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US20230383298A1 - Methods and Compositions for Reducing Immunosupression by Tumor Cells - Google Patents

Methods and Compositions for Reducing Immunosupression by Tumor Cells Download PDF

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US20230383298A1
US20230383298A1 US18/149,520 US202318149520A US2023383298A1 US 20230383298 A1 US20230383298 A1 US 20230383298A1 US 202318149520 A US202318149520 A US 202318149520A US 2023383298 A1 US2023383298 A1 US 2023383298A1
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United States
Prior art keywords
tumor
shrna
cell
cells
nucleic acid
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US18/149,520
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Kai W. Wucherpfennig
Glenn Dranoff
Penghui Zhou
Donald Shaffer
Nir Hacohen
Harvey I. Cantor
Diana Alvarez Arias
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General Hospital Corp
Dana Farber Cancer Institute Inc
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General Hospital Corp
Dana Farber Cancer Institute Inc
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Priority to US18/149,520 priority Critical patent/US20230383298A1/en
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Definitions

  • the instant application contains a Sequence Listing which has been submitted electronically in ST.26 XML format and is hereby incorporated by reference in its entirety.
  • the ST.26 XML, created on Jan. 3, 2023, is named 514293_50010064_SEQ_LISTING_ST26.txt and is 427 KB in size.
  • This invention relates to methods of discovering immunotherapy targets in vivo, therapeutic compositions that modulate immunotherapy targets (e.g., shRNA, immunoresponsive cells expressing shRNA and, in some cases a receptor targeting a cancer cell, e.g., a chimeric antigen receptors (CAR)), and related methods of use.
  • immunotherapy targets e.g., shRNA, immunoresponsive cells expressing shRNA and, in some cases a receptor targeting a cancer cell, e.g., a chimeric antigen receptors (CAR)
  • CAR chimeric antigen receptors
  • Cytotoxic T cells play a central role in immune-mediated control of cancers 1-3 , and monoclonal antibodies that target inhibitory receptors on T cells can induce significant clinical benefit in patients with advanced disease 4-6 .
  • tumors have developed numerous immunosuppressive mechanisms to promote their own growth and to successfully evade the host immune system, effectively blocking the activity of T cells in the tumor microenvironment. This is a central issue in oncology because strong infiltration by CD8 T cells, which have cytotoxic function against tumor cells, is associated with a favorable prognosis in multiple types of human cancer 1,3,11 . This natural defense mechanism is severely blunted in the majority of patients by multiple inhibitory signals emanating from the tumor, its stroma, regulatory T cells and myeloid cell populations.
  • the present disclosure provides targets for inhibiting immunosuppressive pathways used by tumor cells to inactivate and/or suppress immune cells.
  • the disclosure also provides provides compositions and methods related to shRNA with therapeutic potential.
  • the disclosure also provides immunoresponsive cells, including T cells (e.g., cells targeting a tumor antigen) expressing at least one shRNA or other nucleic acid molecule capable of silencing genes that inhibit T cell function.
  • T cells e.g., cells targeting a tumor antigen
  • shRNA or other nucleic acid molecule capable of silencing genes that inhibit T cell function.
  • the disclosure also provides immunoresponsive cells, including T cells, harboring at least one vector expressing a shRNA and at least one chimeric antigen receptor directed to a tumor antigen.
  • the disclosure provides immunoresponsive cells having tumor specificity comprising a vector encoding a shRNA capable of silencing genes that inhibit T cell function.
  • the shRNA sequence reduces the expression of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm 1 g, Blvrb, Tnkl, Prkab2, Trpm7 or Ppp3cc.
  • the shRNA comprises 15 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 604-620 and 653-678.
  • the immunoresponsive cell further comprises a vector encoding a tumor-specific T-cell receptor.
  • the immunoresponsive cell is selected from the group consisting of a tumor-infiltrating lymphocyte (TIL), a Natural Killer T cell (NKT), a cytotoxic T lymphocyte (CTL), and a CD4 T cell.
  • the immunoresponsive cell comprises a vector encoding a CAR, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and a stimulatory domain.
  • the antigen binding domain binds a tumor antigen or pathogen antigen.
  • Exemplary tumor antigens include, for example, prostate-specific membrane antigen (PSMA), Carcinoembryonic Antigen (CEA), CD19, CD20, CD22, ROR1, mesothelin, CD333/IL3Ra, c-Met, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, ERBB2, BIRC5, CEACAM5, WDR46, BAGE, CSAG2, DCT, MAGED4, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE8, IL13RA2, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA9, MAGEA10, MAGEA12, MAGEB1, MAGEB2, MAGEC2, TP53, TYR, TYRP1, SAGE1, SYCP1, SSX2, SSX4, KRAS, PRAME, NRAS, ACTN4, CTNNB1, CASP8, CDC27, CDK4, E
  • the antigen binding domain is an antigen-binding fragment of an antibody (e.g., Fab or a scFv).
  • the intracellular domains of such CARs contain cytoplasmic signaling domains derived from the T cell receptor and costimulatory molecules.
  • the vector is a plasmid, retroviral vector, or lentiviral vector.
  • the disclosure provides isolated nucleic acid molecules encoding a shRNA sequence. In another embodiment, the disclosure provides isolated nucleic acid molecules encoding a CAR. In yet another embodiment, the disclosure provides isolated nucleic acid molecules encoding a CAR and a shRNA sequence.
  • the isolated nucleic acid encodes a shRNA sequence reduces the expression of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, or Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm 1 g, Blvrb, Tnkl, Prkab2, Trpm7 or Ppp3cc.
  • the isolated nucleic acid encodes a shRNA comprising 15 contiguous nucleotides complementary a nucleic
  • the isolated nucleic acid encodes a CAR comprising an antigen binding domain, a transmembrane domain, a stimulatory domain, and a co-stimulatory domain.
  • the antigen binding domain is an antigen-binding fragment of an antibody (e.g., Fab or a scFv).
  • the antigen binding domain is a cytoplasmic signaling domain derived from the T cell receptor and costimulatory molecules.
  • the antigen-binding domain binds tumor antigen (e.g., a tumor antigen associated with a solid tumor, lymphoid tumor, melanoma, carcinoma, sarcomas, adenocarcinoma, lymphoma, leukemia, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer).
  • tumor antigen e.g., a tumor antigen associated with a solid tumor, lymphoid tumor, melanoma, carcinoma, sarcomas, adenocarcinoma, lymphoma, leukemia, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer.
  • the disclosure provides vectors comprising an isolated nucleic acid encoding a shRNA sequence, an isolated nucleic acid encoding a CAR, or an isolated nucleic acid encoding a CAR and a shRNA sequence.
  • the vector is a plasmid, lentiviral vector, retroviral vector, adenoviral vector, adeno-associated viral vector.
  • the shRNA can be operably linked to RNA polymerase II promoter or an RNA polymerase III promoter.
  • the invention provides compositions comprising immunoresponsive cells according to the invention, and a pharmaceutically acceptable carrier.
  • the disclosure provides immunoresponsive cells transfected with a first vector encoding a CAR and a second vector encoding a shRNA sequence.
  • the shRNA sequence reduces the expression of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Map3k3, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm 1 g, Blvrb, Tnkl, Prkab2, Trpm7 or Ppp3cc
  • the shRNA comprise 15 contiguous nucleotides complementary a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 604-620 and 653-678.
  • the immunoresponsive cell further comprises a vector encoding a tumor-specific T-cell receptor.
  • the immunoresponsive cell is selected from the group consisting of a tumor-infiltrating lymphocyte (TIL), a Natural Killer T cell (NKT), a cytotoxic T lymphocyte (CTL), and a CD4 T cell.
  • the disclosure provides methods for treating cancer in a subject, the method comprising administering to the subject an autologous T cell modified to express a tumor-specific T-cell receptor or CAR and an shRNA, wherein the shRNA sequence reduces the expression of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Map3k3, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2,
  • the shRNA sequence comprises 15 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 604-620 and 653-678; and wherein the CAR comprises an antigen binding domain, a transmembrane domain, a stimulatory domain, and a co-stimulatory domain. In some aspects, the CAR comprises an antigen binding domain, a transmembrane domain, a stimulatory domain, and a co-stimulatory domain.
  • the disclosure provides methods for treating cancer in a subject, the method comprising administering to the subject an autologous T cell modified to express a tumor-specific T-cell receptor or CAR and an shRNA of the invention.
  • the disclosure provides methods for treating cancer in a subject in need thereof by silencing genes that inhibit T cell function comprising administering to the subject an immunoresponsive cell comprising a vector, the vector encoding a tumor-specific T-cell receptor or a CAR and a shRNA sequence of the invention.
  • the disclosure provides methods for identifying a gene that inhibits the function of an immunoresponsive T cell, the method comprising providing a population of immunoresponsive T cells harboring vectors expressing a shRNA, contacting the population of immunoresponsive T cells with an immunosuppressive tumor, determining whether a shRNA restores T cell function within the immunosuppressive tumor, and identifying a gene associated with a shRNA that restores T cell function within the tumor as a gene that inhibits the function of tumor-infiltrating T cells.
  • the disclosure provides methods for increasing the immune response in a subject in need thereof, the method comprising administering a therapeutic agent that modulates the activity of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc.
  • a therapeutic agent that modulates the activity of a gene selected from the group consisting of P
  • sequence encoding an shRNA comprises a first sequence comprising 15-25 (15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) nucleotides complementary to any of SEQ ID NOs: 604-620 or SEQ ID NOs: 653-678 and a second sequence that is the reverse complement of the first sequence with one or no mismatches (i.e., is perfectly complementary to the first sequence), and a third sequence of 5-9 nucleotides positioned between the first and second sequences.
  • FIG. 1 is a schematic diagram demonstrating an exemplary approach for in vivo discovery of shRNAs that enhance T cell infiltration and accumulation within the tumor microenvironment.
  • FIG. 2 is a set of graphs showing representative flow cytometry plots of CD8+T cells from Rag1 ⁇ / ⁇ /OT-I TCR transgenic mice following infection with an shRNA vector. Transduction efficiency was determined based on expression of the Thy1.1 reporter encoded by the lentiviral vector. Cytokine-cultured T cells expressing the LacZ control shRNA were then stained with a panel of activation markers (black lines; isotype control, shaded). The majority of infected T cells exhibited a central memory phenotype (CD62L+CD44 + ).
  • FIG. 3 is a set of graphs showing representative flow cytometry plots of OT-I T cells sorted from tumors and secondary lymphoid organs for deep sequencing analysis (dLN, tumor-draining lymph node; irLN, irrelevant lymph node).
  • CD8 + Va2 + Vf35 + Thy 1.1 + cells were sorted and genomic DNA was extracted for PCR amplification of the shRNA cassette.
  • FIG. 4 is a set of graphs showing deep sequencing data from in vivo shRNA pool screen.
  • Upper row sequence reads for all genes in a pool in tumor, irrelevant (irLN) and draining lymph node (dLN); lower row, three individual genes (LacZ, negative control) are plotted in comparison to spleen for tumors, irrelevant lymph nodes (irLN) and tumor-draining lymph nodes (dLN). Sequence reads are plotted for these tissues versus spleen. Dashed lines indicate a deviation by log 2 from diagonal.
  • FIG. 5 is a set of graphs showing deep sequencing data from T cell dysfunction screen.
  • shRNA sequencing reads for genes positive in secondary screen are plotted in comparison to spleen for tumors (red), irrelevant lymph nodes (irLN, blue) and tumor-draining lymph nodes (dLN, green), with dashed lines indicating a deviation of log 2 from the diagonal. Data show enrichment of particular shRNAs representing these genes in tumors compared to spleens or lymph nodes.
  • FIG. 6 is a graph showing flow cytometry based quantification of OT-I CD8 + T cell enrichment in tumors relative to spleen.
  • the percentage of shRNA-expressing OT-I T cells was determined by flow cytometry in tumors/spleens by gating on reporter proteins in CD8 + Va2 + Vf35 + T cells.
  • FIG. 7 is a set of graphs showing representative flow cytometry plots of cell enrichment in tumor transduced with shRNA vectors (LacZ, Akap8I, Smad2, Rbks, Dgkz). The percentage of shRNA-expressing OT-I T cells was determined by flow cytometry in tumors/spleens by gating on reporter proteins in CD8 + Va2 + Vf35 + T cells.
  • FIG. 8 is a set of graphs showing flow cytometry-based quantification of CD4+ and CD8+ T cell enrichment in tumors.
  • shRNA-expressing T cells were identified in tumors and spleens using Thy1.1 reporter (% Thy1.1+CD8 T cells or CD4+ T cells, top and bottom panels).
  • Total numbers of LacZ or Ppp2r2d shRNA-expressing T cells were determined in tumors and spleens 7 days following transfer of 2 ⁇ 106 shRNA-expressing cells (right panels). Fold-enrichment of Ppp2r2d versus LacZ shRNA-expressing T cells in tumors is indicated.
  • FIG. 9 is a graph showing reversal of Ppp2r2d shRNA-mediated T cell expansion in tumors by Ppp2r2d cDNA with a mutated shRNA binding site but preserved protein sequence.
  • the three cell populations were identified based on co-expressed reporters; fold-enrichment was calculated based on percentage of reporter-positive cells in tumors versus spleens.
  • FIG. 10 a describes the generation of mutant Ppp2r2d cDNA with preserved protein sequence but disrupted shRNA binding site.
  • EL4 cells were transduced with mutant or wild type Ppp2r2d cDNA on a vector also containing GFP.
  • GFP-positive cells were sorted to purity and transduced with LacZ or Ppp2r2d shRNA vectors expressing a Thy1.1 reporter.
  • shRNA-transduced (Thy1.1 + ) cells were analyzed by flow cytometry for GFP expression.
  • the Ppp2r2d shRNA reduced GFP levels when wild-type Ppp2r2d, but not when mutant Ppp2r2d was expressed. (SEQ ID NOS: 679-681 shown.)
  • FIG. 10 b demonstrates that expression of Ppp2r2d mutant cDNA prevents phenotype induced by Ppp2r2d shRNA.
  • OT-I T cells were transduced with a vector encoding LacZ shRNA, Ppp2r2d shRNA or Ppp2r2d shRNA plus mutant Ppp2r2d cDNA. The different cell populations were normalized for transduction efficiency and co-injected into B16-Ova tumor bearing mice.
  • FIG. 11 is a graph demonstrating real-time qPCR analysis for Ppp2r2d mRNA levels in OT-I T cells transduced with LacZ shRNA or one of three Ppp2r2d shRNAs identified in the screen.
  • FIG. 12 a is a table demonstrating enrichment of particular shRNAs in tumor versus spleen which was calculated based on deep sequencing results from the secondary screen.
  • FIG. 12 b demonstrates clustering of mean expression levels for mRNAs found to be significantly regulated by T cells in or tumors expressing the LacZ control shRNA or one of five experimental shRNAs. Significant expression differences were defined as an Anova p value ⁇ 0.01 between T cells expressing LacZ control shRNA or one of five experimental shRNAs (Alk, Arhgap5, Egr2, Ptpn2 or Ppp2r2d) (JMP-Genomics 6.0, SAS Institute Inc.). mRNAs significantly regulated in one or more treatment groups are shown after clustering (Fast Ward).
  • FIG. 12 c is a Venn diagram showing overlaps between expression signatures by tumor-infiltrating T cells transduced with one of the five experimental shRNAs (signatures defined as an Anova p ⁇ 0.01 as described above). Indicated are the numbers of overlapping probe IDs for any combination of the 5 signatures, as indicated by the overlapping ovals. The significance of the overlaps versus that expected by random chance (Fishers Exact Test) is shown in the accompanying table.
  • FIG. 13 a is a set of graphs showing representative flow cytometry plots of demonstrating the frequency of Ppp2r2d or LacZ shRNA-transduced CD8 T cells in tumors on day 1.
  • FIG. 13 b are a pair of graphs demonstrating the degree of proliferation (based on CFSE dilution) by Ppp2r2d shRNA-transduced CD8 T cells compared to LacZ shRNA-transduced T cells in tumors on days 1, 3, 5, and 7.
  • FIG. 13 c is a set of graphs demonstrating that Ppp2r2d-silencing inhibits T cell apoptosis upon encounter of tumor cells.
  • CFSE-labeled OT-I T cells were co-cultured with B16-Ova tumor cells for 72 hours. Cells were stained with CD8 and annexin V.
  • FIG. 13 d is a set of graphs demonstrating intracellular staining for anti-apoptotic proteins.
  • OT-I T cells expressing LacZ or Ppp2r2d shRNA were co-cultured with B16-Ova tumor cells for 48 hours and then stained with isotype control (grey) and phospho-AKT (Ser473), phospho-Bad (Ser 112) or B c1-2 antibodies.
  • FIG. 13 e is a graph demonstrating increased IFN- ⁇ secretion by Ppp2r2d-silenced T cells.
  • OT-I T cells isolated from B16-Ova tumor-bearing mice were assayed for IFN- ⁇ expression by intracellular staining.
  • FIG. 13 f is a set of graphs demonstrating Ppp2r2d-silenced T cells expand in tumors even without presentation of tumor antigens by professional antigen presenting cells.
  • LacZ or Ppp2r2d shRNA-expressing OT-I T cells were transferred into day 14 B16-Ova tumor-bearing C57BL/6 or b2m-1-mice.
  • shRNA-expressing T cells were identified based on expression of teal fluorescent protein (TFP) or Thy1.1 (fold enrichment in tumors compared to spleens).
  • TFP teal fluorescent protein
  • Thy1.1 fold enrichment in tumors compared to spleens.
  • FIG. 13 g is a graph demonstrating that Ppp2r2d-silencing inhibits T cell apoptosis upon encounter of tumor cells.
  • CFSE-labeled OT-I T cells were co-cultured with B16-Ova tumor cells for 72 hours (activated caspase-3).
  • FIG. 14 is a set of graphs demonstrating OT-I T cells expressing LacZ or Ppp2r2d shRNAs labeled with CFSE and stimulated with CD3 antibody for 72 h. Cells were then stained with CD8 and annexin V and analyzed by flow cytometry.
  • FIG. 15 is a set of graphs demonstrating accumulation of Ppp2r2d shRNA-expressing T cells in tumors and tumor-draining lymph nodes, but not other secondary lymphoid organs.
  • OT-I T cells expressing Ppp2r2d or LacZ shRNAs were labeled with CFSE and injected into B16-Ova tumor-bearing mice.
  • T cells were isolated from the indicated organs on days 1, 3, 5 and 7 to examine the extent of T cell accumulation based on dilution of the CSFE dye.
  • FIGS. 16 a - c are a set of graphs demonstrating that the silencing of Ppp2r2d enhances anti-tumor activity of CD4 and CD8 T cells.
  • T cells were activated with anti-CD3/CD28 beads, infected with lentiviruses driving LacZ or Ppp2r2d shRNA expression and injected into B16-Ova (a,b) or B16 (c) tumor-bearing mice. Tumor size was measured every three days following T cell transfer using calipers on the two longest axes.
  • a,b CD4 + TRP-1 and/or CD8 + OT-I T cells (2 ⁇ 10 6 ) were transferred (day 12 and 17) into mice bearing day 12 B16-Ova tumors.
  • CD4 + TRP-1 and CD8 + pmel-1 T cells (3 ⁇ 10 6 CD4 + TRP-1 plus 3 ⁇ 10 6 CD8 + pmel-1) were transferred (day 10 and 15) into mice with day 10 B16 tumors.
  • Log-rank (Mantel-Cox) test was performed using GraphPad Prism version 6 comparing survival of mice treated with LacZ versus Ppp2r2d shRNA-expressing T cells.
  • FIG. 18 is a set of graphs demonstrating Ppp2r2d protein quantification by mass spectrometry with labeled synthetic peptides (AQUA, ratio of endogenous to AQUA peptides). Representative data from two independent experiments (a-d); Two-sided student's t-test, * P ⁇ 0.05, ** P ⁇ 0.01; mean+/ ⁇ s.d.
  • FIG. 19 is a graph demonstrating qPCR analysis for Ppp2r2d mRNA in tumor-infiltrating OT-I T cells (day 7).
  • FIG. 20 a are graphs showing representative flow cytometry plots demonstrating proliferation of Ppp2r2d shRNA-expressing T cells in tumors and tumor-draining lymph nodes.
  • OTI T cells expressing Ppp2r2d or LacZ shRNAs were labeled with CFSE and injected into B16-Ova tumor-bearing mice.
  • T cells were isolated from the indicated organs on days 1, 3, 5 and 7 to examine the extent of T cell proliferation based on CFSE dilution. T cells that had not diluted CFSE (nondividing cells) were quantified (right).
  • FIG. 20 b are graphs showing representative flow cytometry plots demonstrating viability of tumor-infiltrating T cells.
  • OT-I T cells expressing Pp2r2d or LacZ shRNAs were injected into B16-Ova tumor-bearing mice. T cells were isolated on day 7 and apoptosis was assessed by intracellular staining with an antibody specific for activated caspase-3 (some T cell death may have been caused by the isolation procedure from tumors).
  • FIGS. 21 a - c are a series of graphs demonstrating ex vivo analysis of cytokine production by tumor-infiltrating OT-I T cells at a single-cell level using a nanowell device (84,672 wells of picoliter volume).
  • a Representative single cells in nanowells and corresponding patterns of cytokine secretion.
  • b Percentage of T cells secreting indicated cytokines.
  • c Cytokine secretion rates calculated from standard curves (mean+/ ⁇ s.d., Mann Whitney test * P ⁇ 0.05).
  • FIG. 22 a is a set of graphs showing representative flow cytometry plots demonstrating that the majority of adoptively transferred OT-I cells have a memory phenotype in lymph nodes but an effector phenotype in tumors.
  • Cytokine pre-treated cells expressing Ppp2r2d or LacZ shRNAs were injected into mice bearing day 14 B16-Ova tumors.
  • T cells were harvested from the indicated organs and stained with CD62L and CD44 antibodies. FACS analysis of shRNA-expressing OT-I cells was performed by gating on CD8/Thy1.1 double-positive cells.
  • FIG. 22 b is a set of graphs showing representative flow cytometry plots demonstrating analysis of exhaustion markers.
  • FIG. 23 a is a set of graphs showing demonstrating intracellular staining for granzyme B by OT-I T cells in tumor-draining lymph nodes and tumors.
  • FIG. 23 b is a pair of images and a graph demonstrating infiltration of shRNA-expressing T cells into tumors.
  • OT-I T cells were transduced with LacZ or Ppp2r2d shRNA vectors encoding a GFP reporter and injected into B16-Ova tumor-bearing mice. After 7 days, tumors were excised and frozen sections stained with anti-GFP and DAPI to enumerate shRNAexpressing OT-I T cells in tumors.
  • FIG. 23 c is a pair of images and a graph demonstrating TUNEL immunohistochemistry performed on tissue sections and apoptotic cells were quantified.
  • the present disclosure is based, in part, on the observation that the regulatory mechanisms that result in loss of T cell function within immunosuppressive tumors can be systematically discovered in vivo using a pooled small hairpin RNA (shRNA) screening approach aimed at identifying genes that block the function of tumor infiltrating T-cells.
  • shRNA small hairpin RNA
  • tumor associated immunosuppressive mechanisms actively block the activity of T cells in the tumor microenvironment.
  • the methods described herein identify shRNAs that enable robust T cell infiltration and accumulation in tumors, despite the multiple inhibitory signals.
  • the methods identify shRNA that silence expression of genes responsible for immunosuppression by tumors, allowing for enhanced T cell infiltration and accumulation in tumors and resistance to apoptosis.
  • the disclosure provides methods for specifically identifying regulatory mechanisms that result in the loss of T cell function within the tumor microenvironment. These methods can include: providing a population of T cells harboring vectors expressing a shRNA; contacting the population of T cells with an immunosuppressive tumor; determining whether a shRNA restores T cell function (e.g., restores ability of T cell to infiltrate and proliferate within the tumor microenvironment) within the immunosuppressive tumor; identifying a gene associated with a shRNA that restores T cell function within the tumor as a gene that inhibits T cell function within the tumor microenvironment.
  • a shRNA restores T cell function e.g., restores ability of T cell to infiltrate and proliferate within the tumor microenvironment
  • the disclosure provides target genes for reducing the immunosuppressive effect of tumors.
  • the expression of the target genes can be reduced in immune cells, e.g., T cells that recognize tumor associated antigens, and the reduction in expression of the target genes can increase the ability of the cells to evade tumor associated immunosuppressive mechanisms.
  • shRNAs that reduce (e.g., silence, eliminate, knock down, knock out, or decrease) expression of genes that impair the function of tumor infiltrating T-cells. These shRNA were identified from the transfer of shRNA transduced T cells into tumors, followed by deep sequencing to quantify the representation of all shRNAs in the tumor and lymphoid organs.
  • shRNA disclosed herein include shRNA that reduce the activity of genes including, for example, Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm 1 g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc.
  • genes including, for example, Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka,
  • the disclosure provides therapeutic compositions (e.g., including isolated nucleic acid molecules, vectors expressing nucleic acid molecules encoding the shRNA) related to the shRNAs that silence expression of genes that block the function of tumor infiltrating T-cells.
  • the disclosure provides modified immunoresponsive cells (e.g., T cells, including Natural Killer T cells (NKT), a cytotoxic T lymphocytes (CTL), and a regulatory T cells) that harbor vectors capable of expressing the shRNA described herein.
  • the modified immunoresponsive cells further harbor a vector capable of expressing a CAR having an antigen binding domain that targets a tumor specific antigen.
  • RNA interference is an effective tool for genome-scale, high throughput analysis of gene function.
  • RNA interference also called post transcriptional gene silencing (PTGS)
  • PTGS post transcriptional gene silencing
  • RNA interfering agent is defined as any agent that interferes with or inhibits expression of a target gene, e.g., a target gene of the invention, by RNA interference (RNAi).
  • RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target gene, e.g., a target gene of the invention, or a fragment thereof, short interfering RNA (siRNA), short hairpin RNA (shRNA), and small molecules which interfere with or inhibit expression of a target gene by RNA interference (RNAi).
  • siRNA short interfering RNA
  • shRNA short hairpin RNA
  • RNAi RNA interference
  • RNA interference is a 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 PTGS of messenger RNA (mRNA) transcribed from that targeted gene, thereby inhibiting expression of the target gene. This process has been described in plants, invertebrates, and mammalian cells. 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.
  • nucleic acid molecules e.g., synthetic siRNAs or RNA interfering agents
  • “inhibition of target gene expression” or “inhibition of marker gene expression” includes any decrease in expression or protein activity or level of the target gene (e.g., a marker gene of the invention) or protein encoded by the target gene, e.g., a marker protein of the invention.
  • the decrease may be of at least 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.
  • siRNA Short interfering RNA
  • small interfering RNA is defined as an agent which functions to inhibit expression of a target gene.
  • RISC RNA-induced silencing complex
  • shRNAs short hairpin RNAs
  • long dsRNAs long dsRNAs
  • short temporal RNAs short temporal RNAs
  • miRNAs micro RNAs
  • shRNA effector molecules either are processed into siRNA, such as in the case of shRNA, or directly aid gene silencing, as in the case of miRNA.
  • the present invention thus encompasses the use of shRNA as well as any other suitable form of RNA to effect posttranscriptional gene silencing by RNAi.
  • Use of shRNA has the advantage over use of chemically synthesized siRNA in that the suppression of the target gene is typically long-term and stable.
  • An siRNA may be chemically synthesized, may be produced by in vitro by transcription, or may be produced within a host cell from expressed shRNA.
  • a siRNA is a small hairpin (also called stem loop) RNA (shRNA).
  • shRNAs are composed of a short (e.g., 19-25 nucleotides) antisense strand, followed by a 5-9 nucleotide loop, and the complementary sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow.
  • shRNAs may be contained in plasmids, retroviruses, and lentiviruses.
  • gene silencing induced by RNA interference 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 introduction of RNA interference.
  • the mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.
  • reduced or “reduce” as used herein generally means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease, or any integer decrease between 10-100% as compared to a reference level.
  • the term “increased” or “increase” as used herein generally means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any integer increase between 10-100% as compared to a reference level, or about a 2-fold, or about a 3-fold, or about a 4-fold, or about a 5-fold or about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • the disclosure provides immunoresponsive cells, including T cells, cytotoxic T cells, tumor-infiltrating lymphocytes (TIL), regulatory (CD4) T cells, and Natural Killer (NKT) cells, expressing at least one of an antigen-recognizing receptor.
  • TIL tumor-infiltrating lymphocytes
  • CD4 regulatory
  • NKT Natural Killer
  • the immunoresponsive cells express at least one tumor specific antigen-recognizing receptor.
  • tumor cell antigen specific T cells, NKT cells, TIL, CTL cells or other immunoresponsive cells are used.
  • Non-limiting examples of immunoresponsive cells include T cells, such as, for example, ⁇ -TCR+ T cells (e.g., CD8+ T cells or CD4+ T cells) ⁇ -TCR+ T cells, tumor-infiltrating lymphocytes (TIL), Natural Killer T cells (NKT), a cytotoxic T lymphocytes (CTL), and a CD4 T cells.
  • TIL tumor-infiltrating lymphocytes
  • NKT Natural Killer T cells
  • CTL cytotoxic T lymphocytes
  • CD4 T cells a CD4 T cells.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences comprising a sequence at least 12, 15, 20 or 25 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 604-620 and 653-678.
  • the shRNA also includes the reverse complement of the contiguous nucleotide sequence and a short sequence located between the two sequences so that the two sequences form a stem loop shRNA that can be processed within a cell provide an siRNA that inhibits the expression of the protein encoded by one of SEQ ID NOs: 604-620 and 653-678, and compositions thereof.
  • Table 1 provides a list of genes identified here as being involved with tumor immunosuppression of T cells.
  • the nucleic acids of the compositions encode the shRNA sequences targeting the sequences provided in Table 2.
  • Table 2 further demonstrates enrichment in tumor versus spleen for the selected shRNA based on deep sequencing analysis (“Enrich Fold”)
  • shRNAs demonstrating an at least ⁇ 3 shRNAs fold enrichment in tumor relative to spleen indicate a more active target sequence region.
  • nucleic acids of the compositions encode the shRNA sequences targeting the human Ppp2r2d and Cb1b sequences provided in Table 2a.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Ppp2r2d target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 372, 373, 374, 375, 376, 377, 378, 378, 379, 380, 381, 382, 383, 384, 385, or 386.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Pp2r2d sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 372, 373, 374, 375, 376, 377, 378, 378, 379, 380, 381, 382, 383, 384, 385, or 386.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Eif2ak3 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 or 147.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Eif2ak3 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 or 147.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Arhgap5 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Arhgap5 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Smad2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, or 490.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Smad2 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, or 490.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Akap81 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Akap81 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Rbks target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, or 445.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Rbks sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, or 445.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Egr2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Egr2 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Dgka target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Dgka sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Cb1b target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Cb1b sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Mdfic target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, or 299.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Mdfic sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, or 299.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Entpdl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, or 162.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Entpdl sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, or 162.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Vamp7 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, or 587.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Vamp7sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, or 587.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Hipkl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, or 222.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Hipkl sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, or 222.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Nuak2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, or 329.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Nuak2 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, or 329.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Alk target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Alk sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Pdzklipltarget sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, or 341.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Pdzkliplsequence that corresponds to a murine target sequence set forth in SEQ ID NO: 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, or 341.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Blvrb target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 52, 53, 54, 55, 56 or 57.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Blvrb that corresponds to a murine target sequence set forth in SEQ ID NO: 52, 53, 54, 55, 56 or 57.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Cdkn2a target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 83, 84, 85, 86 or 87.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Cdkn2a that corresponds to a murine target sequence set forth in SEQ ID NO: 83, 84, 85, 86 or 87.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Fllr target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 175, 176 or 177.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human F11r that corresponds to a murine target sequence set forth in SEQ ID NO: 175, 176 or 177.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Fyn target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 187, 191 or 192.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Fyn that corresponds to a murine target sequence set forth in SEQ ID NO: 187, 191 or 192.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Grk6 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 204, 205, 206 or 207.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Grk6 that corresponds to a murine target sequence set forth in SEQ ID NO: 204, 205, 206 or 207.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Inpp5b target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 232, 234, 235, 236 or 237.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Inpp5b that corresponds to a murine target sequence set forth in SEQ ID NO: 232, 234, 235, 236 or 237.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Impk target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 248, 249, 250, 251 or 252.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Impk that corresponds to a murine target sequence set forth in SEQ ID NO: 248, 249, 250, 251 or 252.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Jun target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 263, 264, 265, 266, 267, 268 or 269.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Jun that corresponds to a murine target sequence set forth in SEQ ID NO: 263, 264, 265, 266, 267, 268 or 269.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Mast2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 281, 282, 283 or 284.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Mast2 that corresponds to a murine target sequence set forth in SEQ ID NO: 281, 282, 283 or 284.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Nptxr target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 311, 312, 313 or 314.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Nptxr that corresponds to a murine target sequence set forth in SEQ ID NO: 311, 312, 313 or 314.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Pkdl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 351, 352, 353, 354, 355 or 356.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Pkdl that corresponds to a murine target sequence set forth in SEQ ID NO: 351, 352, 353, 354, 355 or 356.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Ppm1g target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 367, 368, 369, 370 or 371.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Ppm1g that corresponds to a murine target sequence set forth in SEQ ID NO: 367, 368, 369, 370 or 371.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Ppp3cc target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 399, 400 or 401.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Ppp3cc that corresponds to a murine target sequence set forth in SEQ ID NO: 399, 400 or 401.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Prkab2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 414, 415 or 416.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Prkab2 that corresponds to a murine target sequence set forth in SEQ ID NO: 414, 415 or 416.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Ptpn2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 426, 427, 428, 429 or 430.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Ptpn2 that corresponds to a murine target sequence set forth in SEQ ID NO: 426, 427, 428, 429 or 430.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Rockl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 457, 458, 459 or 460.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Rockl that corresponds to a murine target sequence set forth in SEQ ID NO: 457, 458, 459 or 460.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Sbfl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 470, 471, 472, 473, 474 or 475.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Sbfl that corresponds to a murine target sequence set forth in SEQ ID NO: 470, 471, 472, 473, 474 or 475.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Socsl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 504, 505, 506, 507, 508, 509 or 510.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Socsl that corresponds to a murine target sequence set forth in SEQ ID NO: 504, 505, 506, 507, 508, 509 or 510.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Socs3 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 524, 525, 526, 527 or 528.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Socs3 that corresponds to a murine target sequence set forth in SEQ ID NO: 524, 525, 526, 527 or 528.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Stk17b target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 539, 540, 541, 542 or 543.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Stk17b that corresponds to a murine target sequence set forth in SEQ ID NO: 539, 540, 541, 542 or 543.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Tnkl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 556, 557 or 558.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Tnkl that corresponds to a murine target sequence set forth in SEQ ID NO: 556, 557 or 558.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Trpm7 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 569, 570, 571, 572 or 573.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Trpm7 that corresponds to a murine target sequence set forth in SEQ ID NO: 569, 570, 571, 572 or 573.
  • the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Yesl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 600, 601, 602 or 603.
  • the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Yesl that corresponds to a murine target sequence set forth in SEQ ID NO: 600, 601, 602 or 603.
  • a human sequence that corresponds to a murine target sequence is a sequence which perfectly corresponds to the human gene sequence, and for example, can have none, 1, 2, 3 or 4 nucleotide mismatches with the at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides of the selected murine target sequence.
  • an isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, cDNA, or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote.
  • a virus e.g., a retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes virus
  • an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid.
  • percent sequence identity two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It also will be appreciated that a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over each aligned region. It is noted that the percent identity value is usually rounded to the nearest integer.
  • 78.1%, 78.2%, 78.3%, and 78.4% are rounded down to 78%, while 78.5%, 78.6%, 78.7%, 78.8%, and 78.9% are rounded up to 79%. It is also noted that the length of the aligned region is always an integer.
  • percent sequence identity refers to the degree of identity between any given query sequence and a subject sequence.
  • a percent identity for any query nucleic acid or amino acid sequence, e.g., a transcription factor, relative to another subject nucleic acid or amino acid sequence can be determined as follows.
  • complementary nucleotide sequence also known as an “antisense sequence,” refers to a sequence of a nucleic acid that is completely complementary to the sequence of a “sense” nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence).
  • nucleic acid molecules are provided that comprise a sequence complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides or an entire gene coding strand, or to only a portion thereof.
  • nucleotide sequence refers to a nucleotide sequence of a nucleic acid encoding an identical sequence.
  • siRNA small inhibitory RNA
  • sequences of those nucleic acids will be highly complementary to the mRNA target sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence.
  • sequences of the nucleic acids may be exact matches, i.e. be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch.
  • Highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.
  • vector refers to any viral or non-viral vector, as well as any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form that may or may not be self transmissible or mobilizable, and that can transform prokaryotic or eukaryotic host cells either by integration into the cellular genome or which can exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). Any vector known in the art is envisioned for use in the practice of this invention.
  • Vectors can be viral vectors or non-viral vectors. Should viral vectors be used, it is preferred the viral vectors are replication defective, which can be achieved for example by removing all viral nucleic acids that encode for replication. A replication defective viral vector will still retain its infective properties and enters the cells in a similar manner as a replicating adenoviral vector, however once admitted to the cell a replication defective viral vector does not reproduce or multiply. Vectors also encompass liposomes and nanoparticles and other means to deliver DNA molecule to a cell.
  • viral vectors refers to the use of viruses, or virus-associated vectors as carriers of a nucleic acid construct into a cell. Constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral and lentiviral vectors, for infection or transduction into cells.
  • AAV Adeno-associated virus
  • HSV Herpes simplex virus
  • the vector may or may not be incorporated into the cell's genome.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom,
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • an “Expression vector” is a specialized vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • cosmids e.g., naked or contained in liposomes
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses
  • the disclosure provides modified cells that harbor vectors capable of expressing the shRNA described herein and further modified to express a CAR.
  • the shRNA and the CAR are expressed on the same vector.
  • the shRNA and the CAR are expressed on separate vectors.
  • the modified cells described herein are immunoresponsive cells.
  • the immunoresponsive cells express at least one of an antigen-recognizing receptor.
  • the immunoresponsive cells express at least one of an tumor specific antigen-recognizing receptor.
  • tumor cell antigen specific T cells, NKT cells, TIL, CTL cells or other immunoresponsive cells are used.
  • immunoresponsive cells include T cells, such as, for example, ⁇ -TCR+ T cells (e.g., CD8+ T cells or CD4+ T cells) ⁇ -TCR+ T cells, tumor-infiltrating lymphocytes (TIL), Natural Killer T cells (NKT), a cytotoxic T lymphocytes (CTL), and a CD4 T cells.
  • compositions comprising the immunoresponsive cells of the invention (e.g., T cells, NKT cells, TILs, CTL cells, or their progenitors) can be provided systemically or directly to a subject for the treatment of a cancer.
  • cells of the invention are directly injected into an organ of interest (e.g., an organ affected by a cancer).
  • compositions comprising genetically modified immunoresponsive cells are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature).
  • Expansion and differentiation agents can be provided prior to, during or after administration of the cells to increase production of T cells, NKT cells, TILs, CTL cells in vitro or in vivo.
  • the modified immunoresponsive cells can be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). Usually, at least 1 ⁇ 10 5 cells will be administered, eventually reaching 1 ⁇ 10 10 , or more.
  • Immunoresponsive cells of the invention can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of genetically modified immunoresponsive cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Preferable ranges of purity in populations comprising genetically modified immunoresponsive cells are about 50 to about 55%, about 55 to about 60%, and about 65 to about 70%.
  • the purity is about 70 to about 75%, about 75 to about 80%, about 80 to about 85%; and still more preferably the purity is about 85 to about 90%, about 90 to about 95%, and about 95 to about 100%. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage).
  • the cells can be introduced by injection, catheter, or the like.
  • factors can also be included, including, but not limited to, interleukins, e.g. IL-2, IL-3, IL-6, and IL-11, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM-CSF, interferons, e.g. .gamma.-interferon and erythropoietin.
  • compositions of the invention include pharmaceutical compositions comprising the immunoresponsive cells of the invention or their progenitors and a pharmaceutically acceptable carrier.
  • Administration can be autologous or heterologous.
  • immunoresponsive cells, or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject.
  • the invention provides chimeric antigen receptors (CARs) comprising an antigen binding domain directed to a tumor cell antigen.
  • CAR is an artificially constructed hybrid protein or polypeptide containing an extracellular portion that recognizes a tumor cell antigen (e.g., the antigen binding domains of an antibody (scFv) and a cytoplasmic signaling domain derived from the T cell receptor and costimulatory domain.
  • a tumor cell antigen e.g., the antigen binding domains of an antibody (scFv) and a cytoplasmic signaling domain derived from the T cell receptor and costimulatory domain.
  • Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC—restricted manner, exploiting the antigen-binding properties of monoclonal antibodies.
  • the CAR-modified T-cells have the potential to replicate in vivo and long term persistence allows for sustained tumor control and obviate the need for repeated infusions of antibody. (Kalos M, et al., Sci Transl Med. 2011 Aug. 10; 3(95))
  • the non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.
  • CARs when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
  • TCR T cell receptor
  • a CAR combines the binding site of a molecule that recognizes an antigen being targeted (i.e., an “antigen binding domain”) with one or more domains of conventional immune receptors responsible for initiating signal transduction that leads to lymphocyte activation (e.g., the “stimulatory domain” or “signaling domain”).
  • an antigen binding domain i.e., an “antigen binding domain”
  • one or more domains of conventional immune receptors responsible for initiating signal transduction that leads to lymphocyte activation e.g., the “stimulatory domain” or “signaling domain”.
  • the binding portion used is derived from the structure of the Fab (antigen binding) fragment of a monoclonal antibody (mAb) that has high affinity for the tumor antigen being targeted.
  • Fab antigen binding
  • mAb monoclonal antibody
  • the Fab is the product of two genes, the corresponding sequences are usually combined via a short linker fragment that allows the heavy-chain to fold over the light-chain derived peptides into their native configuration, creating a single-chain fragment variable (scFv) region.
  • Fv or (scFv) antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
  • the binding portion used is derived from a cytoplasmic signaling domain derived from T cell receptor and costimulatory molecules.
  • the signaling portion of CARs contains usually the intracellular domains of the zeta ( ⁇ ) chain of the TCR/CD3 complex 25 or, less commonly, of the gamma ( ⁇ ) chain of the immunoglobulin receptor FccRI 26, 27 or the CD3-epsilon ( ⁇ ) chain, 28 with the transmembrane region being derived from the same molecule.
  • the CARs comprise an antigen binding domain, a transmembrane domain, a stimulatory domain, and a co-stimulatory domain.
  • Further embodiments of the invention provide related nucleic acids, recombinant expression vectors, host cells, populations of cells, antibodies, or antigen binding portions thereof, and pharmaceutical compositions relating to the CARs of the invention.
  • the antigen binding domain binds to a tumor cell antigen.
  • tumor cell antigen or “tumor antigen” as used herein refers to any polypeptide expressed by a tumor that is capable of inducing an immune response.
  • tumor antigens include, for example, prostate-specific membrane antigen (PSMA), Carcinoembryonic Antigen (CEA), CD19, CD20, CD22, ROR1, mesothelin, CD333/IL3Ra, c-Met, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, ERBB2, BIRC5, CEACAM5, WDR46, BAGE, CSAG2, DCT, MAGED4, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE5, IL13RA2, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA9, MAGEA10, MAGEA12, MAGEB1, MAGEB2, MAGEC2, TP53, TYR, TYRP1, SAGE1, SYCP1, SSX2, SSX4, KRAS, PRAME, NRAS, ACTN4, CTNNB1, CASP8, CDC27, CDK
  • PSMA
  • the present invention relates generally to the use of T cells genetically modified to stably express a shRNA of the invention and a desired CAR.
  • T cells expressing a CAR are generally referred to as CAR T cells.
  • T cells expressing a CAR are referred to herein as CAR T cells or CAR modified T cells.
  • the cell can be genetically modified to stably express an antibody binding domain on its surface, conferring novel antigen specificity that is WIC independent.
  • the T cell is genetically modified to stably express a CAR that combines an antigen recognition domain of a specific antibody with an intracellular stimulatory domain (e.g., signaling domain).
  • the CAR in addition to an antigen binding domain can include the intracellular domains of the zeta ( ⁇ ) chain of the TCR/CD3 complex, the gamma ( ⁇ ) chain of the immunoglobulin receptor FccRI26, 27 or the CD3-epsilon ( ⁇ ) chain.
  • the CAR can also include a transmembrane region being from the same molecules or other type I transmembrane proteins such as CD4, CD8 and CD28.
  • the CAR of the invention comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain.
  • the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
  • the cytoplasmic domain can be designed to comprise a stimulatory domain and a costimulatory domain.
  • a CAR can include intracytoplasmatic portion of co-stimulatory molecules, such as CD28, CD134/0X40, CD137/4-1BB, Lck, ICOS or DAP10.
  • the disclosure also relates to a strategy of Adoptive cell therapy (ACT).
  • ACT is a procedure in which therapeutic lymphocytes are administered to patients in order to treat cancer. This approach entails the ex vivo generation of tumor specific T cell lymphocytes and infusing them to patients.
  • the host may be manipulated in other ways which support the take of the T cells and their immune response, for example, preconditioning the host (with radiation or chemotherapy) and administration of lymphocyte growth factors (such as IL-2).
  • lymphocyte growth factors such as IL-2
  • One method for generating such tumor specific lymphocytes involves the expansion of antigen specific T cells.
  • the invention provides generating T cells expressing a shRNA of the invention and a desired CAR directed to a tumor antigen.
  • the modified T cells can be generated by introducing a vector (e.g., plasmid, lentiviral vector, retroviral vector, adenoviral vector, adeno-associated viral vector) encoding both 1) an shRNA capable of reducing expression of a target gene described herein and 2) a desired CAR into the cells.
  • a vector e.g., plasmid, lentiviral vector, retroviral vector, adenoviral vector, adeno-associated viral vector
  • the modified T cells of the invention are able to replicate in vivo resulting in long term persistence that can lead to tumor control.
  • the disclosure provides methods of treating cancer comprising administering a composition capable of silencing genes that inhibit T cell function.
  • the methods relate to administering T cell expressing a shRNA of the invention and a desired CAR directed to a tumor antigen.
  • the T cell to be administered comprises a vector encoding a shRNA of the invention and a desired CAR directed to a tumor antigen.
  • therapeutic compositions disclosed herein can include, in addition to the tumor targeting T cells, compounds, drugs, and/or agents used for the treatment of cancer.
  • Such compounds, drugs, and/or agents can include, for example, chemotherapy drugs, small molecule drugs or antibodies that stimulate the immune response to a given cancer.
  • therapeutic compositions can include, for example, one or more small molecule inhibitors that silence, reduces, eliminates, knocks down, knocks out, or decreases the expression and/or activity of genes selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, F11r, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc.
  • small molecule inhibitors that silence, reduces, eliminate
  • the invention provides one or more inhibitors of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 or Ppp3cc.
  • the invention provides one or more inhibitors of Ppp2r2d.
  • the invention provides one or more inhibitors of Eif2ak3.
  • the invention provides one or more inhibitors of Arhgap5.
  • the invention provides one or more inhibitors of Smad2.
  • the invention provides one or more inhibitors of Akap81.
  • the invention provides one or more inhibitors of Rbks.
  • the invention provides one or more inhibitors of Egr2.
  • the invention provides one or more inhibitors of Dgka.
  • the invention provides one or more inhibitors of Cb1b.
  • the invention provides one or more inhibitors of Map3k3.
  • the invention provides one or more inhibitors vMdfic.
  • the invention provides one or more inhibitors of Entpdl.
  • the invention provides one or more inhibitors of Dgkz.
  • the invention provides one or more inhibitors of Vamp7.
  • the invention provides one or more inhibitors of Nuak2.
  • the invention provides one or more inhibitors of Hipkl.
  • the invention provides one or more inhibitors of Alk.
  • the inhibitor of Alk includes, for example, for example CH5424802 (Hoffmann-La Roche), LDK378 (Novartis), Crizotinib and PF-02341066 (Pfizer) or AP26113 (Ariad Pharmaceuticals).
  • the invention provides one or more inhibitors of Pdzklipl.
  • therapeutic compositions can include, for example, cytokines, chemokines and other biologic signaling molecules, tumor specific vaccines, cellular cancer vaccines (e.g., GM-CSF transduced cancer cells), tumor specific monoclonal antibodies, autologous and allogeneic stem cell rescue (e.g., to augment graft versus tumor effects), other therapeutic antibodies, molecular targeted therapies, anti-angiogenic therapy, infectious agents with therapeutic intent (such as tumor localizing bacteria) and gene therapy.
  • cytokines e.g., chemokines and other biologic signaling molecules
  • tumor specific vaccines e.g., GM-CSF transduced cancer cells
  • tumor specific monoclonal antibodies e.g., GM-CSF transduced cancer cells
  • autologous and allogeneic stem cell rescue e.g., to augment graft versus tumor effects
  • other therapeutic antibodies e.g., to augment graft versus tumor effects
  • molecular targeted therapies e.g., anti-ang
  • compositions disclosed herein can be formulated for use as or in pharmaceutical compositions.
  • Such compositions can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA).
  • FDA Food and Drug Administration
  • Exemplary methods are described in the FDA's CDER Data Standards Manual, version number 004 (which is available at fda.give/cder/dsm/DRG/drg00301.htm).
  • compositions can include an effective amount of one or more peptides.
  • effective amount and “effective to treat,” as used herein, refer to an amount or a concentration of one or more peptides for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome.
  • compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
  • pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
  • methods can include selection of a human subject who has or had a condition or disease (e.g., cancer).
  • suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), and/or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease).
  • subject refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject”, as used herein, refers to a human (e.g., a man, a woman, or a child). Samples for use in the methods can include serum samples, e.g., obtained from the selected subject.
  • subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection.
  • the subject can be confirmed or identified, e.g. by a health care professional, as having had or having a condition or disease.
  • exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, and/or detecting an indication of a positive immune response.
  • multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidate subject and a second party can test the sample.
  • subjects can be selected and/or referred by a medical practitioner (e.g., a general practitioner).
  • subject selection can include obtaining a sample from a selected subject and storing the sample and/or using the in the methods disclosed herein. Samples can include, for example, cells or populations of cells.
  • the disclosure provides methods for increasing the immune response in a subject in need thereof.
  • the disclosure provides therapies that are particularly useful for the treatment of subjects having cancer.
  • the disclosure provides methods of treatment that include administering to a subject a composition disclosed herein.
  • a composition capable of silencing genes that inhibit T cell function e.g., an immunoresponsive T cell expressing a shRNA of the invention and a desired CAR directed to a tumor antigen.
  • T cell is derived from the patient to be treated and has been modified to express the CAR and an shRNA that reduces expression of a target gene described herein.
  • the cancer is a carcinoma, sarcomas, adenocarcinoma, lymphoma, leukemia, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia (including AML, ALL, and CML), and multiple myeloma.
  • solid and lymphoid cancers including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, test
  • the cancer is melanoma.
  • the cancer is a plasma cell malignancy, for example, multiple myeloma (MM) or pre-malignant condition of plasma cells.
  • MM multiple myeloma
  • the subject has been diagnosed as having a cancer or as being predisposed to cancer.
  • cancer refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia (including AML, ALL, and CML), and multiple myeloma.
  • cancer refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast
  • anti-tumor effect refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.
  • treat refers to partially or completely alleviating, inhibiting, ameliorating, and/or relieving the disease or condition from which the subject is suffering. In some instances, treatment can result in the continued absence of the disease or condition from which the subject is suffering.
  • methods include selecting a subject at risk for or with a condition or disease.
  • the subject's condition or disease can be treated with a pharmaceutical composition disclosed herein.
  • methods include selecting a subject with cancer, e.g., wherein the subject's cancer can be treated by increasing T cell accumulation and infiltration within the tumor.
  • treatments methods can include a single administration, multiple administrations, and repeating administration as required for the prophylaxis or treatment of the disease or condition from which the subject is suffering.
  • treatment methods can include assessing a level of disease in the subject prior to treatment, during treatment, and/or after treatment. In some instances, treatment can continue until a decrease in the level of disease in the subject is detected.
  • the subject can be evaluated to detect, assess, or determine their level of disease.
  • treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected.
  • a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • a therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes, antisense oligonucleotides, chemotherapeutic agents and radiation.
  • any of the methods and any of the compositions disclosed herein with conventional cancer therapies and various drugs in order to enhance the efficacy of such therapies through either reducing the doses/toxicity of conventional therapies and/or to increase the sensitivity of conventional therapies.
  • One conventional therapy is the use of radiation therapy.
  • Another conventional therapy is the use of chemotherapeutic drugs that can be divided into: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and antitumour agents. All of these drugs affect cell division or DNA synthesis and function in some way.
  • Other conventional cancer therapies are agents that do not directly interfere with DNA.
  • agents for which to combine with the present invention may include for example “small-molecule” drugs that block specific enzymes involved in cancer cell growth.
  • Monoclonal antibodies, cancer vaccines, angiogenesis inhibitors, and gene therapy are targeted therapies that can also be combined with the compositions and methods disclosed herein because they also interfere with the growth of cancer cells.
  • test compounds e.g., polypeptides, polynucleotides, inorganic or organic large or small molecule test compounds
  • test compounds that silence, reduces, eliminates, knocks down, knocks out, modulates, or decreases the expression and/or activity of genes selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met
  • small molecules refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons.
  • small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da).
  • the small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).
  • test compounds can be, e.g., natural products or members of a combinatorial chemistry library.
  • a set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity.
  • Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the “split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1:60-6 (1997)).
  • a number of small molecule libraries are commercially available. A number of suitable small molecule test compounds are listed in U.S. Pat. No. 6,503,713, incorporated herein by reference in its entirety.
  • Libraries screened using the methods of the present invention can comprise a variety of types of test compounds.
  • a given library can comprise a set of structurally related or unrelated test compounds.
  • the test compounds are peptide or peptidomimetic molecules.
  • the test compounds are nucleic acids.
  • test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound, e.g., a first test compound that is structurally similar to a known natural binding partner of the target polypeptide, or a first small molecule identified as capable of binding the target polypeptide, e.g., using methods known in the art or the methods described herein, and correlating that structure to a resulting biological activity, e.g., a structure-activity relationship study. As one of skill in the art will appreciate, there are a variety of standard methods for creating such a structure-activity relationship.
  • the work may be largely empirical, and in others, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of a small molecule compound or compounds.
  • a general library of small molecules is screened, e.g., using the methods described herein.
  • a test compound is applied to a test sample, e.g., a cell or living tissue or organ, e.g., an eye, and one or more effects of the test compound is evaluated.
  • a test sample e.g., a cell or living tissue or organ, e.g., an eye
  • the test sample is, or is derived from (e.g., a sample taken from) an in vivo model of a disorder as described herein.
  • an animal model e.g., a rodent such as a rat, can be used.
  • Methods for evaluating each of these effects are known in the art. For example, ability to modulate expression of a protein can be evaluated at the gene or protein level, e.g., using quantitative PCR or immunoassay methods. In some embodiments, high throughput methods, e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999,W. H.
  • a candidate compound that has been screened, e.g., in an in vivo model of a disorder, e.g., cancer, and determined to have a desirable effect on the disorder, e.g., on one or more symptoms of the disorder, can be considered a candidate therapeutic agent.
  • Candidate therapeutic agents, once screened in a clinical setting, are therapeutic agents.
  • Candidate compounds, candidate therapeutic agents, and therapeutic agents can be optionally optimized and/or derivatized, and formulated with physiologically acceptable excipients to form pharmaceutical compositions.
  • test compounds identified as “hits” can be selected and systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameter. Such optimization can also be screened for using the methods described herein.
  • the invention includes screening a first library of compounds using a method known in the art and/or described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create a second library of compounds structurally related to the hit, and screening the second library using the methods described herein.
  • cytotoxic T cells play a central role in immune-mediated control of cancers 1-3 , and monoclonal antibodies that target inhibitory receptors on T cells can induce significant clinical benefit in patients with advanced disease 4-6.
  • many of the regulatory mechanisms that result in loss of T cell function within immunosuppressive tumors remain unknown.
  • the inventors demonstrate that such regulatory mechanisms can be systematically discovered in vivo in the tumor microenvironment. The inventors postulated that shRNAs targeting key inhibitors would enable robust T cell infiltration and accumulation in tumors, despite multiple inhibitory signals.
  • candidate shRNA were discovered by transfer of shRNA-transduced T cells into tumor-bearing mice, followed by deep sequencing to quantify the representation of all hairpins in tumors and lymphoid organs.
  • the majority of shRNAs induced T cell accumulation in tumors but not the spleen, demonstrating feasibility of discovering shRNAs with differential action across tissues.
  • One of the targets was Ppp2r2d, a regulatory subunit of the PP2A phosphatase 7 .
  • Control shRNA-transduced T cells underwent apoptosis upon recognition of melanoma cells, while Ppp2r2d shRNA-transduced T cells accumulated in tumors due to enhanced proliferation and resistance to apoptosis. Ppp2r2d shRNA-expressing T cells also significantly delayed tumor growth. This in vivo approach has wide-spread applications to dissect complex immune functions in relevant tissue microenvironments.
  • Immune cells perform complex surveillance functions throughout the body and interact with many different types of cells in distinct tissue microenvironments.
  • Therapeutic targets for modulating immune responses are typically identified in vitro and tested in animal models at a late stage of the process.
  • the inventors have addressed the challenge of how targets for immune modulation can be systematically discovered in vivo. This is a central issue in oncology because strong infiltration by CD8 T cells—which have cytotoxic function against tumor cells—is associated with a favorable prognosis in multiple types of human cancer 1..3.8 .
  • this natural defense mechanism is severely blunted in the majority of patients by multiple inhibitory signals emanating from the tumor, its stroma, regulatory T cells and myeloid cell populations.
  • mice C57BL/6 mice, TRP-1 mice (transgenic mice expressing T-cell receptor (TCR) specific for tyrosinase-related protein 1) 23 , pmel-1 mice (transgenic mice expressing TCR specific for gp100) 18 , and b2m-1-mice 24 were purchased from The Jackson Laboratory.
  • the Rag1 ⁇ / ⁇ OT-I mice 16 were purchased from Taconic Farms, Inc. Mice were bred at the Dana-Farber Cancer Institute animal facility. All experimental procedures were approved by the Dana-Farber Cancer Institute Animal Care and Use Committee.
  • B16 melanomas an aggressive tumor that is difficult to treat, express the surrogate tumor antigen Ovalbumin (Ova), which is recognized by CD8 T cells from OT-I T cell receptor transgenic mice 16, 17 .
  • Ovalbumin Ovalbumin
  • EL4 thymoma 38 and B16-F10 melanoma 15 cells were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 10011 g/ml streptomycin and 10011 g/ml penicillin.
  • Ovalbumin-expressing B16 tumor cells (B16-Ova) were maintained in the same media with addition of 600 m/mL G418 (Invitrogen).
  • shRNAs were selected for 255 genes over-expressed in dysfunctional T cells (anergic or exhausted state).
  • pLKO.3G vector was obtained from The RNAi Consortium.
  • pLKO-Thy1.1, pLKO-Ametrine, pLKO-RFP, pLKO-TFP vectors were modified from pLKO.3G vector by replacing GFP with the corresponding reporter gene.
  • Murine Ppp2r2d and Cb1b sequences targeted by 10 selected shRNAs are provided in Table 3 (listed in order of shRNA activity (highest to lowest)).
  • the LacZ target sequence targeted by a control shRNA is also listed. All other target sequences can be found in Table 2.
  • Antibodies used were specific for CD4, CD8, Va2, V ⁇ 5.1/5.2, Thy1.1, CD25, CD44, CD62L, CD69, CD122, CD127, IFN ⁇ , TNF ⁇ (BioLegend), PD-1, TIM-3, LAG-3, granzyme B, and H-2Kb (BioLegend),Va3.2 (eBioscience), V ⁇ 13, V ⁇ 14 (BD Biosciences), phospho-Akt (Ser473) and phospho-Bad (Ser112) (Cell Signaling). Apoptotic cells were detected by labeling with annexin V (BioLegend) or activated caspase-3 antibody (Cell Signaling). Mouse anti-CD3/CD28 beads were purchased from Invitrogen.
  • B16-Ova melanomas were cut into small pieces in petri dishes containing 5 mL of PBS, 2% FBS and washed with PBS. Tumors were resuspended in 15 mL RPMI supplemented with 2% FBS, 50U/mL Collagenase Type IV (Invitrogen), 20U/mL DNase (Roche), samples incubated at 37° C. for 2 hours and tissue further dissociated using a gentleMACS Dissociator (Miltenyi Biotech). Suspensions were washed three times with PBS and passed through a 70 ⁇ M strainer.
  • Lymphocytes were isolated by density gradient centrifugation and then either analyzed or sorted by flow cytometry using a FACSAria (BD Biosciences). T cell apoptosis. Cytokine pre-treated OT-I cells were transduced with LacZ or Ppp2r2d shRNAs and injected into mice bearing day 14 B16-Ova tumors. After 7 days, intracellular staining was performed using an activated caspase-3 antibody (Cell Signaling) and CD8/Thy1.1 double-positive T cells were gated in the FACS analysis.
  • B16-Ova tumors from mice treated with OT-I T cells expressing LacZ or Ppp2r2d shRNAs (GFP-expressing vector) were cryopreserved in optimal cutting temperature (O.C.T.) compound (Tissue-Tek).
  • 10 ⁇ m-sections from cryopreserved tumors were were permeabilized with 0.2% Triton X-100, fixed in 4% paraformaldehyde and stained with a GFP antibody (Molecular Probes) in combination with DAPI.
  • TUNEL detection sections were stained with TACS 2 TdT Blue Label (Trevigen) based on manufacturer's directions.
  • RNA samples were visualized using a laser-scanning confocal microscope (Leica SP5X) and analyzed with ImageJ software (NIH). qRT-PCR assay.
  • Total RNA was extracted using TRIzol reagent (Invitrogen).
  • RNA was reverse transcribed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).
  • Real time quantitative PCR reactions were performed as triplicates using an ABI 7900HT instrument with SYBR green (ABI). Rp123 levels were used for normalization.
  • Ppp2r2d forward GGAAGCCGACATCATCTCCAC (SEQ ID NO: 622), Ppp2r2d reverse GTGAGCGCGGCCTTTATTCT (SEQ ID NO: 623); Cb1b forward GGTCGCATTTTGGGGATTATTGA (SEQ ID NO: 624), Cb1b reverse TTTGGCACAGTCTTACCACTTT (SEQ ID NO: 625); Rp123 forward CTGTGAAGGGAATCAAGGGA (SEQ ID NO: 626) and Rp123 reverse TGTCGAATTACCACTGCTGG (SEQ ID NO: 627).
  • IL-7/IL-15 cultured OT-I T cells were transduced with one of five experimental shRNAs (Ppp2r2d, Arhgap5, Alk, Egr2, Ptpn2) or a LacZ control shRNA. Infected cells were sorted to purity using GFP encoded by the vector as a reporter. T cells (5 ⁇ 10 6 ) were injected i.v. into mice bearing day 14 B16-Ova tumors. Seven days later, shRNA-expressing OT-I T cells (CD8+GFP+) were isolated from tumors and spleens.
  • shRNA-expressing OT-I T cells CD8+GFP+
  • Antibodies used for T cell activation were anti-mouse CD3 and anti-mouse CD28 (Biolegend).
  • Antibodies used to capture secreted cytokines were anti-mouse IFN ⁇ (Biolegend), anti-mouse IL-2 (Biolegend), anti-mouse TNF ⁇ (Biolegend) and anti-mouse GM-CSF (Biolegend).
  • Detection antibodies were anti-mouse IFN ⁇ (Biolegend), anti-mouse IL-2 (Biolegend), anti-mouse TNF ⁇ (Biolegend) and anti-mouse GM-CSF (Biolegend), and they were fluorescently labeled with appropriate Alexa Fluor dyes (Invitrogen) following manufacturer's instructions.
  • the lipids used to prepare supported bilayers were: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotinyl Cap PE) (Avanti Polar Lipids).
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • Biotinyl Cap PE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotinyl Cap PE) (Avanti Polar Lipids).
  • the array of nanowells was manufactured by injecting polydimethylsiloxane (PDMS, Dow Corning) prepared at a 10:1 base/catalyst weight ratio into a custom-built mold encasing a micropatterned silicon master. Arrays of nanowells were cured at 70° C. for 4-16 h. Each array comprised 72 ⁇ 24 blocks, each containing a 7 ⁇ 7 (50 ⁇ m ⁇ 50 ⁇ m ⁇ 50 ⁇ m) subarray of nanowells (total of 84,672 wells). The PDMS arrays adhered directly to a 3′′ ⁇ 1′′ glass slide forming a 1 mm thick layer.
  • PDMS polydimethylsiloxane
  • lipid bilayers were prepared as described previously14. Bilayers were generated by applying DOPC liposomes containing 2 mol % biotin-Cap-PE lipids on the PDMS array of nanowells. The surfaces were rinsed with deionized water to remove excess liposomes. Before use, the lipid bilayer was blocked with BSA in PBS (100 ⁇ g/mL) for 45 minutes. The bilayer was then incubated with 1 ⁇ g/mL of streptavidin in a solution of 100 ⁇ g/mL BSA in PBS, followed by incubation with biotinylated CD3 and CD28 antibodies. The device was rinsed extensively with PBS before adding the cells.
  • Capture antibodies were diluted in borate buffer (50 mM sodium borate, 8 mM sucrose, and 50 mM NaCl, pH 9.0) to a final concentration of 10 ⁇ g/mL and deposited on the surface of epoxy-modified slides for 1 h at room temperature. Slides were blocked with 3% non-fat milk in PB ST (PBS with 0.05% (v/v) Tween 20) for 30 min at room temperature and washed with PBS before placing them into contact with the PDMS array of nanowells. A suspension of T cells was dispensed onto the surface of the nanowells, modified with a supported lipid bilayer in media and allowed to settle into the wells.
  • borate buffer 50 mM sodium borate, 8 mM sucrose, and 50 mM NaCl, pH 9.0
  • PB ST PBS with 0.05% (v/v) Tween 20
  • the density of suspended cells applied to the array was optimized empirically to maximize well occupancy by single cells (typically ⁇ 30% of wells).
  • a glass slide coated with capture antibodies was then placed onto the loaded array for cytokine capture.
  • the microarray and glass slide were held together by compression in a hybridization chamber (Agilent Technologies, G2534A) and incubated for 1 h at 37° C. with 5% CO 2. The glass slide was then separated from the array and placed in PBS.
  • slides were incubated for 30 min with blocking buffer (PBS, 10 mg/mL BSA, 0.05% (v/v) Tween-20, 2% mouse serum and 2 mM sodium azide), washed with PBST (PBS+0.05% v/v Tween-20), and then incubated with fluorescence detection antibodies at 1 ⁇ g/mL for 45 min at 25° C.
  • the slides were washed with PBST and PBS, rinsed briefly with water, and dried with a N 2 stream. Reference slides were generated at the end of each experiment with the same detection antibodies used on the printed slides.
  • On-chip image-based cytometry T cells were stained with CellMaskTM Plasma Membrane Stain (Invitrogen, Life Technologies) and SYTOX green (for detection of dead cells, Life Technologies). The cell-loaded arrays of nanowells were mounted face-up on the microscope with a coverslip placed on top of the array. Images were acquired on an automated inverted epifluorescence microscope (Carl Zeiss). Transmitted light and epifluoresence micrographs were collected block-by-block (7 ⁇ 7 microwells per block). The resulting collection of images was analyzed using a custom program to determine the number of cells present in each well and the mean fluorescence intensity of each label. Only viable T cells were considered for the analysis. Although the cells expressed GFP, the fluorescence intensity of GFP was negligible under the utilized microscope acquisition setting compared to SYTOX green, enabling identification of dead cells.
  • shRNAs targeting 255 genes over-expressed in dysfunctional T cells (anergic or exhausted state) 31-37 and 1,307 kinase/phosphatase genes ( ⁇ 5 shRNAs per gene) were obtained from The RNAi Consortium (TRC; Broad Institute, Cambridge, MA, USA).
  • TRC RNAi Consortium
  • OT-I T cells isolated by negative selection were cultured with IL-7 (5 ng/mL, Peprotech) and IL-15 (100 ng/mL, Peprotech) in complete RPMI media (RPMI 1640, 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethonal, 2 mM L-glutamine, 100 ⁇ g/ml streptomycin and 100 ⁇ g/ml penicillin).
  • IL-7 5 ng/mL, Peprotech
  • IL-15 100 ng/mL, Peprotech
  • complete RPMI media RPMI 1640, 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethonal, 2 mM L-glutamine, 100 ⁇ g/ml streptomycin and 100 ⁇ g/ml penicillin.
  • OT-I T cells were spin-infected with lentiviral pools (nine lentiviral shRNA pools and a LacZ control shRNA lentiviral vector control) supplemented with protamine sulfate (5 ⁇ g/mL) in 24-well plates coated with retronectin (5 ⁇ g/mL) at a multiplicity of infection (MOI) of 15. Typically, ⁇ 5 ⁇ 10 6 OT-1 T cells were infected for each pool.
  • OT-I cells were cultured with IL-7 (2.5 ng/mL), IL-15 (50 ng/mL) and IL-2 (2 ng/mL) in complete RPMI media.
  • live shRNA-transduced T were enriched using a dead cell removal kit (Miltenyi), and infected cells were positively selected based on Thy1.1 marker (Stemcell Technologies) to 50-60% Thy1.1 positivity.
  • Successful transduction was monitored by surface expression of the Thy1.1 reporter ( FIG. 2 ).
  • T cells (5 ⁇ 10 6 ) were injected i.v. into C57BL/6 mice bearing day 14 B16-Ova tumors (15 mice per shRNA pool)(number of animals chosen to provide sufficient cells for T cell isolation and PCR).
  • Genomic DNA was isolated from 5 ⁇ 10 6 enriched OT-I cells as the start population for deep sequencing. Seven days later, shRNA-expressing T cells (CD8 + Va2 + V(35 + Thy1.1 + ) were isolated by flow cytometry from tumors, spleens, tumor-draining lymph nodes and irrelevant lymph nodes for isolation of genomic DNA, followed by PCR amplification of the shRNA cassette.
  • FIG. 3 Genomic DNA was isolated (Qiagen) and deep-sequencing templates were generated by PCR of the shRNA cassette. Representation of shRNAs in each pool was analyzed by deep sequencing using an Illumina Genome Analyzer 30. Data were normalized using the average reads of control shRNAs in each pool. Kinase/phosphatase genes were selected for the secondary screen based on expression levels in T cells.
  • shRNAs were over-represented in all tested tissues compared to the starting T cell population (e.g. SHP-1), indicative of enhanced proliferation independent of TCR recognition of a tumor antigen.
  • T cell population e.g. SHP-1
  • shRNAs were over-represented in all tested tissues compared to the starting T cell population (e.g. SHP-1), indicative of enhanced proliferation independent of TCR recognition of a tumor antigen.
  • there was a selective loss of shRNAs within tumors e.g. ZAP-70, a critical kinase in the T cell activation pathway.
  • ZAP-70 a critical kinase in the T cell activation pathway
  • lentiviral vectors encoding five different reporter proteins (GFP, TFP, RFP or Ametrine fluorescent proteins, Thy1.1).
  • Cytokine-pretreated OT-I T cells were transduced with lentiviral vectors driving expression of a single shRNA and a reporter protein; 1 ⁇ 10 6 T cells of each population were mixed and co-injected i.v. into C57BL/6 mice bearing day 14 B16-Ova tumors. After seven days T cells were isolated from tumors, spleens and lymph nodes, and the percentage of reporter-positive CD8 + Va2 + Vf35 + T cells was determined by flow cytometry based on co-introduced reporters.
  • T cell accumulation in tumors was >10-fold relative to spleen.
  • the strongest phenotype was observed with shRNAs targeting Ppp2r2d, a regulatory subunit of the PP2A phosphatase7.
  • CD8+OT-I or CD4+ TRP-1 T cells expressing Ppp2r2d or LacZ shRNAs were injected into mice bearing day 14 B16-Ova tumors.
  • shRNA-expressing T cells were identified in tumors and spleens using Thy1.1 reporter ( FIG. 8 , % Thy1.1 + CD8 T cells, left panels).
  • Total numbers of LacZ or Ppp2r2d shRNA-expressing T cells were determined in tumors and spleens 7 days following transfer of 2 ⁇ 10 6 shRNA-expressing cells ( FIG. 8 , right panels). Fold-enrichment of Ppp2r2d versus LacZ shRNA-expressing T cells in tumors is indicated.
  • Ppp2r2d shRNA not only induced accumulation of OT-I CD8 T cells, but also CD4 T cells (from TRP-1 TCR transgenic mice) 23 , with T cell numbers in tumors being significantly higher when Ppp2r2d rather than LacZ shRNA was expressed (36.3-fold for CD8; 16.2-fold for CD4 T cells) ( FIG. 8 ).
  • T cell enrichment in tumors compared to spleen for cells expressing a panel of Ppp2r2d or Cb1b shRNAs ( FIG. 17 , upper panels) Ppp2r2d and Cb1b mRNA levels were also measured by qPCR prior to T cell transfer ( FIG. 17 , lower panels). The strongest T cell enrichment in tumors was observed for shRNAs with >80% knock-down efficiency at the mRNA level (shRNAs #1 and 2 for both Ppp2r2d and Cb1b).
  • CD8 T cell accumulation correlated with the degree of Ppp2r2d knock-down, and two Ppp2r2d shRNAs with the highest in vivo activity induced the lowest levels of Ppp2r2d mRNA ( FIG. 17 ).
  • Ppp2r2d knockdown was also confirmed at the protein level using a quantitative mass spectrometry approach ( FIG. 18 ).
  • a previously reported approach for absolute quantification (AQUA) of proteins from cell lysates by mass spectrometry was used to measure the effect of Ppp2r2d shRNA expression at the protein level (Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W. & Gygi, S. P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. PNAS, 100, 6940-6945 (2003).
  • This strategy is based on a ‘selective reaction monitoring’ approach in which a synthetic peptide with incorporated stable isotopes is used as an internal standard for mass spectrometry analysis.
  • OT-I cells expressing LacZ or Ppp2r2d shRNAs were sorted to purity using FACS.
  • Cells (1 ⁇ 10 6 ) were lysed in 1 ml of MPER extraction reagent (Pierce) containing a Protease Inhibitor Cocktail (Sigma), 1 mM EDTA and 1 mM PMSF for 15 minutes on ice with occasional vortexing. Cell debris was removed by centrifugation and the protein supernatant was filtered (0.2 ⁇ m SpinX centrifuge filter, Costar).
  • Protein concentration was determined by Bradford assay (Biorad) and UV280 nm analysis (Nanodrop instrument); 0.1 mg of cellular protein was separated by SDS-PAGE and stained with Coomassie blue reagent (Pierce). Gel bands corresponding to a MW range of 45-60 kDa were excised followed by in-gel digestion of proteins with trypsin.
  • Eluted peptides were spiked with 300 fmol of isotopically labeled Ppp2r2d (FFEEPEDPSS[13C-15N-R]-OH)(SEQ ID NO: 628) and Actin B (GYSFTTTAE[13C-15N-R]-OH) (SEQ ID NO: 629) peptides (21st Century Biochemicals) for quantification by LC MS/MS (LTQ XL Orbitrap, Thermo Scientific).
  • the Ppp2r2d peptide was chosen from a region of the protein that differs from other regulatory subunits of PP2A.
  • Ppp2r2d shRNA activity was specific because the phenotype was reversed when a mutated Ppp2r2d cDNA (with wild-type protein sequence, but mutated DNA sequence at the shRNA binding site) was co-introduced with the Ppp2r2d shRNA ( FIG. 9 , 10 a - c ).
  • OT-I CD8 T cells over-expressed Ppp2r2d in tumors compared to spleen (in the absence of any shRNA expression), suggesting that it is an intrinsic component of the signaling network inhibiting T cell function in tumors ( FIG. 19 ).
  • Mutant Ppp2r2d cDNA with preserved protein sequence but disrupted shRNA binding site were generated. Wild-type Ppp2r2d cDNA was isolated by RT-PCR using forward primer GGATCCATGGCAGGAGCTGGAGGC (SEQ ID NO: 630) and reverse primer: GCTAGCATTAATTTTGTCCTGGAATATATACAAGTTATTGGTGG (SEQ ID NO: 631).
  • the target sequence of Ppp2r2d shRNA, CCCACATCAGTGCAATGTATT was mutated to TCCCCACCAATGTAACGTGTT (SEQ ID NO: 633) by overlapping PCR (which conserves protein coding sequence) using forward primer: TCCATCCCCACCAATGTAACGTGTTTGTTTACAGCAGCAGCAAGG (SEQ ID NO: 634) and reverse primer: AAACAAACACGTTACATTGGTGGGGATGGAACTCTGCGGCAGTGA (SEQ ID NO: 635).
  • the Ppp2r2d shRNA reduced GFP levels when wild-type Ppp2r2d.
  • the Ppp2r2d shRNA was not able to reduce expression of the GFP reporter in cells expressing the mutant Ppp2r2d cDNA, demonstrating that the shRNA binding site had been successfully mutated. ( FIG. 10 a )
  • Ppp2r2d mutant cDNA also prevents phenotype induced by Ppp2r2d shRNA.
  • FIG. 10 b Ppp2r2d shRNA was cloned into the mutant Ppp2r2d cDNA-2A-GFP construct which resulted in co-expression of Ppp2r2d shRNA and mutated Ppp2r2d cDNA in one vector.
  • OT-I T cells were separately infected with lentiviruses encoding LacZ shRNA (Thy1.1), Ppp2r2d shRNA (Ametrine) or Ppp2r2d shRNA plus mutant Ppp2r2d cDNA (GFP).
  • mice 10 b These three populations there then mixed at the same ratio and injected into mice bearing day 14 B16-Ova tumors.
  • each T cell population was quantified in tumors and spleens by gating on OT-I (CD8 + Va2 + V(35 + )-T cells followed by analysis of populations marked by Thy1.1, Ametrine or GFP expression.
  • FIG. 10 c provides real-time PCR analysis for Ppp2r2d expression in OT-I T cells transduced with LacZ shRNA, Ppp2r2d shRNA, and Ppp2r2d shRNA plus Ppp2r2d mutant cDNA. Also, the Ppp2r2d shRNA with the highest in vivo activity was associated with the lowest levels of Ppp2r2d mRNA ( FIG. 11 ).
  • FIG. 12 a Microarray analysis of tumor-infiltrating T cells expressing experimental or control shRNAs showed that each shRNA induced a distinct set of gene expression changes, with some overlap between particular shRNAs.
  • Two genes (Egr2 and Ptpn2) have known functions in T cells. Enrichment in tumor versus spleen was calculated based on deep sequencing results from the secondary screen.
  • FIG. 12 a Clustering of mean expression levels for mRNAs found to be significantly regulated by T cells in spleens or tumors expressing the LacZ control shRNA or one of five experimental shRNAs.
  • FIG. 12 c is a Venn diagram showing overlaps between expression signatures by tumor-infiltrating T cells transduced with one of the five experimental shRNAs (signatures defined as an Anova p ⁇ 0.01 as described above). Indicated are the numbers of overlapping probe IDs for any combination of the 5 signatures, as indicated by the overlapping ovals. The significance of the overlaps versus that expected by random chance (Fishers Exact Test) is shown in the accompanying table.
  • T cell infiltration into tumors was assessed by transfer of OT-I CD8 T cells labeled with a cytosolic dye, CFSE.
  • OT-I T cells expressing Ppp2r2d or LacZ shRNAs were labeled with CFSE and injected into B16-Ova tumor-bearing mice. Twenty-four hours later transduced T cells were isolated from tumors and spleens and quantified by flow cytometry.
  • OT-I T cells expressing LacZ or Ppp2r2d shRNAs were purified using the Thy1.1 reporter and cultured in complete RPMI media without added cytokines for 24 hours.
  • Live cells isolated by Ficoll density gradient centrifugation (Sigma) were labeled with CFSE (carboxyfluorescein diacetate, succinimidyl ester, Invitrogen), and 2 ⁇ 106 labeled cells were injected into mice bearing day 14 B16-Ova tumors.
  • CFSE dilution was quantified by flow cytometry at 24 hours and days 3, 5 and 7 following transfer.
  • intracellular staining was performed on days 3, 5 and 7 for IFN ⁇ , TNF ⁇ and isotype controls (BD).
  • Ppp2r2d The action of Ppp2r2d was downstream of T cell receptor activation because T cell accumulation was enhanced in tumors and to a lesser extent in tumor-draining lymph nodes. In contrast, no accumulation was observed in irrelevant lymph nodes or the spleen where the relevant antigen is not presented to T cells ( FIG. 15 ).A substantial degree of T cell accumulation was even observed for LacZ shRNA-transduced T cells (complete dilution of CFSE dye by day 7), despite the presence of small numbers of such cells in tumors. This suggested that LacZ shRNA-transduced T cells were lost by apoptosis.
  • OT-I T cells expressing LacZ or Ppp2r2d shRNAs were purified based on Thy1.1 expression and labeled with CFSE, as described above.
  • CFSE labeled OT-I T cells (1 ⁇ 10 5 ) were co-cultured with 5 ⁇ 10 4 B16-Ova cells per well in a 96-well plate for 72 h. Prior to the assay, B16-Ova cells were exposed to 1 ng/mL IFN ⁇ for 48 hours (to induce MHC class I, which is not expressed in vitro) and washed three times. Apoptosis of OT-I T cells was detected by annexin V labeling of CD8+ cells. ( FIG.
  • B16-Ova cells (2 ⁇ 10 5 ) were injected s.c. into female C57BL/6 mice (10 weeks of age). On day 12, mice bearing tumors of similar size were divided into 7 groups (7-8 mice/group).
  • Anti-CD3/CD28 bead activated CD4 TRP-1 or/and CD8 OT-I T cells infected with Ppp2r2d or LacZ shRNA vectors (2 ⁇ 10 6 T cells each) were injected i.v. on days 12 and day 17.
  • mice were treated at day 10 with anti-CD3/CD28 bead activated CD4 TRP-1 and CD8 pmel-1 T cells expressing Ppp2r2d or LacZ shRNAs (3 ⁇ 10 6 T cells each). Tumor size was measured every three days following transfer and calculated as length ⁇ width. Mice with tumors ⁇ 20 mm on the longest axis were sacrificed.
  • Ppp2r2d shRNA-transduced CD8 T cells may be able to proliferate and survive even when they recognize their antigen directly presented by B16-Ova tumor cells.
  • This idea was tested by implantation of tumor cells into b2m ⁇ / ⁇ mice which are deficient in expression of MHC class I proteins. In such mice, only tumor cells but not professional antigen presenting cells of the host could present tumor antigens to T cells.
  • Ppp2r2d shRNA-transduced OT-I CD8 T cells showed massive accumulation within B16-Ova tumors in b2m ⁇ / ⁇ mice ( FIG. 120 while there were very small numbers of T cells in contralateral B16 tumors that lacked expression of the Ova antigen. T cells expressing a Ppp2r2d shRNA could thus effectively proliferate and survive in response to tumor cells, despite a lack of suitable co-stimulatory signals and an inhibitory microenvironment.
  • T cells were activated for 3 hours by CD3/CD28 antibodies on lipid bilayers, followed by 1 hour cytokine capture on antibody-coated slides.
  • CD8 T cells showed a higher secretion rate for IFN ⁇ , IL-2 and GM-CSF, and a larger fraction of T cells more than one cytokine ( FIG. 21 b, c ).
  • the presence of larger numbers of IFN ⁇ -producing T cells was confirmed by intracellular cytokine staining ( FIG. 21 d , FIG. 20 ).
  • PP2A phosphatase is composed of a catalytic and scaffolding subunit, and its substrate specificity is determined by one of many regulatory subunits 7.
  • Ppp2r2d directs PP2A to Cdk1 substrates during interphase and anaphase; it thereby inhibits entry into mitosis and induces exit from mitosis 25 .
  • PP2A plays a gatekeeper role for BAD-mediated apoptosis.
  • Phosphorylated BAD is sequestered in its inactive form in the cytosol by 14-3-3, while dephosphorylated BAD is targeted to mitochondria where it causes cell death by binding Bc1-X L and Bc1-2 26 .
  • PP2A phosphatases have also been shown to interact with the cytoplasmic domains of CD28 and CTLA-4 as well as Carma1 (upstream of the NF- K B pathway), but it is not known which regulatory subunits are required for these activities; Ppp2r2d antibodies suitable for the required biochemical studies are currently not available.
  • B16-Ova tumor cells (2 ⁇ 10 5 ) were injected subcutaneously into female C57BL/6 mice (10 weeks of age). On day 12, mice bearing tumors of similar size were divided into seven groups (7-8 mice/group), either receiving no T cells, 2 ⁇ 10 6 shRNA-transduced TRP-1 CD4 T cells, 2 ⁇ 10 6 shRNA infected OT-I CD8 T cells, or both CD4 and CD8 T cells (days 12 and day 17).
  • anti-CD3/CD28 bead activated CD4 TRP-1 or/and CD8 OT-I T cells infected with Ppp2r2d or LacZ shRNA vectors (2 ⁇ 10 6 T cells each) were injected i.v. on days 12 and day 17.
  • mice were treated at day 10 with anti-CD3/CD28 bead activated CD4 TRP-1 and CD8 pmel-1 T cells expressing Ppp2r2d or LacZ shRNAs (3 ⁇ 106 T cells each). Tumor size was measured every three days following transfer and calculated as length ⁇ width. Mice with tumors ⁇ 20 mm on the longest axis were sacrificed.
  • Ppp2r2d-silencing improved the therapeutic activity of CD4 and CD8 T cells, and a synergistic effect was observed when Ppp2r2d shRNA-transduced CD4 and CD8 T cells were co-administered ( FIG. 16 a, b ).
  • a Ppp2r2d shRNA also enhanced anti-tumor responses when introduced into T cells specific for endogenous tumor antigens (pmel-1 CD8 T cells and TRP-1 CD4 T cells) ( FIG. 16 c ).
  • Ppp2r2d-silenced T cells acquired an effector phenotype in tumors ( FIG. 22 a ) and >30% of the cells expressed granzyme B ( FIG. 23 a ). Consistent with greatly increased numbers of such effector T cells in tumors ( FIG. 23 b ), TUNEL staining demonstrated increased apoptosis in tumors when Ppp2r2d rather than LacZ shRNA expressing T cells were present ( FIG. 23 c ). B16 melanomas are highly aggressive tumors in part because MHC class I expression is very low. Interestingly, Ppp2r2d but not LacZ shRNA-expressing T cells significantly increased MHC class I expression (H-2Kb) by tumor cells ( FIG.
  • FIG. 23 d possibly due to the observed increase in IFN ⁇ secretion by T cells ( FIG. 21 a - c , FIG. 13 e ).
  • a Ppp2r2d shRNA did not reduce expression of inhibitory PD-1 or LAG-3 receptors on tumor-infiltrating T cells, demonstrating that its mechanism of action is distinct from these known negative regulators of T cell function ( FIG. 22 b ). This finding suggests combination approaches targeting these intracellular and cell surface molecules.
  • Targeting of key regulatory switches may offer new approaches to modify the activity of T cells in cancer and other pathologies.
  • the efficacy of such T cell-based therapies could be enhanced by shRNA-mediated silencing of genes that inhibit T cell function in the tumor microenvironment.

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Abstract

The present disclosure provides, in part, methods of discovering immunotherapy targets in vivo, therapeutic compositions (e.g., shRNA, immunoresponsive cells expressing shRNA and/or a chimeric antigen receptors (CAR)), and methods of use thereof.

Description

    RELATED APPLICATIONS
  • This application is a continuation of application Ser. No. 17/102,787, filed Nov. 24, 2020, which is a continuation of application Ser. No. 15/944,330, filed Apr. 3, 2018, now U.S. Pat. No. 10,876,120, which is a division of application Ser. No. 14/897,210, filed Dec. 9, 2015, now U.S. Pat. No. 9,944,931, which claims the benefit under 35 U.S.C. § 371 of International Application No. PCT/US2014/041739, filed Jun. 10, 2014, which claims priority to and the benefit of provisional applications U.S. Ser. No. 61/929,821, filed Jan. 21, 2014, U.S. Ser. No. 61/921,303, filed Dec. 27, 2013 and U.S. Ser. No. 61/833,298, filed Jun. 10, 2013, the contents of all of which are incorporated herein by reference in their entireties.
    • GOVERNMENT SUPPORT
  • This invention was made with government support under grant numbers R01 CA173750, AI073861, and P30 CA014051 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 electronically in ST.26 XML format and is hereby incorporated by reference in its entirety. The ST.26 XML, created on Jan. 3, 2023, is named 514293_50010064_SEQ_LISTING_ST26.txt and is 427 KB in size.
    • TECHNICAL FIELD
  • This invention relates to methods of discovering immunotherapy targets in vivo, therapeutic compositions that modulate immunotherapy targets (e.g., shRNA, immunoresponsive cells expressing shRNA and, in some cases a receptor targeting a cancer cell, e.g., a chimeric antigen receptors (CAR)), and related methods of use.
    • BACKGROUND
  • Cytotoxic T cells play a central role in immune-mediated control of cancers1-3, and monoclonal antibodies that target inhibitory receptors on T cells can induce significant clinical benefit in patients with advanced disease4-6. For survival, tumors have developed numerous immunosuppressive mechanisms to promote their own growth and to successfully evade the host immune system, effectively blocking the activity of T cells in the tumor microenvironment. This is a central issue in oncology because strong infiltration by CD8 T cells, which have cytotoxic function against tumor cells, is associated with a favorable prognosis in multiple types of human cancer1,3,11. This natural defense mechanism is severely blunted in the majority of patients by multiple inhibitory signals emanating from the tumor, its stroma, regulatory T cells and myeloid cell populations.9-11 Various molecular and cellular immunosuppressive mechanisms responsible for tumor evasion have been identified. Certain of these mechanisms target immune antitumor effector cells. However, many of the regulatory mechanisms that result in loss of T cell function within immunosuppressive tumors remain unknown. Improving on the limited success of cancer immunotherapy requires new approaches to inhibit immunosuppressive pathways initiated by tumor cells to evade the host immune system.
    • SUMMARY
  • The present disclosure provides targets for inhibiting immunosuppressive pathways used by tumor cells to inactivate and/or suppress immune cells.
  • The disclosure also provides provides compositions and methods related to shRNA with therapeutic potential.
  • The disclosure also provides immunoresponsive cells, including T cells (e.g., cells targeting a tumor antigen) expressing at least one shRNA or other nucleic acid molecule capable of silencing genes that inhibit T cell function.
  • The disclosure also provides immunoresponsive cells, including T cells, harboring at least one vector expressing a shRNA and at least one chimeric antigen receptor directed to a tumor antigen.
  • In some embodiments, the disclosure provides immunoresponsive cells having tumor specificity comprising a vector encoding a shRNA capable of silencing genes that inhibit T cell function. In some aspects, the shRNA sequence reduces the expression of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm 1 g, Blvrb, Tnkl, Prkab2, Trpm7 or Ppp3cc. In another aspect, the shRNA comprises 15 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 604-620 and 653-678. In some aspects, the immunoresponsive cell further comprises a vector encoding a tumor-specific T-cell receptor. In some aspects, the immunoresponsive cell is selected from the group consisting of a tumor-infiltrating lymphocyte (TIL), a Natural Killer T cell (NKT), a cytotoxic T lymphocyte (CTL), and a CD4 T cell.
  • In some embodiments, the immunoresponsive cell comprises a vector encoding a CAR, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and a stimulatory domain. In some aspects, the antigen binding domain binds a tumor antigen or pathogen antigen. Exemplary tumor antigens include, for example, prostate-specific membrane antigen (PSMA), Carcinoembryonic Antigen (CEA), CD19, CD20, CD22, ROR1, mesothelin, CD333/IL3Ra, c-Met, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, ERBB2, BIRC5, CEACAM5, WDR46, BAGE, CSAG2, DCT, MAGED4, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE8, IL13RA2, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA9, MAGEA10, MAGEA12, MAGEB1, MAGEB2, MAGEC2, TP53, TYR, TYRP1, SAGE1, SYCP1, SSX2, SSX4, KRAS, PRAME, NRAS, ACTN4, CTNNB1, CASP8, CDC27, CDK4, EEF2, FN1, HSPA1B, LPGAT1, ME1, HEAT, TRAPPC1, MUM3, MYO1B, PAPOLG, OS9, PTPRK, TPI1, ADFP, AFP, AIM2, ANXA2, ART4, CLCA2, CPSF1, PPIB, EPHA2, EPHA3, FGF5, CA9, TERT, MGAT5, CEL, F4.2, CAN, ETV6, BIRC7, CSF1, OGT, MUC1, MUC2, MUM1, CTAG1A, CTAG2, CTAG, MRPL28, FOLH1, RAGE, SFMBT1, KAAG1, SART1, TSPYL1, SART3, SOX10, TRG, WT1, TACSTD1, SILV, SCGB2A2, MC1R, MLANA, GPR143, OCA2, KLK3, SUPT7L, ARTC1, BRAF, CASP5, CDKN2A, UBXD5, EFTUD2, GPNMB, NFYC, PRDX5, ZUBR1, SIRT2, SNRPD1, HERV-K-MEL, CXorf61, CCDCl10, VENTXP1, SPA17, KLK4, ANKRD30A, RAB38, CCND1, CYP1B1, MDM2, MMP2, ZNF395, RNF43, SCRN1, STEAP1, 707-AP, TGFBR2, PXDNL, AKAP13, PRTN3, PSCA, RHAMM, ACPP, ACRBP, LCK, RCVRN, RPS2, RPL10A, SLC45A3, BCL2L1, DKK1, ENAH, CSPG4, RGS5, BCR, BCR-ABL, ABL-BCR, DEK, DEK-CAN, ETV6-AML1, LDLR-FUT, NPM1-ALK1, PML-RARA, SYT-SSX1, SYT-SSX2, FLT3, ABL1, AML1, LDLR, FUT1, NPM1, ALK, PML1, RARA, SYT, SSX1, MSLN, UBE2V1, HNRPL, WHSC2, EIF4EBP1, WNK2, OAS3, BCL-2, MCL1, CTSH, ABCC3, BST2, 1VIFGE8, TPBG, FMOD, XAGE1, RPSA, COTL1, CALR3, PA2G4, EZH2, FMNL1, HPSE, APC, UBE2A, BCAP31, TOP2A, TOP2B, ITGB8, RPA1, ABI2, CCNI, CDC2, SEPT2, STAT1, LRP1, ADAM17, JUP, DDR1, ITPR2, HMOX1, TPM4, BAAT, DNAJC8, TAPBP, LGALS3BP, PAGE4, PAK2, CDKN1A, PTHLH, SOX2, SOX11, TRPM8, TYMS, ATIC, PGK1, SOX4, TOR3A, TRGC2, BTBD2, SLBP, EGFR, IER3, TTK, LY6K, IGF2BP3, GPC3, SLC35A4, HSMD, H3F3A, ALDH1A1, MFI2, MMP14, SDCBP, PARP12, MET, CCNB1, PAX3-FKHR, PAX3, FOXO1, XBP1, SYND1, ETV5, HSPA1A, HMHA1, TRIM68, and any combination thereof. In some aspects, the antigen binding domain is an antigen-binding fragment of an antibody (e.g., Fab or a scFv). The intracellular domains of such CARs contain cytoplasmic signaling domains derived from the T cell receptor and costimulatory molecules.
  • In some embodiments, the vector is a plasmid, retroviral vector, or lentiviral vector.
  • In some embodiments, the disclosure provides isolated nucleic acid molecules encoding a shRNA sequence. In another embodiment, the disclosure provides isolated nucleic acid molecules encoding a CAR. In yet another embodiment, the disclosure provides isolated nucleic acid molecules encoding a CAR and a shRNA sequence. In some aspects, the isolated nucleic acid encodes a shRNA sequence reduces the expression of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, or Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm 1 g, Blvrb, Tnkl, Prkab2, Trpm7 or Ppp3cc. In another aspect, the isolated nucleic acid encodes a shRNA comprising 15 contiguous nucleotides complementary a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 604-620 and 653-678.
  • In some embodiments, the isolated nucleic acid encodes a CAR comprising an antigen binding domain, a transmembrane domain, a stimulatory domain, and a co-stimulatory domain. In some embodiments, the antigen binding domain is an antigen-binding fragment of an antibody (e.g., Fab or a scFv). In some embodiments, the antigen binding domain is a cytoplasmic signaling domain derived from the T cell receptor and costimulatory molecules.
  • In some embodiments, the antigen-binding domain binds tumor antigen (e.g., a tumor antigen associated with a solid tumor, lymphoid tumor, melanoma, carcinoma, sarcomas, adenocarcinoma, lymphoma, leukemia, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer).
  • In some embodiments the disclosure provides vectors comprising an isolated nucleic acid encoding a shRNA sequence, an isolated nucleic acid encoding a CAR, or an isolated nucleic acid encoding a CAR and a shRNA sequence. In some aspects, the vector is a plasmid, lentiviral vector, retroviral vector, adenoviral vector, adeno-associated viral vector. The shRNA can be operably linked to RNA polymerase II promoter or an RNA polymerase III promoter.
  • In yet other embodiments, the invention provides compositions comprising immunoresponsive cells according to the invention, and a pharmaceutically acceptable carrier.
  • In some embodiments, the disclosure provides immunoresponsive cells transfected with a first vector encoding a CAR and a second vector encoding a shRNA sequence. In some aspects, the shRNA sequence reduces the expression of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Map3k3, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm 1 g, Blvrb, Tnkl, Prkab2, Trpm7 or Ppp3cc. In another aspect, the shRNA comprise 15 contiguous nucleotides complementary a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 604-620 and 653-678. In some aspects, the immunoresponsive cell further comprises a vector encoding a tumor-specific T-cell receptor. In some aspects, the immunoresponsive cell is selected from the group consisting of a tumor-infiltrating lymphocyte (TIL), a Natural Killer T cell (NKT), a cytotoxic T lymphocyte (CTL), and a CD4 T cell.
  • In some embodiments, the disclosure provides methods for treating cancer in a subject, the method comprising administering to the subject an autologous T cell modified to express a tumor-specific T-cell receptor or CAR and an shRNA, wherein the shRNA sequence reduces the expression of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Map3k3, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 or Ppp3cc. In some aspects, the shRNA sequence comprises 15 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 604-620 and 653-678; and wherein the CAR comprises an antigen binding domain, a transmembrane domain, a stimulatory domain, and a co-stimulatory domain. In some aspects, the CAR comprises an antigen binding domain, a transmembrane domain, a stimulatory domain, and a co-stimulatory domain.
  • In some embodiments, the disclosure provides methods for treating cancer in a subject, the method comprising administering to the subject an autologous T cell modified to express a tumor-specific T-cell receptor or CAR and an shRNA of the invention. In yet another embodiment, the disclosure provides methods for treating cancer in a subject in need thereof by silencing genes that inhibit T cell function comprising administering to the subject an immunoresponsive cell comprising a vector, the vector encoding a tumor-specific T-cell receptor or a CAR and a shRNA sequence of the invention.
  • In some embodiments, the disclosure provides methods for identifying a gene that inhibits the function of an immunoresponsive T cell, the method comprising providing a population of immunoresponsive T cells harboring vectors expressing a shRNA, contacting the population of immunoresponsive T cells with an immunosuppressive tumor, determining whether a shRNA restores T cell function within the immunosuppressive tumor, and identifying a gene associated with a shRNA that restores T cell function within the tumor as a gene that inhibits the function of tumor-infiltrating T cells.
  • In some embodiments, the disclosure provides methods for increasing the immune response in a subject in need thereof, the method comprising administering a therapeutic agent that modulates the activity of a gene selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc.
  • In some cases the sequence encoding an shRNA comprises a first sequence comprising 15-25 (15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) nucleotides complementary to any of SEQ ID NOs: 604-620 or SEQ ID NOs: 653-678 and a second sequence that is the reverse complement of the first sequence with one or no mismatches (i.e., is perfectly complementary to the first sequence), and a third sequence of 5-9 nucleotides positioned between the first and second sequences.
  • Unless otherwise defined, 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 invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
  • Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
    • DESCRIPTION OF DRAWINGS
  • The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
  • FIG. 1 is a schematic diagram demonstrating an exemplary approach for in vivo discovery of shRNAs that enhance T cell infiltration and accumulation within the tumor microenvironment.
  • FIG. 2 is a set of graphs showing representative flow cytometry plots of CD8+T cells from Rag1−/−/OT-I TCR transgenic mice following infection with an shRNA vector. Transduction efficiency was determined based on expression of the Thy1.1 reporter encoded by the lentiviral vector. Cytokine-cultured T cells expressing the LacZ control shRNA were then stained with a panel of activation markers (black lines; isotype control, shaded). The majority of infected T cells exhibited a central memory phenotype (CD62L+CD44+).
  • FIG. 3 is a set of graphs showing representative flow cytometry plots of OT-I T cells sorted from tumors and secondary lymphoid organs for deep sequencing analysis (dLN, tumor-draining lymph node; irLN, irrelevant lymph node). CD8+Va2+Vf35+Thy 1.1+ cells were sorted and genomic DNA was extracted for PCR amplification of the shRNA cassette.
  • FIG. 4 is a set of graphs showing deep sequencing data from in vivo shRNA pool screen. Upper row, sequence reads for all genes in a pool in tumor, irrelevant (irLN) and draining lymph node (dLN); lower row, three individual genes (LacZ, negative control) are plotted in comparison to spleen for tumors, irrelevant lymph nodes (irLN) and tumor-draining lymph nodes (dLN). Sequence reads are plotted for these tissues versus spleen. Dashed lines indicate a deviation by log 2 from diagonal.
  • FIG. 5 is a set of graphs showing deep sequencing data from T cell dysfunction screen. shRNA sequencing reads for genes positive in secondary screen are plotted in comparison to spleen for tumors (red), irrelevant lymph nodes (irLN, blue) and tumor-draining lymph nodes (dLN, green), with dashed lines indicating a deviation of log 2 from the diagonal. Data show enrichment of particular shRNAs representing these genes in tumors compared to spleens or lymph nodes.
  • FIG. 6 is a graph showing flow cytometry based quantification of OT-I CD8+T cell enrichment in tumors relative to spleen. The percentage of shRNA-expressing OT-I T cells was determined by flow cytometry in tumors/spleens by gating on reporter proteins in CD8+Va2+Vf35+T cells. Statistical significance was determined for each experimental shRNA against LacZ shRNA (fold enrichment tumor/spleen) (n=3; * p<0.05, ** p<0.01, Student's t-test).
  • FIG. 7 is a set of graphs showing representative flow cytometry plots of cell enrichment in tumor transduced with shRNA vectors (LacZ, Akap8I, Smad2, Rbks, Dgkz). The percentage of shRNA-expressing OT-I T cells was determined by flow cytometry in tumors/spleens by gating on reporter proteins in CD8+Va2+Vf35+ T cells.
  • FIG. 8 is a set of graphs showing flow cytometry-based quantification of CD4+ and CD8+ T cell enrichment in tumors. shRNA-expressing T cells were identified in tumors and spleens using Thy1.1 reporter (% Thy1.1+CD8 T cells or CD4+ T cells, top and bottom panels). Total numbers of LacZ or Ppp2r2d shRNA-expressing T cells were determined in tumors and spleens 7 days following transfer of 2×106 shRNA-expressing cells (right panels). Fold-enrichment of Ppp2r2d versus LacZ shRNA-expressing T cells in tumors is indicated.
  • FIG. 9 is a graph showing reversal of Ppp2r2d shRNA-mediated T cell expansion in tumors by Ppp2r2d cDNA with a mutated shRNA binding site but preserved protein sequence. The three cell populations were identified based on co-expressed reporters; fold-enrichment was calculated based on percentage of reporter-positive cells in tumors versus spleens.
  • FIG. 10 a describes the generation of mutant Ppp2r2d cDNA with preserved protein sequence but disrupted shRNA binding site. EL4 cells were transduced with mutant or wild type Ppp2r2d cDNA on a vector also containing GFP. GFP-positive cells were sorted to purity and transduced with LacZ or Ppp2r2d shRNA vectors expressing a Thy1.1 reporter. shRNA-transduced (Thy1.1+) cells were analyzed by flow cytometry for GFP expression. The Ppp2r2d shRNA reduced GFP levels when wild-type Ppp2r2d, but not when mutant Ppp2r2d was expressed. (SEQ ID NOS: 679-681 shown.)
  • FIG. 10 b demonstrates that expression of Ppp2r2d mutant cDNA prevents phenotype induced by Ppp2r2d shRNA. OT-I T cells were transduced with a vector encoding LacZ shRNA, Ppp2r2d shRNA or Ppp2r2d shRNA plus mutant Ppp2r2d cDNA. The different cell populations were normalized for transduction efficiency and co-injected into B16-Ova tumor bearing mice. The percentage of each T cell population in tumors and spleens was quantified by gating on CD8+Va2+Vf35+T cells; transduced cells were detected based on expression of Thy1.1 or Ametrine/GFP fluorescent reporters (representative data from 2 independent experiments, n=3 mice per experiment).
  • FIG. 10 c is a graph demonstrating real-time PCR analysis for Ppp2r2d expression in OT-I T cells transduced with LacZ shRNA, Ppp2r2d shRNA, and Ppp2r2d shRNA plus Ppp2r2d mutant cDNA. Data represent biological replicates (n=3), each value represents mean+/−s.d.
  • FIG. 11 is a graph demonstrating real-time qPCR analysis for Ppp2r2d mRNA levels in OT-I T cells transduced with LacZ shRNA or one of three Ppp2r2d shRNAs identified in the screen.
  • FIG. 12 a is a table demonstrating enrichment of particular shRNAs in tumor versus spleen which was calculated based on deep sequencing results from the secondary screen.
  • FIG. 12 b demonstrates clustering of mean expression levels for mRNAs found to be significantly regulated by T cells in or tumors expressing the LacZ control shRNA or one of five experimental shRNAs. Significant expression differences were defined as an Anova p value <0.01 between T cells expressing LacZ control shRNA or one of five experimental shRNAs (Alk, Arhgap5, Egr2, Ptpn2 or Ppp2r2d) (JMP-Genomics 6.0, SAS Institute Inc.). mRNAs significantly regulated in one or more treatment groups are shown after clustering (Fast Ward).
  • FIG. 12 c is a Venn diagram showing overlaps between expression signatures by tumor-infiltrating T cells transduced with one of the five experimental shRNAs (signatures defined as an Anova p<0.01 as described above). Indicated are the numbers of overlapping probe IDs for any combination of the 5 signatures, as indicated by the overlapping ovals. The significance of the overlaps versus that expected by random chance (Fishers Exact Test) is shown in the accompanying table.
  • FIG. 13 a is a set of graphs showing representative flow cytometry plots of demonstrating the frequency of Ppp2r2d or LacZ shRNA-transduced CD8 T cells in tumors on day 1.
  • FIG. 13 b are a pair of graphs demonstrating the degree of proliferation (based on CFSE dilution) by Ppp2r2d shRNA-transduced CD8 T cells compared to LacZ shRNA-transduced T cells in tumors on days 1, 3, 5, and 7.
  • FIG. 13 c is a set of graphs demonstrating that Ppp2r2d-silencing inhibits T cell apoptosis upon encounter of tumor cells. CFSE-labeled OT-I T cells were co-cultured with B16-Ova tumor cells for 72 hours. Cells were stained with CD8 and annexin V.
  • FIG. 13 d is a set of graphs demonstrating intracellular staining for anti-apoptotic proteins. OT-I T cells expressing LacZ or Ppp2r2d shRNA were co-cultured with B16-Ova tumor cells for 48 hours and then stained with isotype control (grey) and phospho-AKT (Ser473), phospho-Bad (Ser 112) or B c1-2 antibodies.
  • FIG. 13 e is a graph demonstrating increased IFN-γ secretion by Ppp2r2d-silenced T cells. OT-I T cells isolated from B16-Ova tumor-bearing mice were assayed for IFN-γ expression by intracellular staining.
  • FIG. 13 f is a set of graphs demonstrating Ppp2r2d-silenced T cells expand in tumors even without presentation of tumor antigens by professional antigen presenting cells. LacZ or Ppp2r2d shRNA-expressing OT-I T cells were transferred into day 14 B16-Ova tumor-bearing C57BL/6 or b2m-1-mice. shRNA-expressing T cells were identified based on expression of teal fluorescent protein (TFP) or Thy1.1 (fold enrichment in tumors compared to spleens).
  • FIG. 13 g is a graph demonstrating that Ppp2r2d-silencing inhibits T cell apoptosis upon encounter of tumor cells. CFSE-labeled OT-I T cells were co-cultured with B16-Ova tumor cells for 72 hours (activated caspase-3).
  • FIG. 14 is a set of graphs demonstrating OT-I T cells expressing LacZ or Ppp2r2d shRNAs labeled with CFSE and stimulated with CD3 antibody for 72 h. Cells were then stained with CD8 and annexin V and analyzed by flow cytometry.
  • FIG. 15 is a set of graphs demonstrating accumulation of Ppp2r2d shRNA-expressing T cells in tumors and tumor-draining lymph nodes, but not other secondary lymphoid organs. OT-I T cells expressing Ppp2r2d or LacZ shRNAs were labeled with CFSE and injected into B16-Ova tumor-bearing mice. T cells were isolated from the indicated organs on days 1, 3, 5 and 7 to examine the extent of T cell accumulation based on dilution of the CSFE dye.
  • FIGS. 16 a-c are a set of graphs demonstrating that the silencing of Ppp2r2d enhances anti-tumor activity of CD4 and CD8 T cells. T cells were activated with anti-CD3/CD28 beads, infected with lentiviruses driving LacZ or Ppp2r2d shRNA expression and injected into B16-Ova (a,b) or B16 (c) tumor-bearing mice. Tumor size was measured every three days following T cell transfer using calipers on the two longest axes. a,b CD4+TRP-1 and/or CD8+OT-I T cells (2×106) were transferred (day 12 and 17) into mice bearing day 12 B16-Ova tumors. Tumor burden (a) and survival (b) were assessed. c, CD4+TRP-1 and CD8+pmel-1 T cells (3×106 CD4+TRP-1 plus 3×106 CD8+pmel-1) were transferred (day 10 and 15) into mice with day 10 B16 tumors. Log-rank (Mantel-Cox) test was performed using GraphPad Prism version 6 comparing survival of mice treated with LacZ versus Ppp2r2d shRNA-expressing T cells.
  • FIG. 17 is a set of graphs demonstrating FACS analysis of T cell enrichment in tumors compared to spleen for cells expressing a panel of Ppp2r2d or Cb1b shRNAs (upper panels). Ppp2r2d and Cb1b mRNA levels were measured by qPCR prior to T cell transfer (lower panels). Data represent biological replicates (n=3), each value represents mean+/−s.d.
  • FIG. 18 is a set of graphs demonstrating Ppp2r2d protein quantification by mass spectrometry with labeled synthetic peptides (AQUA, ratio of endogenous to AQUA peptides). Representative data from two independent experiments (a-d); Two-sided student's t-test, * P<0.05, ** P<0.01; mean+/−s.d.
  • FIG. 19 is a graph demonstrating qPCR analysis for Ppp2r2d mRNA in tumor-infiltrating OT-I T cells (day 7).
  • FIG. 20 a are graphs showing representative flow cytometry plots demonstrating proliferation of Ppp2r2d shRNA-expressing T cells in tumors and tumor-draining lymph nodes. OTI T cells expressing Ppp2r2d or LacZ shRNAs were labeled with CFSE and injected into B16-Ova tumor-bearing mice. T cells were isolated from the indicated organs on days 1, 3, 5 and 7 to examine the extent of T cell proliferation based on CFSE dilution. T cells that had not diluted CFSE (nondividing cells) were quantified (right).
  • FIG. 20 b are graphs showing representative flow cytometry plots demonstrating viability of tumor-infiltrating T cells. OT-I T cells expressing Pp2r2d or LacZ shRNAs were injected into B16-Ova tumor-bearing mice. T cells were isolated on day 7 and apoptosis was assessed by intracellular staining with an antibody specific for activated caspase-3 (some T cell death may have been caused by the isolation procedure from tumors).
  • FIG. 20 c are graphs showing representative flow cytometry plots demonstrating intracellularcytokine staining for IFNγ by LacZ and Ppp2r2d shRNA-expressing T cells harvested from B16-Ova tumors; T cells were labeled with CFSE prior to injection. Data for all experiments are representative of two independent trials. Statistical analysis was performed on biological replicates (n=3); * P<0.05, ** P<0.01, two-sided Student's t-test. Each value represents mean+/−s.d.
  • FIGS. 21 a-c are a series of graphs demonstrating ex vivo analysis of cytokine production by tumor-infiltrating OT-I T cells at a single-cell level using a nanowell device (84,672 wells of picoliter volume). a, Representative single cells in nanowells and corresponding patterns of cytokine secretion. b, Percentage of T cells secreting indicated cytokines. c, Cytokine secretion rates calculated from standard curves (mean+/−s.d., Mann Whitney test * P<0.05).
  • FIG. 22 a is a set of graphs showing representative flow cytometry plots demonstrating that the majority of adoptively transferred OT-I cells have a memory phenotype in lymph nodes but an effector phenotype in tumors. Cytokine pre-treated cells expressing Ppp2r2d or LacZ shRNAs were injected into mice bearing day 14 B16-Ova tumors. On day 7 following transfer, T cells were harvested from the indicated organs and stained with CD62L and CD44 antibodies. FACS analysis of shRNA-expressing OT-I cells was performed by gating on CD8/Thy1.1 double-positive cells.
  • FIG. 22 b is a set of graphs showing representative flow cytometry plots demonstrating analysis of exhaustion markers. OT-I cells were harvested from draining lymph nodes and tumors of mice and stained with antibodies specific for TIM-3, LAG-3, PD-1 and CD25. For all experiments (n=3 biological replicates; * P<0.05, ** P<0.01, Two-sided Student's t-test); each value represents mean+/−s.d.
  • FIG. 23 a is a set of graphs showing demonstrating intracellular staining for granzyme B by OT-I T cells in tumor-draining lymph nodes and tumors.
  • FIG. 23 b is a pair of images and a graph demonstrating infiltration of shRNA-expressing T cells into tumors. OT-I T cells were transduced with LacZ or Ppp2r2d shRNA vectors encoding a GFP reporter and injected into B16-Ova tumor-bearing mice. After 7 days, tumors were excised and frozen sections stained with anti-GFP and DAPI to enumerate shRNAexpressing OT-I T cells in tumors.
  • FIG. 23 c is a pair of images and a graph demonstrating TUNEL immunohistochemistry performed on tissue sections and apoptotic cells were quantified.
  • FIG. 23 d is a set of graphs demonstrating MHC class I expression by tumor cells. Tumors were digested with collagenase and stained with CD45.2 and H-2Kb antibodies. FACS analysis for H-2Kb expression was performed by gating on CD45.2-negative melanoma cells. Datarepresent biological replicates (n=3), each value represents mean+/−s.d.
    •  DETAILED DESCRIPTION
  • The present disclosure is based, in part, on the observation that the regulatory mechanisms that result in loss of T cell function within immunosuppressive tumors can be systematically discovered in vivo using a pooled small hairpin RNA (shRNA) screening approach aimed at identifying genes that block the function of tumor infiltrating T-cells. As described in the background section above, tumor associated immunosuppressive mechanisms actively block the activity of T cells in the tumor microenvironment. The methods described herein identify shRNAs that enable robust T cell infiltration and accumulation in tumors, despite the multiple inhibitory signals. As described below, the methods identify shRNA that silence expression of genes responsible for immunosuppression by tumors, allowing for enhanced T cell infiltration and accumulation in tumors and resistance to apoptosis.
  • In some instances, the disclosure provides methods for specifically identifying regulatory mechanisms that result in the loss of T cell function within the tumor microenvironment. These methods can include: providing a population of T cells harboring vectors expressing a shRNA; contacting the population of T cells with an immunosuppressive tumor; determining whether a shRNA restores T cell function (e.g., restores ability of T cell to infiltrate and proliferate within the tumor microenvironment) within the immunosuppressive tumor; identifying a gene associated with a shRNA that restores T cell function within the tumor as a gene that inhibits T cell function within the tumor microenvironment.
  • The disclosure provides target genes for reducing the immunosuppressive effect of tumors. The expression of the target genes can be reduced in immune cells, e.g., T cells that recognize tumor associated antigens, and the reduction in expression of the target genes can increase the ability of the cells to evade tumor associated immunosuppressive mechanisms.
  • The disclosure provides shRNAs that reduce (e.g., silence, eliminate, knock down, knock out, or decrease) expression of genes that impair the function of tumor infiltrating T-cells. These shRNA were identified from the transfer of shRNA transduced T cells into tumors, followed by deep sequencing to quantify the representation of all shRNAs in the tumor and lymphoid organs. Representative shRNA disclosed herein include shRNA that reduce the activity of genes including, for example, Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm 1 g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc.
  • In some instances, the disclosure provides therapeutic compositions (e.g., including isolated nucleic acid molecules, vectors expressing nucleic acid molecules encoding the shRNA) related to the shRNAs that silence expression of genes that block the function of tumor infiltrating T-cells. In other aspects, the disclosure provides modified immunoresponsive cells (e.g., T cells, including Natural Killer T cells (NKT), a cytotoxic T lymphocytes (CTL), and a regulatory T cells) that harbor vectors capable of expressing the shRNA described herein. In another aspect, the modified immunoresponsive cells further harbor a vector capable of expressing a CAR having an antigen binding domain that targets a tumor specific antigen.
    • RNA Interference
  • One of the most important recent discoveries in biomedical research is the RNA interference (RNAi) pathway, which is used by cells to regulate the activity of many genes. The principles of RNAi have opened many new possibilities for the identification of therapeutic targets. RNA interference (RNAi) is an effective tool for genome-scale, high throughput analysis of gene function. The term “RNA interference” (RNAi), also called post transcriptional gene silencing (PTGS), refers to the biological process in which RNA molecules inhibit gene expression. An “RNA interfering agent” as used herein, is defined as any agent that interferes with or inhibits expression of a target gene, e.g., a target gene of the invention, by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target gene, e.g., a target gene of the invention, or a fragment thereof, short interfering RNA (siRNA), short hairpin RNA (shRNA), and small molecules which interfere with or inhibit expression of a target gene by RNA interference (RNAi).
  • “RNA interference (RNAi)” is a 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 PTGS of messenger RNA (mRNA) transcribed from that targeted gene, thereby inhibiting expression of the target gene. This process has been described in plants, invertebrates, and mammalian cells. 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” or “inhibition of marker gene expression” includes any decrease in expression or protein activity or level of the target gene (e.g., a marker gene of the invention) or protein encoded by the target gene, e.g., a marker protein of the invention. The decrease may be of at least 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. These are the effector molecules for inducing RNAi, leading to posttranscriptional gene silencing with RNA-induced silencing complex (RISC). In addition to siRNA, which can be chemically synthesized, various other systems in the form of potential effector molecules for posttranscriptional gene silencing are available, including short hairpin RNAs (shRNAs), long dsRNAs, short temporal RNAs, and micro RNAs (miRNAs). These effector molecules either are processed into siRNA, such as in the case of shRNA, or directly aid gene silencing, as in the case of miRNA. The present invention thus encompasses the use of shRNA as well as any other suitable form of RNA to effect posttranscriptional gene silencing by RNAi. Use of shRNA has the advantage over use of chemically synthesized siRNA in that the suppression of the target gene is typically long-term and stable. An siRNA may be chemically synthesized, may be produced by in vitro by transcription, or may be produced within a host cell from expressed shRNA.
  • In one embodiment, a siRNA is a small hairpin (also called stem loop) RNA (shRNA). These shRNAs are composed of a short (e.g., 19-25 nucleotides) antisense strand, followed by a 5-9 nucleotide loop, and the complementary sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow. These shRNAs may be contained in plasmids, retroviruses, and lentiviruses.
  • As used herein, “gene silencing” induced by RNA interference 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 introduction of RNA interference. In one preferred embodiment, the mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.
  • The term “reduced” or “reduce” as used herein generally means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease, or any integer decrease between 10-100% as compared to a reference level.
  • The term “increased” or “increase” as used herein generally means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any integer increase between 10-100% as compared to a reference level, or about a 2-fold, or about a 3-fold, or about a 4-fold, or about a 5-fold or about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
    • Immunoresponsive Cells
  • In some embodiments, the disclosure provides immunoresponsive cells, including T cells, cytotoxic T cells, tumor-infiltrating lymphocytes (TIL), regulatory (CD4) T cells, and Natural Killer (NKT) cells, expressing at least one of an antigen-recognizing receptor. In any aspect, the immunoresponsive cells express at least one tumor specific antigen-recognizing receptor. In some aspects, tumor cell antigen specific T cells, NKT cells, TIL, CTL cells or other immunoresponsive cells are used. Non-limiting examples of immunoresponsive cells include T cells, such as, for example, αβ-TCR+ T cells (e.g., CD8+ T cells or CD4+ T cells) γδ-TCR+ T cells, tumor-infiltrating lymphocytes (TIL), Natural Killer T cells (NKT), a cytotoxic T lymphocytes (CTL), and a CD4 T cells.
    • Nucleic Acid Compositions
  • In some embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences comprising a sequence at least 12, 15, 20 or 25 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 604-620 and 653-678. The shRNA also includes the reverse complement of the contiguous nucleotide sequence and a short sequence located between the two sequences so that the two sequences form a stem loop shRNA that can be processed within a cell provide an siRNA that inhibits the expression of the protein encoded by one of SEQ ID NOs: 604-620 and 653-678, and compositions thereof.
  • Table 1 provides a list of genes identified here as being involved with tumor immunosuppression of T cells.
  • TABLE 1
    Human Murine
    Sequence Sequence
    Accession Accession
    Gene Human Gene Sequence No. No.
    Ppp2r2d gtgtccggccaagcggcgccctgaaggcgtgtccggccgcagcttaggctctccgg NM_018461 NM_026391
    gagtccccggagagtaggggcggccggcggcgctagtcttctggggagcgccgg
    gtgcacaccggaccactgcgggaggcctagggccgagggccgaggagctggcct
    gcgcccggcgaccccggcttccctccgcagtcgcccaggcgtcccttcccccctac
    agccgagcggcgccgggcgcaggcgcattggggcccccggcagcccccgcgg
    cccgccccgtccgctgcccgtccgaggaggcggagggcgatgacgtcatcgagc
    ggggcgacgggcattgggcgccattttgaaaagggaaaaaaatccctccccggcg
    gcggcggcggcggcggcggcgccggcggtggtggcggccccggggctgagcg
    ctcggctgcagcggcgcggaggccgtctccctggtctgccgcggtccccgcccgtc
    ccgccgccggctgccatggcaggagccggaggcggcggctgccccgcgggcgg
    caacgacttccagtggtgcttctcgcaggtcaagggggccatcgacgaggacgtgg
    ccgaagcggacatcatttccaccgttgagtttaattactctggagatcttcttgcaacag
    gagacaagggggcagagttgttatttttcagcgtgaacaagagaataaaagccgcc
    ctcattctaggggagaatataatgtttacagcacctttcaaagtcatgaaccggagtttg
    actatttgaaaagtctagaaattgaggaaaaaattaataaaattaggtggttaccacaac
    agaatgctgctcattttctactgtctacaaatgataaaactataaaattatggaaaataag
    tgaacgggataaaagagcagaaggttataacctgaaagacgaagatggaagacttc
    gagacccatttaggatcacggcgctacgggtcccaatattgaagcccatggatcttat
    ggtagaagcgagtccacggcgaatttttgcaaatgctcacacatatcatataaattcca
    tttcagtaaatagtgatcatgaaacatatctttctgcagatgacctgagaattaatttatgg
    cacttagaaatcacagatagaagctttaacatcgtggacatcaagcctgctaacatgg
    aggagctgaccgaagtcatcactgcagccgagttccacccgcaccagtgcaacgtg
    ttcgtctacagcagtagcaaagggaccatccgcctgtgtgacatgcgctcctcggcc
    ctgtgcgacagacactccaagttttttgaagagcctgaagatcccagcagtaggtcctt
    cttctcagaaataatttcatccatatccgatgtaaaattcagtcatagtgggcggtacatg
    atgaccagagactacctgtcggtgaaggtgtgggacctcaacatggagagcaggcc
    ggtggagacccaccaggtccacgagtacctgcgcagcaagctctgctctctctatga
    gaacgactgcatctttgacaagtttgagtgttgctggaacggttcggatagcgccatca
    tgaccgggtcctataacaacttcttcaggatgtttgatagagacacgcggagggatgt
    gaccctggaggcctcgagagagagcagcaaaccgcgcgccagcctcaaaccccg
    gaaggtgtgtacggggggtaagcggaggaaagacgagatcagtgtggacagtctg
    gacttcaacaagaagatcctgcacacagcctggcaccccgtggacaatgtcattgcc
    gtggctgccaccaataacttgtacatattccaggacaaaatcaactagagacgcgaac
    gtgaggaccaagtcttgtcttgcatagttaagccggacatttttctgtcagagaaaagg
    catcattgtccgctccattaagaacagtgacgcacctgctacttcccttcacagacaca
    ggagaaagccgcctccgctggaggcccggtgtggttccgcctcggcgaggcgcga
    gacaggcgctgctgctcacgtggagacgctctcgaagcagagttgacggacactgc
    tcccaaaaggtcattactcagaataaatgtatttatttcagtccgagccttcctttccaattt
    atagaccaaaaaattaacatccaagagaaaagttattgtcagataccgctctttctccaa
    ctttccctctttctctgccatcacacttgggccttcactgcagcgtggtgtggccaccgt
    ccgtgtcctctcggccttcctccgagtccaggtggactctgtggatgtgtggatgtggc
    ccgagcaggctcaggggccccactcacccacagcatccgccgccaccccttcgg
    gtgtgagcgctcaataaaaacaacacactataaagtgtttttaaatccaaaaaaaaaaa
    aaaa (SEQ ID NO: 604)
    Eif2ak3 ggaaagtccaccttccccaacaaggccagcctgggaacatggagtggcagcggcc NM_004836.5 NM_010121.2
    gcagccaatgagagagcaaacgcgcggaaagtttgctcaatgggcgatgtccgag
    ataggctgtcactcaggtggcagcggcagaggccgggctgagacgtggccaggg
    gaacacggctggctgtccaggccgtcggggggcagtagggtccctagcacgtcct
    tgccttcttgggagctccaagcggcgggagaggcaggcgtcagtggctgcgcctcc
    atgcctgcgcgcggggcgggacgctgatggagcgcgccatcagcccggggctgc
    tggtacgggcgctgctgctgctgctgctgctgctggggctcgcggcaaggacggtg
    gccgcggggcgcgcccgtggcctcccagcgccgacggcggaggcggcgttcgg
    cctcggggcggccgctgctcccacctcagcgacgcgagtaccggcggcgggcgc
    cgtggctgcggccgaggtgactgtggaggacgctgaggcgctgccggcagccgc
    gggagagcaggagcctcggggtccggaaccagacgatgagacagagttgcgacc
    gcgcggcaggtcattagtaattatcagcactttagatgggagaattgctgccttggatc
    ctgaaaatcatggtaaaaagcagtgggatttggatgtgggatccggttccttggtgtca
    tccagccttagcaaaccagaggtatttgggaataagatgatcattccttccctggatgg
    agccctcttccagtgggaccaagaccgtgaaagcatggaaacagttcctttcacagtt
    gaatcacttcttgaatcttcttataaatttggagatgatgttgttttggttggaggaaaatct
    ctgactacatatggactcagtgcatatagtggaaaggtgaggtatatctgttcagctctg
    ggttgtcgccaatgggatagtgacgaaatggaacaagaggaagacatcctgcttcta
    cagcgtacccaaaaaactgttagagctgtcggacctcgcagtggcaatgagaagtg
    gaatttcagtgttggccactttgaacttcggtatattccagacatggaaacgagagccg
    gatttattgaaagcacctttaagcccaatgagaacacagaagagtctaaaattatttcag
    atgtggaagaacaggaagctgccataatggacatagtgataaaggtttcggttgctga
    ctggaaagttatggcattcagtaagaagggaggacatctggaatgggagtaccagttt
    tgtactccaattgcatctgcctggttacttaaggatgggaaagtcattcccatcagtctttt
    tgatgatacaagttatacatctaatgatgatgttttagaagatgaagaagacattgtaga
    agctgccagaggagccacagaaaacagtgtttacttgggaatgtatagaggccagct
    gtatctgcagtcatcagtcagaatttcagaaaagtttccttcaagtcccaaggctttgga
    atctgtcactaatgaaaacgcaattattcctttaccaacaatcaaatggaaacccttaatt
    cattctccttccagaactcctgtcttggtaggatctgatgaatttgacaaatgtctcagta
    atgataagttttctcatgaagaatatagtaatggtgcactttcaatcttgcagtatccatat
    gataatggttattatctaccatactacaagagggagaggaacaaacgaagcacacag
    attacagtcagattcctcgacaacccacattacaacaagaatatccgcaaaaaggatc
    ctgttcttcttttacactggtggaaagaaatagttgcaacgattttgttttgtatcatagcaa
    caacgtttattgtgcgcaggcttttccatcctcatcctcacaggcaaaggaaggagtct
    gaaactcagtgtcaaactgaaaataaatatgattctgtaagtggtgaagccaatgaca
    gtagctggaatgacataaaaaactctggatatatatcacgatatctaactgattttgagc
    caattcaatgcctgggacgtggtggctttggagttgtttttgaagctaaaaacaaagta
    gatgactgcaattatgctatcaagaggatccgtctccccaatagggaattggctcggg
    aaaaggtaatgcgagaagttaaagccttagccaagcttgaacacccgggcattgtta
    gatatttcaatgcctggctcgaagcaccaccagagaagtggcaagaaaagatggatg
    aaatttggctgaaagatgaaagcacagactggccactcagctctcctagcccaatgg
    atgcaccatcagttaaaatacgcagaatggatcctttcgctacaaaagaacatattgaa
    atcatagctccttcaccacaaagaagcaggtctttttcagtagggatttcctgtgaccag
    acaagttcatctgagagccagttctcaccactggaattctcaggaatggaccatgagg
    acatcagtgagtcagtggatgcagcatacaacctccaggacagttgccttacagactg
    tgatgtggaagatgggactatggatggcaatgatgaggggcactcctttgaactttgtc
    cttctgaagcttctccttatgtaaggtcaagggagagaacctcctcttcaatagtatttga
    agattctggctgtgataatgcttccagtaaagaagagccgaaaactaatcgattgcata
    ttggcaaccattgtgctaataaactaactgctttcaagcccaccagtagcaaatcttcttc
    tgaagctacattgtctatttctcctccaagaccaaccactttaagtttagatctcactaaaa
    acaccacagaaaaactccagcccagttcaccaaaggtgtatctttacattcaaatgca
    gctgtgcagaaaagaaaacctcaaagactggatgaatggacgatgtaccatagagg
    agagagagaggagcgtgtgtctgcacatcttcctgcagatcgcagaggcagtggag
    tttcttcacagtaaaggactgatgcacagggacctcaagccatccaacatattctttaca
    atggatgatgtggtcaaggttggagactttgggttagtgactgcaatggaccaggatg
    aggaagagcagacggttctgaccccaatgccagcttatgccagacacacaggacaa
    gtagggaccaaactgtatatgagcccagagcagattcatggaaacagctattctcata
    aagtggacatcttttctttaggcctgattctatttgaattgctgtatccattcagcactcag
    atggagagagtcaggaccttaactgatgtaagaaatctcaaatttccaccattatttact
    cagaaatatccttgtgagtacgtgatggttcaagacatgctctctccatcccccatgga
    acgacctgaagctataaacatcattgaaaatgctgtatttgaggacttggactttccagg
    aaaaacagtgctcagacagaggtctcgctccttgagttcatcgggaacaaaacattca
    agacagtccaacaactcccatagccctttgccaagcaattagccttaagttgtgctagc
    aaccctaataggtgatgcagataatagcctacttcttagaatatgcctgtccaaaattgc
    agacttgaaaagtttgttcttcgctcaatttttttgtggactactttttttatatcaaatttaag
    ctggatttgggggcataacctaatttgagccaactcctgagttttgctatacttaaggaa
    agggctatctttgttctttgttagtctcttgaaactggctgctggccaagctttatagccct
    caccatttgcctaaggaggtagcagcaatccctaatatatatatatagtgagaactaaa
    atggatatatttttataatgcagaagaaggaaagtccccctgtgtggtaactgtattgttc
    tagaaatatgctttctagagatatgatgattttgaaactgatttctagaaaaagctgactc
    catttttgtccctgggggtaaattaggaatctgcactattttggaggacaagtagcaca
    aactgtataacggtttatgtccgtagttttatagtcctatttgtagcattcaatagctttattc
    cttagatggttctagggtgggtttacagctttttgtacttttacctccaataaagggaaaat
    gaagctttttatgtaaattggttgaaaggtctagttttgggaggaaaaaagccgtagtaa
    gaaatggatcatatatattacaactaacttcttcaactatggactttttaagcctaatgaaa
    tcttaagtgtcttatatgtaatcctgtaggttggtacttcccccaaactgattataggtaac
    agtttaatcatctcacttgctaacatgtttttatttttcactgtaaatatgtttatgttttatttata
    aaaattctgaaatcaatccatttgggttggtggtgtacagaacacacttaagtgtgttaa
    cttgtgacttctttcaagtctaaatgatttaataaaactttttttaaattaaaaaaaaaaaaa
    aaaaaa (SEQ ID NO: 605)
    Arhgap5 ctcggtgagcgcgccgaggaagagaggcgagcggagagtggaggaggaggcg NM_ NM_009706.2
    gcggcggcgggagcggtccccaggaatgtcgctgccgccgccaccgccggggc 001030055.1
    cgctgccgttgaggaggagacggaggagaccgacgttgttaggaagatgatcccta
    tgatcttgaagatgtttctgcacagaaatgagggaaatacaaagaaccaaatacagttc
    tgaaatttgggatctgtattttgagatgattttattttcagaatgagaagcatatctggttac
    ctttatgaatgtagagacatgagaagagagttatgatggcaaaaaacaaagagcctc
    gtcccccatcctataccatcagtatagttggactctctgggactgaaaaagacaaaggt
    aactgtggagttggaaagtcttgtttgtgcaatagatttgtacgctcaaaagcagatgaa
    tattatccagagcatacttctgtgcttagcaccattgactttggaggacgagtagtaaac
    aatgatcactttttgtactggggtgacataatacaaaatagtgaagatggagtagaatg
    caaaattcatgtcattgaacaaacagagttcattgatgaccagactttcttgcctcatcg
    gagtacgaatttgcaaccatatataaaacgtgcagctgcatctaaattgcagtcagcag
    aaaaactaatgtacatttgcactgatcagctaggcttagaacaagactttgaacagaag
    caaatgcctgaagggaagctcaacgtagatggatttttattatgcattgatgtaagtcaa
    ggatgcaataggaagtttgatgatcaacttaaatttgtgaataacctttttgtccagttatc
    aaaatcaaaaaaacctgtaataatagcagcaactaaatgtgatgaatgcgtggatcatt
    atcttagagaagttcaggcatttgcttcaaataaaaagaaccttcttgtagtggaaacat
    cagcacgatttaatgtcaacattgaaacatgttttactgcactggtacaaatgttggataa
    aactcgtagcaagcctaaaattattccctatttggatgcttataaaacacagagacaact
    tgttgtcacagcaacagataagtttgaaaaacttgtgcagactgtgagagattatcatg
    caacttggaaaactgttagtaataaattaaaaaatcatcctgattatgaagaatacatca
    acttagagggaacaagaaaggccagaaatacattctcaaaacatatagaacaactta
    aacaggaacatataagaaaaaggagagaagagtatataaatactttaccaagagcttt
    taacactcttttgccaaatctagaagagattgaacatttgaattggtcagaagctttgaa
    gttaatggaaaagagagcagatttccagttatgttttgtggtgctagaaaaaactccttg
    ggatgaaactgaccatatagacaaaattaatgataggcggattccatttgacctcctga
    gcactttagaagctgaaaaagtctatcagaaccatgtacagcatctgatatccgagaa
    gaggagggtggaaatgaaggaaaaattcaaaaagactttggaaaaaattcaattcatt
    tcaccagggcagccatgggaggaagttatgtgctttgttatggaggatgaagcctaca
    aatatatcactgaggctgatagcaaagaggtatatggtaggcatcagcgagaaatagt
    tgaaaaagccaaagaagagtttcaagaaatgctttttgagcattctgaacttttttatgatt
    tagatcttaatgcaacacctagttcagataaaatgagtgaaattcatacagttctgagtg
    aagaacctagatataaagctttacagaaacttgcacctgatagggaatcccttctactta
    agcatataggatttgtttatcatcccactaaagaaacatgtcttagtggccaaaattgtac
    agacattaaagtggagcagttacttgctagtagtcttttacagttggatcatggccgctt
    aagattatatcacgatagtaccaatatagataaagttaacctttttattttagggaaggat
    ggccttgcccaagaactagcaaatgagataaggacacaatccactgatgatgagtat
    gccttagatggaaaaatttatgaacttgatcttcggccggttgatgccaaatcgccttac
    tttttgagtcagttatggactgccgcctttaaaccacatgggtgcttctgtgtatttaattcc
    attgagtcattgagttttattggggaatttattgggaaaataagaactgaagcttctcaga
    tcagaaaagataaatacatggctaatcttccatttacattaattctggctaatcagagag
    attccattagtaagaatctaccaattctcaggcaccaagggcagcagttggcaaacaa
    gttgcaatgtccttttgtagatgtacctgctggtacatatcctcgtaaatttaatgaaaccc
    aaataaagcaagctctcagaggagtattggaatcagttaaacacaatttggatgtggtg
    agcccaattcctgccaataaggacttatcagaagctgacttgagaattgtcatgtgcgc
    catgtgtggagatccatttagtgtggatcttattctttcacccttccttgattctcattcttgc
    agtgctgctcaagctggacagaataattccctaatgcttgataaaatcattggtgaaaa
    aaggaggcgaatacagatcacaatattatcataccactcttcaattggagtaagaaaa
    gatgaactagttcatgggtatatattagtttactctgcaaaacggaaagcttcgatggga
    atgcttcgagcatttctatcagaagttcaagacaccattcctgtacagctggtggcagtt
    actgacagccaagcagatttttttgaaaatgaggctatcaaagagttaatgactgaagg
    agaacacattgcaactgagatcactgctaaatttacagcactgtattctttatctcagtat
    catcggcaaactgaggtctttactctgttttttagtgatgttctagagaaaaaaaatatgat
    agaaaattcttatttgtctgataatacaagggaatcaacccatcaaagtgaagatgttttt
    ctaccatctcccagagactgttttccctataataactaccctgattcagatgatgacaca
    gaagcaccacctccttatagtccaattggggatgatgtacagttgcttccaacacctag
    tgaccgttccagatatagattagatttggaaggaaatgagtatcctattcatagtacccc
    aaactgtcatgaccatgaacgcaaccataaagtgcctccacctattaaacctaaacca
    gttgtacctaagacaaatgtgaaaaaactcgatccaaaccttttaaaaacaattgaagc
    tggtattggtaaaaatccaagaaagcagacttcccgggtgcctttggcacatcctgaa
    gatatggatccttcagataactatgcggaacccattgatacaattttcaaacagaaggg
    ctattctgatgagatttatgttgtcccagatgatagtcaaaatcgtattaaaattcgaaact
    catttgtaaataacacccaaggagatgaagaaaatgggttttctgatagaacctcaaaa
    agtcatggggaacggaggccttcaaaatacaaatataaatctaaaaccttgtttagtaa
    agccaagtcatactatagaagaacacattcagatgccagtgatgatgaggctttcacc
    acttctaaaacaaaaagaaaaggaagacatcgtggaagtgaagaagatccacttcttt
    ctcctgttgaaacttggaaaggtggtattgataatcctgcaatcacttctgaccaggagt
    tagatgataagaagatgaagaagaaaacccacaaagtgaaagaagataaaaagca
    gaaaaagaaaactaagaacttcaatccaccaacacgtagaaattgggaaagtaatta
    ctttgggatgcccctccaggatctggttacagctgagaagcccataccactatttgttg
    agaaatgtgtggaatttattgaagatacagggttatgtaccgaaggactctaccgtgtc
    agcgggaataaaactgaccaagacaatattcaaaagcagtttgatcaagatcataata
    tcaatctagtgtcaatggaagtaacagtaaatgctgtagctggagcccttaaagctttct
    ttgcagatctgccagatcctttaattccatattctcttcatccagaactattggaagcagc
    aaaaatcccggataaaacagaacgtcttcatgccttgaaagaaattgttaagaaatttc
    atcctgtaaactatgatgtattcagatacgtgataacacatctaaacagggttagtcagc
    aacataaaatcaacctaatgacagcagacaacttatccatctgtttttggccaaccttga
    tgagacctgattttgaaaatcgagagtttctgtctactactaagattcatcaatctgttgtt
    gaaacattcattcagcagtgtcagtttttcttttacaatggagaaattgtagaaacgacaa
    acattgtggctcctccaccaccttcaaacccaggacagttggtggaaccaatggtgcc
    acttcagttgccgccaccattgcaacctcagctgatacaaccacaattacaaacggat
    cctcttggtattatatgagtaggaagtgattgcaaacaggctggatttggacaaaaagc
    aaatctagacatgcatgtttcagggttcagtagtatacttcatgtttcatacagataattca
    cattcaaaattacattttctctttgaactagatggtattccttattcacttacattacaaatct
    aagaccatgtgataagcatgactggagaggtttaatttttataaacaaaaatagctataa
    agtacaaagctgctgctgcatgcaaccttattgcaatcagtatatcattcctgtggcaatt
    tctgtcaccttatattgtgaataaaatttttctatagaaattaaatgatttaaaaactcaccta
    tatgaaacatttaatgcttttcagcctgctttctggctgattttgttatttgatgtgctaatttg
    ggcaacttaatttacattctggcagtcggtgtagataactaaaagcccagttaagtatttt
    ataatttcaggctactgaggccatgcttgggatgttgtttgaaagaaagaaaaaataca
    cttgacatatttcacatttctgtaccttcatctttacttccaagtaaacccgtggatgatttg
    atgagggataaatgaacctatttcttttacacacataccaaggacatgcttgtggctaaa
    gtgagttgataatgttgtgcaaaggatagttgtcaccaactcatttctttatggtccataat
    gaaataaaaattttgtatactgttaattctgtaaacagatgcatgttcaaaagatctatgat
    ggtcttgtaatcttaatctaatatattttagatattttaattttttccctcttggggaacacattt
    agtatagtgtagaaaatacttccatgacattttcatataaggttatataacttttcatacata
    aacatgaaatttgttgtagaaaattctttaaaccaaacatttaaatctaggacttcaattta
    atttgttccttgaatctatttttatgtggcccttaaaaaatatccaaaaaacccattgctaat
    atagcaataaaaatactttgggtactgacagactctttggagtgtttatattacaaatttgt
    attcatattcttttctgtgatgtgttgtactaaaatccaaaatggcttttgcaccatttttaag
    ccaattttttcctttgatgttggtaccagaattactataagtgactgctgcttttgggggta
    aacattttgttagtgaagataaaaccagaacactaaattatggataaaattttcagaata
    ggtggcacaggtaaatttcactaggttatattttgtgtagtaaagaaaaaaattatttggt
    caatgttatcttaattcatactacaatttaagattatcttatgtgtattatagtaaatagatga
    ttttcagattcaaggctcctaagagtttgatttgctctgttttttcctaaaataaatattgtctc
    tcccaactgttaagttctaggtattgtacttccaattttaacttcagaaccaagatgttggc
    atgaaccaggctgctgttgaagtacatgtatattataaattatcttatttgtgttatactctta
    catgttatcttttctaagaaaacaaagtccctattattcctattgcaaagcacacaggaat
    taagaaagtacagtaatttttaaaaaaaaatccggtaaatgtagtattcttaacctgttcta
    tattacttatacctattgtctatatagctttaatttatagttgtcagtttaactattggcatgtct
    ggcaaagaaaattaaactttaagagttttataaactgtttctaggttgctaaagaatttattt
    ttctactatatatggtatagacaaagcatcaaactatgtacaggaaaaaagcctgactat
    ttctatttggaagtaggctgaaaagagaattttcaaaactgttcgtgtcttcagttcattct
    gtcataactttgctattgtaatatgtgaataccagtttatttaagctgttctcttttatactgta
    ttaatttaatgttcatctgcgtttagtaccatttttgttattaaaactggcatttaccgtttttca
    cattaacccaccttgcaccttcccccaaacttatctccacttttctatgcattctatcattga
    tttgacacacttcatagtgagtcatttaaatactctacgtttggttcaattaaccagtaggtt
    acagttattgaaaattaaagtacagtttaaagctcagtctgttacactgaattgattgtgtt
    tgtttttgccaagggtttagatatgcttttaaatattagaaacatctaagaacagaataac
    ataattaaacttttttctggtaagttactggaaggtttcactgtttagggacctatcatatga
    gacttcttaaaggattaaaagaataggatagtctcataattgtgagtaaacatcaaggc
    attatattttacaatactgaataaaatttcatctacacacatgttgccattgtttcatttaagg
    ttcagtgcttatagttaactacaatattggacctaacaggatctagattagcaatataaag
    aagcatagtggtactctgtttcacactttcagtagatttattagaagtcaaattctattcaa
    cagacacttattaggatatacaactaatttaagaataaaattccaggcacaatatatttttt
    ttaaatggtatttgttagtagtgcttcttccccttaacatttacagtgtaaatactgcaggta
    accgcaatctaagttagccaaaaagcagctttttttcccatactgtatgtaaataatgtag
    acctgggtttttttgtttatttgggtttgtttttttttttgaggtactggaatctaattaatatctc
    ttaggtatcaacaaaagggaacaattggaatgagaatttaggccttagcttccatggtg
    atttttagttttttatacagtaataattgtgatgctatttgtcaactggatataaatacacatat
    aattttaaaaagtcaaaagtgcttttgtttctttgtttaatgtaatttttgtgcttcacctacag
    gatgctgcagtaaattaaatatcagtgaagcttctgatgtataaagaatgctatgaataa
    aacattaagaagctgtgtaattttaagttatagttgcctctatttttaccatttcattggtaaa
    aattagctaatttttttcaagtgaaatgaaaaataaaaatataaatttatcaatatgatgga
    aatcttattaaggagatgtattattgaattttcactgtacctgaaaaggagattcaaaatttt
    ttctggggatgtatataggtgaaaatttgattttttaaattatcaggaaaacaagataatg
    cacagatttctaagactaagatcttacctggatgtgatttttgagctgtggctagacattc
    tttagagccactggaaatattttgaaaactattctagttatagcagagctgctaatattaa
    cgaatatatttgtgtcttcatggtttgtgactattaggccaaattttgtggtatatgttgtca
    gtctggatctggtgaggtctgttcaacatgaatctttgtgttatcttgaatttagtagtttca
    aggtacttaaattcttaacagtttctaatttgtttcaatacatatgggacatggttgattttttt
    actgtattagaactcttggaagttcttagccttttcaggttatgaaatacctgaaagtaaa
    attttctaagatttaataagggaagatactattcaaatcattttcttaggatagcatctttac
    atacaatgagaggattgtacaagcattaatctcatattccaacatccagttacttgatgtg
    atccaagtaccctggtctttttgaagcagttaaaatctaattaattaactttgggagtcttc
    actattcaattgatcctcatcattgtcctatttgcatgactccattttttcctccactatatga
    gttttctttgtcagggggagaggagtgggaagagtcacagaatctcatattcacatctt
    aattaaattgtgtgaaattagtcttttgtggaaattctgtaggcagtatgattttgaaaagc
    taaccaatgataattagcattttagttaatactaaatgcataaaattataacccttgaaatt
    aatttggtgctggcagttctggtttagtcatttttaccagtagttagtagtattaagacctg
    cagtatatgcactttttgagtagctgtcaaataattgtagttgagaaacaacttgtttattct
    cacaattcagattttctattcagttttgtctcaaatagtaagttattgtgaacaatttaataac
    ggccctcctgttctagtttgcctaatattttagttaagatttagtgttttaacctatttttttaag
    tttattttttgtattagattttatttgaataagttatgtgggtttagtaattgacctatttattcatt
    gcttcactaattcatccagattagttttaagtgtgtatatgtatttgctcaccagatcattttc
    ttgggaccttgaactgtgaatgttttgtcctaaccatttaatattttctaggtacttgctgca
    agttcttgaactattttaccagctttaactttggggctcttagtttcttttctccagattcttgtt
    attttattttatccaaataaatatttaggtgttctaagaa (SEQ ID NO: 606)
    Smad2 cggccgggaggcggggcgggccgtaggcaaagggaggtggggaggcggtggc NM_001003652 NM_
    cggcgactccccgcgccccgctcgccccccggcccttcccgcggtgctcggcctc 001252481
    gttcctttcctcctccgctccctccgtcttccatacccgccccgcgcggctttcggccg
    gcgtgcctcgcgccctaacgggcggctggaggcgccaatcagcgggcggcaggg
    tgccagccccggggctgcgccggcgaatcggcggggcccgcggcccagggtgg
    cagggggtctacccgcgcggccgcggcggcggagaagcagctcgccagccag
    cagcccgccagccgccgggaggttcgatacaagaggctgttttcctagcgtggcttg
    ctgcctttggtaagaacatgtcgtccatcttgccattcacgccgccagttgtgaagaga
    ctgctgggatggaagaagtcagctggtgggtctggaggagcaggcggaggagag
    cagaatgggcaggaagaaaagtggtgtgagaaagcagtgaaaagtctggtgaaga
    agctaaagaaaacaggacgattagatgagcttgagaaagccatcaccactcaaaact
    gtaatactaaatgtgttaccataccaagcacttgctctgaaatttggggactgagtacac
    caaatacgatagatcagtgggatacaacaggcctttacagcttctctgaacaaaccag
    gtctcttgatggtcgtctccaggtatcccatcgaaaaggattgccacatgttatatattgc
    cgattatggcgctggcctgatcttcacagtcatcatgaactcaaggcaattgaaaactg
    cgaatatgcttttaatcttaaaaaggatgaagtatgtgtaaacccttaccactatcagag
    agttgagacaccagttttgcctccagtattagtgccccgacacaccgagatcctaaca
    gaacttccgcctctggatgactatactcactccattccagaaaacactaacttcccagc
    aggaattgagccacagagtaattatattccagaaacgccacctcctggatatatcagtg
    aagatggagaaacaagtgaccaacagttgaatcaaagtatggacacaggctctcca
    gcagaactatctcctactactctttcccctgttaatcatagcttggatttacagccagttac
    ttactcagaacctgcattttggtgttcgatagcatattatgaattaaatcagagggttgga
    gaaaccttccatgcatcacagccctcactcactgtagatggctttacagacccatcaaa
    ttcagagaggttctgcttaggtttactctccaatgttaaccgaaatgccacggtagaaat
    gacaagaaggcatataggaagaggagtgcgcttatactacataggtggggaagttttt
    gctgagtgcctaagtgatagtgcaatctttgtgcagagccccaattgtaatcagagata
    tggctggcaccctgcaacagtgtgtaaaattccaccaggctgtaatctgaagatcttca
    acaaccaggaatttgctgctcttctggctcagtctgttaatcagggttttgaagccgtcta
    tcagctaactagaatgtgcaccataagaatgagttttgtgaaagggtggggagcaga
    ataccgaaggcagacggtaacaagtactccttgctggattgaacttcatctgaatgga
    cctctacagtggttggacaaagtattaactcagatgggatccccttcagtgcgttgctc
    aagcatgtcataaagcttcaccaatcaagtcccatgaaaagacttaatgtaacaactctt
    ctgtcatagcattgtgtgtggtccctatggactgtttactatccaaaagttcaagagaga
    aaacagcacttgaggtctcatcaattaaagcaccttgtggaatctgtttcctatatttgaa
    tattagatgggaaaattagtgtctagaaatactctcccattaaagaggaagagaagattt
    taaagacttaatgatgtcttattgggcataaaactgagtgtcccaaaggtttattaataac
    agtagtagttatgtgtacaggtaatgtatcatgatccagtatcacagtattgtgctgtttat
    atacatttttagtttgcatagatgaggtgtgtgtgtgcgctgcttcttgatctaggcaaac
    ctttataaagttgcagtacctaatctgttattcccacttctctgttatttttgtgtgtcttttttaa
    tatataatatatatcaagattttcaaattatttagaagcagattttcctgtagaaaaactaat
    ttttctgccttttaccaaaaataaactcttgggggaagaaaagtggattaacttttgaaat
    ccttgaccttaatgtgttcagtggggcttaaacagtcattctttttgtggttttttgttttttttt
    gtttttttttttaactgctaaatcttattataaggaaaccatactgaaaacctttccaagcct
    cttttttccattcccatttttgtcctcataatcaaaacagcataacatgacatcatcaccagt
    aatagttgcattgatactgctggcaccagttaattctgggatacagtaagaattcatatg
    gagaaagtccctttgtcttatgcccaaatttcaacaggaataattggcttgtataatctag
    cagtctgttgatttatccttccacctcataaaaaatgcataggtggcagtataattattttc
    agggatatgctagaattacttccacatatttatccctttttaaaaaagctaatctataaata
    ccgtttttccaaaggtattttacaatatttcaacagcagaccttctgctcttcgagtagtttg
    atttggtttagtaaccagattgcattatgaaatgggccttttgtaaatgtaattgtttctgca
    aaatacctagaaaagtgatgctgaggtaggatcagcagatatgggccatctgtttttaa
    agtatgttgtattcagtttataaattgattgttattctacacataattatgaattcagaatttta
    aaaattgggggaaaagccatttatttagcaagttttttagcttataagttacctgcagtct
    gagctgttcttaactgatcctggttttgtgattgacaatatttcatgctctgtagtgagagg
    agatttccgaaactctgttgctagttcattctgcagcaaataattattatgtctgatgttga
    ctcattgcagtttaaacatttcttcttgtttgcatcttagtagaaatggaaaataaccactc
    ctggtcgtcttttcataaattttcatatttttgaagctgtctttggtacttgttctttgaaatcat
    atccacctgtctctataggtatcattttcaatactttcaacatttggtggttttctattgggta
    ctccccattttcctatatttgtgtgtatatgtatgtgttcatgtaaatttggtatagtaatttttt
    attcattcaacaaatatttattgttcacctgtttgtaccaggaacttttcttagtctttgggta
    aaggtgaacaagacaactacagttcctgcctttgctgagacagcagttacactaaccc
    ttaattatcttacttgtctatgaaggagataaacagggtactgtactggagaataacaga
    tgggatgcttcaggtaggacatcaaggaaagcctctaaggaaaggatgcatgagcta
    acacctgacattaaagaagcaagccaagtgaggagccaggggagataagcattcct
    ggcaaagagaatagcatcaaatgcaaaaaggttcacactaaaggaaactcctgatta
    ggtattaatgctttatacagaaacctctatacaaatccaaacttgaagatcagaatggtt
    ctacagttcataacattttgaaggtggccttattttgtgatagtctgcttcatgtgattctca
    ctaacatatctccttcctcaacctttgctgtaaaaatttcatttgcaccacatcagtactact
    taatttaacaagcttttgttgtgtaagctctcactgttttagtgccctgctgcttgcttccag
    actttgtgctgtccagtaattatgtcttccactacccatcttgtgagcagagtaaatgtcct
    aggtaataccactatcaggcctgtaggagatactcagtggagcctctgcccttctttttc
    ttacttgagaacttgtaatggtgttagggaacagttgtaggggcagaaaacaactctga
    aagtggtagaaggtcctgatcttggtggttactcttgcattactgtgttaggtcaagcag
    tgcctactatgctgtttcagtagtggagcgcatctctacagttctgatgcgatttttctgta
    cagtatgaaattgggactcaactctttgaaaacacctattgagcagttatacctgttgag
    cagtttacttcctggttgtaattacatttgtgtgaatgtgtttgatgctttttaacgagatgat
    gttttttgtattttatctactgtggcctgattttttttttgttttctgcccctccccccatttatag
    gtgtggttttcatttttctaagtgatagaatcccctctttgttgaatttttgtctttatttaaatta
    gcaacattacttaggatttattcttcacaatactgttaattttctaggaatgatgacctgag
    aaccgaatggccatgctttctatcacatttctaagatgagtaatattttttccagtaggttc
    cacagagacaccttgggggctggcttaggggaggctgttggagttctcactgactta
    gtggcatatttattctgtactgaagaactgcatggggtttcttttggaaagagtttcattgc
    tttaaaaagaagctcagaaagtctttataaccactggtcaacgattagaaaaatataact
    ggatttaggcctaccttctggaataccgctgattgtgctctttttatcctactttaaagaag
    ctttcatgattagatttgagctatatcagttataccgattataccttataatacacattcagtt
    agtaaacatttattgatgcctgttgtttgcccagccactgtgatggatattgaataataaa
    aagatgactaggacggggccctgacccttgagctgtgcttggtcttgtagaggttgtgt
    tttttttcctcaggacctgtcactttggcagaaggaaatctgcctaatttttcttgaaagct
    aaattttctttgtaagtttttacaaattgtttaatacctagttgtattttttaccttaagccacat
    tgagttttgcttgatttgtctgtcttttaaacactgtcaaatgctttcccttttgttaaaattatt
    ttaatttcactttttttgtgcccttgtcaatttaagactaagactttgaaggtaaaacaaaca
    aacaaacatcagtcttagtctcttgctagttgaaatcaaataaaagaaaatatataccca
    gttggtttctctacctcttaaaagcttcccatatatacctttaagatccttctcttttttctttaa
    ctactaaataggttcagcatttattcagtgttagataccctcttcgtctgagggtggcgta
    ggtttatgttgggatataaagtaacacaagacaatcttcactgtacataaaatatgtcttc
    atgtacagtctttactttaaaagctgaacattccaatttgcgccttccctcccaagcccct
    gcccaccaagtatctctttagatatctagtctgtggacatgaacaatgaatacttttttctt
    actctgatcgaaggcattgatacttagacatatcaaacatttcttcctttcatatgctttact
    ttgctaaatctattatattcattgcctgaattttattcttcctttctacctgacaacacacatc
    caggtggtacttgctggttatcctctttcttgttagccttgttttttgttttttttttttttttttgag
    agggagtctcgctctgttgcccaacctggagtgcagtggtgcgatcttggttcactgc
    aagctccgcctcccgggttcacgccatgcttctgcctcagcctcccaagtagctggga
    ctacaggcgcccaccaccacactcggctaattttttgtatttttagtagagacggggttt
    caccgtgttggccaggatggtctcgatctcctgacctcgtgatctgtccacctcggctt
    cccaaagtgctgggattacaggcatgagccaccgcgcccagcctagccatatttttat
    ctgcatatatcagaatgtttctctcctttgaacttattaacaaaaaaggaacatgcttttcat
    acctagagtcctaatttcttcatcatgaaggttgctattcaaattgatcaatcattttaatttt
    acaaatggctcaaaaattctgttcagtaaatgtctttgtgactggcaaatggcataaatta
    tgtttaagattatgaacttttctgacagttgcagccaatgttttccctacgataccagatttc
    catcttggggcatattggattgttgtatttaagacagtcagaataatgatagtgtgtggtc
    tccagaggtagtcagaatcctgctattgagttctttttatatcttccttttcaattttttattac
    cattttgtttgtttagactacactttgtagggattgaggggcaaattatctcttggagtgga
    attcctgtgttttgagccttacaaccaggaaatatgagctatactagatagcctcatgata
    gcatttacgataagaacttatctcgtgtgttcatgtaattttttgagtaggaactgttttatct
    tgaatattgtagctaactatatatagcagaactgcctcagtctttttaagaaggaaataaa
    taatatatgtgtatgaatttatatatacatatacactcatagacaaacttaacagttggggt
    cattctaacagttaaaacaattgttccattgtttaaatctcagatcctggtaaaatgttctta
    atttgtctgtgtacattttcctttcatggacagaccattggagtacattaattttcttaatctg
    ccatttggcagttcatttaatataccattttttggcaacttggtaactaagaatcacagcca
    aaatttgttaacatcaaagaaagctctgccatataccccgttactaaattattatacatcc
    agcagattctgggatgtactaacttagggttaactttgttgttgttgataatactagattgc
    tccctctttaattcttcttctggtgcaaggttgctgcttaagttaccctgggaaatactact
    acaaggtcaaattttctagtatcttacagcctgattgaaggtgattcagatctttgctcaat
    ataaatggattttccaagattctctgggccatccttgacccacaggtgatctcgctgga
    gtatattaacttaacttcagtgccagttggtttggtgccatgagatccataatgaatccag
    aacttcaccattgcttagatataagagtcccttggaagaataatgccactgatgatggg
    ggtcagaaggtgtattaactcaacatagagggcttttagatttttcttcaaaaaaatttcg
    agaaaagtattcttttaccctccaaacagttaacagctcttagtttctccaaatatgctcttt
    gatttacttatttttaattaaagatggtaatttattgaacaatgaaatccgtaatatattgattt
    aaggacaaaagtgaagttttagaattataaaagtacttaaatattatatattttccatttcat
    aattgttttcctttctctgtggctttaaagtttttgactattttacaatgttaatcactaggtaa
    cttgccatatttctggttctatattaagttctatcctttataatgctgttattataaagctggttt
    ttagcatttgtctgtagcaatagaaattttactaagtctctgttctcccagtaagttttttcttt
    tctcagtaagtccctaagaaaacatttgtttgccactcttactattcccaatcttggattgtt
    cgagctgaaaaaaaatttgatgagaaacaggaggatccttttctggtgaatataggttc
    ctgctttaagaatgtggaaatccattgctttatataactaatatacacacagattaattaaa
    attgtgagaaataattcacacatgacaagtaggtaacatgcatgagttttgaatttttttaa
    aaacccaactgtttgacaaaatatagaacccaaattggtactttcttagaccagtgtaac
    ctcacacctcagttttgcttttccaaccctgacttgaaaggcatatttgtatctttttattagt
    gatagtgaagctgtgacactaaccttttatacaaaagagtaaagaaagaaaaactaca
    gcgattaagatgagaacagttctgcagttgttgaactagatcacagcattgtaggcag
    aataaaaaatgttcatatctgagaatattcctttcgccatcttttcccaaggccagacctc
    ctggtggagcacagttaaaagtaacattctgggcctttgtaatcggagggctgtgtctc
    cagctggcagcctttgttttaatatataatgcaggactgtggaaaacagttggcataga
    atattttcacctaaaaaagaaagaaaagacatacaaaactggattaattgcaaaaaga
    gaatacagtaaaataccatataactggacaaagctagaagaacctttagaagatttgtc
    tgaaaacagatttcaagagtgagcttttatacactgctcactaatttgcttgattactacca
    actcttcttaaagttaacacgtttaaggtatttctggacttcctagccttttagcaagcttag
    aggaactagccattagctagtgatgtaaaaatattttggggactgatgcccttaaaggtt
    atgcccttgaaagttcttaccttttctctagtgatattaaggaacgagtgggtagtgttctc
    agggtgaccagctgccctaaagtgcctgggattgagggtttccctggatgcgggact
    ttccctggatacaaaacttttagcagagttttgtatatatgtggatttttctgataagtagca
    catcagaggccttaaccactgcccaaaagcgattctccattgagagtacatatcttgaa
    cttaagaaattcatttgctctgatttttaatcttgtaaagtttttgctaaactcaaaacaagtc
    ccaggcacaccagaaggagctgaccaccttaggtgttcttgtgatttatccttacttccc
    tatgttgtcatagttgcttctaaactcagctgcactatggctgtcaacatttctgatacttat
    tgggatatgtgccatccagtcatttagtactttgaatggaacatgagatttataacacag
    gtaatagctgaaggtaccagtatggtggtgagactcacacttagtgatccagctaagg
    taactgatgttataatggaacagagaagaggccaactagatagctaagttcttctgaac
    ctatgtgtatatgtaagtacaaatcatgcgtccttatggggttaaacttaatctgaaattta
    catttttcatagtaaaaggaaaccaattgttgcagatttcttttcttgtgaggaaatacatg
    gcctttgatgctctggcgtctactgcatttcccagtctgttctgctcgagaagccagaat
    gtgttgttaacatttttccgtgaatgttgtgttaaaatgattaaatgcatcagccaatggca
    agtgaaggaattgggtgtcctgatgcagactgagcagtttctctcaattgtagcctcata
    ctcataaggtgcttaccagctagaacattgagcacgtgaggtgagattttttttctctgat
    ggcattaactttgtaatgcaatatgatggatgcagaccctgttcttgtttccctctggaag
    tccttagtggctgcatccttggtgcactgtgatggagatattaaatgtgttctttgtgagct
    ttcgttctatgattgtcaaaagtacgatgtggttccttttttatttttattaaacaatgagctg
    aggctttattacagctggttttcaagttaaaattgttgaatactgatgtctttctcccaccta
    caccaaatattttagtctatttaaagtacaaaaaaagttctgcttaagaaaacattgcttac
    atgtcctgtgatttctggtcaatttttatatatatttgtgtgcatcatctgtatgtgctttcactt
    tttaccttgtttgctcttacctgtgttaacagccctgtcaccgttgaaaggtggacagtttt
    cctagcattaaaagaaagccatttgagttgtttaccatgttaaaaaaaaaaaaaaaa
    (SEQ ID NO: 607)
    Akap81 gtgtgtggaggggaccctgtggttagcagcagctatcgcagcgtcggatgttcagag NM_014371 NM_017476
    cagcagaagccggcgtcgtcggatgttgtgttgcccgccaccatgagctacacagg
    ctttgtccagggatctgaaaccactttgcagtcgacatactcggataccagcgctcag
    cccacctgtgattatggatatggaacttggaactctgggacaaatagaggctacgagg
    gctatggctatggctatggctatggccaggataacaccaccaactatgggtatggtat
    ggccacttcacactcttgggaaatgcctagctctgacacaaatgcaaacactagtgcc
    tcgggtagcgccagtgccgattccgttttatccagaattaaccagcgcttagatatggt
    gccgcatttggagacagacatgatgcaaggaggcgtgtacggctcaggtggagaaa
    ggtatgactcttatgagtcctgcgactcgagggccgtcctgagtgagcgcgacctgta
    ccggtcaggctatgactacagcgagcttgaccctgagatggaaatggcctatgaggg
    ccaatacgatgcctaccgcgaccagttccgcatgcgtggcaacgacaccttcggtcc
    cagggcacagggctgggcccgggatgcccggagcggccggccaatggcctcag
    gctatgggcgcatgtgggaagaccccatgggggcccggggccagtgcatgtctggt
    gcctctcggctgccctccctcttctcccagaacatcatccccgagtacggcatgttcca
    gggcatgcgaggtgggggcgccttcccgggcggctcccgctttggtttcgggtttgg
    caatggcatgaagcagatgaggcggacctggaagacctggaccacagccgacttc
    cgaaccaagaagaagaagagaaagcagggcggcagtcctgatgagccagatagc
    aaagccacccgcacggactgctcggacaacagcgactcagacaatgatgagggca
    ccgagggggaagccacagagggccttgaaggcaccgaggctgtggagaagggct
    ccagagtggacggagaggatgaggagggaaaagaggatgggagagaagaaggc
    aaagaggatccagagaagggggccctaaccacccaggatgaaaatggccagacc
    aagcgcaagttgcaggcaggcaagaagagtcaggacaagcagaaaaagcggcag
    cgagaccgcatggtggaaaggatccagtttgtgtgttctctgtgcaaataccggacctt
    ctatgaggacgagatggccagccatcttgacagcaagttccacaaggaacactttaa
    gtacgtaggcaccaagctccctaagcagacggctgactttctgcaggagtacgtcac
    taacaagaccaagaagacagaggagctccgaaaaaccgtggaggaccttgatggc
    ctcatccaccaaatctacagagaccaggatctgacccaggaaattgccatggagcatt
    ttgtgaagaaggtggaggcagcccattgtgcagcctgcgacctcttcattcccatgca
    gtttgggatcatccagaagcatctgaagaccatggatcacaaccggaaccgcaggct
    catgatggagcagtccaagaagtcctccctcatggtggcccgcagtattctcaacaac
    aagctcatcagcaagaagctggagcgctacctgaagggcgagaaccctttcaccga
    cagccccgaggaggagaaggagcaggaggaggctgagggcggtgccctggacg
    agggggcgcagggcgaagcggcagggatctcggagggcgcagagggcgtgcc
    ggcgcagcctcccgtgcccccagagccagcccccggggccgtgtcgccgccacc
    gccgccgcccccagaggaggaggaggagggcgccgtgcccttgctgggagggg
    cgctgcaacgccagatccgcggcatcccgggcctcgacgtggaggacgacgagg
    agggcggcgggggcgccccgtgacccgagctcggggcgggcggagcccgcgt
    ggccgaagctggaaaccaaacctaataaagttttcccatcccaccaaaaaaaaaaaa
    aaaaaaaaaa (SEQ ID NO: 608)
    Rbks acctttgagcgatggcggcgtctggggaaccccagaggcagtggcaagaggaggt NM_022128 NM_153196
    ggcggcggtggtagtggtgggctcctgcatgaccgacctggtcagtcttacttctcgtt
    tgccaaaaactggagaaaccatccatggacataagttttttattggctttggagggaaa
    ggtgccaaccagtgtgtccaagctgctcggcttggagcaatgacgtccatggtgtgta
    aggttggcaaagattcttttggcaatgattatatagaaaacttaaaacagaatgatatttc
    tacagaatttacatatcagactaaagatgctgctacaggaactgcttctataattgtcaat
    aatgaaggccagaatatcattgtcatagtggctggagcaaatttacttttgaatacgga
    ggatctgagggcagcagccaatgtcattagcagagccaaagtcatggtctgccagct
    cgaaataactccagcaacttctttggaagccctaacaatggcccgcaggagtggagt
    gaaaaccttgttcaatccagcccctgccattgctgacctggatccccagttctacaccc
    tctcagatgtgttctgctgcaatgaaagtgaggctgagattttaactggcctcacggtg
    ggcagcgctgcagatgctggggaggctgcattagtgctcttgaaaaggggctgcca
    ggtggtaatcattaccttaggggctgaaggatgtgtggtgctgtcacagacagaacct
    gagccaaagcacattcccacagagaaagtcaaggctgtggataccacgggtgctgg
    tgacagctttgtgggagctctggccttctacctggcttactatccaaatctgtccttggaa
    gacatgctcaacagatccaatttcattgcagcagtcagtgtccaggctgcaggaacac
    agtcatcttacccttacaaaaaagaccttccgcttactctgttttgattgctattagtccca
    aaataaatatacctgggaataaaatgtacttgggggtggctgctcctggctaatgcttat
    tagaaaatgtcctcgtcccctttctttgcaaatattagttcttttacgaagtcatcctcaag
    cttcaatttatttataacgatgattcttttgctttccatgcatttgcacaaaacaaccagaat
    taaagattccacaacc (SEQ ID NO: 609)
    Egr2 aactgagcgaggagcaattgattaatagctcggcgaggggactcactgactgttataa NM_000399 NM_010118
    taacactacaccagcaactcctggcttcccagcagccggaacacagacaggagaga
    gtcagtggcaaatagacatttttcttatttcttaaaaaacagcaacttgtttgctacttttatt
    tctgttgatttttttttcttggtgtgtgtggtggttgtttttaagtgtggagggcaaaaggag
    ataccatcccaggctcagtccaacccctctccaaaacggcttttctgacactccaggta
    gcgagggagttgggtctccaggttgtgcgaggagcaaatgatgaccgccaaggcc
    gtagacaaaatcccagtaactctcagtggttttgtgcaccagctgtctgacaacatcta
    cccggtggaggacctcgccgccacgtcggtgaccatctttcccaatgccgaactgg
    gaggcccctttgaccagatgaacggagtggccggagatggcatgatcaacattgac
    atgactggagagaagaggtcgttggatctcccatatcccagcagctttgctcccgtctc
    tgcacctagaaaccagaccttcacttacatgggcaagttctccattgaccctcagtacc
    ctggtgccagctgctacccagaaggcataatcaatattgtgagtgcaggcatcttgca
    aggggtcacttccccagcttcaaccacagcctcatccagcgtcacctctgcctccccc
    aacccactggccacaggacccctgggtgtgtgcaccatgtcccagacccagcctga
    cctggaccacctgtactctccgccaccgcctcctcctccttattctggctgtgcaggag
    acctctaccaggacccttctgcgttcctgtcagcagccaccacctccacctcttcctctc
    tggcctacccaccacctccttcctatccatcccccaagccagccacggacccaggtct
    cttcccaatgatcccagactatcctggattctttccatctcagtgccagagagacctaca
    tggtacagctggcccagaccgtaagccctttccctgcccactggacaccctgcgggt
    gccccctccactcactccactctctacaatccgtaactttaccctggggggccccagt
    gctggggtgaccggaccaggggccagtggaggcagcgagggaccccggctgcct
    ggtagcagctcagcagcagcagcagccgccgccgccgccgcctataacccacacc
    acctgccactgggcccattctgaggcctcgcaagtaccccaacagacccagcaag
    acgccggtgcacgagaggccctacccgtgcccagcagaaggctgcgaccggcgg
    ttctcccgctctgacgagctgacacggcacatccgaatccacactgggcataagccc
    ttccagtgtcggatctgcatgcgcaacttcagccgcagtgaccacctcaccacccata
    tccgcacccacaccggtgagaagcccttcgcctgtgactactgtggccgaaagtttg
    cccggagtgatgagaggaagcgccacaccaagatccacctgagacagaaagagc
    ggaaaagcagtgccccctctgcatcggtgccagccccctctacagcctcctgctctg
    ggggcgtgcagcctgggggtaccctgtgcagcagtaacagcagcagtcttggcgg
    agggccgctcgccccttgctcctctcggacccggacaccttgagatgagactcaggc
    tgatacaccagctcccaaaggtcccggaggccctttgtccactggagctgcacaaca
    aacactaccaccctttcctgtccctctctccctttgttgggcaaagggctttggtggagc
    tagcactgccccctttccacctagaagcaggttcttcctaaaacttagcccattctagtct
    ctcttaggtgagttgactatcaacccaaggcaaaggggaggctcagaaggaggtgg
    tgtggggacccctggccaagagggctgaggtctgaccctgctttaaagggttgtttga
    ctaggttttgctaccccacttccccttattttgacccatcacaggtttttgaccctggatgt
    cagagttgatctaagacgttttctacaataggttgggagatgctgatcccttcaagtgg
    ggacagcaaaaagacaagcaaaactgatgtgcactttatggcttgggactgatttggg
    ggacattgtacagtgagtgaagtatagcctttatgccacactctgtggccctaaaatgg
    tgaatcagagcatatctagttgtctcaacccttgaagcaatatgtattataaactcagag
    aacagaagtgcaatgtgatgggaggaacatagcaatatctgctccttttcgagttgtttg
    agaaatgtaggctattttttcagtgtatatccactcagattttgtgtatttttgatgtacactg
    ttctctaaattctgaatctttgggaaaaaatgtaaagcatttatgatctcagaggttaactt
    atttaagggggatgtacatatattctctgaaactaggatgcatgcaattgtgttggaagt
    gtccttggtgccttgtgtgatgtagacaatgttacaaggtctgcatgtaaatgggttgcc
    ttattatggagaaaaaaatcactccctgagtttagtatggctgtatatttctgcctattaata
    tttggaattttttttagaaagtatatttttgtatgctttgttttgtgacttaaaagtgttacctttg
    tagtcaaatttcagataagaatgtacataatgttaccggagctgatttgtttggtcattag
    ctcttaatagttgtgaaaaaataaatctattctaacgcaaaaccactaactgaagttcag
    ataatggatggtttgtgactatagtgtaaataaatacttttcaacaataaaaaaaaaaaaa
    aa (SEQ ID NO: 610)
    Dgka agttcctgccagtgagtccctaggcctccatctctctcccttgctgtaccaccttcacca NM_001345 NM_016811
    ccatccatgcgaccccaagagccttaatgactctagaagagactccaggcagggga
    agctgaaaggacctttcactccctacttttggccagggccttctgtgccacctgccaag
    accagcaggcctaccctctgaagaggtccaagcaacggaagtactactacgaagct
    gcctttctggccatccttgagaaaaatagacagatggccaaggagaggggcctaata
    agccccagtgattttgcccagctgcaaaaatacatggaatactccaccaaaaaggtca
    gtgatgtcctaaagctcttcgaggatggcgagatggctaaatatgtccaaggagatgc
    cattgggtacgagggattccagcaattcctgaaaatctatctcgaagtggataatgttc
    ccagacacctaagcctggcactgtttcaatcctttgagactggtcactgcttaaatgag
    acaaatgtgacaaaagatgtggtgtgtctcaatgatgtttcctgctacttttcccttctgg
    agggtggtcggccagaagacaagttagaattcaccttcaagctgtacgacacggaca
    gaaatgggatcctggacagctcagaagtggacaaaattatcctacagatgatgcgag
    tggctgaatacctggattgggatgtgtctgagctgaggccgattcttcaggagatgatg
    aaagagattgactatgatggcagtggctctgtctctcaagctgagtgggtccgggctg
    gggccaccaccgtgccactgctagtgctgctgggtctggagatgactctgaaggac
    gacggacagcacatgtggaggcccaagaggttccccagaccagtctactgcaatct
    gtgcgagtcaagcattggtcttggcaaacagggactgagctgtaacctctgtaagtac
    actgttcacgaccagtgtgccatgaaagccctgccttgtgaagtcagcacctatgcca
    agtctcggaaggacattggtgtccaatcacatgtgtgggtgcgaggaggctgtgagt
    ccgggcgctgcgaccgctgtcagaaaaagatccggatctaccacagtctgaccggg
    ctgcattgtgtatggtgccacctagagatccacgatgactgcctgcaagcggtgggc
    catgagtgtgactgtgggctgctccgggatcacatcctgcctccatcttccatctatccc
    agtgtcctggcctctggaccggatcgtaaaaatagcaaaacaagccagaagaccat
    ggatgatttaaatttgagcacctctgaggctctgcggattgaccctgttcctaacaccca
    cccacttctcgtctttgtcaatcctaagagtggcgggaagcaggggcaaagggtgct
    ctggaagttccagtatatattaaaccctcgacaggtgttcaacctcctaaaggatggtc
    ctgagatagggctccgattattcaaggatgttcctgatagccggattttggtgtgtggtg
    gagacggcacagtaggctggattctagagaccattgacaaagctaacttgccagtttt
    gcctcctgttgctgtgttgcccctgggtactggaaatgatctggctcgatgcctaagat
    ggggaggaggttatgaaggacagaatctggcaaagatcctcaaggatttagagatg
    agtaaagtggtacatatggatcgatggtctgtggaggtgatacctcaacaaactgaag
    aaaaaagtgacccagtcccctttcaaatcatcaataactacttctctattggcgtggatg
    cctctattgctcatcgattccacatcatgcgagagaaatatccggagaagttcaacagc
    agaatgaagaacaagctatggtacttcgaatttgccacatctgaatccatcttctcaaca
    tgcaaaaagctggaggagtctttgacagttgagatctgtgggaaaccgctggatctga
    gcaacctgtccctagaaggcatcgcagtgctaaacatccctagcatgcatggtggctc
    caacctctggggtgataccaggagaccccatggggatatctatgggatcaaccagg
    ccttaggtgctacagctaaagtcatcaccgaccctgatatcctgaaaacctgtgtacca
    gacctaagtgacaagagactggaagtggttgggctggagggtgcaattgagatggg
    ccaaatctataccaagctcaagaatgctggacgtcggctggccaagtgctctgagatc
    accttccacaccacaaaaacccttcccatgcaaattgacggagaaccctggatgcag
    acgccctgtacaatcaagatcacccacaagaaccagatgcccatgctcatgggccca
    cccccccgctccaccaatttctttggcttcttgagctaagggggacacccttggcctcc
    aagccagccttgaacccacctccctgtccctggactctactcccgaggctctgtacatt
    gctgccacatactcctgccagcttgggggagtgttccttcaccctcacagtatttattat
    cctgcaccacctcactgttccccatgcgcacacacatacacacaccccaaaacacat
    acattgaaagtgcctcatctgaataaaatgacttgtgtttcccctttgggatctgctaaaa
    aaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO: 611)
    Cb1b ctgggtcctgtgtgtgccacaggggggggtgtccagcgagcggtctcctcctcctg NM_170662 NM_
    ctagtgctgctgcggcgtcccgcggcctccccgagtcgggcgggaggggagagc 001033238
    gggtgtggatttgtcttgacggtaattgttgcgtttccacgtctcggaggcctgcgcgct
    gggttgctccttcttcgggagcgagctgttctcagcgatcccactcccagccggggct
    ccccacacacactgggctgcgtgcgtgtggagtgggacccgcgcacacgcgtgtct
    ctggacagctacggcgccgaaagaactaaaattccagatggcaaactcaatgaatg
    gcagaaaccctggtggtcgaggaggaaatccccgaaaaggtcgaattttgggtatta
    ttgatgctattcaggatgcagttggaccccctaagcaagctgccgcagatcgcagga
    ccgtggagaagacttggaagctcatggacaaagtggtaagactgtgccaaaatccca
    aacttcagttgaaaaatagcccaccatatatacttgatattttgcctgatacatatcagca
    tttacgacttatattgagtaaatatgatgacaaccagaaacttgcccaactcagtgagaa
    tgagtactttaaaatctacattgatagccttatgaaaaagtcaaaacgggcaataagact
    ctttaaagaaggcaaggagagaatgtatgaagaacagtcacaggacagacgaaatc
    tcacaaaactgtcccttatcttcagtcacatgctggcagaaatcaaagcaatctttccca
    atggtcaattccagggagataactttcgtatcacaaaagcagatgctgctgaattctgg
    agaaagttttttggagacaaaactatcgtaccatggaaagtattcagacagtgccttcat
    gaggtccaccagattagctctggcctggaagcaatggctctaaaatcaacaattgattt
    aacttgcaatgattacatttcagtttttgaatttgatatttttaccaggctgtttcagccttgg
    ggctctattttgcggaattggaatttcttagctgtgacacatccaggttacatggcatttct
    cacatatgatgaagttaaagcacgactacagaaatatagcaccaaacccggaagcta
    tattttccggttaagttgcactcgattgggacagtgggccattggctatgtgactgggg
    atgggaatatcttacagaccatacctcataacaagcccttatttcaagccctgattgatg
    gcagcagggaaggattttatctttatcctgatgggaggagttataatcctgatttaactg
    gattatgtgaacctacacctcatgaccatataaaagttacacaggaacaatatgaattat
    attgtgaaatgggctccacttttcagctctgtaagatttgtgcagagaatgacaaagatg
    tcaagattgagccttgtgggcatttgatgtgcacctcttgccttacggcatggcaggag
    tcggatggtcagggctgccctttctgtcgttgtgaaataaaaggaactgagcccataat
    cgtggacccctttgatccaagagatgaaggctccaggtgttgcagcatcattgacccc
    tttggcatgccgatgctagacttggacgacgatgatgatcgtgaggagtccttgatgat
    gaatcggttggcaaacgtccgaaagtgcactgacaggcagaactcaccagtcacat
    caccaggatcctctccccttgcccagagaagaaagccacagcctgacccactccag
    atcccacatctaagcctgccacccgtgcctcctcgcctggatctaattcagaaaggca
    tagttagatctccctgtggcagcccaacgggttcaccaaagtcttctccttgcatggtg
    agaaaacaagataaaccactcccagcaccacctcctcccttaagagatcctcctccac
    cgccacctgaaagacctccaccaatcccaccagacaatagactgagtagacacatc
    catcatgtggaaagcgtgccttccagagacccgccaatgcctcttgaagcatggtgc
    cctcgggatgtgtttgggactaatcagcttgtgggatgtcgactcctaggggagggct
    ctccaaaacctggaatcacagcgagttcaaatgtcaatggaaggcacagtagagtgg
    gctctgacccagtgcttatgcggaaacacagacgccatgatttgcctttagaaggagc
    taaggtcttttccaatggtcaccttggaagtgaagaatatgatgttcctccccggctttct
    cctcctcctccagttaccaccctcctccctagcataaagtgtactggtccgttagcaaat
    tctctttcagagaaaacaagagacccagtagaggaagatgatgatgaatacaagattc
    cttcatcccaccctgtttccctgaattcacaaccatctcattgtcataatgtaaaacctcct
    gttcggtcttgtgataatggtcactgtatgctgaatggaacacatggtccatcttcagag
    aagaaatcaaacatccctgacttaagcatatatttaaagggagatgtttttgattcagcct
    ctgatcccgtgccattaccacctgccaggcctccaactcgggacaatccaaagcatg
    gttcttcactcaacaggacgccctctgattatgatcttctcatccctccattaggtgaaga
    tgcttttgatgccctccctccatctctcccacctcccccacctcctgcaaggcatagtct
    cattgaacattcaaaacctcctggctccagtagccggccatcctcaggacaggatcttt
    ttcttcttccttcagatccctttgttgatctagcaagtggccaagttcctttgcctcctgcta
    gaaggttaccaggtgaaaatgtcaaaactaacagaacatcacaggactatgatcagc
    ttccttcatgttcagatggttcacaggcaccagccagaccccctaaaccacgaccgcg
    caggactgcaccagaaattcaccacagaaaaccccatgggcctgaggcggcattgg
    aaaatgtcgatgcaaaaattgcaaaactcatgggagagggttatgcctttgaagaggt
    gaagagagccttagagatagcccagaataatgtcgaagttgcccggagcatcctccg
    agaatttgccttccctcctccagtatccccacgtctaaatctatagcagccagaactgta
    gacaccaaaatggaaagcaatcgatgtattccaagagtgtggaaataaagagaactg
    agatggaattcaagagagaagtgtctcctcctcgtgtagcagcttgagaagaggcttg
    ggagtgcagcttctcaaaggagaccgatgcttgctcaggatgtcgacagctgtggctt
    ccttgtttttgctagccatatttttaaatcagggttgaactgacaaaaataatttaaagacg
    tttacttcccttgaactttgaacctgtgaaatgctttaccttgtttacagtttggcaaagttg
    cagtttgttcttgtttttagtttagttttgttttggtgttttgatacctgtactgtgttcttcacag
    accctttgtagcgtggtcaggtctgctgtaacatttcccaccaactctcttgctgtccaca
    tcaacagctaaatcatttattcatatggatctctaccatccccatgccttgcccaggtcca
    gttccatttctctcattcacaagatgctttgaaggttctgattttcaactgatcaaactaatg
    caaaaaaaaaaaagtatgtattcttcactactgagtttcttctttggaaaccatcactattg
    agagatgggaaaaacctgaatgtataaagcatttatttgtcaataaactgccttttgtaa
    ggggttttcacataacata (SEQ ID NO: 612)
    Mdfic cccaggccggctctggcctcctgacccagacagcgcagggcgcgagggatcgcg NM_001166345 NM_175088
    cggccgagcccgggtcgcgccgctcccagcatcggggccgctagccaagagttcg
    aggccttcccgatccggatgtgatgaaaaagagcaacagagggagaagtgtttcag
    gattgtaggagtggaagaggggaaagagaggcagagagggggaaggccccctc
    gcaggggagccggctggagtgagctggctggaaagagggggcggagtgcgcgg
    agtcagagccgccaccgctgccgcagttgccgccactgcggcgtctgggctgagc
    cggagggaggcgggaggacgcgcaggggcggccgccgccgtcgtcaggccac
    cggggcgaaaatgcggccgctgccggaggctcgctaactttccggggcggaagag
    gaggaggaggaggaggaaggggcttggagcgactacggggggatgcggagaag
    cagtcagttccctgcacccagcacctcacagcccttcctccgtgcgccctgccgggc
    ggcgagctaggcggcagcggcgcggcgcgggctcggcggagcggcccatgtcc
    ggcgcgggcgaagccctcgctcccgggcccgtggggccgcagcgcgtggccga
    ggcgggcggcggccagctgggctccacagcccagggaaaatgtgataaagacaat
    actgagaaagatataactcaagctaccaatagccacttcacacatggagagatgcaa
    gaccagtccatttggggaaatccttcggatggtgaactcattagaacccaacctcagc
    gcttgcctcagcttcagacttcagcccaggtgccaagtggtgaggaaataggcaaga
    taaagaacggccacacaggtctgagcaatggaaatggaattcaccacggggccaaa
    cacggatccgcagataatcgcaaactttcagcacctgtttctcaaaaaatgcatagaaa
    aattcagtccagcttgtctgtaaacagcgatatcagtaagaagagcaaagtaaatgct
    gtcttttcccaaaagacaggctcttcacctgaagattgttgtgtccactgtatcctggctt
    gcttgttctgcgaattcctgaccctttgcaacattgtcctgggacaagcgtcatgtggca
    tctgcacctcagaagcctgctgctgttgctgtggtgacgagatgggggatgattgtaa
    ctgcccttgtgatatggactgtggcatcatggatgcctgttgtgaatcatcagactgctt
    ggaaatctgtatggaatgctgtggaatttgttttccttcataaatatttatcttttgtttgtgtt
    aaaactggagagtgtttaaaaatttccttttggggggaagaaaagcacattgtaagatt
    ctcatgaaacaacatggaatttgcactgttaactcattattgtaagtaatctctgaaagcc
    tttttactttaaccaaatctacatggtttaatatgtgaaattttaactactttaactagttttata
    aatttcttaatatgttacaataacttagggacattttgacaccccccttcccaaatgttaaa
    tgccttctcctttttaccgatatttctgtttcttttaaccgttctcaggagcactttgctccaa
    atatattatttttcagtgtgtatttaaacgaggcagtttattttgatatgtatctattcatgatt
    gaaaggaagcagtcttggccaggcacggtggcttacacctgtaaccctggcattttg
    ggaggccaaggtgggcagattgcctgagctcaggagttcgagaccagccagggca
    acatggtgaaaccccatctctactaaaatacaaaaagttagctgggcttggcggtgtg
    cgcctgtagtcccagctactcaggaggctgaggcaggagaattgcttgaacccgag
    aggcggaagttgcagtgagccgagattgtgccactgaactccaacctgcactccagc
    ctgggcaacagagcgagactccatctctaaataaataaataaataaataaataaataa
    ataaataaataaacaaaccagtctttattttaaaagaaactttaggaaacaaacccacat
    aatagttgggaaccagtgttgatctctctcccttaccttctccacttgttcaacagactct
    gaatgccgactgtgtggactctcttcctcagactgtggggacagatacaattccactcc
    tgtccacaggaacatgagatttagcagactaaggagatctgtaaagaatgaaccatac
    cacaaggcatactgaagtgaggattataagagaaataaactcaaaatgctgttggaat
    atgcagagaattgctaccagaatattcagtaaggtttcagggagaatgtggcatttgag
    gactctcttagaatgagtgattcacctgctatttaaatgaattatttagatttttgacaaaga
    tttaggtggacaccctaaactgtgtgtgcctttaaccagttaaaagaacagtgccttcag
    catacttttttattagttgtaggaatacagctttttgaaaaagctataaagtttaaattaacta
    aaaatatgcattttcttacacataatttaaatgttatcatacttttttgatgaaaacataatgc
    cttagtaaaatagctctatttaataaagaagattgagtactctgacacatttcatttaaatta
    ggaaatttttaatattaaaatcccagtgttctgagttattgaaaggctttcttttattttgaga
    gctttaggtctttttgggatgagaacattttagttgtttagtttgtttcttaagcagtgctattt
    tttgtaaacacagataaatggaaaccattcttttcaatgcagaagaaatctagatatccc
    ctactgtgaccaaatttctgtattacgattttatgttaaattaaactaatatggcaggttata
    atgatccttaagtgtaaagaaatcagtcaattacaagagtaattgtatagttattgagacc
    tatagtgtgtggcttagatgaaagggagagtaaattttcataccatgctctctcctactca
    gtttgatctctctaaaattgtagtttggtttgatttaatataattcttagtagaaattttgaaag
    tatgctttgggattaataattatttttaatttttctggctgaatatcaaattgatagtaacaac
    agaagcataattttaggaaggctttcgcaaacctagccttttaagagaggtttttaacct
    gaagcatgagaatatatcacctgtggtttttcctttgagatgaaacgtagtttctagttata
    tcattacttaaagggcttaaaaagaaaaaacttagcaaacttttgaatctttcttttattgct
    atttacacatacatacacacatacaaaacctttaaattttgggatctgaatataattctggt
    aaacagctgtcttcatttttctcctctaaagaacttaattcatttgttacataaaatataagg
    aaatctttatactattttacagtaaccacaatctaaatatttacatatacccaaaattaactt
    atgctcatatattaggatgtgagaatatcatctgtttatggacacatgaaacctcctaatg
    acctggaattgttagaatatttgactttttatatgcaaagtttttcaaccaagtggtttgtcta
    atatttaaacatgtactggcacaatttgtgatgaaaatattagcacatttgcaataatgttt
    ctccataacagagaatgttaatggataccagaattttatttttgtatttatgttcatagtactt
    ttcctcttgtctactccagacagttattccataaagcatttgtataattaaaaggaaaaca
    gaaaaaggaaaagtaggcaaatgtgaaaatagtttcaatatatcttatgatttcttaatgt
    aaaatgttttgttgaagtatatggctatcatgactaagtgctagaatttatagttacaggc
    ggtgtccttttaaatgtggaaaggcttttaaaatattttaaaactggacctgtattatcctg
    aatacactattttgaaaatttttaaaaatgacttctttattttgctttaccgtatgtttatatcta
    attgacatattgactaatgtttgaaagaattcaaccataagttaaaatctgaaggttatctt
    tatcatgtttcatccctgtctgaagatttcctagtcttcttatgtaaatcacatgactcatgtc
    cgtaaatgaactatgaaagatatcgatcagtttatgatcattgacatgtgatttcaaaaca
    cagtgttcttttaaaaatctataatatgtcaaaatacaagttttttttttttacatcgttttagta
    agttaatttcatttatttactttggagctatatttccacttagaaaaactaaggtaattttaca
    atatatgctgagattaaaaaccaaggtaaaaatgatcaaacatatatgaaattgagtctt
    agatttaatgaatttcactcgaaaataaatgatcagaagaattttcatctaaggcataga
    gtggcgaaatttttgtaaatgctcgcagttagcatctaactaaaacaatacagtatgactt
    tatttaggagaaggctttttatttagaaaattattttttcatttttacagtgtatcaactgtatc
    cattttcctcacctggatagtcaatgttatctgagcagttcaaggagtaaccaaggcaa
    ccttatgtaataactttccattctttatccatacaaactctttcagtgccctagattctaatgt
    tataaacgtcaaacatcactgcccaacataaataagactcgagacttattaacataaat
    aagtatcttgccttcttgaatgctagttaaatgcttagatttacctaactgcctaatgaatc
    aggttatttgttaataagattatttttcaaattatttaagacctttatgccccttccaattactt
    gtgatttgtaggcctgtaggattgttgcatctaatctgactggcaacagaaaatgtcatc
    aaatactataatatccattttttttcttttgcactaatacaacagaacatatcatttttgtttta
    aacaatggttaatatattaatagggtttgttccacacttactatttatagtttttataatcaag
    cattgggtattaaaagagaatcctttcaacccttcatcttcgtatgcttatacaataaattg
    cagtgagtgt (SEQ ID NO: 613)
    Entpd1 agggaagaagggagaaagagagagagatttgaatatacattgcttcaaggatgcaa NM_001776 NM_009848
    aaaattacaacctggaaaaggcttcgagtaactttaggaaaatgagctgctggactcc
    tcagtcaatctgtcctttctagtcaatgaaaaagacagggtttgaggttccttccgaaac
    ggggccggctaatttagcccctcccacgagcccaagggtctgttatatctctgtttcctt
    gaggacctctctcacggagacggaccacagcaagcagaggctgggggggggaaa
    gacgaggaaagaggaggaaaacaaaagctgctacttatggaagatacaaaggagt
    ctaacgtgaagacattttgctccaagaatatcctagccatccttggcttctcctctatcat
    agctgtgatagctttgcttgctgtggggttgacccagaacaaagcattgccagaaaac
    gttaagtatgggattgtgctggatgcgggttcttctcacacaagtttatacatctataagt
    ggccagcagaaaaggagaatgacacaggcgtggtgcatcaagtagaagaatgcag
    ggttaaaggtcctggaatctcaaaatttgttcagaaagtaaatgaaataggcatttacct
    gactgattgcatggaaagagctagggaagtgattccaaggtcccagcaccaagaga
    cacccgtttacctgggagccacggcaggcatgcggttgctcaggatggaaagtgaa
    gagttggcagacagggttctggatgtggtggagaggagcctcagcaactaccccttt
    gacttccagggtgccaggatcattactggccaagaggaaggtgcctatggctggatt
    actatcaactatctgctgggcaaattcagtcagaaaacaaggtggttcagcatagtccc
    atatgaaaccaataatcaggaaacctttggagctttggaccttgggggagcctctaca
    caagtcacttttgtaccccaaaaccagactatcgagtccccagataatgctctgcaattt
    cgcctctatggcaaggactacaatgtctacacacatagcttcttgtgctatgggaagga
    tcaggcactctggcagaaactggccaaggacattcaggttgcaagtaatgaaattctc
    agggacccatgctttcatcctggatataagaaggtagtgaacgtaagtgacctttacaa
    gaccccctgcaccaagagatttgagatgactcttccattccagcagtttgaaatccagg
    gtattggaaactatcaacaatgccatcaaagcatcctggagctcttcaacaccagttac
    tgcccttactcccagtgtgccttcaatgggattttcttgccaccactccagggggattttg
    gggcattttcagctttttactttgtgatgaagtttttaaacttgacatcgagaaagtctctca
    ggaaaaggtgactgagatgatgaaaaagttctgtgctcagccttgggaggagataaa
    aacatcttacgctggagtaaaggagaagtacctgagtgaatactgcttttctggtacct
    acattctctccctccttctgcaaggctatcatttcacagctgattcctgggagcacatcc
    atttcattggcaagatccagggcagcgacgccggctggactttgggctacatgctga
    acctgaccaacatgatcccagctgagcaaccattgtccacacctctctcccactccac
    ctatgtcttcctcatggttctattctccctggtccttttcacagtggccatcataggcttgct
    tatctttcacaagccttcatatttctggaaagatatggtatagcaaaagcagctgaaatat
    gctggctggagtgaggaaaaaaatcgtccagggagcattttcctccatcgcagtgttc
    aaggccatccttccctgtctgccagggccagtcttgacgagtgtgaagcttccttggct
    tttactgaagcctttcttttggaggtattcaatatcctttgcctcaaggacttcggcagata
    ctgtctctttcatgagtttttcccagctacacctttctcctttgtactttgtgcttgtataggttt
    taaagacctgacacctttcataatctttgctttataaaagaacaatattgactttgtctaga
    agaactgagagtcttgagtcctgtgataggaggctgagctggctgaaagaagaatct
    caggaactggttcagttgtactctttaagaacccctttctctctcctgtttgccatccatta
    agaaagccatatgatgcctttggagaaggcagacacacattccattcccagcctgctc
    tgtgggtaggagaattttctacagtaggcaaatatgtgctaaagccaaagagttttataa
    ggaaatatatgtgctcatgcagtcaatacagttctcaatcccacccaaagcaggtatgt
    caataaatcacatattcctaggtgatacccaaatgctacagagtggaacactcagacct
    gagatttgcaaaaagcagatgtaaatatatgcattcaaacatcagggcttactatgagg
    taggtggtatatacatgtcacaaataaaaatacagttacaactcagggtcacaaaaaat
    gcatcttccaatgcatatttttattatggtaaaatatacataaatataattcaccattttaaca
    tttaattcatattaaatacgtacaaatcagtgacatttagtacattcacagtgttgtgccac
    catcaccactatttagttccagaacatttgcatcatcaatacattgtctagagacaagact
    atcctgggtaggcagaaaccatagatcttttgtgtttacagctatggaaaccaactgtac
    cataaagatagttcactgagttttaaagccaagccacatcttatttttccaaggtttaattt
    agtgagagggcagcattagtgtggagtggcatgcttttgccctatcgtggaatttacac
    atcagaatgtgcaggatccaagtctgaaagtgttgccacccgtcacacaacatgggct
    ttgtttgcttattccatgaagcagcagctatagaccttaccatggaaacatgaagagac
    cctgcacccctttccttaaggattgctgcaagagttacctgttgagcaggattgactggt
    gatgtttcattctgaccttgtcccaagctctccatctctagatctggggactgactgttga
    gctgatggggaaagaaaagctctcacacaaaccggaagccaaatgtcccctatctct
    tgaatgatcaagtcacttttgacaacatccaggtgaatataaaaacttaataaagctgtg
    gaaaggaactcttaatcttcttttctgctacttaggttaaattcactagatcttgattaggaa
    tcaaaattcgaattgggacatgttcaaattctttcttgtggtagttgcctatactgtcatcg
    ctgctgttggttgagcatttgtggtgtaccacgctgtgtgctcaagggtattacattcatc
    ttctcatttaatcctcacaacaatctgaagaaggtaggtattacaattcccacttcataga
    aacagaaactgaggttcagagaggttaagtcatttgcccaaatggctgagccaaagc
    ctaccatgtacctaacctttattttctttcccgaacataccaggctgtctcctcataacttc
    caagcatgcacttaaaactccacatgaatacaaggttcatgggacttggtattcataga
    aagggaggcagaaagctggtctgttcctgataggcttgtaatttaatatcattctgttcat
    gtgctttggatggaagcacatctggcatatgatgctaatcagtggttcccatacccctg
    gcttcctaattttaatgtttgctcacagcatagtagattgacatcaaatagtggccgatga
    tgatgaaaataaaggtcaaataagttgagccaataacagccgcttttttccttctgtctgc
    gtatacaaagcactgtcatgcacacaatctattctgaccctcacaacaacccataagg
    gtgtaaatagtatttccattttacaaatgaggatcacacaaactactacatggcagagca
    gatactccaactcatgtcttctggttgaagcctattgctttttcttttctaaacactttccctc
    agcaagttggaattagacttcacaagtctccttcagagaacacaaatcttttcttattcca
    ttcctgtttggttgcctacgtccaatctccccctccccagagatgccaaaaaaaaaatc
    ctttaaggtatttgggagccaaactcaacttgttaaaatctcaaattatggagacaatca
    gcagacacaacctaaccccaattattttggcaggaaggttggtttagaggcagatcca
    gcaatctgctttgggccactctgggtggggtaggtgaaataagattggtcactgttaac
    taattttaatattggattggccattggttatcactgattaccattctcccctggattttcacc
    caggactcaaaacttggttctgctaaccctgttcctttatgaggaaccttttaaagattcc
    tttataaggtgggagttttttttctatgaacctataggggagaaaaaagatcagcagaag
    tcattacttttttttttttttttttttttttttgagagagagtctcactccattgcccaggctggag
    tgcagtggtgctatctcggctcactgcaacctccgcctcctgggttcaagcaattctcct
    gcctcagcctcccgagtagctgggattgcaggtgcccaccaccacacccggctaatt
    tttgtatttttagtaaagacagggtttcaccatgttggccaggctggtctccaactcccaa
    tctcaggtgatcctattgcctcgggctcccaaagtgctgggattacaggagtgagcca
    ccatgcctggccagaagtggttacttctgtagacaaaagaataatgctacttaatcagg
    ctttctgtgtgacaagaaagagaaagaaaataaagaagtttcaattcatccaattcttaa
    taagaaatatgtaaataaaattttttaaaattacacttcattttaatgttgtatcagtcaaggt
    ccctgcaagagatggatggtatggtacactcaaactgggtaacacaggagagttttca
    gaaagcaactaaatccaaaatactatcaaggaatcaatataaaaattgttaatatttttct
    catactaaattttcaaaatattttgtgtctattacatttacagcacatcttaattaggactag
    ctgtgtgttcacctcacatgtggcttgtagctaccatactggacagcacatgtccaaaa
    aaatacacgtaaagttaaagtttaaaagacacaggaactaagccctcattgtctttccct
    tgggaggtagtttaaagagctatagatgctgtaacattcttgctattatttattatatatgac
    attattcctaaaaaagcttttgagatcctaggttgtattcctcaggttttgttgccttcccat
    gaagatgtgaaggcagggatgcctgttattcagtccaagatgcatgacaagagacctt
    gggaaagtttcatctggatttaaagattaattcttgatgcttacattccatactcaaaatgt
    aaatttgaatattaaaataaagatgattttttttttggagctagtcttgctctgttgcccagg
    ctggaatgcagtggcatgatcatggctcactgcagcctcgacctcccaagctcaagc
    aaggctacaggtgtgcacctaagtagctaggactacaggtgtgcaccaccatgtcta
    gctatttttttttctgtagagacagggttttcctatgttgtccaggctggtctcgaactcctg
    ccctcaagcaatcctcctgccttggcctcccaaagtgttgagattacaggcgtaagcc
    actgcacctggccaagatgaatattttaatagctcacagaacaaagtttgccacataat
    gataaaattactatgaaaatatattccctttattgtcagtttaaaagatgaactgagtttca
    cccaaactggtctggcccctctctgattcaaataccaatagttgctctgattcaaattcca
    actgttagaacatgacagctgctcataactagctttgcttactaaccatgtttctttccattt
    gtattaggtcctttactttttataacagcctcaaagtttcatgaattgctgcagtaaacattg
    attttcatgtttgtgagtctgcaagccagctgggcagctctacttcaggtggtaagggtg
    gatcagacctattccatatacctcttgttctccttgtccagtggtttctagggatatgttctc
    atgatgaaccccgcagaggctcgtgaaagtgagaggaaactaggatgcctcttaag
    gtcttggtcaggatggggtctcctgtcacttctgtcacaggctattgtaagtcatatgag
    caagctcaataaaatataaacaagtcagataaacagtgggaggaatggcaaagtcat
    atggccaaggccatgagtgattaattttaacacaggaaaaaagtaaagcattaaatgc
    gattatttaatatacaatgtcttattaactgaaatataaaatgtgtttactgtaaaatataatc
    tgtttatctcaccaaagaaatattatctttaaaaaatgtcattacttctaagacatcatcagt
    ctgcaacttctttccatagccttaatcaggatgctgtggcagctcccacattagcctcgc
    attctaaactggtagatgtcctaggaaaccatacatctatgtatttttcttattttatacgttt
    aggacaatgtatagctaattacccaactttttatttgcatacaaatctaatacaactgaac
    acaatcagttttatcacaggtataatggatttttcaatagtgaggaggtgcctccatgag
    ccttctctttagaaaagtggcattcaagactcttcatttgaagtgaagattgctatgtctttt
    gcattgctctattttacataaattaagttataaattgacactataatcaactgacaccatga
    tcagtgatgatgatcaccctcatcagcactagagttgacttgtttttataacccctttgcat
    gtatgttgaatagcaaagttcatcagagaacatgtattagtcaatggtaagtaagatact
    ctcatctaagaaataacatcacctcttctaatgaagttctaagaagagagggaagaaa
    aagtcttgggagctagtcagggaatagtgtgtatttgcaattacctaaactgaactctac
    cattactcctaacccagttcctcctcctgtgttttacatgattaatgccacccctgcctcaa
    tgaaccaagatcagctccatcactgggacctccccattctgcctgtgcaatattttttttt
    ttatttctccttctaatattactgttattgctccagtaaagagctgtaatatattttacctgga
    ctgataccaggaatggtggtgttgcttccaatctgttgctgctagattaatctttgcaaag
    cacaggcttaatttcattgctgctcaactaaaaccactggtggctttccattgcctacaa
    aataaagtcaacctccccatcagacattcaaggctttcaatgatccatggccgccagct
    ctctccaggctcatatcccactccactcctctgatgtttcctacactacactacactatac
    tacactacagccaggtagaatgactgttcacccaacaccactcaggttgtcttctcaac
    ttggaatactcttgcaccttcaaagctcatttcaaatgccccttcatttgtgaagccttctc
    caaatttccaagtcagaatgtctcttccttgtgctaccacaaccctttaactgagcctcca
    ttagtgcactgagaccattctgttcagtgtctgggtgaagcttcctggtgaaaaatatgtt
    acctatttctttctgaaaagttggattcagggatattatcacggacctaaggtaatagttct
    agccaacctccctgtccactgccaggccgactacaaacccttctgttgctggcgagct
    ggtccgcaccactagttctgcttcactctatttatctcttgatgtaaccatcttctttctcca
    ggttttaagaaccagcccaactcctggttccctgatgaagcttttattcccctagccaca
    tggaacttttcctttttggaacatgcctttagtttctgtgtagtttgccatgcagcacttcatt
    gtacacattattaaaacagaattttaaggattagaatgaaccttaaaagatcatgcatctc
    aaaatttaatgtacatacaaattacccagggattttgttgaaataaaaattatttaattttaa
    ttaatataaataattcagtaggtctggggtgaggcctgaggttttacatttccaacaagct
    gccaggtaaagccaatacatctgtccaggaatcacactttgcgtatcaaaggtctagat
    gacattatcattccaaagagtttcttttacaggctctcagatcagtgttcatccactacctg
    actactgtcattcacaggcattctgttccacagcaggccagctaacgtggtatttacaa
    agctcactcctcttatacaacaatccaagtgtttcttttgtcagttgtctgtgccccagga
    gatccctctctgccttgccttgccctctgcctttggagaccagcacctcatactcagtga
    aggcctggagtgcttaagagggatttcttccagctctcttgccctggtcttcagtgtatta
    gatgtattacctccatgctctcagtagaggcccataggaaagagtaggtaggttatgc
    cagctcacacgcatcctttaaaaatggtttagaagtttagctggtttcttattactcctgtct
    atggatgtttccttctgtcactctactagggatgaaacagctaatcatgttcaatagttac
    atttagattggtttttaaaaactatgattgtattagttcgtttccatgctgctgataaagacat
    atctgagactggaaacaaaaagggtttaattggacttacagttccacatggctgggga
    ggcctcaaaatcaggtgggaggcaaaaggtacttcttacgtggtggcatcaagagca
    aaatgaggaagaagcaaaagcagaaactcttcataaacccaccagatcttgtgggac
    ttattatcacgagaatagcacagaaaagactggcctccatgattcaattacctcccact
    gcgtccctcccacaacatgtgggaattctgggagatacaattcaagttgagatttgggt
    ggggacacagccaaaccatatcattcctccctgggctcctccaaatttcataatcctca
    catttcaaaaccaatcattccttcccaacagttccccaaagtcttaactcatttcagcatta
    acccaaaagtccacagtccaaagtctcatctgagacaaggcaagtcccttccacttac
    aagcctgtaaaagcaagctagttacctcctagatacaatggggggtacaggtattggg
    taaatacagctgttccaaatgagagaaattggccaaaacaaaggggttacagggtcc
    atgcaagtctgaaatccagtggggcagtcaaattttaaagctccataatgatctcctttg
    actccatgtctcacattcaggtcatgctgatgcaagagataggttcccatggtcttgtgc
    agctccgcccctgtggctttgcagagtacagcctccctcctggctgctttctcaggctg
    atgttgagtgtctgtagcttttccaggcacaagatgcaagttggtggttgatctaccattc
    tggggtctaccattctggggtctaccgttctgggactgtggccttcttctcacagctcca
    ctaggcagtgccccaacagggactctgtgtgggggctctgccccacatttcccttcca
    cactgccctaggagaggttccccatgagggctctgcccctgcagcaaacttttgcctg
    gacatccaggtgtttccatatatattctgaaatctaggcagaggttcccaaatctcaattc
    ttgacatctctgcacccacaggctcaacatcacatggaagctgccaatgcttggggcc
    tctaccctctgaagccacagcccaagctctatgttggctcctttcagccatggctggag
    cagctgggacacagggcaccaagtccctaggctgcacacagcacagagaccctgg
    gcccagcccacaaaaccactttttcctcctgggcctctgggcctgtgatgggagggg
    ctgccatgaaggtctctgacatgacctggagacattttccccatggtcttggggattaa
    cattaggctccttgctgcttatgcaaatttctgcagccagcttgaatttctccttaaaaaaa
    atgggtttttcttttctactgcatcatcaggctgcagattttccacatttatgctcttgtttcc
    cttttaaaacagaatgtttttaacagcacccaagtcaccttttgaatgctttgctgcttaga
    aatttattccaccagataccctaagtcatctctctcaagctctaagttccacaaatctcta
    gggcaagggtgaaatgctgccagtctccttgctaaaacataacaagggtcacctttac
    ttcagttcccaacaaggtcttcatctccatctgagaccacctcagcctggaccttattgtt
    catatcactatcagtatttttgtcaatgccattcacagtctctaggaggttccaaactttcc
    tacattttcctatcttcttctgagccctccagattatttcaacacccagttccaaagttgctt
    ccacattttcgggtatcttttcagcaatgccccactctactggtactattagtccattttcat
    gctgctgataaagacatacctgagactgggaacaaaaagaggtttaattggacttata
    gttccacctggctggggaggcctcagaatcatggcaggaggtgaaaggcatttctta
    cacggcagcagcaagagaaaaatgaagaagcagcaaaagcagaaacccctgata
    aaaccatcagatctcgtgagacttattcactatcacaagaatagcatgggaaagacca
    gcccccttgattcaattacctccccctgggtcctgtgggaattctggaaggtacaattca
    agttgagatttgggtggggacacagccaaaccatatcaatgattttgtactttaaccag
    ctgaatggaagtacaatctcttgctatatgacacaataattatttgcaaaatgagtaaac
    atatcataaggaaattatttttacaaggtttgaaacctgaaatgcagtctattatcatacat
    aactaaaaatagagcctcaataaacagattcccagttttgaaaatgcaacatttgtactc
    cacattgtcagttttcttaggtatatttataaatactcctataaaaatgtaaagaaacacat
    aatgtagattgctaattttataataacacaagttgattttgacatccaacttattaattatga
    aatgacttttggcctagtaacaatgaaaatgggggcaaatacagataaatggtaattctt
    agaatgaactactcagcaccaattctaagtttttcttgatggtaaatcataatgttcccttt
    ctcctcggttctgcaatctataggcataccataattgtaatcaatagcttaaaaatatgtct
    ctctgtcctattctgtatctgtatctcttggatttttacctttgcaatagtcaactgaaccatc
    ttcttggagtactcatgaagatggaagtctacatggagaatacaggatgaatccactct
    gtctcctgcagtgaagtctgtttgaaggatgtatttggctgtcttctggacaggccattct
    aataacagaaacaaacaagttattttaaaacttattggaatattcaaatattaaccaaagt
    agaaaaatataatacacatccatgtgcccatcacagaacttcactgattatcatcattta
    gccagtcttgaagaagcaagtgctaattacaatcacaaatgaaacaagattcagactt
    catgaagagcactgcgctataataaaagaagaaatgagcacatacattcttttactgac
    agtcaaatggtgaaggtgggcagaatcattatgtgatgcaacatggcaaaagtataca
    gacagtgcatccagaggaaggcaccttgctgaatgactagaatggaagtaggagac
    attttgcaggcccccttcatcctgcagggagaaccagaaccacagcagctctatttgc
    ctattcctctttaaattacaaagttaaaatttgggagtagtagaaaatcaattggttatctta
    tagagtctcctagaatatttcattggcattgagaaggtggaaaatgcaaattatatacttt
    aaaatgtaatttttgcttttcacatatgcttaaagcctaaaacctcttaataaacttcttctga
    aatata (SEQ ID NO: 614)
    Dgkz ggagagtgtctctaaggtgacactcgggtgcgcggcagcagcggcggttgcagga NM_201532 NM_138306
    gctcgctctccgcccgggctccggctccgctccagccgtccggggggcgccgcgg
    cgcgcagagcgcagcaccccgactccagccaggagcccccgcccccccggagc
    gcaggaggaccccggcccgcctctcccaggcgcagcgcccagcatctcgctgctc
    ctgtcgtctaagcgtcggcgtcgctagggacctgcggaacccggcgctcccctccct
    ccccgcctcgcgtccccggcccgggcggactggagactcgaacttgagcgggtgc
    ccgaaaggccgcaggagccgcgggcggaaggggccgcacgatggccgaggg
    gcagggcggcggagggcagcgctgggactgggctggcggcggccgggcagcc
    gaggaggaggtggtgcggcggcgatgccggcgcggggaggaggcccaggtcgc
    gcagccctggcccgagggttcccggggcacggccgctgggcccccggtggagga
    gcgtttccgccagctgcacctacgaaagcaggtgtcttacaggaaagccatcaccaa
    gtcgggcctccagcacctggccccccctccgcccacccctggggccccgtgcagc
    gagtcagagcggcagatccggagtacagtggactggagcgagtcagcgacatatg
    gggagcacatctggttcgagaccaacgtgtccggggacttctgctacgttggggagc
    agtactgtgtagccaggatgctgaagtcagtgtctcgaagaaagtgcgcagcctgca
    agattgtggtgcacacgccctgcatcgagcagctggagaagataaatttccgctgtaa
    gccgtccttccgtgaatcaggctccaggaatgtccgcgagccaacctttgtacggca
    ccactgggtacacagacgacgccaggacggcaagtgtcggcactgtgggaaggg
    attccagcagaagttcaccttccacagcaaggagattgtggccatcagctgctcgtgg
    tgcaagcaggcataccacagcaaggtgtcctgcttcatgctgcagcagatcgagga
    gccgtgctcgctgggggtccacgcagccgtggtcatcccgcccacctggatcctcc
    gcgcccggaggccccagaatactctgaaagcaagcaagaagaagaagagggcat
    ccttcaagaggaagtccagcaagaaagggcctgaggagggccgctggagaccctt
    catcatcaggcccaccccctccccgctcatgaagcccctgctggtgtttgtgaacccc
    aagagtgggggcaaccagggtgcaaagatcatccagtctttcctctggtatctcaatc
    cccgacaagtcttcgacctgagccagggagggcccaaggaggcgctggagatgta
    ccgcaaagtgcacaacctgcggatcctggcgtgcgggggcgacggcacggtggg
    ctggatcctctccaccctggaccagctacgcctgaagccgccaccccctgttgccatc
    ctgcccctgggtactggcaacgacttggcccgaaccctcaactggggtgggggcta
    cacagatgagcctgtgtccaagatcctctcccacgtggaggaggggaacgtggtac
    agctggaccgctgggacctccacgctgagcccaaccccgaggcagggcctgagg
    accgagatgaaggcgccaccgaccggttgcccctggatgtcttcaacaactacttca
    gcctgggctttgacgcccacgtcaccctggagttccacgagtctcgagaggccaacc
    cagagaaattcaacagccgctttcggaataagatgttctacgccgggacagctttctct
    gacttcctgatgggcagctccaaggacctggccaagcacatccgagtggtgtgtgat
    ggaatggacttgactcccaagatccaggacctgaaaccccagtgtgttgttttcctgaa
    catccccaggtactgtgcgggcaccatgccctggggccaccctggggagcaccac
    gactttgagccccagcggcatgacgacggctacctcgaggtcattggcttcaccatg
    acgtcgttggccgcgctgcaggtgggcggacacggcgagcggctgacgcagtgtc
    gcgaggtggtgctcaccacatccaaggccatcccggtgcaggtggatggcgagcc
    ctgcaagcttgcagcctcacgcatccgcatcgccctgcgcaaccaggccaccatggt
    gcagaaggccaagcggcggagcgccgcccccctgcacagcgaccagcagccgg
    tgccagagcagttgcgcatccaggtgagtcgcgtcagcatgcacgactatgaggcc
    ctgcactacgacaaggagcagctcaaggaggcctctgtgccgctgggcactgtggt
    ggtcccaggagacagtgacctagagctctgccgtgcccacattgagagactccagc
    aggagcccgatggtgctggagccaagtccccgacatgccagaaactgtcccccaa
    gtggtgcttcctggacgccaccactgccagccgcttctacaggatcgaccgagccca
    ggagcacctcaactatgtgactgagatcgcacaggatgagatttatatcctggaccct
    gagctgctgggggcatcggcccggcctgacctcccaacccccacttcccctctcccc
    acctcaccctgctcacccacgccccggtcactgcaaggggatgctgcaccccctca
    aggtgaagagctgattgaggctgccaagaggaacgacttctgtaagctccaggagct
    gcaccgagctgggggcgacctcatgcaccgagacgagcagagtcgcacgctcctg
    caccacgcagtcagcactggcagcaaggatgtggtccgctacctgctggaccacgc
    ccccccagagatccttgatgcggtggaggaaaacggggagacctgtttgcaccaag
    cagcggccctgggccagcgcaccatctgccactacatcgtggaggccggggcctc
    gctcatgaagacagaccagcagggcgacactccccggcagcgggctgagaaggc
    tcaggacaccgagctggccgcctacctggagaaccggcagcactaccagatgatcc
    agcgggaggaccaggagacggctgtgtagcgggccgcccacgggcagcaggag
    ggacaatgcggccaggggacgagcgccttccttgcccacctcactgccacattcca
    gtgggacggccacggggggacctaggccccagggaaagagccccatgccgccc
    cctaaggagccgcccagacctagggctggactcaggagctgggggggcctcacct
    gttcccctgaggaccccgccggacccggaggctcacagggaacaagacacggct
    gggttggatatgcctttgccggggttctggggcagggcgctccctggccgcagcag
    atgccctcccaggagtggaggggctggagagggggaggccttcgggaagaggctt
    cctgggccccctggtcttcggccgggtccccagcccccgctcctgccccaccccac
    ctcctccgggcttcctcccggaaactcagcgcctgctgcacttgcctgccctgccttg
    cttggcacccgctccggcgaccctccccgctcccctgtcatttcatcgcggactgtgc
    ggcctggggggggggggggactctcacggtgacatgtttacagctgggtgtgac
    tcagtaaagtggatttttttttctttaaaaaaaa (SEQ ID NO: 615)
    Vamp7 attggaggagcgctcccactcccaagaggccacgcgtagacggggcgcttcatgc NM_005638 NM_011515
    ggaagtcagcggcgtccggtcccagcctcctctgggagcgggcagttggcgaccct
    gcactgacccgcgtccctccgtcccgagcccgcgcgccctcagagggtgcccgga
    cagactgaagccatggcgattctttttgctgttgttgccagggggaccactatccttgcc
    aaacatgcttggtgtggaggaaacttcctggaggtgacagagcagattctggctaag
    ataccttctgaaaataacaaactaacgtactcacatggcaattatttgtttcattacatctg
    ccaagacaggattgtatatctttgtatcactgatgatgattttgaacgttcccgagccttta
    attttctgaatgagataaagaagaggttccagactacttacggttcaagagcacagac
    agcacttccatatgccatgaatagcgagttctcaagtgtcttagctgcacagctgaagc
    atcactctgagaataagggcctagacaaagtgatggagactcaagcccaagtggatg
    aactgaaaggaatcatggtcagaaacatagatctggtagctcagcgaggagaaagat
    tggaattattgattgacaaaacagaaaatcttgtggattcttctgtcaccttcaaaactac
    cagcagaaatcttgctcgagccatgtgtatgaagaacctcaagctcactattatcatcat
    catcgtatcaattgtgttcatctatatcattgtttcacctctctgtggtggatttacatggcc
    aagctgtgtgaagaaataggaaagaagaagttaccattaaccaaggatatgagagaa
    caaggagttaaaagcaatccatgtgactcaagcctttcacatactgacagatggtatct
    gccagtctcttcaaccctcttctcactttttaaaatcttgttccatgcctccaggtttatcttt
    gtcttatctaccagtttattcctgtgaacttcagattgaaccattcattgcagcagtagcct
    taaaaaggcttttgtttatttctttggtttgttaactagtgtcatctatttagagaaacattttt
    gtttttaattgctcaaagctgtcgccgctagtcttatgagctatctactaaaactatggag
    aaactttgtatgtgcacacaaaagtattcaagagacagtattgctaacatctcatcttaat
    gtcttttgttattgagaagttttaggtgcttcaaaacaatataaatggataatagttgttattt
    ggggaattgtaatgatgttggtgctgcttccttctaagagctcagacaagtaaagtatg
    aaacattcttatttcagttagatggggaacattttgctagcccattagaagcacacagaa
    ttatccttgtcctcctaatattgactttcaggaataaagttcagtgtgctgatcattcacaat
    acagtggatagcttgatatcttctgttttcccattgcagttgatttgagaagatgaaggttt
    aaatattgttgaaagttgcagttttttaaatgtgttcctttttcttctgtgaatatttagggcaa
    tcgtgtcgctaatagaatatgtagtagagggggggggaggtaaattcctctgacttgc
    caaagaaaaagaagggaaccacagtggatatgctagcattttagctgtgcaaaggga
    ggtagtgtgggaaaagtgtttccattctgggaaaagcccaaaccgaatacggtcagc
    agtcaactccagggtttgggcttgattcctgttgaataatagttttgagcattctttgtggtt
    aaataaattcttaaatctgcctagttttgatgaattcttttgtgaaacttgaaagagaatag
    acagtatgacatatagaattaatacaaaacagtttaacaaccatttaactgcagtgtaag
    aaaattggactgtaatcatatcgctactggcatctgttatctagtatgcatttctggtgtgt
    atctgaaaggaagacattttctaccctagatccaattgcatttatttatcaataagtgccat
    taaattgaaattatattacattttacactttctcaatgaatgaacaaattagtctgtagaatc
    tagccacctgtttagcctagtcatgtgccttgaacatatatgtgtcccataatctggctca
    tggtacctgttcttctatccaaacctttcaattcatgctacctgattcatttatttgacataga
    tcttaggcccacttgaactcttttcttgtttatctagcatagcacaaacgtttttccagtcttc
    tttatcaacactaatgcctcttaattgcatcagtatttcctattggaaaatacatctgttcca
    gaaaaacatttggcattcctgaataatttccaaatgtttttaatccaaagaaaaaggttta
    aagcttatttccctttcttatacacacctgaataaaattgatgtgcatgttttagggatcaat
    tacctaactgttccttggtctatttatgtataagaatgctttttaaagcacatgtctcatttta
    aatgacgcacaaactgaagatgttaataaaatttaagagtaatacaatgaaaaaa
    (SEQ ID NO: 616)
    Hipk1 gcagagtctgcagtgcggaggggggggaagtccaggccccgcactcgatccac NM_198268 NM_010432
    gctggctccctacggaggcccacctactcgaggcccaccgactcctactgcaatcag
    tactatgcgatcgtcctagagagtccattcagctgcacttccgcctcagtatggcatca
    cagctgcaagtgttttcgcccccatcagtgtcgtcgagtgccttctgcagtgcgaaga
    aactgaaaatagagccctctggctgggatgtttcaggacagagtagcaacgacaaat
    attatacccacagcaaaaccctcccagccacacaagggcaagccaactcctctcacc
    aggtagcaaatttcaacatccctgcttacgaccagggcctcctcctcccagctcctgc
    agtggagcatattgttgtaacagccgctgatagctcgggcagtgctgctacatcaacc
    ttccaaagcagccagaccctgactcacagaagcaacgtttctttgcttgagccatatca
    aaaatgtggattgaaacgaaaaagtgaggaagttgacagcaacggtagtgtgcagat
    catagaagaacatccccctctcatgctgcaaaacaggactgtggtgggtgctgctgc
    cacaaccaccactgtgaccacaaagagtagcagttccagcggagaaggggattacc
    agctggtccagcatgagatcctttgctctatgaccaatagctatgaagtcttggagttcc
    taggccgggggacatttggacaggtggctaagtgctggaagaggagcaccaagga
    aattgtggctattaaaatcttgaagaaccacccctcctatgccagacaaggacagattg
    aagtgagcatcctttcccgcctaagcagtgaaaatgctgatgagtataattttgtccgtt
    catacgagtgctttcagcataagaatcacacctgccttgtttttgaaatgttggagcaga
    acttatatgattttctaaagcaaaacaaatttagcccactgccactcaagtacatcagac
    caatcttgcagcaggtggccacagccttgatgaagctcaagagtcttggtctgatcca
    cgctgaccttaagcctgaaaacatcatgctggttgatccagttcgccagccctaccga
    gtgaaggtcattgactttggttctgctagtcacgtttccaaagctgtgtgctcaacctact
    tacagtcacgttactacagagctcctgaaattattcttgggttaccattttgtgaagctatt
    gatatgtggtcactgggctgtgtgatagctgagctgttcctgggatggcctctttatcct
    ggtgcttcagaatatgatcagattcgttatatttcacaaacacaaggcttgccagctgaa
    tatcttctcagtgccggaacaaaaacaaccaggtttttcaacagagatcctaatttggg
    gtacccactgtggaggcttaagacacctgaagaacatgaactggagactggaataaa
    atcaaaagaagctcggaagtacatttttaattgcttagatgacatggctcaggtgaatat
    gtctacagacctggagggaacagacatgttggcagagaaggcagaccgaagagaa
    tacattgatctgttaaagaaaatgctcacaattgatgcagataagagaattacccctcta
    aaaactcttaaccatcagtttgtgacaatgactcaccttttggattttccacatagcaatc
    atgttaagtcttgttttcagaacatggagatctgcaagcggagggttcacatgtatgata
    cagtgagtcagatcaagagtcccttcactacacatgttgccccaaatacaagcacaaa
    tctaaccatgagcttcagcaatcagctcaatacagtgcacaatcaggccagtgttctag
    cttccagttctactgcagcagctgctactctttctctggctaattcagatgtctcactacta
    aactaccagtcagctttgtacccatcatctgctgcaccagttcctggagttgcccagca
    gggtgtttccttgcagcctggaaccacccagatttgcactcagacagatccattccaac
    agacatttatagtatgtccacctgcgtttcaaactggactacaagcaacaacaaagcat
    tctggattccctgtgaggatggataatgctgtaccgattgtaccccaggcaccagctg
    ctcagccactacagattcagtcaggagttctcacgcagggaagctgtacaccactaat
    ggtagcaactctccaccctcaagtagccaccatcacaccgcagtatgcggtgcccttt
    actctgagctgcgcagccggccggccggcgctggttgaacagactgccgctgtact
    gcaggcgtggcctggagggactcagcaaattctcctgccttcaacttggcaacagttg
    cctggggtagctctacacaactctgtccagcccacagcaatgattccagaggccatg
    gggagtggacagcagctagctgactggaggaatgcccactctcatggcaaccagta
    cagcactatcatgcagcagccatccttgctgactaaccatgtgacattggccactgctc
    agcctctgaatgttggtgttgcccatgttgtcagacaacaacaatccagttccctccctt
    cgaagaagaataagcagtcagctccagtctcttccaagtcctctctagatgttctgcctt
    cccaagtctattctctggttgggagcagtcccctccgcaccacatcttcttataattcctt
    ggtccctgtccaagatcagcatcagcccatcatcattccagatactcccagccctcctg
    tgagtgtcatcactatccgaagtgacactgatgaggaagaggacaacaaatacaagc
    ccagtagctctggactgaagccaaggtctaatgtcatcagttatgtcactgtcaatgatt
    ctccagactctgactcttctttgagcagcccttattccactgataccctgagtgctctccg
    aggcaatagtggatccgttttggaggggcctggcagagttgtggcagatggcactgg
    cacccgcactatcattgtgcctccactgaaaactcagcttggtgactgcactgtagcaa
    cccaggcctcaggtctcctgagcaataagactaagccagtcgcttcagtgagtgggc
    agtcatctggatgctgtatcacccccacagggtatcgagctcaacgcggggggacc
    agtgcagcacaaccactcaatcttagccagaaccagcagtcatcggcggctccaac
    ctcacaggagagaagcagcaacccagccccccgcaggcagcaggcgtttgtggcc
    cctctctcccaagccccctacaccttccagcatggcagcccgctacactcgacaggg
    cacccacaccttgccccggcccctgctcacctgccaagccaggctcatctgtatacgt
    atgctgccccgacttctgctgctgcactgggctcaaccagctccattgctcatcttttctc
    cccacagggttcctcaaggcatgctgcagcctataccactcaccctagcactttggtg
    caccaggtccctgtcagtgttgggcccagcctcctcacttctgccagcgtggcccctg
    ctcagtaccaacaccagtttgccacccaatcctacattgggtcttcccgaggctcaaca
    atttacactggatacccgctgagtcctaccaagatcagccagtattcctacttatagttg
    gtgagcatgagggaggaggaatcatggctaccttctcctggccctgcgttcttaatatt
    gggctatggagagatcctcctttaccctcttgaaatttcttagccagcaacttgttctgca
    ggggcccactgaagcagaaggtttttctctgggggaacctgtctcagtgttgactgca
    ttgttgtagtcttcccaaagtttgccctatttttaaattcattatttttgtgacagtaattttggt
    acttggaagagttcagatgcccatcttctgcagttaccaaggaagagagattgttctga
    agttaccctctgaaaaatattttgtctctctgacttgatttctataaatgcttttaaaaacaa
    gtgaagcccctctttatttcattttgtgttattgtgattgctggtcaggaaaaatgctgata
    gaaggagttgaaatctgatgacaaaaaaagaaaaattactttttgtttgtttataaactca
    gacttgcctattttattttaaaagcggcttacacaatctcccttttgtttattggacatttaaa
    cttacagagtttcagttttgttttaatgtcatattatacttaatgggcaattgttatttttgcaa
    aactggttacgtattactctgtgttactattgagattctctcaattgctcctgtgtttgttata
    aagtagtgtttaaaaggcagctcaccatttgctggtaacttaatgtgagagaatccatat
    ctgcgtgaaaacaccaagtattctttttaaatgaagcaccatgaattcttttttaaattatttt
    ttaaaagtctttctctctctgattcagcttaaatttttttatcgaaaaagccattaaggtggtt
    attattacatggtggtggtggttttattatatgcaaaatctctgtctattatgagatactggc
    attgatgagctttgcctaaagattagtatgaattttcagtaatacacctctgttttgctcatc
    tctcccttctgttttatgtgatttgtttggggagaaagctaaaaaaacctgaaaccagata
    agaacatttcttgtgtatagcttttatacttcaaagtagcttcctttgtatgccagcagcaa
    attgaatgctctcttattaagacttatataataagtgcatgtaggaattgcaaaaaatatttt
    aaaaatttattactgaatttaaaaatattttagaagttttgtaatggtggtgttttaatatttta
    cataattaaatatgtacatattgattagaaaaatataacaagcaatttttcctgctaaccca
    aaatgttatttgtaatcaaatgtgtagtgattacacttgaattgtgtacttagtgtgtatgtg
    atcctccagtgttatcccggagatggattgatgtctccattgtatttaaaccaaaatgaac
    tgatacttgttggaatgtatgtgaactaattgcaattatattagagcatattactgtagtgct
    gaatgagcaggggcattgcctgcaaggagaggagacccttggaattgttttgcacag
    gtgtgtctggtgaggagtttttcagtgtgtgtctcttccttccctttcttcctccttcccttatt
    gtagtgccttatatgataatgtagtggttaatagagtttacagtgagcttgccttaggatg
    gaccagcaagcccccgtggaccctaagttgttcaccgggatttatcagaacaggatta
    gtagctgtattgtgtaatgcattgttctcagtttccctgccaacattgaaaaataaaaaca
    gcagcttttctcctttaccaccacctctacccctttccattttggattctcggctgagttctc
    acagaagcattttccccatgtggctctctcactgtgcgttgctaccttgcttctgtgagaa
    ttcaggaagcaggtgagaggagtcaagccaatattaaatatgcattcttttaaagtatgt
    gcaatcacttttagaatgaatttttttttccttttcccatgtggcagtccttcctgcacatagt
    tgacattcctagtaaaatatttgcttgttgaaaaaaacatgttaacagatgtgtttatacca
    aagagcctgttgtattgcttaccatgtccccatactatgaggagaagttttgtggtgccg
    ctggtgacaaggaactcacagaaaggtttcttagctggtgaagaatatagagaagga
    accaaagcctgttgagtcattgaggcttttgaggtttcttttttaacagcttgtatagtcttg
    gggcccttcaagctgtgaaattgtccttgtactctcagctcctgcatggatctgggtcaa
    gtagaaggtactggggatggggacattcctgcccataaaggatttggggaaagaag
    attaatcctaaaatacaggtgtgttccatctgaattgaaaatgatatatttgagatataattt
    taggactggttctgtgtagatagagatggtgtcaaggaggtgcaggatggagatggg
    agatttcatggagcctggtcagccagctctgtaccaggttgaacaccgaggagctgtc
    aaagtatttggagtttcttcattgtaaggagtaagggcttccaagatggggcaggtagt
    ccgtacagcctaccaggaacatgttgtgttttctttattttttaaaatcattatattgagttgt
    gttttcagcactatattggtcaagatagccaagcagtttgtataatttctgtcactagtgtc
    atacagttttctggtcaacatgtgtgatctttgtgtctcctttttgccaagcacattctgattt
    tcttgttggaacacaggtctagtttctaaaggacaaattttttgttccttgtcttttttctgtaa
    gggacaagatttgttgtttttgtaagaaatgagatgcaggaaagaaaaccaaatcccat
    tcctgcaccccagtccaataagcagataccacttaagataggagtctaaactccacag
    aaaaggataataccaagagcttgtattgttaccttagtcacttgcctagcagtgtgtggc
    tttaaaaactagagatttttcagtcttagtctgcaaactggcatttccgattttccagcata
    aaaatccacctgtgtctgctgaatgtgtatgtatgtgctcactgtggctttagattctgtcc
    ctggggttagccctgttggccctgacaggaagggaggaagcctggtgaatttagtga
    gcagctggcctgggtcacagtgacctgacctcaaaccagcttaaggctttaagtcctc
    tctcagaacttggcatttccaacttcttcctttccgggtgagagaagaagcggagaag
    ggttcagtgtagccactctgggctcatagggacacttggtcactccagagtttttaatag
    ctcccaggaggtgatattattttcagtgctcagctgaaataccaaccccaggaataaga
    actccatttcaaacagttctggccattctgagcctgcttttgtgattgctcatccattgtcct
    ccactagaggggctaagcttgactgcccttagccaggcaagcacagtaatgtgtgttt
    tgttcagcattattatgcaaaaattcactagttgagatggtttgttttaggataggaaatga
    aattgcctctcagtgacaggagtggcccgagcctgcttcctattttgattttttttttttttaa
    ctgatagatggtgcagcatgtctacatggttgtttgttgctaaactttatataatgtgtggtt
    tcaattcagcttgaaaaataatctcactacatgtagcagtacattatatgtacattatatgt
    aatgttagtatttctgctttgaatccttgatattgcaatggaattcctactttattaaatgtatt
    tgatatgctagttattgtgtgcgatttaaactttttttgctttctccctttttttggttgtgcgctt
    tcttttacaacaagcctctagaaacagatagtttctgagaattactgagctatgtttgtaat
    gcagatgtacttagggagtatgtaaaataatcattttaacaaaagaaatagatatttaaa
    atttaatactaactatgggaaaagggtccattgtgtaaaacatagtttatctttggattcaa
    tgtttgtctttggttttacaaagtagcttgtattttcagtattttctacataatatggtaaaatg
    tagagcaattgcaatgcatcaataaaatgggtaaattttctgacttatgtggctgtttttga
    cttctgttataggatataaaggggatcaataaatgacatctttgaaagtgaaaa (SEQ
    ID NO: 617)
    Nuak2 gtgctttactgcgcgctctggtactgctgtggctccccgtcctggtgcgggacctgtgc NM_030952 NM_
    cccgcgcttcagccctccccgcacagcctactgattcccctgccgcccttgctcacct 001195025
    cctgctcgccatggagtcgctggttttcgcgcggcgctccggccccactccctcggc
    cgcagagctagcccggccgctggcggaagggctgatcaagtcgcccaagccccta
    atgaagaagcaggcggtgaagcggcaccaccacaagcacaacctgcggcaccgc
    tacgagttcctggagaccctgggcaaaggcacctacgggaaggtgaagaaggcgc
    gggagagctcggggcgcctggtggccatcaagtcaatccggaaggacaaaatcaa
    agatgagcaagatctgatgcacatacggagggagattgagatcatgtcatcactcaa
    ccaccctcacatcattgccatccatgaagtgtttgagaacagcagcaagatcgtgatc
    gtcatggagtatgccagccggggcgacctttatgactacatcagcgagcggcagca
    gctcagtgagcgcgaagctaggcatttcttccggcagatcgtctctgccgtgcactatt
    gccatcagaacagagttgtccaccgagatctcaagctggagaacatcctcttggatgc
    caatgggaatatcaagattgctgacttcggcctctccaacctctaccatcaaggcaagt
    tcctgcagacattctgtgggagccccctctatgcctcgccagagattgtcaatgggaa
    gccctacacaggcccagaggtggacagctggtccctgggtgttctcctctacatcctg
    gtgcatggcaccatgccctttgatgggcatgaccataagatcctagtgaaacagatca
    gcaacggggcctaccgggagccacctaaaccctctgatgcctgtggcctgatccgg
    tggctgttgatggtgaaccccacccgccgggccaccctggaggatgtggccagtca
    ctggtgggtcaactggggctacgccacccgagtgggagagcaggaggctccgcat
    gagggtgggcaccctggcagtgactctgcccgcgcctccatggctgactggctccg
    gcgttcctcccgccccctcctggagaatggggccaaggtgtgcagcttcttcaagca
    gcatgcacctggtgggggaagcaccacccctggcctggagcgccagcattcgctca
    agaagtcccgcaaggagaatgacatggcccagtctctccacagtgacacggctgat
    gacactgcccatcgccctggcaagagcaacctcaagctgccaaagggcattctcaa
    gaagaaggtgtcagcctctgcagaaggggtacaggaggaccctccggagctcagc
    ccaatccctgcgagcccagggcaggctgccccgctgctccccaagaagggcattct
    caagaagccccgacagcgcgagtctggctactactcctctcccgagcccagtgaatc
    tggggagctcttggacgcaggcgacgtgtttgtgagtggggatcccaaggagcaga
    agcctccgcaagcttcagggctgctcctccatcgcaaaggcatcctcaaactcaatg
    gcaagttctcccagacagccttggagctcgcggcccccaccaccttcggctccctgg
    atgaactcgccccacctcgccccctggcccgggccagccgaccctcaggggctgt
    gagcgaggacagcatcctgtcctctgagtcctttgaccagctggacttgcctgaacgg
    ctcccagagcccccactgcggggctgtgtgtctgtggacaacctcacggggcttgag
    gagcccccctcagagggccctggaagctgcctgaggcgctggcggcaggatccttt
    gggggacagctgcttttccctgacagactgccaggaggtgacagcgacctaccgac
    aggcactgagggtctgctcaaagctcacctgagtggagtaggcattgccccagccc
    ggtcaggctctcagatgcagctggttgcaccccgaggggagatgccttctcccccac
    ctcccaggacctgcatcccagctcagaaggctgagagggtttgcagtggagccctg
    agcagggctggatatgggaagtaggcaaatgaaatgcgccaagggttcagtgtctgt
    cttcagccctgctgaacgaagaggatactaaagagaggggaacgggaatgcccgc
    gacagagtccacattgcctgtttcttgtgtacatgggggggccacagagacctggaa
    agagaactctcccagggcccatctcctgcatcccatgaatactctgtacacatggtgc
    cttctaaggacagctccttccctactcattccctgcccaagtggggccagacctctttac
    acacacattcccgttcctaccaaccaccagaactggatggtggcacccctaatgtgca
    tgaggcatcctgggaatggtctggagtaacgcttcgttatttttatttttatttttatttatttat
    ttatttttttgagacggagtttcgctcttggtgcccaggctagagtgcaatggcgcgatc
    tcagctcacctcaacctccgcctcccgggttcaagcgattctcctgcctcagcctccct
    agtagctgggattacaggcgcccgccaccatgcccggctaattttgtatttttagtaga
    gacagggtttctccatgttggtcaggctggtctcaaactcccgacctcaggtgatccac
    ccacctcggcctcccaaagtgctgggattacaggcgtgagccaccgcgccccacct
    aacccttccttatttagcctaggagtaagagaacacaatctctgtttcttcaatggttctct
    tcccttttccatcctccaaacctggcctgagcctcctgaagttgctgctgtgaatctgaa
    agacttgaaaagcctccgcctgctgtgtggacttcatctcaaggggcccagcctcctc
    tggactccaccttggacctcagtgactcagaacttctgcctctaagctgctctaaagtc
    cagactatggatgtgttctctaggccttcaggactctagaatgtccatatttatttttatgtt
    cttggctttgtgttttaggaaaagtgaatcttgctgttttcaataatgtgaatgctatgttct
    gggaaaatccactatgacatctaagttttgtgtacagagagatatttttgcaactatttcc
    acctcctcccacaaccccccacactccactccacactcttgagtctctttacctaatggt
    ctctacctaatggacctccgtggccaaaaagtaccattaaaaccagaaaggtgattgg
    aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID
    NO: 618)
    Alk agctgcaagtggcgggcgcccaggcagatgcgatccagcggctctgggggcggc NM_004304 NM_007439
    agcggtggtagcagctggtacctcccgccgcctctgttcggagggtcgcggggcac
    cgaggtgctttccggccgccctctggtcggccacccaaagccgcgggcgctgatga
    tgggtgaggagggggcggcaagatttcgggcgcccctgccctgaacgccctcagc
    tgctgccgccggggccgctccagtgcctgcgaactctgaggagccgaggcgccgg
    tgagagcaaggacgctgcaaacttgcgcagcgcgggggctgggattcacgcccag
    aagttcagcaggcagacagtccgaagccttcccgcagcggagagatagcttgagg
    gtgcgcaagacggcagcctccgccctcggttcccgcccagaccgggcagaagag
    cttggaggagccaaaaggaacgcaaaaggcggccaggacagcgtgcagcagctg
    ggagccgccgttctcagccttaaaagttgcagagattggaggctgccccgagaggg
    gacagaccccagctccgactgcggggggcaggagaggacggtacccaactgcca
    cctcccttcaaccatagtagttcctctgtaccgagcgcagcgagctacagacggggg
    cgcggcactcggcgcggagagcgggaggctcaaggtcccagccagtgagcccag
    tgtgcttgagtgtctctggactcgcccctgagcttccaggtctgtttcatttagactcctg
    ctcgcctccgtgcagttgggggaaagcaagagacttgcgcgcacgcacagtcctct
    ggagatcaggtggaaggagccgctgggtaccaaggactgttcagagcctcttcccat
    ctcggggagagcgaagggtgaggctgggcccggagagcagtgtaaacggcctcc
    tccggcgggatgggagccatcgggctcctgtggctcctgccgctgctgctttccacg
    gcagctgtgggctccgggatggggaccggccagcgcgcgggctccccagctgcg
    gggccgccgctgcagccccgggagccactcagctactcgcgcctgcagaggaag
    agtctggcagttgacttcgtggtgccctcgctcttccgtgtctacgcccgggacctact
    gctgccaccatcctcctcggagctgaaggctggcaggcccgaggcccgcggctcg
    ctagctctggactgcgccccgctgctcaggttgctggggccggcgccgggggtctc
    ctggaccgccggttcaccagccccggcagaggcccggacgctgtccagggtgctg
    aagggcggctccgtgcgcaagctccggcgtgccaagcagttggtgctggagctgg
    gcgaggaggcgatcttggagggttgcgtcgggccccccggggaggcggctgtgg
    ggctgctccagttcaatctcagcgagctgttcagttggtggattcgccaaggcgaagg
    gcgactgaggatccgcctgatgcccgagaagaaggcgtcggaagtgggcagaga
    gggaaggctgtccgcggcaattcgcgcctcccagccccgccttctcttccagatcttc
    gggactggtcatagctccttggaatcaccaacaaacatgccttctccttctcctgattatt
    ttacatggaatctcacctggataatgaaagactccttccctttcctgtctcatcgcagcc
    gatatggtctggagtgcagctttgacttcccctgtgagctggagtattcccctccactgc
    atgacctcaggaaccagagctggtcctggcgccgcatcccctccgaggaggcctcc
    cagatggacttgctggatgggcctggggcagagcgttctaaggagatgcccagagg
    ctcctttctccttctcaacacctcagctgactccaagcacaccatcctgagtccgtggat
    gaggagcagcagtgagcactgcacactggccgtctcggtgcacaggcacctgcag
    ccctctggaaggtacattgcccagctgctgccccacaacgaggctgcaagagagat
    cctcctgatgcccactccagggaagcatggttggacagtgctccagggaagaatcg
    ggcgtccagacaacccatttcgagtggccctggaatacatctccagtggaaaccgca
    gcttgtctgcagtggacttctttgccctgaagaactgcagtgaaggaacatccccagg
    ctccaagatggccctgcagagctccttcacttgttggaatgggacagtcctccagcttg
    ggcaggcctgtgacttccaccaggactgtgcccagggagaagatgagagccagat
    gtgccggaaactgcctgtgggtttttactgcaactttgaagatggcttctgtggctggac
    ccaaggcacactgtcaccccacactcctcaatggcaggtcaggaccctaaaggatg
    cccggttccaggaccaccaagaccatgctctattgctcagtaccactgatgtccccgc
    ttctgaaagtgctacagtgaccagtgctacgtttcctgcaccgatcaagagctctccat
    gtgagctccgaatgtcctggctcattcgtggagtcttgaggggaaacgtgtccttggtg
    ctagtggagaacaaaaccgggaaggagcaaggcaggatggtctggcatgtcgccg
    cctatgaaggcttgagcctgtggcagtggatggtgttgcctctcctcgatgtgtctgac
    aggttctggctgcagatggtcgcatggtggggacaaggatccagagccatcgtggct
    tttgacaatatctccatcagcctggactgctacctcaccattagcggagaggacaagat
    cctgcagaatacagcacccaaatcaagaaacctgtttgagagaaacccaaacaagg
    agctgaaacccggggaaaattcaccaagacagacccccatctttgaccctacagttc
    attggctgttcaccacatgtggggccagcgggccccatggccccacccaggcacag
    tgcaacaacgcctaccagaactccaacctgagcgtggaggtggggagcgagggcc
    ccctgaaaggcatccagatctggaaggtgccagccaccgacacctacagcatctcg
    ggctacggagctgctgggggaaaggcgggaagaacaccatgatgcggtcccac
    ggcgtgtctgtgctgggcatcttcaacctggagaaggatgacatgctgtacatcctgg
    ttgggcagcagggagaggacgcctgccccagtacaaaccagttaatccagaaagtc
    tgcattggagagaacaatgtgatagaagaagaaatccgtgtgaacagaagcgtgcat
    gagtgggcaggaggcggaggaggagggggtggagccacctacgtatttaagatga
    aggatggagtgccggtgcccctgatcattgcagccggaggtggtggcagggcctac
    ggggccaagacagacacgttccacccagagagactggagaataactcctcggttct
    agggctaaacggcaattccggagccgcaggtggtggaggtggctggaatgataac
    acttccttgctctgggccggaaaatctttgcaggagggtgccaccggaggacattcct
    gcccccaggccatgaagaagtggggggggagacaagagggggtttcggagggg
    gtggaggggggtgctcctcaggtggaggaggcggaggatatataggcggcaatgc
    agcctcaaacaatgaccccgaaatggatggggaagatggggtttccttcatcagtcc
    actgggcatcctgtacaccccagctttaaaagtgatggaaggccacggggaagtga
    atattaagcattatctaaactgcagtcactgtgaggtagacgaatgtcacatggaccct
    gaaagccacaaggtcatctgcttctgtgaccacgggacggtgctggctgaggatgg
    cgtctcctgcattgtgtcacccaccccggagccacacctgccactctcgctgatcctct
    ctgtggtgacctctgccctcgtggccgccctggtcctggctttctccggcatcatgatt
    gtgtaccgccggaagcaccaggagctgcaagccatgcagatggagctgcagagcc
    ctgagtacaagctgagcaagctccgcacctcgaccatcatgaccgactacaacccca
    actactgctttgctggcaagacctcctccatcagtgacctgaaggaggtgccgcgga
    aaaacatcaccctcattcggggtctgggccatggcgcctttggggaggtgtatgaag
    gccaggtgtccggaatgcccaacgacccaagccccctgcaagtggctgtgaagac
    gctgcctgaagtgtgctctgaacaggacgaactggatttcctcatggaagccctgatc
    atcagcaaattcaaccaccagaacattgttcgctgcattggggtgagcctgcaatccct
    gccccggttcatcctgctggagctcatggggggggagacctcaagtccttcctccg
    agagacccgccctcgcccgagccagccctcctccctggccatgctggaccttctgca
    cgtggctcgggacattgcctgtggctgtcagtatttggaggaaaaccacttcatccac
    cgagacattgctgccagaaactgcctcttgacctgtccaggccctggaagagtggcc
    aagattggagacttcgggatggcccgagacatctacagggcgagctactatagaaa
    gggaggctgtgccatgctgccagttaagtggatgcccccagaggccttcatggaag
    gaatattcacttctaaaacagacacatggtcctttggagtgctgctatgggaaatcttttc
    tcttggatatatgccataccccagcaaaagcaaccaggaagttctggagtttgtcacca
    gtggaggccggatggacccacccaagaactgccctgggcctgtataccggataatg
    actcagtgctggcaacatcagcctgaagacaggcccaactttgccatcattttggaga
    ggattgaatactgcacccaggacccggatgtaatcaacaccgctttgccgatagaata
    tggtccacttgtggaagaggaagagaaagtgcctgtgaggcccaaggaccctgagg
    gggttcctcctctcctggtctctcaacaggcaaaacgggaggaggagcgcagccca
    gctgccccaccacctctgcctaccacctcctctggcaaggctgcaaagaaacccaca
    gctgcagagatctctgttcgagtccctagagggccggccgtggaagggggacacgt
    gaatatggcattctctcagtccaaccctccttcggagttgcacaaggtccacggatcca
    gaaacaagcccaccagcttgtggaacccaacgtacggctcctggtttacagagaaac
    ccaccaaaaagaataatcctatagcaaagaaggagccacacgacaggggtaacctg
    gggctggagggaagctgtactgtcccacctaacgttgcaactgggagacttccggg
    ggcctcactgctcctagagccctcttcgctgactgccaatatgaaggaggtacctctgt
    tcaggctacgtcacttcccttgtgggaatgtcaattacggctaccagcaacagggcttg
    cccttagaagccgctactgcccctggagctggtcattacgaggataccattctgaaaa
    gcaagaatagcatgaaccagcctgggccctgagctcggtcgcacactcacttctcttc
    cttgggatccctaagaccgtggaggagagagaggcaatggctccttcacaaaccag
    agaccaaatgtcacgttttgttttgtgccaacctattttgaagtaccaccaaaaaagctgt
    attttgaaaatgctttagaaaggttttgagcatgggttcatcctattctttcgaaagaaga
    aaatatcataaaaatgagtgataaatacaaggcccagatgtggttgcataaggtttttat
    gcatgtttgttgtatacttccttatgcttctttcaaattgtgtgtgctctgcttcaatgtagtca
    gaattagctgcttctatgtttcatagttggggtcatagatgtttccttgccttgttgatgtgg
    acatgagccatttgaggggagagggaacggaaataaaggagttatttgtaatgacta
    aaa (SEQ ID NO: 619)
    Pdzklip1 gcccgtcttcgtgtctcctccctccctcgccttcctccttcctagctcctctcctccaggg NM_005764 NM_
    ccagactgagcccaggttgatttcaggcggacaccaatagactccacagcagctcca 001164557
    ggagcccagacaccggcggccagaagcaaggctaggagctgctgcagccatgtc
    ggccctcagcctcctcattctgggcctgctcacggcagtgccacctgccagctgtca
    gcaaggcctggggaaccttcagccctggatgcagggccttatcgcggtggccgtgtt
    cctggtcctcgttgcaatcgcctttgcagtcaaccacttctggtgccaggaggagccg
    gagcctgcacacatgatcctgaccgtcggaaacaaggcagatggagtcctggtggg
    aacagatggaaggtactcttcgatggcggccagtttcaggtccagtgagcatgagaa
    tgcctatgagaatgtgcccgaggaggaaggcaaggtccgcagcaccccgatgtaac
    cttctctgtggctccaaccccaagactcccaggcacatgggatggatgtccagtgcta
    ccacccaagccccctccttctttgtgtggaatctgcaatagtgggctgactccctccag
    ccccatgccggccctacccgcccttgaagtatagccagccaaggttggagctcaga
    ccgtgtctaggttggggctcggctgtggccctggggtctcctgctcagctcagaaga
    gccttctggagaggacagtcagctgagcacctcccatcctgctcacacgtccttcccc
    ataactatggaaatggccctaatttctgtgaaataaagactttttgtatttctggggctga
    ggctcagcaacagcccctcaggcttccagtga (SEQ ID NO: 620)
    Inpp5b aaatgtagtcactgtcccggaacctggggcagcggagtcccgtgcgccctgtggtg NM_005540 NM_008385
    acagctcaggagggtgtgtgcgctcagcaggggccagcatggaccagtctgtggc
    aatccaggagacgctggctgagggggaatactgcgtcatcgcggtgcaaggtgtgc
    tgtgtgagggggacagccggcagagccgcctcctgggactcgtgcgctaccgcct
    ggagcacggcggccaggaacacgctctcttcctctatacgcaccggaggatggcca
    ttaccggggacgatgtctctctggaccagatagtgccagtctcgcgggattttacgctg
    gaagaagtgtccccagatggtgaactctacatccttggctcagatgtgaccgtccagc
    tggacacagcagagcttagcctcgtattccaactgccctttggttcacaaaccaggat
    gttcctccacgaagttgccagggcctgtccaggcttcgattctgcgacccgggatcct
    gaattcctgtggctgtctcggtataggtgcgcagagctggagctggagatgccaacg
    ccgcgcggttgtaactcggccctagttacctggccagggtacgcgacaattggcgga
    ggtggttctaactttgatggtttgagaccaaatgggaagggagtgcctatggaccaaa
    gctccaggggtcaagataaaccagaaagcttgcaaccaagacagaataaatccaag
    tccgaaattactgacatggttcgctcctccactatcacagtgtcggacaaggctcatatt
    ttatccatgcagaagtttggactgcgagatacaattgtgaaatcacatctactacagaa
    agaagaggattacacctatatccagaacttcaggttttttgcgggaacatacaatgtaa
    atgggcagtcccccaaagaatgcctccggctgtggctgagcaatggtatccaggcc
    ccagatgtctattgtgtagggttccaggagcttgatctgagtaaggaagcttttttctttc
    acgataccccaaaggaggaagagtggttcaaagctgtgtcagagggtcttcatccag
    atgccaaatatgcaaaggtgaagcttatccgactggttgggattatgctgctgttatatg
    tcaaacaggagcatgcagcttatatctcagaagtggaagccgagactgtggggaca
    ggaatcatggggaggatgggcaacaagggaggcgtggcgatcaggttccagttcc
    acaacaccagcatctgcgttgtgaattctcacttggcagcccacattgaagagtatga
    gaggaggaaccaggactataaggacatttgttctcgaatgcagttttgtcagcctgac
    ccaagccttccccctctcaccatcagcaaccatgatgtgatcttgtggctgggggacc
    tcaactacaggatagaagagctggatgtggaaaaagtgaaaaagctcatcgaagag
    aaggactttcaaatgctgtatgcatatgatcagctgaaaattcaggtggccgcaaaga
    ctgtctttgaaggcttcacagagggtgagctcacattccagcctacttacaagtatgata
    cgggctctgacgactgggataccagtgagaagtgccgtgctcctgcctggtgtgatc
    ggattctctggaaagggaagaacatcactcagctgagttaccagagccacatggccc
    tgaagaccagtgaccacaagcctgtcagctcagtgtttgacatcggggtgagggtcg
    taaatgacgagctttaccggaagacactggaggaaattgttcgctccctggataagat
    ggaaaatgccaacattccttctgtgtccctgtccaagcgagagttctgttttcagaatgt
    gaagtacatgcaattgaaagtagaatcctttacaattcataatggacaagtaccctgtca
    ttttgaattcatcaacaagcctgatgaagagtcttactgtaagcagtggctgaatgccaa
    ccccagcagaggcttcctcctgccagattctgatgttgagattgacttggagctcttcgt
    aaataagatgacagctacaaagctcaactcgggtgaagacaaaattgaggacattct
    ggttctgcacttggacaggggaaaggattactttttgtctgtgtctgggaactacctgcc
    cagctgttttgggtctcccattcatacactgtgttacatgagagagccaatcttggacct
    accacttgaaaccattagtgagctgactctgatgccagtatggactggagatgatggg
    agccagttggatagccccatggaaatccccaaagagctctggatgatggttgattacc
    tgtaccgaaatgctgtccagcaggaagatctgtttcagcaaccaggcctgaggtcag
    aatttgaacatatcagggactgcttggatactggaatgattgataacctctctgccagca
    atcattctgtagccgaagccctgctgcttttcctggagagccttccagagcctgtcatct
    gttacagcacctaccataactgcttggagtgttctggcaactacacagcaagcaaaca
    ggtcatttctactctccccatattccacaaaaatgtcttccactacttgatggcgtttttgc
    gagaactgctgaaaaattcagcaaaaaatcatttggatgagaatattctagctagcata
    tttggcagcttattgcttcgaaacccagctggtcaccaaaagcttgatatgacagagaa
    gaagaaggctcaagaatttattcaccagttcctctgcaacccactctgagcctctctctc
    ctcctattttacttgaggctgccaattaccagccccacctgtttcagctcaagagatgcc
    ttaagataattatgtgaggccacttggtagcaagaatggcagctatttcctgagcctagt
    accccaattaagcccaccattggttagcacactcagcgctgtgagtcgtgaagacac
    gggagaaaatccaccataataaaactgacattcaattttcaactttagttatttaacacag
    atttttttattttttatttttttttattttgagacggagttttgctctgtcgcgcagggtggagtg
    cggtggcacgatctcggctcactgcaacctctgcctcctgggtgcaagcaattatcct
    gcctcagcctcccgagtagctgggactgcaggcacacactgccacgcccagctaat
    tttttgcattttagtagagacggggtttcaccgtgttgcccaggctgttctaaaactcctg
    aactcaggtaatctgcctgcctcggcctccccaagtgctaggattacagatgtgagcc
    accacgcccggccttttttttttttttttcttttttgagatggagtttcactcttgttgcccagg
    ctggagtgcgttggcgtggtcttggctcactgcaacctctgcctccttggttcaagcaa
    ttctcctgcctcagcctctcgagtagctgggattataggcgtccgccaccatgcctggc
    taatttttttgtgtgtttttagtatagacacggtttcaccatgttggccaggctggtctcgaa
    tgcctggcctcaggtgatccacctgccttggcctcccaaagtgctgggattacaggca
    tgaaccaccacgcctggcctaaaatgtttttaaataactgtacttgtactcactcacccta
    cctccagggcatagtcagtctgggctgagatccccatgatcagatatttgatggaaag
    tcctgaaaggccaatgagttggatggcaagaatgcaggcagaagctgctggataaa
    ataggctacagccacctcagatgctttcagtgctctgtctgaggatgtgtatatgcatat
    gcaaactcgacccccgttcctgcccagataatggctcaataactctgaggctggttgc
    tcagcctctgagggcaatacaggcatttaaaaaattaaaatgaccaggcacagtggct
    cacgcctgtaatctcggcactttgggagactgaggtgggagcatcacttgagaccag
    gagtttgggaccaggctgggcaacacagggagaccccctctctacaaaaacattttta
    aaaaattagctgggtgtggtgatgcatgcctgtggtcccagttacttgggaggctgac
    gtgggtggctcacttgagcacaggagtttgaggctgcagtgacctatgaccacatca
    ctgtacgccagcccgggtgagagagggagaccccgtctctaaaaataaaatgtaaa
    atcactgaaaaaatgagtgttcggtgaaacaagtgggattttctgggccagcaagtctt
    ccaaactgtatatgatgcatcctgtctccatgtgtaatatattttaatgataaatgtattttta
    acagtgaaaaaaaaaaaaaaa (SEQ ID NO: 653)
    Socs1 ggcagctgcacggctcctggccccggagcatgcgcgagagccgccccggagcgc NM_003745 NM_
    cccggagccccccgccgtcccgcccgcggcgtcccgcgccccgccgccagcgca 001271603
    cccccggacgctatggcccacccctccggctggccccttctgtaggatggtagcaca
    caaccaggtggcagccgacaatgcagtctccacagcagcagagccccgacggcg
    gccagaaccttcctcctcttcctcctcctcgcccgcggcccccgcgcgcccgcggcc
    gtgccccgcggtcccggccccggcccccggcgacacgcacttccgcacattccgtt
    cgcacgccgattaccggcgcatcacgcgcgccagcgcgctcctggacgcctgcgg
    attctactgggggcccctgagcgtgcacggggcgcacgagcggctgcgcgccgag
    cccgtgggcaccttcctggtgcgcgacagccgccagcggaactgctttttcgccctta
    gcgtgaagatggcctcgggacccacgagcatccgcgtgcactttcaggccggccg
    ctttcacctggatggcagccgcgagagcttcgactgcctcttcgagctgctggagcac
    tacgtggcggcgccgcgccgcatgctgggggccccgctgcgccagcgccgcgtg
    cggccgctgcaggagctgtgccgccagcgcatcgtggccaccgtgggccgcgag
    aacctggctcgcatccccctcaaccccgtcctccgcgactacctgagctccttcccctt
    ccagatttgaccggcagcgcccgccgtgcacgcagcattaactgggatgccgtgtta
    ttttgttattacttgcctggaaccatgtgggtaccctccccggcctgggttggagggag
    cggatgggtgtaggggcgaggcgcctcccgccctcggctggagacgaggccgca
    gaccccttctcacctcttgagggggtcctccccctcctggtgctccctctgggtccccc
    tggttgttgtagcagcttaactgtatctggagccaggacctgaactcgcacctcctacc
    tcttcatgtttacatatacccagtatctttgcacaaaccaggggttgggggagggtctct
    ggctttatttttctgctgtgcagaatcctattttatattttttaaagtcagtttaggtaataaac
    tttattatgaaagtttttttttt (SEQ ID NO: 654)
    Jun gacatcatgggctatttttaggggttgactggtagcagataagtgttgagctcgggctg NM_002228 NM_010591
    gataagggctcagagttgcactgagtgtggctgaagcagcgaggcgggagtggag
    gtgcgcggagtcaggcagacagacagacacagccagccagccaggtcggcagta
    tagtccgaactgcaaatcttattttcttttcaccttctctctaactgcccagagctagcgcc
    tgtggctcccgggctggtgtttcgggagtgtccagagagcctggtctccagccgccc
    ccgggaggagagccctgctgcccaggcgctgttgacagcggcggaaagcagcgg
    tacccacgcgcccgccgggggaagtcggcgagcggctgcagcagcaaagaacttt
    cccggctgggaggaccggagacaagtggcagagtcccggagcgaacttttgcaag
    cctttcctgcgtcttaggcttctccacggcggtaaagaccagaaggcggcggagagc
    cacgcaagagaagaaggacgtgcgctcagcttcgctcgcaccggttgttgaacttgg
    gcgagcgcgagccgcggctgccgggcgccccctccccctagcagcggaggagg
    ggacaagtcgtcggagtccgggcggccaagacccgccgccggccggccactgca
    gggtccgcactgatccgctccgcggggagagccgctgctctgggaagtgagttcgc
    ctgcggactccgaggaaccgctgcgcccgaagagcgctcagtgagtgaccgcgac
    ttttcaaagccgggtagcgcgcgcgagtcgacaagtaagagtgcgggaggcatctt
    aattaaccctgcgctccctggagcgagctggtgaggagggcgcagcggggacgac
    agccagcgggtgcgtgcgctcttagagaaactttccctgtcaaaggctccgggggg
    cgcgggtgtcccccgcttgccagagccctgttgcggccccgaaacttgtgcgcgca
    gcccaaactaacctcacgtgaagtgacggactgttctatgactgcaaagatggaaac
    gaccttctatgacgatgccctcaacgcctcgttcctcccgtccgagagcggaccttat
    ggctacagtaaccccaagatcctgaaacagagcatgaccctgaacctggccgaccc
    agtgggagcctgaagccgcacctccgcgccaagaactcggacctcctcacctcgcc
    cgacgtggggctgctcaagctggcgtcgcccgagctggagcgcctgataatccagt
    ccagcaacgggcacatcaccaccacgccgacccccacccagttcctgtgccccaa
    gaacgtgacagatgagcaggagggcttcgccgagggcttcgtgcgcgccctggcc
    gaactgcacagccagaacacgctgcccagcgtcacgtcggcggcgcagccggtca
    acggggcaggcatggtggctcccgcggtagcctcggtggcagggggcagcggca
    gcggcggcttcagcgccagcctgcacagcgagccgccggtctacgcaaacctcag
    caacttcaacccaggcgcgctgagcagcggggggggcgccctcctacggcgc
    ggccggcctggcctttcccgcgcaaccccagcagcagcagcagccgccgcacca
    cctgccccagcagatgcccgtgcagcacccgcggctgcaggccctgaaggagga
    gcctcagacagtgcccgagatgcccggcgagacaccgcccctgtcccccatcgac
    atggagtcccaggagcggatcaaggggagaggaagcgcatgaggaaccgcatc
    gctgcctccaagtgccgaaaaaggaagctggagagaatcgcccggctggaggaaa
    aagtgaaaaccttgaaagctcagaactcggagctggcgtccacggccaacatgctc
    agggaacaggtggcacagcttaaacagaaagtcatgaaccacgttaacagtgggtg
    ccaactcatgctaacgcagcagttgcaaacattttgaagagagaccgtcgggggctg
    aggggcaacgaagaaaaaaaataacacagagagacagacttgagaacttgacaag
    ttgcgacggagagaaaaaagaagtgtccgagaactaaagccaagggtatccaagtt
    ggactgggttgcgtcctgacggcgcccccagtgtgcacgagtgggaaggacttggc
    gcgccctcccttggcgtggagccagggagcggccgcctgcgggctgccccgcttt
    gcggacgggctgtccccgcgcgaacggaacgttggacttttcgttaacattgaccaa
    gaactgcatggacctaacattcgatctcattcagtattaaaggggggagggggaggg
    ggttacaaactgcaatagagactgtagattgcttctgtagtactccttaagaacacaaa
    gcggggggagggttggggaggggcggcaggagggaggtttgtgagagcgaggc
    tgagcctacagatgaactctttctggcctgccttcgttaactgtgtatgtacatatatatat
    tttttaatttgatgaaagctgattactgtcaataaacagcttcatgcctttgtaagttatttctt
    gtttgtttgtttgggtatcctgcccagtgttgtttgtaaataagagatttggagcactctga
    gtttaccatttgtaataaagtatataatttttttatgttttgtttctgaaaattccagaaaggat
    atttaagaaaatacaataaactattggaaagtactcccctaacctcttttctgcatcatctg
    tagatactagctatctaggtggagttgaaagagttaagaatgtcgattaaaatcactctc
    agtgcttcttactattaagcagtaaaaactgttctctattagactttagaaataaatgtacc
    tgatgtacctgatgctatggtcaggttatactcctcctcccccagctatctatatggaatt
    gcttaccaaaggatagtgcgatgtttcaggaggctggaggaaggggggttgcagtg
    gagagggacagcccactgagaagtcaaacatttcaaagtttggattgtatcaagtggc
    atgtgctgtgaccatttataatgttagtagaaattttacaataggtgcttattctcaaagca
    ggaattggtggcagattttacaaaagatgtatccttccaatttggaatcttctctttgacaa
    ttcctagataaaaagatggcctttgcttatgaatatttataacagcattcttgtcacaataa
    atgtattcaaataccaaaaaaaaaaaaaaaaa (SEQ ID NO: 655)
    Nptxr cggccgcggcgacagctccagctccggctccggctccggctccggctccggctcc NM_014293 NM_030689
    cgcgcctgccccgctcggcccagcgcgcccgggctccgcgccccgaccccgccg
    ccgcgcctgccgggggcctcgggcgcccccgccgcccgcctcacgctgaagttcc
    tggccgtgctgctggccgcgggcatgctggcgttcctcggtgccgtcatctgcatcat
    cgccagcgtgcccctggcggccagcccggcgcgggcgctgcccggcggcgccg
    acaatgcttcggtcgcctcgggcgccgccgcgtccccgggcccgcagcggagcct
    gagcgcgctgcacggcgcgggcggttcagccgggccccccgcgctgcccgggg
    cacccgcggccagcgcgcacccgctgccgcccgggcccctgttcagccgcttcct
    gtgcacgccgctggctgctgcctgcccgtcgggggcccagcagggggacgcggc
    gggcgctgcgccgggcgagcgcgaagagctgctgctgctgcagagcacggccga
    gcagctgcgccagacggcgctgcagcaggaggcgcgcatccgcgccgaccagg
    acaccatccgtgagctcaccggcaagctgggccgctgcgagagcggcctgccgcg
    cggcctccagggcgccgggccccgccgcgacaccatggccgacgggccctggg
    actcgcctgcgctcattctggagctggaggacgccgtgcgcgccctgcgggaccgc
    atcgaccgcctggagcaggagcttccagcccgtgtgaacctctcagctgccccagc
    cccagtctctgctgtgcccaccggcctacactccaagatggaccagctggaggggc
    agctgctggcccaggtgctggcactggagaaggagcgtgtggccctcagccacag
    cagccgccggcagaggcaggaagtggaaaaggagttggacgtcctgcagggtcgt
    gtggctgagctggagcacgggtcctcagcctacagtcctccagatgccttcaagatc
    agcatccccatccgtaacaactacatgtacgcccgcgtgcggaaggctctgcccga
    gctctacgcattcaccgcctgcatgtggctgcggtccaggtccagcggcaccggcc
    agggcacccccttctcctactcagtgcccgggcaggccaacgagattgtactgctag
    aggcgggccatgagcccatggagctgctgatcaacgacaaggtggcccagctgcc
    cctgagcctgaaggacaatggctggcaccacatctgcatcgcctggaccacaaggg
    atggcctatggtctgcctaccaggacggggagctgcagggctccggtgagaacctg
    gctgcctggcaccccatcaagcctcatgggatccttatcttgggccaggagcaggat
    accctgggtggccggtttgatgccacccaggcctttgtcggtgacattgcccagtttaa
    cctgtgggaccacgccctgacaccagcccaggtcctgggcattgccaactgcactg
    cgccactgctgggcaacgtccttccctgggaagacaagttggtggaggcctttgggg
    gtgcaacaaaggctgccttcgatgtctgcaaggggagggccaaggcatgaggggc
    cacctcatccagggcccctcccttgcctgccactttggggacttgaggggggtcatat
    tccctcctcagcctgcccacgcactggccttccctcctgccccactcctggctgtgcct
    cccatttcccctcacctgtacccacacctccagaatgccctgccctgcgagtgtgtccc
    ctgtccccacctgagtggggaggagcgtctcaagtgaacagtgggagcctgcccac
    ctggcactgcactggagttgtctcttaccccaccctccctgcccatcaactgtatctgat
    ttcactaattttgacagcacccccagtagggtaggattgtgtatgagggggaccccac
    tatctcagtggtgggggtggccgcccgcccccttgtcccccatgcaacaggcccagt
    ggcttccccttcagggccacaacaggctgtagaaggggatgacgaggacatcaga
    ggttagacttaccctcctccctctttccaccagctgccagtcaagggcagtgggatctc
    gatggagcctccccccccccccacccatgcctccctcttcctcctctttcctcctctcttt
    gtgtgtagcggtttgaatgttggttccatgcctggcccagccccacctcagtctccagg
    acattcctttcccagctccagcctggagggaaggggacaaagaccccaggaggcc
    aaagggctgcagtcaccccttgtgctcacccatagtgatggccactggtatagtcatc
    gctctccctccatgccaaggacaggacttggaccgcttcagcctgggctgggagca
    gccctaaggtagaggcctcatggcccaggagaccccacctctggcagagccacatt
    acctaccctgtgcatggtcctggggcagcaaggaagaagctcagagggtggggag
    aagcatgaagcagtgagcagagcactgggtgagagggagaagaccttggttcctag
    ccagccctgctaatgtgctgtgtggccttctgtaagtccctgccctctctgggcctggc
    cttcctcattcgtgagctgaggccctcgctttggtcatttgctctccagattgggtgtgag
    cttctctgtgattccaggtggatatgtggggaaagctctggtgaccctgggcttcgcag
    gggtagatcccaggactcggcagtggatgggatgcagccagtcatgggttagggtc
    agcagagactcagagtccagggcaaggttcaaggcagactaacctcatgcatggatt
    gtaaaaaaccagctccctttggatcaacccagcctggcacccttgcctgtctgagagt
    gtctcaaagggctgatggcttcctggtccccttgagtcatcaccagcttccccaagag
    agtgtcagaatcttaagagctgagaggccgggcacggtggctcacgcctgtaatccc
    agcactttgggaggctgagacaggcagatcacttgaggtcaggagttcgaagtcag
    cctggccaacgtggtgaaaccccatcttcactaaaaatacaaaacttagctggttaggt
    ggtgcatgcctgtagtcccagctactcgggaggccgaggcagaagaatctcttgaac
    tgaggaggtggaggttgcagtgagccgagatcacgccattgcactccagcctgggc
    aacagagcaagactccatctcaaaaaaataataataatcttaaagatgagaaaagcca
    ccccatctggcaccacagctgcatcttgcttgtgagaaatggggaagagttcaggga
    ggacacgtgacctgcacaggatcacagagcatggggcagagccaggactagagct
    cagggcatctgactccctcttcagtgttcttccccctccatgttgcctgcccctgaagac
    ctttgagttcagtctacacctaagcaggtagacatccgcgaggtcagatgctttccaac
    atgacacctgaacatcttcctttatgcaacacccaaacatcttggcatccccaccccag
    gaagtgcggggaggaggttatgatccctgggcgcttcggcagaatggagagctga
    ggtgtccctcccctgctagtcacctaccaggtgtctgagcagctgcatgctccctggct
    caagtgggcactgtaccttttgcctgcctttttgttccctatctccactccctgaggccac
    ttagcctgagacatgatgcaagagctgcaggccggggggctcagtgccatggaagc
    tactccaagttgcattgcctcccgcgcccagatcctgctttccatttcgagaacataaat
    agattgcccagcccctccagtacaatcccactggaagaaaaggcaatggcgggctt
    cagccagacctgctgagacctaggttgccacggtaacagccaaagacatcaaccca
    agtgctgggtcaagtgtctcatcatactggcactgttgctggggtgacggcagaattc
    agaacttcaatttcagtgacgccaagcttgatgtgtttctgttattgttttgaagaaggtag
    ctcttgtggaggacttgggagaaggatggggtcttaggaaggaggtgacagcacttg
    catggtcacttgagcccacacacacgctcaaccccaagtcctttatgctttgtcacagt
    gaagatgagacctctgacgtccaagccttgttcctgtgctgcatcacccactcagcctt
    ccaaagggaacaggaacaaatttccccagcaccactgtttgggtcccgcttttcctatc
    ttctgctgcccctgagcacatccaagcagacagggaaagaggagtcagacatggcc
    cagtcacatcctgagctgctcctggctgataaccacgatggagcccgtgtttgtcctgc
    catctggcactgcactgagtgtggcacaggcaccgtcctgttgatctcacaacacagt
    tctaagttaggacgttcttggctccgttagacaggtgaggaaactggggcacagaga
    ggtgatgtcatctgcctggtgtcaatcagctagcaagtgatggagcccagatttcaaa
    ccaaagggggttacgtccaggggctgagttcccactcacctgtgtagagtgccatct
    gggcaccattgctccagacgtgttccgacccctttcccagcccacagggcttgaagt
    gaaggaacagaggcaggggggggccagccccagggccagggtccccttggtga
    agccgtgccagggggctcagctgcttcagggaatgtgtccctcccaccatgggcca
    gagcttcagcccttctttagctcagctagagttcacaggagagccaaaaaagaaaag
    gaagctgagcatctcccgagtcctgggcagggaaggggagggaaattgctgcttct
    ccaactcttgcttggggccaagccctgcaccagttgcttcccagctgttatctgccaga
    tcttcccatcttgtggcatgtggtgcccccaccaacatcccaaggggaccaatcccctt
    gccaccactttgcatcacctgggaccacagatttggacaggaagggctctgagaaga
    ggccaaagccctcattttacagatgaggaagctgaagcccggggggggagcgac
    cctcaaggccacccagctggacacgggagacttgagcccagccttctgactgcattc
    agccctctctaggacgcagcagcctctccccagcactgagtcccccctcctttgtgtgt
    cccagcacccttggcctgagtaaacttggaaaggggctccctcccagagaagggac
    tactctcttcacccctttattccagctgcctgccaccccagacccccacctcccaccct
    gacccccgacccctgggtggggaaggggctcacatgggcccaggctgagtgtgag
    tgagcatgtcaagttgtctgacactgtgacattagtgcaccctactgacaacccctccc
    cagccttgcccctttctcctctccctgttttgtacataaattgacatgagctgcaacatgt
    gtgcgtgtgtgtgcgtgtgtgtgtgtgtgtatgtgtgtgtgatctgtgtcatggttttgttac
    ctttttgtttttgtaaacttgaatgttcaaaataaacatgctgtttactctgagaaaaaaaaa
    aaaaaaa (SEQ ID NO: 656)
    Socs3 gcggctccgacttggactccctgctccgctgctgccgcttcggccccgcacgcagcc NM_003955 NM_007707
    agccgccagccgcccgcccggcccagctcccgccgcggccccttgccgcggtcc
    ctctcctggtcccctcccggttggtccgggggtgcgcagggggcagggcgggcgc
    ccaggggaagctcgagggacgcgcgcgcgaaggctcctttgtggacttcacggcc
    gccaacatctgggcgcagcgcgggccaccgctggccgtctcgccgccgcgtcgcc
    ttggggacccgagggggctcagccccaaggacggagacttcgattcgggaccagc
    cccccgggatgcggtagcggccgctgtgcggaggccgcgaagcagctgcagccg
    ccgccgcgcagatccacgctggctccgtgcgccatggtcacccacagcaagtttcc
    cgccgccgggatgagccgccccctggacaccagcctgcgcctcaagaccttcagct
    ccaagagcgagtaccagctggtggtgaacgcagtgcgcaagctgcaggagagcg
    gcttctactggagcgcagtgaccggggcgaggcgaacctgctgctcagtgccgag
    cccgccggcacctttctgatccgcgacagctcggaccagcgccacttcttcacgctc
    agcgtcaagacccagtctgggaccaagaacctgcgcatccagtgtgaggggggca
    gcttctctctgcagagcgatccccggagcacgcagcccgtgccccgcttcgactgcg
    tgctcaagctggtgcaccactacatgccgccccctggagccccctccttcccctcgcc
    acctactgaaccctcctccgaggtgcccgagcagccgtctgcccagccactccctgg
    gagtccccccagaagagcctattacatctactccgggggcgagaagatccccctggt
    gttgagccggcccctctcctccaacgtggccactcttcagcatctctgtcggaagacc
    gtcaacggccacctggactcctatgagaaagtcacccagctgccggggcccattcg
    ggagttcctggaccagtacgatgccccgctttaaggggtaaagggcgcaaagggca
    tgggtcgggagaggggacgcaggcccctctcctccgtggcacatggcacaagcac
    aagaagccaaccaggagagagtcctgtagctctggggggaaagagggcggacag
    gcccctccctctgccctctccctgcagaatgtggcaggcggacctggaatgtgttgga
    gggaagggggagtaccacctgagtctccagcttctccggaggagccagctgtcctg
    gtgggacgatagcaaccacaagtggattctccttcaattcctcagcttcccctctgcct
    ccaaacaggggacacttcgggaatgctgaactaatgagaactgccagggaatcttca
    aactttccaacggaacttgtttgctctttgatttggtttaaacctgagctggttgtggagcc
    tgggaaaggtggaagagagagaggtcctgagggccccagggctgcgggctggcg
    aaggaaatggtcacaccccccgcccaccccaggcgaggatcctggtgacatgctcc
    tctccctggctccggggagaagggcttggggtgacctgaagggaaccatcctggta
    ccccacatcctctcctccgggacagtcaccgaaaacacaggttccaaagtctacctg
    gtgcctgagagcccagggcccttcctccgttttaagggggaagcaacatttggaggg
    gatggatgggctggtcagctggtctccttttcctactcatactataccttcctgtacctgg
    gtggatggagcgggaggatggaggagacgggacatctttcacctcaggctcctggt
    agagaagacaggggattctactctgtgcctcctgactatgtctggctaagagattcgc
    cttaaatgctccctgtcccatggagagggacccagcataggaaagccacatactcag
    cctggatgggtggagaggctgagggactcactggagggcaccaagccagcccac
    agccagggaagtggggagggggggcggaaacccatgcctcccagctgagcactg
    ggaatgtcagcccagtaagtattggccagtcaggcgcctcgtggtcagagcagagc
    caccaggtcccactgccccgagccctgcacagccctccctcctgcctgggggggg
    aggctggaggtcattggagaggctggactgctgccaccccgggtgctcccgctctg
    ccatagcactgatcagtgacaatttacaggaatgtagcagcgatggaattacctggaa
    cagttttttgtttttgtttttgtttttgtttttgtgggggggggcaactaaacaaacacaaagt
    attctgtgtcaggtattgggctggacagggcagttgtgtgttggggtggtttttttctctat
    ttttttgtttgtttcttgttttttaataatgtttacaatctgcctcaatcactctgtcttttataaag
    attccacctccagtcctctctcctcccccctactcaggcccttgaggctattaggagatg
    cttgaagaactcaacaaaatcccaatccaagtcaaactttgcacatatttatatttatattc
    agaaaagaaacatttcagtaatttataataaagagcactattttttaatgaaaaac
    (SEQ ID NO: 657)
    F11r gaggcagctcctgtggggaaaggcgccagtgcgccgaggcggggagtggcggc NM_016946 NM_172647
    ggggtaacacctggccgaggtgactcgttctgaagagcagcggttccttacaccaat
    cggaacgtgcaggggggggagctggccaatcaggcgcggagggcggggccgg
    gcggggttccacctggcggctggctctcagtcccctcgctgtagtcgcggagctgtg
    tctgttcccaggagtccttcggcggctgttgtgtcgggagcctgatcgcgatggggac
    aaaggcgcaagtcgagaggaaactgttgtgcctcttcatattggcgatcctgttgtgct
    ccctggcattgggcagtgttacagtgcactcttctgaacctgaagtcagaattcctgag
    aataatcctgtgaagttgtcctgtgcctactcgggcttttcttctccccgtgtggagtgga
    agtttgaccaaggagacaccaccagactcgtttgctataataacaagatcacagcttc
    ctatgaggaccgggtgaccttcttgccaactggtatcaccttcaagtccgtgacacgg
    gaagacactgggacatacacttgtatggtctctgaggaaggcggcaacagctatggg
    gaggtcaaggtcaagctcatcgtgcttgtgcctccatccaagcctacagttaacatccc
    ctcctctgccaccattgggaaccgggcagtgctgacatgctcagaacaagatggttc
    cccaccttctgaatacacctggttcaaagatgggatagtgatgcctacgaatcccaaa
    agcacccgtgccttcagcaactcttcctatgtcctgaatcccacaacaggagagctgg
    tctttgatcccctgtcagcctctgatactggagaatacagctgtgaggcacggaatgg
    gtatgggacacccatgacttcaaatgctgtgcgcatggaagctgtggagcggaatgt
    gggggtcatcgtggcagccgtccttgtaaccctgattctcctgggaatcttggtttttgg
    catctggtttgcctatagccgaggccactttgacagaacaaagaaagggacttcgagt
    aagaaggtgatttacagccagcctagtgcccgaagtgaaggagaattcaaacagac
    ctcgtcattcctggtgtgagcctggtcggctcaccgcctatcatctgcatttgccttactc
    aggtgctaccggactctggcccctgatgtctgtagtttcacaggatgccttatttgtcttc
    tacaccccacagggccccctacttcttcggatgtgtttttaataatgtcagctatgtgccc
    catcctccttcatgccctccctccctttcctaccactgctgagtggcctggaacttgttta
    aagtgtttattccccatttctttgagggatcaggaaggaatcctgggtatgccattgactt
    cccttctaagtagacagcaaaaatggcgggggtcgcaggaatctgcactcaactgcc
    cacctggctggcagggatctttgaataggtatcttgagcttggttctgggctctttccttg
    tgtactgacgaccagggccagctgttctagagcgggaattagaggctagagcggct
    gaaatggttgtttggtgatgacactggggtccttccatctctggggcccactctcttctgt
    cttcccatgggaagtgccactgggatccctctgccctgtcctcctgaatacaagctga
    ctgacattgactgtgtctgtggaaaatgggagctcttgttgtggagagcatagtaaattt
    tcagagaacttgaagccaaaaggatttaaaaccgctgctctaaagaaaagaaaactg
    gaggctgggcgcagtggctcacgcctataatcccagaggctgaggcaggcggatc
    acctgaggtcaggagttcaagatcagcctgaccaacatggagaaaccctactaaaaa
    tacaaagttagccaggcatagtggtgcatgcctgtaatcccagctgctcaggagcctg
    gcaacaagagcaaaactccagctcaaaaaaaaaaagaaagaaaagaaagctggag
    ctggtggcttaggccatcacccttcccttggctggaactactggacagacccttttgag
    atgtgcctgtggtgctgtggagatgtgtgtagtggtcttagctctttgttgagcttgtgtgt
    gtgttgtgtagtcttagctgtatgctgaaattgggcgtgtgttggagggcttcttagctctt
    tggtgagattgtatttctatgtgtttgtatcagctgaatgttgctggaaataaaaccttggtt
    tgtcaaggctcttttttgtgggaagtaagtaggggaaaaggtctttgagggttcctagg
    ctcctttgtacaacaggaaaatgcctcaaagccttgcttcccagcaacctggggctgg
    ttcccagtgcctggtcctgccccttcctggttcttatctcaaggcagagcttctgaatttc
    aggccttcattccagagccctcttgtggccaggccttcctttgctggaggaaggtaca
    cagggtgaagctgatgctgtacttgggggatctccttggcctgttccaccaagtgaga
    gaaggtacttactcttgtacctcctgttcagccaggtgcattaacagacctccctacag
    ctgtaggaactactgtcccagagctgaggcaaggggatttctcaggtcatttggagaa
    caagtgctttagtagtagtttaaagtagtaactgctactgtatttagtggggtggaattca
    gaagaaatttgaagaccagatcatgggtggtctgcatgtgaatgaacaggaatgagc
    cggacagcctggctgtcattgctttcttcctccccatttggacccttctctgcccttacatt
    tttgtttctccatctaccaccatccaccagtctatttattaacttagcaagaggacaagta
    aagggccctcttggcttgattttgcttctttctttctgtggaggatatactaagtgcgacttt
    gccctatcctatttggaaatccctaacagaattgagttttctattaaggatccaaaaaga
    aaaacaaaatgctaatgaagccatcagtcaagggtcacatgccaataaacaataaatt
    ttccagaagaaatgaaatccaactagacaaataaagtagagcttatgaaatggttcagt
    aaagatgagtttgttgttttttgttttgttttgttttgtttttttaaagacggagtctcgctctgt
    cacccaggctggagtgcagtggtatgatcttggctcactgtaacctccgcctcccggg
    ttcaagccattctcctgcctcagtctcctgagtagctgggattacgggtgcgtgccacc
    atgcctggctaatttttgtgtttttagtagagacagggtttcaccatgttggtcgggctgg
    tctcaaactcctgacctcttgatccgcctgccttggcctcccaaagtgatgggattaca
    gatgtgagccaccgtgcctagccaaggatgagatttttaaagtatgtttcagttctgtgt
    catggttggaagacagagtaggaaggatatggaaaaggtcatggggaagcagagg
    tgattcatggctctgtgaatttgaggtgaatggttccttattgtctaggccacttgtgaag
    aatatgagtcagttattgccagccttggaatttacttctctagcttacaatggaccttttga
    actggaaaacaccttgtctgcattcactttaaaatgtcaaaactaatttttataataaatgtt
    tattttcacattgagtttgtttaaatcctgaagttcttaccttaagagaattgggactcctag
    agtgattggacattcaaaatattcctgatagtcttgttaattaagagattaggatatctttc
    cattaccttgataattacgttttaatttagcttttttcattggcctgtgtttaaatgcaaataac
    cccacaatggacatttcctatgttaaagtgacatttaggggataaaaaatgagagcagt
    tccatggattttggtgtttcccctgagacatgaactcagcataatctgggataaaatgatt
    gagtgttaaggatgtgtttgttgttcctgtcgtttttttattttcttcaaagtatacaacatggt
    ttgatatgcacatacatttgtgtaatgattgccatggtcaattaacacatcaccatttttgtg
    tgtgtgtgtgtgtgtgtgtgtgagggagtcttgctccgttgccaggctggagtgcaatg
    gtacaaccttggctcactgcaacctccacctcctgggttcaagcaattctcttgcctcag
    cctcctaagtagctgggactataggcgtgtgccaccatgcccagctaatttttgtattttt
    agtagagacggggtttcaccatgttggccaggatgatctcgatcccttgacctcatgat
    ccgcccacctcggcctcccaaagtgctgggattacaggcgtgagtcactgcacccg
    gccacatcacctcccatgttctatcttacgtattcagaacttgttcatcttgtaactgaaag
    cgtgtaccctttgaccaacactgtttttcctgtcttaacaggatctacagatcaaggaca
    ggggaggggatagtggaggaaaacggagttagtctgtttctaaatgaggggacagt
    atgtttcttggggcctgaggacagcttaataaagtagacaaatgaagaaaaacaacaa
    tttgcattaaaaaatatccaattcttta (SEQ ID NO: 658)
    Fyn agagcatcagcaagagtagcagcgagcagccgcgctggtggcggcggcgcgtcg NM_002037 NM_
    ttgcagttgcgccatctgtcaggagcggagccggcgaggagggggctgccgcggg 001122892
    cgaggaggaggggtcgccgcgagccgaaggccttcgagacccgcccgccgccc
    ggcggcgagagtagaggcgaggttgttgtgcgagcggcgcgtcctctcccgcccg
    ggcgcgccgcgcttctcccagcgcaccgaggaccgcccgggcgcacacaaagcc
    gccgcccgcgccgcaccgcccggcggccgccgcccgcgccagggagggattcg
    gccgccgggccggggacaccccggcgccgccccctcggtgctctcggaaggccc
    accggctcccgggcccgccggggaccccccggagccgcctcggccgcgccgga
    ggagggcggggagaggaccatgtgagtgggctccggagcctcagcgccgcgca
    gtttttttgaagaagcaggatgctgatctaaacgtggaaaaagaccagtcctgcctctg
    ttgtagaagacatgtggtgtatataaagtttgtgatcgttggcggacattttggaatttag
    ataatgggctgtgtgcaatgtaaggataaagaagcaacaaaactgacggaggagag
    ggacggcagcctgaaccagagctctgggtaccgctatggcacagaccccacccctc
    agcactaccccagcttcggtgtgacctccatccccaactacaacaacttccacgcagc
    cgggggccaaggactcaccgtctttggaggtgtgaactcttcgtctcatacggggac
    cttgcgtacgagaggaggaacaggagtgacactctttgtggccctttatgactatgaa
    gcacggacagaagatgacctgagttttcacaaaggagaaaaatttcaaatattgaaca
    gctcggaaggagattggtgggaagcccgctccttgacaactggagagacaggttac
    attcccagcaattatgtggctccagttgactctatccaggcagaagagtggtactttgg
    aaaacttggccgaaaagatgctgagcgacagctattgtcctttggaaacccaagagg
    tacctttcttatccgcgagagtgaaaccaccaaaggtgcctattcactttctatccgtgat
    tgggatgatatgaaaggagaccatgtcaaacattataaaattcgcaaacttgacaatg
    gtggatactacattaccacccgggcccagtttgaaacacttcagcagcttgtacaacat
    tactcagagagagctgcaggtctctgctgccgcctagtagttccctgtcacaaaggga
    tgccaaggcttaccgatctgtctgtcaaaaccaaagatgtctgggaaatccctcgaga
    atccctgcagttgatcaagagactgggaaatgggcagtttggggaagtatggatggg
    tacctggaatggaaacacaaaagtagccataaagactcttaaaccaggcacaatgtc
    ccccgaatcattccttgaggaagcgcagatcatgaagaagctgaagcacgacaagc
    tggtccagctctatgcagtggtgtctgaggagcccatctacatcgtcaccgagtatatg
    aacaaaggaagtttactggatttcttaaaagatggagaaggaagagctctgaaattac
    caaatcttgtggacatggcagcacaggtggctgcaggaatggcttacatcgagcgca
    tgaattatatccatagagatctgcgatcagcaaacattctagtggggaatggactcatat
    gcaagattgctgacttcggattggcccgattgatagaagacaatgagtacacagcaa
    gacaaggtgcaaagttccccatcaagtggacggcccccgaggcagccctgtacgg
    gaggttcacaatcaagtctgacgtgtggtcttttggaatcttactcacagagctggtcac
    caaaggaagagtgccatacccaggcatgaacaaccgggaggtgctggagcaggtg
    gagcgaggctacaggatgccctgcccgcaggactgccccatctctctgcatgagctc
    atgatccactgctggaaaaaggaccctgaagaacgccccacttttgagtacttgcaga
    gcttcctggaagactactttaccgcgacagagccccagtaccaacctggtgaaaacc
    tgtaaggcccgggtctgcggagagaggccttgtcccagaggctgccccacccctcc
    ccattagctttcaattccgtagccagctgctccccagcagcggaaccgcccaggatc
    agattgcatgtgactctgaagctgacgaacttccatggccctcattaatgacacttgtcc
    ccaaatccgaacctcctctgtgaagcattcgagacagaaccttgttatttctcagacttt
    ggaaaatgcattgtatcgatgttatgtaaaaggccaaacctctgttcagtgtaaatagtt
    actccagtgccaacaatcctagtgctttccttttttaaaaatgcaaatcctatgtgattttaa
    ctctgtcttcacctgattcaactaaaaaaaaaaaagtattattttccaaaagtggcctcttt
    gtctaaaacaataaaattttttttcatgttttaacaaaaaccaatcaggacaggtgtttgttt
    ttgttttcttttttataaatatgaatatatataatatatatgtccctgtacatatacaatgtggg
    tgctaatgtggagactgtggccggcctgagccaccaagctgcgggacccagaggg
    aggattttactgcaagtcagcatcaaagcaccggtgttattctgaaaacaccagtggc
    ctcatttttggcttttgcaaagcatgaattttttcatttggattgcactttcctggttcatgact
    gtacctgtaggtggttgttactttgactcttttcaggaaccaccccccaagctgaatttac
    aagttctgttagcactatttgcttcaacttactgcgatttgttctcaaaacttaaaaataag
    caagcaaatggctgatactaccaagagaactggaagatggataccacacaaacttctt
    gtataaaaatatgaatgctgaaatgtttcagacatttttaatttaataaacctgtaaccaca
    tttaagtgatctaaaacccatagcattgtagtcatggcaacccgctaaactttctcatgc
    aactaaaatttctgggggaaatgaggggggggttgtacatttcccattgtaaaataag
    tgttttaaatgtcctgtactgctaacgaatgactttctatatgtccaggagttctccagtgg
    aataactatgcactactttacatttcatggggatgcacaaaaacaaaaaagtattacattt
    ttagttgctgtttgtaccaaccttaaattacatatgtttaacaacaacaaatcaaaaatcct
    atttctattgagtttttaatactgactagcaactctgaagtcttaattccttttttgttatgattt
    atttgtgagtttacatttttaaattgtttaactttcttaatttagtaattaaaaagagagcatttt
    acatttgaa (SEQ ID NO: 659)
    Ype12 gccgcggcggtggcggagactgtggctttaagagcgtgccgggagcccgagccc NM_001005404 NM_
    cagccgggccgcgcttcgccgctgcgcaccccagcggagccaagccccacgctg 001005341
    gccggacagggccgcctgtcgccgggctgctgagaactagccctagacctctgcgt
    gagggttcttctgccgaagacatcaccagtgtgtggagcctgccacacccacccgct
    gccaaaccacggcctttacctgtgtcttccggtgtttcccgtgcgacccatcctgtggg
    agtgcctcgtgggctgccccagagttcaccccacactcagcagcaccaatggtgaa
    gatgacaagatcgaagactttccaggcatatctgccctcctgccaccggacctacag
    ctgcattcactgcagagctcacttggccaatcatgatgaactaatttccaagtcattcca
    aggaagtcaaggacgagcatacctctttaactcagtagttaatgtgggctgtgggcct
    gcagaagagcgagtgttgctaacaggactgcatgcagtcgcagacatttactgtgaa
    aactgcaaaaccactctgggctggaaatacgaacatgcttttgaaagcagccagaaa
    tataaagaaggcaaatacatcattgaactagcacacatgatcaaggacaatggctgg
    gactgattggacagcatctacccaacccagtgtccacgtgaacgccattcaaccgaa
    cattcttcccaagcgtgagagagtgactgacacttggttccatccatttaggggccttg
    ccatccggggcatcctcccaccctgacgccatctttctggtgaccggcctctaaatcg
    ctgtctctctgtctctttgctttgtatctgtttgtgagttgatcctggcttctctctctgttctag
    ttttggctgaaaacaaaacaacaaaaggaacagatccttgaccgcatggcggcagcc
    caccttggtaagggccccagggcccatgcgagagctgcctgatggcctcttgtcagg
    agagcagtggcacgggggcgtgaggaagagggaaaggggaaactctaagggtcc
    tggcgcggggaaggggtggaagggtggaggtaggaacaaaattgcgccgctcctg
    gagacctgataacttaggcttgaaataattgacttgtctaaaaggacaaagagaaaaa
    aaaaatacctcatgactgcattctctctgactagaagcttctgttcctgacaccaaatgt
    gccaggttagcaaatgagcacaagatgtggccctgattctagttggtggggcaaggg
    cctggttctcctgggctgagtgggggagtgtcctggcagcagcgagtgacctgggc
    agtggccaggtgggtgcgatgactctgatgcctcactcagtctctgggcaatcatcat
    ctttgcctctagccaccgtagataaggtgtgaagggactgctgtttgcaatgggcttac
    catccaaatatcccaaaggctttgaccagcaaccaagtaaaatcagtaattgaggaga
    gcagggcacaaaggggctgcagtttgggagctcctgaagaaatggctcagatattg
    agtcagagaaataaaaagtaggatcagttagcaattctaactgcccttccttctgaccc
    ctcataagaggagtgtggtgagggaggggactgggtaggggtcatcccaggagga
    ggggtttacattggaaccagttcaggttcggtgcatctttcctcttcggttttacagtggc
    ttccgtgggatcgtcaatttcttgttcttagagtttcgggtgtttttctccagtcttgttactgt
    agactgtagaaagcacgggccccaggctctgagcttagtaataacctggctggtaga
    ttcctcatgcccctaattgtcccacttaggcctgaatgtcttgcatggagagaaatctcct
    gtcagtgtggtccagcagcagggaggagttctgcccaaattccgatatcaccccttcc
    cccatccaagcatccttcgattagggaagtggagagcacatccctgtaaggcccata
    agagaaagaggagtttgttacatttaatcaacactgtgaagtctgttctacagcaattca
    gccattacacagtatatgactgaaactcatttaactgggttaatttcatttcttagactgaa
    tatattattgttaagatacgtgtgcgtgttaggtaattctcagcatctcctccaagtaggc
    cgaccttctcggaaaattcaccctaaaagtctcacaaaagaatgagttcatggggaga
    ttctgtaaagtgatgaactgagatgaaagcagccaacagcccaggagcttttcagaat
    agcgtctgcagcagaaccagtttccattcagagcgcgtccttggtggaaatgcttttttg
    tgtgtctccacgcgctgatggtggaatgggagccccaagacgtgtgggcttagaaat
    caacttttgttccccaaggcttcttgtccagatctttccagtgctttcatagccctgggag
    atcaagttgttctccccactttactgcaaggtagactgaagttcagaagaaatactgaat
    ttctgctcccagaagaatagtttctctggctcacaggcccaagttctcaatgaaatcgttt
    tttaactttcacattcctaagctggcttcccggcacagaagccatggatttcccctctctc
    ccttccccctcctcaaggaaatagtcttcctttatggattttcattggactctttcctcagc
    gattgtcctggctgtttattgatagtccttcccataagaaaatggggttaaacatggggt
    aggtattttgtctttcaaactacaaatggaatgtggtgacataaactagacatggggtgc
    cctcaagtttccaaggggaccaatgtgccactgttcttccttggggatgaggcctttga
    ctgttggatggatcagagcaggctccagtcagaccctggttctgaatgttttttttttcgg
    tgactatccagtgagccttcagtgggtgcaaggcgccatacttgctgtgagagagctg
    agtagagtgttggtttttccataactacagggggaaaaaaagtcattaggctttcccttt
    gtgtcagtgaaaccaaaagtgcttcttacaacgttcgctctgttcatgggttgtctatcta
    acattgagcagcattggagaggccacagctgagctatggagatgctaaattaactcat
    ggcctcagtcagttcattctttaatttcctcaccaaattattgacttagagcataaccaaa
    gacctcattcattcaccccaggtgggttggggtaattggagtttgttggtgaagtttggg
    ggcggggtgttgggagtagagacagggtaaggggacgtgagaaaggaaaaggca
    tgaagttctatacctcagccagcagctgccttcgtttggaactgaagtccagccagca
    gactctctagctccatctcccctgtgccaccctaggtcatatgaccttggccaccttgg
    agtagacccagacccctcgggacccgggacattagtctcaggctgctgatggattga
    tttgacatgaaccaaacacagccaaactcgatacccacaagctgtcagctgaacctg
    actgagtgttcttcctgagttcacgaggataggctagagtgcatttttactggtggatca
    gtgtgtgcgaaagagatgaccctttataaagagattttcaagtggatatatataaaaga
    aacagttgcttgtaaaatatacttttgtaaataatatttaattttttaaataatatatttggtgct
    gttttctcagatcccctgagagcactttttattttccttttaaattctatggtttcctttgcattt
    cttgaagtatattttaagggaaacagtgatcaccaatacatgttttcagttttttttttttttaa
    ggtctctatcactttaatctggatcaaggctttgaagcaatgcctctctgcattttttcccc
    agtggaacagactctgcagtacattaatcaggttgagaattgaaatattttcttgcatca
    gtattggctagaaaagaaaataaataaaaccaagttaatttagtagtaacaacttacagt
    gattcttcctgttggaagaatttccaacaaatcagaatcacgtttttagttgtgcgtgtgc
    gcgcacacgtgtgtaaaaagcactttcgattgtgcctcctgttttctcgagtggggaca
    ctttaactacagtttacacctcgggcgcataaagtttttcttctctttctctctggttgtttct
    gtttctgagtggaccaacagcagaacccacgaggatttgttttgagtatggagctgttg
    cgggtttgctcctttttcttgctttgcgtgctcagtttttacagactgtaaaggagatgtgtt
    gtttgtgaagatggagcagagtcaaatctgtgcttctaactgagatgagagtgtattaat
    cacgtatcgcagggctccagctgttttagaagccacatcatgttaaacattaactggttt
    ggattaaaagaacattaatattataatacacatatcttagtggtaaacagctttttttttttaa
    ggtcagattgcctcaggtttagaaagaggctgagaaatcaaatcttgaacacaatcaa
    cttacatattttaaaggaatctgcctcaaatgagaaaatatgctagttatctagatagagg
    aaagagatatttacttttttaaaaattaaaatagttatgaaatctggcagaaaaggtaaag
    cctagaagaaactatgaaagctattctcatgttaccaaattctatctgcgcatatgtttttg
    tataacatttcggtgacagtgggagtcggttccctttcccaacctgcagagactatcttc
    caatacagaatctgtctatttatgcttgtgtttacaaactgtatttgttgggtttgggtttttgt
    tttctttggtggcatttttcaggtcactttgcttctataacaaaggtaattgttttcaaataatt
    tgtcttcaccttttcctgtatttgtacatagtgattcagtattagagaaaagtgcattgtttct
    gtcatatttccaatctgtgttggtgctcatttgagaaaataaaagttttcaaatattaactct
    taaaaaaaaaa (SEQ ID NO: 660)
    Pkd1 ccctcccctcccgatcctcatccccttgccctcccccagcccagggacttttccggaa NM_002742 NM_008858
    agtttttattttccgtctgggctctcggagaaagaagctcctggctcagcggctgcaaa
    actttcctgctgccgcgccgccagcccccgccctccgctgcccggccctgcgcccc
    gccgagcgatgagcgcccctccggtcctgcggccgcccagtccgctgctgcccgt
    ggcggcggcagctgccgcagcggccgccgcactggtcccagggtccgggcccg
    ggcccgcgccgttcttggctcctgtcgcggccccggtcgggggcatctcgttccatct
    gcagatcggcctgagccgtgagccggtgctgctgctgcaggactcgtccggggact
    acagcctggcgcacgtccgcgagatggcttgctccattgtcgaccagaagttccctg
    aatgtggtttctacggaatgtatgataagatcctgctttttcgccatgaccctacctctga
    aaacatccttcagctggtgaaagcggccagtgatatccaggaaggcgatcttattgaa
    gtggtcttgtcagcttccgccacctttgaagactttcagattcgtccccacgctctctttgt
    tcattcatacagagctccagctttctgtgatcactgtggagaaatgctgtgggggctgg
    tacgtcaaggtcttaaatgtgaagggtgtggtctgaattaccataagagatgtgcattta
    aaatacccaacaattgcagcggtgtgaggcggagaaggctctcaaacgtttccctca
    ctggggtcagcaccatccgcacatcatctgctgaactctctacaagtgcccctgatga
    gccccttctgcaaaaatcaccatcagagtcgtttattggtcgagagaagaggtcaaatt
    ctcaatcatacattggacgaccaattcaccttgacaagattttgatgtctaaagttaaagt
    gccgcacacatttgtcatccactcctacacccggcccacagtgtgccagtactgcaag
    aagcttctgaaggggcttttcaggcagggcttgcagtgcaaagattgcagattcaact
    gccataaacgttgtgcaccgaaagtaccaaacaactgccttggcgaagtgaccatta
    atggagatttgcttagccctggggcagagtctgatgtggtcatggaagaagggagtg
    atgacaatgatagtgaaaggaacagtgggctcatggatgatatggaagaagcaatgg
    tccaagatgcagagatggcaatggcagagtgccagaacgacagtggcgagatgca
    agatccagacccagaccacgaggacgccaacagaaccatcagtccatcaacaagc
    aacaatatcccactcatgagggtagtgcagtctgtcaaacacacgaagaggaaaagc
    agcacagtcatgaaagaaggatggatggtccactacaccagcaaggacacgctgc
    ggaaacggcactattggagattggatagcaaatgtattaccctctttcagaatgacaca
    ggaagcaggtactacaaggaaattcctttatctgaaattttgtctctggaaccagtaaaa
    acttcagctttaattcctaatggggccaatcctcattgtttcgaaatcactacggcaaatg
    tagtgtattatgtgggagaaaatgtggtcaatccttccagcccatcaccaaataacagt
    gttctcaccagtggcgttggtgcagatgtggccaggatgtgggagatagccatccag
    catgcccttatgcccgtcattcccaagggctcctccgtgggtacaggaaccaacttgc
    acagagatatctctgtgagtatttcagtatcaaattgccagattcaagaaaatgtggaca
    tcagcacagtatatcagatttttcctgatgaagtactgggttctggacagtttggaattgtt
    tatggaggaaaacatcgtaaaacaggaagagatgtagctattaaaatcattgacaaatt
    acgatttccaacaaaacaagaaagccagcttcgtaatgaggttgcaattctacagaac
    cttcatcaccctggtgttgtaaatttggagtgtatgtttgagacgcctgaaagagtgtttg
    ttgttatggaaaaactccatggagacatgctggaaatgatcttgtcaagtgaaaaggg
    caggttgccagagcacataacgaagtttttaattactcagatactcgtggctttgcggc
    accttcattttaaaaatatcgttcactgtgacctcaaaccagaaaatgtgttgctagcctc
    agctgatccttttcctcaggtgaaactttgtgattttggttttgcccggatcattggagag
    aagtctttccggaggtcagtggtgggtacccccgcttacctggctcctgaggtcctaa
    ggaacaagggctacaatcgctctctagacatgtggtctgttggggtcatcatctatgta
    agcctaagcggcacattcccatttaatgaagatgaagacatacacgaccaaattcaga
    atgcagctttcatgtatccaccaaatccctggaaggaaatatctcatgaagccattgatc
    ttatcaacaatttgctgcaagtaaaaatgagaaagcgctacagtgtggataagaccttg
    agccacccttggctacaggactatcagacctggttagatttgcgagagctggaatgca
    aaatcggggagcgctacatcacccatgaaagtgatgacctgaggtgggagaagtat
    gcaggcgagcaggggctgcagtaccccacacacctgatcaatccaagtgctagcca
    cagtgacactcctgagactgaagaaacagaaatgaaagccctcggtgagcgtgtca
    gcatcctctgagttccatctcctataatctgtcaaaacactgtggaactaataaatacata
    cggtcaggtttaacatttgccttgcagaactgccattattttctgtcagatgagaacaaa
    gctgttaaactgttagcactgttgatgtatctgagttgccaagacaaatcaacagaagc
    atttgtattttgtgtgaccaactgtgttgtattaacaaaagttccctgaaacacgaaacttg
    ttattgtgaatgattcatgttatatttaatgcattaaacctgtctccactgtgcctttgcaaat
    cagtgtttttcttactggagcttcattttggtaagagacagaatgtatctgtgaagtagttc
    tgtttggtgtgtcccattggtgttgtcattgtaaacaaactcttgaagagtcgattatttcc
    agtgttctatgaacaactccaaaacccatgtgggaaaaaaatgaatgaggagggtag
    ggaataaaatcctaagacacaaatgcatgaacaagttttaatgtatagttttgaatccttt
    gcctgcctggtgtgcctcagtatatttaaactcaagacaatgcacctagctgtgcaaga
    cctagtgctcttaagcctaaatgccttagaaatgtaaactgccatatataacagatacatt
    tccctctttcttataatactctgttgtactatggaaaatcagctgctcagcaacctttcacct
    ttgtgtatttttcaataataaaaaatattcttgtcaaaa (SEQ ID NO: 661)
    Ptpn2 gctcgggcgccgagtctgcgcgctgacgtccgacgctccaggtactttccccacgg NM_002828 NM_008977
    ccgacagggcttggcgtgggggcggggcgcggcgcgcagcgcgcatgcgccgc
    agcgccagcgctctccccggatcgtgcggggcctgagcctctccgccggcgcagg
    ctctgctcgcgccagctcgctcccgcagccatgcccaccaccatcgagcgggagtt
    cgaagagttggatactcagcgtcgctggcagccgctgtacttggaaattcgaaatga
    gtcccatgactatcctcatagagtggccaagtttccagaaaacagaaatcgaaacaga
    tacagagatgtaagcccatatgatcacagtcgtgttaaactgcaaaatgctgagaatg
    attatattaatgccagtttagttgacatagaagaggcacaaaggagttacatcttaacac
    agggtccacttcctaacacatgctgccatttctggcttatggtttggcagcagaagacc
    aaagcagttgtcatgctgaaccgcattgtggagaaagaatcggttaaatgtgcacagt
    actggccaacagatgaccaagagatgctgtttaaagaaacaggattcagtgtgaagc
    tcttgtcagaagatgtgaagtcgtattatacagtacatctactacaattagaaaatatcaa
    tagtggtgaaaccagaacaatatctcactttcattatactacctggccagattttggagtc
    cctgaatcaccagcttcatttctcaatttcttgtttaaagtgagagaatctggctccttgaa
    ccctgaccatgggcctgcggtgatccactgtagtgcaggcattgggcgctctggcac
    cttctctctggtagacacttgtcttgttttgatggaaaaaggagatgatattaacataaaa
    caagtgttactgaacatgagaaaataccgaatgggtcttattcagaccccagatcaact
    gagattctcatacatggctataatagaaggagcaaaatgtataaagggagattctagta
    tacagaaacgatggaaagaactttctaaggaagacttatctcctgcctttgatcattcac
    caaacaaaataatgactgaaaaatacaatgggaacagaataggtctagaagaagaa
    aaactgacaggtgaccgatgtacaggactttcctctaaaatgcaagatacaatggagg
    agaacagtgagagtgctctacggaaacgtattcgagaggacagaaaggccaccac
    agctcagaaggtgcagcagatgaaacagaggctaaatgagaatgaacgaaaaaga
    aaaaggtggttatattggcaacctattctcactaagatggggtttatgtcagtcattttggt
    tggcgcttttgttggctggacactgttttttcagcaaaatgccctataaacaattaattttg
    cccagcaagcttctgcactagtaactgacagtgctacattaatcataggggtttgtctgc
    agcaaacgcctcatatcccaaaaacggtgcagtagaatagacatcaaccagataagt
    gatatttacagtcacaagcccaacatctcaggactcttgactgcaggttcctctgaacc
    ccaaactgtaaatggctgtctaaaataaagacattcatgtttgttaaaaactggtaaatttt
    gcaactgtattcatacatgtcaaacacagtatttcacctgaccaacattgagatatccttt
    atcacaggatttgtttttggaggctatctggattttaacctgcacttgatataagcaataaa
    tattgtggttttatctacgttattggaaagaaaatgacatttaaataatgtgtgtaatgtata
    atgtactattgacatgggcatcaacacttttattcttaagcatttcagggtaaatatatttta
    taagtatctatttaatcttttgtagttaactgtactttttaagagctcaatttgaaaaatctgtt
    actaaaaaaataaattgtatgtcgattgaattgtactggatacattttccatttttctaaaga
    gaagtttgatatgagcagttagaagttggaataagcaatttctactatatattgcatttcttt
    tatgttttacagttttccccattttaaaaagaaaagcaaacaaagaaacaaaagtttttcct
    aaaaatatctttgaaggaaaattctccttactgggatagtcaggtaaacagttggtcaag
    actttgtaaagaaattggtttctgtaaatcccattattgatatgtttatttttcatgaaaatttc
    aatgtagttggggtagattatgatttaggaagcaaaagtaagaagcagcattttatgatt
    cataatttcagtttactagactgaagttttgaagtaaacacttttcagtttctttctacttcaa
    taaatagtatgattatatgcaaaccttacattgtcattttaacttaatgaatattttttaaagc
    aaactgtttaatgaatttaactgctcatttgaatgctagctttcctcagatttcaacattcca
    ttcagtgtttaatttgtcttacttaaacttgaaattgttgttacaaatttaattgctaggaggc
    atggatagcatacattattatggatagcataccttatttcagtggttttcaaactatgctcat
    tggatgtccaggtgggtcaagaggttactttcaaccacagcatctctgccttgtctcttta
    tatgccacataagatttctgcataaggcttaagtattttaaagggggcagttatcatttaa
    aaacagtttggtcgggcgcggtggctcatgcctgtaatcccagcactttgggaggctg
    aagtgggcagatcacctgaggtcaggagttcaagaccagcctggccaacgtggtga
    aacaccatctctactaaaaatgcaaaaattagctgggcatggtggagggcacctgtaa
    tctcagctactcaggaggctgaggtaggagaattgcttgaacccaggagatggaggt
    tgcagtgagctgagatcacgtcactgcactccagccagggcgacagagcgagactc
    catctcaaaagaaacaaacaaaaaaaacagtttgggccgggtgtggtggctcacgct
    tgtaatcccagcacttcggaaggccaagggggcggatcacgaggtcaagagatg
    gagactgtcctggccaacatggtgaaatcccttctttactaaaaatacaaaaattatctg
    ggcgtggtggtgcatgcctgtagtcccagctccttgggaggctaaggcaggagaatc
    acttgaacccgggaggcagaggttgcagtgagccgagattgcaccactgcactcca
    gcctggcaacagagcaagacttcgtctc (SEQ ID NO: 662)
    Grk6 cggctggctgcggcggccggggaggccggggaggccgcggcgcggtcactgcg NM_001004106 NM_
    agccgagccgagccgcgccgagccgcgccgatcgccatccggcctcggcactcg 001038018
    cgcgcgatcccggccggcggcgcggcccgggggccaggcggcgccacagcc
    catggagctcgagaacatcgtagcgaacacggtgctactcaaggcccgggaaggt
    ggcggtggaaatcgcaaaggcaaaagcaagaaatggcggcagatgctccagttcc
    ctcacatcagccagtgcgaagagctgcggctcagcctcgagcgtgactatcacagc
    ctgtgcgagcggcagcccattgggcgcctgctgttccgagagttctgtgccacgagg
    ccggagctgagccgctgcgtcgccttcctggatggggtggccgagtatgaagtgac
    cccggatgacaagcggaaggcatgtgggcggcagctaacgcagaattttctgagcc
    acacgggtcctgacctcatccctgaggtcccccggcagctggtgacgaactgcacc
    cagcggctggagcagggtccctgcaaagaccttttccaggaactcacccggctgac
    ccacgagtacctgagcgtggccccttttgccgactacctcgacagcatctacttcaac
    cgtttcctgcagtggaagtggctggaaaggcagccagtgaccaaaaacaccttcag
    gcaataccgagtcctgggcaaaggtggctttggggaggtgtgcgcctgccaggtgc
    gggccacaggtaagatgtatgcctgcaagaagctagagaaaaagcggatcaagaa
    gcggaaaggggaggccatggcgctgaacgagaagcagatcctggagaaagtgaa
    cagtaggtttgtagtgagcttggcctacgcctatgagaccaaggacgcgctgtgcctg
    gtgctgacactgatgaacgggggcgacctcaagttccacatctaccacatgggccag
    gctggcttccccgaagcgcgggccgtcttctacgccgccgagatctgctgtggcctg
    gaggacctgcaccgggagcgcatcgtgtacagggacctgaagcccgagaacatctt
    gctggatgaccacggccacatccgcatctctgacctgggactagctgtgcatgtgcc
    cgagggccagaccatcaaagggcgtgtgggcaccgtgggttacatggctccggag
    gtggtgaagaatgaacggtacacgttcagccctgactggtgggcgctcggctgcctc
    ctgtacgagatgatcgcaggccagtcgcccttccagcagaggaagaagaagatcaa
    gcgggaggaggtggagcggctggtgaaggaggtccccgaggagtattccgagcg
    cttttccccgcaggcccgctcactttgctcacagctcctctgcaaggaccctgccgaa
    cgcctggggtgtcgtgggggcagtgcccgcgaggtgaaggagcaccccctctttaa
    gaagctgaacttcaagcggctgggagctggcatgctggagccgccgttcaagcctg
    acccccaggccatttactgcaaggatgttctggacattgaacagttctctacggtcaag
    ggcgtggagctggagcctaccgaccaggacttctaccagaagtttgccacaggcag
    tgtgcccatcccctggcagaacgagatggtggagaccgagtgcttccaagagctga
    atgtctttgggctggatggctcagttcccccagacctggactggaagggccagccac
    ctgcacctcctaaaaagggactgctgcagagactcttcagtcgccaagattgctgtgg
    aaactgcagcgacagcgaggaagagctccccacccgcctctagcccccagcccga
    ggcccccaccagcagttggcggtagcagctactccgagcgccgtttacagttttgca
    cagtgatcttccccattgtccactcaagtcgtggcctggggaacacagacggagctgt
    ccccagtgtcctccgtccctcagcccctggcctggctgagtttggcagggcctgggc
    catccctgggacaaaggtgcgtcccttcagctcttctccgtggagctcggggctttctg
    tatttatgtatttgtacgaatgtatatagcgaccagagcattcttaattcccgccgcagac
    ctggcgcccccgccttggctcctgggggcagccagccctggctgggagagcggga
    gctggcagaggagccactgccaaactcaaggctcctctggcccagcttggatggct
    gagggtggtcacacccctgagccttcagcactgtgctggccaccccggcctctgagt
    aagactcgtgcctccccctgctgccctgggctcaggctgctaccctctggggcccaa
    agctgtcccttctcagtgcttgtcagcgctgggtctggggcctctgtatgccctaggcc
    tgtgccaaagtggccagagattgggctgcctgtgatacccatcagcccactgccccg
    gccggcccagataggtctgcctctgccttccagctcccacagcctggtccctgatact
    gggctctgtcctgcagacacctctttcagaaacgcccaagcccagcccctaggagg
    gggtggggcatccctggtcaaccctcaaacattccggactcccctcataacaataga
    cacatgtgcccagcaataatccgccccttcctgtgtgcgcctgtggggtgcgtgcgc
    gcgcgtgtgtacctgtgtgggtgaaggggatagggcgaggctgtgcctgtgcccca
    ggtcccagccctggcccttcccagactgtgatggccatcctggtcccagtgttagggt
    agcatgggattacagggccctgttttttccatatttaaagccaatttttattactcgttttgtc
    caacgtaa (SEQ ID NO: 663)
    Cdkn2a cgagggctgcttccggctggtgcccccgggggagacccaacctggggcgacttca NM_000077 NM_
    ggggtgccacattcgctaagtgctcggagttaatagcacctcctccgagcactcgctc 001040654
    acggcgtccccttgcctggaaagataccgcggtccctccagaggatttgagggaca
    gggtcggagggggctcttccgccagcaccggaggaagaaagaggaggggctgg
    ctggtcaccagagggtggggcggaccgcgtgcgctcggcggctgcggagagggg
    gagagcaggcagcgggcggcggggagcagcatggagccggcggggggagca
    gcatggagccttcggctgactggctggccacggccgcggcccggggtcgggtaga
    ggaggtgcgggcgctgctggaggcgggggcgctgcccaacgcaccgaatagtta
    cggtcggaggccgatccaggtcatgatgatgggcagcgcccgagtggcggagctg
    ctgctgctccacggcgcggagcccaactgcgccgaccccgccactctcacccgac
    ccgtgcacgacgctgcccgggagggcttcctggacacgctggtggtgctgcaccg
    ggccggggcgcggctggacgtgcgcgatgcctggggccgtctgcccgtggacctg
    gctgaggagctgggccatcgcgatgtcgcacggtacctgcgcgcggctgcggggg
    gcaccagaggcagtaaccatgcccgcatagatgccgcggaaggtccctcagacatc
    cccgattgaaagaaccagagaggctctgagaaacctcgggaaacttagatcatcagt
    caccgaaggtcctacagggccacaactgcccccgccacaacccaccccgctttcgt
    agttttcatttagaaaatagagcttttaaaaatgtcctgccttttaacgtagatatatgcctt
    cccccactaccgtaaatgtccatttatatcattttttatatattcttataaaaatgtaaaaaa
    gaaaaacaccgcttctgccttttcactgtgttggagttttctggagtgagcactcacgcc
    ctaagcgcacattcatgtgggcatttcttgcgagcctcgcagcctccggaagctgtcg
    acttcatgacaagcattttgtgaactagggaagctcaggggggttactggcttctcttg
    agtcacactgctagcaaatggcagaaccaaagctcaaataaaaataaaataattttcat
    tcattcactcaaaaaaaaaaaaaa (SEQ ID NO: 664)
    Sbf1 gggcgggccggctggctgggaagatggcggcgggaacctgggccgccgccgcc NM_002972 NM_
    gccgccgccgccgccgcggagcgaaccaggggtgtccggggtgcgcggtccag 001170561
    ggccggggccgggccatgagcgcgccgtcctcgagtccccgagccgcggagccc
    gcccgcgcccctcgggccgccccgcgtccctcgccatggcgcggctcgcggacta
    cttcgtgctggtggcgttcgggccgcacccgcgcgggagtggggaaggccagggc
    cagattctgcagcgcttcccagagaaggactgggaggacaacccattcccccaggg
    catcgagctgttttgccagcccagcgggtggcagctgtgtcccgagaggaatccacc
    gaccttctttgttgctgtcctcaccgacatcaactccgagcgccactactgcgcctgctt
    gaccttctgggagccagcggagccttcacaggaaacgacgcgcgtggaggatgcc
    acagagagggaggaagagggggatgagggaggccagacccacctgtctcccaca
    gcacctgccccatctgcccagctgtttgcaccgaagacgctggtactggtgtcgcga
    ctcgaccacacggaggtgttcaggaacagccttggcctcatctatgccatccacgtg
    gagggcctgaatgtgtgcctggagaacgtgattgggaacctgctgacgtgcactgtg
    cccctggctgggggctcgcagaggacgatctctttgggggctggtgaccggcaggt
    catccagactccactggccgactcgctgcccgtcagccgctgcagcgtggccctgct
    cttccgccagctaggcatcaccaacgtgctgtctttgttctgtgccgccctcacggagc
    acaaggttctcttcctgtcccggagctaccagcggctcgccgatgcctgtaggggcct
    cctggcactgctgtttcctctcagatacagcttcacctatgtgcccatcctgccggctca
    gctgctggaggtcctcagcacacccacgcccttcatcattggggtcaacgcggcctt
    ccaggcagagacccaggagctgctcgatgtgattgttgctgatctggatggagggac
    ggtcaccattcctgagtgtgtgcacattccacccttgccagagccactgcagagtcag
    acgcacagtgtgctgagcatggtcctggacccggagctggagttggctgacctcgc
    cttccctccgcccacgacatccacctcctccctgaagatgcaggacaaggagctgcg
    cgcggtcttcctgcggctgttcgctcagctgctgcagggctatcgctggtgcctgcac
    gtcgtgcgcatccacccggagcctgtcatccgcttccataaggcagccttcctgggc
    cagcgtgggctggtagaggacgatttcctgatgaaggtgctggagggcatggccttt
    gctggctttgtgtcagagcgtggggtcccataccgccctacggacctgttcgatgagc
    tggtggcccacgaggtggcaaggatgcgggcggatgagaaccacccccagcgtgt
    cctgcgtcacgtccaggaactggcagagcagctctacaagaacgagaacccgtacc
    cagccgtggcgatgcacaaggtacagaggcccggtgagagcagccacctgcgac
    gggtgccccgacccttcccccggctggatgagggcaccgtgcagtggatcgtggac
    caggctgcagccaagatgcagggtgcacccccagctgtgaaggccgagaggagg
    accaccgtgccctcagggccccccatgactgccatactggagcggtgcagtgggct
    gcatgtcaacagcgcccggcggctggaggttgtgcgcaactgcatctcctacgtgttt
    gaggggaaaatgcttgaggccaagaagctgctcccagccgtgttgagggccctgaa
    ggggcgagctgcccgccgctgcctcgcccaggagctgcacctgcatgtgcagcag
    aaccgtgcggtcctggaccaccagcagtttgactttgtcgtccgtatgatgaactgctg
    cctgcaggactgcacttctctggacgagcatggcattgcggcggctctgctgcctctg
    gtcacagccttctgccggaagctgagcccgggggtgacgcagtttgcatacagctgt
    gtgcaggagcacgtggtgtggagcacgccacagttctgggaggccatgttctatgg
    ggatgtgcagactcacatccgggccctctacctggagcccacggaggacctggccc
    ccgcccaggaggttggggaggcaccttcccaggaggacgagcgctctgccctaga
    cgtggcttctgagcagcggcgcttgtggccaactctgagtcgtgagaagcagcagg
    agctggtgcagaaggaggagagcacggtgttcagccaggccatccactatgccaac
    cgcatgagctacctcctcctgcccctggacagcagcaagagccgcctacttcggga
    gcgtgccgggctgggcgacctggagagcgccagcaacagcctggtcaccaacag
    catggctggcagtgtggccgagagctatgacacggagagcggcttcgaggatgca
    gagacctgcgacgtagctggggctgtggtccgcttcatcaaccgctttgtggacaag
    gtctgcacggagagtggggtcaccagcgaccacctcaaggggctgcatgtcatggt
    gccagacattgtccagatgcacatcgagaccctggaggccgtgcagcgggagagc
    cggaggctgccgcccatccagaagcccaagctgctgcggccgcgcctgctgccgg
    gtgaggagtgtgtgctggacggcctgcgcgtctacctgctgccggatgggcgtgag
    gagggcgcggggggcagtgctgggggaccagcattgctcccagctgagggcgcc
    gtcttcctcaccacgtaccgggtcatcttcacggggatgcccacggaccccctggttg
    gggagcaggtggtggtccgctccttcccggtggctgcgctgaccaaggagaagcg
    catcagcgtccagacccctgtggaccagctcctgcaggacgggctccagctgcgct
    cctgcacattccagctgctgaaaatggcctttgacgaggaggtggggtctgacagcg
    ccgagctcttccgtaagcagctgcataagctgcggtacccgccggacatcagggcc
    acctttgcgttcaccttgggctctgcccacacacctggccggccaccgcgagtcacc
    aaggacaagggtccttccctcagaaccctgtcccggaacctggtcaagaacgccaa
    gaagaccatcgggcggcagcatgtcactcgcaagaagtacaacccccccagctgg
    gagcaccggggccagccgccccctgaggaccaggaggacgagatctcagtgtcg
    gaggagctggagcccagcacgctgaccccgtcctcagccctgaagccctccgacc
    gcatgaccatgagcagcctggtggaaagggcttgctgtcgcgactaccagcgcctc
    ggtctgggcaccctgagcagcagcctgagccgggccaagtctgagcccttccgcat
    ttctccggtcaaccgcatgtatgccatctgccgcagctacccagggctgctgatcgtg
    ccccagagtgtccaggacaacgccctgcagcgcgtgtcccgctgctaccgccagaa
    ccgcttccccgtggtctgctggcgcagcgggcggtccaaggcggtgctgctgcgct
    ctggaggcctgcatggcaaaggtgtcgtcggcctcttcaaggcccagaacgcacctt
    ctccaggccagtcccaggcggactcgagtagcctggagcaggagaagtacctgca
    ggctgtggtcagctccatgccccgctacgccgacgcgtcgggacgcaacacgctta
    gcggcttctcctcagcccacatgggcagtcacgttcccagccccagagccagggtc
    accacgctgtccaaccccatggcggcctcggcctccagacggaccgcaccccgag
    gtaagtggggcagtgtccggaccagtggacgcagcagtggccttggcaccgatgtg
    ggctcccggctagctggcagagacgcgctggccccaccccaggccaacgggggc
    cctcccgacccgggcttcctgcgtccgcagcgagcagccctctatatccttggggac
    aaagcccagctcaagggtgtgcggtcagaccccctgcagcagtgggagctggtgc
    ccattgaggtattcgaggcacggcaggtgaaggctagcttcaagaagctgctgaaag
    catgtgtcccaggctgccccgctgctgagcccagcccagcctccttcctgcgctcact
    ggaggactcagagtggctgatccagatccacaagctgctgcaggtgtctgtgctggt
    ggtggagctcctggattcaggctcctccgtgctggtgggcctggaggatggctggga
    catcaccacccaggtggtatccttggtgcagctgctctcagaccccttctaccgcacg
    ctggagggctttcgcctgctggtggagaaggagtggctgtccttcggccatcgcttca
    gccaccgtggagctcacaccctggccgggcagagcagcggcttcacacccgtcttc
    ctgcagttcctggactgcgtacaccaggtccacctgcagttccccatggagtttgagtt
    cagccagttctacctcaagttcctcggctaccaccatgtgtcccgccgtttccggacct
    tcctgctcgactctgactatgagcgcattgagctggggctgctgtatgaggagaagg
    gggaacgcaggggccaggtgccgtgcaggtctgtgtgggagtatgtggaccggct
    gagcaagaggacgcctgtgttccacaattacatgtatgcgcccgaggacgcagagg
    tcctgcggccctacagcaacgtgtccaacctgaaggtgtgggacttctacactgagg
    agacgctggccgagggccctccctatgactgggaactggcccaggggccccctga
    acccccagaggaagaacggtctgatggaggcgctccccagagcaggcgccgcgt
    ggtgtggccctgttacgacagctgcccggggcccagcctgacgccatctcacgcc
    tgctggaggagctgcagaggctggagacagagttgggccaacccgctgagcgctg
    gaaggacacctgggaccgggtgaaggctgcacagcgcctcgagggccggccaga
    cggccgtggcacccctagctccctccttgtgtccaccgcaccccaccaccgtcgctc
    gctgggtgtgtacctgcaggaggggcccgtgggctccaccctgagcctcagcctgg
    acagcgaccagagtagtggctcaaccacatccggctcccgtcaggctgcccgccgc
    agcaccagcaccctgtacagccagttccagacagcagagagtgagaacaggtccta
    cgagggcactctgtacaagaagggggccttcatgaagccttggaaggcccgctggt
    tcgtgctggacaagaccaagcaccagctgcgctactacgaccaccgtgtggacaca
    gagtgcaagggtgtcatcgacttggcggaggtggaggctgtggcacctggcacgcc
    cactatgggtgcccctaagactgtggacgagaaggccttctttgacgtgaagacaac
    gcgtcgcgtttacaacttctgtgcccaggacgtgccctcggcccagcagtgggtgga
    ccggatccagagctgcctgtcggacgcctgagcctcccagccctgcccggctgctct
    gcttccggtcgttaccgaccactaggggtgggcagggccgccccggccatgtttaca
    gccccggccctcgacagtattgaggccccgagcccccagcacttgtgtgtacagcc
    cccgtccccgccccgccccgcccggccggccctaacttattttggcgtcacagctga
    gcaccgtgccgggaggtggccaaggtacagcccgcaatgggcctgtaaatagtcc
    ggccccgtcagcgtgtgctggtccagccagcggctgcaggcgagtttctagaacca
    gagtctatataaagagagaactaacgccacgctcctgtgcctgccttccccactcccc
    ggctgcctgctctcggcctacccagagggtcccatctgcccctatccaggcccacct
    ggcgggaggttggcatctttctcgtgagcctctcctggtgcctgggtccacccagctc
    ggcctgcatgtccctgggagtgactttgctctgggggcggatcgagcaggaggcttc
    actggggacttgcttgattccctccacgcctcagggctggtctaggggccggcacgg
    ctggagaggaagcccccatccctacccaggggatgcagaagctgacctcacagag
    gcttgggggtgaaagggtgggtggtcatttgaccccagaaggctgttgcaggtccag
    aggacacttgaggtggacgtcagtttctggctagacccgagctgaagggatggagg
    ccggaggcgggggggggggggggacagtgggctcccaggggaatgcaggttga
    ccacatctggctcctgccaggcaacgagcagcatctggcagagtaaggggccaac
    gcccatgggggatggaccctctcagttcttgggaattctgccccaaaagtcctttccct
    ggggtctcagagggcccccgtccttcccttcttggtgtcactgtggcccctcactgctc
    ttttcctattcaaacctgagtcccaccaggcccagggcttcacctgctgagctgttgtgt
    ccttgcctgtgacgaggcctggccaggggtgcaggagcagaaggtggggagggtt
    atagacgctgcaaaggccaagagaacatctgagagtggcagctggtgacctggcca
    gaggggctggtgaggggcagagaacctggctagaggctgggtccctcaggtggtc
    ctctcaggtgggaggcgagcagcaggtgtgggtgaggggaaggttctgatgacag
    ctgcagaggcagggcccagtgctggcaggtggggggccaagaccctcccctggtg
    ggacgttgaagccaaggatggccttggaccctgtcaggcccagcatggtcccgcca
    cctcccccaccccacaggtggtgttgggacacctgggcgagatgtgagggtgggct
    cacttgagccactgaaaccagccaggtcttccctcaggccggacagatggcgcctg
    accgaagttcctggcacctggaaaacccacaggtcagagtaaggggagaaaggac
    cctgccctccctgttccacgtctgtggggggagaggacaaatgccaggcacagggt
    aggcggcgagaacaaggcactcaatgtgtagctggggcagagactcggcctctgg
    ggagctgagcgggttccctccacccccaaccgtggtggaaagacaagctcgctgg
    ggcgggggggggtctggtctccacctgcccctcccactcagccactgaggacaag
    gtggggcccaggcttctgggagggggagctggcacaaaaggaagtcctggggttg
    atgtgtttgagcgttaggcgaagtggttccccccatcccccaaacggaaaaatgtcag
    tatttgctaagctgtagagacctgatgccgtgatgtggcctgttccgcctccacccatta
    cacggggataacgctggggggtggcgggcccacaaaagaggtgctggaggagac
    tctcccacccctggccgggccggggctttggggccggaaggttcacagtacgcggt
    ttgtccgaacgtcacggcttttattgggagttgggggtttggggtgccctgtcaggtga
    tcagaacattaaaaatggactcaacgtaaaaaaaaaaaaaaaaaa (SEQ ID
    NO: 665)
    Lpmk gccgtcagggccccagggagcgcggggcgccgctgctgctgttcttcggctcggtt NM_152230 NM_027184
    ctgtctaccgggcagcgccggggccggcggctgcggcggcagaggaacaggag
    ccgggagccgcgttccgccgagagttgggcagaggagcgcccgcgccccggcg
    gcgtcatgggccccctccccgcgcttcagagggcaccagccgcgggaacccccg
    ggcctcctcgcgcccgagcctgagcgaccctgggttctccggcgccccctccctc
    gccctattttttttcctactctcgctgccgttaccgcttctgctctccgttatggcaacaga
    gccaccatcccccctccgggtcgaggcgccgggccccccagaaatgcggacctca
    ccggcgatcgagtccacccctgagggcaccccgcagccgggggcggcagactc
    cgcttcctcaacggctgcgtgcccctctcgcatcaggtggccgggcacatgtacggg
    aaggacaaagtgggtatactgcaacatccagatggcacagttttgaaacagttacaac
    cacctccaaggggcccaagagagctggaattctataatatggtttatgctgctgactgt
    tttgatggtgttcttctagagctacgaaaatatttgccaaaatattatggcatctggtcacc
    tcccactgcaccaaacgatttatacctaaaactggaagatgtgacccataaatttaataa
    gccctgtataatggatgtaaagatagggcaaaaaagctatgatccttttgcctcatctga
    gaagattcagcaacaggtcagcaagtacccattaatggaagagattgggttcttggtg
    cttggcatgagggtttatcatgttcattccgatagctatgagacagaaaaccagcattac
    ggaagaagcttaacaaaagaaactataaaggatggagtctccagattttttcataatgg
    gtactgcttaagaaaagatgctgttgctgccagtattcagaagattgagaaaattctgc
    agtggtttgaaaaccagaagcagcttaatttttacgcaagttcattactctttgtttatgaa
    ggttcatctcagccaaccactacaaaattgaatgacagaactttggcagaaaagttttt
    gtccaaaggacaactgtcagacacagaagtactagagtacaataataactttcatgtgt
    taagttccacagctaatggaaaaatagagtcttcagtgggcaaaagcttgtccaagat
    gtatgcgcgtcacaggaaaatatatacaaaaaagcatcacagtcagacttcattgaaa
    gttgaaaatctggagcaagacaatgggggaaaagcatgtcacaggaacatttaaat
    ggaaatgtactttcccaactggaaaaagttttctaccatcttcccactggttgccaagag
    attgctgaagtagaagtgcgaatgatagattttgctcatgtgttccctagcaacacaata
    gatgagggatatgtttatgggctaaagcatttaatttctgtacttcgaagtattttagacaa
    ttgaatcctctgttgcagtctttttaaggggtgggccaatcataatgaagaggggcagt
    caatatctgcacctttaatgctatgtaaaaaatttgtattatgagtcgacattttatttgtcttt
    atacttttggaagaatggttaacttttttataatcttactcaggaaaactaactatttgttcat
    tagaaaactatgaagaataaagaaacttaggaatgttaagcagggaatgtggtggtac
    atggcttaaacatcttttttggctcaagcaaaatgcaaaccattattcagtcattaagagtt
    tagttagctttctgtagccaattcatgaaatctctgtccacccagccttgacaatgagcc
    atatctaaaatattacattattagaacacctaccaaaatctcgaaagcacaggttgatgt
    ccttagtattgctatgtatgaagttactaaaactggagaaaattctacttcagaaataagt
    actgtttaggttttatattaaaagttcagaccagcatatcaaagggtgctccttagtgaaa
    tgatttagaattgttgcattccaaaagcaggttttctctttaatttttacatctctctctcaaa
    atattatacttcatgaaaaagacaattgatgtggatgacaacaacaaagtcttgaaatta
    agggcacactaattgtccttactggggttaggggaagagagatattattttcaaggaac
    aaaatattttcctttacaatctttcattcatgagaaaattggaatataaatttattacattgtg
    aaagtatcataaaccatatacctttgtatctaaatgcagcttcaaaaaagtaaataattga
    agttttatttctcctctaaataacttgaatttttttctttaaaaatttatgtatttatatgtcccca
    tttagttaagtggtagtgtaaatgtatgttgttaaaaacagtttctcagaattatagtaagc
    aatgaaagacaatatctaattaggttgttatcaaaaatactgtgtgtaaattagtccgtaa
    tatagggtttggtgcgtatctatattcatgcttctatttcactcttcctcaaaacagttttatat
    tatgttgaccagtgaaattgtaacttaatttcatggggacaggggcagtgctacagttcc
    tggaaaaattagatttgtattatctttgtttcacacccaccaccttaaaaaaaaatcaacta
    gttatttgtcatttaaaacatttaaaactttgagtcttcaaatacatttgatgttaatgctgcc
    attacttgcacttccattcactaataacatttctaggtagttatcagttttgtcatattcctgg
    aaaatattttggggttgtaaattctttctcctctttttcttctggagttacaaattgaatttttaa
    atccgagcacctttattgtggtgtggagaaaattatcacaattttatgtttattttaccttctc
    agccttctctgagggcactttgcaaatacctgagtccaaacagaagtaccaactaaat
    gctctatgaactctatccttagtaaatctattaaacctgaataatttaaaagatcatgttcat
    tttgtaatagcaaaatttgattttaattttttatttagaattggtgtatttatcatagggacttcc
    aatttttcttcactttttgaatggatattggctatagttttatgttttaacgggaatgaatttca
    agtcataataatcagaatttttagttttacttttttcttttacaatatggattttgttgttatttgg
    atagtggttcaataaatcttaagctcagataattaaacactattttgaatcttaacaagata
    ctgaggctttttttgtatgggatgatatcaacctatgtacaatgaatttaataaacttaagt
    attgtcagattttttgcacattttagctcaataaaatcttaatgttcaagatttttttatctgcat
    ttggaaatacaattttgtaaaatcaatgtcttacctttttgatacaatagatcatgttttgtttt
    taataaagcaagaagcccttttatctgttgtttttcagggaagggattaacatttaattctg
    tttgtttacatttgttatcattgttatccaatgctcattttatgttgctttataagtaggcttagg
    tataacagaataagtatctgtttatctaatctacatgtgactatcttagtctctctcggtcac
    ttaatattatgctgaaatttaccactgtggggatgaatgatcgctattcaccaagtatattt
    gaacatgtaaatgcttaagaaataagcataatgcggatatagtttgggttaataggattc
    tcatagttttttttcccctatgaaacataagtaatgattttagtgtatttcttatggaatacact
    catttaaaaaggactttaagaaattgtggatgtgaataatacctttctctaataaaaattta
    aattgtataatagttttataatatttacattaattgatattttaatatggatagacattgcatag
    attcaaataaattaaaatcaatgataaatgctaaatattttatctaaatagtttttcaagaaa
    cagttatggaaatgtgtatattaaatggctctaatgtggagcttgtggtatttcaactcagt
    attcattattagttgtgtgtctggaaagattgtacttacttttcctctttacactacagtttgct
    cttatggggctctaaactgtttaactgaagaaccttcgtctgtattttgattgagcataattt
    agtattttatgatttccaagatgatgttcttatgtctatcaagtctatgtatcaaatttataac
    atcatttaagaaaaaggaatttccacagatacttcagttgcaattttttgtttcatgctactg
    aaaatacatttgtttctaggggttggaatattatagaagatgtaggatgaaagaaaacg
    atagaacaacgaaagaattctgtttatgaaattacaggaattgtgtccactatggtaaag
    cattgtcattttagtacattttctcttagtagtttggcattttatactttaaaacttgttttgcttt
    aaaaattgtttataatgcttaccttctttctccagtgcctttagtcttgatttgatatgtttgta
    ccctcagttaccctttctattacatgtttttgatgttttcatagcctaggaaacatcgattcct
    ttttaataattgtcaatctgattatttaaagaggtaacaattatctgttaatgctttggaaaa
    acaagtagggttgcctttggaggccaggcttcttagttcattcaaaaatattccttggatt
    tatgccatgtattaagcatttttagcccccagtattacaactgtgaaccaaacggataag
    gccctaaccattttcagcattctctttggatggggtgggattggggacttaattaaaata
    gagatatagaaaaataggcatctaaataagataataagtgtggggttgaaatgaagca
    tctaacaatagttgaagttagaagtaatattttacagtattgtaacctctatttaagtttggg
    tattagttacagatagcataaaaaagccttaatttttcactttccttgctggcaaaggtaca
    tttatttagactgtccatttaaagtaatgtttaacataaacattactgtgaaaaacattccat
    tacatattcccaagcaaatgagctgcatcttctttactgtattttacaatttagtacaacagt
    tttaggcctcaatcttaacatcactggtattttaaatttggcaatgaatatgaaattactttt
    gacttacagattgattatattattactttgaaaatgcattaatttcttagaaaagtttggagc
    ctctatctttttttgagttaatacttaaattctcattacttatattaatagcctgtactaagtga
    aaatattatttatgcaagtaaacaagtcactataggcttttaagacttttctttaattttagat
    tttgtcatcaaagtttaaattttttacctactgtccacttaaatataatttaacagtttgtaaag
    tgaaatagttttaagtatgatgtatgatgcacctgcatataaatgaaaatggcgtgcaca
    aagacactttactatgggaactgtactggaagatttatgaaagcatgtgaaattgcacct
    aaaattgtgttattagtgactataagcagcaatgctaaatttattgtacttgatgaatgaat
    gtatttagtcacagttactttggtttaaatgtataaatgtctttagggtttttttttaaatgtgtt
    tgtaatttgtactattgtgggggtatacttggactgcaggggttattgtcaatgtgtgattt
    gtgtttttattttatagaatcatctaatgtgatataccaatttttataagtgatatttacataatt
    ctaataactgtatatttgacaacctattaaaatgttttgcattggaa (SEQ ID NO:
    666)
    Rock1 gctggttccccttccgagcgtccgcgccccgcatgcgcagtctgccccggcggtctc NM_005406 NM_009071
    cgtttgtttgaacaggaaggcggacatattagtccctctcagcccccctcgccccacc
    ccccaggcattcgccgccgcgactcgccctttccccggctgggaccgcagcccctc
    ccagaagctcccccatcagcagccgccgggacccaactatcgtcttcctcttcgccc
    gctctccagcctttcctctgctaagtctccatcgggcatcgacctcgccctgccccacc
    ggacaccgtagcagcagccccagcagcgacgggacaaaatgggagagtgaggct
    gtcctgcgtggaccagctcgtggccgagactgatcggtgcgtcgggccgggccga
    gtagagccggggacgcggggctagaccgtctacagcgcctctgagcggagcggg
    cccggcccgtggcccgagcggcggccgcagctggcacagctcctcacccgccctt
    tgctttcgcctttcctcttctccctcccttgttgcccggagggagtctccaccctgcttctc
    tttctctacccgctcctgcccatctcgggacggggacccctccatggcgacggggc
    cggggcccgctagactgaagcacctcgccggagcgacgaggctggtggcgacgg
    cgctgtcggctgtcgtgaggggctgccgggtgggatgcgactttgggcgtccgagc
    ggctgtgggtcgctgttgcccccggcccggggtctggagagcggaggtcccctcag
    tgaggggaagacgggggaaccgggcgcacctggtgaccctgaggttccggctcct
    ccgccccgcggctgcgaacccaccgcggaggaagttggttgaaattgctttccgctg
    ctggtgctggtaagagggcattgtcacagcagcagcaacatgtcgactggggacag
    ttttgagactcgatttgaaaaaatggacaacctgctgcgggatcccaaatcggaagtg
    aattcggattgtttgctggatggattggatgctttggtatatgatttggattttcctgcctta
    agaaaaaacaaaaatattgacaactttttaagcagatataaagacacaataaataaaat
    cagagatttacgaatgaaagctgaagattatgaagtagtgaaggtgattggtagaggt
    gcatttggagaagttcaattggtaaggcataaatccaccaggaaggtatatgctatgaa
    gcttctcagcaaatttgaaatgataaagagatctgattctgcttttttctgggaagaaagg
    gacatcatggcttttgccaacagtccttgggttgttcagcttttttatgcattccaagatga
    tcgttatctctacatggtgatggaatacatgcctggtggagatcttgtaaacttaatgag
    caactatgatgtgcctgaaaaatgggcacgattctatactgcagaagtagttcttgcatt
    ggatgcaatccattccatgggttttattcacagagatgtgaagcctgataacatgctgct
    ggataaatctggacatttgaagttagcagattttggtacttgtatgaagatgaataagga
    aggcatggtacgatgtgatacagcggttggaacacctgattatatttcccctgaagtatt
    aaaatcccaaggtggtgatggttattatggaagagaatgtgactggtggtcggttggg
    gtatttttatacgaaatgcttgtaggtgatacacctttttatgcagattctttggttggaactt
    acagtaaaattatgaaccataaaaattcacttacctttcctgatgataatgacatatcaaa
    agaagcaaaaaaccttatttgtgccttccttactgacagggaagtgaggttagggcga
    aatggtgtagaagaaatcaaacgacatctcttcttcaaaaatgaccagtgggcttggg
    aaacgctccgagacactgtagcaccagttgtacccgatttaagtagtgacattgatact
    agtaattttgatgacttggaagaagataaaggagaggaagaaacattccctattcctaa
    agctttcgttggcaatcaactaccttttgtaggatttacatattatagcaatcgtagatactt
    atcttcagcaaatcctaatgataacagaactagctccaatgcagataaaagcttgcag
    gaaagtttgcaaaaaacaatctataagctggaagaacagctgcataatgaaatgcagt
    taaaagatgaaatggagcagaagtgcagaacctcaaacataaaactagacaagata
    atgaaagaattggatgaagagggaaatcaaagaagaaatctagaatctacagtgtctc
    agattgagaaggagaaaatgttgctacagcatagaattaatgagtaccaaagaaaag
    ctgaacaggaaaatgagaagagaagaaatgtagaaaatgaagtttctacattaaagg
    atcagttggaagacttaaagaaagtcagtcagaattcacagcttgctaatgagaagct
    gtcccagttacaaaagcagctagaagaagccaatgacttacttaggacagaatcgga
    cacagctgtaagattgaggaagagtcacacagagatgagcaagtcaattagtcagtt
    agagtccctgaacagagagttgcaagagagaaatcgaattttagagaattctaagtca
    caaacagacaaagattattaccagctgcaagctatattagaagctgaacgaagagac
    agaggtcatgattctgagatgattggagaccttcaagctcgaattacatctttacaaga
    ggaggtgaagcatctcaaacataatctcgaaaaagtggaaggagaaagaaaagag
    gctcaagacatgcttaatcactcagaaaaggaaaagaataatttagagatagatttaaa
    ctacaaacttaaatcattacaacaacggttagaacaagaggtaaatgaacacaaagta
    accaaagctcgtttaactgacaaacatcaatctattgaagaggcaaagtctgtggcaat
    gtgtgagatggaaaaaaagctgaaagaagaaagagaagctcgagagaaggctgaa
    aatcgggttgttcagattgagaaacagtgttccatgctagacgttgatctgaagcaatct
    cagcagaaactagaacatttgactggaaataaagaaaggatggaggatgaagttaag
    aatctaaccctgcaactggagcaggaatcaaataagcggctgttgttacaaaatgaatt
    gaagactcaagcatttgaggcagacaatttaaaaggtttagaaaagcagatgaaaca
    ggaaataaatactttattggaagcaaagagattattagaatttgagttagctcagcttac
    gaaacagtatagaggaaatgaaggacagatgcgggagctacaagatcagcttgaag
    ctgagcaatatttctcgacactttataaaacccaggtaaaggaacttaaagaagaaatt
    gaagaaaaaaacagagaaaatttaaagaaaatacaggaactacaaaatgaaaaaga
    aactcttgctactcagttggatctagcagaaacaaaagctgagtctgagcagttggcg
    cgaggccttctggaagaacagtattttgaattgacgcaagaaagcaagaaagctgctt
    caagaaatagacaagagattacagataaagatcacactgttagtcggcttgaagaag
    caaacagcatgctaaccaaagatattgaaatattaagaagagagaatgaagagctaa
    cagagaaaatgaagaaggcagaggaagaatataaactggagaaggaggaggaga
    tcagtaatcttaaggctgcctttgaaaagaatatcaacactgaacgaacccttaaaaca
    caggctgttaacaaattggcagaaataatgaatcgaaaagattttaaaattgatagaaa
    gaaagctaatacacaagatttgagaaagaaagaaaaggaaaatcgaaagctgcaac
    tggaactcaaccaagaaagagagaaattcaaccagatggtagtgaaacatcagaag
    gaactgaatgacatgcaagcgcaattggtagaagaatgtgcacataggaatgagctt
    cagatgcagttggccagcaaagagagtgatattgagcaattgcgtgctaaacttttgg
    acctctcggattctacaagtgttgctagttttcctagtgctgatgaaactgatggtaacct
    cccagagtcaagaattgaaggttggctttcagtaccaaatagaggaaatatcaaacga
    tatggctggaagaaacagtatgttgtggtaagcagcaaaaaaattttgttctataatgac
    gaacaagataaggagcaatccaatccatctatggtattggacatagataaactgtttca
    cgttagacctgtaacccaaggagatgtgtatagagctgaaactgaagaaattcctaaa
    atattccagatactatatgcaaatgaaggtgaatgtagaaaagatgtagagatggaac
    cagtacaacaagctgaaaaaactaatttccaaaatcacaaaggccatgagtttattcct
    acactctaccactttcctgccaattgtgatgcctgtgccaaacctctctggcatgtttttaa
    gccaccccctgccctagagtgtcgaagatgccatgttaagtgccacagagatcactta
    gataagaaagaggacttaatttgtccatgtaaagtaagttatgatgtaacatcagcaag
    agatatgctgctgttagcatgttctcaggatgaacaaaaaaaatgggtaactcatttagt
    aaagaaaatccctaagaatccaccatctggttttgttcgtgcttcccctcgaacgctttct
    acaagatccactgcaaatcagtctttccggaaagtggtcaaaaatacatctggaaaaa
    ctagttaaccatgtgactgagtgccctgtggaatcgtgtgggatgctacctgataaacc
    aggcttctttaaccatgcagagcagacaggctgtttctttgacacaaatatcacaggctt
    cagggttaagattgctgtttttctgtccttgctttggcacaacacactgagggttttttttatt
    gcgggtttgcctacaggtagattagattaattattactatgtaatgcaagtacagttggg
    ggaaagcttaggtagatatattttttttaaaaggtgctgcctttttggatttataagaaaat
    gcctgtcagtcgtgatagaacagagttttcctcatatgagtaagaggaagggactttca
    ctttcaagtggaacagccatcactatcaagatcagctcatggaaggagtaaagaaaat
    atctcaaaatgagacaaactgaagttttgttttttttttaatgacttaagtttttgtgctcttgc
    aagactatacaaaactattttaagaaagcagtgatatcacttgaacttcagtgccctcac
    tgtagaatttaaaagccttactgttgattgcccatgttggacttgatggagaaattaaata
    tctttcattatgctttacaaaatactgtatatgtttcagcaagtttggggaatgggagagg
    acaaaaaaaagttacatttaatctatgcatttttgccaagccatattgagttattttactact
    agagacattaggaaactaactgtacaaaagaaccaagtttaaaagcattttgtggggt
    acatcatttctataattgtataatgtatttctttgtggttttaaatgataaagacattaagttaa
    caaacatataagaaatgtatgcactgtttgaaatgtaaattattcttagaacactttcaatg
    ggggttgcattgtccttttagtgccttaatttgagataattattttactgccatgagtaagta
    tagaaatttcaaaaaatgtattttcaaaaaattatgtgtgtcagtgagtttttcattgataatt
    ggtttaatttaaaatatttagaggtttgttggactttcataaattgagtacaatctttgcatca
    aactacctgctacaataatgactttataaaactgcaaaaaatgtagaaggttgcaccaa
    cataaaaaggaaatatggcaatacatccatgatgttttccagttaacataggaattacca
    gataaatactgttaaactcttgtccagtaacaagagttgattcatatggacagtatgattt
    attgtttatttttttaaccaaatacctcctcagtaatttataatggctttgcagtaatgtgtatc
    agataagaagcactggaaaaccgatcgtctctaggatgatatgcatgtttcaagtggta
    ttgaaagccgcactgatggatatgtaataataaacatatctgttattaatatactaatgact
    ctgtgctcatttaatgagaaataaaagtaatttatggatgggtatctttaatttttactgcaa
    tgtgttttctcatggctgaaatgaatggaaaacatacttcaaattagtctctgattgtatat
    aaatgtttgtgaaattccatggttagattaaagtgtatttttaaaagataaaa (SEQ ID
    NO: 667)
    Stk17b gaacggcgatgccccagacgcggctgcagttttcaaaccgcgactgcaagcttcgg NM_004226 NM_133810
    tagtcctctccgctgctgtcgccaggagtcacttcacgagaagccaggtcacaaccgt
    cggcccttgtctggaaaagtaaaagtggatcctgccacgttcggagctccctggcgc
    ctcgcccggctggagctagagaactcgtcctgtggcggcccccggcgtggggcgg
    gacagcggccccctggagggggcagtcccgggagaacctgcggcggccggagc
    ggtaaaaataagtgactaaagaagcagacctgggaatcacctaacatgtcgaggag
    gagatttgattgccgaagtatttcaggcctactaactacaactcctcaaattccaataaa
    aatggaaaactttaataatttctatatacttacatctaaagagctagggagaggaaaattt
    gctgtggttagacaatgtatatcaaaatctactggccaagaatatgctgcaaaatttcta
    aaaaagagaagaagaggacaggattgtcgagcagaaattttacacgagattgctgtg
    cttgaattggcaaagtcttgtccccgtgttattaatcttcatgaggtctatgaaaatacaa
    gtgaaatcattttgatattggaatatgctgcaggtggagaaattttcagcctgtgtttacct
    gagttggctgaaatggtttctgaaaatgatgttatcagactcattaaacaaatacttgaa
    ggagtttattatctacatcagaataacattgtacaccttgatttaaagccacagaatatatt
    actgagcagcatataccctctcggggacattaaaatagtagattttggaatgtctcgaa
    aaatagggcatgcgtgtgaacttcgggaaatcatgggaacaccagaatatttagctcc
    agaaatcctgaactatgatcccattaccacagcaacagatatgtggaatattggtataat
    agcatatatgttgttaactcacacatcaccatttgtgggagaagataatcaagaaacata
    cctcaatatttctcaagttaatgtagattattcggaagaaactttttcatcagtttcacagct
    ggccacagactttattcagagccttttagtaaaaaatccagagaaaagaccaacagca
    gagatatgcctttctcattcttggctacagcagtgggactttgaaaacttgtttcaccctg
    aagaaacttccagttcctctcaaactcaggatcattctgtaaggtcctctgaagacaag
    acttctaaatcctcctgtaatggaacctgtggtgatagagaagacaaagagaatatccc
    agaggatagcagcatggtttccaaaagatttcgtttcgatgactcattacccaatcccc
    atgaacttgtttcagatttgctctgttagcacttttttctttgactcatttggactgaatttgaa
    attttatatccactccagtgagattatgatttgtagcttcatatatgacatgtttatattgtaa
    atgcacttttccatggaataatttagggaagtgttttaatgttaaattactagttgctagcat
    gttatgatttcatatcctgagatagctctgcagataagaaaatatttaaatatatgacaaa
    aagtaaaattgtacatgtgagtttacatgttaatgaaataattcaacttcaaatgaacttac
    cagaatgttttgcatatcaacaaaaaaagtggcttgagttttattatagttggtgtaaact
    gaacacagtgaagacattggaatttaataggttctctctctaaggtgactcttataccat
    gcctctatcaacataatttgtttaggaaagcagtatgaagtttaagccaaaataatttcta
    ctttatagatgctcaagagacattttacaattgaaaatgtctttcaattacaaatattttgaa
    acttcgtaagattttcattctctgtggtctgttatatgagagagatcctttaactagagcaa
    agagggagttagaaacctgatcagggatattctttacaagttggagcagaggaaaga
    gtagcatgccttcgtattttaacgcaaatgtctttttcctcctcccaacctacttgagatct
    gataaggtctggaagatggagatatttggtatgcaagtgtagagttttttaatcctccag
    aatttctagagtagaagatacttaggtatagttaaatattctgtatttttagtcaaacatattt
    attaattgaatatagaagaaaatgttgacacactcagacagcttactgaattttagatgtc
    ttctgcatcttagaatacaagccagtcattcagagttctaaaagtatgcataaaaaattac
    agcaccggtaggtctattaacacagtgcccgagtcagcggtagcaagactgatgtga
    tcataaaacatgacatcaggctcgtctgaagttcttgtgtgaaattcctagtgagtgagg
    aggctcagcttaaagccatctgcagagtggcccctcattgtggtcttttgctgggacca
    atgcaagagactagggagagcaaaatgtttgcttatggctagagactatatccagccc
    taatgatggggaaagttagtccttttcgggtaatcttttatgaattttcacctgatgaccgt
    tatattggtctgttatcatgttacgataactgtgatctcatgaccatgttgctgtatcagaa
    gaaatagtttgacaaatggtaacaacaacctgatgttccccctttagacctttaacttctc
    aaaattttggtaagtttccaaattctttaataataacttaaaactttttgaataactatcaggt
    cactttatttgaccacatggtgaattcctttaatgtcttcagcatttgttaaggaaaagtttt
    ctctacttgtgtgtgtatgtgtgcacatgtgtgtatgtacaggtgtatgtatatatctataga
    tagatacaatacattctttagacacttttcaagattctttgctgtggtatattgtgctcaact
    caggtgccaaaggagctttttttttttttttttttttttgagatggagttttgctctgtctctcag
    gctggagtgcagtggcatgatctcagctcacggcaacctctgcctcccgggttcaag
    caattctcctgtctcagcctcctgagtagttgggattacaggcgcatgccaccgtgccc
    agctaatttttgtatttttagtagagacggggtttcaccatgttggccaggctggtcacaa
    actcctgacttcaagtgatccacccgcctcggcctcccaaagtgctgggattacaggc
    gtgagccactgcgcccccgcccaggagctcttttcttatgacatataaattatgacattt
    atattctttatatgactttatgttctcttcttatgacatttaaattctttaagtagtttgttggtcc
    aataaactagacgttgtataatctaaattgagcccttgtatatctaaaactgatgagttgtt
    tctaaattgttgattgtccatttacttgcctttggtattaagataatgcaagtaaagtttagta
    agtcattggataatgaaatgattatgtttctgaagaccatattatatttttaatttttagagga
    atcatgccatcccccaaaaaatcaagaaatatttgaattttaaattataagttcatttgtta
    aaagacatttttacaaatgtctgaaaatcttaaaatactttacatctacctttaagtagtag
    aatacagagctgtaaatttccatgccttttttcctgatattaagttttatagtaaaaaagca
    actagtgattgcacaaagaatataaaaatccactctttttacaaaggtgtgaatttaaata
    acgttattgattggaatatgaaaatagaccaatcatttaagagctttttagcaaatgattc
    aattcttactctttttctcccaagattgaaaagcataatgtatttctctaaagtaggaatcta
    gagagcccctgtgagtggacaaatgtcagtaacacttgaacacatgagaagataagt
    gttatgttgtgataatttaaagttaaatttgctttttgggtaggatccctaaatagatgggat
    ttttaaatagatgatatatagatgacaattgcaattgtcattttaattattttccctacagtaa
    agaacctagctctgagcagtgaaattgtaatggcactttaaaggaagtaagccgttaa
    ctgttctctagtggagcgatctccaactgttttggcactagggacgggttttgtggaaga
    aaatttttccacaggactgggggtttagggggatggtttcaggatgattcaagtacatta
    catttatcattagattctcataaggagcatgcaacctagatctcttgcacgtgtggttcac
    agcaggattcgagctcctttgagaatctaatgccatggctgatctaacaggaaactga
    gctcaggcagtaatgcttggcaccgccccccaccttctatgcagcccggtcgtggcc
    tggggactggggacccctgctctagtcagtaataaggtacttgtgccagaatataaat
    caacacattgcttcctttatcaaagaagtcttgttatttaaaaaaagtcaactgagccagt
    atgattagtgatgtaattgattttcattctggcacaagcctctttcattctggacagctcac
    aaatagttaatggaccatgctttgaatagccttcctctaagcaacatttataaatactgat
    attttagaactgtttacatttcttctgtttatttttgaattttcagtttgatatcttgtccttattcat
    tgttgtataaacaactgtactttaatttcaagtagtattaaaagtatttcacttcagtttggg
    gggattattatcaatttataattttataaaagtattttaaagaataattgtaaattttccataaa
    ttacaacttcctgccatattttattaaataataatcttgcttaaggcatatagacagacatta
    ttatgagtattccagtaaaaaaaatctacatcaacttgaccattctggctaaaaattaaaa
    agcacttttttatatctgtggttgtcatttgtttcaaagcatttctaaatttattgttcttaaaag
    tatgtctgcatgttctagcctttgacctaggtcatctatgaaccctctttgtgtctaataaac
    atatctgtaaaggcaaaaaaaaaaaaaaaaaa (SEQ ID NO: 668)
    Mast2 taggcaggcggctgagccggcggcgggtggcctgcccaacgtgtgctgggtggg NM_015112 NM_
    agaaggcgaggcgtcagcgatgctgtctcttccgtgaggagcgcagaggaggtcg 001042743
    cggcgccggaggccccagaaggctcgaaggcgccgcgggctggggtcggtggc
    ttagggagcccgtccggccatggtggccgcgggtggtggttggcgcggctgcgctg
    cggcccggggcagtgcggagccgggacagtcgcggcgctgacgcccgcgggcc
    ccagctgcagatatgaagcggagccgctgccgcgaccgaccgcagccgccgccg
    cccgaccgccgggaggatggagttcagcgggcagcggagctgtctcagtctttgcc
    gccgcgccggcgagcgccgcccgggaggcagcggctggaggagcggacgggc
    cccgcggggcccgagggcaaggagcaggatgtagtaactggagttagtcccctgct
    cttcaggaaactcagtaatcctgacatattttcatccactggaaaagttaaacttcagcg
    acaactgagtcaggatgattgtaagttatggagaggaaacctggccagctctctatcg
    ggtaagcagctgctccctttgtccagcagtgtacatagcagtgtgggacaggtgactt
    ggcagtcgtcaggagaagcatcaaacctggttcgaatgagaaaccagtcccttggac
    agtctgcaccttctcttactgctggcctgaaggagttgagccttccaagaagaggcag
    cttttgtcggacaagtaaccgcaagagcttgattgtgacctctagcacatcacctacac
    taccacggccacactcaccactccatggccacacaggtaacagtcctttggacagcc
    cccggaatttctctccaaatgcacctgctcacttttcttttgttcctgcccgtaggactgat
    gggcggcgctggtctttggcctctttgccctcttcaggatatggaactaacactcctag
    ctccactgtctcatcatcatgctcctcacaggaaaagctgcatcagttgcctttccagcc
    tacagctgatgagctgcactttttgacgaagcatttcagcacagagagcgtaccagat
    gaggaaggacggcagtccccagccatgcggcctcgctcccggagcctcagtcccg
    gacgatccccagtatcctttgacagtgaaataataatgatgaatcatgtttacaaagaaa
    gattcccaaaggccaccgcacaaatggaagagcgactagcagagtttatttcctcca
    acactccagacagcgtgctgcccttggcagatggagccctgagctttattcatcatca
    ggtgattgagatggcccgagactgcctggataaatctcggagtggcctcattacatca
    caatacttctacgaacttcaagataatttggagaaacttttacaagatgctcatgagcgc
    tcagagagctcagaagtggcttttgtgatgcagctggtgaaaaagctgatgattatcat
    tgcccgcccagcacgtctcctggaatgcctggagtttgaccctgaagagttctaccac
    cttttagaagcagctgagggccacgccaaagagggacaagggattaaatgtgacatt
    ccccgctacatcgttagccagctgggcctcacccgggatcccctagaagaaatggc
    ccagttgagcagctgtgacagtcctgacactccagagacagatgattctattgagggc
    catggggcatctctgccatctaaaaagacaccctctgaagaggacttcgagaccatta
    agctcatcagcaatggcgcctatggggctgtatttctggtgcggcacaagtccacccg
    gcagcgctttgccatgaagaagatcaacaagcagaacctgatcctacggaaccagat
    ccagcaggccttcgtggagcgtgacatactgactttcgctgagaacccctttgtggtc
    agcatgttctgctcctttgataccaagcgccacttgtgcatggtgatggagtacgttgaa
    gggggagactgtgccactctgctgaagaatattggggccctgcctgtggacatggtg
    cgtctatactttgcggaaactgtgctggccctggagtacttacacaactatggcatcgt
    gcaccgtgacctcaagcctgacaacctcctaattacatccatggggcacatcaagctc
    acggactttggactgtccaaaattggcctcatgagtctgacaacgaacttgtatgagg
    gtcatattgaaaaggatgcccgggaattcctggacaagcaggtatgcgggacccca
    gaatacattgcgcctgaggtgatcctgcgccagggctatgggaagccagtggactg
    gtgggccatgggcattatcctgtatgagttcctggtgggctgcgtccctttttttggagat
    actccggaggagctctttgggcaggtgatcagtgatgagattgtgtggcctgagggt
    gatgaggcactgcccccagacgcccaggacctcacctccaaactgctccaccagaa
    ccctctggagagacttggcacaggcagtgcctatgaggtgaagcagcacccattcttt
    actggtctggactggacaggacttctccgccagaaggctgaatttattcctcagttgga
    gtcagaggatgatactagctattttgacacccgctcagagcgataccaccacatggac
    tcggaggatgaggaagaagtgagtgaggatggctgccttgagatccgccagttctct
    tcctgctctccaaggttcaacaaggtgtacagcagcatggagcggctctcactgctcg
    aggagcgccggacaccacccccgaccaagcgcagcctgagtgaggagaaggag
    gaccattcagatggcctggcagggctcaaaggccgagaccggagctgggtgattg
    gctcccctgagatattacggaagcggctgtcggtgtctgagtcatcccacacagaga
    gtgactcaagccctccaatgacagtgcgacgccgctgctcaggcctcctggatgcgc
    ctcggttcccggagggccctgaggaggccagcagcaccctcaggaggcaaccac
    aggagggtatatgggtcctgacacccccatctggagagggggtatctgggcctgtca
    ctgaacactcaggggagcagcggccaaagctggatgaggaagctgttggccggag
    cagtggttccagtccagctatggagacccgaggccgtgggacctcacagctggctg
    agggagccacagccaaggccatcagtgacctggctgtgcgtagggcccgccaccg
    gctgctctctggggactcaacagagaagcgcactgctcgccctgtcaacaaagtgat
    caagtccgcctcagccacagccctctcactcctcattccttcggaacaccacacctgc
    tccccgttggccagccccatgtccccacattctcagtcgtccaacccatcatcccggg
    actcttctccaagcagggacttcttgccagcccttggcagcatgaggcctcccatcatc
    atccaccgagctggcaagaagtatggcttcaccctgcgggccattcgcgtctacatg
    ggtgactccgatgtctacaccgtgcaccatatggtgtggcacgtggaggatggaggt
    ccggccagtgaggcagggcttcgtcaaggtgacctcatcacccatgtcaatgggga
    acctgtgcatggcctggtgcacacggaggtggtagagctgatcctgaagagtggaa
    acaaggtggccatttcaacaactcccctggagaacacatccattaaagtggggccag
    ctcggaagggcagctacaaggccaagatggcccgaaggagcaagaggagccgc
    ggcaaggatgggcaagaaagcagaaaaaggagctccctgttccgcaagatcacca
    agcaagcatccctgctccacaccagccgcagcctttcttcccttaaccgctccttgtca
    tcaggggagagtgggccaggctctcccacacacagccacagcctttccccccgatc
    tcccactcaaggctaccgggtgacccccgatgctgtgcattcagtgggagggaattc
    atcacagagcagctcccccagctccagcgtgcccagttccccagccggctctgggc
    acacacggcccagctccctccacggtctggcacccaagctccaacgccagtaccgc
    tctccacggcgcaagtcagcaggcagcatcccactgtcaccactggcccacacccc
    ttctcccccacccccaacagcttcacctcagcggtccccatcgcccctgtctggccat
    gtagcccaggcctttcccacaaagcttcacttgtcacctcccctgggcaggcaactct
    cacggcccaagagtgcggagccaccccgttcaccactactcaagagggtgcagtc
    ggctgagaaactggcagcagcacttgccgcctctgagaagaagctagccacttctcg
    caagcacagccttgacctgccccactctgaactaaagaaggaactgccgcccaggg
    aagtgagccctctggaggtagttggagccaggagtgtgctgtctggcaagggggcc
    ctgccagggaagggggtgctgcagcctgctccctcacgggccctaggcaccctcc
    ggcaggaccgagccgaacgacgggagtcgctgcagaagcaagaagccattcgtg
    aggtggactcctcagaggacgacaccgaggaagggcctgagaacagccagggtg
    cacaggagctgagcttggcacctcacccagaagtgagccagagtgtggcccctaaa
    ggagcaggagagagtggggaagaggatcctttcccgtccagagaccctaggagcc
    tgggcccaatggtcccaagcctattgacagggatcacactggggcctcccagaatg
    gaaagtcccagtggtccccacaggaggctcgggagcccacaagccattgaggagg
    ctgccagctcctcctcagcaggccccaacctaggtcagtctggagccacagacccc
    atccctcctgaaggttgctggaaggcccagcacctccacacccaggcactaacagc
    actttctcccagcacttcgggactcacccccaccagcagttgctctcctcccagctcca
    cctctgggaagctgagcatgtggtcctggaaatcccttattgagggcccagacaggg
    catccccaagcagaaaggcaaccatggcaggtgggctagccaacctccaggatttg
    gaaaacacaactccagcccagcctaagaacctgtctcccagggagcaggggaaga
    cacagccacctagtgcccccagactggcccatccatcttatgaggatcccagccagg
    gctggctatgggagtctgagtgtgcacaagcagtgaaagaggatccagccctgagc
    atcacccaagtgcctgatgcctcaggtgacagaaggcaggacgttccatgccgagg
    ctgccccctcacccagaagtctgagcccagcctcaggaggggccaagaaccaggg
    ggccatcaaaagcatcgggatttggcattggttccagatgagcttttaaagcaaacata
    gcagttgtttgccatttcttgcactcagacctgtgtaatatatgctcctggaaaccatcaa
    aaaaaaaaaaaaaaaa (SEQ ID NO: 669)
    Pdp1 agagtgggcaggccgggggtgagggctcgcgctccgggagctgcacggggctgc NM_001161779 NM_
    gtggaaagagcgccgagcggtggcgtcgttgtcgccccctcctcgtcgggaagaat 001098231
    cgtttggtctcctgccgtgcccggttcgtattccctactccctgccacgagccgccccg
    tccgggatcctccacccgtccaaagttgtgagggggcgccgggcgtgctcgcggat
    cggcggccgcgggcgtgcggagggctggacgagccctggagcgccaggagaat
    gtgtgtgtgtcccgggcccagacgaattggaatcccagtcagaagttccagcctgcc
    actgttctctgatgccatgccagcaccaactcaactgttttttcctctcatccgtaactgtg
    aactgagcaggatctatggcactgcatgttactgccaccacaaacatctctgttgttcct
    catcgtacattcctcagagtcgactgagatacacacctcatccagcatatgctacctttt
    gcaggccaaaggagaactggtggcagtacacccaaggaaggagatatgcttccac
    accacagaaattttacctcacacctccacaagtcaatagcatccttaaagctaatgaat
    acagtttcaaagtgccagaatttgacggcaaaaatgtcagttctatccttggatttgaca
    gcaatcagctgcctgcaaatgcacccattgaggaccggagaagtgcagcaacctgc
    ttgcagaccagagggatgcttttgggggtttttgatggccatgcaggttgtgcttgttcc
    caggcagtcagtgaaagactcttttattatattgctgtctctttgttaccccatgagacttt
    gctagagattgaaaatgcagtggagagcggccgggcactgctacccattctccagtg
    gcacaagcaccccaatgattactttagtaaggaggcatccaaattgtactttaacagctt
    gaggacttactggcaagagcttatagacctcaacactggtgagtcgactgatattgat
    gttaaggaggctctaattaatgccttcaagaggcttgataatgacatctccttggaggc
    gcaagttggtgatcctaattcttttctcaactacctggtgcttcgagtggcattttctggag
    ccactgcttgtgtggcccatgtggatggtgttgaccttcatgtggccaatactggcgat
    agcagagccatgctgggtgtgcaggaagaggacggctcatggtcagcagtcacgct
    gtctaatgaccacaatgctcaaaatgaaagagaactagaacggctgaaattggaaca
    tccaaagagtgaggccaagagtgtcgtgaaacaggatcggctgcttggcttgctgat
    gccatttagggcatttggagatgtaaagttcaaatggagcattgaccttcaaaagaga
    gtgatagaatctggcccagaccagttgaatgacaatgaatataccaagtttattcctcct
    aattatcacacacctccttatctcactgctgagccagaggtaacttaccaccgattaag
    gccacaggataagtttctggtgttggctactgatgggttgtgggagactatgcataggc
    aggatgtggttaggattgtgggtgagtacctaactggcatgcatcaccaacagccaat
    agctgttggtggctacaaggtgactctgggacagatgcatggccttttaacagaaagg
    agaaccaaaatgtcctcggtatttgaggatcagaacgcagcaacccatctcattcgcc
    acgctgtgggcaacaacgagtttgggactgttgatcatgagcgcctctctaaaatgctt
    agtcttcctgaagagcttgctcgaatgtacagagatgacattacaatcattgtagttcag
    ttcaattctcatgttgtaggggcgtatcaaaaccaagaatagtgagtggctctttcactg
    gcaattctcaaatgatatacatttaaagggcagattttttaaaaagatactactataataa
    acatttccagttggtcattctaagcatttacccttttgatactctagctagtcaggtactcc
    aaattgactttgcagcagggtggcagggtcaggagagtctggtcctgcctagctcag
    atttcatggcacctgcacttgaagcaagtcacttctttatcacaggtgtcttgaaacatta
    gcttcttttaccaacctgagaaaattaggatgacctggcaaataagatcttgaataggc
    caaaagcaagtatcttgctgtgtgtagtctcttggttaaagtgaagaaacagtactgttc
    acacctttcttcactgagattccagtgtacatgagaacatatatttattgcatgattttctag
    atacacagtctatgcattattcatatacatttattttagcctaaagtggttttcaaatccagtt
    cttcaagccataaatgaccaagatccaagcaatctgaatttgtttttgtgattatttgactg
    gaatgcttcttaagtggaataactatactccgttatccacccgatttcctaatgtaattgaa
    agattttctattttgccacacacttggagacaataagggtttttagttttatctactcttctatt
    gaagttaaagaaagaaaaaaagatttttttatttgtattaatgaaaagctttagtttaaaat
    aaggagatccagaataaaaagaagagactgatctcttcaattattgtcatctgtagcca
    ccagcacatcactcttatgtaatccccaaaggcttggcatgccgtaagtgtgtggtgg
    gtagactgctgccggggaatcgtacttcttatttagtaatgataagacttttcattatttttg
    gaattttaaagatgacataaataagtttaaatatcaatttggggagtaaggtttaatattgc
    catcgggtattgagacaggaggaagtttctgtttttctccatttagacataggtcaattaa
    aatatttgggtttaaaatgactaaatgctttaaacatattgtagcttaagatatatgtgttaa
    gatatatacatgagaaactttaaaaggtaactactgtgcatgcctgatgcttaatagaat
    acttagtggcatcaaatgtttgcagcagtctccataattatattcagtcccttctaatactg
    tatcaatgtaaatgaaataaatatattcaaattggctttttgatatgcatcaagtggcatttt
    gttcctgtgtttaatagtgatctgtatacagctgtgcacatattgtcatcacttattctagca
    tcactgttaaggctgtgattatgtttgatattcacctggattttaatacaagccaatatcag
    cttcccattgtgtaataacttgggtgtttaggagtcttttcacattttttggggatatgaact
    agatgttcaagaactccttctggactgtggatactgaatcagtgtactattggctgcaga
    atttgtttcaattgaaaatagactcaggaagattgctgctcagaatatcatataatgtttatt
    ttttgaggtgtttttgtttttatttgtgtgtttttttttttttaagtcagcttggaacttttttcctgg
    gtagtatttgggagagggaaaggctgtactatatatttatttctaaatgttttgactgggc
    atttttcttttaatgaaatatgtggactgctctagcaaaccctattttcagctactatttgaat
    attcttgaacaccaccactgaagagtttcatatacaccaaataatgtctcatctctatagt
    acagggaatataaaattggtttcctgtggtcatgatcaagatagtagtattattacacaa
    gaaacttggtctgcagtctggaagcttgtctgctctatagaaatgaaaatgcagcatga
    agttgacattgtggaaatgaaagtaattgggtattagaaatctgaaagtactgtcatcta
    aaagcaattgtgattttattgtaattggttgtcactgttgtacggtgtctagaattaaagaa
    tacatgtaaactttcatggtatttagcctttcttaaatttttttaaaatttaaactttctaaccta
    tgtattcaacttctgtatttatatttaatcagtggttcatgttatataatacacccttaactagt
    taaatggaatgttggtatggtacagagtaccatattgctaagaaaactgtcttataaaag
    atgtatatgtgtgaagacatgaaagtttaatgtacagaatggttggagaaatgcctatg
    gtgaattaaagcttcatatctgctttctgaaaaaaaaaaaaaaa (SEQ ID NO:
    670)
    Yes1 ggaggaggtggagagtgaggccgaggcgtggggagcccgggaactccctcctcc NM_005433 NM_009535
    tgaagtaacgcgtcccgggccggctctgccgtcgttgctgccgccgggcgccccgg
    gacgaggaggtggaggagggagagggcccgcgggcctcgcctccgccctccgc
    cacctcgagctgcggtagcagcgactcatgagagcgcggccggaggacagatttg
    ataatgggctgcattaaaagtaaagaaaacaaaagtccagccattaaatacagacctg
    aaaatactccagagcctgtcagtacaagtgtgagccattatggagcagaacccacta
    cagtgtcaccatgtccgtcatcttcagcaaagggaacagcagttaatttcagcagtcttt
    ccatgacaccatttggaggatcctcaggggtaacgccttttggaggtgcatcttcctca
    ttttcagtggtgccaagttcatatcctgctggtttaacaggtggtgttactatatttgtggc
    cttatatgattatgaagctagaactacagaagacctttcatttaagaagggtgaaagatt
    tcaaataattaacaatacggaaggagattggtgggaagcaagatcaatcgctacagg
    aaagaatggttatatcccgagcaattatgtagcgcctgcagattccattcaggcagaa
    gaatggtattttggcaaaatggggagaaaagatgctgaaagattacttttgaatcctgg
    aaatcaacgaggtattttcttagtaagagagagtgaaacaactaaaggtgcttattccct
    ttctattcgtgattgggatgagataaggggtgacaatgtgaaacactacaaaattagga
    aacttgacaatggtggatactatatcacaaccagagcacaatttgatactctgcagaaa
    ttggtgaaacactacacagaacatgctgatggtttatgccacaagttgacaactgtgtg
    tccaactgtgaaacctcagactcaaggtctagcaaaagatgcttgggaaatccctcga
    gaatctttgcgactagaggttaaactaggacaaggatgtttcggcgaagtgtggatgg
    gaacatggaatggaaccacgaaagtagcaatcaaaacactaaaaccaggtacaatg
    atgccagaagctttccttcaagaagctcagataatgaaaaaattaagacatgataaact
    tgttccactatatgctgttgtttctgaagaaccaatttacattgtcactgaatttatgtcaaa
    aggaagcttattagatttccttaaggaaggagatggaaagtatttgaagcttccacagc
    tggttgatatggctgctcagattgctgatggtatggcatatattgaaagaatgaactatat
    tcaccgagatcttcgggctgctaatattcttgtaggagaaaatcttgtgtgcaaaatagc
    agactttggtttagcaaggttaattgaagacaatgaatacacagcaagacaaggtgca
    aaatttccaatcaaatggacagctcctgaagctgcactgtatggtcggtttacaataaa
    gtctgatgtctggtcatttggaattctgcaaacagaactagtaacaaagggccgagtg
    ccatatccaggtatggtgaaccgtgaagtactagaacaagtggagcgaggatacag
    gatgccgtgccctcagggctgtccagaatccctccatgaattgatgaatctgtgttgga
    agaaggaccctgatgaaagaccaacatttgaatatattcagtccttcttggaagactac
    ttcactgctacagagccacagtaccagccaggagaaaatttataattcaagtagcctat
    tttatatgcacaaatctgccaaaatataaagaacttgtgtagattttctacaggaatcaaa
    agaagaaaatcttctttactctgcatgtttttaatggtaaactggaatcccagatatggttg
    cacaaaaccacttttttttccccaagtattaaactctaatgtaccaatgatgaatttatcag
    cgtatttcagggtccaaacaaaatagagctaagatactgatgacagtgtgggtgacag
    catggtaatgaaggacagtgaggctcctgcttatttataaatcatttcctttctttttttccc
    caaagtcagaattgctcaaagaaaattatttattgttacagataaaacttgagagataaa
    aagctataccataataaaatctaaaattaaggaatatcatgggaccaaataattccattc
    cagttttttaaagtttcttgcatttattattctcaaaagttttttctaagttaaacagtcagtat
    gcaatcttaatatatgctttcttttgcatggacatgggccaggtttttcaaaaggaatataa
    acaggatctcaaacttgattaaatgttagaccacagaagtggaatttgaaagtataatg
    cagtacattaatattcatgttcatggaactgaaagaataagaactttttcacttcagtcctt
    ttctgaagagtttgacttagaataatgaaggtaactagaaagtgagttaatcttgtatga
    ggttgcattgattttttaaggcaatatataattgaaactactgtccaatcaaaggggaaat
    gttttgatctttagatagcatgcaaagtaagacccagcattttaaaagccctttttaaaaa
    ctagacttcgtactgtgagtattgcttatatgtccttatggggatgggtgccacaaatag
    aaaatatgaccagatcagggacttgaatgcacttttgctcatggtgaatatagatgaac
    agagaggaaaatgtatttaaaagaaatacgagaaaagaaagtgaaagttttacaagtt
    agagggatggaaggtaatgtttaatgttgatgtcatggagtgacagaatggctttgctg
    gcactcagagctcctcacttagctatattctgagactttgaagagttataaagtataacta
    taaaactaatttttcttacacactaaatgggtatttgttcaaaataatgaagttatggcttca
    cattcattgcagtgggatatggtttttatgtaaaacatttttagaactccagttttcaaatca
    tgtttgaatctacattcacttttttttgttttcttttttgagacggagtctcgctctgtcgccca
    ggctggagtgcagtggcgcgatctcggctcactgcaagctctgcctcccaggttcac
    accattctcctgcctcagcctcccgagtagctgggactacaggtgcccaccaccacg
    cctggctagttttttgtatttttagtagagacgcagtttcaccgtgttagccaggatggtct
    cgatctcctgaccttgtgatctgcccgcctcggcctcccaaagtgctgggattacagg
    cgtgagccaccgcgcccagcctacattcacttctaaagtctatgtaatggtggtcatttt
    ttcccttttagaatacattaaatggttgatttggggaggaaaacttattctgaatattaacg
    gtggtgaaaaggggacagtttttaccctaaagtgcaaaagtgaaacatacaaaataag
    actaatttttaagagtaactcagtaatttcaaaatacagatttgaatagcagcattagtgg
    tttgagtgtctagcaaaggaaaaattgatgaataaaatgaaggtctggtgtatatgtttta
    aaatactctcatatagtcacactttaaattaagccttatattaggcccctctattttcaggat
    ataattcttaactatcattatttacctgattttaatcatcagattcgaaattctgtgccatggc
    atatatgttcaaattcaaaccatttttaaaatgtgaagatggacttcatgcaagttggcag
    tggttctggtactaaaaattgtggttgttttttctgtttacgtaacctgcttagtattgacact
    ctctaccaagagggtcttcctaagaagagtgctgtcattatttcctcttatcaacaacttg
    tgacatgagattttttaagggctttatgtgaactatgatattgtaatttttctaagcatattca
    aaagggtgacaaaattacgtttatgtactaaatctaatcaggaaagtaaggcaggaaa
    agttgatggtattcattaggttttaactgaatggagcagttccttatataataacaattgtat
    agtagggataaaacactaacttaatgtgtattcattttaaattgttctgtatttttaaattgcc
    aagaaaaacaactttgtaaatttggagatattttccaacagcttttcgtcttcagtgtctta
    atgtggaagttaacccttaccaaaaaaggaagttggcaaaaacagccttctagcacac
    ttttttaaatgaataatggtagcctaaacttaatatttttataaagtattgtaatattgttttgtg
    gataattgaaataaaaagttctcattgaatgcacctattaatcgttttagttgctattcatatt
    ctcattcgttttttaaaaactgatatattctgaatttattcttccattgagaaaaaaatgttca
    gttacttgtaactactgagcagaatttaatcaatcctttattaaattcagaacattattgaa
    (SEQ ID NO: 671)
    Met gccctcgccgcccgcggcgccccgagcgctttgtgagcagatgcggagccgagtg NM_001127500 NM_008591
    gagggcgcgagccagatgcggggcgacagctgacttgctgagaggaggcgggg
    aggcgcggagcgcgcgtgtggtccttgcgccgctgacttctccactggttcctgggc
    accgaaagataaacctctcataatgaaggcccccgctgtgcttgcacctggcatcctc
    gtgctcctgtttaccttggtgcagaggagcaatggggagtgtaaagaggcactagca
    aagtccgagatgaatgtgaatatgaagtatcagcttcccaacttcaccgcggaaacac
    ccatccagaatgtcattctacatgagcatcacattttccttggtgccactaactacatttat
    gttttaaatgaggaagaccttcagaaggttgctgagtacaagactgggcctgtgctgg
    aacacccagattgtttcccatgtcaggactgcagcagcaaagccaatttatcaggagg
    tgtttggaaagataacatcaacatggctctagttgtcgacacctactatgatgatcaact
    cattagctgtggcagcgtcaacagagggacctgccagcgacatgtctttccccacaat
    catactgctgacatacagtcggaggttcactgcatattctccccacagatagaagagc
    ccagccagtgtcctgactgtgtggtgagcgccctgggagccaaagtcctttcatctgt
    aaaggaccggttcatcaacttctttgtaggcaataccataaattcttcttatttcccagatc
    atccattgcattcgatatcagtgagaaggctaaaggaaacgaaagatggttttatgttttt
    gacggaccagtcctacattgatgttttacctgagttcagagattcttaccccattaagtat
    gtccatgcctttgaaagcaacaattttatttacttcttgacggtccaaagggaaactctag
    atgctcagacttttcacacaagaataatcaggttctgttccataaactctggattgcattc
    ctacatggaaatgcctctggagtgtattctcacagaaaagagaaaaaagagatccac
    aaagaaggaagtgtttaatatacttcaggctgcgtatgtcagcaagcctggggcccag
    cttgctagacaaataggagccagcctgaatgatgacattcttttcggggtgttcgcaca
    aagcaagccagattctgccgaaccaatggatcgatctgccatgtgtgcattccctatca
    aatatgtcaacgacttcttcaacaagatcgtcaacaaaaacaatgtgagatgtctccag
    catttttacggacccaatcatgagcactgctttaataggacacttctgagaaattcatca
    ggctgtgaagcgcgccgtgatgaatatcgaacagagtttaccacagctttgcagcgc
    gttgacttattcatgggtcaattcagcgaagtcctcttaacatctatatccaccttcattaa
    aggagacctcaccatagctaatcttgggacatcagagggtcgcttcatgcaggttgtg
    gtttctcgatcaggaccatcaacccctcatgtgaattttctcctggactcccatccagtgt
    ctccagaagtgattgtggagcatacattaaaccaaaatggctacacactggttatcact
    gggaagaagatcacgaagatcccattgaatggcttgggctgcagacatttccagtcct
    gcagtcaatgcctctctgccccaccctttgttcagtgtggctggtgccacgacaaatgt
    gtgcgatcggaggaatgcctgagcgggacatggactcaacagatctgtctgcctgca
    atctacaaggttttcccaaatagtgcaccccttgaaggagggacaaggctgaccatat
    gtggctgggactttggatttcgg
    aggaataataaatttgatttaaagaaaactagagttctccttggaaatgagagctgcac
    cttgactttaagtgagagcacgatgaatacattgaaatgcacagttggtcctgccatga
    ataagcatttcaatatgtccataattatttcaaatggccacgggacaacacaatacagta
    cattctcctatgtggatcctgtaataacaagtatttcgccgaaatacggtcctatggctg
    gtggcactttacttactttaactggaaattacctaaacagtgggaattctagacacatttc
    aattggtggaaaaacatgtactttaaaaagtgtgtcaaacagtattcttgaatgttatacc
    ccagcccaaaccatttcaactgagtttgctgttaaattgaaaattgacttagccaaccga
    gagacaagcatcttcagttaccgtgaagatcccattgtctatgaaattcatccaaccaa
    atcttttattagtacttggtggaaagaacctctcaacattgtcagttttctattttgctttgcc
    agtggtgggagcacaataacaggtgttgggaaaaacctgaattcagttagtgtcccg
    agaatggtcataaatgtgcatgaagcaggaaggaactttacagtggcatgtcaacatc
    gctctaattcagagataatctgttgtaccactccttccctgcaacagctgaatctgcaac
    tccccctgaaaaccaaagcctttttcatgttagatgggatcctttccaaatactttgatctc
    atttatgtacataatcctgtgtttaagccttttgaaaagccagtgatgatctcaatgggca
    atgaaaatgtactggaaattaagggaaatgatattgaccctgaagcagttaaaggtga
    agtgttaaaagttggaaataagagctgtgagaatatacacttacattctgaagccgtttt
    atgcacggtccccaatgacctgctgaaattgaacagcgagctaaatatagagtggaa
    gcaagcaatttcttcaaccgtccttggaaaagtaatagttcaaccagatcagaatttcac
    aggattgattgctggtgttgtctcaatatcaacagcactgttattactacttgggtttttcct
    gtggctgaaaaagagaaagcaaattaaagatctgggcagtgaattagttcgctacgat
    gcaagagtacacactcctcatttggataggcttgtaagtgcccgaagtgtaagcccaa
    ctacagaaatggtttcaaatgaatctgtagactaccgagctacttttccagaagatcagt
    ttcctaattcatctcagaacggttcatgccgacaagtgcagtatcctctgacagacatgt
    cccccatcctaactagtggggactctgatatatccagtccattactgcaaaatactgtcc
    acattgacctcagtgctctaaatccagagctggtccaggcagtgcagcatgtagtgatt
    gggcccagtagcctgattgtgcatttcaatgaagtcataggaagagggcattttggttg
    tgtatatcatgggactttgttggacaatgatggcaagaaaattcactgtgctgtgaaatc
    cttgaacagaatcactgacataggagaagtttcccaatttctgaccgagggaatcatca
    tgaaagattttagtcatcccaatgtcctctcgctcctgggaatctgcctgcgaagtgaa
    gggtctccgctggtggtcctaccatacatgaaacatggagatcttcgaaatttcattcg
    aaatgagactcataatccaactgtaaaagatcttattggctttggtcttcaagtagccaa
    aggcatgaaatatcttgcaagcaaaaagtttgtccacagagacttggctgcaagaaac
    tgtatgctggatgaaaaattcacagtcaaggttgctgattttggtcttgccagagacatg
    tatgataaagaatactatagtgtacacaacaaaacaggtgcaaagctgccagtgaagt
    ggatggctttggaaagtctgcaaactcaaaagtttaccaccaagtcagatgtgtggtcc
    tttggcgtgctcctctgggagctgatgacaagaggagccccaccttatcctgacgtaa
    acacctttgatataactgtttacttgttgcaagggagaagactcctacaacccgaatact
    gcccagaccccttatatgaagtaatgctaaaatgctggcaccctaaagccgaaatgc
    gcccatccttttctgaactggtgtcccggatatcagcgatcttctctactttcattgggga
    gcactatgtccatgtgaacgctacttatgtgaacgtaaaatgtgtcgctccgtatccttct
    ctgttgtcatcagaagataacgctgatgatgaggtggacacacgaccagcctccttct
    gggagacatcatagtgctagtactatgtcaaagcaacagtccacactttgtccaatggt
    tttttcactgcctgacctttaaaaggccatcgatattctttgctcttgccaaaattgcactat
    tataggacttgtattgttatttaaattactggattctaaggaatttcttatctgacagagcat
    cagaaccagaggcttggtcccacaggccacggaccaatggcctgcagccgtgaca
    acactcctgtcatattggagtccaaaacttgaattctgggttgaattttttaaaaatcaggt
    accacttgatttcatatgggaaattgaagcaggaaatattgagggcttcttgatcacag
    aaaactcagaagagatagtaatgctcaggacaggagcggcagccccagaacaggc
    cactcatttagaattctagtgtttcaaaacacttttgtgtgttgtatggtcaataacatttttc
    attactgatggtgtcattcacccattaggtaaacattcccttttaaatgtttgtttgttttttga
    gacaggatctcactctgttgccagggctgtagtgcagtggtgtgatcatagctcactgc
    aacctccacctcccaggctcaagcctcccgaatagctgggactacaggcgcacacc
    accatccccggctaatttttgtattttttgtagagacggggttttgccatgttgccaaggct
    ggtttcaaactcctggactcaagaaatccacccacctcagcctcccaaagtgctagga
    ttacaggcatgagccactgcgcccagcccttataaatttttgtatagacattcctttggtt
    ggaagaatatttataggcaatacagtcaaagtttcaaaatagcatcacacaaaacatgt
    ttataaatgaacaggatgtaatgtacatagatgacattaagaaaatttgtatgaaataatt
    tagtcatcatgaaatatttagttgtcatataaaaacccactgtttgagaatgatgctactct
    gatctaatgaatgtgaacatgtagatgttttgtgtgtatttttttaaatgaaaactcaaaata
    agacaagtaatttgttgataaatatttttaaagataactcagcatgtttgtaaagcaggat
    acattttactaaaaggttcattggttccaatcacagctcataggtagagcaaagaaagg
    gtggatggattgaaaagattagcctctgtctcggtggcaggttcccacctcgcaagca
    attggaaacaaaacttttggggagttttattttgcattagggtgtgttttatgttaagcaaa
    acatactttagaaacaaatgaaaaaggcaattgaaaatcccagctatttcacctagatg
    gaatagccaccctgagcagaactttgtgatgcttcattctgtggaattttgtgcttgctac
    tgtatagtgcatgtggtgtaggttactctaactggttttgtcgacgtaaacatttaaagtgt
    tatattttttataaaaatgtttatttttaatgatatgagaaaaattttgttaggccacaaaaac
    actgcactgtgaacattttagaaaaggtatgtcagactgggattaatgacagcatgattt
    tcaatgactgtaaattgcgataaggaaatgtactgattgccaatacaccccaccctcatt
    acatcatcaggacttgaagccaagggttaacccagcaagctacaaagagggtgtgtc
    acactgaaactcaatagttgagtttggctgttgttgcaggaaaatgattataactaaaag
    ctctctgatagtgcagagacttaccagaagacacaaggaattgtactgaagagctatt
    acaatccaaatattgccgtttcataaatgtaataagtaatactaattcacagagtattgta
    aatggtggatgacaaaagaaaatctgctctgtggaaagaaagaactgtctctaccag
    ggtcaagagcatgaacgcatcaatagaaagaactcggggaaacatcccatcaacag
    gactacacacttgtatatacattcttgagaacactgcaatgtgaaaatcacgtttgctattt
    ataaacttgtccttagattaatgtgtctggacagattgtgggagtaagtgattcttctaag
    aattagatacttgtcactgcctatacctgcagctgaactgaatggtacttcgtatgttaat
    agttgttctgataaatcatgcaattaaagtaaagtgatgcaacatcttgtaaaaaaaaaa
    aaaaaaaaaa (SEQ ID NO: 672)
    Ppm1g agttgctaaggaaatgactgcccgcagcgcctggccccgccgcgcaggccgggcg NM_177983 NM_008014
    gggtctggagcggcgccgtttccgcttccgctccctcacagctcccgtcccgttaccg
    cctcctggccggcctcgcgcctttcaccggcaccttgcgtcggtcgcgccgcgggg
    cctgctcctgccgcgcgcacccccggggcttcggctccggcacgggtcgcgccca
    gctttcctgcacctgaggccgccggccagccgccgccatgggtgcctacctctccca
    gcccaacacggtgaagtgctccggggacggggtcggcgccccgcgcctgccgct
    gccctacggcttctccgccatgcaaggctggcgcgtctccatggaggatgctcacaa
    ctgtattcctgagctggacagtgagacagccatgttttctgtctacgatggacatggag
    gggaggaagttgccttgtactgtgccaaatatcttcctgatatcatcaaagatcagaag
    gcctacaaggaaggcaagctacagaaggctttagaagatgccttcttggctattgacg
    ccaaattgaccactgaagaagtcattaaagagctggcacagattgcagggcgaccc
    actgaggatgaagatgaaaaagaaaaagtagctgatgaagatgatgtggacaatga
    ggaggctgcactgctgcatgaagaggctaccatgactattgaagagctgctgacacg
    ctacgggcagaactgtcacaagggccctccccacagcaaatctggaggtgggaca
    ggcgaggaaccagggtcccagggcctcaatggggaggcaggacctgaggactca
    actagggaaactccttcacaagaaaatggccccacagccaaggcctacacaggcttt
    tcctccaactcggaacgtgggactgaggcaggccaagttggtgagcctggcattccc
    actggtgaggctgggccttcctgctcttcagcctctgacaagctgcctcgagttgctaa
    gtccaagttctttgaggacagtgaggatgagtcagatgaggcggaggaagaagagg
    aagacagtgaggaatgcagcgaggaagaggatggctacagcagtgaggaggcag
    agaatgaggaagatgaggatgacaccgaggaggctgaagaggacgatgaagaag
    aagaagaagagatgatggtgccagggatggaaggcaaagaggagcctggctctga
    cagtggtacaacagcggtggtggccctgatacgagggaagcagttgattgtagcca
    acgcaggagactctcgctgtgtggtatctgaggctggcaaagctttagacatgtcctat
    gatcacaaaccagaggatgaagtagaactagcacgcatcaagaatgctggtggcaa
    ggtcaccatggatgggcgagtcaacgggggcctcaacctctccagagccattgggg
    accacttctataagagaaacaagaacctgccacctgaggaacagatgatttcagccct
    tcctgacatcaaggtgctgactctcactgacgaccatgaattcatggtcattgcctgtga
    tggcatctggaatgtgatgagcagccaggaagttgtagatttcattcaatcaaagatca
    gccagcgtgatgaaaatggggagcttcggttattgtcatccattgtggaagagctgct
    ggatcagtgcctggcaccagacacttctggggatggtacagggtgtgacaacatgac
    ctgcatcatcatttgcttcaagccccgaaacacagcagagctccagccagagagtgg
    caagcgaaaactagaggaggtgctctctactgagggggctgaagaaaatggcaaca
    gcgacaagaagaagaaggccaagcgagactagcagtcatccagacccctgcccac
    ctagactgttttctgagccctccggacctgagactgagttttgtctttttcctttagccttag
    cagtgggtatgaggtgtgcagggggagctgggtggcttcactccgcccattccaaag
    agggctctccctccacactgcagccgggagcctctgctgtccttcccagccgcctctg
    ctcctcgggctcatcaccggttctgtgcctgtgctctgttgtgttggagggaaggactg
    gcggttctggtttttactctgtgaactttatttaaggacattcttttttattggcggctccatg
    gccctcggccgcttgcacccgctctctgttgtacactttcaatcaacactttttcagacta
    aaggccaaaacctaa (SEQ ID NO: 673)
    Blvrb Ggcgtggcccttcgagccagctccgccccgttgttcctggcttgagtagggcagag NM_000713 NM_144923
    agcaccgcccagcagccagtgggttcccgcgcgtgccgagactctgaggccttgca
    cccccacgatcccgtacgatggccgtcaagaagatcgcgatcttcggcgccactgg
    ccagaccgggctcaccaccctggcgcaggcggtgcaagcaggttacgaagtgaca
    gtgctggtgcgggactcctccaggctgccatcagaggggccccggccggcccacg
    tggtagtgggagatgttctgcaggcagccgatgtggacaagaccgtggctgggcag
    gacgctgtcatcgtgctgctgggcacccgcaatgacctcagtcccacgacagtgatg
    tccgagggcgcccggaacattgtggcagccatgaaggctcatggtgtggacaaggt
    cgtggcctgcacctcggctttcctgctctgggaccctaccaaggtgcccccacgact
    gcaggctgtgactgatgaccacatccggatgcacaaggtgctgcgggaatcaggcc
    tgaagtacgtggctgtgatgccgccacacataggagaccagccactaactggggcg
    tacacagtgaccctggatggacgagggccctcaagggtcatctccaaacatgacctg
    ggccatttcatgctgcgctgcctcaccaccgatgagtacgacggacacagcacctac
    ccctcccaccagtaccagtagcactctgtccccatctgggagggtggcattctggga
    catgaggagcaaaggaagggggcaataaatgttgagccaagagcttcaaattactct
    agagaaaccgacaaaaaaaaaaaaaaaaaa (SEQ ID NO: 674)
    Tnk1 ggaactcggggtgcggccctcgccggccccgggccagcggccaggtccccgccc NM_001251902 NM_031880
    tccgcgggatttactcctgtcccgcctcctcggatttagcccaggcagcctgggaggt
    tccgcagtcgccgcttccgccttgaccaggtggagctggagacctggtctctctagg
    gcctaccctgagctcaccatctgaaggagagtgccatcatccttaggaactccttctcc
    agacatgcttcctgaggctggctccctgtggctactgaagctgctccgggacatccag
    ttggcccagttttactggcccatccttgaggagcttaatgtcactcggccagagcactt
    cgactttgtaaagcctgaggacctggacggcattggcatgggccggcctgcccagc
    gcagactgtccgaagctctgaaaaggctacgttctgggcctaagtctaagaactgggt
    ctacaagatccttggaggttttgcccctgagcacaaggagcccaccctgccctcgga
    cagcccacggcacctccctgagccagaggggggcctcaagtgtctgatcccagag
    ggtgctgtttgcagaggggagctgctgggttcaggctgcttcggtgtggtgcaccga
    gggctgtggacgctgcccagtggcaagagtgtcccagtggctgtcaagtccctccg
    ggtaggtcccgaaggcccgatgggcacagaactgggggacttcctgcgagaggta
    tcggtcatgatgaacttggagcacccacacgtgctgcgtctgcacggccttgtactgg
    gccagcctctgcagatggtgatggagctggcgccactgggctccctgcacgcgcgc
    ctaacggccccggccccgacacccccgctgctcgtggccctgctctgcctcttcctg
    cggcagctggcgggagccatggcgtacctgggggcccgcgggctggtgcaccga
    gacctcgctacgcgcaacctactgctggcgtcgccgcgcaccatcaaggtggctga
    cttcgggctggtgcggcctctgggcggtgcccggggccgctacgtcatgggcggg
    ccccgccctatcccctacgcctggtgtgccccagagagcctgcgccacggagcctt
    ctcgtctgcctcggacgtgtggatgtttggggtgacgctgtgggagatgttctccggg
    ggcgaggaaccctgggccggggtcccaccgtacctcatcctgcagcggctggagg
    acagagcccggctgcctaggcctcccctctgctccagggccctctactccctcgcctt
    gcgctgctgggccccccaccctgccgaccggcctagcttttcccacctggaggggc
    tgctgcaagaggccgggccttcggaagcatgttgtgtgagggatgtcacagaacca
    ggcgccctgaggatggagactggtgaccccatcacagtcatcgagggcagctcctc
    tttccacagccccgactccacaatctggaagggccagaatggtcgcaccttcaaagt
    gggcagcttcccagcctcggcagtgacgctggcagatgcggggggcttgccagcc
    acccgtccagtccacagaggcacccctgcccggggagatcaacacccaggaagca
    tagatggagacagaaagaaggcaaatctttgggatgcgcccccagcacggggcca
    gaggaggaacatgcccctggagaggatgaaaggcatttccaggagtctggagtcag
    ttctgtccctcggtcctcgtcccacagggggtggttcaagcccccctgaaattcgaca
    agccagagctgtgccccagggacctccaggcctgcctccacgcccacctttatcctc
    tagctctcctcagcccagccagccctctagggagaggcttccctggcccaaaagaaa
    acccccacacaatcaccccatgggaatgcctggagcccgtaaagccgctgccctct
    ctggaggcctcttgtccgatcctgagttgcagaggaagattatggaggtggagctga
    gtgtgcatggggtcacccaccaggagtgccagacagcactaggagccactggggg
    agatgtggtttctgccatccggaacctcaaggtagatcagctcttccacctgagtagcc
    ggtccagagctgactgctggcgcatcctggagcattaccagtgggacctctcagctg
    ccagccgctatgtcctggccaggccctgagctcagcttctgcgggcacagacacca
    gcatgaaaagcctaggcccctgagggcctggccacatgggaccaagcggaacca
    gaacaaggtcccgacaggggtagacgttccacctggggagatcccacctgccgtag
    gcacatggaggaggagcccagagttgggcactggcaaatgtctcctccctcccatg
    ctccttggcttctgaaggctgaagctcctttggctgggccaagaaggatctagtctgcc
    cactacattctcaaacaagaggacttggaggaaaagagctgctatacatcatatgcag
    aggaagcttctacgcgctagagaggatcaaggggccacactggaccatgtgaacag
    ccatcctgaactgccatcagctaccacactggactctgcagggcagccatcctggat
    gatggaagccaccatattgacttggggtataggcccaaactgccttcgtttggtccag
    ggccatcgtgggtgatgacgattgctctcttgcactcaaggacatttgatgctggtagt
    atggattatgagatggactagcccctgccccagcccagctctcacattcccctttgttttt
    tcccataccaactgcttctaccctcccctattacatacatctttcaatgtccaaaaagttac
    aaagtttatatgaatgtaacatataaaaaaa (SEQ ID NO: 675)
    Prkab2 actgggcggactccgcgccgccggccttgtagccattttaggaggaatcgctggtcg NM_005399 NM_182997
    ccagcgaggggtgcggcttcaatttcaataactttattggtggcctgatctgcagaaca
    gccatcacatcagtggcccttggaggagggagcgcatcgcccgaggtggtccccg
    acgagctgcagccatgggaaacaccaccagcgaccgggtgtccggggagcgcca
    cggcgccaaggctgcacgctccgagggcgcaggcggccatgccccggggaagg
    agcacaagatcatggtggggagtacggacgaccccagcgtgttcagcctccctgac
    tccaagctccctggggacaaagagtttgtatcatggcagcaggatttggaggactcc
    gtaaagcccacacagcaggcccggcccactgttatccgctggtctgaaggaggcaa
    ggaggtcttcatctctgggtccttcaacaattggagcaccaagattccactgattaaga
    gccataatgactttgttgccatcctggacctccctgagggagagcaccaatacaagtt
    ctttgtggatggacagtgggttcatgatccatcagagcctgtggttaccagtcagcttg
    gcacaattaacaatttgatccatgtcaagaaatctgattttgaggtgttcgatgctttaaa
    gttagattctatggaaagttctgagacatcttgtagagacctttccagctcacccccagg
    gccttatggtcaagaaatgtatgcgtttcgatctgaggaaagattcaaatccccaccca
    tccttcctcctcatctacttcaagttattcttaacaaagacactaatatttcttgtgacccag
    ccttactccctgagcccaaccatgttatgctgaaccatctctatgcattgtccattaagg
    acagtgtgatggtccttagcgcaacccatcgctacaagaagaagtatgttactactctg
    ctatacaagcccatttgaagggatcccttcttgcctctaaggattcaggagaagcatct
    cccttgcatttctggactgaaccagtcttacctgagactggaaggctgatttgctttgag
    gctgatatgtgtgtttcagagcctctgagtaggatgctctgcttttgcatttgattgcagat
    gagagctttatgagttcacggaatttattttaagaaaaaaaaatatacatatgagaagaa
    ggtaaatggaagcctcctagccccagctagaagtattgtttctgcctgtgggttttcacc
    aagacctgtttgggggcgctgcaggaataactatataggaagatttttcctaaaatgaa
    agaacagcaaactcttaggatccttgttgggtggagattctatcactgctaccttggctc
    tccaaggaatgggcttgtgctagaccgctgccctacttaacagctgcctcattgcaag
    ggcagtttttcttgcatgggttctctatattcccagagtatgtggcacaatctgtgttgttta
    tatgataccagatgccccacaagaacccttattcctctcatttcacattcttcctttaatag
    cctccttcagatcccatacctgacccctctctaacacaaaacttattgggtaagtgacttt
    gaaaagttttgtggcacctgacccaccccagacactagggctatcagaaggtctcctt
    tttagcccagcacaggcccaggccactttgtcgtgtttgttttaacttctaaagaaaatat
    gtttcagcattataagaaaggcagaatgcagaacacctacatttttgttttagtttggtgc
    caaggctcaggctgtattggcaaattcccgaaagttttcccactttgcctggccctgctt
    ctgtcttttctttctcagtaaacagttctgaaggcaggagtggaacccgggagtattttc
    atgtctttcatccttgaaagatttttatgtgcctgcattttttttttaattaaaaaatgccttttc
    attggtcttaagagaccgcattggagaatttcaggcttttgataaatgcttcttcaaagag
    attttcttctctagtctagccttccacattcttagattaatatggccaaccctgtacacatca
    ctacactaaacactgctctagataaactgctcaagttcatttaactcatttgatgcaccta
    aaggggttcctcattttaaagatttgttaggccaagaagcaagagagtattcctagtatt
    cccaaccatgaaaagtatcattctttgcaccaaatgttaacaaaatcattttgttctcctgc
    ctcttctttttaaaggtgtttgatgattaagtggggtcactgaattccatttgtggactgaa
    aagtattcaatccacttttggggttcagagataaaacattttttcccaagtagctggggct
    cttccattttgcagataagtcaaataatcaacactaaaggaggctaaactgttgatgaat
    gagagactccctgactgctcagatgaccctagccacactgaaagggcacctacagg
    tcagtttagctacctcctgtctttcccatgcaaagctgataacacagttgtctttggacttg
    tagacctcttggattccaggtgtgatggagtaaagtgtgggattgttgttttgctgggat
    gcaaataactaaatgctttggtggttaattgctaagagtaaatactactttagccatccaa
    ggccaccttctgcagcaaaaggcttttgtggagaaccttttatgttcccaaccactttttg
    aatggtgtgccatttaaaaatccaggccagatcctattataaccaactctcaggatttac
    agccttcagttgtactagaattttgtttttatccaatactcattaaataagtgggccacttag
    gaagattcaaaatcttggttattacatgaagtttgttatatttcttgtcaacagtattgaaat
    gtaatatgtatgtgttcatgtatgaaaatttttactccacacaggtgtttcagtagagtggg
    gcaggaaaagagatctcttcgatttctttcaggcctgaggcttttgtgaaatgcgtcagc
    cccctgtgacagtaggttttgatgctagtgatcttcagatctttctctctggaaatgtgca
    gagagtgtcagtttcccaagttctgaggtaactctcagcccagatgtgaaatgggagc
    ctaccagctggtatagaagggaatgggtaggaggcactgggtgctgactcattcagc
    actgtcccttttctatactgctgatacatcccatggttctgagaagccttatctcagtctatt
    tggaagagagggaggaagagaaggaagtaacccaaagtactactcatttatcattgt
    atattgattagttaaagggataattaatttaatgctgaggagagtttgacagattttgaaa
    atgagtaaaggcaaaaaaaatttttttagcctttattttgcttttgggaattttacagagtca
    aagtaggcagaataagaaaatagttcttcaggagggccgacctttaaagaacttcaac
    atagtttcggaattgtggggaagagaagagtgactgagctgagaagtaataatagaat
    aaagggttgagtaacttacaactgaaaatgatctcttttaaaaagaaattaaatcagaca
    ccacatggtggtgtccttggatctcactgtacagaattagcagtgtataaccatcttctct
    tttcatcttgttccaattctctcctctttcctttccattctgctttaagctcatgtgtcaggcag
    actttaccagagtgtcagacattacctaaaacacatacgttagccatgctgctggtatg
    gagaaattccacaccatgattattagcctcctttaagctgaatgggatttaaccattctag
    gcaacacccctgaagggcatacctaacctcaatagtgttggcttttaaaacgtatgtttg
    tatggtagagaaactttgtaaaagaagaatccaagagaagtttgtgaggatcctacaa
    acccaggcccactcactttgctctaattctttctagtatcttgtagatctaatgggtctggg
    ataaaaactttgaaaagtgtcaatattccatgtatgctgctgaaatgaagttaagtttgga
    aagaagtgatacctctagactgggtttatattaatctgggatataaatgaagaagacata
    ctaatagaactccttgcttttaattggggaaatagggctttaataattttgacctcaactaa
    aaatgatatgcaatagtctctgtgtgtgtttgaaatacattgtgttctcagagatttctacat
    tctcacgttctagtgatttggggcatgggcttaatagcagatgtacagtgtattcctgcat
    tattgtgattccccttaaagcccagttcttgctgtcttctaccaggggctgctgactccag
    ttacccatggaatgcaggacctgggaggggtagccattagggtctttcaaaactctttg
    gatctaagcatttgtctctccttaagtgccaatcacaattggatatggaaggactgtgatt
    tctgcaatgaacccaaacttttagagtaaaaagccaaatttaaattataagaaagaagg
    gaaaaaagagaaaaactcaagtctattacttgtagagtccaattcttagcaatggaatc
    gctctaggattctagtttgggctttgtctggatttgcttttctcagttgtgctttgaagtgaat
    aagctttgttacaaattaattttttattagttccaatattagttggagttaacttgaattgattg
    tatgtagcacagcacttttgcagtaagattggtgtgaaatactaaacactatggattttgt
    aggtgtcaggttaaatggtcaagggatacctacattaagtcatatattaggtattgatga
    tcttacttcttttctgttcccctgtacaaaacacttacctaacccagcttgtggttttaggac
    agccaaagctcactgttgttggttagtcctaatcactacacgggtctcataaatgagact
    tgtttgaattttggtacattggagcatgttggttggtattacacggcagcatttcgaatga
    gtgcagctctgtgtctgtcagaaaggagagataagactactttgaagggaattaaatat
    gtgagtcctctttttaatggtgctttttgtaacctttaatgctgaggtacagagctgcttttc
    aatatttcataaaggagtggcagacaagagtggattttaaagctgttcttcaaacgtaat
    ttgtcactggactctgacacacctggaaattatatgatatgatacatacagaaatgttgt
    gggttttttccataaaactttaataaaagtattatacagcaataaaaaaaaaaaaaaa
    (SEQ ID NO: 676)
    Trpm7 gcgccgctcacgtggtccgtccccagccccgtcgccggcggaggcgggcgcggg NM_017672 NM_021450
    cgcgtccctgtggccagtcacccggaggagttggtcgcacaattatgaaagactcgg
    cttctgctgctagcgccggagctgagttagttctgagaaggtttccctgggcgttccttg
    tccggcggcctctgctgccgcctccggagacgcttcccgatagatggctacaggcc
    gcggaggaggaggaggtggagttgctgcccttccggagtccgccccgtgaggaga
    atgtcccagaaatcctggatagaaagcactttgaccaagagggaatgtgtatatattat
    accaagttccaaggaccctcacagatgccttccaggatgtcaaatttgtcagcaactc
    gtcaggtgtttttgtggtcgcttggtcaagcaacatgcttgttttactgcaagtcttgccat
    gaaatactcagatgtgaaattgggtgaccattttaatcaggcaatagaagaatggtctg
    tggaaaagcatacagaacagagcccaacggatgcttatggagtcataaattttcaagg
    gggttctcattcctacagagctaagtatgtgaggctatcatatgacaccaaacctgaag
    tcattctgcaacttctgcttaaagaatggcaaatggagttacccaaacttgttatctctgt
    acatgggggcatgcagaaatttgagcttcacccacgaatcaagcagttgcttggaaa
    aggtcttattaaagctgcagttacaactggagcctggattttaactggaggagtaaaca
    caggtgtggcaaaacatgttggagatgccctcaaagaacatgcttccagatcatctcg
    aaagatttgcactatcggaatagctccatggggagtgattgaaaacagaaatgatcttg
    ttgggagagatgtggttgctccttatcaaaccttattgaaccccctgagcaaattgaatg
    ttttgaataatctgcattcccatttcatattggtggatgatggcactgttggaaagtatgg
    ggcggaagtcagactgagaagagaacttgaaaaaactattaatcagcaaagaattca
    tgctaggattggccagggtgtccctgtggtggcacttatatttgagggtgggccaaat
    gttatcctcacagttcttgaataccttcaggaaagcccccctgttccagtagttgtgtgtg
    aaggaacaggcagagctgcagatctgctagcgtatattcataaacaaacagaagaa
    ggagggaatcttcctgatgcagcagagcccgatattatttccactatcaaaaaaacatt
    taactttggccagaatgaagcacttcatttatttcaaacactgatggagtgcatgaaaag
    aaaggagcttatcactgttttccatattgggtcagatgaacatcaagatatagatgtagc
    aatacttactgcactgctaaaaggtactaatgcatctgcatttgaccagcttatccttaca
    ttggcatgggatagagttgacattgccaaaaatcatgtatttgtttatggacagcagtgg
    ctggttggatccttggaacaagctatgcttgatgctcttgtaatggatagagttgcatttg
    taaaacttcttattgaaaatggagtaagcatgcataaattccttaccattccgagactgg
    aagaactttacaacactaaacaaggtccaactaatccaatgctgtttcatcttgttcgag
    acgtcaaacagggaaatcttcctccaggatataagatcactctgattgatataggactt
    gttattgaatatctcatgggaggaacctacagatgcacctatactaggaaacgttttcga
    ttaatatataatagtcttggtggaaataatcggaggtctggccgaaatacctccagcag
    cactcctcagttgcgaaagagtcatgaatcttttggcaatagggcagataaaaaggaa
    aaaatgaggcataaccatttcattaagacagcacagccctaccgaccaaagattgata
    cagttatggaagaaggaaagaagaaaagaaccaaagatgaaattgtagacattgatg
    atccagaaaccaagcgctttccttatccacttaatgaacttttaatttgggcttgccttatg
    aagaggcaggtcatggcccgttttttatggcaacatggtgaagaatcaatggctaaag
    cattagttgcctgtaagatctatcgttcaatggcatatgaagcaaagcagagtgacctg
    gtagatgatacttcagaagaactaaaacagtattccaatgattttggtcagttggccgtt
    gaattattagaacagtccttcagacaagatgaaaccatggctatgaaattgctcacttat
    gaactgaagaactggagtaattcaacctgccttaagttagcagtttcttcaagacttaga
    ccttttgtagctcacacctgtacacaaatgttgttatctgatatgtggatgggaaggctg
    aatatgaggaaaaattcctggtacaaggtcatactaagcattttagttccacctgccata
    ttgctgttagagtataaaactaaggctgaaatgtcccatatcccacaatctcaagatgct
    catcagatgacaatggatgacagcgaaaacaactttcagaacataacagaagagatc
    cccatggaagtgtttaaagaagtacggattttggatagtaatgaaggaaagaatgaga
    tggagatacaaatgaaatcaaaaaagcttccaattacgcgaaagttttatgccttttatc
    atgcaccaattgtaaaattctggtttaacacgttggcatatttaggatttctgatgctttata
    catttgtggttcttgtacaaatggaacagttaccttcagttcaagaatggattgttattgctt
    atatttttacttatgccattgagaaagtccgtgagatctttatgtctgaagctgggaaagt
    aaaccagaagattaaagtatggtttagtgattacttcaacatcagtgatacaattgccat
    aatttctttcttcattggatttggactaagatttggagcaaaatggaactttgcaaatgcat
    atgataatcatgtttttgtggctggaagattaatttactgtcttaacataatattttggtatgt
    gcgtttgctagattttctagctgtaaatcaacaggcaggaccttatgtaatgatgattgg
    aaaaatggtggccaatatgttctacattgtagtgattatggctcttgtattacttagttttgg
    tgttcccagaaaggcaatactttatcctcatgaagcaccatcttggactcttgctaaaga
    tatagtttttcacccatactggatgatttttggtgaagtttatgcatacgaaattgatgtgtg
    tgcaaatgattctgttatccctcaaatctgtggtcctgggacgtggttgactccatttcttc
    aagcagtctacctctttgtacagtatatcattatggttaatcttcttattgcatttttcaacaa
    tgtgtatttacaagtgaaggcaatttccaatattgtatggaagtaccagcgttatcatttta
    ttatggcttatcatgagaaaccagttctgcctcctccacttatcattcttagccatatagttt
    ctctgttttgctgcatatgtaagagaagaaagaaagataagacttccgatggaccaaa
    acttttcttaacagaagaagatcaaaagaaacttcatgattttgaagagcagtgtgttga
    aatgtatttcaatgaaaaagatgacaaatttcattctgggagtgaagagagaattcgtgt
    cacttttgaaagagtggaacagatgtgcattcagattaaagaagttggagatcgtgtca
    actacataaaaagatcattacaatcattagattctcaaattggccatttgcaagatctttca
    gccctgacggtagatacattaaaaacactcactgcccagaaagcgtcggaagctagc
    aaagttcataatgaaatcacacgagaactgagcatttccaaacacttggctcaaaacct
    tattgatgatggtcctgtaagaccttctgtatggaaaaagcatggtgttgtaaatacactt
    agctcctctcttcctcaaggtgatcttgaaagtaataatccttttcattgtaatattttaatga
    aagatgacaaagatccccagtgtaatatatttggtcaagacttacctgcagtaccccag
    agaaaagaatttaattttccagaggctggttcctcttctggtgccttattcccaagtgctg
    tttcccctccagaactgcgacagagactacatggggtagaactcttaaaaatatttaata
    aaaatcaaaaattaggcagttcatctactagcataccacatctgtcatccccaccaacc
    aaattttttgttagtacaccatctcagccaagttgcaaaagccacttggaaactggaacc
    aaagatcaagaaactgtttgctctaaagctacagaaggagataatacagaatttggag
    catttgtaggacacagagatagcatggatttacagaggtttaaagaaacatcaaacaa
    gataaaaatactatccaataacaatacttctgaaaacactttgaaacgagtgagttctctt
    gctggatttactgactgtcacagaacttccattcctgttcattcaaaacaagcagaaaaa
    atcagtagaaggccatctaccgaagacactcatgaagtagattccaaagcagctttaa
    taccggattggttacaagatagaccatcaaacagagaaatgccatctgaagaaggaa
    cattaaatggtctcacttctccatttaagccagctatggatacaaattactattattcagct
    gtggaaagaaataacttgatgaggttatcacagagcattccatttacacctgtgcctcc
    aagaggggagcctgtcacagtgtatcgtttggaagagagttcacccaacatactaaat
    aacagcatgtcttcttggtcacaactaggcctctgtgccaaaatagagtttttaagcaaa
    gaggagatgggaggaggtttacgaagagctgtcaaagtacagtgtacctggtcaga
    acatgatatcctcaaatcagggcatctttatattatcaaatcttttcttccagaggtggtta
    atacatggtcaagtatttacaaagaagatacagttctgcatctctgtctgagagaaattc
    aacaacagagagcagcacaaaagcttacgtttgcctttaatcaaatgaaacccaaatc
    cataccatattctccaaggttccttgaagttttcctgctgtattgccattcagcaggacag
    tggtttgctgtggaagaatgtatgactggagaatttagaaaatacaacaataataatgg
    agatgagattattccaactaatactctggaagagatcatgctagcctttagccactgga
    cttacgaatatacaagaggggagttactggtacttgatttgcaaggtgttggtgaaaatt
    tgactgacccatctgtgataaaagcagaagaaaagagatcctgtgatatggtttttggc
    ccagcaaatctaggagaagatgcaattaaaaacttcagagcaaaacatcactgtaatt
    cttgctgtagaaagcttaaacttccagatctgaagaggaatgattatacgcctgataaa
    attatatttcctcaggatgagccttcagatttgaatcttcagcctggaaattccaccaaag
    aatcagaatcaactaattctgttcgtctgatgttataatattaatattactgaatcattggttt
    tgcctgcacctcacagaaatgttactgtgtcacttttccctcgggaggaaattgtttggta
    atatagaaaggtgtatgcaagttgaatttgctgactccagcacagttaaaaggtcaatat
    tcttttgacctgattaatcagtcagaaagtccctataggatagagctggcagctgagaa
    attttaaaggtaattgataattagtatttataactttttaaagggctctttgtatagcagagg
    atctcatttgactttgttttgatgagggtgatgctctctcttatgtggtacaataccattaac
    caaaggtaggtgtccatgcagattttattggcagctgttttattgccattcaactaggga
    aatgaagaaatcacgcagccttttggttaaatggcagtcaaaattttcctcagtgtattta
    gtgtgttcagtgatgatatcactggttcccaactagatgcttgttggccacgggaaggg
    aaatgacttgttctaattctaggttcacagaggtatgagaagcctgaactgaagaccatt
    ttcaagagggacggtatttatgaatcagggttaggctccatatttaaagatagagccag
    tttttttttttaaatagaacccaaattgtgtaaaaatgttaattgggttttttaaacattgttttat
    caagtcactgttaagtagaagaaagccatggtaaactgatacataacctaaattataaa
    agcagaaacctaactcactcgtcaagggaagttaccttttgaggaaagttaaagtactt
    ttttccctatctgtatctatagcaacaacccagaacttacaaacttctccaaagattttatt
    gattgttatatcaaatcagaatgtaaacatgaactcttgcatatatttaaaattgtgttgga
    acatttgaacatgaatgctgtttgtggtacttaagaaattaattcagttggattatcattatg
    tgatactggcagattgcagtgcaaccttatgccaataaaatgtaatttaacagccccag
    atattgttgaatattcaacaataacaagaaaagcttttcatctaagttttatgctttaatttttt
    ttctttttttttctttttcttttgtttccttggtactaattttaatttttatttggaagggagcagtat
    aaagcttatttgtatttagtagtgtatctcatagatacagacaaggcaagagatgataag
    ctgtttaaatagtgtttaatattgattgggggggggagaaagaaaaagtgtattacttaa
    agatactatatacgttttgtatatcattaaatctttaaaagaaatgaaataaatttattgttta
    cagatgtttagtgagtttaatcattctgaaaaattatctgacattttcagggtgtcaatttga
    gtatcagtttttttaaatgaaccatttgtatacctgtgcttttgatctcctgtcctgtacaatg
    tttaaattaatactgatttcttactgtcttcttagaaatctgttttttgttaggccaaaaaagg
    gcaatatgggctgtctgttgatttttaattttatattgattattttcacaggattataatagtag
    ctatacttttttttttttttttttttttgagacggagtctcgctctgttgcttgggctggagtgca
    gtggtgcgatctcagctcaccacaaccgccgccttccgggtttaagtgattctcctgcc
    tcagcctcccgagtagctgggactacaggcacacgccaccatgcccagctaattttta
    tatttttagtagagacagggtttcactatgttggccagtgtggtcacaaactcctgacctt
    gtgagccaccgcacctggctgctaacacttatttagtgcctactgtgtaccagacatta
    ctctaagtatttcacatatattaacctacttaatccttataacaatgttataaagaaataggt
    gttattatcctgttttgcagatttgaaagtcaaggtgctagagaggtaaagtaacgtcca
    taagattcttacgtttatttaataataagtagcaacggtaggatttgaacccaggctggct
    gcctttcatctatactgtttttgttttgttttgttttgttttgttttgttttgtttgtcttggtggggc
    atggtggctcatgcctgtaatcccagcacttcgggaggccaaggcaggtggatcact
    tgggctcaggagtttgagaccagcctgggcaacatggcaaaatcctatctctgctaaa
    aaaaaaaatacaaaaattaggccaggtgcagtggctcatgcctgtaatcccagcactt
    tgggaggccaaggtgggcggatcacaaggtcaggagttcgagaccagcctgacca
    acatagtgaaaccccgtctctactaaaaatacaaaaaattagctgggcatggcggtga
    gtgcctgtaatcccagctactcaggagtctgaggcaggagaattgcttgaacctggg
    aggtggaggttgcagtgagctgagatcgtgccattgcgctccagcctgggcaacagt
    gcgagactccgtcaaaaaaaaaaaaataactggatgtgatggtgtgcacctgtagttc
    cagctacttgggagactgaggtgggaggatcacttgagcctgggagactgaggcag
    cagtgagctgagatcatgccactgctttccaacctgggcaacagagtgagatcctgtc
    tcagaaagaaaaaaaaaaaaaagacaacctcttgctctgttgcccaggctggagtgt
    agtagcgtgatcatagctcactgcagccgtaaactcctgggctcaagcaatcctcctg
    ccactgcctcttgattaggtggaaccacaggcatgcaccaccacacgtacctaatttta
    tatatatatttttttatttttcatttttatttatttttgtttttttgagttgaagtctcactctgttgcc
    caggccggagtacagtggcacaatcttggctcactgcaacctctgcctcccaagatc
    aagcaattctcgtgcttcagcctccaaagtagctgagattacaggtacccaccataatg
    cctggctgatttttgtatttttcgtagagacaaggtttcaccttgttggccaggctgatctc
    aaactcctgacctcaagtgatccacctcccccggctacccaaagtactgggattatag
    gtgtgagccaccatgcctgggtaacacccaactaattttaaatatatattttgtagagat
    ggggtctagccttgttgcccacgctggtctcaaattcctgggctcaagtgatcctctcg
    cctgagcttcccaaagtggtagaattgcaggcatgaattgctgcacccagcctcatct
    gtgctgtgaattatgtgctgtattgactctcaagcatgatgaccattggtggtttctgtac
    catttcctgttactttactgaaacacacctactccattaacttcttgggttaagtctagaaa
    gtaacagtttacttgtaaaccacatttcttatccccaataagtatttttttaagattattaaag
    ttcattattactaccctatgatgtgaaagtgtcatttgcttaatctttttaattttttattctcaa
    cctcatcttactgaagagaataaaactcttttaccatattcttaaaatgtggaattctcggc
    caggtgcagtggctcacgcctgtaattccatcactttgggaggccaaggtgggtggat
    catctgaggtcaggagttcaagaccagcctggccaacatggtgaaaccccgtctcta
    ctaaaaatacaaaaattatctgggtgtggtggcgcgtgcctgtaggcccagctactca
    ggaggctgaggcaggagaattgcttgaacccaagaggtggaggttgcagtgagcct
    agattgctgccactgcactccagcctgggtgacagcagaactctgtctcaaaaaaaa
    gatgtggaattcttttctgcaaatgttctctaatagtataccttcttcagtctgtcgatatatg
    tatgctattattttacaagtaatacatgttgattgtattggaaattatagaaaagattatattg
    gattgtttagaaaatatttttaaatgtgaagaaaaatataaaaattactcccttgttccactt
    tccccactctcaagtcagactatgttgttttcatagttagtagctagcagtctaccccact
    agattatatgcttcacagagggaagggaccctcaagacttcactggattgagtagcac
    ccaataccttgcttgctgcctggtttgtgatgggcatactgtaagaaaaaaaaatctgaa
    tgacaaaatgtttttccataataccagacttcctcttgaagagatgggtcgtaatgttgta
    gtcttacatgcttacgtagacaatcaaagcaagaatactcaataaatggctatttaccac
    ttgaaagaaa (SEQ ID NO: 677)
    Ppp3cc aaggcggaagggtggggagggcggcgctcggggcgggaggcccggccgggtc NM_001243974 NM_008915
    cgctaggacagcggggccgctgggaagttgtgagagcggcgctcgggggcgcgc
    ttgcgtgcacgagggcccgggccgcgagcagccgcggccgtcccggtcgccacc
    cttagcagcggtcgcggtcggtgccgaagcggtgttccccgccttagccgctggcg
    cctcccaagagagcggccggtgggccctcgtcctgtcagtggcgtcggaggccgg
    cgctgcggtggccgcgcccttctggtgctcggacaccgctgaggagccggggccg
    ggcacggctggctgacggctccgggcagctaaggctgcccgaggagaaggcggc
    ggccgcggcgtaggcgcacgtccggcgggctcctggagcctggaggaggccga
    ggggaccatgtccgggaggcgcttccacctctccaccaccgaccgcgtcatcaaag
    ctgtcccctttcctccaacccaacggcttactttcaaggaagtatttgagaatgggaaa
    cctaaagttgatgttttaaaaaaccatttggtaaaggaaggacgactggaagaggaag
    tagccttaaagataatcaatgatggggctgccatcctgaggcaagagaagactatgat
    agaagtagatgctccaatcacagtatgtggtgatattcatggacaattctttgacctaat
    gaagttatttgaagttggaggatcacctagtaacacacgctacctctttctgggtgacta
    tgtggacagaggctatttcagtatagagtgtgtgctgtatttatggagtttaaagattaat
    catcccaaaacattgtttctgcttcggggaaatcatgaatgcaggcatcttacagactat
    ttcaccttcaaacaggaatgtcgaatcaaatattcggaacaggtgtatgatgcctgtatg
    gagacatttgactgtcttcctcttgctgccctcttaaaccagcagtttctctgtgtacatgg
    aggaatgtcacctgaaattacttctttagatgacattaggaaattagacaggtttacgga
    acctcccgcctttggacctgtgtgtgacctgctttggtctgatccctcagaggattatgg
    caatgagaagaccttggagcactatacccacaacactgtccgagggtgctcttatttct
    acagttaccctgcagtttgtgaatttttgcagaacaataatttactatcaattatcagagcc
    catgaagcccaagatgctgggtatcgaatgtacaggaagagccaagccacaggcttt
    ccatcacttattacaattttctctgcccccaattacctagatgtctataacaataaagctgc
    tgtgttgaaatatgaaaacaatgtcatgaatatcaggcagtttaactgttctccacaccc
    ctactggcttccaaactttatggatgttttcacatggtctttgccttttgttggggaaaaag
    tcacagagatgctggtaaatgtgctcaacatatgctctgatgacgaactgatttctgatg
    atgaagcagaagatcactacattccaagctatcagaaaggaagcactacagttcgtaa
    ggagatcatcaggaataagatcagagccattgggaagatggcacgggtcttttcaatt
    cttcggcaagaaagtgagagtgtgctgactctcaagggcctgactcccacaggcaca
    ctccctctgggcgtcctctcaggaggcaagcagactatcgagacagccacagtaga
    agcggtagaggcccgggaagccatcagagggttctcgcttcagcacaagatccgg
    agttttgaagaagcgcgaggtctggaccgaattaatgagcgaatgccaccccgaaa
    ggatagcatacacgctggtgggccaatgaaatctgtaacctcagcacactcacatgct
    gcgcacaggagcgaccaagggaagaaagcccattcatgacttagagtcctgccgtg
    gctcaggtggatctaaaactcaagaacaaattctatttatttattattggaaaatgaaaag
    caactcaaaacaacttcaacgtggaggtgcatttataattcagtctgcatttattctgtaa
    aaaggtggctgttttataaattcttttaatttatgttcaatatatataaaaagtgcatctgtttt
    gtttttcccttttttctccataattttaagaaatgaatctgattgttgtcaacacatttgtgaag
    tcttgtgctataaaggggaacttcccctaataaaagggccttggaaacctcaaacctg
    ggtttctgacttgaaaaaaaaaaaaaaa (SEQ ID NO: 678)
  • In some aspects, the nucleic acids of the compositions encode the shRNA sequences targeting the sequences provided in Table 2. Table 2 further demonstrates enrichment in tumor versus spleen for the selected shRNA based on deep sequencing analysis (“Enrich Fold”)
  • TABLE 2
    Mouse Mouse SEQ Human
    Gene Gene shRNA shRNA  ID Enrich Gene Human
    Symbol ID Clone ID Target Sequence NO: Fold Symbol Gene ID
    Akap8l 54194 ND000290 CGAAACCGCAGGCTTATGATG 1 0.5 AKAP8L 26993
    Akap8l 54194 ND000285 CAGACTGCTCAGACAACAGTG 2 0.7 AKAP8L 26993
    Akap8l 54194 TRCN0000288034 CCACAAGGAACACTTCAAATA 3 1.0 AKAP8L 26993
    Akap81 54194 ND000291 AGACCTCTACCGGTCAAGCTA 4 1.1 AKAP8L 26993
    Akap8l 54194 ND000286 ATAGAGGCTACGAGAACTATG 5 1.4 AKAP8L 26993
    Akap8l 54194 TRCN0000288033 CCAGAACATCATACCCGAGTA 6 1.6 AKAP8L 26993
    Akap81 54194 ND000289 TTAGATATGATGCCGCACTTG 7 1.7 AKAP8L 26993
    Akap81 54194 TRCN0000088483 CCCACCTGTGATTATGGATAT 8 1.8 AKAP8L 26993
    Akap81 54194 ND000288 GGCGAGAATCCTTTCACTGAC 9 1.9 AKAP8L 26993
    Akap8l 54194 TRCN0000088486 CGAGAACTATGGTTATGGCTA 10 2.1 AKAP8L 26993
    Akap8l 54194 ND000292 CAAATACCGGACCTTCTATGA 11 2.8 AKAP8L 26993
    Akap81 54194 TRCN0000307538 GATATCTGAAGGGCGAGAATC 12 3.8 AKAP8L 26993
    Akap8l 54194 TRCN0000307539 ACCGGTCAAGCTATGACTATG 13 4.4 AKAP8L 26993
    Akap8l 54194 ND000287 TTGGATTTGGCAATGGCATGA 14 7.1 AKAP8L 26993
    Akap81 54194 TRCN0000088487 CCGAAACCACTTTGCAGTCTA 15 11.8 AKAP8L 26993
    Alk 11682 TRCN0000361004 ACCTAGAGGAGAATCACTTTA 16 0.2 ALK 238
    Alk 11682 TRCN0000023725 GCCTTCATGGAAGGGATATTT 17 0.4 ALK 238
    Alk 11682 TRCN0000361067 CGGGCCTGTATACCGGATAAT 18 0.7 ALK 238
    Alk 11682 TRCN0000361003 GTGGAGCCACCTACGTGTTTA 19 0.9 ALK 238
    Alk 11682 ND000299 GGAATCTGACCTGGACGATGA 20 1.0 ALK 238
    Alk 11682 ND000293 CTTCGTTGTACCCTCGCTCTT 21 1.1 ALK 238
    Alk 11682 ND000298 GAAGGGATATTTACCTCTAAA 22 1.3 ALK 238
    Alk 11682 TRCN0000023728 CCGGGATATTGCTGCTAGAAA 23 1.7 ALK 238
    Alk 11682 TRCN0000023724 GCATCGCATTGGAGGCTATAA 24 2.1 ALK 238
    Alk 11682 ND000297 GGGCCTGTATACCGGATAATG 25 2.4 ALK 238
    Alk 11682 TRCN0000023726 CGGAGGATATATAGGTGGCAA 26 2.9 ALK 238
    Alk 11682 ND000300 ATCGAATACGGTCCAGTAGTA 27 3.4 ALK 238
    Alk 11682 ND000296 TGCTTCCGCGTAGTCAGAAAT 28 3.8 ALK 238
    Alk 11682 ND000294 CCTGCGGCAATGTCAACTATG 29 9.4 ALK 238
    Alk 11682 TRCN0000023727 CCCGAACGTCAACTATGGTTA 30 9.5 ALK 238
    Alk 11682 ND000295 GGCGAGGAGACGATTCTTGAA 31 13.5 ALK 238
    Arhgap5 11855 TRCN0000321111 TGGTACATATCCTCGTAAATT 32 0.5 ARHGAP5 394
    Arhgap5 11855 TRCN0000360350 ATTGCAATCAGTATATCATTC 33 0.8 ARHGAP5 394
    Arhgap5 11855 TRCN0000360421 GATCATGAACGTAACCATAAA 34 1.2 ARHGAP5 394
    Arhgap5 11855 TRCN0000360349 TGATAATAGCAGCAACTAAAT 35 1.3 ARHGAP5 394
    Arhgap5 11855 TRCN0000321112 AGCATGACTGGAGAGGTTTAA 36 1.4 ARHGAP5 394
    Arhgap5 11855 TRCN0000321110 TGATAGTCAGAATCGAATTAT 37 1.4 ARHGAP5 394
    Arhgap5 11855 TRCN0000321109 GAACTGGTTCATGGGTATATA 38 1.5 ARHGAP5 394
    Arhgap5 11855 TRCN0000012706 GCAAGCTCTAAGAGGAGTATT 39 3.6 ARHGAP5 394
    Arhgap5 11855 TRCN0000012707 CCTGATCCTTTGATTCCATAT 40 6.0 ARHGAP5 394
    Arhgap5 11855 TRCN0000321181 ACAGATCCTCTTGGTATTATA 41 8.3 ARHGAP5 394
    Arhgap5 11855 TRCN0000012703 GCACGATTTAATGTCAACATT 42 15.7 ARHGAP5 394
    Blvrb 233016 ND000310 CTCAGTCCCACTACAGTAATG 43 0.8 BLVRB 645
    Blvrb 233016 ND000308 TGACCACATCCGGATGCATAA 44 1.0 BLVRB 645
    Blvrb 233016 ND000306 GCCTCACCACCAATGAGTATG 45 1.2 BLVRB 645
    Blvrb 233016 ND000309 TGAGAAATGACACAAATAGAG 46 1.2 BLVRB 645
    Blvrb 233016 ND000303 TGCAAGAGTCAGGGCTGAAAT 47 1.3 BLVRB 645
    Blvrb 233016 ND000301 GGAAGCTGTCATCGTGCTACT 48 1.5 BLVRB 645
    Blvrb 233016 ND000304 GCATAAGATTCTGCAAGAGTC 49 1.9 BLVRB 645
    Blvrb 233016 TRCN0000042385 CCTCAGTCCCACTACAGTAAT 50 2.2 BLVRB 645
    Blvrb 233016 ND000302 TCGAGGGTCATATCCAAGCAT 51 2.4 BLVRB 645
    Blvrb 233016 TRCN0000324726 GAACATCGTGACAGCCATGAA 52 3.0 BLVRB 645
    Blvrb 233016 TRCN0000042384 CCAATGAGTATGACGGACACA 53 3.1 BLVRB 645
    Blvrb 233016 ND000307 GAGGGTCATGCATCCTGAGAA 54 3.1 BLVRB 645
    Blvrb 233016 ND000305 TAGGAGACCAACCACTAACTG 55 5.3 BLVRB 645
    Blvrb 233016 TRCN0000324662 GCTGAAATACGTGGCAGTGAT 56 5.3 BLVRB 645
    Blvrb 233016 TRCN0000042386 CGGATGCATAAGATTCTGCAA 57 8.0 BLVRB 645
    Cblb 208650 ND000027 TCTACATCGATAGTCTCATGA 58 0.7 CBLB 868
    Cblb 208650 TRCN0000244603 CTACACCTCACGATCATATAA 59 0.9 CBLB 868
    Cblb 208650 TRCN0000244605 TGAGCGAGAATGAGTACTTTA 60 0.9 CBLB 868
    Cblb 208650 ND000026 ATCGAACATCCCAGATTTAGG 61 1.0 CBLB 868
    Cblb 208650 ND000029 TAAAGTGTACTGGTCCATTAG 62 1.4 CBLB 868
    Cblb 208650 TRCN0000244607 CTTGTACTCCAGTACCATAAT 63 1.5 CBLB 868
    Cblb 208650 ND000028 GTATGAGACAGAAGGACTGAG 64 1.5 CBLB 868
    Cblb 208650 TRCN0000244604 CCAGATTTAGGCATCTATTTG 65 1.6 CBLB 868
    Cblb 208650 ND000031 TCAGCACTTGAGACTTATATT 66 1.7 CBLB 868
    Cblb 208650 ND000024 TACACCTCACGATCATATAAA 67 2.1 CBLB 868
    Cblb 208650 ND000033 AACACAGACGCCATGATTTGC 68 5.1 CBLB 868
    Cblb 208650 ND000032 AAGATGTCAAGATTGAGCCTT 69 5.3 CBLB 868
    Cblb 208650 TRCN0000244606 CCCTGATTTAACCGGATTATG 70 6.1 CBLB 868
    Cblb 208650 ND000030 AGCCAGGTCCAATTCCATTTC 71 10.0 CBLB 868
    Cblb 208650 ND000025 CGAGCGATCCGGCTCTTTAAA 72 10.8 CBLB 868
    Cdkn2a 12578 ND000317 CTTGGTGAAGTTCGTGCGATC 73 0.6 CDKN2A 1029
    Cdkn2a 12578 TRCN0000257162 CGCTCTGGCTTTCGTGAACAT 74 0.8 CDKN2A 1029
    Cdkn2a 12578 TRCN0000362594 GATGATGATGGGCAACGTTCA 75 0.9 CDKN2A 1029
    Cdkn2a 12578 TRCN0000231228 TCCCAAGAGCAGAGCTAAATC 76 0.9 CDKN2A 1029
    Cdkn2a 12578 TRCN0000362666 TCTTGGTGAAGTTCGTGCGAT 77 1.0 CDKN2A 1029
    Cdkn2a 12578 TRCN0000362596 ACGGGCATAGCTTCAGCTCAA 78 1.1 CDKN2A 1029
    Cdkn2a 12578 TRCN0000222730 GCTCGGCTGGATGTGCGCGAT 79 1.1 CDKN2A 1029
    Cdkn2a 12578 TRCN0000231225 TTGAGGCTAGAGAGGATCTTG 80 1.2 CDKN2A 1029
    Cdkn2a 12578 TRCN0000222731 CATCAAGACATCGTGCGATAT 81 2.1 CDKN2A 1029
    Cdkn2a 12578 TRCN0000077815 GTGAACATGTTGTTGAGGCTA 82 2.3 CDKN2A 1029
    Cdkn2a 12578 TRCN0000077816 GTCTTTGTGTACCGCTGGGAA 83 3.3 CDKN2A 1029
    Cdkn2a 12578 TRCN0000362595 CTAGCGATGCTAGCGTGTCTA 84 4.1 CDKN2A 1029
    Cdkn2a 12578 TRCN0000222729 GTGATGATGATGGGCAACGTT 85 5.6 CDKN2A 1029
    Cdkn2a 12578 TRCN0000231226 GCTCAACTACGGTGCAGATTC 86 6.9 CDKN2A 1029
    Cdkn2a 12578 TRCN0000231227 TCAAGACATCGTGCGATATTT 87 7.2 CDKN2A 1029
    Dgka 13139 TRCN0000024825 GAGCTAAGTAAGGTGGTATAT 88 0.7 DGKA 1606
    Dgka 13139 TRCN0000368765 GCGATGTACTGAAGGTCTTTG 89 0.7 DGKA 1606
    Dgka 13139 ND000059 TCAGTGATGTGTACTGCTACT 90 0.8 DGKA 1606
    Dgka 13139 ND000054 GTATATCTCGACCGATGGTTC 91 1.0 DGKA 1606
    Dgka 13139 TRCN0000378505 TGATGCGAGTGGCCGAATATC 92 1.1 DGKA 1606
    Dgka 13139 TRCN0000024828 CCTAGGATTTGAACAATTCAT 93 1.2 DGKA 1606
    Dgka 13139 ND000058 AAAGATTCTCAAGGATATAGA 94 1.6 DGKA 1606
    Dgka 13139 ND000056 GAGGGATGTTCCATCACCTTC 95 1.9 DGKA 1606
    Dgka 13139 ND000053 TACAGACATCCTTACACAACC 96 2.0 DGKA 1606
    Dgka 13139 TRCN0000024824 GCCGAATATCTAGACTGGGAT 97 3.4 DGKA 1606
    Dgka 13139 TRCN0000024827 CGGCTGGAAGTGGTAGGAATA 98 3.5 DGKA 1606
    Dgka 13139 ND000055 GTTCCTCAGTTCCGGATATTG 99 5.0 DGKA 1606
    Dgka 13139 TRCN0000024826 CCTGAGCTGTAACTTCTGTAA 100 6.8 DGKA 1606
    Dgka 13139 ND000057 TGCGAACAGAGCATTAGCCTT 101 7.8 DGKA 1606
    Dgka 13139 TRCN0000361167 TGTTCCTCAGTTCCGGATATT 102 10.2 DGKA 1606
    Dgkz 104418 ND000063 CACCTTCCACAGCAAGGAGAT 103 0.4 DGKZ 8525
    Dgkz 104418 ND000061 ATCGTGGTGCATACCCAATGC 104 0.4 DGKZ 8525
    Dgkz 104418 TRCN0000278613 CCTGGATGTCTTTAACAACTA 105 0.7 DGKZ 8525
    Dgkz 104418 ND000060 CGAGTAGTGTGTGACGGAATG 106 0.9 DGKZ 8525
    Dgkz 104418 ND000065 CACATCTGGTTTGAGACCAAC 107 1.4 DGKZ 8525
    Dgkz 104418 TRCN0000278690 GAGAAGTTCAACAGCCGCTTT 108 1.6 DGKZ 8525
    Dgkz 104418 ND000069 ACTGTGCAGGCACCATGCCCT 109 2.0 DGKZ 8525
    Dgkz 104418 ND000068 AGAAGCTGTTCAGATCTAGGG 110 2.8 DGKZ 8525
    Dgkz 104418 TRCN0000297512 GTGGACTTCAAAGAATTCATT 111 3.6 DGKZ 8525
    Dgkz 104418 ND000064 ACTACGAGGCTCTACATTATG 112 5.2 DGKZ 8525
    Dgkz 104418 ND000067 AGTACATAATTTGAGGATTCT 113 5.5 DGKZ 8525
    Dgkz 104418 TRCN0000278682 CGAGGCTCTACATTATGACAA 114 6.0 DGKZ 8525
    Dgkz 104418 TRCN0000278614 CCTGTAAGATCGTGGTGCATA 115 6.4 DGKZ 8525
    Dgkz 104418 ND000062 GAAACCGCAGTGCATCGTCTT 116 7.7 DGKZ 8525
    Dgkz 104418 ND000066 CAGCATCACGGATTCGAATTG 117 14.0 DGKZ 8525
    Egr2 13654 TRCN0000218224 AGGATCCTTCAGCATTCTTAT 118 0.4 EGR2 1959
    Egr2 13654 ND000075 AGCTCTGGCTGACACACCAG 119 0.6 EGR2 1959
    Egr2 13654 TRCN0000081682 CCAGGATCCTTCAGCATTCTT 120 0.6 EGR2 1959
    Egr2 13654 TRCN0000081678 GCTGTATATTTCTGCCTATTA 121 1.3 EGR2 1959
    Egr2 13654 TRCN0000235777 ACTATTGTGGCCGCAAGTTTG 122 1.3 EGR2 1959
    Egr2 13654 TRCN0000235775 AGCGGGTACTACCGTTTATTT 123 1.6 EGR2 1959
    Egr2 13654 TRCN0000235778 CTGTATATTTCTGCCTATTAA 124 2.4 EGR2 1959
    Egr2 13654 ND000073 GTGACCACCTTACTACTCACA 125 3.2 EGR2 1959
    Egr2 13654 ND000074 GTTTGCCAGGAGTGACGAAAG 126 3.9 EGR2 1959
    Egr2 13654 TRCN0000081681 CCTTCACCTACATGGGCAAAT 127 4.0 EGR2 1959
    Egr2 13654 TRCN0000081680 CCAGAAGGTATCATCAATATT 128 5.1 EGR2 1959
    Egr2 13654 TRCN0000081679 CCACTCTCTACCATCCGTAAT 129 5.2 EGR2 1959
    Egr2 13654 ND000072 CCGTGCCAGAGAGATCCACAC 130 5.6 EGR2 1959
    Egr2 13654 ND000071 CAATAGGTTGGGAGTTGCTGA 131 8.6 EGR2 1959
    Egr2 13654 TRCN0000235776 ACTCTCTACCATCCGTAATTT 132 10.2 EGR2 1959
    Eif2ak3 13666 TRCN0000321872 CCATGAGTTCATCTGGAACAA 133 0.4 EIF2AK3 9451
    Eif2ak3 13666 ND000328 CATAGCTCCTTCTCCTGAAAG 134 0.9 EIF2AK3 9451
    Eif2ak3 13666 ND000332 GATGACTGCAATTACGCTATC 135 1.1 EIF2AK3 9451
    Eif2ak3 13666 ND000325 GTCGCCATTTATGTCGGTAGT 136 1.1 EIF2AK3 9451
    Eif2ak3 13666 ND000326 TGGAAACAACTACTCCCATAA 137 1.1 EIF2AK3 9451
    Eif2ak3 13666 TRCN0000321873 GTGACCCATCTGCACTAATTT 138 1.3 EIF2AK3 9451
    Eif2ak3 13666 ND000329 GCATGATGGCAACCATTATGT 139 1.3 EIF2AK3 9451
    Eif2ak3 13666 ND000330 ATCCCGATATCTAACAGATTT 140 1.6 EIF2AK3 9451
    Eif2ak3 13666 ND000333 TGTCGCCGATGGGATAGTGAT 141 1.9 EIF2AK3 9451
    Eif2ak3 13666 TRCN0000321805 GCCACTTTGAACTTCGGTATA 142 2.0 EIF2AK3 9451
    Eif2ak3 13666 TRCN0000028759 CCATACGATAACGGTTACTAT 143 4.8 EIF2AK3 9451
    Eif2ak3 13666 TRCN0000321806 CCTCTACTGTTCACTCAGAAA 144 5.8 EIF2AK3 9451
    Eif2ak3 13666 ND000327 CATACGATAACGGTTACTATC 145 5.9 EIF2AK3 9451
    Eif2ak3 13666 ND000331 CGTGACCCATCTGCACTAATT 146 7.3 EIF2AK3 9451
    Eif2ak3 13666 TRCN0000028799 GCCTGTTTGATGATACAAGTT 147 13.4 EIF2AK3 9451
    Entpd1 12495 ND000082 GAATGTAAGTGAGCTCTATGG 148 0.3 ENTPD1 953
    Entpd1 12495 TRCN0000222348 CCGAACTGATACCAACATCCA 149 0.4 ENTPD1 953
    Entpd1 12495 TRCN0000222346 CCCATGCTTTAACCCAGGATA 150 0.4 ENTPD1 953
    Entpd1 12495 TRCN0000222345 CCTTGGTTTCACCTCTATCTT 151 0.8 ENTPD1 953
    Entpd1 12495 TRCN0000222344 CCAAGGACATTCAGGTTTCAA 152 0.9 ENTPD1 953
    Entpd1 12495 ND000085 CAGGAACAGAGTTGGCTAAGC 153 1.0 ENTPD1 953
    Entpd1 12495 ND000078 TTAACCCAGGATACGAGAAGG 154 1.1 ENTPD1 953
    Entpd1 12495 ND000081 ACTATCTCAGCCATGGCTTTG 155 1.2 ENTPD1 953
    Entpd1 12495 ND000077 TTCAAGTGGTGGCGTCCTTAA 156 1.3 ENTPD1 953
    Entpd1 12495 ND000076 GACTTTGGGCTACATGCTGAA 157 1.4 ENTPD1 953
    Entpd1 12495 ND000080 GGCATGCGCTTGCTTAGAATG 158 1.9 ENTPD1 953
    Entpd1 12495 ND000084 GCACTGGAGACTACGAACAGT 159 1.9 ENTPD1 953
    Entpd1 12495 ND000083 GTGGATTACTATTAACTATCT 160 6.5 ENTPD1 953
    Entpd1 12495 TRCN0000222347 GCTCCTGGGAACAGATTCATT 161 7.3 ENTPD1 953
    Entpd1 12495 ND000079 ACCATTTGATCAGTTTCGAAT 162 13.3 ENTPD1 953
    F11r 16456 TRCN0000284518 GCTGATTCCCAGGACTATATT 163 0.6 F11R 50848
    F11r 16456 TRCN0000124868 GTATCGCTGTATAACTATGTA 164 0.6 F11R 50848
    F11r 16456 ND000093 ATTGACCTGCACCTACTCT 165 0.6 F11R 50848
    F11r 16456 ND000094 GCCGGGAGGAAACTGTTGT 166 0.6 F11R 50848
    F11r 16456 TRCN0000271840 CCTGGTTCAAGGACGGGATAT 167 0.7 F11R 50848
    F11r 16456 TRCN0000271841 TTCGGTGTACACTGCTCAATC 168 0.7 F11R 50848
    F11r 16456 TRCN0000271792 CACCGGGTAAGAAGGTCATTT 169 0.9 F11R 50848
    F11r 16456 ND000088 ACTTGCATGGTCTCCGAGGAA 170 0.9 F11R 50848
    F11r 16456 ND000086 GTAACACTGATTCTCCTTGGA 171 1.0 F11R 50848
    F11r 16456 ND000090 GTTATAACAGCCAGATCACAG 172 1.1 F11R 50848
    F11r 16456 ND000092 TAGCTGCACAGGATGCCTTCA 173 1.3 F11R 50848
    F11r 16456 ND000087 GGTTTGCCTATAGCCGTGGAT 174 1.9 F11R 50848
    F11r 16456 TRCN0000271794 CCTATAGCCGTGGATACTTTG 175 4.3 F11R 50848
    F11r 16456 ND000091 CTCCGTTGTCCATTTGCCTTA 176 4.6 F11R 50848
    F11r 16456 ND000089 CCACCCTCTGAATATTCCTGG 177 6.8 F11R 50849
    Fyn 14360 TRCN0000023383 CATCCCGAACTACAACAACTT 178 0.7 FYN 2534
    Fyn 14360 TRCN0000023381 CCTTTGGAAACCCAAGAGGTA 179 0.9 FYN 2534
    Fyn 14360 TRCN0000361148 TCTGAGACAGAAGCGTGTTAT 180 1.4 FYN 2534
    Fyn 14360 TRCN0000023379 GCTCGGTTGATTGAAGACAAT 181 1.4 FYN 2534
    Fyn 14360 TRCN0000361213 TTGACAATGGTGGATACTATA 182 1.9 FYN 2534
    Fyn 14360 TRCN0000361149 TCTTCACCTGATTCAACTAAA 183 1.9 FYN 2534
    Fyn 14360 TRCN0000023382 GCTCTGAAGTTGCCAAACCTT 184 2.0 FYN 2534
    Fyn 14360 TRCN0000361212 CACTGTTTGTGGCGCTTTATG 185 2.3 FYN 2534
    Fyn 14360 TRCN0000361152 CATCGAGCGCATGAATTATAT 186 2.9 FYN 2534
    Fyn 14360 TRCN0000023380 CCTGTATGGAAGGTTCACAAT 187 6.5 FYN 2534
    Fyn 14360 ND000111 TCGATGTTATGTCAAAGGCC 188 0.5 FYN 2534
    Fyn 14360 ND000112 ACCACACAAACTTCCTGTAT 189 0.7 FYN 2534
    Fyn 14360 ND000115 ACAGCTCCTGTCCTTTGGAAA 190 1.0 FYN 2534
    Fyn 14360 ND000113 GCAGCGAAACTGACAGAGGAG 191 4.1 FYN 2534
    Fyn 14360 ND000114 ACACTGTTTGTGGCGCTTTAT 192 4.4 FYN 2534
    Grk6 26385 ND000356 TGACTACCACAGCCTATGTGA 193 0.5 GRK6 2870
    Grk6 26385 TRCN0000022851 CGAGAAACAGATCTTGGAGAA 194 0.6 GRK6 2870
    Grk6 26385 ND000355 CTAACCTTGCTTAGCAACTGT 195 0.6 GRK6 2870
    Grk6 26385 ND000359 AGGAATGAGCGCTACACGTTC 196 1.0 GRK6 2870
    Grk6 26385 TRCN0000022853 TCTTGGAGAAAGTGAACAGTA 197 1.1 GRK6 2870
    Grk6 26385 TRCN0000022850 GCGCCTGTTATTTCGTGAGTT 198 1.1 GRK6 2870
    Grk6 26385 TRCN0000361581 GAACAGTTCTCTACAGTTAAA 199 1.1 GRK6 2870
    Grk6 26385 ND000354 CAGGCTATTTATTGCAAGGAT 200 1.2 GRK6 2870
    Grk6 26385 ND000357 GAGCTTAGCCTACGCCTATGA 201 1.3 GRK6 2870
    Grk6 26385 TRCN0000022852 GCAAAGGCAAGAGCAAGAAAT 202 1.3 GRK6 2870
    Grk6 26385 TRCN0000361580 CCATGGCTCTCAACGAGAAAC 203 2.7 GRK6 2870
    Grk6 26385 ND000358 TCTATGCTGCTGAGATCTGCT 204 4.2 GRK6 2870
    Grk6 26385 TRCN0000361508 GCCGACTAATGCAGAACTTTC 205 4.5 GRK6 2870
    Grk6 26385 ND000360 CGCCTGTTATTTCGTGAGTTC 206 5.8 GRK6 2870
    Grk6 26385 TRCN0000022849 CGCCGACTAATGCAGAACTTT 207 11.0 GRK6 2870
    Hipk1 15257 ND000371 CTACCTGCAATCACGCTACTA 208 0.3 HIPK1 204851
    Hipk1 15257 ND000374 AGCGGAGGGTTCACATGTATG 209 0.4 HIPK1 204851
    Hipk1 15257 TRCN0000361231 CAACCAGTACAGCACTATTAT 210 0.4 HIPK1 204851
    Hipk1 15257 TRCN0000361237 TACCCTTTCTCTGGCTAATTC 211 0.7 HIPK1 204851
    Hipk1 15257 TRCN0000368011 AGCCTGAAGGCGAGGTCTAAT 212 1.1 HIPK1 204851
    Hipk1 15257 ND000376 CATTGGCACCCGTACTATCAT 213 1.1 HIPK1 204851
    Hipk1 15257 TRCN0000023157 GCTTCAGAATACGATCAGATT 214 1.2 HIPK1 204851
    Hipk1 15257 ND000375 GAAGACTCTTAACCACCAATT 215 1.8 HIPK1 204851
    Hipk1 15257 TRCN0000361233 ATACGATCAGATTCGCTATAT 216 1.9 HIPK1 204851
    Hipk1 15257 ND000372 CTGTCATACATTTGGTCTCTT 217 2.7 HIPK1 204851
    Hipk1 15257 ND000377 GCTACTAGCCCTGAGTTCTTA 218 3.4 HIPK1 204851
    Hipk1 15257 TRCN0000361232 TATAACTTTGTCCGTTCTTAT 219 4.5 HIPK1 204851
    Hipk1 15257 ND000373 CTCGCTGCTAAACTACCAATC 220 6.3 HIPK1 204851
    Hipk1 15257 ND000378 GCCAATCATCATTCCAGATAC 221 6.7 HIPK1 204851
    Hipk1 15257 TRCN0000023154 CGCTCCAAATACAAGCACAAA 222 12.3 HIPK1 204851
    Inpp5b 16330 TRCN0000080903 GCTTAGAGGTTCCTGGATAAA 223 0.5 INPP5B 3633
    Inpp5b 16330 TRCN0000080906 CCTTTGGTTCACACACCAGAA 224 0.7 INPP5B 3633
    Inpp5b 16330 ND000130 CTGTTAGTGACCTGACGTTGA 225 0.8 INPP5B 3633
    Inpp5b 16330 TRCN0000305895 ATATTCTAGCTAGCATATTTG 226 0.8 INPP5B 3633
    Inpp5b 16330 TRCN0000311434 GGCCAGAGTTTGACCATATAA 227 1.4 INPP5B 3633
    Inpp5b 16330 ND000131 GAGTCCTTCACGATTCATAAT 228 1.4 INPP5B 3633
    Inpp5b 16330 TRCN0000080905 CGGATCTCCTATCCATACATT 229 1.5 INPP5B 3633
    Inpp5b 16330 ND000128 GTATCGGACAAGGCTCACATT 230 1.6 INPP5B 3633
    Inpp5b 16330 ND000129 TTCGAGACACAATCGTGAGAT 231 1.9 INPP5B 3633
    Inpp5b 16330 ND000127 CTGTCCAAGCCGCAAACATGT 232 3.1 INPP5B 3633
    Inpp5b 16330 ND000133 CTCAAGCTTGTATTCCAACTT 233 4.3 INPP5B 3633
    Inpp5b 16330 ND000132 ATATAAGGGACTGTCTAGATA 234 4.6 INPP5B 3633
    Inpp5b 16330 TRCN0000080904 CGAGTCCTTCACGATTCATAA 235 6.2 INPP5B 3633
    Inpp5b 16330 TRCN0000080907 CCGAGTCCTTCACGATTCATA 236 8.1 INPP5B 3633
    Inpp5b 16330 ND000134 CGTCCGACTGGTTGGGATTAT 237 9.5 INPP5B 3633
    Ipmk 69718 TRCN0000024840 CCCAGATGGTACAGTTCTGAA 238 0.5 IPMK 253430
    Ipmk 69718 ND000384 CGAGGCTCTGTGGGTTCTATA 239 0.5 IPMK 253430
    Ipmk 69718 TRCN0000360733 TTGCCGTGCTTCGGAGTATTT 240 0.6 IPMK 253430
    Ipmk 69718 TRCN0000360808 GATGCGATTGCCGCCAGTATT 241 0.7 IPMK 253430
    Ipmk 69718 TRCN0000024839 CCTAACGAAAGAGACCCTGAA 242 0.8 IPMK 253430
    Ipmk 69718 ND000383 ATTGCCGTGCTTCGGAGTATT 243 1.1 IPMK 253430
    Ipmk 69718 ND000380 AGCGGAAGTACGGATGATAGA 244 1.3 IPMK 253430
    Ipmk 69718 TRCN0000360807 GAGGCTCTGTGGGTTCTATAT 245 1.4 IPMK 253430
    Ipmk 69718 ND000379 TGCCCAAATACTACGGCGTCT 246 1.7 IPMK 253430
    Ipmk 69718 TRCN0000024843 CGGCAAGGACAAAGTGGGCAT 247 2.9 IPMK 253430
    Ipmk 69718 ND000381 CTAGCAACACAGTCGATGAGG 248 3.2 IPMK 253430
    Ipmk 69718 TRCN0000360732 ACCAAACGATGTGTACCTAAA 249 4.0 IPMK 253430
    lpmk 69718 TRCN0000024841 ACCCTGTATAATGGACGTGAA 250 4.1 IPMK 253430
    Ipmk 69718 ND000382 CCTGTATAATGGACGTGAAGA 251 4.7 IPMK 253430
    Ipmk 69718 TRCN0000024842 CACCAAACGATGTGTACCTAA 252 6.9 IPMK 253430
    Jun 16476 TRCN0000229526 GAACAGGTGGCACAGCTTAAG 253 0.5 JUN 3725
    Jun 16476 TRCN0000042693 CGGCTACAGTAACCCTAAGAT 254 0.5 JUN 3725
    Jun 16476 TRCN0000055205 CTACGCCAACCTCAGCAACTT 255 0.7 JUN 3725
    Jun 16476 TRCN0000055206 CGGTGCCTACGGCTACAGTAA 256 0.8 JUN 3725
    Jun 16476 TRCN0000042695 GCTTAAGCAGAAAGTCATGAA 257 0.9 JUN 3725
    Jun 16476 TRCN0000360499 AGCGCATGAGGAACCGCATTG 258 0.9 JUN 3725
    Jun 16476 TRCN0000360498 CCTATCGACATGGAGTCTCAG 259 0.9 JUN 3725
    Jun 16476 TRCN0000042697 GAAGCGCATGAGGAACCGCAT 260 1.0 JUN 3725
    Jun 16476 TRCN0000360511 ATTCGATCTCATTCAGTATTA 261 1.1 JUN 3725
    Jun 16476 TRCN0000360572 GGATCGCTCGGCTAGAGGAAA 262 1.2 JUN 3725
    Jun 16476 TRCN0000055207 GCGGATCAAGGCAGAGAGGAA 263 3.1 JUN 3725
    Jun 16476 TRCN0000229528 GGCATGTGCTGTGATCATTTA 264 3.2 JUN 3725
    Jun 16476 TRCN0000042694 ACGCAGCAGTTGCAAACGTTT 265 3.3 JUN 3725
    Jun 16476 TRCN0000055203 GCGGGCTAACTGCAATAAGAT 266 5.2 JUN 3725
    Jun 16476 TRCN0000229525 CAGTAACCCTAAGATCCTAAA 267 5.5 JUN 3725
    Jun 16476 TRCN0000229527 GCTAACGCAGCAGTTGCAAAC 268 5.8 JUN 3725
    Jun 16476 TRCN0000218856 GAAAGTCATGAACCACGTTAA 269 6.4 JUN 3725
    Mast2 17776 TRCN0000225743 AGCAACAACAGGAAGGTATAT 270 0.4 MAST2 23139
    Mast2 17776 TRCN0000022896 GCATCCACGAACAAGACCATA 271 0.7 MAST2 23139
    Mast2 17776 TRCN0000225741 TTGAGACCAAGCGTCACTTAT 272 1.0 MAST2 23139
    Mast2 17776 ND000396 CCGCAAGAGCTTGATTGTAAC 273 1.2 MAST2 23139
    Mast2 17776 TRCN0000022898 GCTGGTTCTGAAGAGTGGAAA 274 1.2 MAST2 23139
    Mast2 17776 ND000392 GATATTACGGAAGCGGTTATC 275 1.3 MAST2 23139
    Mast2 17776 ND000393 ACGAATACCACGGTCCCAAAT 276 1.4 MAST2 23139
    Mast2 17776 TRCN0000218393 GTGGAAACAAGGTATCAATTT 277 1.5 MAST2 23139
    Mast2 17776 ND000397 GAAGTGTGCTATCCGGGAAAG 278 1.6 MAST2 23139
    Mast2 17776 ND000395 GCCTCATTACGTCACACTATT 279 1.6 MAST2 23139
    Mast2 17776 TRCN0000022895 CCTCATTACGTCACACTATTT 280 1.9 MAST2 23139
    Mast2 17776 TRCN0000225742 ACTTGTATGAGGGTCATATTG 281 4.1 MAST2 23139
    Mast2 17776 TRCN0000022897 CGAATGAGAAACCAATCCCTT 282 4.2 MAST2 23139
    Mast2 17776 ND000394 GCATCAAACCTGGTTCGAATG 283 4.3 MAST2 23139
    Mast2 17776 TRCN0000022894 CCCTGTCAACAAAGTAATCAA 284 5.1 MAST2 23139
    Mdfic 16543 TRCN0000237997 GGAGGAAACAGGCAAGATAAA 285 0.2 MDFIC 29969
    Mdfic 16543 TRCN0000237994 TGATGCGGGACCAGTCCATTT 286 0.4 MDFIC 29969
    Mdfic 16543 ND000148 TGTAATGAGGACAATACGGAG 287 0.4 MDFIC 29969
    Mdfic 16543 TRCN0000362432 TCCTGACCCTCTGCAACATTG 288 0.6 MDFIC 29969
    Mdfic 16543 TRCN0000237996 TGACATGGACTGCGGCATCAT 289 0.8 MDFIC 29969
    Mdfic 16543 TRCN0000095981 CGAAGCATGTAATGAGGACAA 290 1.0 MDFIC 29969
    Mdfic 16543 TRCN0000095982 GACATCAGTAAGAAGAGTAAA 291 1.1 MDFIC 29969
    Mdfic 16543 TRCN0000237998 TGCCAAGTGACAGGTTATAAA 292 1.1 MDFIC 29969
    Mdfic 16543 TRCN0000095983 TGCAACATTGTCCTGGGACAA 293 1.5 MDFIC 29969
    Mdfic 16543 TRCN0000237995 ATCGTCAGACTGTCTAGAAAT 294 1.6 MDFIC 29969
    Mdfic 16543 TRCN0000095980 CCGTGGAGAATCACAAGATAT 295 2.6 MDFIC 29969
    Mdfic 16543 TRCN0000362509 GTTTATCTATTGGAGGTTAAA 296 4.4 MDFIC 29969
    Mdfic 16543 ND000147 GAAGAGTAAAGTAAATGCTGT 297 5.1 MDFIC 29969
    Mdfic 16543 TRCN0000095979 CGCCGGATGTATGTGGTTTAA 298 7.2 MDFIC 29969
    Mdfic 16543 TRCN0000362431 GCCGGATGTATGTGGTTTAAT 299 10.0 MDFIC 29969
    Nptxr 73340 TRCN0000219475 CTTGGTCTCTCCCATCATATA 300 0.5 NPTXR 23467
    Nptxr 73340 ND000150 ACAGCAACTGGCACCATATCT 301 0.8 NPTXR 23467
    Nptxr 73340 TRCN0000219474 GATACCTTGGGAGGCCGATTT 302 0.8 NPTXR 23467
    Nptxr 73340 ND000155 GGCCAATGAGATCGTGCTTCT 303 1.0 NPTXR 23467
    Nptxr 73340 ND000154 GTAGCCTTTGACCCTCAAATC 304 1.0 NPTXR 23467
    Nptxr 73340 ND000152 CAATGGAGCTGCTGATCAACG 305 1.0 NPTXR 23467
    Nptxr 73340 TRCN0000219472 GACAGCAACTGGCACCATATC 306 1.1 NPTXR 23467
    Nptxr 73340 ND000158 TTGGTCTCTCCCATCATATAC 307 1.3 NPTXR 23467
    Nptxr 73340 ND000159 ATACCTTGGGAGGCCGATTTG 308 1.3 NPTXR 23467
    Nptxr 73340 ND000153 CCTGTCAGTTTCAGGACTTTG 309 2.0 NPTXR 23467
    Nptxr 73340 ND000156 TCCGCAACAACTACATGTACG 310 2.1 NPTXR 23467
    Nptxr 73340 ND000157 ATAAGCTGGTAGAGGCCTTTG 311 3.9 NPTXR 23467
    Nptxr 73340 ND000149 CGGTGCCGTCATCTGCATCAT 312 6.6 NPTXR 23467
    Nptxr 73340 TRCN0000219473 CAAGCCACACGGCATCCTTAT 313 7.0 NPTXR 23467
    Nptxr 73340 ND000151 TCAAGCCACACGGCATCCTTA 314 7.2 NPTXR 23467
    Nuak2 74137 ND000434 TTGGACTTGCCTGAACGTCTT 315 0.2 NUAK2 81788
    Nuak2 74137 TRCN0000361872 TTTGACGGGCAGGATCATAAA 316 0.4 NUAK2 81788
    Nuak2 74137 TRCN0000024271 GCCAATGGAAACATCAAGATT 317 0.7 NUAK2 81788
    Nuak2 74137 TRCN0000361873 GTGTAGTGACTGCCATTATTT 318 0.7 NUAK2 81788
    Nuak2 74137 ND000436 CCAAGGTGTGCAGCTTCTTCA 319 1.6 NUAK2 81788
    Nuak2 74137 ND000431 CCTGATCCGGTGGCTGTTAAT 320 1.7 NUAK2 81788
    Nuak2 74137 TRCN0000378457 GGGCTCATCAAGTCGCCTAAA 321 1.8 NUAK2 81788
    Nuak2 74137 TRCN0000024270 CCGAAAGGCATTCTCAAGAAA 322 2.1 NUAK2 81788
    Nuak2 74137 TRCN0000024273 GTCGCCTAAACCTCTGATGAA 323 2.1 NUAK2 81788
    Nuak2 74137 TRCN0000024272 CCGAGGCGATCTGTATGATTA 324 2.1 NUAK2 81788
    Nuak2 74137 TRCN0000378409 GAAGTCTCGACAGCGTGAATC 325 2.8 NUAK2 81788
    Nuak2 74137 ND000435 TCGGACCGCTGTTTGACTTCA 326 2.8 NUAK2 81788
    Nuak2 74137 ND000433 TAGCAGCAAGATTGTGATTGT 327 4.5 NUAK2 81788
    Nuak2 74137 ND000432 AGTCTCGACAGCGTGAATCTG 328 5.4 NUAK2 81788
    Nuak2 74137 TRCN0000024269 CCCAAGGAAAGGCATCCTTAA 329 13.1 NUAK2 81788
    Pdzklip1 67182 TRCN0000244507 GATGGCAGATACTCCTCAATG 330 0.4 PDZK1IP1 10158
    Pdzklip1 67182 ND000172 GGGAATGGATGGCAGATACTC 331 0.5 PDZK1IP1 10158
    Pdzklip1 67182 ND000176 CTCCCTCACCTCTCTAGAATC 332 0.6 PDZK1IP1 10158
    Pdzklip1 67182 ND000170 TGCAATCGTCTTCGCCGTCAA 333 0.8 PDZK1IP1 10158
    Pdzklip1 67182 ND000173 CATTGCTGTCGCTGTGTTCTT 334 1.2 PDZK1IP1 10158
    Pdzklip1 67182 TRCN0000244505 ACAAGAATGCCTACGAGAATG 335 1.7 PDZK1IP1 10158
    Pdzklip1 67182 ND000174 TTCTTGGTCCTTGTTGCAATC 336 2.0 PDZK1IP1 10158
    Pdzklip1 67182 TRCN0000244509 GGAGCACAGTGATGATCATTG 337 2.5 PDZK1IP1 10158
    Pdzklip1 67182 ND000171 ACTGCTCTACAGGAATCTACT 338 2.5 PDZK1IP1 10158
    Pdzklip1 67182 ND000175 CTGTCAACAAGGTCTAGGAAA 339 4.8 PDZK1IP1 10158
    Pdzklip1 67182 TRCN0000244508 CCTCATTGCTGTCGCTGTGTT 340 6.3 PDZK1IP1 10158
    Pdzklip1 67182 TRCN0000244506 TCTACAGGAATCTACTGAAAC 341 12.9 PDZK1IP1 10158
    Pkd1 18763 ND000445 CAAGTCCTATGACCCTAATTT 342 0.5 PKD1 5310
    Pkd1 18763 TRCN0000304664 GGTGGACACCACTCAGTATTA 343 0.8 PKD1 5310
    Pkd1 18763 TRCN0000072086 CCAACTCAACATCACCGTAAA 344 0.8 PKD1 5310
    Pkd1 18763 TRCN0000304612 ACACAATACCACGCATATTTA 345 0.9 PKD1 5310
    Pkd1 18763 ND000447 GGCCGCTTCAAATATGAAATA 346 1.2 PKD1 5310
    Pkd1 18763 ND000444 TTCACTAGGAGTGGCATATTC 347 1.3 PKD1 5310
    Pkd1 18763 ND000442 CATCTATAAGGGTAGTCTTTC 348 1.4 PKD1 5310
    Pkd1 18763 ND000441 GTTATTACCTCTCTTGTTTCT 349 1.8 PKD1 5310
    Pkd1 18763 ND000446 GTAGTCTACCCTGTCTATTTG 350 2.9 PKD1 5310
    Pkd1 18763 TRCN0000072084 GCCCTGTACCTTTCAACCAAT 351 4.9 PKD1 5310
    Pkd1 18763 ND000443 CATGTCATCGAGTACTCTTTA 352 6.2 PKD1 5310
    Pkd1 18763 TRCN0000304611 CAACTGATGGTGTCCTATATA 353 7.7 PKD1 5310
    Pkd1 18763 TRCN0000072085 CCATCATTGAAGGTGGCTCAT 354 8.9 PKD1 5310
    Pkd1 18763 TRCN0000072087 GCTTCACTACTCTTCCTGCTT 355 9.9 PKD1 5310
    Pkd1 18763 TRCN0000331808 CGCTCGCACTTTCAGCAATAA 356 47.6 PKD1 5310
    Ppm1g 14208 TRCN0000326875 GAGGATGATAAAGACAAAGTA 357 0.3 PPM1G 5496
    Ppm1g 14208 TRCN0000326874 GCTTTCCTCAGCCCATTACAA 358 0.5 PPM1G 5496
    Ppm1g 14208 ND000458 GAGATGATGGTCCCTGGAATG 359 0.8 PPM1G 5496
    Ppm1g 14208 TRCN0000375841 TGACCACAGAGGAAGTCATTA 360 1.1 PPM1G 5496
    Ppm1g 14208 TRCN0000081212 GATGCCTTCTTGGCTATTGAT 361 1.1 PPM1G 5496
    Ppm1g 14208 TRCN0000306418 CCATGGATGGACGAGTCAATG 362 1.2 PPM1G 5496
    Ppm1g 14208 ND000460 TGACGCGATATGGGCAGAACT 363 1.2 PPM1G 5496
    Ppm1g 14208 ND000464 GCTACCATGACTATTGAAGAG 364 1.3 PPM1G 5496
    Ppm1g 14208 ND000462 TGGCAAAGCTTTAGATATGTC 365 2.1 PPM1G 5496
    Ppm1g 14208 ND000465 CATGGATGGACGAGTCAATGG 366 2.9 PPM1G 5496
    Ppm1g 14208 TRCN0000081210 CTTCGGTTATTGTCATCCATT 367 3.0 PPM1G 5496
    Ppm1g 14208 ND000459 TGCCTGTGCTCTGTTGTGTTG 368 3.6 PPM1G 5496
    Ppm1g 14208 ND000461 CAAATTAGTGAGCCCGGTACT 369 6.2 PPM1G 5496
    Ppm1g 14208 TRCN0000081209 GCCTTGTACTGTGCCAAATAT 370 7.1 PPM1G 5496
    Ppm1g 14208 ND000463 CATGACGTGCATCATCATTTG 371 8.5 PPM1G 5496
    Ppp2r2d 52432 ND000490 ACTTCGAGACCCATTTAGAAT 372 0.7 PPP2R2D 55844
    Ppp2r2d 52432 ND000488 CAGAAGATCCCAGCAGTAGAT 373 0.9 PPP2R2D 55844
    Ppp2r2d 52432 TRCN0000080899 GCCACCAATAACTTGTATATA 374 1.0 PPP2R2D 55844
    Ppp2r2d 52432 TRCN0000430828 ATAGTGATCATGAAACATATC 375 1.3 PPP2R2D 55844
    Ppp2r2d 52432 ND000487 ATATGTACGCCGGTCAATTAG 376 1.4 PPP2R2D 55844
    Ppp2r2d 52432 TRCN0000425449 ATGCTCATACATATCACATAA 377 1.5 PPP2R2D 55844
    Ppp2r2d 52432 TRCN0000427220 TCATCTCCACCGTTGAGTTTA 378 1.6 PPP2R2D 55844
    Ppp2r2d 52432 ND000491 GATCTGAGAATTAACCTATGG 379 1.7 PPP2R2D 55844
    Ppp2r2d 52432 TRCN0000080901 CCATTTAGAATTACGGCACTA 380 1.9 PPP2R2D 55844
    Ppp2r2d 52432 TRCN0000080902 CGGTTCAGACAGTGCCATTAT 381 2.0 PPP2R2D 55844
    Ppp2r2d 52432 ND000489 CACCGTTGAGTTTAACTACTC 382 4.0 PPP2R2D 55844
    Ppp2r2d 52432 ND000486 GCTCAATAAAGGCCATTACTC 383 4.9 PPP2R2D 55844
    Ppp2r2d 52432 TRCN0000431278 GAGAATTAACCTATGGCATTT 384 8.3 PPP2R2D 55844
    Ppp2r2d 52432 ND000492 CCACAGTGGTCGATACATGAT 385 16.3 PPP2R2D 55844
    Ppp2r2d 52432 TRCN0000080900 CCCACATCAGTGCAATGTATT 386 17.2 PPP2R2D 55844
    Ppp3cc 19057 ND000512 CCCGAGGTCTAGACCGAATTA 387 0.1 PPP3CC 5533
    Ppp3cc 19057 ND000510 TCACAGTGTGTGGTGATGTTC 388 0.4 PPP3CC 5533
    Ppp3cc 19057 TRCN0000012695 GCTGTATCTATGGAGCTTAAA 389 0.4 PPP3CC 5533
    Ppp3cc 19057 TRCN0000012693 CCTATGAGCAAATCACATTTA 390 0.4 PPP3CC 5533
    Ppp3cc 19057 ND000511 AGGAATGTCGGATCAAGTATT 391 0.7 PPP3CC 5533
    Ppp3cc 19057 TRCN0000012694 CGGCTAACTTTGAAGGAAGTT 392 0.9 PPP3CC 5533
    Ppp3cc 19057 TRCN0000012696 CGGATGAAGAAATGAACGTAA 393 1.2 PPP3CC 5533
    Ppp3cc 19057 ND000508 ACCTAGTAATACTCGCTACCT 394 1.4 PPP3CC 5533
    Ppp3cc 19057 ND000513 CTGTATCTATGGAGCTTAAAG 395 1.6 PPP3CC 5533
    Ppp3cc 19057 ND000515 AGAAATGAACGTAACCGATGA 396 1.8 PPP3CC 5533
    Ppp3cc 19057 ND000514 CAAACAACTTAAACTTGGAGG 397 2.4 PPP3CC 5533
    Ppp3cc 19057 ND000507 TGTAATTCAGTCGCATTTATT 398 2.6 PPP3CC 5533
    Ppp3cc 19057 ND000506 GGACAATTCTTTGACCTGATG 399 4.2 PPP3CC 5533
    Ppp3cc 19057 TRCN0000012697 CGAGGTCTAGACCGAATTAAT 400 4.3 PPP3CC 5533
    Ppp3cc 19057 ND000509 TTCCGTCACTTATTACGATTT 401 4.4 PPP3CC 5533
    Prkab2 108097 ND000529 CTGTGGTTACCAGTCAGCTTG 402 0.2 PRKAB2 5565
    Prkab2 108097 TRCN0000025112 GTATGTCACCACGCTGCTGTA 403 0.4 PRKAB2 5565
    Prkab2 108097 ND000527 CCCTCACCTACTCCAAGTTAT 404 0.7 PRKAB2 5565
    Prkab2 108097 TRCN0000361908 TATGAGTTCACGGAGTTTATT 405 0.7 PRKAB2 5565
    Prkab2 108097 TRCN0000025111 CGCAACCCATCGCTACAAGAA 406 0.8 PRKAB2 5565
    Prkab2 108097 TRCN0000025109 CATCGCTACAAGAAGAAGTAT 407 0.9 PRKAB2 5565
    Prkab2 108097 ND000528 CAATTGGAGCACCAAGATCCC 408 1.1 PRKAB2 5565
    Prkab2 108097 ND000530 AGTGGGTTCATGATCCGTCAG 409 1.1 PRKAB2 5565
    Prkab2 108097 ND000526 ACCGTTATCCGCTGGTCTGAA 410 1.8 PRKAB2 5565
    Prkab2 108097 TRCN0000361952 GATCTGAGGAGAGATTCAAAT 411 2.0 PRKAB2 5565
    Prkab2 108097 TRCN0000361953 CTTAACAAGGACACGAATATT 412 2.3 PRKAB2 5565
    Prkab2 108097 TRCN0000361910 CTCTGATAAAGAGTCATAATG 413 2.6 PRKAB2 5565
    Prkab2 108097 TRCN0000025110 CGCTGCTGTATAAGCCCATCT 414 4.1 PRKAB2 5565
    Prkab2 108097 ND000525 CTTACGGTCAAGAAATGTATG 415 4.8 PRKAB2 5565
    Prkab2 108097 TRCN0000025113 CATTAAGGACAGTGTGATGGT 416 7.0 PRKAB2 5565
    Ptpn2 19255 ND000532 TCCGAACACATGCTGCCATTT 417 0.5 PTPN2 5771
    Ptpn2 19255 TRCN0000029891 GCCAAGATTGACAGACACCTA 418 1.0 PTPN2 5771
    Ptpn2 19255 TRCN0000279253 AGACTATTCTGCAGCTATAAA 419 1.0 PTPN2 5771
    Ptpn2 19255 TRCN0000029893 CCGTTATACTTGGAAATTCGA 420 1.0 PTPN2 5771
    Ptpn2 19255 TRCN0000279254 AGTATCGAATGGGACTTATTC 421 1.2 PTPN2 5771
    Ptpn2 19255 ND000534 TTATATTAATGCCAGCTTAGT 422 1.4 PTPN2 5771
    Ptpn2 19255 ND000531 ATGTTCATGACTTGAGACTAT 423 1.7 PTPN2 5771
    Ptpn2 19255 TRCN0000279329 ATATGATCACAGTCGTGTTAA 424 2.2 PTPN2 5771
    Ptpn2 19255 TRCN0000279252 CGGTGGAAAGAACTTTCTAAA 425 2.2 PTPN2 5771
    Ptpn2 19255 ND000533 CCATATCTCACTTCCATTATA 426 4.7 PTPN2 5771
    Ptpn2 19255 TRCN0000279330 TCTCCTACATGGCCATAATAG 427 5.0 PTPN2 5771
    Ptpn2 19255 TRCN0000029890 CGGTGGAAAGAACTTTCTAAA 428 5.1 PTPN2 5771
    Ptpn2 19255 ND000535 TATCGAATGGGACTTATTCAG 429 5.5 PTPN2 5771
    Ptpn2 19255 TRCN0000029892 CCTGTCTTGTTCTGATGGAAA 430 7.4 PTPN2 5771
    Rbks 71336 ND000536 TCGCTGCAGTCAGTGTACAGG 431 0.4 RBKS 611132
    Rbks 71336 ND000543 GGCCTTCTACCTGGCTTACTA 432 0.6 RBKS 611132
    Rbks 71336 ND000537 CTGCAATGATTCTCCTAGAAC 433 0.9 RBKS 611132
    Rbks 71336 ND000544 AGTGGTGGGTTCCTGCATGAC 434 0.9 RBKS 611132
    Rbks 71336 ND000539 ATATGCCAGCTAGAAATAAGC 435 1.1 RBKS 611132
    Rbks 71336 TRCN0000078936 GTGATGATATGCCAGCTAGAA 436 1.2 RBKS 611132
    Rbks 71336 ND000538 CATATTTCTACAGAGTTTACA 437 1.7 RBKS 611132
    Rbks 71336 TRCN0000078934 TCAATAATGAAGGCCAGAATA 438 1.9 RBKS 611132
    Rbks 71336 ND000545 GCTGCCAGGTTGTGGTCATCA 439 2.7 RBKS 611132
    Rbks 71336 TRCN0000078937 TGATGATATGCCAGCTAGAAA 440 4.0 RBKS 611132
    Rbks 71336 ND000541 CAAGGTTGGCAACGATTCTTT 441 4.1 RBKS 611132
    Rbks 71336 ND000542 GAGCCTGTTCCAAAGCACATT 442 5.0 RBKS 611132
    Rbks 71336 TRCN0000078935 CCAAAGCACATTCCCACTGAA 443 5.7 RBKS 611132
    Rbks 71336 ND000540 CATTAGCCGAGCCAAAGTGAT 444 12.8 RBKS 611132
    Rbks 71336 TRCN0000078933 GCCTCCATAATTGTCAATAAT 445 13.9 RBKS 611132
    Rock1 19877 ND000568 CATACTGTTAGTCGGCTTGAA 446 0.6 ROCK1 6093
    Rock1 19877 ND000567 ATGACATGCAAGCGCAATTGG 447 0.7 ROCK1 6093
    Rock1 19877 ND000565 GCCTACAGGTAGATTAGATTA 448 0.9 ROCK1 6093
    Rock1 19877 ND000569 AGTTCAATTGGTGAGGCATAA 449 1.0 ROCK1 6093
    Rock1 19877 TRCN0000361452 CTAGCAAAGAGAGTGATATTG 450 1.2 ROCK1 6093
    Rock1 19877 TRCN0000022901 CCTGGTTTATGATTTGGATTT 451 1.6 ROCK1 6093
    Rock1 19877 TRCN0000022900 CGGGAGTTACAAGATCAACTT 452 1.7 ROCK1 6093
    Rock1 19877 TRCN0000022902 CCGTGCAAAGTAAGTTACGAT 453 1.8 ROCK1 6093
    Rock1 19877 TRCN0000022899 GCAGAAATAATGAATCGCAAA 454 2.0 ROCK1 6093
    Rock1 19877 ND000566 ATCAAGATCAGATCGTGGAAG 455 2.2 ROCK1 6093
    Rock1 19877 TRCN0000361453 TTCAATTGGTGAGGCATAAAT 456 2.3 ROCK1 6093
    Rock1 19877 TRCN0000022903 GCAGTGTCTCAAATTGAGAAA 457 4.1 ROCK1 6093
    Rock1 19877 TRCN0000361455 TGTGGGATGCTACCTGATAAA 458 4.4 ROCK1 6093
    Rock1 19877 TRCN0000361522 CTACAGGTAGATTAGATTAAT 459 5.6 ROCK1 6093
    Rock1 19877 TRCN0000361521 CAACTTTCTAAGCAGATATAA 460 6.5 ROCK1 6093
    Sbf1 77980 ND000571 CAGTATGTTACTCGTAAGAAG 461 0.2 SBF1 6305
    Sbf1 77980 TRCN0000081099 GCAGTATGTTACTCGTAAGAA 462 0.4 SBF1 6305
    Sbf1 77980 ND000575 TGCTAAGTIGTTTCTAGAACC 463 0.8 SBF1 6305
    Sbf1 77980 ND000570 CGATACTATGACCACCGAATG 464 0.8 SBF1 6305
    Sbf1 77980 TRCN0000081101 CGAGAGGAATCCACCAACTTT 465 0.9 SBF1 6305
    Sbf1 77980 TRCN0000081102 GCGATACTATGACCACCGAAT 466 1.5 SBF1 6305
    Sbf1 77980 ND000578 CTAACTTATTGTGGTGTCATG 467 1.5 SBF1 6305
    Sbf1 77980 ND000574 TCTTGCTGGACTCTGATTATG 468 1.6 SBF1 6305
    Sbf1 77980 ND000572 GGCTAGATGAGGGCACAATTC 469 2.2 SBF1 6305
    Sbf1 77980 ND000573 GAAGACAACACGTCGCGTTTA 470 3.1 SBF1 6305
    Sbf1 77980 ND000577 TACGGAATTGCATCTCCTATG 471 3.2 SBF1 6305
    Sbf1 77980 TRCN0000081098 CACGCGGACATCTATGACAAA 472 4.8 SBF1 6305
    Sbf1 77980 ND000579 TTACCACATACCGCGTCATCT 473 5.6 SBF1 6305
    Sbf1 77980 TRCN0000081100 CCCTACAGCAATGTGTCCAAT 474 6.0 SBF1 6305
    Sbf1 77980 ND000576 GACTTTGTCGTCCGCATGATG 475 6.9 SBF1 6305
    Smad2 17126 ND000208 AGATCAGTGGGACACAACAGG 476 0.4 SMAD2 4087
    Smad2 17126 TRCN0000089336 TGGTGTTCAATCGCATACTAT 477 1.0 SMAD2 4087
    Smad2 17126 ND000205 GTAATTACATCCCAGAAACAC 478 1.1 SMAD2 4087
    Smad2 17126 TRCN0000089334 CGGTTAGATGAGCTTGAGAAA 479 1.2 SMAD2 4087
    Smad2 17126 TRCN0000089333 CCAGTAGTAGTGCCTGAAGTA 480 1.2 SMAD2 4087
    Smad2 17126 ND000207 TAACCCGAATGTGCACCATAA 481 1.2 SMAD2 4087
    Smad2 17126 ND000199 CCCAACTGTAACCAGAGATAC 482 1.4 SMAD2 4087
    Smad2 17126 TRCN0000089335 CCACTGTAGAAATGACAAGAA 483 1.5 SMAD2 4087
    Smad2 17126 ND000200 CCTCCGTCGTAGTATTCATGT 484 1.9 SMAD2 4087
    Smad2 17126 ND000201 GCCAGTGGTGAAGAGACTTCT 485 1.9 SMAD2 4087
    Smad2 17126 ND000203 CTCGGCACACGGAGATTCTAA 486 6.7 SMAD2 4087
    Smad2 17126 ND000204 GACAGTATCCCAAAGGTTATT 487 7.1 SMAD2 4087
    Smad2 17126 ND000202 GAGTGCGCTTGTATTACATAG 488 7.1 SMAD2 4087
    Smad2 17126 TRCN0000089337 CTAAGTGATAGTGCAATCTTT 489 19.3 SMAD2 4087
    Smad2 17126 ND000206 TGCCTAAGTGATAGTGCAATC 490 30.3 SMAD2 4087
    Socs1 12703 ND000214 TTTCGAGCTGCTGGAGCACTA 491 0.6 SOCS1 8651
    Socs1 12703 ND000219 TCGAGCTGCTGGAGCACTACG 492 1.2 SOCS1 8651
    Socs1 12703 TRCN0000231240 TCGCCAACGGAACTGCTTCTT 493 1.4 SOCS1 8651
    Socs1 12703 ND000218 ACTTCTGGCTGGAGACCTCAT 494 1.5 SOCS1 8651
    Socs1 12703 TRCN0000067420 GCGAGACCTTCGACTGCCTTT 495 1.7 SOCS1 8651
    Socs1 12703 TRCN0000067418 CGACACTCACTTCCGCACCTT 496 1.8 SOCS1 8651
    Socs1 12703 ND000220 CTACCTGAGTTCCTTCCCCTT 497 1.8 SOCS1 8651
    Socs1 12703 TRCN0000231238 TTCCGCTCCCACTCCGATTAC 498 1.8 SOCS1 8651
    Socs1 12703 TRCN0000231241 TAACCCGGTACTCCGTGACTA 499 1.9 SOCS1 8651
    Socs1 12703 ND000216 TACTCCGTGACTACCTGAGTT 500 2.4 SOCS1 8651
    Socs1 12703 ND000211 CTTCCGCTCCCACTCCGATTA 501 2.6 SOCS1 8651
    Socs1 12703 TRCN0000067422 GCGCGACAGTCGCCAACGGAA 502 2.7 SOCS1 8651
    Socs1 12703 TRCN0000231239 TGGACGCCTGCGGCTTCTATT 503 2.9 SOCS1 8651
    Socs1 12703 TRCN0000067419 CGCATCCCTCTTAACCCGGTA 504 3.4 SOCS1 8651
    Socs1 12703 ND000212 TACATATTCCCAGTATCTTTG 505 3.6 SOCS1 8651
    Socs1 12703 TRCN0000231242 GCGCCTTATTATTTCTTATTA 506 4.1 SOCS1 8651
    Socs1 12703 TRCN0000067421 CCGTGACTACCTGAGTTCCTT 507 5.8 SOCS1 8651
    Socs1 12703 ND000215 GGAGGGTCTCTGGCTTCATTT 508 7.8 SOCS1 8651
    Socs1 12703 ND000213 TTCGCGCTCAGCGTGAAGATG 509 8.4 SOCS1 8651
    Socs1 12703 ND000217 ATCCCTCTTAACCCGGTACTC 510 8.5 SOCS1 8651
    Socs3 12702 ND000222 CGAGAAGATTCCGCTGGTACT 511 0.3 SOCS3 9021
    Socs3 12702 TRCN0000067472 GCTGCAGGAGAGCGGATTCTA 512 0.4 SOCS3 9021
    Socs3 12702 TRCN0000231180 GGCTAGGAGACTCGCCTTAAA 513 0.7 SOCS3 9021
    Socs3 12702 TRCN0000067468 GCTAGGAGACTCGCCTTAAAT 514 0.8 SOCS3 9021
    Socs3 12702 ND000227 GAGAGCTTACTACATCTATTC 515 0.9 SOCS3 9021
    Socs3 12702 ND000221 GGGAGTTCCTGGATCAGTATG 516 1.0 SOCS3 9021
    Socs3 12702 TRCN0000067470 CAAGAGAGCTTACTACATCTA 517 1.1 SOCS3 9021
    Socs3 12702 TRCN0000231179 CAGTATGATGCTCCACTTTAA 518 1.2 SOCS3 9021
    Socs3 12702 ND000223 CAAGCTGGTGCACCACTACAT 519 1.3 SOCS3 9021
    Socs3 12702 ND000224 ACCTGGACTCCTATGAGAAAG 520 1.4 SOCS3 9021
    Socs3 12702 TRCN0000067471 CTTCTTCACGTTGAGCGTCAA 521 1.6 SOCS3 9021
    Socs3 12702 ND000228 TCGGGAGTTCCTGGATCAGTA 522 1.7 SOCS3 9021
    Socs3 12702 ND000226 TGCAGGAGAGCGGATTCTACT 523 1.9 SOCS3 9021
    Socs3 12702 ND000225 CCTGGTGGGACAATACCTTTG 524 3.3 SOCS3 9021
    Socs3 12702 TRCN0000067469 GATCAGTATGATGCTCCACTT 525 4.6 SOCS3 9021
    Socs3 12702 TRCN0000231176 TCTTCACGTTGAGCGTCAAGA 526 4.7 SOCS3 9021
    Socs3 12702 TRCN0000231177 CGCTTCGACTGTGTACTCAAG 527 4.9 SOCS3 9021
    Socs3 12702 ND000229 GGAGCAAAAGGGTCAGAGGGG 528 5.3 SOCS3 9021
    Stk17b 98267 ND000590 AGTGGGACTTTGGAAGCTTGT 529 0.3 STK17B 9262
    Stk17b 98267 ND000597 CATCTGGACTGACTCGGAAAT 530 0.5 STK17B 9262
    Stk17b 98267 ND000596 ATGCTGCGGGTGGAGAAATTT 531 0.6 STK17B 9262
    Stk17b 98267 ND000588 TATCTGAATATTTCTCAAGTG 532 0.6 STK17B 9262
    Stk17b 98267 ND000593 TTTACCTGAGTTAGCCGAAAT 533 0.7 STK17B 9262
    Stk17b 98267 ND000589 GTTAACTCATACATCACCATT 534 1.1 STK17B 9262
    Stk17b 98267 ND000594 CCTATACCATAACTCTATTAC 535 1.3 STK17B 9262
    Stk17b 98267 ND000592 CTCAACTATGATCCCATTACC 536 1.3 STK17B 9262
    Stk17b 98267 ND000591 AGACCTCCAAGTCCTCCTGTA 537 1.4 STK17B 9262
    Stk17b 98267 TRCN0000024255 GCTGTGGTTAGACAATGTATA 538 1.6 STK17B 9262
    Stk17b 98267 ND000595 TATTGGCATAATAGCGTATAT 539 3.6 STK17B 9262
    Stk17b 98267 TRCN0000024256 GCTTGTTTCATCCTGAGGAAA 540 4.0 STK17B 9262
    Stk17b 98267 TRCN0000024258 TCCTCAACTATGATCCCATTA 541 4.2 STK17B 9262
    Stk17b 98267 TRCN0000024254 GCAGAAGCTAAGGACGAATTT 542 4.4 STK17B 9262
    Stk17b 98267 TRCN0000024257 CAGAATAACATTGTTCACCTT 543 6.4 STK17B 9262
    Tnk1 83813 ND000599 TGCCCAGCGCAGACTTAATGA 544 0.3 TNK1 8711
    Tnk1 83813 TRCN0000023704 CGTGACACTCTGGGAAATGTT 545 0.6 TNK1 8711
    Tnk1 83813 ND000602 GTGTCCCACCATATCTCATCC 546 0.7 TNK1 8711
    Tnk1 83813 ND000600 AGTAGCAATACCGGATCACTG 547 0.7 TNK1 8711
    Tnk1 83813 TRCN0000023706 GCGGGAAGTATCTGTCATGAT 548 0.8 TNK1 8711
    Tnk1 83813 ND000603 AGAGGATGCGAGGCATTTCCA 549 1.1 TNK1 8711
    Tnk1 83813 ND000601 GGACAGAGAGAAGGCAACGTT 550 1.1 TNK1 8711
    Tnk1 83813 TRCN0000361891 AGAATTGGGTGTACAAGATAC 551 1.3 TNK1 8711
    Tnk1 83813 TRCN0000023707 CCACCTATTATCTGCAACTCT 552 1.6 TNK1 8711
    Tnk1 83813 TRCN0000023705 GCCTCTGATGTGTGGATGTTT 553 1.7 TNK1 8711
    Tnk1 83813 TRCN0000361890 TGCAGAGGATGCGAGGCATTT 554 1.8 TNK1 8711
    Tnk1 83813 TRCN0000361889 TGGCGTGACACTCTGGGAAAT 555 2.0 TNK1 8711
    Tnk1 83813 TRCN0000023708 CAGACTTAATGAAGCCCTGAA 556 5.2 TNK1 8711
    Tnk1 83813 TRCN0000361892 GTGTTGTACATCGAGGGTTAT 557 5.2 TNK1 8711
    Tnk1 83813 ND000598 CCAGAACTTCGGCGTACAAGA 558 7.6 TNK1 8711
    Trpm7 58800 ND000607 GAAGTATCAGCGGTATCATTT 559 0.4 TRPM7 54822
    Trpm7 58800 TRCN0000274774 ATGGATTGTTATCGCTTATAT 560 0.7 TRPM7 54822
    Trpm7 58800 ND000606 GCTTGGAAAGGGTCTTATTAA 561 0.9 TRPM7 54822
    Trpm7 58800 ND000608 ATTGAATCCCTTGAGCAAATT 562 0.9 TRPM7 54822
    Trpm7 58800 TRCN0000274712 CCTTATCAAACCCTATTGAAT 563 1.1 TRPM7 54822
    Trpm7 58800 TRCN0000274773 CCAAAGATCAAGAACCCATTT 564 1.2 TRPM7 54822
    Trpm7 58800 ND000604 TAGAGGTAATGTTCTCATTGA 565 1.2 TRPM7 54822
    Trpm7 58800 ND000610 ACCGGATTGGTTACGAGATAG 566 1.5 TRPM7 54822
    Trpm7 58800 TRCN0000274772 ACCTGGTGCAGGACCATTAAC 567 1.7 TRPM7 54822
    Trpm7 58800 ND000605 TAGACTTTCTAGCCGTAAATC 568 2.9 TRPM7 54822
    Trpm7 58800 TRCN0000274711 CTAGACTTTCTAGCCGTAAAT 569 3.1 TRPM7 54822
    Trpm7 58800 TRCN0000023957 CCTCAGGATGAGTCATCAGAT 570 3.5 TRPM7 54822
    Trpm7 58800 TRCN0000023956 CCTGGTATAAGGTCATATTAA 571 4.9 TRPM7 54822
    Trpm7 58800 TRCN0000023955 GCTCAGAATCTTATTGATGAT 572 5.3 TRPM7 54822
    Trpm7 58800 ND000609 GCCCTAACAGTAGATACATTG 573 5.9 TRPM7 54822
    Vamp7 20955 TRCN0000115068 CTTACTCACATGGCAATTATT 574 0.6 VAMP7 6845
    Vamp7 20955 TRCN0000380436 GCACAACTGAAGCATCACTCT 575 0.8 VAMP7 6845
    Vamp7 20955 TRCN0000336075 GCACAAGTGGATGAACTGAAA 576 0.9 VAMP7 6845
    Vamp7 20955 TRCN0000336077 TTACGGTTCAAGAGCACAAAC 577 1.0 VAMP7 6845
    Vamp7 20955 TRCN0000380733 TAAGAGCCTAGACAAAGTGAT 578 1.0 VAMP7 6845
    Vamp7 20955 ND000255 AGCCATGTGTATGAAGAATAT 579 1.2 VAMP7 6845
    Vamp7 20955 ND000258 TCCAGGAGCCCATACAAGTAA 580 1.4 VAMP7 6845
    Vamp7 20955 ND000256 ATAAACTAACTTACTCACATG 581 1.5 VAMP7 6845
    Vamp7 20955 TRCN0000336014 GCCGCCACATTTCGTTGTAAA 582 1.8 VAMP7 6845
    Vamp7 20955 TRCN0000353419 GCACTTCCTTATGCTATGAAT 583 1.9 VAMP7 6845
    Vamp7 20955 TRCN0000115066 GCCTTAAGATATGCAATGTTA 584 2.2 VAMP7 6845
    Vamp7 20955 ND000257 CTGAAAGGAATAATGGTCAGA 585 4.0 VAMP7 6845
    Vamp7 20955 ND000259 CTCCTTGTAAATGATACACAA 586 9.8 VAMP7 6845
    Vamp7 20955 TRCN0000353291 CTTTGCCTGTCATATAGTTTG 587 10.5 VAMP7 6845
    Vamp7 20955 TRCN0000115069 TCGAGCCATGTGTATGAAGAA 588 11.3 VAMP7 6845
    Yes1 22612 ND000617 ATCCCTAGCAATTACGTAGTG 589 0.5 YES1 7525
    Yes1 22612 TRCN0000339152 TGGTTATATCCCTAGCAATTA 590 0.5 YES1 7525
    Yes1 22612 ND000614 TATGCTTCACTCGGCATGTTT 591 0.6 YES1 7525
    Yes1 22612 ND000616 ATTCCAGATACGGTTACTCAA 592 0.6 YES1 7525
    Yes1 22612 ND000613 TTTAAGAAGGGTGAACGATTT 593 0.7 YES1 7525
    Yes1 22612 ND000612 CACGACCAGAGCTCAGTTTGA 594 0.8 YES1 7525
    Yes1 22612 ND000615 CAGGTATGGTAAACCGTGAAG 595 0.8 YES1 7525
    Yes1 22612 ND000611 GGAGTGGAACATGCTACAGTT 596 1.0 YES1 7525
    Yes1 22612 ND000618 CCTCATTCTCAGTGGTGTCAA 597 2.6 YES1 7525
    Yes1 22612 ND000619 TCGAGAATCATTGCGACTAGA 598 2.8 YES1 7525
    Yes1 22612 TRCN0000339083 CCAGGTACAATGATGCCAGAA 599 2.8 YES1 7525
    Yes1 22612 TRCN0000339150 GCGGAAAGATTACTTCTGAAT 600 3.9 YES1 7525
    Yes1 22612 TRCN0000023616 GCTGCTCTGTATGGTCGATTT 601 4.1 YES1 7525
    Yes1 22612 TRCN0000023618 CCTTGTATGATTATGAAGCTA 602 5.4 YES1 7525
    Yes1 22612 TRCN0000023617 GCCAGTCATTATGGAGTGGAA 603 9.7 YES1 7525
  • shRNAs demonstrating an at least ≥3 shRNAs fold enrichment in tumor relative to spleen indicate a more active target sequence region.
  • In some aspects, the nucleic acids of the compositions encode the shRNA sequences targeting the human Ppp2r2d and Cb1b sequences provided in Table 2a.
  • TABLE 2a
    # Gene Human shRNA Target Sequence
    1 Ppp2r2d CCCGCACCAGTGCAACGTGTT
    (SEQ ID NO: 636)
    2 Ppp2r2d TCATAGTGGGCGGTACATGAT
    (SEQ ID NO: 637)
    3 Ppp2r2d GAGAATTAATTTATGGCACTT
    (SEQ ID NO: 638)
    4 Ppp2r2d CCATTTAGGATCACGGCGCTA
    (SEQ ID NO: 639)
    5 Ppp2r2d ATAGTGATCATGAAACATATC
    (SEQ ID NO: 375)
    6 Ppp2r2d GCCACCAATAACTTGTACATA
    (SEQ ID NO: 640)
    7 Ppp2r2d CGGTTCGGATAGCGCCATCAT
    (SEQ ID NO: 641)
    8 Ppp2r2d TCATTTCCACCGTTGAGTTTA
    (SEQ ID NO: 642)
    9 Ppp2r2d ATGCTCACACATATCATATAA
    (SEQ ID NO: 643)
    1 Cblb CGGGCAATAAGACTCTTTAA
    (SEQ ID NO: 644)
    2 Cblb TGCCCAGGTCCAGTTCCATTTC
    (SEQ ID NO: 645)
    3 Cblb TCCTGATTTAACTGGATTATG
    (SEQ ID NO: 646)
    4 Cblb ATCAAACATCCCTGACTTAAG
    (SEQ ID NO: 647)
    5 Cblb CTACACCTCATGACCATATAA
    (SEQ ID NO: 648)
    6 Cblb TACACCTCATGACCATATAAA
    (SEQ ID NO: 649)
    7 Cblb TCAGTGAGAATGAGTACTTTA
    (SEQ ID NO: 650)
    8 Cblb CCTGACTTAAGCATATATTTA
    (SEQ ID NO: 651)
    9 Cblb TCTACATTGATAGCCTTATGA
    (SEQ ID NO: 652)
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Ppp2r2d target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 372, 373, 374, 375, 376, 377, 378, 378, 379, 380, 381, 382, 383, 384, 385, or 386.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Pp2r2d sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 372, 373, 374, 375, 376, 377, 378, 378, 379, 380, 381, 382, 383, 384, 385, or 386.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Eif2ak3 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 or 147.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Eif2ak3 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 or 147.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Arhgap5 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Arhgap5 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Smad2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, or 490.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Smad2 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, or 490.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Akap81 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Akap81 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Rbks target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, or 445.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Rbks sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, or 445.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Egr2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Egr2 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Dgka target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Dgka sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Cb1b target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Cb1b sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Mdfic target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, or 299.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Mdfic sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, or 299.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Entpdl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, or 162.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Entpdl sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, or 162.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Vamp7 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, or 587.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Vamp7sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, or 587.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Hipkl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, or 222.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Hipkl sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, or 222.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Nuak2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, or 329.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Nuak2 sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, or 329.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Alk target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Alk sequence that corresponds to a murine target sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Pdzklipltarget sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, or 341.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Pdzkliplsequence that corresponds to a murine target sequence set forth in SEQ ID NO: 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, or 341.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Blvrb target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 52, 53, 54, 55, 56 or 57.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Blvrb that corresponds to a murine target sequence set forth in SEQ ID NO: 52, 53, 54, 55, 56 or 57.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Cdkn2a target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 83, 84, 85, 86 or 87.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Cdkn2a that corresponds to a murine target sequence set forth in SEQ ID NO: 83, 84, 85, 86 or 87.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Fllr target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 175, 176 or 177.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human F11r that corresponds to a murine target sequence set forth in SEQ ID NO: 175, 176 or 177.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Fyn target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 187, 191 or 192.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Fyn that corresponds to a murine target sequence set forth in SEQ ID NO: 187, 191 or 192.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Grk6 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 204, 205, 206 or 207.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Grk6 that corresponds to a murine target sequence set forth in SEQ ID NO: 204, 205, 206 or 207.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Inpp5b target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 232, 234, 235, 236 or 237.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Inpp5b that corresponds to a murine target sequence set forth in SEQ ID NO: 232, 234, 235, 236 or 237.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to an Impk target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 248, 249, 250, 251 or 252.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Impk that corresponds to a murine target sequence set forth in SEQ ID NO: 248, 249, 250, 251 or 252.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Jun target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 263, 264, 265, 266, 267, 268 or 269.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Jun that corresponds to a murine target sequence set forth in SEQ ID NO: 263, 264, 265, 266, 267, 268 or 269.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Mast2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 281, 282, 283 or 284.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Mast2 that corresponds to a murine target sequence set forth in SEQ ID NO: 281, 282, 283 or 284.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Nptxr target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 311, 312, 313 or 314.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Nptxr that corresponds to a murine target sequence set forth in SEQ ID NO: 311, 312, 313 or 314.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Pkdl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 351, 352, 353, 354, 355 or 356.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Pkdl that corresponds to a murine target sequence set forth in SEQ ID NO: 351, 352, 353, 354, 355 or 356.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Ppm1g target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 367, 368, 369, 370 or 371.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Ppm1g that corresponds to a murine target sequence set forth in SEQ ID NO: 367, 368, 369, 370 or 371.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Ppp3cc target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 399, 400 or 401.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Ppp3cc that corresponds to a murine target sequence set forth in SEQ ID NO: 399, 400 or 401.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Prkab2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 414, 415 or 416.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Prkab2 that corresponds to a murine target sequence set forth in SEQ ID NO: 414, 415 or 416.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Ptpn2 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 426, 427, 428, 429 or 430.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Ptpn2 that corresponds to a murine target sequence set forth in SEQ ID NO: 426, 427, 428, 429 or 430.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Rockl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 457, 458, 459 or 460.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Rockl that corresponds to a murine target sequence set forth in SEQ ID NO: 457, 458, 459 or 460.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Sbfl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 470, 471, 472, 473, 474 or 475.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Sbfl that corresponds to a murine target sequence set forth in SEQ ID NO: 470, 471, 472, 473, 474 or 475.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Socsl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 504, 505, 506, 507, 508, 509 or 510.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Socsl that corresponds to a murine target sequence set forth in SEQ ID NO: 504, 505, 506, 507, 508, 509 or 510.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Socs3 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 524, 525, 526, 527 or 528.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Socs3 that corresponds to a murine target sequence set forth in SEQ ID NO: 524, 525, 526, 527 or 528.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Stk17b target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 539, 540, 541, 542 or 543.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Stk17b that corresponds to a murine target sequence set forth in SEQ ID NO: 539, 540, 541, 542 or 543.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Tnkl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 556, 557 or 558.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Tnkl that corresponds to a murine target sequence set forth in SEQ ID NO: 556, 557 or 558.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Trpm7 target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 569, 570, 571, 572 or 573.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Trpm7 that corresponds to a murine target sequence set forth in SEQ ID NO: 569, 570, 571, 572 or 573.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding shRNA sequences complementary to a Yesl target sequence identical to at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides set forth in SEQ ID NO: 600, 601, 602 or 603.
  • In other embodiments, the disclosure provides isolated nucleic acids encoding a shRNA comprising a sequence complementary to a human Yesl that corresponds to a murine target sequence set forth in SEQ ID NO: 600, 601, 602 or 603.
  • In any embodiment, a human sequence that corresponds to a murine target sequence is a sequence which perfectly corresponds to the human gene sequence, and for example, can have none, 1, 2, 3 or 4 nucleotide mismatches with the at least 12, at least 15, at least 20, or at least 25 contiguous nucleotides of the selected murine target sequence.
  • An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, cDNA, or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, adeno-associated virus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.
  • In calculating percent sequence identity, two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It also will be appreciated that a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over each aligned region. It is noted that the percent identity value is usually rounded to the nearest integer. For example, 78.1%, 78.2%, 78.3%, and 78.4% are rounded down to 78%, while 78.5%, 78.6%, 78.7%, 78.8%, and 78.9% are rounded up to 79%. It is also noted that the length of the aligned region is always an integer.
  • As used herein, the term “percent sequence identity” refers to the degree of identity between any given query sequence and a subject sequence. A percent identity for any query nucleic acid or amino acid sequence, e.g., a transcription factor, relative to another subject nucleic acid or amino acid sequence can be determined as follows.
  • As used herein, the term “complementary nucleotide sequence,” also known as an “antisense sequence,” refers to a sequence of a nucleic acid that is completely complementary to the sequence of a “sense” nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). Herein, nucleic acid molecules are provided that comprise a sequence complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides or an entire gene coding strand, or to only a portion thereof.
  • As used herein, the term “correspond to a nucleotide sequence” refers to a nucleotide sequence of a nucleic acid encoding an identical sequence. In some instances, when antisense nucleotides (nucleic acids) or siRNA's (small inhibitory RNA) hybridize to a target sequence a particular antisense or small inhibitory RNA (siRNA) sequence is substantially complementary to the target sequence, and thus will specifically bind to a portion of an mRNA encoding polypeptide. As such, typically the sequences of those nucleic acids will be highly complementary to the mRNA target sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence. In many instances, it may be desirable for the sequences of the nucleic acids to be exact matches, i.e. be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch. Highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.
  • As used herein, the term “vector” refers to any viral or non-viral vector, as well as any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form that may or may not be self transmissible or mobilizable, and that can transform prokaryotic or eukaryotic host cells either by integration into the cellular genome or which can exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). Any vector known in the art is envisioned for use in the practice of this invention.
  • Vectors can be viral vectors or non-viral vectors. Should viral vectors be used, it is preferred the viral vectors are replication defective, which can be achieved for example by removing all viral nucleic acids that encode for replication. A replication defective viral vector will still retain its infective properties and enters the cells in a similar manner as a replicating adenoviral vector, however once admitted to the cell a replication defective viral vector does not reproduce or multiply. Vectors also encompass liposomes and nanoparticles and other means to deliver DNA molecule to a cell.
  • The term “viral vectors” refers to the use of viruses, or virus-associated vectors as carriers of a nucleic acid construct into a cell. Constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral and lentiviral vectors, for infection or transduction into cells. The vector may or may not be incorporated into the cell's genome.
  • “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system, Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. Thus, an “Expression vector” is a specialized vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • In some aspects, the disclosure provides modified cells that harbor vectors capable of expressing the shRNA described herein and further modified to express a CAR. In one aspect the shRNA and the CAR are expressed on the same vector. In another aspect, the shRNA and the CAR are expressed on separate vectors.
  • In some embodiments, the modified cells described herein are immunoresponsive cells. In some aspects, the immunoresponsive cells express at least one of an antigen-recognizing receptor. In any aspect, the immunoresponsive cells express at least one of an tumor specific antigen-recognizing receptor. In some aspects, tumor cell antigen specific T cells, NKT cells, TIL, CTL cells or other immunoresponsive cells are used. Non-limiting examples of immunoresponsive cells include T cells, such as, for example, αβ-TCR+ T cells (e.g., CD8+ T cells or CD4+ T cells) γδ-TCR+ T cells, tumor-infiltrating lymphocytes (TIL), Natural Killer T cells (NKT), a cytotoxic T lymphocytes (CTL), and a CD4 T cells.
  • Compositions comprising the immunoresponsive cells of the invention (e.g., T cells, NKT cells, TILs, CTL cells, or their progenitors) can be provided systemically or directly to a subject for the treatment of a cancer. In one embodiment, cells of the invention are directly injected into an organ of interest (e.g., an organ affected by a cancer). Alternatively, compositions comprising genetically modified immunoresponsive cells are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature). Expansion and differentiation agents can be provided prior to, during or after administration of the cells to increase production of T cells, NKT cells, TILs, CTL cells in vitro or in vivo.
  • The modified immunoresponsive cells can be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). Usually, at least 1×105 cells will be administered, eventually reaching 1×1010, or more. Immunoresponsive cells of the invention can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of genetically modified immunoresponsive cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Preferable ranges of purity in populations comprising genetically modified immunoresponsive cells are about 50 to about 55%, about 55 to about 60%, and about 65 to about 70%. More preferably the purity is about 70 to about 75%, about 75 to about 80%, about 80 to about 85%; and still more preferably the purity is about 85 to about 90%, about 90 to about 95%, and about 95 to about 100%. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage).
  • The cells can be introduced by injection, catheter, or the like. If desired, factors can also be included, including, but not limited to, interleukins, e.g. IL-2, IL-3, IL-6, and IL-11, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM-CSF, interferons, e.g. .gamma.-interferon and erythropoietin.
  • Compositions of the invention include pharmaceutical compositions comprising the immunoresponsive cells of the invention or their progenitors and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, immunoresponsive cells, or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject.
  • Chimeric Antigen Receptors
  • In some instances, the invention provides chimeric antigen receptors (CARs) comprising an antigen binding domain directed to a tumor cell antigen. A CAR is an artificially constructed hybrid protein or polypeptide containing an extracellular portion that recognizes a tumor cell antigen (e.g., the antigen binding domains of an antibody (scFv) and a cytoplasmic signaling domain derived from the T cell receptor and costimulatory domain. (Kalos M, et al., Sci Transl Med. 2011 Aug. 10; 3(95)) Kalos et al. describes the generation of CART cells that target CD19 and demonstrates the CAR modified T-cells mediated potent antitumor effect in chronic lymphocytic leukemia patients. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC—restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The CAR-modified T-cells have the potential to replicate in vivo and long term persistence allows for sustained tumor control and obviate the need for repeated infusions of antibody. (Kalos M, et al., Sci Transl Med. 2011 Aug. 10; 3(95)) The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains. CAR-modified T cells are described in detail in WO2012/079000 and WO2012/09999 and in Milone et al. 2009 Mol. Ther. 17:1453.
  • A CAR combines the binding site of a molecule that recognizes an antigen being targeted (i.e., an “antigen binding domain”) with one or more domains of conventional immune receptors responsible for initiating signal transduction that leads to lymphocyte activation (e.g., the “stimulatory domain” or “signaling domain”).
  • In some embodiments, the binding portion used is derived from the structure of the Fab (antigen binding) fragment of a monoclonal antibody (mAb) that has high affinity for the tumor antigen being targeted. Because the Fab is the product of two genes, the corresponding sequences are usually combined via a short linker fragment that allows the heavy-chain to fold over the light-chain derived peptides into their native configuration, creating a single-chain fragment variable (scFv) region.
  • Fv or (scFv) antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
  • In some embodiments, the binding portion used is derived from a cytoplasmic signaling domain derived from T cell receptor and costimulatory molecules.
  • In some embodiments, the signaling portion of CARs contains usually the intracellular domains of the zeta (ζ) chain of the TCR/CD3 complex25 or, less commonly, of the gamma (γ) chain of the immunoglobulin receptor FccRI26, 27 or the CD3-epsilon (ε) chain,28 with the transmembrane region being derived from the same molecule.
  • In some aspects, the CARs comprise an antigen binding domain, a transmembrane domain, a stimulatory domain, and a co-stimulatory domain. Further embodiments of the invention provide related nucleic acids, recombinant expression vectors, host cells, populations of cells, antibodies, or antigen binding portions thereof, and pharmaceutical compositions relating to the CARs of the invention.
  • In one aspect, the antigen binding domain binds to a tumor cell antigen. The term “tumor cell antigen” or “tumor antigen” as used herein refers to any polypeptide expressed by a tumor that is capable of inducing an immune response. Non-limiting examples of tumor antigens include, for example, prostate-specific membrane antigen (PSMA), Carcinoembryonic Antigen (CEA), CD19, CD20, CD22, ROR1, mesothelin, CD333/IL3Ra, c-Met, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, ERBB2, BIRC5, CEACAM5, WDR46, BAGE, CSAG2, DCT, MAGED4, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE5, IL13RA2, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA9, MAGEA10, MAGEA12, MAGEB1, MAGEB2, MAGEC2, TP53, TYR, TYRP1, SAGE1, SYCP1, SSX2, SSX4, KRAS, PRAME, NRAS, ACTN4, CTNNB1, CASP8, CDC27, CDK4, EEF2, FN1, HSPA1B, LPGAT1, ME1, HHAT, TRAPPC1, MUM3, MYO1B, PAPOLG, PTPRK, TPI1, ADFP, AFP, AIM2, ANXA2, ART4, CLCA2, CPSF1, PPIB, EPHA2, EPHA3, FGF5, CA9, TERT, MGAT5, CEL, F4.2, CAN, ETV6, BIRC7, CSF1, OGT, MUC1, MUC2, MUM1, CTAG1A, CTAG2, CTAG, MRPL28, FOLH1, RAGE, SFMBT1, KAAG1, SART1, TSPYL1, SART3, SOX10, TRG, WT1, TACSTD1, SILV, SCGB2A2, MC1R, MLANA, GPR143, OCA2, KLK3, SUPT7L, ARTC1, BRAF, CASP5, CDKN2A, UBXD5, EFTUD2, GPNMB, NFYC, PRDX5, ZUBR1, SIRT2, SNRPD1, HERV-K-MEL, CXorf61, CCDCl10, VENTXP1, SPA17, KLK4, ANKRD30A, RAB38, CCND1, CYP1B1, MDM2, MMP2, ZNF395, RNF43, SCRN1, STEAP1, 707-AP, TGFBR2, PXDNL, AKAP13, PRTN3, PSCA, RHAMM, ACPP, ACRBP, LCK, RCVRN, RPS2, RPL10A, SLC45A3, BCL2L1, DKK1, ENAH, CSPG4, RGS5, BCR, BCR-ABL, ABL-BCR, DEK, DEK-CAN, ETV6-AML1, LDLR-FUT, NPM1-ALK1, PML-RARA, SYT-SSX1, SYT-SSX2, FLT3, ABL1, AML1, LDLR, FUT1, NPM1, ALK, PML1, RARA, SYT, SSX1, MSLN, UBE2V1, HNRPL, WHSC2, EIF4EBP1, WNK2, OAS3, BCL-2, MCL1, CTSH, ABCC3, BST2, MFGE8, TPBG, FMOD, XAGE1, RPSA, COTL1, CALR3, PA2G4, EZH2, FMNL1, HPSE, APC, UBE2A, BCAP31, TOP2A, TOP2B, ITGB8, RPA1, ABI2, CCNI, CDC2, SEPT2, STAT1, LRP1, ADAM17, JUP, DDR1, ITPR2, HMOX1, TPM4, BAAT, DNAJC8, TAPBP, LGALS3BP, PAGE4, PAK2, CDKN1A, PTHLH, SOX2, SOX11, TRPM8, TYMS, ATIC, PGK1, SOX4, TOR3A, TRGC2, BTBD2, SLBP, EGFR, IER3, TTK, LY6K, IGF2BP3, GPC3, SLC35A4, HSMD, H3F3A, ALDH1A1, MFI2, MMP14, SDCBP, PARP12, MET, CCNB1, PAX3-FKHR, PAX3, FOXO1, XBP1, SYND1, ETV5, HSPA1A, HMHA1, TRIM68 and any combination thereof.
  • The present invention relates generally to the use of T cells genetically modified to stably express a shRNA of the invention and a desired CAR. T cells expressing a CAR are generally referred to as CAR T cells. T cells expressing a CAR are referred to herein as CAR T cells or CAR modified T cells. Preferably, the cell can be genetically modified to stably express an antibody binding domain on its surface, conferring novel antigen specificity that is WIC independent. In some instances, the T cell is genetically modified to stably express a CAR that combines an antigen recognition domain of a specific antibody with an intracellular stimulatory domain (e.g., signaling domain). Thus, in addition to an antigen binding domain the CAR can include the intracellular domains of the zeta (ζ) chain of the TCR/CD3 complex, the gamma (γ) chain of the immunoglobulin receptor FccRI26, 27 or the CD3-epsilon (ε) chain. The CAR can also include a transmembrane region being from the same molecules or other type I transmembrane proteins such as CD4, CD8 and CD28.
  • In one embodiment, the CAR of the invention comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain.
  • In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In another embodiment, the cytoplasmic domain can be designed to comprise a stimulatory domain and a costimulatory domain.
  • A CAR can include intracytoplasmatic portion of co-stimulatory molecules, such as CD28, CD134/0X40, CD137/4-1BB, Lck, ICOS or DAP10.
  • The disclosure also relates to a strategy of Adoptive cell therapy (ACT). ACT is a procedure in which therapeutic lymphocytes are administered to patients in order to treat cancer. This approach entails the ex vivo generation of tumor specific T cell lymphocytes and infusing them to patients. In addition to the lymphocyte infusion the host may be manipulated in other ways which support the take of the T cells and their immune response, for example, preconditioning the host (with radiation or chemotherapy) and administration of lymphocyte growth factors (such as IL-2). One method for generating such tumor specific lymphocytes involves the expansion of antigen specific T cells.
  • In one embodiment, the invention provides generating T cells expressing a shRNA of the invention and a desired CAR directed to a tumor antigen. The modified T cells can be generated by introducing a vector (e.g., plasmid, lentiviral vector, retroviral vector, adenoviral vector, adeno-associated viral vector) encoding both 1) an shRNA capable of reducing expression of a target gene described herein and 2) a desired CAR into the cells. The modified T cells of the invention are able to replicate in vivo resulting in long term persistence that can lead to tumor control.
  • In one aspect, the disclosure provides methods of treating cancer comprising administering a composition capable of silencing genes that inhibit T cell function. In one embodiment, the methods relate to administering T cell expressing a shRNA of the invention and a desired CAR directed to a tumor antigen. In one aspect the T cell to be administered comprises a vector encoding a shRNA of the invention and a desired CAR directed to a tumor antigen.
    • Pharmaceutical Formulations
  • In some instances, therapeutic compositions disclosed herein can include, in addition to the tumor targeting T cells, compounds, drugs, and/or agents used for the treatment of cancer. Such compounds, drugs, and/or agents can include, for example, chemotherapy drugs, small molecule drugs or antibodies that stimulate the immune response to a given cancer. In other instances, therapeutic compositions can include, for example, one or more small molecule inhibitors that silence, reduces, eliminates, knocks down, knocks out, or decreases the expression and/or activity of genes selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, F11r, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc. Accordingly, the invention provides one or more inhibitors of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 or Ppp3cc.
  • In one aspect, the invention provides one or more inhibitors of Ppp2r2d.
  • In another aspect, the invention provides one or more inhibitors of Eif2ak3.
  • In another aspect, the invention provides one or more inhibitors of Arhgap5.
  • In another aspect, the invention provides one or more inhibitors of Smad2.
  • In another aspect, the invention provides one or more inhibitors of Akap81.
  • In another aspect, the invention provides one or more inhibitors of Rbks.
  • In another aspect, the invention provides one or more inhibitors of Egr2.
  • In another aspect, the invention provides one or more inhibitors of Dgka.
  • In another aspect, the invention provides one or more inhibitors of Cb1b.
  • In another aspect, the invention provides one or more inhibitors of Map3k3.
  • In another aspect, the invention provides one or more inhibitors vMdfic.
  • In another aspect, the invention provides one or more inhibitors of Entpdl.
  • In another aspect, the invention provides one or more inhibitors of Dgkz.
  • In another aspect, the invention provides one or more inhibitors of Vamp7.
  • In another aspect, the invention provides one or more inhibitors of Nuak2.
  • In another aspect, the invention provides one or more inhibitors of Hipkl.
  • In another aspect, the invention provides one or more inhibitors of Alk. In one embodiment, the inhibitor of Alk includes, for example, for example CH5424802 (Hoffmann-La Roche), LDK378 (Novartis), Crizotinib and PF-02341066 (Pfizer) or AP26113 (Ariad Pharmaceuticals).
  • In another aspect, the invention provides one or more inhibitors of Pdzklipl.
  • In some instances, therapeutic compositions can include, for example, cytokines, chemokines and other biologic signaling molecules, tumor specific vaccines, cellular cancer vaccines (e.g., GM-CSF transduced cancer cells), tumor specific monoclonal antibodies, autologous and allogeneic stem cell rescue (e.g., to augment graft versus tumor effects), other therapeutic antibodies, molecular targeted therapies, anti-angiogenic therapy, infectious agents with therapeutic intent (such as tumor localizing bacteria) and gene therapy.
  • In some instances, therapeutic compositions disclosed herein can be formulated for use as or in pharmaceutical compositions. Such compositions can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA's CDER Data Standards Manual, version number 004 (which is available at fda.give/cder/dsm/DRG/drg00301.htm).
  • In some instances, pharmaceutical compositions can include an effective amount of one or more peptides. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more peptides for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome.
  • The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
    • Methods
  • In some instances, methods can include selection of a human subject who has or had a condition or disease (e.g., cancer). In some instances, suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), and/or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease).
  • The term “subject,” as used herein, refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject”, as used herein, refers to a human (e.g., a man, a woman, or a child). Samples for use in the methods can include serum samples, e.g., obtained from the selected subject.
  • In some instances, subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection. In some instances, the subject can be confirmed or identified, e.g. by a health care professional, as having had or having a condition or disease. In some instances, exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, and/or detecting an indication of a positive immune response. In some instances multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidate subject and a second party can test the sample. In some instances, subjects can be selected and/or referred by a medical practitioner (e.g., a general practitioner). In some instances, subject selection can include obtaining a sample from a selected subject and storing the sample and/or using the in the methods disclosed herein. Samples can include, for example, cells or populations of cells.
    • Methods of Use
  • In some embodiments, the disclosure provides methods for increasing the immune response in a subject in need thereof. The disclosure provides therapies that are particularly useful for the treatment of subjects having cancer. In some instances, the disclosure provides methods of treatment that include administering to a subject a composition disclosed herein.
  • Provided herein are methods for treating and/or preventing cancer or symptoms of cancer in a subject comprising administering to the subject a therapeutically effective amount of a composition capable of silencing genes that inhibit T cell function (e.g., an immunoresponsive T cell expressing a shRNA of the invention and a desired CAR directed to a tumor antigen). In some cases the T cell is derived from the patient to be treated and has been modified to express the CAR and an shRNA that reduces expression of a target gene described herein.
  • In some embodiments, the cancer is a carcinoma, sarcomas, adenocarcinoma, lymphoma, leukemia, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia (including AML, ALL, and CML), and multiple myeloma. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is a plasma cell malignancy, for example, multiple myeloma (MM) or pre-malignant condition of plasma cells. In some embodiments the subject has been diagnosed as having a cancer or as being predisposed to cancer.
  • As used herein, “cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia (including AML, ALL, and CML), and multiple myeloma.
  • The term “anti-tumor effect” as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.
  • The terms “treat” or “treating,” as used herein, refers to partially or completely alleviating, inhibiting, ameliorating, and/or relieving the disease or condition from which the subject is suffering. In some instances, treatment can result in the continued absence of the disease or condition from which the subject is suffering.
  • In general, methods include selecting a subject at risk for or with a condition or disease. In some instances, the subject's condition or disease can be treated with a pharmaceutical composition disclosed herein. For example, in some instances, methods include selecting a subject with cancer, e.g., wherein the subject's cancer can be treated by increasing T cell accumulation and infiltration within the tumor.
  • In some instances, treatments methods can include a single administration, multiple administrations, and repeating administration as required for the prophylaxis or treatment of the disease or condition from which the subject is suffering. In some instances treatment methods can include assessing a level of disease in the subject prior to treatment, during treatment, and/or after treatment. In some instances, treatment can continue until a decrease in the level of disease in the subject is detected.
  • Following administration, the subject can be evaluated to detect, assess, or determine their level of disease. In some instances, treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected.
  • Upon improvement of a patient's condition (e.g., a change (e.g., decrease) in the level of disease in the subject), a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • It is also within the scope of the present invention to combine any of the methods and any of the compositions disclosed herein with one or more therapeutic agents. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes, antisense oligonucleotides, chemotherapeutic agents and radiation.
  • It is also within the scope of the present invention to combine any of the methods and any of the compositions disclosed herein with conventional cancer therapies and various drugs in order to enhance the efficacy of such therapies through either reducing the doses/toxicity of conventional therapies and/or to increase the sensitivity of conventional therapies. One conventional therapy is the use of radiation therapy. Another conventional therapy is the use of chemotherapeutic drugs that can be divided into: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and antitumour agents. All of these drugs affect cell division or DNA synthesis and function in some way. Other conventional cancer therapies are agents that do not directly interfere with DNA. Examples of such agents for which to combine with the present invention may include for example “small-molecule” drugs that block specific enzymes involved in cancer cell growth. Monoclonal antibodies, cancer vaccines, angiogenesis inhibitors, and gene therapy are targeted therapies that can also be combined with the compositions and methods disclosed herein because they also interfere with the growth of cancer cells.
    • Methods of Screening Test Compounds
  • Included herein are methods for screening test compounds, e.g., polypeptides, polynucleotides, inorganic or organic large or small molecule test compounds, to identify agents useful in the treatment of cancer e.g., test compounds that silence, reduces, eliminates, knocks down, knocks out, modulates, or decreases the expression and/or activity of genes selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc.
  • As used herein, “small molecules” refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).
  • The test compounds can be, e.g., natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the “split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). In addition, a number of small molecule libraries are commercially available. A number of suitable small molecule test compounds are listed in U.S. Pat. No. 6,503,713, incorporated herein by reference in its entirety.
  • Libraries screened using the methods of the present invention can comprise a variety of types of test compounds. A given library can comprise a set of structurally related or unrelated test compounds. In some embodiments, the test compounds are peptide or peptidomimetic molecules. In some embodiments, the test compounds are nucleic acids.
  • In some embodiments, the test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound, e.g., a first test compound that is structurally similar to a known natural binding partner of the target polypeptide, or a first small molecule identified as capable of binding the target polypeptide, e.g., using methods known in the art or the methods described herein, and correlating that structure to a resulting biological activity, e.g., a structure-activity relationship study. As one of skill in the art will appreciate, there are a variety of standard methods for creating such a structure-activity relationship. Thus, in some instances, the work may be largely empirical, and in others, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of a small molecule compound or compounds. For example, in one embodiment, a general library of small molecules is screened, e.g., using the methods described herein.
  • In some embodiments, a test compound is applied to a test sample, e.g., a cell or living tissue or organ, e.g., an eye, and one or more effects of the test compound is evaluated. In a cultured or primary cell for example, the ability of the test compound to silence, reduces, eliminates, knocks down, knocks out, modulates, or decreases the expression and/or activity of genes selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc.
  • In some embodiments, the test sample is, or is derived from (e.g., a sample taken from) an in vivo model of a disorder as described herein. For example, an animal model, e.g., a rodent such as a rat, can be used.
  • Methods for evaluating each of these effects are known in the art. For example, ability to modulate expression of a protein can be evaluated at the gene or protein level, e.g., using quantitative PCR or immunoassay methods. In some embodiments, high throughput methods, e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999,W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485):1760-1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect an effect on Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc activity or gene expression.
  • A test compound that has been screened by a method described herein and determined to silence, reduces, eliminates, knocks down, knocks out, or decreases the expression and/or activity of genes selected from the group consisting of Ppp2r2d, Eif2ak3, Arhgap5, Smad2, Akap81, Rbks, Egr2, Dgka, Cb1b, Mdfic, Entpdl, Dgkz, Vamp7, Hipkl, Nuak2, Alk, Pdzklipl, Inpp5b, Socsl, Jun, Nptxr, Socs3, Fllr, Fyn, Ype12, Pkdl, Grk6, Cdkn2a, Sbfl, Ipmk, Rockl, Stk17b, Mast2, Pdpl, Yesl, Met, Ppm1g, Blvrb, Tnkl, Prkab2, Trpm7 and Ppp3cc, can be considered a candidate compound. A candidate compound that has been screened, e.g., in an in vivo model of a disorder, e.g., cancer, and determined to have a desirable effect on the disorder, e.g., on one or more symptoms of the disorder, can be considered a candidate therapeutic agent. Candidate therapeutic agents, once screened in a clinical setting, are therapeutic agents. Candidate compounds, candidate therapeutic agents, and therapeutic agents can be optionally optimized and/or derivatized, and formulated with physiologically acceptable excipients to form pharmaceutical compositions.
  • Thus, test compounds identified as “hits” (e.g., test compounds that inhibiting immunosuppressive pathways used by tumor cells to inactivate and/or suppress immune cells) in a first screen can be selected and systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameter. Such optimization can also be screened for using the methods described herein. Thus, in one embodiment, the invention includes screening a first library of compounds using a method known in the art and/or described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create a second library of compounds structurally related to the hit, and screening the second library using the methods described herein.
    • Examples
  • The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
  • Recent work has shown that cytotoxic T cells play a central role in immune-mediated control of cancers1-3, and monoclonal antibodies that target inhibitory receptors on T cells can induce significant clinical benefit in patients with advanced disease 4-6. However, many of the regulatory mechanisms that result in loss of T cell function within immunosuppressive tumors remain unknown. In the following examples, the inventors demonstrate that such regulatory mechanisms can be systematically discovered in vivo in the tumor microenvironment. The inventors postulated that shRNAs targeting key inhibitors would enable robust T cell infiltration and accumulation in tumors, despite multiple inhibitory signals. Using a pool shRNA screening approach aimed at identifying genes that block the function of tumor-infiltrating CD8 T cells, candidate shRNA were discovered by transfer of shRNA-transduced T cells into tumor-bearing mice, followed by deep sequencing to quantify the representation of all hairpins in tumors and lymphoid organs. The majority of shRNAs induced T cell accumulation in tumors but not the spleen, demonstrating feasibility of discovering shRNAs with differential action across tissues. One of the targets was Ppp2r2d, a regulatory subunit of the PP2A phosphatase7. Control shRNA-transduced T cells underwent apoptosis upon recognition of melanoma cells, while Ppp2r2d shRNA-transduced T cells accumulated in tumors due to enhanced proliferation and resistance to apoptosis. Ppp2r2d shRNA-expressing T cells also significantly delayed tumor growth. This in vivo approach has wide-spread applications to dissect complex immune functions in relevant tissue microenvironments.
  • Immune cells perform complex surveillance functions throughout the body and interact with many different types of cells in distinct tissue microenvironments. Therapeutic targets for modulating immune responses are typically identified in vitro and tested in animal models at a late stage of the process. Here the inventors have addressed the challenge of how targets for immune modulation can be systematically discovered in vivo. This is a central issue in oncology because strong infiltration by CD8 T cells—which have cytotoxic function against tumor cells—is associated with a favorable prognosis in multiple types of human cancer1..3.8. Unfortunately, this natural defense mechanism is severely blunted in the majority of patients by multiple inhibitory signals emanating from the tumor, its stroma, regulatory T cells and myeloid cell populations.9-11
  • Pooled shRNA libraries have been shown to be powerful discovery tools12-14. The inventors reasoned that shRNAs capable of restoring CD8 T cell function can be systematically discovered in vivo by taking advantage of the extensive proliferative capacity of T cells following triggering of the T cell receptor by a tumor-associated antigen. When introduced into T cells, only a small subset of shRNAs from a pool will restore T cell proliferation resulting in their enrichment within tumors. Over-representation of active shRNAs within each pool can be quantified by deep sequencing of the shRNA cassette from tumors and secondary lymphoid organs (FIG. 1 ).
  • Experimental animals. C57BL/6 mice, TRP-1 mice (transgenic mice expressing T-cell receptor (TCR) specific for tyrosinase-related protein 1)23, pmel-1 mice (transgenic mice expressing TCR specific for gp100)18, and b2m-1-mice24 were purchased from The Jackson Laboratory. The Rag1−/− OT-I mice 16 were purchased from Taconic Farms, Inc. Mice were bred at the Dana-Farber Cancer Institute animal facility. All experimental procedures were approved by the Dana-Farber Cancer Institute Animal Care and Use Committee.
  • Cell lines. B16 melanomas, an aggressive tumor that is difficult to treat, express the surrogate tumor antigen Ovalbumin (Ova), which is recognized by CD8 T cells from OT-I T cell receptor transgenic mice16, 17. EL4 thymoma38 and B16-F10 melanoma15 cells were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 10011 g/ml streptomycin and 10011 g/ml penicillin. Ovalbumin-expressing B16 tumor cells (B16-Ova) were maintained in the same media with addition of 600 m/mL G418 (Invitrogen).
  • Vectors and shRNA Sequences. shRNAs were selected for 255 genes over-expressed in dysfunctional T cells (anergic or exhausted state). pLKO.3G vector was obtained from The RNAi Consortium. pLKO-Thy1.1, pLKO-Ametrine, pLKO-RFP, pLKO-TFP vectors were modified from pLKO.3G vector by replacing GFP with the corresponding reporter gene. Murine Ppp2r2d and Cb1b sequences targeted by 10 selected shRNAs are provided in Table 3 (listed in order of shRNA activity (highest to lowest)). The LacZ target sequence targeted by a control shRNA is also listed. All other target sequences can be found in Table 2.
  • TABLE 3
    # Gene Clone ID Murine shRNA Target Sequence
    LacZ TRCN0000072227 GCGCTAATCACGACGCGCTGT
    (SEQ ID NO: 621)
     1 Ppp2r2d TRCN0000080900 CCCACATCAGTGCAATGTATT
    (SEQ ID NO: 386)
     2 Ppp2r2d ND000492 CCACAGTGGTCGATACATGAT
    (SEQ ID NO: 385)
     3 Ppp2r2d TRCN0000431278 GAGAATTAACCTATGGCATTT
    (SEQ ID NO: 384)
     4 Ppp2r2d ND000486 GCTCAATAAAGGCCATTACTC
    (SEQ ID NO: 383)
     5 Ppp2r2d TRCN0000080901 CCATTTAGAATTACGGCACTA
    (SEQ ID NO: 380)
     6 Ppp2r2d TRCN0000430828 ATAGTGATCATGAAACATATC
    (SEQ ID NO: 375)
     7 Ppp2r2d TRCN0000080899 GCCACCAATAACTTGTATATA
    (SEQ ID NO: 374)
     8 Ppp2r2d TRCN0000080902 CGGTTCAGACAGTGCCATTAT
    (SEQ ID NO: 381)
     9 Ppp2r2d TRCN0000427220 TCATCTCCACCGTTGAGTTTA
    (SEQ ID NO: 378)
    10 Ppp2r2d TRCN0000425449 ATGCTCATACATATCACATAA
    (SEQ ID NO: 377)
     1 Cblb ND000025 CGAGCGATCCGGCTCTTTAAA
    (SEQ ID NO: 72)
     2 Cblb ND000030 AGCCAGGTCCAATTCCATTTC
    (SEQ ID NO: 71)
     3 Cblb TRCN0000244606 CCCTGATTTAACCGGATTATG
    (SEQ ID NO: 70)
     4 Cblb ND000026 ATCGAACATCCCAGATTTAGG
    (SEQ ID NO: 61)
     5 Cblb TRCN0000244603 CTACACCTCACGATCATATAA
    (SEQ ID NO: 59)
     6 Cblb ND000024 TACACCTCACGATCATATAAA
    (SEQ ID NO: 67)
     7 Cblb TRCN0000244605 TGAGCGAGAATGAGTACTTTA
    (SEQ ID NO: 60)
     8 Cblb TRCN0000244604 CCAGATTTAGGCATCTATTTG
    (SEQ ID NO: 65)
     9 Cblb TRCN0000244607 CTTGTACTCCAGTACCATAAT
    (SEQ ID NO: 63)
    10 Cblb ND000027 TCTACATCGATAGTCTCATGA
    (SEQ ID NO: 58)
  • Antibodies and flow cytometry. Single-cell suspensions were stained in PBS, 2% FBS with labeled antibodies at 4° C. for 20 minutes, followed by two washes with ice-cold PBS, 2% FBS. Cells were analyzed/sorted using a FACSAria (BD Biosciences) and FlowJo software (TriStar). Antibodies used were specific for CD4, CD8, Va2, Vβ5.1/5.2, Thy1.1, CD25, CD44, CD62L, CD69, CD122, CD127, IFNγ, TNFα (BioLegend), PD-1, TIM-3, LAG-3, granzyme B, and H-2Kb (BioLegend),Va3.2 (eBioscience), Vβ13, Vβ14 (BD Biosciences), phospho-Akt (Ser473) and phospho-Bad (Ser112) (Cell Signaling). Apoptotic cells were detected by labeling with annexin V (BioLegend) or activated caspase-3 antibody (Cell Signaling). Mouse anti-CD3/CD28 beads were purchased from Invitrogen.
  • T cell isolation from tumors. B16-Ova melanomas were cut into small pieces in petri dishes containing 5 mL of PBS, 2% FBS and washed with PBS. Tumors were resuspended in 15 mL RPMI supplemented with 2% FBS, 50U/mL Collagenase Type IV (Invitrogen), 20U/mL DNase (Roche), samples incubated at 37° C. for 2 hours and tissue further dissociated using a gentleMACS Dissociator (Miltenyi Biotech). Suspensions were washed three times with PBS and passed through a 70 μM strainer. Lymphocytes were isolated by density gradient centrifugation and then either analyzed or sorted by flow cytometry using a FACSAria (BD Biosciences). T cell apoptosis. Cytokine pre-treated OT-I cells were transduced with LacZ or Ppp2r2d shRNAs and injected into mice bearing day 14 B16-Ova tumors. After 7 days, intracellular staining was performed using an activated caspase-3 antibody (Cell Signaling) and CD8/Thy1.1 double-positive T cells were gated in the FACS analysis.
  • Immunofluorescence and immunohistochemistry. B16-Ova tumors from mice treated with OT-I T cells expressing LacZ or Ppp2r2d shRNAs (GFP-expressing vector) were cryopreserved in optimal cutting temperature (O.C.T.) compound (Tissue-Tek). 10 μm-sections from cryopreserved tumors were were permeabilized with 0.2% Triton X-100, fixed in 4% paraformaldehyde and stained with a GFP antibody (Molecular Probes) in combination with DAPI. For TUNEL detection, sections were stained with TACS 2 TdT Blue Label (Trevigen) based on manufacturer's directions. Samples were visualized using a laser-scanning confocal microscope (Leica SP5X) and analyzed with ImageJ software (NIH). qRT-PCR assay. Total RNA was extracted using TRIzol reagent (Invitrogen). RNA was reverse transcribed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Real time quantitative PCR reactions were performed as triplicates using an ABI 7900HT instrument with SYBR green (ABI). Rp123 levels were used for normalization. The following primers were used: Ppp2r2d forward GGAAGCCGACATCATCTCCAC (SEQ ID NO: 622), Ppp2r2d reverse GTGAGCGCGGCCTTTATTCT (SEQ ID NO: 623); Cb1b forward GGTCGCATTTTGGGGATTATTGA (SEQ ID NO: 624), Cb1b reverse TTTGGCACAGTCTTACCACTTT (SEQ ID NO: 625); Rp123 forward CTGTGAAGGGAATCAAGGGA (SEQ ID NO: 626) and Rp123 reverse TGTCGAATTACCACTGCTGG (SEQ ID NO: 627).
  • Microarray Analysis. IL-7/IL-15 cultured OT-I T cells were transduced with one of five experimental shRNAs (Ppp2r2d, Arhgap5, Alk, Egr2, Ptpn2) or a LacZ control shRNA. Infected cells were sorted to purity using GFP encoded by the vector as a reporter. T cells (5×106) were injected i.v. into mice bearing day 14 B16-Ova tumors. Seven days later, shRNA-expressing OT-I T cells (CD8+GFP+) were isolated from tumors and spleens. Cells were sorted twice to high purity and total RNA was extracted using TRIzol reagent (Invitrogen) for Affymetrix gene expression profiling (Mouse Genome 430 2.0 Arrays). Arrays for each shRNA were done in triplicate (6 mice per group).
    • Nanowell Analysis of Cytokine Production at a Single Cell Level
  • Materials. Antibodies used for T cell activation were anti-mouse CD3 and anti-mouse CD28 (Biolegend). Antibodies used to capture secreted cytokines were anti-mouse IFNγ (Biolegend), anti-mouse IL-2 (Biolegend), anti-mouse TNFα (Biolegend) and anti-mouse GM-CSF (Biolegend). Detection antibodies were anti-mouse IFNγ (Biolegend), anti-mouse IL-2 (Biolegend), anti-mouse TNFα (Biolegend) and anti-mouse GM-CSF (Biolegend), and they were fluorescently labeled with appropriate Alexa Fluor dyes (Invitrogen) following manufacturer's instructions. The lipids used to prepare supported bilayers were: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotinyl Cap PE) (Avanti Polar Lipids).
  • Fabrication of PDMS arrays of nanowells and preparation of supported lipid bilayers. The array of nanowells was manufactured by injecting polydimethylsiloxane (PDMS, Dow Corning) prepared at a 10:1 base/catalyst weight ratio into a custom-built mold encasing a micropatterned silicon master. Arrays of nanowells were cured at 70° C. for 4-16 h. Each array comprised 72×24 blocks, each containing a 7×7 (50 μm×50 μm×50 μm) subarray of nanowells (total of 84,672 wells). The PDMS arrays adhered directly to a 3″×1″ glass slide forming a 1 mm thick layer. Supported lipid bilayers were prepared as described previously14. Bilayers were generated by applying DOPC liposomes containing 2 mol % biotin-Cap-PE lipids on the PDMS array of nanowells. The surfaces were rinsed with deionized water to remove excess liposomes. Before use, the lipid bilayer was blocked with BSA in PBS (100 μg/mL) for 45 minutes. The bilayer was then incubated with 1 μg/mL of streptavidin in a solution of 100 μg/mL BSA in PBS, followed by incubation with biotinylated CD3 and CD28 antibodies. The device was rinsed extensively with PBS before adding the cells.
  • Microengraving. Capture antibodies were diluted in borate buffer (50 mM sodium borate, 8 mM sucrose, and 50 mM NaCl, pH 9.0) to a final concentration of 10 μg/mL and deposited on the surface of epoxy-modified slides for 1 h at room temperature. Slides were blocked with 3% non-fat milk in PB ST (PBS with 0.05% (v/v) Tween 20) for 30 min at room temperature and washed with PBS before placing them into contact with the PDMS array of nanowells. A suspension of T cells was dispensed onto the surface of the nanowells, modified with a supported lipid bilayer in media and allowed to settle into the wells. The density of suspended cells applied to the array was optimized empirically to maximize well occupancy by single cells (typically ˜30% of wells). After incubation of the cell-loaded wells, a glass slide coated with capture antibodies was then placed onto the loaded array for cytokine capture. The microarray and glass slide were held together by compression in a hybridization chamber (Agilent Technologies, G2534A) and incubated for 1 h at 37° C. with 5% CO 2. The glass slide was then separated from the array and placed in PBS. After microengraving, slides were incubated for 30 min with blocking buffer (PBS, 10 mg/mL BSA, 0.05% (v/v) Tween-20, 2% mouse serum and 2 mM sodium azide), washed with PBST (PBS+0.05% v/v Tween-20), and then incubated with fluorescence detection antibodies at 1 μg/mL for 45 min at 25° C. The slides were washed with PBST and PBS, rinsed briefly with water, and dried with a N2 stream. Reference slides were generated at the end of each experiment with the same detection antibodies used on the printed slides. For reference slides, antibodies were diluted in water, spotted onto blank poly-L-lysine slides (1 μL/spot), and the reference slides were dried under vacuum. Slides were scanned using a Genepix 4200AL microarray scanner (Molecular Devices). The median fluorescence intensity of each spot was extracted using Genepix Pro.
  • On-chip image-based cytometry. Before imaging, T cells were stained with CellMask™ Plasma Membrane Stain (Invitrogen, Life Technologies) and SYTOX green (for detection of dead cells, Life Technologies). The cell-loaded arrays of nanowells were mounted face-up on the microscope with a coverslip placed on top of the array. Images were acquired on an automated inverted epifluorescence microscope (Carl Zeiss). Transmitted light and epifluoresence micrographs were collected block-by-block (7×7 microwells per block). The resulting collection of images was analyzed using a custom program to determine the number of cells present in each well and the mean fluorescence intensity of each label. Only viable T cells were considered for the analysis. Although the cells expressed GFP, the fluorescence intensity of GFP was negligible under the utilized microscope acquisition setting compared to SYTOX green, enabling identification of dead cells.
  • Data analysis. Data extracted from both on-chip cytometry and printed cytokines were matched in Microsoft Excel using unique identifiers assigned to each well within the array. The dataset was filtered to include wells containing only single cells. To compensate from signal bleed-through and convert the measured fluorescence intensity for the captured cytokines from a given cell into a rate of secretion, the data from standard calibration curves (from reference slides) prepared with known amounts of detection antibodies was used to convert measured intensities to a number of molecules, as described previously (Han, Q., et.al., Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving. Lab Chip 10, 1391-1400, doi:10.1039/b926849a (2010).
    • Example 1: In vivo RNAi Discovery of Immunotherapy Targets
  • Two large primary screens were performed, with the first focusing on genes over-expressed in dysfunctional T cells (T cell anergy or exhaustion; 255 genes, 1,275 shRNAs divided into two pools), and the second on kinases/phosphatases (1,307 genes, 6,535 shRNAs divided into seven pools) (Table 4). In these primary screens, each gene was represented by ˜5 shRNAs.
  • TABLE 4
    T cell Kinase/ shRNA
    Dysfunction Phosphatase Enrichment
    1st Genes 255 1307   4-10x: 123
    Screen shRNAs 1275 6535 10-20x: 17
    Candidate 32 82  >20x: 1
    Genes
    2nd Genes 32 43   4-10x: 191
    Screen shRNAs 480 645 10-20x: 27
    Candidate 17 26  >20x: 1
    Genes
  • shRNAs targeting 255 genes over-expressed in dysfunctional T cells (anergic or exhausted state)31-37 and 1,307 kinase/phosphatase genes (˜5 shRNAs per gene) were obtained from The RNAi Consortium (TRC; Broad Institute, Cambridge, MA, USA). Nine pools were created and shRNAs subcloned into the pLKO-Thy1.1 lentiviral vector. Each pool also contained 85 negative-control shRNAs (number of shRNAs: GFP, 24; LacZ, 20; luciferase 25; RFP 16). OT-I T cells isolated by negative selection (Stemcell Technologies) were cultured with IL-7 (5 ng/mL, Peprotech) and IL-15 (100 ng/mL, Peprotech) in complete RPMI media (RPMI 1640, 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethonal, 2 mM L-glutamine, 100 μg/ml streptomycin and 100 μg/ml penicillin). On day 2, OT-I T cells were spin-infected with lentiviral pools (nine lentiviral shRNA pools and a LacZ control shRNA lentiviral vector control) supplemented with protamine sulfate (5 μg/mL) in 24-well plates coated with retronectin (5 μg/mL) at a multiplicity of infection (MOI) of 15. Typically, ˜5×106 OT-1 T cells were infected for each pool.
  • Following infection, OT-I cells were cultured with IL-7 (2.5 ng/mL), IL-15 (50 ng/mL) and IL-2 (2 ng/mL) in complete RPMI media. On day 5, live shRNA-transduced T were enriched using a dead cell removal kit (Miltenyi), and infected cells were positively selected based on Thy1.1 marker (Stemcell Technologies) to 50-60% Thy1.1 positivity. Successful transduction was monitored by surface expression of the Thy1.1 reporter (FIG. 2 ). T cells (5×106) were injected i.v. into C57BL/6 mice bearing day 14 B16-Ova tumors (15 mice per shRNA pool)(number of animals chosen to provide sufficient cells for T cell isolation and PCR). Genomic DNA was isolated from 5×106 enriched OT-I cells as the start population for deep sequencing. Seven days later, shRNA-expressing T cells (CD8+Va2+V(35+Thy1.1+) were isolated by flow cytometry from tumors, spleens, tumor-draining lymph nodes and irrelevant lymph nodes for isolation of genomic DNA, followed by PCR amplification of the shRNA cassette. (FIG. 3 ) Genomic DNA was isolated (Qiagen) and deep-sequencing templates were generated by PCR of the shRNA cassette. Representation of shRNAs in each pool was analyzed by deep sequencing using an Illumina Genome Analyzer 30. Data were normalized using the average reads of control shRNAs in each pool. Kinase/phosphatase genes were selected for the secondary screen based on expression levels in T cells.
  • For certain genes, shRNAs were over-represented in all tested tissues compared to the starting T cell population (e.g. SHP-1), indicative of enhanced proliferation independent of TCR recognition of a tumor antigen. For other genes, there was a selective loss of shRNAs within tumors (e.g. ZAP-70, a critical kinase in the T cell activation pathway). We focused our analysis on genes whose shRNAs showed substantial over-representation in tumor but not spleen, a secondary lymphoid organ. Substantial T cell accumulation in tumors was observed for a number of shRNAs, despite the immunosuppressive environment. For secondary screens, we created focused pools in which each candidate gene was represented by ˜15 shRNAs.
  • Primary data from this analysis are shown for three genes in FIG. 4 : LacZ (negative control), Cb1b (an E3 ubiquitin ligase that induces T cell receptor internalization)19 and Ppp2r2d (not previously studied in T cells). For both Ppp2r2d and Cb1b, five shRNAs were substantially increased in tumors (red) compared to spleen, while no enrichment was observed for LacZ shRNAs. Overall, 43 genes met the following criteria: ≥_4-fold enrichment for 3 or more shRNAs in tumors compared to spleen (Table 5, FIG. 4 , FIG. 5 ). The set included gene products previously identified as inhibitors of T cell receptor signaling (including Cb1b, Dgka, Dgkz, Ptpn2) as well as other well-known inhibitors of T cell function (e.g. Smad2, Socsl, Socs3, Egr2), validating our approach (Table 5, Table 6).20-22 Table 5 describes the functional classification of candidate genes from the secondary screen.
  • TABLE 5
    Function Genes
    Inhibition of TCR signaling Cbib, Dgka, Dgkz, Fyn, Inpp5b,
    Ppp3cc, Ptpn2, Stk17b, Tnk1
    Phosphoinositol metabolism Dgka, Dgkz, Impk, Inpp5b, Sbf1
    Inhibitory cytokine signaling Smad2, Socs1, Socs 3
    pathways
    AMP signaling, inhibition of Entpd1, Prkab2, Nuak
    mTOR
    Cell cycle Cdkn2a, Pkd1, Ppp2r2d
    Actin and microtubules Arhgap5, Mast2, Rock 1
    Potential nuclear functions Blvrb, Egr2, Impk, Jun, Ppm1g
    Role in cancel cells Alk, Arhgap5, Eif2ak3, Hipk1, Met,
    Nuak, Pdzk1ip, Rock1, Yes1
  • Secondary screens were performed focusing on genes whose shRNAs showed substantial over-representation in tumor but not spleen, a secondary lymphoid organ. Substantial T cell accumulation in tumors was observed for a number of shRNAs, despite the immunosuppressive environment. For these secondary screens, ˜10 additional shRNAs were synthesized for each gene (IDT) for a total of ˜15 shRNAs per gene. These focused pools contained 85 negative-control shRNAs. Two control shRNAs (one for RFP, one for luciferase) showed some enrichment in tumors relative to spleen (4.0 and 5.1-fold, respectively). Cut-off in the secondary screen was defined as ≥3 shRNAs with ≥4 fold enrichment in tumor relative to spleen. Screening results were validated at a cellular level by introducing individual shRNAs into T cells, along with a reporter protein (GFP, TFP, RFP or Ametrine fluorescent proteins, Thy1.1). This approach enabled simultaneous testing of five shRNAs in an animal (three mice per group). Proliferation of shRNA-transduced T cells was visualized based on CFSE dilution after 24 hours as well as 3, 5 and 7 days. In addition, intracellular staining was performed on days 3, 5 and 7 for IFNγ, TNFα and isotype controls. Results from the primary and secondary screen of T cell dysfunction pool shRNA library are provided in Table 6. Genes for which at least 3 shRNAs showed >4 fold enrichment in tumors are listed, along with a brief description of their function. Results from secondary screen of kinase and phosphatase shRNA libraries are shown in Table 7.
  • TABLE 6
    Total # Enrichment
    Symbol shRNAs (fold) Function
    Dgkz
    6 5.2-14.0 Phosphorylates and thereby inactivates DAG
    Egr2
    6 4.0-10.2 Transcription factor involved in T cell
    unresponsiveness, expression of Cblb
    Smad2
    5 6.7-30.3 TGF beta signaling pathway
    Cblb
    5 4.1-10.8 E3 ubiquitin ligase (degradation of TCR and signaling
    molecules; ko mice reject tumors)
    Inpp5b 5 4.3-9.5  Inositol polyphosphate-5-phosphatase, hydrolyzes PIP2
    Socs1
    5 4.1-8.5  Inhibitor of cytokine signaling
    Jun
    5 5.2-6.4  Persistent AP-1 activation in tumor-infiltrating T cells
    leads to upregulated PD-1
    Entpd1 4 6.5-13.3 Extracellular degradation of ATP to AMP (an inhibitory
    signal through AMP kinase)
    Vamp7 4 4.0-11.3 Vesicle associated transmembrane protein
    Dgka
    4 5.0-10.2 Phosphorylates and thereby inactivates DAG
    Mdfic
    4 4.4-10.0 Inhibits viral gene expression, interacts with cyclin T1
    and T2
    Nptxr
    4 4.0-7.2  Pentraxin Receptor
    F11r
    4 4.6-6.8  Cell migration
    Socs3
    4 4.6-6.3  Inhibitor of cytokine signaling
    Pdzk1ip1
    3 4.8-12.9 Pdzk1 interacting protein, expression correlates with
    tumor progression
    Fyn
    3 4.1-6.5  Inhibits activation of resting T cells (through Csk)
    Ypel2 3 4.6-5.1  Function unknown
  • TABLE 7
    Total # Enrichment
    Symbol shRNAs (fold) Function
    Rbks 6 4.0-12.8 Ribokinase carbohydrate metabolism
    Pkd1 6 4.9-9.9  Cell cycle arrest (activates JAK/STAT pathway)
    Ppp2r2d 5 4.0-17.2 Regulatory subunit of PP2A phosphatase
    Eif2ak3 5 4.8-13.4 ER stress sensor, resistance of cancer cells to
    chemotherapy
    Ptpn2 5 4.7-7.4  Inhibitor of T cell and cytokine signaling
    Hipk1 4 4.5-12.3 Interacts with p53 and c-myb, knockout mice develop
    fewer carcinogen-induced tumors
    Grk6 4 4.2-11   Regulator of particular G-protein coupled receptors
    Cdkn2a 4 4.1-7.2  G1 cell cycle arrest and apoptosis in T cells
    Sbf1 4 4.8-6.9  Activates MTMR2, which dephosphorylates PI(3)P and
    PI(3,5)P2
    Ipmk 4 4.0-6.9  Inositol polyphosphate kinase, nuclear functions such as
    chromatin remodeling
    Rock1 4 4 4.1-6.5    Rho kinase, inhibitors have shown activity in mouse
    models of cancer
    Stk17b 4 4.0-6.4  Inhibitor of T cell signaling forms complex with protein
    kinase D
    Mast2 4 4.1-5.1  Microtubule-associated serine/threonine kinase
    Arhgap5 3 6.0-15.7 Negative regulator of Rho GTPases, inhibition can reduce
    cancer cell invasion
    Alk 3 9.6-13.5 Anaplastic lymphoma kinase (translocation of
    nucleophosmin and ALK in ALCL)
    Nuak 3 4.5-13.1 Member of AMP-activated protein kinase-related kinase
    family, oncogene in melanoma
    Akap81 3 4.4-11.8 A-kinase anchoring protein, recruits cAMP-dependent
    protein kinase (PKA) to chromatin
    Pdp1 3 4.1-9.8  Pyruvate dehydrogenase phosphatase 1, regulation of
    glucose metabolism
    Yes1 3 5.4-9.7  Src family kinase, oncogene in several tumors
    Met 3 4.1-8.9  Receptor tyrosine kinase, involved in hepatocellular and
    other cancers
    Ppm1g 3 6.2-8.2  Dephosphorylates spliceosome substrates and histones
    H2A-H2B
    Blvrb 3 5.3-8.0  Biliverdin reductase, also transcription factor, arrest of
    cell cycle
    Tnk1 3 5.2-7.6  Downregulates Ras pathway (phosphorylation of Grb2),
    inhibition of NF-KB pathway
    Prkab2 3 4.1-7.0  Subunit of AMP kinase, inhibits fatty acid synthesis and
    mTOR pathway
    Trpm7 3 4.9-5.9  Ion channel and serine-threonine kinase
    Ppp3cc 3 4.2-4.4  Regulatory subunit of calcineurin (phosphatase in T cell
    receptor signaling)
    • Example 2: shRNA-Driven Expansion of CD4 and CD8 T Cells in B16 Melanomas
  • Positive shRNAs from deep sequencing analysis were cloned into lentiviral vectors encoding five different reporter proteins (GFP, TFP, RFP or Ametrine fluorescent proteins, Thy1.1). Cytokine-pretreated OT-I T cells were transduced with lentiviral vectors driving expression of a single shRNA and a reporter protein; 1×106 T cells of each population were mixed and co-injected i.v. into C57BL/6 mice bearing day 14 B16-Ova tumors. After seven days T cells were isolated from tumors, spleens and lymph nodes, and the percentage of reporter-positive CD8+Va2+Vf35+ T cells was determined by flow cytometry based on co-introduced reporters. Fold-enrichment in tumors compared to spleen was calculated based on the percentage of OT-I T cells in each organ expressing a particular reporter. When the control LacZ shRNA was expressed in CD8 OT-I T cells, the frequency of shRNA-expressing CD8 OT-I T cells was lower in tumors compared to spleen (˜2-fold). In contrast, experimental shRNAs induced accumulation of CD8 OT-I T cells in tumors but not the spleen (FIG. 6 , FIG. 7 ). For seven of these shRNAs (e.g., Ppp2r2D, Eif2ak3, Arhgap5, Smad2, Akap8I, Rbks and Egr2), T cell accumulation in tumors was >10-fold relative to spleen. The strongest phenotype was observed with shRNAs targeting Ppp2r2d, a regulatory subunit of the PP2A phosphatase7.
  • CD8+OT-I or CD4+ TRP-1 T cells expressing Ppp2r2d or LacZ shRNAs were injected into mice bearing day 14 B16-Ova tumors. shRNA-expressing T cells were identified in tumors and spleens using Thy1.1 reporter (FIG. 8 , % Thy1.1+CD8 T cells, left panels). Total numbers of LacZ or Ppp2r2d shRNA-expressing T cells were determined in tumors and spleens 7 days following transfer of 2×106 shRNA-expressing cells (FIG. 8 , right panels). Fold-enrichment of Ppp2r2d versus LacZ shRNA-expressing T cells in tumors is indicated. Ppp2r2d shRNA not only induced accumulation of OT-I CD8 T cells, but also CD4 T cells (from TRP-1 TCR transgenic mice)23, with T cell numbers in tumors being significantly higher when Ppp2r2d rather than LacZ shRNA was expressed (36.3-fold for CD8; 16.2-fold for CD4 T cells) (FIG. 8 ).
  • T cell enrichment in tumors compared to spleen for cells expressing a panel of Ppp2r2d or Cb1b shRNAs (FIG. 17 , upper panels) Ppp2r2d and Cb1b mRNA levels were also measured by qPCR prior to T cell transfer (FIG. 17 , lower panels). The strongest T cell enrichment in tumors was observed for shRNAs with >80% knock-down efficiency at the mRNA level ( shRNAs # 1 and 2 for both Ppp2r2d and Cb1b). CD8 T cell accumulation correlated with the degree of Ppp2r2d knock-down, and two Ppp2r2d shRNAs with the highest in vivo activity induced the lowest levels of Ppp2r2d mRNA (FIG. 17 ).
  • Ppp2r2d knockdown was also confirmed at the protein level using a quantitative mass spectrometry approach (FIG. 18 ). A previously reported approach for absolute quantification (AQUA) of proteins from cell lysates by mass spectrometry was used to measure the effect of Ppp2r2d shRNA expression at the protein level (Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W. & Gygi, S. P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. PNAS, 100, 6940-6945 (2003). This strategy is based on a ‘selective reaction monitoring’ approach in which a synthetic peptide with incorporated stable isotopes is used as an internal standard for mass spectrometry analysis.OT-I cells expressing LacZ or Ppp2r2d shRNAs were sorted to purity using FACS. Cells (1×106) were lysed in 1 ml of MPER extraction reagent (Pierce) containing a Protease Inhibitor Cocktail (Sigma), 1 mM EDTA and 1 mM PMSF for 15 minutes on ice with occasional vortexing. Cell debris was removed by centrifugation and the protein supernatant was filtered (0.2 μm SpinX centrifuge filter, Costar). Protein concentration was determined by Bradford assay (Biorad) and UV280 nm analysis (Nanodrop instrument); 0.1 mg of cellular protein was separated by SDS-PAGE and stained with Coomassie blue reagent (Pierce). Gel bands corresponding to a MW range of 45-60 kDa were excised followed by in-gel digestion of proteins with trypsin. Eluted peptides were spiked with 300 fmol of isotopically labeled Ppp2r2d (FFEEPEDPSS[13C-15N-R]-OH)(SEQ ID NO: 628) and Actin B (GYSFTTTAE[13C-15N-R]-OH) (SEQ ID NO: 629) peptides (21st Century Biochemicals) for quantification by LC MS/MS (LTQ XL Orbitrap, Thermo Scientific). The Ppp2r2d peptide was chosen from a region of the protein that differs from other regulatory subunits of PP2A. Initially, a LC-MS/MS run of a LacZ shRNA sample was analyzed to localize the Ppp2r2d and Actin B peptides that were being monitored. The absolute quantification AQUA peptides co-eluted with the corresponding endogenous peptides from the reverse-phase column, yet their higher MW (10 Da) enabled the ratio of peak intensity for endogenous and AQUA peptides to be determined using abundant peptide fragment ions. Triplicate samples were analyzed by SDS-PAGE—LC-MS/MS and statistical significance was determined using Graphpad Prism 6.0 software using a two-sided Student t-test (F test, * p=0.0062).
  • The specificity of Ppp2r2d shRNA was determined. Ppp2r2d shRNA activity was specific because the phenotype was reversed when a mutated Ppp2r2d cDNA (with wild-type protein sequence, but mutated DNA sequence at the shRNA binding site) was co-introduced with the Ppp2r2d shRNA (FIG. 9, 10 a-c). Furthermore, OT-I CD8 T cells over-expressed Ppp2r2d in tumors compared to spleen (in the absence of any shRNA expression), suggesting that it is an intrinsic component of the signaling network inhibiting T cell function in tumors (FIG. 19 ).
  • OT-I T cells transduced with lentiviral vectors driving expression of LacZ shRNA, Ppp2r2d shRNA, Ppp2r2d shRNA. Mutant Ppp2r2d cDNA with preserved protein sequence but disrupted shRNA binding site were generated. Wild-type Ppp2r2d cDNA was isolated by RT-PCR using forward primer GGATCCATGGCAGGAGCTGGAGGC (SEQ ID NO: 630) and reverse primer: GCTAGCATTAATTTTGTCCTGGAATATATACAAGTTATTGGTGG (SEQ ID NO: 631). The target sequence of Ppp2r2d shRNA, CCCACATCAGTGCAATGTATT (SEQ ID NO: 632) was mutated to TCCCCACCAATGTAACGTGTT (SEQ ID NO: 633) by overlapping PCR (which conserves protein coding sequence) using forward primer: TCCATCCCCACCAATGTAACGTGTTTGTTTACAGCAGCAGCAAGG (SEQ ID NO: 634) and reverse primer: AAACAAACACGTTACATTGGTGGGGATGGAACTCTGCGGCAGTGA (SEQ ID NO: 635). (FIG. 10 a ) Both wild-type and mutant Ppp2r2d cDNAs were cloned into a modified pLKO.3 vector with a 2A ribosomal skip peptide-GFP sequence (resulting in stoichiometric Ppp2r2d and GFP expression in cells). Constructs were introduced into EL4 thymoma cells. GFP-expressing EL4 cells were sorted to purity and then transduced with LacZ or Ppp2r2d shRNA lentiviral vectors driving expression of a Thy1.1 reporter. shRNA-transduced (Thy1.1+) cells were analyzed by flow cytometry for GFP expression. The Ppp2r2d shRNA reduced GFP levels when wild-type Ppp2r2d. The Ppp2r2d shRNA was not able to reduce expression of the GFP reporter in cells expressing the mutant Ppp2r2d cDNA, demonstrating that the shRNA binding site had been successfully mutated. (FIG. 10 a )
  • Expression of Ppp2r2d mutant cDNA also prevents phenotype induced by Ppp2r2d shRNA. (FIG. 10 b ) Ppp2r2d shRNA was cloned into the mutant Ppp2r2d cDNA-2A-GFP construct which resulted in co-expression of Ppp2r2d shRNA and mutated Ppp2r2d cDNA in one vector. OT-I T cells were separately infected with lentiviruses encoding LacZ shRNA (Thy1.1), Ppp2r2d shRNA (Ametrine) or Ppp2r2d shRNA plus mutant Ppp2r2d cDNA (GFP). (FIG. 10 b ) These three populations there then mixed at the same ratio and injected into mice bearing day 14 B16-Ova tumors. On day 7, each T cell population was quantified in tumors and spleens by gating on OT-I (CD8+Va2+V(35+)-T cells followed by analysis of populations marked by Thy1.1, Ametrine or GFP expression. The percentage of each T cell population in tumors and spleens was quantified by gating on Va2+Vf35+T cells; transduced cells were detected based on expression of Thy1.1 or Ametrine/GFP fluorescent reporters and the results are shown in FIG. 10 b . (representative data from 2 independent experiments, n=3 mice per experiment).
  • FIG. 10 c provides real-time PCR analysis for Ppp2r2d expression in OT-I T cells transduced with LacZ shRNA, Ppp2r2d shRNA, and Ppp2r2d shRNA plus Ppp2r2d mutant cDNA. Also, the Ppp2r2d shRNA with the highest in vivo activity was associated with the lowest levels of Ppp2r2d mRNA (FIG. 11 ).
  • Microarray analysis of tumor-infiltrating T cells expressing experimental or control shRNAs showed that each shRNA induced a distinct set of gene expression changes, with some overlap between particular shRNAs (FIG. 12 a-c ). Two genes (Egr2 and Ptpn2) have known functions in T cells. Enrichment in tumor versus spleen was calculated based on deep sequencing results from the secondary screen. (FIG. 12 a ) Clustering of mean expression levels for mRNAs found to be significantly regulated by T cells in spleens or tumors expressing the LacZ control shRNA or one of five experimental shRNAs. (FIG. 12 b ) Significant expression differences were defined as an Anova p value <0.01 between T cells expressing LacZ control shRNA or one of five experimental shRNAs (Alk, Arhgap5, Egr2, Ptpn2 or Ppp2r2d) (JMP-Genomics 6.0, SAS Institute Inc.). mRNAs significantly regulated in one or more treatment groups are shown after clustering (Fast Ward). FIG. 12 c is a Venn diagram showing overlaps between expression signatures by tumor-infiltrating T cells transduced with one of the five experimental shRNAs (signatures defined as an Anova p<0.01 as described above). Indicated are the numbers of overlapping probe IDs for any combination of the 5 signatures, as indicated by the overlapping ovals. The significance of the overlaps versus that expected by random chance (Fishers Exact Test) is shown in the accompanying table.
    • Example 3: Changes in T Cell Function Induced by Ppp2r2d
  • For this example, the cellular mechanisms driving T cell accumulation by a Ppp2r2d shRNA in tumors—specifically T cell infiltration, accumulation and apoptosis were examined. T cell infiltration into tumors was assessed by transfer of OT-I CD8 T cells labeled with a cytosolic dye, CFSE. OT-I T cells expressing Ppp2r2d or LacZ shRNAs were labeled with CFSE and injected into B16-Ova tumor-bearing mice. Twenty-four hours later transduced T cells were isolated from tumors and spleens and quantified by flow cytometry. OT-I T cells expressing LacZ or Ppp2r2d shRNAs were purified using the Thy1.1 reporter and cultured in complete RPMI media without added cytokines for 24 hours. Live cells isolated by Ficoll density gradient centrifugation (Sigma) were labeled with CFSE (carboxyfluorescein diacetate, succinimidyl ester, Invitrogen), and 2×106 labeled cells were injected into mice bearing day 14 B16-Ova tumors. CFSE dilution was quantified by flow cytometry at 24 hours and days 3, 5 and 7 following transfer. In addition, intracellular staining was performed on days 3, 5 and 7 for IFNγ, TNFα and isotype controls (BD). No differences were observed in the frequency of Ppp2r2d or LacZ shRNA-transduced CD8 T cells in tumors on day 1, arguing against a substantial effect on T cell infiltration (FIG. 13 a ). However, analysis of later time points (days 3 and 5) demonstrated a higher degree of proliferation (based on CFSE dilution) by Ppp2r2d compared to LacZ shRNA-transduced T cells (FIG. 13 b , FIG. 20 a ). Ppp2r2d shRNA-transduced T cells also produced higher levels of interferon-γ, a cytokine critical for anti-tumor immunity (FIG. 13 e ). The action of Ppp2r2d was downstream of T cell receptor activation because T cell accumulation was enhanced in tumors and to a lesser extent in tumor-draining lymph nodes. In contrast, no accumulation was observed in irrelevant lymph nodes or the spleen where the relevant antigen is not presented to T cells (FIG. 15 ).A substantial degree of T cell accumulation was even observed for LacZ shRNA-transduced T cells (complete dilution of CFSE dye by day 7), despite the presence of small numbers of such cells in tumors. This suggested that LacZ shRNA-transduced T cells were lost by apoptosis. Indeed, a larger percentage of tumor-infiltrating T cells were labeled with an antibody specific for active caspase-3 when the LacZ control shRNA (rather than Ppp2r2d shRNA) was expressed (FIG. 13 g , FIG. 20 b ). Furthermore, co-culture of CD8 T cells with B16-Ova tumor cells showed that the majority of LacZ shRNA expressing T cells became apoptotic (65.7%) while most Ppp2r2d shRNA-transduced T cells were viable (89.5%, FIG. 13 c ).
  • OT-I T cells expressing LacZ or Ppp2r2d shRNAs were purified based on Thy1.1 expression and labeled with CFSE, as described above. CFSE labeled OT-I T cells (1×105) were co-cultured with 5×104 B16-Ova cells per well in a 96-well plate for 72 h. Prior to the assay, B16-Ova cells were exposed to 1 ng/mL IFNγ for 48 hours (to induce MHC class I, which is not expressed in vitro) and washed three times. Apoptosis of OT-I T cells was detected by annexin V labeling of CD8+ cells. (FIG. 13 c ) Intracellular staining of phospho-AKT (Ser473), phopsho-B ad (Ser 112), Bc1-2 and isotype control was performed at 48 hours using a BD intracellular staining kit. Co-culture of CD8 T cells with B16-Ova tumor cells indeed showed that the majority of LacZ shRNA expressing T cells were apoptotic (65.7%) while the majority of Ppp2r2d shRNA-transduced T cells were viable (89.5%, FIG. 13 c ). A similar phenotype was observed when Ppp2r2d and LacZ shRNA-expressing T cells were stimulated with immobilized CD3 antibody in the absence of CD28 costimulation (FIG. 14 ). Specifically, B16-Ova cells (2×105) were injected s.c. into female C57BL/6 mice (10 weeks of age). On day 12, mice bearing tumors of similar size were divided into 7 groups (7-8 mice/group). Anti-CD3/CD28 bead activated CD4 TRP-1 or/and CD8 OT-I T cells infected with Ppp2r2d or LacZ shRNA vectors (2×106 T cells each) were injected i.v. on days 12 and day 17. For the treatment of B16 tumors, mice were treated at day 10 with anti-CD3/CD28 bead activated CD4 TRP-1 and CD8 pmel-1 T cells expressing Ppp2r2d or LacZ shRNAs (3×106 T cells each). Tumor size was measured every three days following transfer and calculated as length×width. Mice with tumors ≥20 mm on the longest axis were sacrificed.
  • These results suggested the possibility that Ppp2r2d shRNA-transduced CD8 T cells may be able to proliferate and survive even when they recognize their antigen directly presented by B16-Ova tumor cells. This idea was tested by implantation of tumor cells into b2m−/− mice which are deficient in expression of MHC class I proteins. In such mice, only tumor cells but not professional antigen presenting cells of the host could present tumor antigens to T cells. Indeed, Ppp2r2d shRNA-transduced OT-I CD8 T cells showed massive accumulation within B16-Ova tumors in b2m−/− mice (FIG. 120 while there were very small numbers of T cells in contralateral B16 tumors that lacked expression of the Ova antigen. T cells expressing a Ppp2r2d shRNA could thus effectively proliferate and survive in response to tumor cells, despite a lack of suitable co-stimulatory signals and an inhibitory microenvironment.
  • Ex vivo analysis of tumor-infiltrating T cells at a single-cell level using a nanowell device also demonstrated that Ppp2r2d silencing increased cytokine production by T cells (FIG. 21 a-c ). T cells were activated for 3 hours by CD3/CD28 antibodies on lipid bilayers, followed by 1 hour cytokine capture on antibody-coated slides. CD8 T cells showed a higher secretion rate for IFNγ, IL-2 and GM-CSF, and a larger fraction of T cells more than one cytokine (FIG. 21 b, c ). The presence of larger numbers of IFNγ-producing T cells was confirmed by intracellular cytokine staining (FIG. 21 d , FIG. 20 ).
  • PP2A phosphatase is composed of a catalytic and scaffolding subunit, and its substrate specificity is determined by one of many regulatory subunits 7. Ppp2r2d directs PP2A to Cdk1 substrates during interphase and anaphase; it thereby inhibits entry into mitosis and induces exit from mitosis25. PP2A plays a gatekeeper role for BAD-mediated apoptosis. Phosphorylated BAD is sequestered in its inactive form in the cytosol by 14-3-3, while dephosphorylated BAD is targeted to mitochondria where it causes cell death by binding Bc1-X L and Bc1-2 26. PP2A phosphatases have also been shown to interact with the cytoplasmic domains of CD28 and CTLA-4 as well as Carma1 (upstream of the NF-KB pathway), but it is not known which regulatory subunits are required for these activities; Ppp2r2d antibodies suitable for the required biochemical studies are currently not available.
    • Example 4: Silencing of Ppp2r2d Enhances Anti-Tumor Activity of CD4 and CD8 T Cells
  • The ability of a Ppp2r2d shRNA to enhance the efficacy of adoptive T cell therapy was assessed. B16-Ova tumor cells (2×105) were injected subcutaneously into female C57BL/6 mice (10 weeks of age). On day 12, mice bearing tumors of similar size were divided into seven groups (7-8 mice/group), either receiving no T cells, 2×106 shRNA-transduced TRP-1 CD4 T cells, 2×106 shRNA infected OT-I CD8 T cells, or both CD4 and CD8 T cells (days 12 and day 17). According to group, anti-CD3/CD28 bead activated CD4 TRP-1 or/and CD8 OT-I T cells infected with Ppp2r2d or LacZ shRNA vectors (2×106 T cells each) were injected i.v. on days 12 and day 17. For the treatment of B16 tumors, mice were treated at day 10 with anti-CD3/CD28 bead activated CD4 TRP-1 and CD8 pmel-1 T cells expressing Ppp2r2d or LacZ shRNAs (3×106 T cells each). Tumor size was measured every three days following transfer and calculated as length×width. Mice with tumors ≥20 mm on the longest axis were sacrificed. Ppp2r2d-silencing improved the therapeutic activity of CD4 and CD8 T cells, and a synergistic effect was observed when Ppp2r2d shRNA-transduced CD4 and CD8 T cells were co-administered (FIG. 16 a, b ). A Ppp2r2d shRNA also enhanced anti-tumor responses when introduced into T cells specific for endogenous tumor antigens (pmel-1 CD8 T cells and TRP-1 CD4 T cells) (FIG. 16 c ).
  • Ppp2r2d-silenced T cells acquired an effector phenotype in tumors (FIG. 22 a ) and >30% of the cells expressed granzyme B (FIG. 23 a ). Consistent with greatly increased numbers of such effector T cells in tumors (FIG. 23 b ), TUNEL staining demonstrated increased apoptosis in tumors when Ppp2r2d rather than LacZ shRNA expressing T cells were present (FIG. 23 c ). B16 melanomas are highly aggressive tumors in part because MHC class I expression is very low. Interestingly, Ppp2r2d but not LacZ shRNA-expressing T cells significantly increased MHC class I expression (H-2Kb) by tumor cells (FIG. 23 d ), possibly due to the observed increase in IFNγ secretion by T cells (FIG. 21 a-c , FIG. 13 e ). A Ppp2r2d shRNA did not reduce expression of inhibitory PD-1 or LAG-3 receptors on tumor-infiltrating T cells, demonstrating that its mechanism of action is distinct from these known negative regulators of T cell function (FIG. 22 b ). This finding suggests combination approaches targeting these intracellular and cell surface molecules.
  • These results establish the feasibility of in vivo discovery of novel targets for immunotherapy in complex tissue microenvironments. The inventors have shown that it is possible to discover genes with differential action across tissues, as exemplified by T cell accumulation in tumors compared to secondary lymphoid organs. For genes with tissue-selective action, T cell accumulation and survival are likely to be under the control of the T cell receptor and therefore do not occur in tissues lacking presentation of a relevant antigen. Many variations of the approach presented here can be envisioned to investigate control of particular immune cell functions in vivo. For example, fluorescent reporters for expression of cytokines or cytotoxic molecules (granzyme B, perforin) could be integrated into our approach to discover genes that control critical T cell effector functions in tumors.
  • Targeting of key regulatory switches may offer new approaches to modify the activity of T cells in cancer and other pathologies. The efficacy of such T cell-based therapies could be enhanced by shRNA-mediated silencing of genes that inhibit T cell function in the tumor microenvironment.
    • OTHER EMBODIMENTS
  • It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
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Claims (20)

1. An isolated nucleic acid encoding a chimeric antigen receptor (CAR) and a sequence encoding an shRNA,
the shRNA comprises 15 contiguous nucleotides complementary a nucleic acid sequence of SEQ ID NO: 612, and
wherein the CAR comprising an antigen binding domain, a transmembrane domain, a stimulatory domain, and a co-stimulatory domain.
2. The isolated nucleic acid of claim 1, wherein the shRNA sequence reduces expression of Cb1b.
3. The isolated nucleic acid of claim 1, wherein the antigen binding domain is an antigen-binding fragment of an antibody.
4. The isolated nucleic acid of claim 3, wherein the antigen-binding fragment is a Fab or scFv.
5. The isolated nucleic acid of claim 1, wherein the antigen-binding domain binds tumor antigen.
6. The isolated nucleic acid of claim 5, wherein the tumor antigen is associated with a melanoma, carcinoma, sarcomas, adenocarcinoma, lymphoma, leukemia, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer.
7. The isolated nucleic acid of claim 5, wherein the tumor antigen is associated with a solid tumor or lymphoid tumor.
8. A vector comprising the nucleic acid of claim 1.
9. An immunoresponsive cell comprising the nucleic acid of claim 1.
10. The immunoresponsive cell of claim 9, wherein the immunoresponsive cell is tumor specific.
11. The immunoresponsive cell of claim 10, wherein the immunoresponsive cell is selected from the group consisting of a tumorinfiltrating lymphocyte (TIL), a Natural Killer T cell (NKT), a cytotoxic T lymphocyte (CTL), and a CD4 T cell.
12. The immunoresponsive cell of claim 9, wherein the CAR is directed to a tumor antigen comprising prostate-specific membrane antigen (PSMA).
13. A human T cell harboring the nucleic acid molecule of claim 1.
14. A method for preparing an immunoresponsive cell having tumor specificity and increased resistance to immunosuppression, comprising:
providing an immunoresponsive cell having tumor specificity; and
introducing into the cell a vector comprising a sequence encoding a shRNA,
wherein the shRNA comprises 15 contiguous nucleotides complementary a nucleic acid sequence of SEQ ID NO: 612.
15. The method of claim 14, wherein the immunoresponsive cell is selected from the group consisting of a tumor-infiltrating lymphocyte (TIL), a Natural Killer T cell (NKT), a cytotoxic T lymphocyte (CTL), and a CD4T cell.
16. The method of claim 14, wherein the immunoresponsive cell expresses a tumor-specific T-cell receptor.
17. The method of claim 14, wherein the immunoresponsive cell comprises a vector encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and a stimulatory domain.
18. The method of claim 17, wherein the CAR is directed to a tumor antigen comprising prostate-specific membrane antigen (PSMA).
19. The method of claim 14, wherein the shRNA sequence reduces expression of Cb1b.
20. The isolated nucleic acid molecule of claim 1, wherein the sequence encoding the shRNA comprises a first sequence comprising 15-25 nucleotides complementary to SEQ ID NO: 612 and a second sequence that is the reverse complement of the first sequence with one or no mismatches, and a third sequence of 5-9 nucleotides positioned between the first and second sequences.
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