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FIG. 78 W O 2012/048316 A2 Llll II II 11III II I II III III I III III II I II

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FIG. 78 W O 2012/048316 A2 Llll II II 11III II I II III III I III III II I II (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 12 April 2012 (12.04.2012) WO 2012/048316 A2 (51) International Patent Classification: Cambridge, Massachusetts 02140 (US). SHARMA, So- A61K 48/00 (2006.01) nia [CA/US]; 48 Beacon Street, Boston, Massachusetts 02108 (US). (21) International Application Number: PCT/US20 11/055561 (74) Agents: RESNICK, David et al; Nixon Peabody LLP, 100 Summer Street, Boston, Massachusetts 02 110-213 1 (22) International Filing Date: (US). 10 October 201 1 (10.10.201 1) (81) Designated States (unless otherwise indicated, for every (25) Filing Language: English kind of national protection available): AE, AG, AL, AM, (26) Publication Language: English AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (30) Priority Data: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, 61/391,445 8 October 2010 (08.10.2010) US HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (71) Applicant (for all designated States except US): IM¬ KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, MUNE DISEASE INSTITUTE, INC. [US/US]; CLSB ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 3rd Floor, 3 Blackfan Circle, Boston, Massachusetts NO, NZ, OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, 021 15 (US). RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (72) Inventors; and ZM, ZW. (75) Inventors/Applicants (for US only): RAO, Anjana [US/ US]; 19 Agassiz Street, Cambridge, Massachusetts 02 140 (84) Designated States (unless otherwise indicated, for every (US). HOGAN, Patrick [US/US]; 19 Agassiz Street, kind of regional protection available): ARIPO (BW, GH, [Continued on next page] (54) Title: REGULATORS OF NFAT AND/OR STORE-OPERATED CALCIUM ENTRY (57) Abstract: Embodiments of the in ventions relate to modulating NFAT ac tivity, modulating store-operated Ca + entry into a cell and treating and/or pre venting hyperactivity or inappropriate immune response by inhibiting the ex pression or activities of septin 4 (SEPT 4) and septin 5 (SEPT 5) proteins in volved in the calcineurin/NFAT axis and T-cell activation. siControl siSEPT4 siSEPT4 oligo#3 oligo#4 < FIG. 78 w o 2012/048316 A2 llll II II 11III II I II III III I III III II I II GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, Published: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, — without international search report and to be republished RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, upon receipt of that report (Rule 48.2(g)) DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, — with sequence listing part of description (Rule 5.2(a)) SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). REGULATORS OF NFAT AND/OR STORE-OPERATED CALCIUM ENTRY CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application No. 61/391,445 filed on October 8, 2010, the contents of which are incorporated herein by reference in its entirety. GOVERNMENT SUPPORT [0002] This invention was made with Government support under contract Nos. AI40127 and GM075256, awarded by the National Institutes of Health. The Government has certain rights in the invention. FIELD OF THE INVENTION [0003] The invention relates to modulating nuclear factors of activated T-cell (NFAT) and/or store operated Ca2+ entry (SOCE) in cells, in particular T cells. The invention relates to the regulation of the activation of T cells and the modulation of immune responses. BACKGROUND OF INVENTION [0004] The calcium/calcineurin-dependent NFAT family is thought to have arisen following the recombination of an ancient precursor with a el domain about 500 million years ago, producing a new group of signaling and transcription factors (the NFAT genes) found only in the genomes of vertebrates. The family of NFAT transcription factor consists of five members NFAT1, NFAT2, NFAT3, NFAT4 and NFAT5. The NFAT proteins are activated by an increase in intracellular calcium levels, e.g., by means of store-operated calcium entry (SOCE) into a cell (see Fig. 1). SUMMARY OF THE INVENTION [0005] Embodiments of the invention are based on the discovery that several hundred genes in the human and mouse genomes whose gene products directly and/or indirectly modulate nuclear factors of activated T cell (NFAT) activation and/or modulate the store- operated Ca2+ entry (SOCE) into a cell. For example, the SEPT 4, SEPT 5 and UEV3 gene product modulate SOCE into a cell, modulate NFAT nuclear translocation and the consequential T cell activation. [0006] NFAT is a family of transcription factors that normally reside in the cytoplasm when inactive. When activated by dephosphorylation by calcineurin, the NFATs can translocate into the nucleus and "turn on" specific gene transcription. The inventors developed a cell-based reporter system for screening for modulators of NFAT and/or SOCE into a cell, with NFAT nuclear translocation as the readout for scoring a modulator. The cell-based reporter system comprises a mammalian cell co-expressing a NFAT-GFP, a STIMl-RFP, and an Orail-FLAG. The markers: GFP, RFP and FLAG-tag facilitate the visual localization of the respectively expressed proteins within the cell compartments. Thapsigargin (TG), a tight-binding inhibitor of sarco/endoplasmic reticulum Ca + ATPase, was used to deplete the Ca + in the endoplasmic reticulum and initiate SOCE, which in turn leads to NFAT dephosphorylation and NFAT nuclear translocation. The inventors used the cytoplasm-to-nuclear translocation of NFAT-GFP as their assay readout. [0007] The inventors performed a large scale high-throughput siRNA screening of the human and mouse genome for genes that modulate NFAT nuclear translocation and/or SOCE. Genes that modulate the NFAT nuclear translocation and/or SOCE can either up-regulate (i. e. promote) or down-regulate (i.e., inhibit) NFAT nuclear translocation and/or SOCE. NFAT nuclear translocation and/or SOCE are necessary for the activation of T cells, the proliferation of activated T cells, and for maintaining the immune response involving T- and B- cells in the body. In addition, the NFAT translocation is associated with multiple signaling pathways such as the MAP kinase, WNT, and NOTCH signaling pathways. As such, NFATs directly and/or indirectly play important roles in cell proliferation and regeneration, cancer, angiogenesis, cardiovascular diseases, diabetes, neural regeneration, bone diseases and T cell adaptation to name a few. Therefore, identification of the modulator genes of NFAT nuclear translocation and/or SOCE allows therapeutic regulation of the immune system, immune responses and other disease conditions associated with NFATs. [0008] The inventors found that the inhibition of the SEPT 4 and SEPT 5 gene expressions by RNA interference methods greatly reduced NFAT nuclear translocation in their assay system (see Figs. 75B, 78) and also SOCE in the affected cell (see Figs.53, 58, 70, 73, 75C, 77B). Septin proteins have functions in cytokinesis, membrane remodeling and compartmentalization in a cell. [0009] The inventors also found that the inhibition of the UEV3 gene expression by RNA interference methods greatly reduced NFAT nuclear translocation in their assay system (see Figs. 25, 35, 36) and also SOCE in the affected cell (see Figs. 24-26, 31). [0010] Inhibition of genes that up-regulate NFAT nuclear translocation and/or SOCE can help inhibit T-cell activation and immune response associated with hyperactivity or inappropriate activity of the immune system. Conversely, inhibition of genes that down-regulate NFAT nuclear translocation and/or SOCE can help increase T cells activation and immune responses associated with immune deficiency disease or conditions. [0011] Secondary and tertiary screens of the hits from primary screens were conducted. Secondary and tertiary screens comprise Ca + influx as readout for scoring. [0012] Accordingly, provided herein is a pharmaceutical composition comprising an agent that inhibits the function of a septin protein and/or the expression of a septin gene, and a pharmaceutically acceptable carrier. In one embodiment of the pharmaceutical composition, the septin is a septin 4. Other not limiting examples of septins include septin 2, 4, 5, 6, 7, and 9. In another embodiment, the pharmaceutical composition further comprises an agent that inhibits the function of a UEV3 protein and/or the expression of a UEV3 gene. [0013] In one embodiment of the pharmaceutical composition, the agent inhibits the function of at least two septin proteins and/or the expression of at least two septin genes. In one embodiment, the at least two septins are septin 4 and 5. In another embodiment, the at least two septins are septin 3 and 4. In another embodiment, the pharmaceutical composition further comprises an agent that inhibits the function of a UEV3 protein and/or the expression of a UEV3 gene. [0014] In another embodiment, the pharmaceutical composition comprises a first agent that inhibits the function of a first septin protein and/or the expression of a first septin gene, a second agent that inhibits the function of a second septin protein and/or the expression of a second septin gene, and a pharmaceutically acceptable carrier. In one embodiment of the pharmaceutical composition, the first septin is a septin 4 and the second septin is a septin 5. In another embodiment, the pharmaceutical composition further comprises an agent that inhibits the function of a UEV3 protein and/or the expression of a UEV3 gene.
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