PL-50076
O17
X
−2.62
−2.27
W
SLC36A1
206358
NM_078483
M-007550-00


PL-50054
L23
X
−2.77
−2.46
W
SLC6A14
11254
NM_007231
M-007601-00


PL-50019
H08
X
−2.11
−1.95
W
SLC6A2
6530
NM_001043
M-007602-00


PL-50019
H06
X
−2.68
−2.10
W
SLC6A4
6532
NM_001045
M-007604-00


PL-50003
G19
X
−1.94
−2.19
W
SMG1
23049
NM_014006
M-005033-00


PL-50063
D20
X
−2.37
−2.17
W
SMOC2
64094
NM_022138
M-013886-00


PL-50051
D08
X
−0.53
−2.93
W
SNAP23
8773
NM_003825
M-017545-00


PL-50066
N08
X
−2.00
−3.51
W
SNX27
81609
NM_030918
M-017346-00


PL-50009
P21
X
−2.18
−2.36
W
SP4
6671
NM_003112
M-006562-00


PL-50057
I08
X
−2.55
−1.44
W
SPINK4
27290
NM_014471
M-020235-00


PL-50050
N12
X
−2.03
−2.92
W
SPINT1
6692
NM_003710
M-004578-00


PL-50055
P09
X
−2.01
−2.06
W
SR140
23350
XM_031553
M-023607-00


PL-50050
J16
X
−1.80
−3.15
W
SSA2
6738
NM_004600
M-017733-00


PL-50005
C04
X
−2.07
−3.32
W
SSTR2
6752
NM_001050
M-005728-01


PL-50005
A20
X
−2.44
−2.38
W
SSTR4
6754
NM_001052
M-005730-02


PL-50009
P10
X
−2.02
−2.97
W
SSX1
6756
NM_005635
M-019194-00


PL-50439
E21
X
−2.83
−2.58
W
STAMBPL1
57559
NM_020799
M-005783-01


PL-50057
E15
X
−2.76
−2.12
W
STEAP
26872
NM_012449
M-003713-00


PL-50052
G04
X
−3.23
−2.14
W
STOML1
9399
NM_004809
M-009360-00


PL-50058
A05
X
−1.40
−2.62
W
STOML2
30968
NM_013442
M-020518-00


PL-50057
M06
X
−3.18
−1.68
W
SULT1C2
27233
NM_006588
M-010391-00


PL-50050
P19
X
−2.48
−1.21
W
SUMO2
6613
NM_006937
M-016450-00


PL-50050
F02
X
−2.15
−2.47
W
SYCP1
6847
NM_003176
M-019171-00


PL-50007
F02
X
−1.95
−2.02
W
SYNCRIP
10492
NM_006372
M-016218-00


PL-50055
A16
X
−1.90
−2.68
W
SYNE2
23224
NM_015180
M-019259-00


PL-50069
B05
X
−1.91
−2.45
W
SYTL4
94121
NM_080737
M-007111-00


PL-50077
G16
X
−2.35
−4.18
W
TAB3
257397
NM_152787



PL-50077
E08
X
−1.65
−2.60
W
TAS2R45
259291
NM_176886



PL-50012
G18
X
−3.34
−2.54
W
TBCC
6903
NM_003192
M-011401-00


PL-50003
O04
X
−2.57
−2.29
W
TESK2
10420
NM_007170
M-005044-00


PL-50062
A07
X
−3.15
−2.06
W
TEX13B
56156
NM_031273
M-013485-00


PL-50009
B20
X
−2.23
−2.23
W
TGIF2LY
90655
NM_139214
M-017279-00


PL-50050
B02
X
−1.78
−2.85
W
TGM3
7053
NM_003245
M-010088-00


PL-50007
B06
X
−2.51
−2.28
W
TIMELESS
8914
NM_003920
M-019488-00


PL-50058
F14
X
−2.28
−2.99
W
TMEM14C
51522
NM_016462
M-020269-00


PL-50070
C21
X
−2.29
−2.46
W
TNFRSF13C
115650
NM_052945
M-013424-00


PL-50018
A19
X
−2.73
−1.44
W
TNFSF13B
10673
NM_006573
M-017586-00


PL-50068
B04
X
−2.26
−2.71
W
TNKS1BP1
85456
NM_033396
M-015106-00


PL-50008
A15
X
−3.66
−2.06
W
TNXB
7148
NM_019105
M-008106-00


PL-50018
C19
X
−2.25
−2.22
W
TOLLIP
54472
NM_019009
M-016930-00


PL-50064
I10
X
−2.96
−2.82
W
TORC3
64784
NM_022769
M-014210-00


PL-50015
O08
X
−2.51
−1.97
W
TRIM22
10346.00
NM_006074
M-006927-01


PL-50015
F23
X
−2.38
−2.30
W
TRIM33
51592.00
NM_015906
M-005392-02


PL-50053
I20
X
−2.24
−3.53
W
TSPAN-1
10103
NM_005727
M-003719-00


PL-50012
J09
X
−2.74
−1.51
W
TULP1
7287
NM_003322
M-011413-00


PL-50062
C20
X
−2.31
−2.28
W
TULP4
56995
NM_020245
M-013785-00


PL-50066
N17
X
−2.02
−2.71
W
TXNDC
81542
NM_030755
M-010675-00


PL-50012
J17
X
−2.09
−2.45
W
TYMS
7298
NM_001071
M-004717-01


PL-50050
N20
X
−4.03
−1.70
W
UAP1
6675
NM_003115
M-017160-00


PL-50012
L13
X
−2.86
−2.62
W
UBE2L6
9246
NM_004223
M-008569-00


PL-50058
B08
X
−1.72
−2.24
W
UFM1
51569
NM_016617
M-021005-00


PL-50071
B19
X
−1.54
−2.68
W
UNQ2446
123904
NM_198443
M-027207-00


PL-50080
C20
X
−4.44
−2.16
W
UNQ2492
377841
NM_198585
M-027275-00


PL-50078
M23
X
−2.63
−1.54
W
UNQ3033
284415
NM_198481
M-027236-00


PL-50080
D14
X
−2.57
−2.61
W
UNQ9370
400454
NM_207447
M-032131-00


PL-50064
F15
X
−2.44
−2.66
W
UPF3B
65109
NM_023010
M-012871-00


PL-50070
M13
X
−2.79
−1.27
W
VEST1
116328
NM_052958
M-015175-00


PL-50076
N14
X
−2.58
−2.97
W
VGLL2
245806
NM_153453
M-015963-00


PL-50005
H07
X
−2.62
−3.09
W
VN1R4
317703
NM_173857
M-017651-00


PL-50019
F05
X
−2.18
−2.02
W
VPS13A
23230
NM_015186
M-012878-00


PL-50061
B02
X
−2.23
−1.62
W
VPS35
55737
NM_018206
M-010894-00


PL-50064
C18
X
−2.66
−2.10
W
WARP
64856
NM_022834
M-016331-00


PL-50072
B08
X
−2.36
−2.10
W
WFDC3
140686
NM_181522
M-013334-00


PL-50016
F06
X
−3.36
−4.06
W
WNT7B
7477.00
NM_058238
M-003722-02


PL-50063
B10
X
−3.24
−2.28
W
XYLT2
64132
NM_022167
M-013040-00


PL-50003
E06
X
−3.18
−2.63
W
ZAK
51776
NM_133646
M-005068-00


PL-50063
K05
X
−1.49
−2.73
W
ZBTB2
57621
NM_020861
M-014129-00


PL-50021
B08
X
−2.06
−2.17
W
ZBTB7
51341
NM_015898
M-020818-00


PL-50061
O10
X
−1.65
−2.48
W
ZCCHC8
55596
NM_017612
M-021026-00


PL-50072
B18
X
−1.71
−2.12
W
ZFP28
140612
NM_020828
M-014089-00


PL-50010
C23
X
−2.43
−2.86
W
ZFP67
51043
NM_015872
M-020934-00


PL-50059
G23
X
−2.13
−1.92
W
ZFR
51663
NM_016107
M-019266-00


PL-50051
I10
X
−1.86
−4.52
W
ZNF192
7745
NM_006298
M-020154-00


PL-50058
A11
X
−2.88
−2.74
W
ZNF295
49854
NM_020727
M-013945-00


PL-50055
M17
X
−2.52
−1.51
W
ZNF297B
23099
NM_014007
M-020320-00


PL-50062
P19
X
−3.24
−2.42
W
ZNF304
57343
NM_020657
M-020719-00


PL-50056
M18
X
−2.34
−2.31
W
ZNF324
25799
NM_014347
M-006964-00


PL-50061
B09
X
−1.88
−2.35
W
ZNF334
55713
NM_018102
M-017955-00


PL-50057
D02
X
−2.35
−2.62
W
ZNF354C
30832
NM_014594
M-014199-00


PL-50068
A18
X
−2.28
−2.49
W
ZNF496
84838
NM_032752
M-014983-00


PL-50070
C15
X
−2.56
−3.03
W
ZNF501
115560
NM_145044
M-007118-00


PL-50068
B07
X
−3.37
−2.84
W
ZNF503
84858
NM_032772
M-015846-00


PL-50057
G12
X
−1.43
−2.54
W
ZNF544
27300
NM_014480
M-020223-00


PL-50073
P08
X
−2.79
−2.65
W
ZNF570
148268
NM_144694



PL-50078
K23
X
−2.22
−1.70
W
ZNF615
284370
NM_198480
M-032239-00


PL-50057
B18
X
−1.29
−5.41
W
ZNRD1
30834
NM_014596
M-017359-00





Stock_ID
Row
Type
Z score_A
Z score_B
Screen+
Gene Symbol
Accession #
Catalog #
Description





PL-50001
A05
X
2.49
1.61
*W
AAK1
22848
NM_014911
M-005300-00


PL-50001
F04
X
2.28
2.34
*W
GSK3A
2931
NM_019884
M-003009-01


PL-50004
I14
X
2.20
1.88
*W
FLJ10060
55065
NM_017986
M-010712-00


PL-50005
B15
X
2.18
2.38
*W
TAS2R16
50833
NM_016945
M-013103-00


PL-50005
F21
X
2.02
2.31
*W
VN1R1
57191
NM_020633
M-013177-00


PL-50008
A12
X
2.19
1.82
*W
BHLHB2
8553
NM_003670
M-010318-00


PL-50010
A12
X
2.15
1.98
*W
B4GALT7
11285
NM_007255
M-012387-00


PL-50010
J15
X
2.19
2.10
*W
CTPS
1503
NM_001905
M-006644-00


PL-50013
F18
X
2.13
1.81
*W
ATP2A3
489
NM_005173
M-006114-01


PL-50016
A10
X
2.61
2.25
*W
KCNH7
90134.00
NM_033272
M-006237-01


PL-50016
J16
X
2.24
1.89
*W
TRADD
8717.00
NM_003789
M-004452-00


PL-50019
M11
X
2.37
2.46
*W
CDC10
989
NM_001788
M-011607-00


PL-50019
I08
X
2.22
2.02
*W
PKP1
5317
NM_000299
M-012545-00


PL-50019
G10
X
2.30
1.86
*W
RPS5
6193
NM_001009
M-010498-01


PL-50019
L06
X
2.05
1.96
*W
SLC22A12
116085
NM_144585
M-007446-01


PL-50020
C14
X
1.95
2.54
*W
ACYP1
97
XM_352906
M-009937-00


PL-50020
L12
X
2.05
1.76
*W
BCL2L1
598
NM_138578
M-003458-00


PL-50020
C05
X
2.29
1.14
*W
CACNA1D
776
NM_000720
M-006124-01


PL-50021
G12
X
2.27
1.23
*W
CYB5
1528
NM_001914
M-019621-00


PL-50022
G19
X
2.14
2.75
*W
FLJ25952
253832
NM_153251
M-016758-00


PL-50022
K11
X
2.08
1.67
*W
GAA
2548
NM_000152
M-008881-00


PL-50022
K13
X
2.20
2.98
*W
GAB3
139716
NM_080612
M-015239-00


PL-50022
M13
X
1.86
2.83
*W
GATA6
2627
NM_005257
M-008351-01


PL-50023
E05
X
2.44
1.66
*W
HNRPA1
3178
NM_031157
M-008221-01


PL-50024
G11
X
2.34
2.76
*W
RPS16
6217
NM_001020
M-013627-00


PL-50024
N06
X
3.90
3.36
*M
XPO1
7514
NM_003400
M-003030-01


PL-50047
D11
X
2.55
2.13
*W
AP2M1
1173
NM_004068
M-008170-00


PL-50047
O13
X
3.41
2.59
*W
ATP2B1
490
NM_001682
M-006115-00


PL-50047
B15
X
2.22
1.89
*W
CEACAM3
1084
NM_001815
M-019510-00


PL-50048
P21
X
2.81
2.50
*W
FADS3
3995
NM_021727
M-008483-00


PL-50048
K19
X
2.52
1.91
*W
GGTL3
2686
NM_052830
M-005886-01


PL-50048
C10
X
2.29
2.46
*W
HNRPL
3191
NM_001533
M-011293-00


PL-50049
B11
X
2.20
2.20
*W
POLR2I
5438
NM_006233
M-012248-00


PL-50050
M10
X
2.37
2.17
*W
RPL28
6158
NM_000991
M-011145-00


PL-50050
K08
X
2.01
1.96
*W
RPLP2
6181
NM_001004
M-004314-00


PL-50050
I18
X
2.12
1.46
*W
RPS3A
6189
NM_001006
M-013603-00


PL-50053
C13
X
2.35
2.10
*W
PLEKHM1
9842
NM_014798
M-023203-00


PL-50055
B10
X
2.03
2.04
*W
RBM9
23543
NM_014309
M-020616-00


PL-50060
N02
X
2.09
1.47
*W
FLJ10774
55226
NM_024662
M-014402-00


PL-50061
B15
X
1.98
2.01
*W
FLJ10534
55720
NM_018128
M-017111-00


PL-50064
B04
X
2.31
1.97
*W
VIK
79027
NM_024061
M-012894-00


PL-50065
I02
X
2.24
1.89
*W
FLJ22222
79701
NM_024648



PL-50065
B16
X
2.69
1.76
*W
POF1B
79983
NM_024921



PL-50066
N10
X
1.71
2.25
*W
FIP1L1
81608
NM_030917
M-014670-00


PL-50066
I11
X
2.35
1.19
*W
FLJ22173
80111
NM_025041
M-014563-00


PL-50066
P07
X
2.55
2.66
*W
STMN4
81551
NM_030795
M-016810-00


PL-50066
E04
X
2.46
1.37
*W
ULBP1
80329
NM_025218
M-014611-00


PL-50067
H12
X
2.15
2.13
*W
C6ORF125
84300
NM_032340
M-021290-00


PL-50067
C10
X
2.15
2.00
*W
KFZP564O052
84060
NM_032120
M-014780-00


PL-50067
I14
X
2.06
1.83
*W
KRTAP1-5
83895
NM_031957
M-013515-00


PL-50069
B09
X
2.16
2.15
*W
ARHGAP12
94134
NM_018287
M-008729-00


PL-50073
F08
X
2.07
2.01
*W
FLJ33084
149483
NM_152500



PL-50076
O11
X
2.22
2.00
*W
KIAA2018
205717
XM_291062
M-023709-00


PL-50076
I18
X
2.04
2.03
*W
SPAS1
219938
NM_174927
M-017842-00


PL-50076
D09
X
1.68
2.01
*W
TDRD6
221400
XM_166443
M-025108-00


PL-50078
A18
X
1.96
2.05
*W
FLJ46536
285180
NM_198483
M-027237-00


PL-50080
G07
X
2.01
1.75
*W
GLTDC1
360203
NM_182974
M-019460-00


PL-50083
K07
X
1.90
2.53
*W
LOC219612
0
XM_168585
M-025428-00


PL-50085
H09
X
2.13
1.86
*W
LOC402280
0
XM_377946
M-028792-00


PL-50085
H10
X
1.79
2.08
*W
LOC402489
0
XM_379819
M-029023-00


PL-50086
H15
X
2.05
2.08
*W
LOC389777
0
XM_374300
M-029575-00


PL-50086
H20
X
2.17
2.28
*W
LOC401638
0
XM_377109
M-029788-00


PL-50088
H17
X
1.43
2.56
*W
LOC390937
0
XM_372730



PL-50088
H13
X
2.23
1.75
*W
LOC401915
0
XM_377529



PL-50439
A09
X
1.74
2.27
*W
COPS5
10987
NM_006837
M-005814-01


PL-50439
I21
X
2.55
2.65
*W
UBE2J2
118424
NM_058167
M-008614-00


PL-50439
D19
X
2.18
2.60
*W
USP35
57558
XM_290527
M-006083-01



















TABLE 2





GeneSymbol
EntrezGeneID
Genbank Acc. No.
NFAT Score


















AB026190
27252
NM_014458
W


ABCC13
150000
NM_138726
M


ABLIM2
84448
NM_032432
W


ACLY
47
NM_001096
W


ACTB
60
NM_001101
M


ACY1L2
135293
XM_072402
W


ADAM10
102
NM_001110
M


ADAMTS5
11096
NM_007038
S


ADCY4
196883
NM_139247
W


ADK
132
NM_001123
W


ADRA2B
151
NM_000682
W


AFG3L1
172
NM_001132
M


AGK
55750
NM_018238
W


AKAP11
11215
NM_016248
W


AKR1CL1
340811
XM_291723
W


AKR1CL2
83592
NM_031436
M


ALCAM
214
NM_001627
M


ALS2CR13
150864
NM_173511
W


ALS2CR15
130026
NM_138468
M


AMH
268
NM_000479
W


AMIGO2
347902
NM_181847
W


ANC_2H01
51193
NM_016331
M


ANKFX1
51479
NM_016376
W


ANKK1
255239
NM_178510
W


ANKMX2
57037
NM_020319
W


ANKRD9
122416
NM_152326
M


AP1S3
130340
NM_178814
W


AP3B2
8120
NM_004644
W


APG16L
55054
NM_017974
W


APH-1A
51107
NM_016022
M


APOBEC1
339
NM_001644
W


APOL4
80832
NM_030643
M


APXL
357
NM_001649
W


AQR
9716
NM_014691
W


ARCN1
372
NM_001655
S


ARHGAP15
55843
NM_018460
W


ARHGAP17
55114
NM_018054
W


ARHGDIA
396
NM_004309
W


ARL10C
55207
NM_018184
W


ARL11
115761
NM_138450
M


ARL5C
390790
XM_372668
M


ARMC2
84071
NM_032131
W


ARPP-21
10777
NM_016300
W


ARS2
51593
NM_015908
W


ASB10
136371
NM_080871
W


ASB4
51666
NM_016116
S


ASMTL
8623
NM_004192
W


ASTL
431705
NM_001002036
W


ATP10D
57205
NM_020453
W


ATP5L2
267020
NM_198822
M


ATP5S
27109
NM_015684
W


ATP6V0D1
9114
NM_004691
M


ATP6V1D
51382
NM_015994
M


AZ2
64343
NM_022461
W


BACE1
23621
NM_012104
M


BATF
10538
NM_006399
M


BBX
56987
NM_020235
W


BC-2
27243
NM_014453
W


BCL2L12
83596
NM_052842
M


BEST3
84821
NM_032735
W


BEXL1
56271
XM_043653
W


BFSP1
631
NM_001195
W


BG1
23205
NM_015162
S


BGN
633
NM_001711
M


BIG1
10565
NM_006421
S


BIN3
55909
NM_018688
M


BMP15
9210
NM_005448
W


BMP4
652
NM_001202
S


BMPER
168667
NM_133468
W


BMSC-UBP
84993
NM_032907
S


BRD8
10902
NM_006696
W


BRP44L
51660
NM_016098
M


BRUNOL6
60677
NM_052840
W


BSCL2
26580
NM_032667
W


BTBD11
121551
NM_152322
M


BTN3A3
10384
NM_006994
W


C10ORF53
282966
NM_182554
M


C10ORF56
219654
NM_153367
M


C10ORF59
55328
NM_018363
W


C10ORF81
79949
NM_024889
M


C10ORF94
93426
NM_130784
W


C11ORF17
56672
NM_020642
W


C13ORF10
64062
NM_022118
W


C13ORF12
51371
NM_015932
M


C14ORF11
55837
NM_018453
M


C14ORF126
112487
NM_080664
W


C14ORF147
171546
NM_138288
W


C14ORF43
91748
NM_194278
W


C14ORF73
91828
XM_040910
W


C14ORF8
122664
NM_173846
W


C14ORF92
9878
XM_375045
W


C15ORF24
56851
NM_020154
S


C16ORF23
79006
NM_024042
W


C18ORF34
374864
NM_198995
W


C19ORF13
26065
NM_015578
S


C19ORF24
55009
NM_017914
W


C1ORF123
54987
NM_017887
M


C20ORF104
51230
NM_016436
S


C20ORF96
140680
NM_153269
M


C21ORF107
54014
NM_018963
W


C21ORF45
54069
NM_018944
W


C21ORF49
54067
NM_001006116
S


C21ORF6
10069
NM_016940
W


C21ORF84
114038
NM_153752
W


C3ORF6
152137
NM_174908
M


C4ORF8
8603
NM_003704
W


C5ORF11
167410
NM_153234
W


C6ORF115
58527
XM_371848
M


C6ORF191
253582
XM_173166
S


C6ORF51
112495
NM_138408
W


C6ORF57
135154
NM_145267
W


C6ORF59
79992
NM_024929
W


C6ORF84
22832
XM_376518
S


C8ORF4
56892
NM_020130
W


C9ORF11
54586
XM_035953
M


C9ORF138
158297
NM_153707
M


C9ORF150
286343
NM_203403
W


C9ORF71
169693
XM_376874
M


C9ORF72
203228
NM_018325
S


C9ORF79
286234
NM_178828
M


C9ORF84
158401
NM_173521
W


CABLES2
81928
NM_031215
W


CACNA1A
773
NM_000068
W


CACNG4
27092
NM_014405
W


CADPS
8618
NM_003716
W


CARD12
58484
NM_021209
W


CASC1
55259
NM_018272
S


CAV3
859
NM_001234
W


CBLL1
79872
NM_024814
S


CBLN2
147381
NM_182511
W


CBX6
23466
NM_014292
W


CCDC125
202243
NM_176816
M


CCK
885
NM_000729
M


CCL11
6356
NM_002986
M


CCNB2
9133
NM_004701
S


CCNK
8812
NM_003858
M


CCR6
1235
NM_004367
W


CCRN4L
25819
NM_012118
M


CD151
977
NM_004357
W


CD1E
913
NM_030893
W


CD209L
10332
NM_014257
M


CD3G
917
NM_000073
W


CD5
921
NM_014207
W


CD74
972
NM_004355
W


CDC27
996
NM_001256
S


CDC2L5
8621
NM_003718
M


CDC42EP5
148170
NM_145057
W


CDH9
1007
NM_016279
W


CENTG1
116986
NM_014770
W


CFL2
1073
NM_021914
W


CGI-04
51067
NM_015936
S


CHCHD5
84269
NM_032309
W


CHD4
1108
NM_001273
W


CHERP
10523
NM_006387
W


CHFR
55743
NM_018223
W


CHRM3
1131
NM_000740
W


CHRNA4
1137
NM_000744
W


CIRBP
1153
NM_001280
M


CKN1
1161
NM_000082
W


CLCN4
1183
NM_001830
W


CLDN22
53842
XM_210581
S


CLPS
1208
NM_001832
M


CLUL1
27098
NM_014410
W


CMAS
55907
NM_018686
M


CMRF-35H
11314
NM_007261
W


CNTN3
5067
XM_039627
M


COMT
1312
NM_000754
W


COPA
1314
NM_004371
S


COPB1
1315
NM_016451
S


COPB2
9276
NM_004766
S


COPE
11316
NM_007263
S


COPG
22820
NM_016128
S


COPZ1
22818
NM_016057
S


COX8A
1351
NM_004074
W


CPEB4
80315
NM_030627
S


CPT2
1376
NM_000098
M


CRBN
51185
NM_016302
W


CRHR2
1395
NM_001883
W


CRLF3
51379
NM_015986
M


CRSP2
9282
NM_004229
W


CRSP3
9439
NM_004830
W


CRSP6
9440
NM_004268
W


CRSP9
9443
NM_004270
M


CRXBA2
1412
NM_005209
W


CRXBB1
1414
NM_001887
W


CRXBB3
1417
NM_004076
W


CRXGC
1420
NM_020989
W


CSAD
51380
NM_015989
W


CSE1L
1434
NM_001316
S


CST7
8530
NM_003650
W


CYLC1
1538
XM_088636
W


CYP1A1
1543
NM_000499
W


CYP2S1
29785
NM_030622
S


CYP3A5
1577
NM_000777
W


CYT19
57412
NM_020682
S


D2S448
7837
XM_056455
W


D4ST1
113189
NM_130468
S


DAAM1
23002
NM_014992
W


DACH1
1602
NM_004392
M


DBI
1622
NM_020548
W


DC2
58505
NM_021227
W


DDX46
9879
NM_014829
W


DDX53
168400
NM_182699
M


DGCR6L
85359
NM_033257
M


DHPS
1725
NM_001930
W


DHRS4
10901
NM_021004
M


DHRS4L2
317749
NM_198083
M


DHRS9
10170
NM_005771
M


DIABLO
56616
NM_019887
S


DIPA
11007
NM_006848
S


DISP2
85455
NM_033510
M


DJ383J4.3
91687
XM_371328
M


DKFZP434B1231
91156
NM_178275
W


DKFZP547E1010
26097
NM_015607
M


DKFZP564D1378
84064
NM_032124
M


DKFZP566D1346
81573
NM_030816
M


DKFZP686P0288
285190
NM_182588
W


DKFZP761B1514
84248
NM_032288
W


DLAT
1737
NM_001931
W


DNAJC5G
285126
NM_173650
M


DNM1L
10059
NM_005690
W


DONSON
29980
NM_145794
M


DRPLA
1822
NM_001940
M


DSEL
92126
NM_032160
M


DSG4
147409
NM_177986
M


DUSP12
11266
NM_007240
M


DUSP16
80824
NM_030640
W


DUSP18
150290
NM_152511
W


DUX1
26584
NM_012146
W


DUX5
26581
NM_012149
W


DVL3
1857
NM_004423
W


E(X)2
56943
NM_020189
W


E2IG2
51287
NM_016565
W


EBPL
84650
NM_032565
W


EG1
80306
NM_025205
S


EGLN3
112399
NM_022073
M


EHD2
30846
NM_014601
M


ELMOD1
55531
NM_018712
S


ELXS
25909
NM_015446
S


EML4
27436
NM_019063
M


EPB41L5
57669
NM_020909
W


EPO
2056
NM_000799
M


EPSTI1
94240
NM_033255
M


ERBB4
2066
NM_005235
M


EREG
2069
NM_001432
W


ERK8
225689
NM_139021
W


ESRRBL1
55081
NM_018010
W


EVI5
7813
NM_005665
W


F11R
50848
NM_016946
M


FAM108C1
58489
XM_051862
S


FAM14A
83982
NM_032036
W


FAM171A2
284069
XM_208993
S


FAM23B
0
XM_291726
W


FAM31C
79958
NM_024898
M


FAM38A
9780
NM_014745
W


FAM57B
83723
NM_031478
W


FAS
355
NM_000043
S


FASTKD5
60493
NM_021826
M


FBXL20
84961
NM_032875
W


FBXL3P
26223
NM_012159
W


FBXO11
80204
NM_012167
M


FBXO22
26263
NM_012170
W


FBXO46
23403
XM_371179
W


FBXO5
26271
NM_012177
S


FCGR3A
2214
NM_000569
W


FCHSD2
9873
NM_014824
M


FGF14
2259
NM_004115
W


FGF7
2252
NM_002009
W


FGFR2
2263
NM_000141
M


FGFR4
2264
NM_002011
M


FKBP1C
135521
XM_059776
W


FLJ10159
55084
NM_018013
W


FLJ10352
55125
NM_018069
W


FLJ10613
54552
NM_019067
W


FLJ10759
55223
NM_018207
M


FLJ10826
55239
NM_018233
M


FLJ11126
55308
NM_018332
W


FLJ11127
54491
NM_019018
M


FLJ11193
55322
NM_018356
W


FLJ12517
65094
NM_023007
W


FLJ14299
80139
NM_025069
S


FLJ20152
54463
NM_019000
W


FLJ20257
56257
NM_019606
M


FLJ20280
54876
NM_017741
M


FLJ20291
54883
NM_017748
M


FLJ20321
54897
NM_017766
M


FLJ20485
54517
NM_019042
W


FLJ20509
54956
NM_017851
W


FLJ20519
54964
NM_017860
W


FLJ20534
54969
NM_017867
S


FLJ20618
55000
NM_017903
M


FLJ20793/TMX3
54495
NM_019022
S


FLJ20972
80098
NM_025030
W


FLJ21415
79794
NM_024738
S


FLJ21687
79917
NM_024859
M


FLJ21986
79974
NM_024913
M


FLJ22531
79703
NM_024650
M


FLJ22688
80199
NM_025129
W


FLJ23554
79864
NM_024806
W


FLJ25286
153443
NM_152546
W


FLJ25555
124930
NM_152345
M


FLJ30656
124801
NM_152344
S


FLJ32356
144717
NM_144671
W


FLJ32421
148362
NM_144695
W


FLJ32569
148811
NM_152491
W


FLJ32682
220081
NM_182542
W


FLJ32734
146849
NM_144681
W


FLJ32743
220136
NM_145020
M


FLJ33516
139221
NM_152423
M


FLJ33814
150275
NM_173510
W


FLJ33817
124997
NM_152348
M


FLJ34690
284034
NM_182567
W


FLJ35757
162333
NM_152598
W


FLJ35838
163479
NM_173532
W


FLJ35843
160762
NM_152591
W


FLJ35961
127294
NM_152372
W


FLJ36070
284358
NM_182574
S


FLJ36754/P18SRP
285672
NM_173829
S


FLJ36878
284114
NM_178518
W


FLJ38379
285097
NM_178530
W


FLJ38984
127703
NM_152374
W


FLJ39117
126638
XM_371312
W


FLJ39155
133584
NM_152403
W


FLJ40160
128209
NM_173484
W


FLJ40172
285051
NM_173649
M


FLJ40311
124535
XM_064190
S


FLJ42953
400892
NM_207474
W


FLJ42957
400077
NM_207436
W


FLJ43965
389206
NM_207406
W


FLJ44290
375347
NM_198564
M


FLJ44313
400658
NM_207460
M


FLJ45121
400556
NM_207451
M


FLJ45803
399948
NM_207429
W


FLJ46354
374977
NM_198547
W


FLJ46365
401459
NM_207504
S


FLJ46481
389197
NM_207405
W


FOXB1
27023
NM_012182
W


FOXK2
3607
NM_004514
S


FOXP2
93986
NM_014491
W


FOXP4
116113
NM_138457
W


FRMPD1
22844
NM_014907
M


FRRS1
0
XM_372784
W


FSHPRH1
2491
NM_006733
W


FSIP1
161835
NM_152597
S


FTH1
2495
NM_002032
W


FXC1
26515
NM_012192
W


FXYD2
486
NM_001680
M


FYN
2534
NM_002037
W


GABRB1
2560
NM_000812
W


GAF1
26056
NM_015470
S


GART
2618
NM_000819
W


GBP1
2633
NM_002053
W


GBP5
115362
NM_052942
M


GCAT
23464
NM_014291
M


GDNF
2668
NM_000514
W


GGA1
26088
NM_001001560
M


GGA3
23163
NM_014001
M


GJB3
2707
NM_024009
W


GL004
56947
NM_020194
W


GLMN
11146
NM_053274
M


GLT1D1
144423
NM_144669
M


GMFG
9535
NM_004877
W


GNAQ
2776
NM_002072
W


GOLGA6
55889
NM_018652
W


GORASP1
64689
NM_031899
W


GOSR2
9570
NM_004287
S


GOT1
2805
NM_002079
W


GPD1
2819
NM_005276
M


GPD1L
23171
NM_015141
S


GPHA2
170589
NM_130769
M


GPKOW
27238
NM_015698
W


GPM6B
2824
NM_005278
W


GPR101
83550
NM_054021
W


GPR114
221188
NM_153837
W


GPR14
2837
NM_018949
M


GPR23
2846
NM_005296
M


GPR50
9248
NM_004224
W


GPR56
9289
NM_005682
W


GPR73L1
128674
NM_144733
W


GRB7
2886
NM_005310
W


GRID1
2894
XM_043613
W


GRID2
2895
NM_001510
W


GRK4
2868
NM_005307
M


GRK7
131890
NM_139209
W


GRSP1
23150
XM_114303
S


GSR
2936
NM_000637
M


GSTM2
2946
NM_000848
M


GTPBP1
9567
NM_004286
W


GUCA1B
2979
NM_002098
S


H2AFZ
3015
NM_002106
W


H6PD
9563
NM_004285
W


HBB
3043
NM_000518
S


HBE1
3046
NM_005330
W


HBXIP
10542
NM_006402
W


HCFC1
3054
NM_005334
W


HD
3064
NM_002111
W


HDAC3
8841
NM_003883
W


HEMGN
55363
NM_018437
W


HERV-FRD
405754
NM_207582
W


HES2
54626
XM_375684
S


HIST1H1B
3009
NM_005322
W


HIST1H2AL
8332
NM_003511
W


HIST1H3B
8358
NM_003537
W


HIST1H4A
8359
NM_003538
W


HMG4L
128872
NM_178467
M


HMP19
51617
NM_015980
M


HOXA7
3204
NM_006896
W


HOXB8
3218
NM_024016
W


HOXC8
3224
NM_022658
W


HOXD4
3233
NM_014621
M


HRASLS2
54979
NM_017878
W


HS747E2A
25770
NM_015370
W


HSCARG
57407
NM_020677
W


HSD11B2
3291
NM_000196
M


HSPB9
94086
NM_033194
W


HTR1A
3350
NM_000524
W


HUNK
30811
NM_014586
W


HXAL2
8692
NM_003773
W


HXAL4
23553
NM_012269
M


IDH3G
3421
NM_004135
W


IFRG15
64163
NM_022347
W


IGF1R
3480
NM_000875
S


IGSF8
93185
NM_052868
W


IL10RB
3588
NM_000628
W


IL15RA
3601
NM_002189
W


IL17
3605
NM_002190
W


IL17F
112744
NM_052872
W


IL1F9
56300
NM_019618
W


IL1RAPL1
11141
NM_014271
M


IL1RL1
9173
NM_003856
W


IL20RA
53832
NM_014432
M


IL22
50616
NM_020525
W


IL8RB
3579
NM_001557
W


IL9
3578
NM_000590
M


INM01
157695
NM_175075
S


INSIG1
3638
NM_005542
W


INSM1
3642
NM_002196
W


INTERSEX
55588
XM_290829
W


IRF7
3665
NM_001572
W


IRX1
79192
XM_380171
W


ITIH5
80760
NM_030569
M


ITSN2
50618
NM_006277
W


JARID1D
8284
NM_004653
M


JIK
51347
NM_016281
M


JM1
28952
NM_014008
W


JM11
90060
NM_033626
W


JMJD2B
23030
NM_015015
M


JPH2
57158
NM_020433
M


JUB
84962
NM_198086
W


KALRN
8997
NM_003947
W


KBTBD7
84078
NM_032138
W


KCNC4
3749
NM_004978
W


KCNH4
23415
NM_012285
W


KCNIP2
30819
NM_014591
M


KCNJ3
3760
NM_002239
W


KCNK9
51305
NM_016601
M


KCNN4
3783
NM_002250
S


KCTD14
65987
NM_023930
W


KDR
3791
NM_002253
W


KEAP1
9817
NM_012289
W


KIAA0217
23185
XM_040265
W


KIAA0284
283638
XM_208766
S


KIAA0303
23227
XM_291141
M


KIAA0527
26032
XM_171054
M


KIAA0540
23218
XM_291064
M


KIAA0542
9814
XM_038520
W


KIAA0701
23074
XM_045423
S


KIAA0841
23354
XM_049237
M


KIAA0980
22981
NM_025176
W


KIAA1012
22878
NM_014939
M


KIAA1068
23386
NM_015332
W


KIAA1189
57471
XM_371576
W


KIAA1194
57472
NM_015455
W


KIAA1280
55841
NM_015691
M


KIAA1361
57551
XM_290796
W


KIAA1510
57642
NM_020882
M


KIAA1549
57670
XM_371956
W


KIAA1573
57685
NM_020925
W


KIAA1726
85463
XM_370654
S


KIAA1862
84626
XM_044212
M


KIAA1971
123720
XM_058720
S


KIAA1987
170951
XM_375298
W


KIF11
3832
NM_004523
W


KIF13B
23303
NM_015254
M


KIR2DL4
3805
NM_002255
W


KLHL11
55175
NM_018143
M


KLRC3
3823
NM_002261
M


KPNB1
3837
NM_002265
S


KRTAP21-2
337978
NM_181617
M


KRTAP4-5
85289
NM_033188
M


KRTAP9-4
85280
NM_033191
W


KRTHA5
3886
NM_002280
M


L1TD1
54596
NM_019079
M


LACE1
246269
NM_145315
W


LAP1B
26092
NM_015602
M


LAP3
51056
NM_015907
W


LASP1
3927
NM_006148
W


LCN10
414332
NM_001001712
W


LEPRE1
64175
NM_022356
W


LGI2
55203
NM_018176
W


LIM
10611
NM_006457
W


LIMCH1
22998
XM_044461
M


LMAN1L
79748
NM_021819
S


LMF2
91289
NM_033200
W


LMNB1
4001
NM_005573
S


LMO7
4008
NM_005358
W


LMTK3
114783
XM_055866
W


LNX2
222484
NM_153371
W


LOC113828
113828
NM_138435
W


LOC116064
116064
XM_057296
M


LOC116068
116068
XM_371760
W


LOC120376
120376
XM_071712
W


LOC124402
124402
NM_145253
W


LOC125893
125893
XM_064856
W


LOC126520
126520
XM_59051
W


LOC131873
131873
XM_067585
M


LOC134145
134145
NM_199133
W


LOC144097
144097
NM_138471
W


LOC145414
0
XM_085138
W


LOC146443
146443
XM_378558
W


LOC146713
146713
XM_378712
W


LOC146795
146795
XM_378701
S


LOC146909
146909
XM_085634
M


LOC149643
0
XM_086616
W


LOC151484
151484
XM_379159
W


LOC152877
0
XM_094066
W


LOC153328
153328
NM_145282
W


LOC153441
153441
XM_087671
M


LOC154222
154222
XM_379456
W


LOC154907
0
XM_088072
W


LOC155036
155036
XM_376722
W


LOC158796
0
XM_088677
W


LOC159090
159090
NM_145284
W


LOC162427
162427
NM_178126
W


LOC163223
163223
NM_001001411
M


LOC164153
164153
NM_203412
M


LOC195977
195977
XM_113625
S


LOC196394
196394
NM_207337
M


LOC200493
0
XM_115715
W


LOC200933
200933
XM_117294
M


LOC201475
201475
XM_113967
S


LOC202051
202051
XM_114430
W


LOC205251
205251
NM_174925
W


LOC254808
254808
XM_374069
M


LOC254897
0
XM_170950
W


LOC254938
254938
XM_173120
M


LOC256085
256085
XM_172389
M


LOC283152
283152
XM_378314
W


LOC283677
283677
XM_208778
M


LOC283914
283914
XM_378589
M


LOC283989
283989
NM_207346
W


LOC284058
284058
NM_015443
W


LOC284361
284361
NM_175063
W


LOC284371
284371
XM_209155
M


LOC284390
284390
XM_371138
W


LOC284661
284661
XM_378832
W


LOC284739
284739
NM_207349
W


LOC284825
284825
XM_375935
W


LOC285194
285194
XM_379207
W


LOC285248
0
XM_211816
W


LOC285636
285636
NM_175921
M


LOC285671
285671
NM_178532
M


LOC286076
286076
XM_209889
S


LOC338734
0
XM_290547
W


LOC338750
338750
XM_291974
M


LOC338756
0
XM_291989
W


LOC338829
338829
XM_292122
M


LOC339951
339951
XM_293656
M


LOC340109
340109
XM_379322
W


LOC340318
340318
XM_290401
M


LOC340591
340591
XM_291346
M


LOC340765
340765
XM_291704
M


LOC341356
0
XM_292023
W


LOC343578
343578
XM_293123
S


LOC345643
345643
XM_293918
S


LOC345651
0
XM_293924
W


LOC345711
345711
XM_293937
M


LOC347454
347454
XM_293380
S


LOC375133
375133
NM_199345
W


LOC375295
375295
XM_374020
M


LOC386597
386597
XM_379073
W


LOC387761
387761
XM_373495
S


LOC387784
0
XM_373506
W


LOC387810
0
XM_373513
W


LOC387825
0
XM_370668
W


LOC387845
0
XM_370684
W


LOC387914
0
XM_370718
W


LOC388298
0
XM_370992
W


LOC388381
388381
XM_371053
M


LOC388418
388418
XM_373748
M


LOC388432
0
XM_371086
W


LOC388469
388469
XM_371111
M


LOC388585
0
XM_371215
W


LOC388697
0
XM_373868
W


LOC388807
388807
XM_373922
M


LOC388847
388847
XM_371424
M


LOC389000
0
XM_371534
W


LOC389067
0
XM_374021
W


LOC389070
0
XM_374022
W


LOC389102
0
XM_371623
W


LOC389107
389107
XM_371626
M


LOC389153
0
XM_374053
W


LOC389224
389224
XM_374086
S


LOC389273
0
XM_374115
W


LOC389319
389319
XM_374134
M


LOC389370
0
XM_374162
W


LOC389386
0
XM_371818
W


LOC389416
0
XM_371837
W


LOC389541
0
XM_371939
W


LOC389705
389705
XM_372076
M


LOC389727
0
XM_372092
W


LOC389753
0
XM_372112
W


LOC389950
0
XM_372307
W


LOC390377
0
XM_372486
W


LOC390530
390530
XM_372543
M


LOC390734
390734
XM_372640
M


LOC391209
0
XM_372840
W


LOC391426
391426
XM_372950
M


LOC392549
392549
XM_373373
M


LOC392702
0
XM_374730
W


LOC392726
0
XM_374734
W


LOC392791
0
XM_374752
W


LOC399786
0
XM_378236
W


LOC399920
0
XM_378300
W


LOC399959
399959
XM_378316
M


LOC399968
399968
XM_374945
M


LOC400047
400047
XM_378363
S


LOC400092
0
XM_378398
W


LOC400479
0
XM_375282
W


LOC400619
0
XM_378703
W


LOC400622
400622
XM_375491
M


LOC400687
400687
XM_375602
S


LOC400688
400688
XM_375603
M


LOC400740
0
XM_378840
W


LOC400877
400877
XM_379025
M


LOC400939
400939
XM_379072
S


LOC401155
401155
XM_379276
S


LOC401169
0
XM_379306
W


LOC401175
0
XM_379317
W


LOC401286
0
XM_376555
W


LOC401293
401293
XM_376558
M


LOC401314
0
XM_376586
W


LOC401316
0
XM_376587
W


LOC401317
0
XM_379479
W


LOC401321
0
XM_379483
W


LOC401322
401322
XM_376591
M


LOC401518
0
XM_379638
W


LOC401548
0
XM_376902
W


LOC401552
0
XM_379668
W


LOC401624
401624
XM_377073
M


LOC401720
401720
XM_377265
M


LOC401778
401778
XM_377343
M


LOC402148
0
XM_377818
W


LOC402251
402251
XM_377933
M


LOC402382
402382
XM_378090
S


LOC402477
0
XM_379803
W


LOC402515
0
XM_380112
W


LOC402521
0
XM_379848
W


LOC402537
0
XM_380120
W


LOC402556
0
XM_379877
W


LOC402560
0
XM_380127
W


LOC402586
0
XM_380138
W


LOC402587
0
XM_380139
W


LOC402625
0
XM_379975
W


LOC402641
0
XM_379995
W


LOC404785
404785
NM_207513
W


LOC51054
51054
NM_015899
W


LOC51066
51066
NM_015931
W


LOC51333
51333
NM_016643
W


LOC51693
51693
NM_016209
W


LOC57168
57168
NM_020437
M


LOC88523
88523
NM_033111
S


LOC90120
90120
XM_379680
M


LOC92689
92689
NM_138389
W


LOC96597
96597
XM_378655
W


LOR
4014
NM_000427
W


LPO
4025
NM_006151
W


LTBP3
4054
NM_021070
W


LU
4059
NM_005581
W


LXNX1
66004
NM_177477
M


LXZL1
84569
NM_032517
M


LY64
4064
NM_005582
W


M96
22823
NM_007358
W


MAD2L2
10459
NM_006341
W


MAGEL2
54551
NM_019066
W


MAP4
4134
NM_002375
M


MAPBPIP
28956
NM_014017
S


MAPK13
5603
NM_002754
W


MAPRE2
10982
NM_014268
W


MASP1
5648
NM_001879
W


MBP
4155
NM_002385
M


MBTPS2
51360
NM_015884
W


MC4R
4160
NM_005912
W


MCC
4163
NM_002387
W


MCRS1
10445
NM_006337
W


MDGA1
266727
NM_153487
W


MDH1B
130752
XM_059468
W


MDS1
4197
NM_004991
S


MED19
219541
NM_153450
M


MEF2A
4205
NM_005587
W


MEF2B
4207
NM_005919
W


MET
4233
NM_000245
M


MFSD11
79157
NM_024311
W


MFSD3
113655
NM_138431
S


MGAT4B
11282
NM_014275
M


MGC11266
79172
NM_024322
M


MGC14126
84984
NM_032898
W


MGC15882
84970
NM_032884
M


MGC16279
85002
NM_032916
M


MGC16372
92749
NM_145038
W


MGC16491
115572
NM_052943
M


MGC16597
339230
XM_375500
W


MGC17337
91283
NM_080655
W


MGC21394
404203
NM_205841
W


MGC23918
151903
NM_144716
W


MGC23937
139596
NM_145052
W


MGC26856
256710
NM_152779
W


MGC2941
79142
NM_024297
M


MGC33584
285971
NM_173680
M


MGC33887
201134
NM_145036
M


MGC39633
153733
NM_152549
W


MGC39696
255193
NM_152771
M


MGC41945
138724
NM_203299
W


MGC4238
84292
NM_032332
W


MGC4734
138065
NM_145051
M


MGC50559
254013
NM_173802
W


MGC52000
375260
NM_198943
W


MGC87042
256227
NM_207342
M


MICAL3
57553
XM_032997
W


MIG12
58526
NM_021242
W


MIRAB13
85377
NM_033386
W


MLL
4297
NM_005933
M


MLL4
9757
NM_014727
M


MLR2
84458
XM_050988
M


MLSTD1
55711
NM_018099
M


MMP24
10893
NM_006690
W


MO25
51719
NM_016289
W


MORF4L1
10933
NM_006791
W


MRC2
9902
NM_006039
W


MRPL48
51642
NM_016055
W


MRPS21
54460
NM_018997
W


MRPS6
64968
NM_032476
M


MRS2L
57380
NM_020662
M


MSL3L1
10943
NM_078628
S


MT1A
4489
NM_005946
W


MTFMT
123263
NM_139242
W


MTMR6
9107
NM_004685
S


MTMR9
66036
NM_015458
W


MTRF1L
54516
NM_019041
W


MYADM
91663
NM_138373
M


MYBL2
4605
NM_002466
M


MYC
4609
NM_002467
M


MYH1
4619
NM_005963
W


MYLIP
29116
NM_013262
W


MYO5C
55930
NM_018728
W


MYO9A
4649
NM_006901
M


MYST3
7994
NM_006766
W


NAP1L4
4676
NM_005969
W


NAPA
8775
NM_003827
M


NAPG
8774
NM_003826
W


NBPF10
388776
XM_371384
M


NCB5OR
51167
NM_016230
W


NCBP2
22916
NM_007362
M


NCF4
4689
NM_000631
W


NCOA5
57727
NM_020967
W


NDEL1
81565
NM_030808
W


NDRG1
10397
NM_006096
M


NDUFA5
4698
NM_005000
M


NDUFB9
4715
NM_005005
W


NDUFC1
4717
NM_002494
W


NDUFS1
4719
NM_005006
W


NDUFS6
4726
NM_004553
W


NEBL
10529
NM_006393
M


NET-5
10867
NM_006675
W


NEU4
129807
NM_080741
W


NEURL
9148
NM_004210
S


NFATC2
4773
NM_012340
W


NFKB2
4791
NM_002502
W


NFS1
9054
NM_021100
W


NHLH2
4808
NM_005599
W


NIPA
51530
NM_016478
S


NIPA2
81614
NM_030922
S


NOLC1
9221
NM_004741
M


NOPE
57722
NM_020962
W


NPEPPS
9520
NM_006310
M


NPFF
8620
NM_003717
W


NPY2R
4887
NM_000910
W


NPY5R
4889
NM_006174
W


NRAS
4893
NM_002524
M


NRM
11270
NM_007243
W


NUCB1
4924
NM_006184
W


NUP107
57122
NM_020401
W


NUP133
55746
NM_018230
W


NUP160
23279
XM_113678
S


NUP205
23165
XM_058073
M


NUP54
53371
NM_017426
M


NUP62
23636
NM_012346
S


NUP93
9688
NM_014669
M


NUPL1
9818
NM_014089
M


NXD-TSP1
84654
NM_032567
M


NXF2
56001
NM_017809
W


NXF5
55998
NM_032946
W


NXT1
29107
NM_013248
W


NYD-SP28
85478
NM_033124
W


OFD1
8481
NM_003611
W


OKL38
29948
NM_013370
M


OPN3
23596
NM_014322
W


OR2A2
442361
NM_001005480
M


OR2B3
442184
NM_001005226
S


OR3A4
390756
NM_001005334
W


OR4A5
81318
NM_001005272
W


OR4K15
81127
NM_001005486
M


OR5B2
390190
NM_001005566
W


OR5K4
403278
NM_001005517
S


OR5M11
219487
NM_001005245
W


OR6C74
254783
NM_001005490
W


Orai1
84876
NM_032790
M


ORC3L
23595
NM_012381
W


OSM
5008
NM_020530
M


OSTM1
28962
NM_014028
S


OTOR
56914
NM_020157
W


P4HA2
8974
NM_004199
S


PADI3
51702
NM_016233
W


PAGE-5
90737
NM_130467
W


PAI-RBP1
26135
NM_015640
W


PAK1IP1
55003
NM_017906
W


PAQR10
221938
NM_198403
W


PASD1
139135
NM_173493
M


PAWR
5074
NM_002583
W


PAX2
5076
NM_000278
M


PCBP1
5093
NM_006196
W


PCDH11X
83259
NM_032971
M


PCDH11X
27328
NM_014522
S


PCDHB13
56123
NM_018933
M


PCDHGB7
56099
NM_018927
S


PCNP
57092
NM_020357
W


PCOLCE
5118
NM_002593
W


PCOLN3
5119
NM_002768
S


PDCD1LG2
80380
NM_025239
S


PDE6A
5145
NM_000440
W


PDF
64146
NM_022341
W


PDHA2
5161
NM_005390
W


PDP2
57546
NM_020786
W


PELO
53918
NM_015946
S


PEPP3
22874
NM_014935
M


PERLD1
93210
NM_033419
W


PEX11A
8800
NM_003847
S


PEX11G
92960
NM_080662
M


PEX26
55670
NM_017929
W


PEX3
8504
NM_003630
M


PFKFB3
5209
NM_004566
W


PHF13
148479
NM_153812
W


PHF17
79960
NM_024900
M


PHF2
5253
NM_005392
M


PHYHIPL
84457
NM_032439
W


PIGW
284098
NM_178517
W


PIK3CB
5291
NM_006219
S


PIK3R2
5296
NM_005027
S


PIK3R3
8503
NM_003629
W


PIK4CA
5297
NM_002650
M


PIK4CB
5298
NM_002651
W


PILRA
29992
NM_013439
S


PIPOX
51268
NM_016518
W


PIPPIN
27254
NM_014460
S


PJA1
64219
NM_022368
M


PKD1L1
168507
NM_138295
W


PLA2G4D
283748
NM_178034
M


PLAC8
51316
NM_016619
M


PMCA4
493
NM_001684
W


PMCH
5367
NM_002674
M


PNLIP
5406
NM_000936
W


PNLIPRP1
5407
NM_006229
W


PNUTL2
5414
NM_004574
S


POLG
5428
NM_002693
M


POLH
5429
NM_006502
M


PON3
5446
NM_000940
M


PPP1R13B
23368
NM_015316
M


PPP1R9B
84687
NM_032595
M


PPP3CA
5530
NM_000944
S


PPP3R1
5534
NM_000945
M


PRDX3
10935
NM_006793
W


PRKACA
5566
NM_002730
W


PRKWNK2
65268
NM_006648
M


PROK1
84432
NM_032414
W


PROL5
26952
NM_012390
W


PRPS1L1
221823
NM_175886
W


PRPSAP2
5636
NM_002767
S


PRRT1
80863
NM_030651
M


PRSS1
5644
NM_002769
M


PSG3
5671
NM_021016
W


PTD004
29789
NM_013341
W


PTD008
51398
NM_016145
M


PTPN13
5783
NM_006264
M


PTRH1
138428
XM_059972
W


PTX1
51290
NM_016570
W


PXGO1
26108
NM_015617
M


PXK
54899
NM_017771
M


QP-C
27089
NM_014402
S


RABGAP1
23637
NM_012197
W


RABGGTB
5876
NM_004582
S


RABL2A
11159
NM_007082
W


RAD9B
144715
NM_152442
S


RAI14
26064
NM_015577
S


RAN
5901
NM_006325
S


RANBP2
5903
NM_006267
W


RANBP2L1
84220
NM_005054
M


RAP1GA1
5909
NM_002885
M


RASD2
23551
NM_014310
W


RASL10B
91608
NM_033315
W


RBM27
54439
XM_291128
W


RBM5
10181
NM_005778
W


RCE1
9986
NM_005133
W


RCOR1
23186
NM_015156
M


RDH12
145226
NM_152443
W


RDH5
5959
NM_002905
W


REPIN1
29803
NM_013400
W


REV3L
5980
NM_002912
M


RFPL3
10738
NM_006604
W


RGS7
6000
NM_002924
M


RIOK3
8780
NM_003831
M


RKHD2
51320
NM_016626
M


RLN3
117579
NM_080864
M


RNF13
11342
NM_007282
W


RNF159
84333
NM_032373
M


RNF185
91445
NM_152267
M


RNF32
140545
NM_030936
M


RNPEPL1
57140
NM_018226
M


RORB
6096
NM_006914
W


RP26
375298
NM_201548
W


RPGR
6103
NM_000328
S


RPIB9
154661
NM_138290
W


RPL3L
6123
NM_005061
W


RPS6KA2
6196
NM_021135
W


RRAS2
22800
NM_012250
M


RRH
10692
NM_006583
W


RRM2
6241
NM_001034
M


RX1
11017
NM_006857
S


SAA2
6289
NM_030754
W


SALPR
51289
NM_016568
W


SAST
22983
NM_014975
M


SATB1
6304
NM_002971
W


SCA7
6314
NM_000333
W


SCFD1
23256
NM_016106
M


SCG3
29106
NM_013243
W


SCML1
6322
NM_006746
M


SEC13L1
6396
NM_030673
S


SEC22L1
9554
NM_004892
W


SECISBP2
79048
NM_024077
W


SELENBP1
8991
NM_003944
S


SENP1
29843
NM_014554
W


SENP6
26054
NM_015571
S


SENP7
57337
NM_020654
W


SENP8
123228
NM_145204
W


SERPINA12
145264
NM_173850
M


SERPINA9
327657
NM_175739
M


SERPINB1
1992
NM_030666
M


SERPINE1
5054
NM_000602
M


SEZ6L
23544
NM_021115
W


SF4
57794
NM_172231
W


SFPQ
6421
NM_005066
M


SFRS2
6427
NM_003016
M


SFRS3
6428
NM_003017
S


SFRS7
6432
NM_006276
W


SFT2D1
113402
NM_145169
S


SFXN 5
94097
NM_144579
M


SHD
56961
NM_020209
W


SIAHBP1
22827
NM_014281
M


SIGLEC8
27181
NM_014442
S


SIRT7
51547
NM_016538
S


SKIP
51763
NM_016532
S


SLAMF6
114836
NM_052931
W


SLC12A9
56996
NM_020246
W


SLC22A1LS
5003
NM_007105
W


SLC25A23
79085
NM_024103
W


SLC25A3
5250
NM_002635
M


SLC30A5
64924
NM_022902
S


SLC36A1
206358
NM_078483
W


SLC38A6
145389
NM_153811
M


SLC41A3
54946
NM_017836
M


SLC6A14
11254
NM_007231
W


SLC6A2
6530
NM_001043
W


SLC6A4
6532
NM_001045
W


SLCO2B1
11309
NM_007256
S


SMAD2
4087
NM_005901
M


SMARCD3
6604
NM_003078
M


SMC5L1
23137
NM_015110
M


SMG1
23049
NM_014006
W


SMOC2
64094
NM_022138
W


SNAP23
8773
NM_003825
W


SNRP70
6625
NM_003089
M


SNRPC
6631
NM_003093
S


SNX27
81609
NM_030918
W


SOX8
30812
NM_014587
S


SP4
6671
NM_003112
W


SPINK4
27290
NM_014471
W


SPINT1
6692
NM_003710
W


SPTLC2
9517
NM_004863
S


SR140
23350
XM_031553
W


SREBF1
6720
NM_004176
M


SRP46
10929
NM_032102
M


SSA2
6738
NM_004600
W


SSTR2
6752
NM_001050
W


SSTR4
6754
NM_001052
W


SSX1
6756
NM_005635
W


STAM
8027
NM_003473
M


STAMBPL1
57559
NM_020799
W


STEAP
26872
NM_012449
W


STIM1
6786
NM_003156
S


STIM2
57620
NM_020860
M


STOML1
9399
NM_004809
W


STOML2
30968
NM_013442
W


STX18
53407
NM_016930
M


STXBP2
6813
NM_006949
M


SULT1C2
27233
NM_006588
W


SUMO2
6613
NM_006937
W


SV2C
22987
XM_043493
S


SYCP1
6847
NM_003176
W


SYNCRIP
10492
NM_006372
W


SYNE2
23224
NM_015180
W


SYT15
83849
NM_181519
S


SYTL4
94121
NM_080737
W


T2BP
92610
NM_052864
M


TAB3
257397
NM_152787
W


TAS2R45
259291
NM_176886
W


TBCC
6903
NM_003192
W


TBK1
29110
NM_013254
M


TCEB2
6923
NM_007108
S


TDRKH
11022
NM_006862
M


TESK2
10420
NM_007170
W


TEX13B
56156
NM_031273
W


TEX14
56155
NM_031272
M


TFCP2L3
79977
NM_024915
M


TFPI2
7980
NM_006528
S


TGIF2LX
90655
NM_139214
W


TGM3
7053
NM_003245
W


THG-1
81628
NM_030935
M


TIMELESS
8914
NM_003920
W


TIPARP
25976
NM_015508
M


TLR6
10333
NM_006068
M


TMEM11
8834
NM_003876
W


TMEM110
375346
NM_198563
S


TMEM14C
51522
NM_016462
W


TMEM187
8269
NM_003492
W


TMEM199
147007
NM_152464
W


TMEM43
79188
NM_024334
S


TMEM63B
55362
XM_371822
W


TMP21
10972
NM_006827
M


TNFRSF13C
115650
NM_052945
W


TNFRSF18
8784
NM_004195
M


TNFSF13B
10673
NM_006573
W


TNIK
23043
XM_039796
S


TNKS1BP1
85456
NM_033396
W


TNXB
7148
NM_019105
W


TOE1
114034
NM_025077
M


TOLLIP
54472
NM_019009
W


TOR1AIP2
163590
NM_145034
W


TORC3
64784
NM_022769
W


TPTE
7179
NM_013315
M


TRAPPC1
58485
NM_021210
M


TRIM22
10346
NM_006074
W


TRIM3
10612
NM_006458
M


TRIM33
51592
NM_015906
W


TRIM59
286827
NM_173084
M


TRNT1
51095
NM_016000
M


TROAP
10024
NM_005480
S


TSPAN-1
10103
NM_005727
W


TULP1
7287
NM_003322
W


TULP4
56995
NM_020245
W


TXK
7294
NM_003328
M


TXMS
7298
NM_001071
W


TXNDC
81542
NM_030755
W


TXNDC15
79770
NM_024715
S


TXNRD2
10587
NM_006440
W


UAP1
6675
NM_003115
W


UBAP1
51271
NM_016525
M


UBC
7316
NM_021009
M


UBE2L6
9246
NM_004223
W


UEV3
55293
NM_018314
S


UFM1
51569
NM_016617
W


UHSKERB
57830
NM_021046
S


UMPS
7372
NM_000373
M


UNQ2446
123904
NM_198443
W


UNQ2492
377841
NM_198585
W


UNQ3033
284415
NM_198481
W


UNQ9370
400454
NM_207447
W


UPF3B
65109
NM_023010
W


USP13
8975
NM_003940
M


VAX2
25806
NM_012476
M


VCX2
51480
NM_016378
M


VEST1
116328
NM_052958
W


VGF
7425
NM_003378
M


VGLL2
245806
NM_153453
W


VMP
140767
NM_080723
S


VN1R4
317703
NM_173857
W


VPS13A
23230
NM_015186
W


VPS28
51160
NM_016208
M


VPS35
55737
NM_018206
W


WARP
64856
NM_022834
W


WFDC3
140686
NM_181522
W


WNT7B
7477
NM_058238
W


WRNIP1
56897
NM_020135
S


XKR5
389610
NM_207411
M


XKRX2
353515
NM_001002906
M


XKRY
9082
NM_004677
S


XPO7
23039
NM_015024
M


XYLT2
64132
NM_022167
W


YIPF3
25844
NM_015388
M


ZADH1
145482
NM_152444
M


ZAK
51776
NM_133646
W


ZBTB2
57621
NM_020861
W


ZBTB7
51341
NM_015898
W


ZCCHC8
55596
NM_017612
W


ZDHHC2
51201
NM_016353
M


ZFP28
140612
NM_020828
W


ZFP67
51043
NM_015872
W


ZFR
51663
NM_016107
W


ZNF143
7702
NM_003442
M


ZNF148
7707
NM_021964
S


ZNF157
7712
NM_003446
M


ZNF192
7745
NM_006298
W


ZNF267
10308
NM_003414
M


ZNF289
84364
NM_032389
S


ZNF295
49854
NM_020727
W


ZNF297B
23099
NM_014007
W


ZNF304
57343
NM_020657
W


ZNF324
25799
NM_014347
W


ZNF334
55713
NM_018102
W


ZNF342
162979
NM_145288
S


ZNF354C
30832
NM_014594
W


ZNF496
84838
NM_032752
W


ZNF501
115560
NM_145044
W


ZNF503
84858
NM_032772
W


ZNF512
84450
NM_032434
M


ZNF544
27300
NM_014480
W


ZNF568
374900
NM_198539
M


ZNF570
148268
NM_144694
W


ZNF615
284370
NM_198480
W


ZNF706
51123
NM_016096
M


ZNFN1A4
64375
NM_022465
M


ZNRD1
30834
NM_014596
W


ZSWIM1
90204
NM_080603
M


ZYX
7791
NM_003461
M


ZZEF1
23140
NM_015113
S


ZZZ3
26009
NM_015534
M




















TABLE 3





GeneSymbol
EntrezGeneID
Genbank Acc. No.
NFAT Score
Calcium Hit



















ABCC13
150000
NM_138726
M



ACSBG1
23205
NM_015162
S
X


ACTB
60
NM_001101
M
X


ADAMTS5
11096
NM_007038
M



AFG3L1
172
NM_001132
M
X


AKR1CL2
83592
NM_031436
M



ALCAM
214
NM_001627
M
X


ANKRD58
347454
XM_293380
S



ANKRD9
122416
NM_152326
M



ANTXRL
195977
XM_113625
S



APH1A
51107
NM_016022
M



APOL4
80832
NM_030643
M



ARCN1
372
NM_001655
S



ARL5C
390790
XM_372668
M
X


AS3MT
57412
NM_020682
S



ASB4
51666
NM_016116
S



ASPHD2
57168
NM_020437
M



ATN1
1822
NM_001940
M
X


ATP5L2
267020
NM_198822
M
X


ATP6V0D1
9114
NM_004691
M
X


ATP6V1D
51382
NM_015994
M



BCDIN3
56257
NM_019606
M



BGN
633
NM_001711
M



BMP4
652
NM_001202
S



BREA2
286076
XM_209889
S



BRP44L
51660
NM_016098
M



C10orf53
282966
NM_182554
M



C10orf56
219654
NM_153367
M



C12orf49
79794
NM_024738
S



C19orf34
255193
NM_152771
M



C19orf56
51398
NM_016145
M



C1orf123
54987
NM_017887
M
X


C1orf77
26097
NM_015607
M



C20orf95
343578
XM_293123
S



C20orf96
140680
NM_153269
M
X


C21orf49
54067
NM_001006116
S



C4orf27
54969
NM_017867
S



C4orf30
54876
NM_017741
M



C5orf14
79770
NM_024715
S



C6orf115
58527
XM_371848
M



C6orf191
253582
XM_173166
S
X


C8orf42
157695
NM_175075
S
X


C9orf11
54586
XM_035953
M



C9orf138
158297
NM_153707
M



C9orf71
169693
XM_376874
M



C9orf72
203228
NM_018325
S



CA5BL
340591
XM_291346
M



CASC1
55259
NM_018272
S



CBLL1
79872
NM_024814
S



CCDC11
220136
NM_145020
M



CCDC125
202243
NM_176816
M
X


CCDC46
201134
NM_145036
M



CCDC49
54883
NM_017748
M



CCDC50
152137
NM_174908
M



CCDC85B
11007
NM_006848
S



CCK
885
NM_000729
M



CCL11
6356
NM_002986
M



CCNB2
9133
NM_004701
S
X


CCNK
8812
NM_003858
M



CDC27
996
NM_001256
S



CDC2L5
8621
NM_003718
M



CENPO
79172
NM_024322
M



CHMP1A
5119
NM_002768
S



CHST14
113189
NM_130468
S



CIRBP
1153
NM_001280
M



CLDN22
53842
XM_210581
S



CLEC4M
10332
NM_014257
M



CLPS
1208
NM_001832
M



CMAS
55907
NM_018686
M



CNTN3
5067
XM_039627
M
X


COL20A1
57642
NM_020882
M



COPA
1314
NM_004371
S
X


COPB1
1315
NM_016451
S
X


COPB2
9276
NM_004766
S
X


COPE
11316
NM_007263
S
X


COPG
22820
NM_016128
S
X


COPZ1
22818
NM_016057
S
X


CPEB4
80315
NM_030627
S
X


CPT2
1376
NM_000098
M
X


CRLF3
51379
NM_015986
M



CYP2S1
29785
NM_030622
S



DDX53
168400
NM_182699
M



DENND1C
79958
NM_024898
M



DGCR6L
85359
NM_033257
M



DHRS4
10901
NM_021004
M



DHRS4L2
317749
NM_198083
M



DHRS9
10170
NM_005771
M



DIABLO
56616
NM_019887
S



DISP2
85455
NM_033510
M



DKFZP686A01247
22998
XM_044461
M
X


DNAJC5G
285126
NM_173650
M
X


DONSON
29980
NM_145794
M



DSEL
92126
NM_032160
M



DSG4
147409
NM_177986
M



DUSP12
11266
NM_007240
M



EHD2
30846
NM_014601
M



ELMOD1
55531
NM_018712
S
X


EPO
2056
NM_000799
M



ERBB4
2066
NM_005235
M



F11R
50848
NM_016946
M



FAM105A
54491
NM_019018
M



FAM108C1
58489
XM_051862
S
X


FAM46B
115572
NM_052943
M



FAS
355
NM_000043
S
X


FASTKD5
60493
NM_021826
M
X


FBXO11
80204
NM_012167
M



FBXO45
200933
XM_117294
M



FBXO5
26271
NM012177
S
X


FLJ21986
79974
NM_024913
M
X


FLJ 30698
400687
XM_375602
S
X


FLJ 36070
284358
NM_182574
S



FLJ40172
285051
NM_173649
M



FLJ41047
399968
XM_374945
M



FLJ44290
375347
NM_198564
M



FLJ44313
400658
NM_207460
M



FLJ45121
400556
NM_207451
M



FLJ46365
401459
NM_207504
S



FRMD4B
23150
XM_114303
S



FRMPD1
22844
NM_014907
M
X


FSIP1
161835
NM_152597
S



FXYD2
486
NM_001680
M



GBP5
115362
NM_052942
M



GGA1
26088
NM_001001560
M



GGA3
23163
NM_014001
M
X


GLMN
11146
NM_053274
M



GLT1D1
144423
NM_144669
M
X


GOSR2
9570
NM_004287
S
X


GPD1
2819
NM_005276
M
X


GPD1L
23171
NM_015141
S
X


GPR23
2846
NM_005296
M
X


GRK4
2868
NM_005307
W



GSR
2936
NM_000637
M



GSTM2
2946
NM_000848
M
X


GUCA1B
2979
NM_002098
S



HBB
3043
NM_000518
S



HDHD2
84064
NM_032124
M



HSD11B2
3291
NM_000196
M



HYAL4
23553
NM_012269
M



ICA1L
130026
NM_138468
M



IGF1R
3480
NM_145574, NM_000875
S



IL20RA
53832
NM_014432
M



IL9
3578
NM_000590
M
X


ITIH5
80760
NM_030569
M



JPH2
57158
NM_020433
M
X


KCNIP2
30819
NM_014591
M
X


KCNK9
51305
NM_016601
M



KCNN4
3783
NM_002250
S
X


KIAA0284
283638
XM_208766
S
X


KIF13B
23303
NM_015254
M



KLHL11
55175
NM_018143
M



KLRC3
3823
NM_002261
M



KRBA1
84626
XM_044212
M



KRT35
3886
NM_002280
M
X


KRTAP21-2
337978
NM_181617
M
X


KRTAP5-8
57830
NM_021046
S
X


L1TD1
54596
NM_019079
M
X


LASP1
3927
NM_006148
W



LMAN1L
79748
NM_021819
S
X


LMNB1
4001
NM_005573
S
X


LOC131873
131873
XM_067585
M



LOC146795
146795
XM_378701
S



LOC153441
153441
XM_087671
M



LOC254938
254938
XM_173120
M



LOC283914
283914
XM_378589
M
X


LOC284371
284371
XM_209155
M



LOC285636
285636
NM_175921
M



LOC338750
338750
XM_291974
M



LOC338829
338829
XM_292122
M
X


LOC340318
340318
XM 290401
M



LOC340765
340765
XM_291704
M



LOC345643
345643
XM_293918
S



LOC345711
345711
XM_293937
M
X


LOC375295
375295
XM_374020
M



LOC387761
387761
XM_373495
S



LOC388381
388381
XM_371053
M
X


LOC388418
388418
XM_373748
M



LOC388469
388469
XM_371111
M
X


LOC388776
388776
XM_371384
M



LOC388807
388807
XM_373922
M
X


LOC389107
389107
XM_371626
M
X


LOC389224
389224
XM_374086
S



LOC389319
389319
XM_374134
M
X


LOC390734
390734
XM_372640
M



LOC391426
391426
XM_372950
M
X


LOC392549
392549
XM_373373
M
X


LOC399959
399959
XM_378316
M



LOC400622
400622
XM_375491
M



LOC400688
400688
XM_375603
M



LOC400877
400877
XM_379025
M
X


LOC400939
400939
XM_379072
S
X


LOC401155
401155
XM_379276
S
X


LOC401293
401293
XM_376558
M



LOC401322
401322
XM_376591
M
X


LOC401624
401624
XM_377073
M
X


LOC401720
401720
XM_377265
M



LOC401778
401778
XM_377343
M
X


LOC402251
402251
XM_377933
M



LOC402382
402382
XM_378090
S



LOC90120
90120
XM_379680
M



LRRC58
116064
XM_057296
M



LSM12
124801
NM_152344
S



LSM14A
26065
NM_015578
S



LYN X1
66004
NM_177477
M
X


LYZL1
84569
NM_032517
M
X


MAP4
4134
NM_002375
M



MAPBP IP
28956
NM_014017
S



MAST4
23227
XM_291141
M



MBP
4155
NM_002385
M



MED19
219541
NM_153450
M
X


M ED28
80306
NM_025205
S



MGAT4B
11282
NM_014275
M



MGC34829
284069
XM_208993
S
X


MGC87042
256227
NM_207342
M



MICAL3
57553
XM_032997
W



MLSTD1
55711
NM_018099
M



MRPS6
64968
NM_032476
M



MRS2L
57380
NM_020662
M
X


MTMR6
9107
NM_004685
S



MYADM
91663
NM_138373
M



MYO9A
4649
NM_006901
M
X


NAPA
8775
NM_003827
M
X


NAPG
8774
NM_003826




NDRG1
10397
NM_006096
M



NDUFA5
4698
NM_005000
M
X


NEBL
10529
NM_006393
W



NEURL
9148
NM_004210
S



NIPA2
81614
NM_030922
S
X


NRAS
4893
NM_002524
M



NRSN1
140767
NM_080723
S



OR2A2
442361
NM_001005480 
M



OR2B3
442184
NM_001005226
S



OR4K15
81127
NM_001005486 
M



OR5K4
403278
NM_001005517 
S



OSM
5008
NM_020530
M



OSTM1
28962
NM_014028
S
X


P4 HA2
8974
NM_004199
S



PASD1
139135
NM_173493
M
X


PCDH11X
27328
NM_014522
S



PCDH11Y
83259
NM_032971
M



PCDHB13
56123
NM_018933
M



PCDHGB7
56099
NM_018927
S



PDCD1LG2
80380
NM_025239
S



PELO
53918
NM_015946
S



PEX11A
8800
NM_003847
S



PEX3
8504
NM_003630
M



PHF23
79142
NM_024297
M



PIK3R2
5296
NM_005027
S



PIK4CA
5297
NM_002650
M
X


PILRA
29992
NM_013439
S
X


PJA1
64219
NM_022368
M
X


PLA2G4D
283748
NM_178034
M



PLEKHA6
22874
NM_014935
M



PMCH
5367
NM_002674
M



POLG
5428
NM_002693
M



POMP
51371
NM_015932
M



PPP1R9B
84687
NM_032595
M



PPP3CA
5530
NM_000944
S



PPP3R1
5534
NM_000945
M



PRRT1
80863
NM_030651
M
X


PRSS1
5644
NM_002769
M
X


PTPN13
5783
NM_006264
M



RAB11FIP5
26056
NM_015470
S



RAB12
201475
XM_113967
S



RABGGTB
5876
NM_004582
S



RAD9B
144715
NM_152442
S
X


RAI14
26064
NM_015577
S



RAP1GAP
5909
NM_002885
M



REV3L
5980
NM_002912
M



RGS7
6000
NM_002924
M



RIOK3
8780
NM_003831
M



RLN3
117579
NM_080864
M



RNF180
285671
NM_178532
M



RNF185
91445
NM_152267
M
X


RNF32
140545
NM_030936
M



RNPEPL1
57140
NM_018226
M
X


RP11-298P3.3
88523
NM_033111
S



RPGR
6103
NM_000328
S
X


RY1
11017
NM_006857
S



SEC13
6396
NM_030673
S



SELENBP1
8991
NM_003944
S



SENP6
26054
NM_015571
S



SEPT4
5414
NM_004574
S
X


SERPINA12
145264
NM_173850
M



SERPINA9
327657
NM_175739
M



SERPINB1
1992
NM_030666
M



SERPINE1
5054
NM_000602
M



SFXN5
94097
NM_144579
M
X


SIGLEC8
27181
NM_014442
S



SLC25A3
5250
NM_002635
M



SLC30A5
64924
NM_022902
S



SLC38A6
145389
NM_153811
M



SLC41A3
54946
NM_017836
M
X


SPTLC2
9517
NM_004863
S
X


STAM
8027
NM_003473
M
X


STIM1
6786
NM_003156
S
X


STIM2
57620
NM_020860
M
X


STX18
53407
NM_016930
M



STXBP2
6813
NM_006949
M
X


SUSD5
26032
XM_171054
M



SV2C
22987
XM_043493
S



SYT15
83849
NM_181519
S



TBK1
29110
NM_013254
M



TDRKH
11022
NM_006862
M



TEX14
56155
NM_031272
M



TFPI2
7980
NM_006528
S



TIFA
92610
NM_052864
M



TLR6
10333
NM_006068
M



TMED10
10972
NM_006827
M
X


TMEM110
375346
NM_198563
S
X


TMEM142A
84876
NM_032790
M
X


TMEM43
79188
NM_024334
S



TNFRSF18
8784
NM_004195
M
X


TNIK
23043
XM_039796
S



TOLLIP
54472
NM_019009
W



TOR1AIP1
26092
NM_015602
M



TPTE
7179
NM_013315
M



TRIM59
286827
NM_173084
M
X


TRNT1
51095
NM_016000
M



TROAP
10024
NM_005480
S



TUG1
55000
NM_017903
M



TXNDC10
54495
NM_019022
S



UBAP1
51271
NM_016525
M



UBC
7316
NM_021009
M
X


UBL4B
164153
NM_203412
M



UBL7
84993
NM_032907
S



UEVLD
55293
NM_018314
S
X


UQCRQ
27089
NM_ 014402 
S



USP13
8975
NM_003940
M



VGF
7425
NM_003378
M



VPS28
51160
NM_016208
M



WDR81
124997
NM_152348
M



WHDC1
123720
XM_058720
S



WNK2
65268
NM_006648
M



XKR5
389610
NM_207411
M
X


XKRY
9082
NM_004677
S



XKRY2
353515
NM_001002906
M



YARS2
51067
NM_015936
S



ZC3H12C
85463
XM_370654
S



ZDHHC2
51201
NM_016353
M



ZNF289
84364
NM_032389
S
X


ZNF706
51123
NM_016096
M
X


ZYX
7791
NM_003461
M



ZZEF1
23140
NM_015113
S
X


ZZZ3
26009
NM_015534
M





















TABLE 4







ACSBG 1
DNAJC5G
IL9
MRS2L
RPGR
UEVLD


ACTB
ELMOD1
JPH2
MYO9A
SEPT4/PNUTL2
XKR5


ALCAM
FAM108C1 
KCNIP2
NAPA
SFXN5
ZNF289


ATN1
FAS
KCNN4
NDUFA5
SLC41A3
ZNF706


ATPVOD1
FASTKD5
KIAA0284
NIPA2
SPTLC2
ZZEF1


C1ORF123
FBXO5
KRT35
OSTM 1
STAM



C20ORF96
FLJ21986
KRTAP21-2
PASD1
STIM1



C6ORF191
FRMPD1
KRTAP5-8
PI K4CA
STIM2



C80RF42
GGA3
L1TD1
PI LRA
STXBP2



CCDC125
GLT1D1
LMAN1L
PJA1
TMED10



CCNB2
GOSR2
LMNB1
PRRT1
TMEM110



CNTN3
GPD1
LOC338829
PRSS1
TMEM142A



CPEB4
GPD1L
LOC388381
RAD9B
TNFRSF18



CPT2
GPR23
LYZL1
RNF185
TRIM59



DKFZP686A01247
GSTM2
MGC34829
RNPEPLI
UBC


















TABLE 5





Gene Name/Gene Symbol
Description
GeneID

















TRIM59
tripartite motif-containing 59
286827


SPTLC2
serine palmitoyltransferase, long chain base subunit 2
9517


PRRT1 = C6ORF31
proline-rich transmembrane protein 1
80863


TMEM110 = MGC52022
transmembrane protein 110
375346


FASTKD5 = FLJ3149
FAST kinase domains 5
60493


GPR23 = LPAR4
lysophosphatidic acid receptor 4
2846


SLC41A3
solute carrier family 41, member 3
54946


ATP6V0D1
ATPase, H+ transporting
9114


KIAA0284
hypothetical protein LOC2836382
283638


PILRA
paired immunoglobin-like type 2 receptor alpha
29992


RAD9B
RAD9 homolog B (S. pombe)
144715


UHSKERB = KRTAP5-8
keratin associated protein 5-8
57830


GSTM2
glutathione S-transferase mu 2
2946


KRTHA5 = KRT35
keratin 35
3886


KRTAP21-2
keratin associated protein 21-2
337978


PCOLN3 = CHMP1A
involved in multivesicular body sorting of proteins to lysosomes
5119


PRSS1
protease, serine, 1 (trypsin 1) = can this be correct?
5644


CPT2
original designation CPT2B09
1376


GOSR2
carnitine palmitoyltransferase 2
9570


C6ORF191
chromosome 6 open reading frame 191
253582


USP13
ubiquitin specific peptidase 13 (isopeptidase T-3)
8975


UEV3 = UEVLD
UEV and lactate/malate dehyrogenase domains
55293


FBXO5
F-box protein 5
26271


PNUTL2 = Sept. 4 & 5
Septin 4 and Septin 5
5414


TRIM3
tripartite motif-containing 3
10612


MYO9A
myosin IXA
4649


PJA1
praja ring finger 1
64219


RNPEPLI
arginyl aminopeptidase (aminopeptidase B)-like 1
57140


FASTKD5 = FLJ3149
FAST kinase domains 5
60493


C1ORF123 = FLJ20580
FLJ20580
54987


MICAL3
microtubule associated monoxygenase
57553


ALCAM
activated leukocyte cell adhesion molecule
214


FRMPD1
FERM and PDZ domain containing 1
22844


CCNB2
cyclin B2
9133


DNAJC5G
DnaJ (Hsp40) homolog, subfamily C, member 5 gamma
285126


IL9
interleukin 9
3578


LOC338829
RefSed status: withdrawn (discontinued June 2009)



PIK4CA
phosphatidylinositol 4-kinase, catalytic, alpha
5297


RPGR
retinitis pigmentosa GTPase regulator
6103


FLJ21986 = C70RF58
C7ORF58
79974


FAS = TNFRSF6
TNF receptor superfamily, member 6
355


XKR5 = UNQ2754
XK, Kell blood group complex subunit-related family, member 5
389610


ZNF289
ARFGAP2 ADP-ribosylation factor GTPase activating protein 2
84364


NDUFA5
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5, 13kC
4698


CBLL1
Cas-Br-M (murine) ecotropic retroviral transforming sequence-like
79872


KCNIP2
Kv channel interacting protein 2
30819


TMED10 = TMP21
transmembrane emp24-like trafficking protein 10 (yeast)
10972


UBC
ubiquitin C
7316


ACSBG1 = BG1
acyl-CoA synthetase bubblegum family member 1
23205


SFXN5
sideroflexin 5
94097


LOC388381
C17orf98
388381


L1TD1 = FLJ10884
LINE-1 type transposase domain containing 1
54596


STXBP2
syntaxin binding protein 2
6813


LYZL1
lysozyme-like 1
84569


ZNF706 = L0051123
zinc finger protein 706
51123

## Claims

1. A method of modulating NFAT activity in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the expression of a TMEM110 gene, wherein the agent is a nucleic acid inhibitor which inhibits gene expression.

2. The method of claim 1, wherein the nucleic acid inhibitor is an siRNA or shRNA.

3. A method of modulating store-operated Ca2+ entry into a cell, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the expression of a TMEM110 gene, wherein the agent is a nucleic acid inhibitor which inhibits gene expression.

4. A method of treating and/or preventing hyperactivity or inappropriate immune response in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that inhibits the expression of a TMEM110 gene, wherein the hyperactivity or inappropriate immune response in a subject is associated with acute and chronic immune diseases selected from a group consisting of asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, autoimmune thyroiditis, hemolytic anemia, multiple sclerosis, transplant graft rejections and graft-versus-host disease, and wherein the agent is a nucleic acid inhibitor which inhibits gene expression.

5. The method of claim 4, wherein the nucleic acid inhibitor is an siRNA or shRNA.