WO2024092092A2

WO2024092092A2 - Compositions and methods for allogeneic immunotherapies

Info

Publication number: WO2024092092A2
Application number: PCT/US2023/077856
Authority: WO
Inventors: Michael Bethune; Michael Yi; Nhung Nguyen
Original assignee: Cargo Therapeutics, Inc.
Priority date: 2022-10-27
Filing date: 2023-10-26
Publication date: 2024-05-02
Also published as: WO2024092092A3

Abstract

The present application provides compositions, methods, pharmaceuticals, kits comprising recombinant polynucleotides comprising sequence encoding mutant or truncated RFX polypeptides for allogeneic immunotherapies and/or transplantations are provided herein.

Description

COMPOSITIONS AND METHODS FOR ALLOGENEIC IMMUNOTHERAPIES

CROSS REFERENCE

[0001] This application claims priority to U.S. Provisional Application No. 63/381,192, filed on October 27, 2022, the entirety of which is hereby incorporated by reference herein.

BACKGROUND OF THE DISCLOSURE

[0002] Immunotherapies that bolster a subject’s immune armament against infections, autoimmune conditions or cancer have been generated to express recombinant proteins on the surface of transgenic T cell receptors such as chimeric antigen receptors. The use of genetically modified T cell receptors (TCR), chimeric antigen receptor T-cells (CAR T-cell) or allogeneic cells holds significant promise in medicine for their use in adoptive immunotherapy. Broadly speaking, adoptive immunotherapies involve the transfer of autologous exogenously produced antigen-specific T cell receptors. In this process, customized T-lymphocytes recovered from the subject are engineered to produce proteins on T cell surface receptor. Subsequently, modified T cell, such as for example, CAR-T cells, are expanded in vitro in order to amplify the number of cells if necessary. Such engineered T cells e.g. CAR T-lymphocytes, are infused back into the patient to potentially function as expected. However, when or if the T cells or lymphocytes are isolated from a tumor mass (i.e. tumor infiltrating T lymphocytes, or TILs), for example, the process of TIL isolation and expansion may lead to loss of antitumor potency due to exhaustion of T cells limits their clinical applications. Therefore, while these therapies have proven effective in the treatment of certain diseases, autologous therapies face substantial technical and logistic hurdles to practical application; generation of autologous therapies requires expensive dedicated facilities, expert personnel and must be generated in a short time following a patient's diagnosis. In many cases, pretreatment of the patient has resulted in very low T cells or cells with degraded immune function. Moreover, since each patient's autologous cell preparation is effectively a new T cell/CAR-T cell product, generating cells may cause substantial variations in efficacy, safety, long wait times for subjects and high costs. Because of these hurdles, the development of off-the-shelf standardized therapy in which non-autologous/ allogeneic immunotherapeutic cells could be pre -manufactured, characterized in detail and be readily available is an attractive alternative approach. Nonetheless, because allogeneic immunotherapies involve recovery and use of cells from a genetically different subject, the use of allogeneic therapies present major challenges.

[0003] One of the challenges of allogeneic therapies (allograft) or immunotherapies lies in the immune reaction in a recipient following transfer of the allogeneic cells. Transfer of the modified T/allogeneic cells/CAR-T cells or allograft into a host e.g. an immune -competent host inevitably triggers an immune reaction that may cascade and result in rejection of the donor graft cells and/or damage to host tissues. Due to gender or HLA/MHC disparity between the recipient host and the donor, the grafted allogeneic cells may be rapidly rejected by the host's immune system leading to host-derived allogeneic graft rejection. In host-derived allogeneic graft rejection, the recipient’s immune system recognizes the allogeneic graft cells e.g. CAR-T cells or transplant and destroys the modified T/allogeneic cells/CAR-T cells. Additionally, graft T cells expressing functional T cell receptor may react to epitopes expressed on host tissues, leading to graft versus host disease (GVHD). GVHD occurs due to the presence of immunocompetent T lymphocytes in the graft cells attacking the tissue of the recipient host due to histocompatibility difference; the transplant or graft cells see the recipient host as an antigen and starts destroying the host system. To reduce host-derived allogeneic graft rejection, the recipient host’s immune can be suppressed prior to transfer of allogeneic grafts or cell transfusions. Glucocorticoid steroids are widely used therapeutically for immunosuppression, however, treatment of T cells with glucocorticoid steroids results in reduced levels of cytokine production leading to T cell anergy and reduced T cell activation. Use of other therapies such as Alemtuzumab, may lead to rapid depletion of circulating lymphocytes and monocytes but such harsh lymphodepletion treatment in subjects receiving adoptive immunotherapies may have a detrimental effect on transferred immunotherapeutic cells such as therapeutic T lymphocytes (e.g. CAR- T cells).

[0004] Adoptive immunotherapies that utilize standardized allogeneic (allograft) cell therapies e.g. modified T cell therapy hold great promise, but before such therapies can become routine or standardized immunotherapies, the risks associated with alloreactivity and the resultant host-mediated rejection or the potentially deadly GVHD must be overcome.

SUMMARY OF THE DISCLOSURE

[0005] To progress the promise of using allogeneic immunotherapies as standard treatments, the present disclosure described herein compositions, methods of use thereof ameliorates or prevents or avoids allogeneic graft rejection by a recipient host when present disclosures are provided simultaneously during provision of allogeneic transplantation and/or treatments. Such standardized allogeneic immunotherapies could be generated from any number of cells such as T cells, embryonic/fetal/adult hematopoietic stem cells (HSC), induced pluripotent stem cells (e.g. human iPSC), chimeric antigen receptors T cells, natural killer cells, placental cells, umbilical cells, gamma delta T cells or any number of modified or unmodified cells, such as for example, immune cells, including any living cells which are designed to express the compositions, methods, kits, pharmaceuticals disclosed herein.

[0006] Disclosed herein are recombinant nucleic acids that may be expressed in grafted cells to improve the efficacy of grafted cell implantation, while decreasing the activation of host immune responses and/or the activation of graft cell immune responses. Disclosed herein are approaches to decrease immune responsiveness by disrupting expression of graft cell regulatory genes that mediate the major histocompatibility complex proteins. Therefore, the promise of off-the-shelf or universal allogeneic cell immunotherapy products in effectively treating diseases such as, without limitations, cancer, demonstrates an urgent medical need exists for strategies to engineer cells that do not activate the host/graft immune response.

[0007] Provided herein is a composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFX5 polypeptide, wherein the truncated RFX5 polypeptide has a length of at most 359 amino acids. [0008] Also provided herein is a composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFX5 polypeptide, wherein the truncated RFX5 polypeptide comprises a truncation of at least 257 amino acids.

[0009] Also provided herein is a composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFX5 polypeptide, wherein the truncated RFX5 polypeptide comprises an N-terminal truncation.

[0010] Also provided herein is a composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFX5 polypeptide, wherein the truncated RFX5 polypeptide comprises an amino acid sequence consisting of amino acids 1-198 of SEQ ID NO:2.

[0011] Also provided herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide, and wherein the cell expresses endogenous RFX5. In some embodiments, the mutant or truncated RFX5 polypeptide comprises from 150-250 amino acids, from 175-225 amino acids, from 190-210 amino acids, or about 200 amino acids. In some embodiments, the mutant RFX5 polypeptide is a truncated RFX5 polypeptide. In some embodiments, the cell expresses endogenous RFXANK and endogenous RFXAP. In some embodiments the mutant RFX5 polypeptide interacts with endogenous RFXANK, endogenous RFXAP or a combination thereof. In some embodiments, the mutant RFX5 polypeptide interacts with endogenous RFXANK and endogenous RFXAP in the cell to form a complex. In some embodiments, expression of the mutant RFX5 polypeptide inhibits or interferes with MHC expression in the cell. In some embodiments, an expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is less than an expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is at least 25% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is at least 50% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the MHC is an endogenous MHC. In some embodiments, the MHC is an MHC Class I protein. In some embodiments, the MHC is an MHC Class II protein. In some embodiments, the MHC comprises an MHC Class I protein and an MHC Class II protein. In some embodiments, the MHC Class I protein is encoded by an HLA-A, HLA-B or HLA-C gene. In some embodiments, the MHC Class II protein is encoded by an HLA-DR, HLA-DP, or HLA-DQ gene. In some embodiments, disclosed herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide, wherein the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is substantially the same as the expression level of the MHC in a RFX5 knockout cell. In some embodiments, disclosed herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide, wherein the truncated RFX5 polypeptide comprises a PX2LPX5X6 motif; wherein X2 is any amino acid, X5 is any amino acid, and Xe is isoleucine (I) or leucine (L). In some embodiments, disclosed herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide, wherein, the mutant or truncated RFX5 lacks an NFY binding domain.

[0012] Also provided herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide, wherein the mutant or truncated RFX5 polypeptide comprises: a DNA binding domain; a C-terminal truncation; an RFXAP binding site; an RFXANK binding site; and wherein the truncated RFX5 lacks an NFY binding domain. In some embodiments, disclosed herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant or truncated RFX5 polypeptide, wherein, the mutant or truncated RFX5 polypeptide is a dominant negative RFX5 polypeptide (RFX5 DN). In some embodiments, the truncated RFX5 polypeptide lacks a nuclear localization signal (NLS).

[0013] Also disclosed herein in some embodiments, is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide, wherein the cell is an allogeneic cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a NK cell. In some embodiments, the cell is a gamma delta T cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, the cell is an embryonic or adult hematopoietic stem cell (HSC). In some embodiments, the T cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the T cell is a chimeric antigen receptor T (CAR-T) cell. In some embodiments, the cell is a host cell. In some embodiments, the host is a human host. In some embodiments, disclosed herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide, wherein the cell is a population of cells. In some embodiments, the population of cells comprises at least IxlO⁵ cells. In some embodiments, disclosed herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide, further comprises a recombinant nucleic acid comprising a sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises: (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain. In some embodiments, the antigen binding domain is an anti-CD19 binding domain. In some embodiments, the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYN SALKS and YAMDYWG, respectively. In some embodiments, the antigen binding domain is an anti-CD22 binding domain. In some embodiments, the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. In some embodiments, the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7- H3, GPC2, L1CAM, EGFR, mesothelin, MART-1, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE- 6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, Kiras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp- 175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, R0R1, and GD2. In some embodiments, the intracellular domain of the CAR comprises an intracellular signaling domain from CD3zeta, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3E, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70. In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28. In some embodiments, the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28. In some embodiments, the composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide and further comprises a recombinant nucleic acid wherein the recombinant nucleic acid is a vector. In some embodiments, the recombinant nucleic acid is a viral vector. In some embodiments, the recombinant nucleic acid is a plasmid vector. In some embodiments, the recombinant nucleic acid is an RNA or a DNA.

[0014] Also provided herein is a pharmaceutical composition comprising a composition disclosed herein, and a pharmaceutically acceptable excipient, diluent or carrier.

[0015] Also provided herein is a method of modulating MHC expression comprising expressing the mutant or truncated RFX5 polypeptide encoded by the nucleic acid sequence of the recombinant nucleic acid of the composition disclosed herein in a cell.

[0016] Also provided herein is a method of modulating MHC expression comprising inhibiting MHC expression. In some embodiments, inhibiting or modulating MHC expression comprises the mutant or truncated RFX5 polypeptide binding to a promoter for an MHC in the cell.

[0017] Also provided herein is a method comprising a cell comprising the composition disclosed herein and further comprising a population of T cells, wherein a higher percentage of T cells in the population of T cell survive when administered to a subject compared to the percentage of T cells that survive in a population of T cells that do not express the mutant or truncated RFX5 polypeptide when administered to the subject. In some embodiments, provided herein is a method comprising a cell comprising the composition disclosed herein, wherein the cell further comprises a population of T cells, and wherein a percentage of T cells in the population of T cell that survive when administered to a subject is about 50%-120% of the T cells that survive in a population of RFX5 knockout T cells when administered to the subject. In some embodiments, the subject comprises alloreactive T cells. Also provided herein is a method comprising a cell comprising compositions disclosed herein and further comprising T cells, wherein the number of T cells in the population of T cell that survive when administered to a subject is at least 1.5 fold higher than the number of T cells T cells that survive in a population of T cells that do not express the mutant or truncated RFX5 polypeptide when administered to the subject. In some embodiments, the number of T cells in the population of T cell that survive when administered to a subject is at least 2 or 3 or 4 or 5 fold higher than the number of T cells T cells that survive in a population of T cells that do not express the mutant or truncated RFX5 polypeptide when administered to the subject.

[0018] Also provided herein is a method comprising a recombinant nucleic acid encoding a mutant or truncated RFX5 polypeptide, wherein the mutant or truncated RFX5 polypeptide inhibits binding or recruitment of RFXANK and/or RFXAP to a promoter for an MHC in the cell. Also provided herein is a method of inhibiting binding or recruitment of endogenous RFX5 to a promoter for an MHC, wherein the method comprises contacting the promoter for an MHC in a cell with mutant or truncated RFX5 polypeptide. In some embodiments, the mutant or truncated RFX5 polypeptide competes with endogenous RFX5 for a promoter for an MHC in the cell.

[0019] Also provided herein is a method comprising a cell comprising the compositions disclosed herein wherein the methods comprise treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the subject is a human subject. In some embodiments, the disease or condition is cancer or an auto-immune disease or condition. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and/or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC). Also provided herein is a method comprising a cell comprising the compositions disclosed herein wherein the methods comprise treating an autoimmune disease or condition, wherein the autoimmune disease is an inflammatory condition.

[0020] Also provided herein is a vector comprising a sequence encoding a recombinant nucleic acid comprising a mutant RFX5 polypeptide, wherein the vector further comprising a sequence encoding a chimeric antigen receptor (CAR).

[0021] Also provided herein is a composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFXANK polypeptide, wherein the truncated RFXANK polypeptide has a length of less than 260 amino acids. In some embodiments, the truncated RFXANK polypeptide further comprises a mutation. In some embodiments, the mutation is a mutation at a position corresponding to position 121 of SEQ ID NO: 18 and/or 224 of SEQ ID NO: 16.

[0022] Also provided herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFXANK polypeptide, and wherein the cell expresses endogenous RFXANK. In some embodiments, the mutant RFXANK polypeptide is a truncated RFXANK polypeptide. In some embodiments, the mutant or truncated RFXANK polypeptide lacks one or more ankyrin repeat domains and/or lacks one or more domains that bind to CIITA and/or NLRC5. In some embodiments, the truncated RFXANK polypeptide comprises 3 or fewer ankyrin repeat domains. In some embodiments, the truncated RFXANK polypeptide comprises 2 or fewer ankyrin repeat domains. In some embodiments, the truncated RFXANK polypeptide comprises 1 ankyrin repeat domain. In some embodiments, the truncated RFXANK polypeptide lacks an ankyrin repeat domain. In some embodiments, the mutant RFXANK polypeptide is a truncated RFXANK. In some embodiments, the mutant RFXANK comprises a mutation at a position corresponding to position 121 of SEQ ID NO: 18 and/or 224 of SEQ ID NO: 16. In some embodiments, the mutation is selected from the group consisting of a D121V mutation, a Y224A mutation and a combination thereof.

[0023] Also provided herein is a composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFXANK polypeptide, wherein expression of the mutant RFXANK polypeptide inhibits or interferes with expression of an MHC in the cell. In some embodiments, expression level of an MHC in the cell expressing the mutant RFXANK polypeptide is less than an expression level of the MHC in a cell not expressing the mutant RFXANK polypeptide. In some embodiments, the MHC is an endogenous MHC. In some embodiments, the MHC is an MHC Class I protein. In some embodiments, the MHC is an MHC Class II protein. In some embodiments, the MHC Class II protein is encoded by an HLA-A, HLA-B, or HLA-C gene. In some embodiments, the MHC Class II protein is encoded by an HLA-DR, HLA-DP, or HLA-DQ gene. Also provided herein is a composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFXANK polypeptide, wherein the expression level of an MHC in the cell expressing the mutant RFXANK polypeptide is substantially the same as the expression level of the MHC in a RFXANK knockout cell. Also provided herein is a composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFXANK polypeptide, wherein the mutant RFXANK polypeptide is a dominant negative RFXANK polypeptide (RFXANK DN). Also provided herein is a composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFXANK polypeptide, wherein the truncated RFXANK polypeptide has a length of less than 260 amino acids. In some embodiments, the mutant or truncated RFXANK polypeptide has a length of less than 150, 125 or 100 amino acids. In some embodiments, the mutant or truncated RFXANK polypeptide has a length of about 122 amino acids.

INCORPORATION BY REFERENCE

[0024] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0026] FIG. 1 is an exemplary illustration of native regulatory factors completely bound to and by RFX5 in the cognate promoter region that are essential for the transcription of MHC class I/II and the incomplete binding that occurs when a variant of truncated RFX5 competes for and successfully binds within the MHC transcriptional regulatory region thereby blocking expression of MHC protein genes and MHC class I/II proteins.

[0027] FIG. 2 is an exemplary schematic illustrating an overview of the elements and interacting factors governing transcriptional regulation of HLA/MHC class I and HLA/MHC class II.

[0028] FIG. 3 is an exemplary schematic of full length, wild-type RFX5 (616 amino acids) and truncated RFX5 polypeptides of varying length containing the indicated domains. DNA binding domain (DBD); NFY binding domain (NFY BD); nuclear localization sequence (NLS).

[0029] FIG. 4 is an exemplary schematic of wild-type RFXANK, truncated RFXANK polypeptides and a Y224A mutant RFXANK polypeptide. The Y224A mutant RFXANK polypeptide competes for and binds in the MHC promoter region thereby decreasing MHC class I/II expression.

[0030] FIG. 5 depicts exemplary graphs of MHC expression data obtain using of four RFX5 truncations that abrogate expression of both MHC I and II (left; 1371, 1372, 1372), or MHC II alone (right; 1484). To evaluate the modulation of MHC expression following binding of truncated versions of RFX5, a positive control, RFX5 knockout construct (RFX5 KO) was included. Two additional controls, a negative control (NTD) and a transduction marker (BFP) were also included. Truncated RFX5 (1-198 amino acids) lacking the NFY binding domain (1371) abrogated MHC Class I/II to levels phenotypically comparable to those of the RFX5 KO construct.

[0031] FIG. 6 depicts an exemplary graph of data obtained using various truncated RFX5 constructs. The effect of truncated RFX5 on the expression of MHC is shown; percent (%) HLA mean fluorescent intensity (MFI) was determined for the HLA-ABC (MHC I; % HLA-ABC MFI) and % HLA-DR, DP and DQ (MHC II; %HLA- DRDPDQ MFI) gene expression. To evaluate MHC modulated expression, a positive control, RFX5 knockout construct (RFX5 KO) was included. Two additional controls, a negative control (NTD) and a transduction marker (BFP) were also included. As shown, truncated RFX5 (1-198 amino acids) lacking the NFY binding domain (1371) abrogated MHC Class I/II to levels phenotypically comparable to those of the RFX5 KO construct.

[0032] FIG 7 depicts exemplary FACS graphs of MHC-I expression vs BFP (transduction marker). The results indicate that the downmodulation of truncated RFX5 did not significantly impact MHC I expression suggesting MHC expression was minimally dependent on transduction levels of the RFX5 regulatory factor (BFP; transduction marker). Truncated RFX5 (1-198 amino acids), the dominant negative RFX5, (RFX5 DN), showed decreased MHC I expression, even at lower transduction (lower BFP MFI). When Simian viral vector nuclear localization signal (SV40 NLS (PKKRKV) was fused to the C-terminal, higher expression was required for MHC I reduction.

[0033] FIG. 8 depicts an exemplary graph of data showing the impact of RFX5 dominant negative (DN) on graft survival when allogeneic T cells were co-cultured with graft T cells for 48 hours in a 1-way T cell mixed lymphocyte reaction. % survival of cells expressing RFX5 dominant negative (DN) was comparable to that for RFX5 knockout (KO) cells, and was significantly higher than survival of unmodified controls. Addback of the RFX5 nuclear localization signal (NLS) or addition of an SV40 NLS to RFX5 DN resulted in decreased RFX5 DN efficacy (data not shown).

[0034] FIG. 9 depicts an exemplary graph of data showing the impact of RFX5 dominant negative (DN) on graft survival when allogeneic NK cells were co-cultured with graft T cells for 48 hours in a 1-way NK cell mixed lymphocyte reaction. % survival of cells expressing RFX5 dominant negative (DN) was comparable to that for RFX5 knockout (KO) cells and was significantly higher than survival of 2m KO cells.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0035] Disclosed herein are provided compositions, kits and methods of use comprising a cell comprising a recombinant polynucleic acid comprising a sequence encoding a dominant negative (DN) regulatory factor X (RFX) polypeptide, such as a truncated RFX polypeptide or a mutated RFX polypeptide. In some embodiments, the DN RFX polypeptide ("DN RFX polypeptide" used interchangeably with "truncated RFX polypeptide" and mutant RFX polypeptide") encoded by the recombinant polynucleic acid can be expressed in a cell. In some embodiments, a DN RFX polypeptide disclosed herein can be expressed in a cell expressing a corresponding wild-type RFX polypeptide. In some embodiments, the DN RFX polypeptide comprises a truncated Regulatory factor X 5 (RFX5) polypeptide or a truncated RFXANK (RFX ankyrin containing protein) polypeptide. In some embodiments, the DN RFX polypeptide can be expressed in a cell to generate an allogeneic immunotherapy. In some embodiments, the DN RFX polypeptide can be expressed in a cell expressing a chimeric antigen receptor (CAR). In some embodiments, a cell, such as a T cell, comprising a recombinant polynucleic acid encoding the DN RFX polypeptide can be formulated as a pharmaceutical composition and/or used to treat a disease or condition, such as cancer. In some embodiments, a cell, such as a T cell, comprising a recombinant polynucleic acid encoding the DN RFX polypeptide and a CAR can be formulated as a pharmaceutical composition and/or used to treat a disease or condition, such as cancer when expressed in the cell expressing a chimeric antigen receptor (CAR).

[0036] In some embodiments, a truncated RFX polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncated RFXANK polypeptide. In some embodiments, a truncated RFX polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncated RFXB polypeptide. In some embodiments, a truncated RFX polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncated RFXAP polypeptide.

[0037] In some embodiments, the truncated RFX polypeptide is a truncated RFX5 polypeptide. In some instances, a truncated RFX5 polypeptide comprises a truncation at the N-terminus or the C-terminus. In some embodiments, a DN RFX5 polypeptide disclosed herein can be expressed in a cell expressing a wild-type RFX5 polypeptide.

[0038] In some embodiments, the DN RFX polypeptide is a DN RFXANK polypeptide. In some embodiments, a DN RFXANK polypeptide is a truncated RFX polypeptide. In some embodiments, a DN RFXANK polypeptide is a mutant RFXANK polypeptide. For example, a DN RFXANK polypeptide can be a RFXANK polypeptide with a Y224A mutation. In some instances, a truncated RFXANK polypeptide comprises a truncation at the N-terminus or the C-terminus. In some embodiments, a DN RFXANK polypeptide disclosed herein can be expressed in a cell expressing a wild-type RFXANK polypeptide. In some embodiments, a DN RFXANK polypeptide can be expressed in a cell expressing wild-type RFXANK and can retain a capacity to interact with other native polypeptides in the RFX complex, e.g. wild-type RFX5 and/or RFXAP.

[0039] Wild-type RFX5 can bind to wild-type RFXANK and RFXAP to form the RFX complex which may bind an XI regulatory element and can interacts with NLRC5 and CREB 1/ATF/NFY which can promote expression of HLA I genes. In some instances, a DN RFX5 polypeptide provided herein, such as a truncated or mutant RFX5 polypeptide, can bind to an NLRC5 and/or CREB 1/ATF/NFY regulatory region of an MHC promoter. For example, binding of the DN RFX5 polypeptide to XI can influence binding and/or interaction of NLRC5 and/or CREB 1/ATF/NFY regulatory regions in an MHC promoter and can affect expression of HLA I genes. In some instances, a DN RFX5 polypeptide provided herein can bind to an XI regulatory region influencing binding and/or interaction of a CIITA and/or CREB 1/ATF/NFY regulatory regions of an MHC promoter. For example, binding of the DN RFX5 polypeptide and interaction with CIITA and/or CREB 1/ATF/NFY regulatory regions in an MHC promoter can affect expression of HLA II genes. In some embodiments, the DN RFX5 provided herein can bind to RFXB/ANK/AP to form an RFX complex. For example, a DN RFX5 polypeptide disclosed herein can retain a capacity to bind to a DNA-binding domain on an XI box. For example, a DN RFX5 polypeptide disclosed herein can retain a capacity to bind to a regulatory region in the MHC promoter. In some embodiments, the DN RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can bind competitively, to the cognate, endogenous binding site of a native RFX5 polypeptide in a cell to negatively affect transcriptional regulation in the cell. For example, binding of the DN RFX5 polypeptide to the cognate, endogenous binding site of the native RFX5 can modulate an MHC promoter in the cell. In some embodiments, the binding site of the native RFX5 in the cell can be an endogenous site in the MHC promoter region. In some embodiments, binding of DN RFX5 polypeptide to the endogenous site of native RFX5 polypeptide enables DN RFX5 to interact with regulatory factors (or transcription factors) within the MHC promoter region. In some embodiments, binding of DN RFX5 encoded by sequences of the recombinant polynucleotides can inhibit complete binding and engagement of the multimers within the MHC promoter. Truncated RFX5 polypeptides (tRFX5) encoded by sequences of the recombinant polynucleotides can interact with wild-type RFXAP/ANK polypeptides and bind to the SXY (XI) region where the complex comprising truncated RFX5 polypeptide/RFXAP/ANK may be unable to for example, engage the NF-Y molecule. In some embodiments, the truncated RFX5 polypeptide encoded by sequences of the recombinant polynucleotides can interact with native RFXAP/ANK complex to promote inhibition of a functional MHC promoter. In some embodiments, interaction of truncated RFX5 polypeptide with native RFXAP/ANK and/or other factors abrogates transcription from the MHC promoter in a dominant negative fashion.

[0040] In some instances, a DN RFXANK polypeptide, such as a truncated or mutant RFXANK polypeptide, encoded by sequences of the recombinant polynucleotides can interact via the ankyrin repeats, with NLRC5 and CREB1/ATF/NFY regulatory region of the MHC promoter. For example, the DN RFXANK polypeptide can bind to native RFX5/RFXB/AP polypeptide and can interact with NLRC5 and CREB1/ATF/NFY regulatory region in the MHC promoter which can affect expression of HLA I genes. In some embodiments, a DN RFXANK polypeptide can interact with and can bind to native RFX5/RFXB/AP polypeptides to interact with CIITA and CREB1/ATF/NFY regulatory region of the MHC promoter. For example, a DN RFXANK polypeptide can bind to native RFX5/RFXB/AP polypeptides and can interact with CIITA and CREB1/ATF/NFY regulatory region in the MHC promoter which can affect expression of HLA II genes. In some embodiments, a DN RFXANK encoded by sequences of the recombinant polynucleotides can bind to native RFX5/RFXB/AP polypeptides to form an RFX complex. For example, a DN RFXANK polypeptide can retain the capacity to bind and interact with native RFX5/RFXB/RFXAP in a regulatory region in the MHC promoter of the cell. In some embodiments, a DN RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can bind and interact competitively, to the cognate, endogenous binding site of a native RFXANK polypeptide in a cell to negatively affect transcriptional regulation in the cell. For example, the binding of a DN RFXANK polypeptide to the cognate, endogenous binding site of the native RFXANK can modulate an MHC promoter in the cell. For example, the binding site of the native RFXANK in the cell can be an endogenous site in the MHC promoter region. In some embodiments, binding of a DN RFXANK polypeptide to the endogenous site of native RFXANK polypeptide enables a DN RFXANK to interact with regulatory factors e.g. native RFX5 and RFXB/AP (or transcription factors) within the MHC promoter region.

[0041] In some embodiments, the compositions, methods, kits, pharmaceuticals disclosed herein e.g., truncated RFX polypeptide comprising a sequence encoding a recombinant polynucleotide e.g. comprising truncated or mutant RFX5 protein, mutant or truncated RFXANK protein can be expressed and used in allogeneic cells- based therapies. For example, allogeneic cells comprising compositions, kits, methods of use, or pharmaceuticals disclosed herein can provide robust, safe, off-the-shelf global allogeneic cell therapies.

-I l [0042] In some embodiments, a DN RFX polypeptide, e.g. a truncated or mutant RFX5 polypeptide or a truncated or mutant RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can be used to generate an allogeneic cell therapy. For example, a truncated RFX5 polypeptide, or a truncated RFXANP polypeptide or a combination of gene products comprising genetic modifications comprising a truncated, mutated, or a variant of an RFX polypeptide can be used alone or in combinations with any locus of the gene product to generate allogeneic cell therapies comprising for example, a T cell therapy, a chimeric antigen T cell- associated therapy (CAR-T), or comprising an embryonic or adult hematopoietic stem cell-associated therapy ((HSC) e.g. HSC transplantation for treating a disease, a condition, or any monogenic disease such as for example, sickle cell disease), or comprising an induced pluripotent stem cell (iPSC e.g. an animal -associated iPSC, e.g. a human-associated iPSC) therapy, or comprising a chimeric antigen-associated (CAR) therapy. For example, a DN RFX polypeptide encoded by a sequence of a recombinant polynucleotide can be used to generate allogeneic cell therapies e.g., an iPSC-associated therapy/treatment comprising for example, beta islet cells-associated iPSC, or natural killer (NK) cells or chimeric antigen NK cells-associated iPSC, or retinal cells- associated iPSC, or neural cells-associated iPSC, or cardiomyocytes-associated iPSC, or CAR T cell-associated iPSC, or macrophage-associated iPSC, or B cells-associated iPSC, or T cell or T lymphocyte-associated iPSC or innate lymphoid cells (ILCs)-associated iPSC, or mesenchymal stem cells (MSC)-associated iPSC; or placenta cells-associated iPSC or fetal cells-associated iPSC or an allogeneic cell derived therapy that is an iPSC-derived cell comprising a mammalian cell e.g. a human cell.

[0043] Provided herein is a cell comprising a recombinant nucleic acid comprising a sequencing encoding a truncated RFX5 polypeptide, such as for example truncated RFX5 polypeptide or truncated RFXANK polypeptide, can provide dominant negative when expressed in a cell. In some embodiments, a cell expressing a truncated or mutant DN RFX polypeptide encoded by a sequence of the recombinant polynucleotide can modulate MHC/HLA gene expression in a dominant negative manner. For example, a DN RFX polypeptide expressed in a cell can inhibit the function of a wild-type RFX polypeptide. For example, a cell expressing a truncated RFX5 DN or a truncated RFXANK DN encoded by a sequence of the recombinant polynucleotide can modulate MHC/HLA genes in a dominant negative manner. In some instances, an allogeneic cell can express a mutant or truncated RFX5 DN or truncated RFXANK DN which can abrogate an HLA/MHC barrier encountered in allogeneic cell therapies discussed above for example, allogeneic cell-derived therapies comprising e.g. HSC, CARs therapies, iPSC therapies, CAR-T therapies. In some embodiments, these allogeneic cell therapies can mediate MHC/HLA-associated barriers in allograft or allotransplant therapies. For example, these allogeneic cell can mediate host-derived allotransplant or allograft rejection. For example, these allogeneic cell therapies can mediate recipient-derived graft vs host disease (GVHD). For example, these allogeneic cell therapies can mediate an HLA/MHC-mediated barrier that can be associated with using autologous cell therapies, allogeneic cell therapies, allograft or allotransplants, grafts of any kind or any HLA/MHC-derived barrier that can be encountered during transplant of biomaterials into a living system such as an animal, such as a human. [0044] In some embodiments, a cell e.g. an allogeneic cell comprising a truncated RFX polypeptide DN (e.g. truncated RFX5 DN or truncated or mutant RFXANK DN) comprising a sequence encoding a recombinant polynucleotide may be generated from a chimeric antigen receptor, a hematopoietic stem cell, or an induced pluripotent stem cell (e.g. a human iPSC) derived from any living cell, for example, a living cell from a mammal/animal such as a human cell.

[0045] In some embodiments, a cell e.g. an allogeneic cell comprising the compositions, kits, methods of use or pharmaceuticals disclosed herein can be administered to a subject or individual in need thereof to treat or ameliorate, reduce, inhibit, block, neutralize, interfere, abrogate or prevent a disease, a disorder, an infection or a condition or syndrome or symptom. In some embodiments, a cell e.g. an allogeneic cell comprising the compositions, kits, methods of use or pharmaceuticals disclosed herein can be administered to a subject or individual in need thereof to treat (used interchangeably with ameliorate, reduce, inhibit, block, neutralize, interfere, abrogate or prevent) a condition, disease, disorder, infection, syndrome or symptom while preventing the activity of MHC promoter thereby providing the treatment and with a respite against MHC/HLA associated barriers. Such treatment, prophylaxis, prevention or administration may be targeted to one, two, three or more conditions, infections, symptoms, diseases, syndromes in the subject in need thereof in any dosage required and administered in any form to bring about such a relief or treatment or prophylaxis or change. Non-limiting example of indications comprising conditions comprising cancers, infections, tissue or cell-specific disorders or diseases or disorders of the immune system such as those associated with inflammatory reactions, immune reactions, as well as many other indications known and unknown presently but for which the administering brings relief, a prophylactic treatment, curative treatment, preventative treatment, alleviation of symptoms or any kind of distress, bodily or otherwise to the subject is need is hereby claimed. The subject can be treated wherein the administration of the claimed disclosures leads to at least one or more symptoms, disorders, conditions, syndromes, diseases, infections to become better or to be reduced, relieved, prevented, treated, cured, alleviated or eliminated or placed in a condition that is different from before the administering of any of the disclosed inventions.

Definitions

[0046] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”

[0047] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the disclosure. [0048] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0049] “Percent (%) sequence identity” or “homology” with respect to the nucleic acid or amino acid sequences identified herein is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.

[0050] ‘ ‘Percent (%) identity” with respect to the nucleic acid or amino acid sequences identified herein is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.

[0051] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of subranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the recombinant polypeptides, methods and other aspects belong. Although any recombinant polypeptides methods and other aspects similar or equivalent to those described herein can also be used in the practice or testing of the recombinant polypeptides, methods and other aspects, representative illustrative recombinant polypeptides, methods and other aspects are now described.

[0053] As used herein, the terms “polynucleotides,” “nucleic acids,” and “oligonucleotide,” are used interchangeably. As used herein recombinant nucleic acids, recombinant polynucleotides are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides, or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. Polynucleotides may have any three-dimensional structure and may perform any known function or unknown function. Various nonlimiting examples of polynucleotides includes: non-coding or coding regions of a gene fragment, or a gene, locus (loci) that is defined from linkage analysis, exons or introns, messenger RNA, transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA or RNA of any sequences, nucleic acid probes or primers. There are many forms and descriptions denoting a polypeptide as disclosed herein. Therefore, a polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may exist in a cell-free environment. A polynucleotide may be a gene or fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure, and may perform any function, known or unknown. A polynucleotide may comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5- bromouracil, peptide nucleic acid, xeno nucleic acid, morpholines, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g. rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, eDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell- free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non- nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0054] As used herein, the term “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Also “polypeptides” may denote an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.

[0055] Variants of the amino acid sequences described herein may be included in various embodiments. The term "variant" refers to a protein or polypeptide in which one or more amino acid substitutions, deletions, and/or insertions are present as compared to the amino acid sequence of a protein or polypeptide, and the term includes naturally occurring allelic variants and alternative splice variants of a protein or polypeptide. The term "variant" includes the replacement of one or more amino acids in an amino acid sequence with a similar or homologous amino acid(s) or a dissimilar amino acid(s). Some variants include alanine substitutions at one or more amino acid positions in an amino acid sequence. Other substitutions include conservative substitutions that have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein.

[0056] A “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.

[0057] A “fragment” is a truncated form of a native biologically active protein that, in some instances, retains at least a portion of the therapeutic and/or biological activity. A “variant” is a protein with sequence homology to the native biologically active protein that, in some instances, retains at least a portion of the therapeutic and/or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the reference biologically active protein. As used herein, the term “biologically active protein moiety” includes proteins modified, as for example, by site directed mutagenesis, insertions, or accidentally through mutations.

[0058] As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids. As used herein, the term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T), wherein the redundancy for the genetic code is included in its entirety for all amino acid codons.

[0059] As used herein, the term “non-naturally occurring,” as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally-occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned. [0060] As used herein, the terms “gene” or “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof.

[0061] “Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.

[0062] “Homology” or “homologous” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores. Preferably, polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity to those sequences.

[0063] The term “binding domain”, as used herein, refers to a molecule, such as a protein, or polypeptide sequence, which specifically binds to a target.

[0064] As used herein, a subject is “in need of’ a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

[0065] As used herein, the term “operably connected” or “operably linked” refers to positioning of components such that they function in their intended manner. For example, the components can be operably connected by a fusion, a linker, and/or a spacer.

[0066] As used herein, “specifically binds” means that the binding domain preferentially binds the corresponding target over other targets. In some embodiments, “specifically binds” means that the binding domains have a higher affinity for the target than for other targets.

[0067] As used herein, a “therapeutically effective amount” or “therapeutically effective number” of an agent is an amount or number sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of an agent means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer, an infection, an autoimmune disease, an inflammatory disorder or disease, an immune reaction syndrome or symptom, condition or disease. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques.

[0068] As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” is used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions disclosed herein may be administered to a subject at risk of developing a particular disease, condition, disorder, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0069] The terms “administering,” “introducing” and “transplanting” are used interchangeably in the context of the placement of the recombinant polypeptides, nucleic acids, and/or gene editing molecules, and/or recombinant cells of the disclosure into a subject, by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced. The recombinant polypeptides, nucleic acids, and/or gene editing molecules, or recombinant cells of the disclosure can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the patient, i.e., long-term engraftment. For example, in some embodiments, described herein, an effective amount of the recombinant polypeptides, nucleic acids, and/or gene editing molecules, or recombinant cells is administered via a systemic route of administration, such as an intraperitoneal or intravenous route.

[0070] The terms “individual”, “subject,” “host” and “patient” are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human being.

[0071] The term “donor” is used to refer to an individual that is not the patient. In some embodiments, the donor is an individual who does not have or is not suspected of having the medical condition to be treated. In some embodiments, multiple donors, e.g., two or more donors, can be used. In some embodiments, each donor used is an individual who does not have or is not suspected of having the medical condition to be treated.

[0072] When provided therapeutically, the recombinant polypeptides, nucleic acids, and/or gene editing molecules, or recombinant cells of the disclosure may be provided at (or after) the onset of a symptom or indication of a medical condition, e.g., upon the onset of disease.

[0073] In some embodiments, the cells being administered according to the compositions and methods described herein comprise allogeneic T cells. In some embodiments, the cells being administered according to the compositions and methods described herein comprise T cells obtained from one or more donors. In some embodiments, the cell population being administered can be allogeneic immune cells, T cells, blood cells, hematopoietic stem cells, hematopoietic progenitor cells, embryonic stem cells, or induced embryonic stem cells.

[0074] A cell composition can be administered by any appropriate route that results in effective treatment in the subject, i.e. administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, i.e. at least 1 x 10⁴ cells are delivered to the desired site for a period of time. Modes of administration include injection, infusion, instillation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrastemal injection and infusion. In some embodiments, the route is intravenous. For the delivery of cells, administration by injection of a cell comprising truncated RFX polypeptides can be made.

[0075] The phrases “systemic administration,” “administered systemically”, “peripheral administration” and “administered peripherally” refer to the administration of a population of recombinant cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes.

[0076] “As used herein, "percent (%) identity," refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of another reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences may be disregarded for comparison purposes). Alignment, for purposes of determining percent identity, may be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software and any other software to perform polypeptide alignment of for example, amino acid sequences, or nucleotide sequences as determined to be necessary and using appropriate parameters for determining alignment, including algorithms needed to achieve maximal alignment over the length of the sequences being compared. Percent identity of two sequences may be calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides in the same position of the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence. Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured. In some embodiments, a truncated RFX protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence described herein.

[0077] A “vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function fortranscription and/or translation of the DNA or RNA and any derivative of nucleic acids including for example constructs such as siRNA, antisense, miRNA and the like. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide (s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product. [0078] As used herein, a “therapeutic effect”, may refer to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. The efficacy of a treatment comprising a composition for the treatment of a medical condition can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” if any one or all of the signs or symptoms of, as but one example, tumor size is reduced (e.g., reduced by at least 10%), or other clinically accepted symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0079] As used herein the terms “therapeutically effective amount” and “therapeutically effective dose”, may refer to an amount a biologically active protein such as for example, the therapeutic cells e.g. allogeneic cells that can comprise compositions, methods of use, kits, or pharmaceuticals comprising the mutant or truncated RFX5 DN protein, mutant or truncated RFXANK DN protein or any mutant or truncated RFX protein of the RFX complex disclosed herein, when used either alone or as a part of other treatment, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject using any one of the methods to administer therapeutics. Such effect need not be absolute to be beneficial. In some embodiments, therapeutically effective dose regimen may refer to a schedule for consecutively administered doses of a biologically active cell e.g. cell therapies comprising the compositions, kits, methods of use or pharmaceuticals herein either alone or as a part of other treatment regimen, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition without the effect being necessarily absolute to be beneficial.

[0080] As disclosed herein are experimental procedures that may provide descriptions of tests, assays and experimentations that can use general techniques. Such general techniques as used in the present disclosure may be publicly available, for example such techniques as described herein may employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3^rd edition, Cold Spring Harbor Laboratory Press, 2001; “Current protocols in molecular biology”, F. M. Ausubel, et al. eds., 1987; the series “Methods in Enzymology,” Academic Press, San Diego, Calif.; “PCR 2: a practical approach”, M. J. MacPherson, B. D. Hames and G. R. Taylor eds., Oxford University Press, 1995; “Antibodies, a laboratory manual” Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988; “Goodman & Gilman's The Pharmacological Basis of Therapeutics,” 11^th Edition, McGraw-Hill, 2005; and Freshney, R. I., “Culture of Animal Cells: A Manual of Basic Technique,” 4^th edition, John Wiley & Sons, Somerset, N J, 2000, the contents of which are incorporated in their entirety herein by reference.

[0081] As used herein, the term “recombinant” as applied to a polynucleotide means that the polynucleotide, polypeptides is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other recombinant DNA/RNA/Polypeptide technological procedures that result in a construct that can potentially be expressed in a host cell.

Overview: immunotherapies

[0082] The present disclosure provides compositions, methods of use, kits or pharmaceutical that can be applied widely in the transplant, allograft, allogeneic or autologous cell therapies.

[0083] Allogeneic cell therapies offer alternative approach to the design of a ready-made and may be an attractive option to autologous cell therapies. Allogeneic cell therapies can comprise for example, allogeneic chimeric antigen receptor (CAR) T cell therapies, adult or embryonic hematopoietic stem cell transplants, bone barrow transplants, induced pluripotent stem cell therapies (iPSC), such as human-derived iPSC, and any number of allogeneic cell therapeutics. Because allogeneic cell therapies might be pre-manufactured as a readymade therapy for multiple recipients in centralized batched production runs, the economic burden can be reduced compared to personalized autologous therapies generated for individual patients. Presently, there are no approved or certified allogeneic cell therapies.

[0084] Provided herein are compositions, methods of use, kits and pharmaceuticals that can be used to generate a universal allogeneic cell therapeutic that can provide non-alloreactivity in the donor host T cells. For example, expression of the present compositions in a recombinant cell can provide a shield which may alleviate destruction of e.g. graft cells by the host cells.

[0085] Present allogeneic cell therapies/infusions suffer from a risk of alloreactivity in the host. The cells of the immune system are trained early to differentiate between self and non-self (antigens), however, the ability to recognize the antigenic cell depends on the human leukocyte antigens (HLA) / major histocompatibility complex (MHC) proteins. MHC/HLA gene products can be critical for developing adaptive immunity e.g. defense against infections. However, successful allogeneic cell therapies or transplantation depend on a match between the donor’s and recipient’s human leukocyte antigens (HLA) since mismatch in MHC/HLA class I/II genes can be a life-threatening or risky cause of graft rejection issues such as for example, graft versus host disease (GVHD) and/or host-mediated graft rejection.

[0086] Host cells such as the dendritic cells of the recipient host may indirectly recognizes and/or acquire allogeneic MHC peptides from immune cells or they may recognize or acquire MHC peptides of the donor allogeneic cells which may stimulate T cell to direct an immune response against the cells. In some embodiments, the present disclosure comprises compositions that can modulate T cell response in the donor or allogeneic cell such that allogeneic cells expressing disclosed composition may not activate the T cell immune response of the host. In some embodiments, the present disclosure is directed at compositions that can modulate host-mediated rejection to overcome HLA/MHC-derived barriers in transplantation/graft or allogeneic cell therapies. [0087] The present disclosure provides a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding truncated regulatory factor x 5 (RFX5) polypeptide that can block transcription of HLA/MHC gene transcription. For example, the present disclosure provides composition that may block T cell response by interfering with recognition by the CD4⁺ T cell receptor (TCRa ) of a cognate antigen presented by self-major histocompatibility complex (MHC) class II on a professional antigen presenting cell (APC). For example, the present disclosure provides a composition that may block T cell response by interfering with recognition by the TCRa0 of a cognate antigen presented by self MHC class II on a B lymphocyte, a dendritic cell or a macrophage. In some embodiments, the present disclosure provides a composition that can disrupt donor T cells alloreactivity toward minor mismatched proteins in the host and can abrogate cell activation by allopeptides presented by host MHC. For example, the compositions of the present disclosure can block MHC class II dependent GVHD in donor CD4⁺ T cell which can otherwise be triggered by host APCs or which can be initiated by other MHC class II-expressing host cells in the absence of the composition(s) of the present disclosure.

[0088] In some embodiments, the present disclosure obliterates barriers that presently accompany use of allogeneic cell therapies and/or allograft transplants. For example, by overcoming HLA/MHC-derived barrier, the compositions of the present disclosure can be utilized to pre-manufacture off-the-shelf cells modulation of graft rej ection without the costly side effects that presently accompany clinical use of allogeneic cell therapies.

Universal (non-alloreactive) allogeneic cell therapies

[0089] Overcoming barriers to allogeneic cell therapies include, but are not limited to, disruption of genes involved in the recognition of T cell surface antigens, or MHC-bound antigens which can interrupt allogeneic cell therapies. T cell surface antigens e.g. MHC-bound antigens can be interrupted using drugs to render infused allogeneic T cells resistant to lymphodepleting chemotherapy or antibody therapy or by controlling for example, HLA/MHC genes in the allogeneic cells using modification tools such as for example, without limitation, RNA interference (RNAi), short hairpin RNA (shRNA), small interfering RNA (siRNA), meganucleases (MN), zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein (CRISPR) for example CRISPR/Cas9, etcetera.

[0090] The present disclosure provides non-gene editing modulations comprising a composition that can block an immune reaction from the host toward the allogeneic cell, e.g. by blocking T cell receptor (TCRa0) activation, and/or inhibit HLA/MHC gene expression. For example, the compositions/methods of use/pharmaceuticals/kit can trigger disruption of HLA/MHC transcription which may prevent a TCRap from recognizing an HLA/MHC-bound antigen in the recipient. For example, disrupting TCRa0 or blocking HLA/MHC transcription can alleviate the risk of donor-mediated GVHD and/or alleviating host-mediated rejection encountered when patients receive allogeneic cell transplants/infusions.

[0091] The present disclosure obviate the need for in vivo genetic modifications entirely and instead provides a composition comprising truncated RFX polypeptides wherein expression of the truncated RFX polypeptides e.g. truncated or mutant RFX5/RFXANK polypeptide can modulate an MHC promoter via a dominant negative expression thereby disrupting TCRa0 from being activated e.g. due to expression of the truncated polypeptide in a recombinant cell. Truncated RFX polypeptides

[0092] The present disclosure provides a composition comprising truncations of an RFX polypeptide encoded by sequence of recombinant polynucleotides. In one aspect, the disclosure provides a composition comprising a cell comprising a truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide. In one aspect, the disclosure provides a composition comprising a cell comprising a truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide. In some embodiments, disclosed herein is a genetic truncation (or mutation) that can be used to produce a portion or mutant of a gene product. In some embodiments disclosed herein, is a truncation that can be used to create and produce a fragment of a gene product. In some embodiments disclosed herein is a truncation that can used to target a portion of a protein or polypeptide. For example, a truncation of an RFX5 polypeptide or RFXANK polypeptide encoded by a sequence comprising a recombinant polynucleotide disclosed herein can target the N-terminal domain of the protein or the C- terminal domain. In some embodiments, a truncation in an RFX5 polypeptide or an RFXANK polypeptide can target a non-coding region or a coding region. For example, the truncation in an RFX polypeptide can be designed to target an amino acid codon change e.g. to generate a premature stop codon, a missense codon or a nonsynonymous codon change in the polypeptide wherein the change in the amino acid codon can interferes with the protein function. For example, such a truncation in the RFX5 polypeptide or RFXANK polypeptide may comprise a truncation in the N or C-terminal domain. In some embodiments, the truncation can interrupt the function of the gene product. For example, truncations in the RFX5 polypeptide or RFXANK polypeptide can target partial disruption in protein expression wherein the protein produced comprises non-functional domains that retain capacity to interact with proximal proteins. In some embodiments, a truncation in RFX5 polypeptide or RFXANK polypeptide can comprise a truncation, or a mutation, or a deletion, or an insertion, or a wherein such truncation can generate a variant as compared to the wild-type form. For example, a truncation in the RFX5 polypeptide or RFXANK polypeptide can comprise a gene variant, or a protein variant or an mRNA variant.

[0093] In some embodiments, the recombinant polynucleotide comprises a sequence encoding truncated RFX5 polypeptide wherein the truncated RFX5 polypeptide has a length of at most 359 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide has a length of about 350 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide has a length of about 340 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide has a length of about 330 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide has a length of about 320 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide has a length of about 310 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide has a length of about 300 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide has a length of about 270 amino acids. In some embodiments, the truncated RFX5 polypeptide encoded by a recombinant polynucleotide has a length of about 260 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide has a length of about 200 to about 250 amino acids.

[0094] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation at the C-terminal domain. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation comprising a missing portion of the C-terminal domain. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a missing portion of the C-terminal wherein the truncated RFX5 polypeptide can be a variant or mutant RFX5 polypeptide that is different from a wild-type RFX5 polypeptide. For example, the truncated polypeptide encoded by a sequence of the recombinant polypeptide can comprise a truncation of at least 257 amino acids. In some embodiments, the truncated RFX5 polypeptide can comprise a truncation of at least 259 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation of at least 270 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation of at least 280 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation of at least 300 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation of at least 320 amino acids. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation of at least 330 to 358 amino acids.

[0095] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polypeptide can comprise a mutant RFX5 polypeptide. In some embodiments, the mutant or truncated RFX5 polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a length from about 150-250 amino acids. In some embodiments, the mutant or truncated RFX5 polypeptide can comprise a length of about 150, 160, 170, 180, 190, 200, 220 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at least 150 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at leastl60 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at least 170 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at least 180 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at least 90 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at least 200 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at least 220 amino acids. In some embodiments, the mutant or truncated RFX5 polypeptide can comprise a length from 175-225 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of from about 180-200 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of about 175 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of about 185 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of about 190 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of about 195. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at least 175 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at least 180 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of at least 198-200 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of about 200 amino acids. In some embodiments, the mutant or truncated RFX5 polypeptide can comprise a length from 190-210 amino acids. For example, the mutant or truncated RFX5 polypeptide can comprise a length of about 210-350 amino acids.

[0096] For example, the truncated RFX5 encoded by a sequence of the recombinant polypeptide can be a variant of a native RFX5 polypeptide.

[0097] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncated C-terminal domain such that the truncation in the C-terminus makes it impossible to bind or corporate with an NF-Y.l molecule. For example, the truncated RFX5 comprising the truncated C-terminal domain can interact with native RFXB/AP/ANK to form an RFX complex which can inhibit or be hindered to interact with other regulatory factors e.g. NF-Y.l molecule in the MHC promoter.

[0098] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a wild-type C-terminal domain. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a native C-terminal domain comprising the C-terminal site that can mediate cooperative binding between an RFX complex and an NF-Y.l molecule.

[0099] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polypeptide can comprise an N-terminal truncation. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation comprising at least one or more amino acids at the N-terminal domain. In some embodiments, truncated RFX5 polypeptide can comprise a truncation comprising at least 2, 5, 10, 15, 20 or more amino acids at the N-terminal domain. In some embodiments, truncated RFX5 polypeptide can comprise a truncation comprising at least, 2 or more amino acids at the N- terminal domain. In some embodiments, truncated RFX5 polypeptide can comprise a truncation comprising at least 5 or more amino acids at the N-terminal domain. In some embodiments, truncated RFX5 polypeptide can comprise a truncation comprising at least 10 or more amino acids at the N-terminal domain. In some embodiments, truncated RFX5 polypeptide can comprise a truncation comprising at least 15 or more amino acids at the N-terminal domain. In some embodiments, truncated RFX5 polypeptide can comprise a truncation comprising at least 20 or more amino acids at the N-terminal domain. In some embodiments, truncated RFX5 polypeptide can comprise a truncation comprising at least 50 or more amino acids at the N-terminal domain. In some embodiments, the truncated RFX5 polypeptide can comprise a truncation at the N-terminal domain comprising a mutation, a deletion, an insertion, or a single nucleotide polymorphism.

[0100] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polypeptide can comprise an N-terminal that is a wild-type N-terminus or a native N-terminus. For example, truncated RFX5 polypeptide encoded by a sequence of the recombinant polypeptide can comprise an N-terminus comprising a region that may be required for binding to and interacting with other RFX polypeptides to comprise an RFX complex. For example, a region of the N- terminus of truncated RFX5 polypeptide can be necessary for dimer formation, or association with RFXB/AP/ANK polypeptides wherein the dimer formation can assemble an RFX complex on the MHC promoter. For example, a region of the N-terminus of truncated RFX5 polypeptide encoded by a sequence of the recombinant polypeptide can be necessary for the assembly of the

RFX complex to bind to the regulatory XI box target site in the MHC class I or class II promoter region.

[0101] Disclosed herein are compositions, kits, methods of use, pharmaceuticals, cells comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFXANK polypeptide, wherein the truncated RFXANK polypeptide has a length of less than 260 amino acids. For example, truncated RFXANK polypeptide can comprise a length of less than 255 amino acids. For example, truncated RFXANK polypeptide can comprise a length of less than 250 amino acids. For example, truncated RFXANK polypeptide can comprise a length of less than 245 amino acids. For example, truncated RFXANK polypeptide can comprise a length of less than 240 amino acids. For example, truncated RFXANK polypeptide can comprise a length of less than 235 amino acids. For example, truncated RFXANK polypeptide can comprise a length of less than 230 amino acids.

[0102] In some embodiments, the truncation can target a region of an RFX complex comprising truncations or mutations in the RFXANK polypeptide. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polypeptide can comprise a mutant RFXANK polypeptide. In some embodiments, the mutant or truncated RFXANK polypeptide can comprise a sequence length of less than 150 ammo acids. In some embodiments, the mutant or truncated RFXANK polypeptide a length of less than 145 ammo acids. In some embodiments, the mutant or truncated RFXANK polypeptide a length of less than 140 ammo acids. In some embodiments, the mutant or truncated RFXANK polypeptide a length of less than 135 ammo acids. In some embodiments, the mutant or truncated RFXANK polypeptide a length of less than 130 ammo acids. In some embodiments, the mutant or truncated RFXANK polypeptide a length of less than 125 ammo acids. In some embodiments, the mutant or truncated RFXANK polypeptide a length of less than 120 ammo acids. In some embodiments, the mutant or truncated RFXANK polypeptide a length of less than 115 ammo acids. In some embodiments, the mutant or truncated RFXANK polypeptide a length of less than 100 amino acids.

[0103] In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polypeptide can comprise a mutant or truncated RFXANK polypeptide having a length of about 122 amino acids. For example, the mutant RFXANK polypeptide can comprise a length of about 100-120 amino acids. For example, the mutant RFXANK polypeptide can comprise a length of about 100 amino acids. For example, the mutant RFXANK polypeptide can comprise a length of about 105 amino acids. For example, the mutant RFXANK polypeptide can comprise a length of about 110 amino acids. For example, the mutant RFXANK polypeptide can comprise a length of about 115 amino acids. For example, the mutant RFXANK polypeptide can comprise a length of about 120 amino acids. For example, the mutant RFXANK polypeptide can comprise a length of at most 122 amino acids.

[0104] In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation at the N-terminal domain. For instance, the truncated RFXANK polypeptide may lack one or more ankyrin repeat domains and/or lacks one or more domains that bind to CIITA and/or NLRC5. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation at the C-terminal domain. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a truncation at the N- and C-terminal domain.

[0105] In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise at least 3 ankyrin repeat domains. In some embodiments, the truncated RFXANK polypeptide can comprise at least 2 ankyrin repeat domains at the C-terminus. In some embodiments, the truncated RFXANK polypeptide can comprise 2 or fewer ankyrin repeat domains. In some embodiments, the truncated RFXANK polypeptide can comprise 1 ankyrin repeat domain. In some embodiments, the truncated

RFXANK polypeptide can comprise 1 ankyrin repeat domain at the C-terminus. In some embodiments, the truncated RFXANK polypeptide can comprise 3 or fewer ankyrin repeat domains. In some embodiments, the truncated RFXANK polypeptide can comprise 2 or fewer ankyrin repeat domains. In some embodiments, the truncated RFXANK polypeptide lacks an ankyrin repeat domain.

[0106] In some embodiments, the mutant RFXANK polypeptide is a truncated RFXANK polypeptide. In some embodiments, the mutant RFXANK polypeptide comprises a mutation at a position corresponding to position 121 and/or 224 of SEQ ID NO: 16. In some embodiments, encoded by a sequence of the recombinant polynucleotide comprises a mutation selected from the group consisting of a D121V mutation, a Y224A mutation and a combination thereof.

[0107] In some embodiments, a truncated or mutant RFX5 polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence in Table 1.

[0108] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 1. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 2. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 3. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 4. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 6. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 7. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a

- l- sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 8. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 9. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 10. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 11. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to atruncated RFX5 polypeptide sequence from SEQ ID NO: 12. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 13. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a DNA binding domain (DBD), a NK decoy and a nuclear localization signal (nls) sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 5. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a simian virus (SV40) nuclear localization signal (nls) and a porcine teschovirus-1 2A (P2A) sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 14.

[0109] In some embodiments, the recombinant polynucleotide comprising a sequence encoding truncated RFX5 polypeptide can be a DNA sequence. In some embodiments, the recombinant polynucleotide comprising a sequence encoding truncated RFX5 polypeptide can be an mRNA sequence. For instance, the recombinant polynucleotide comprising a sequence encoding truncated RFX5 polypeptide can be a nucleic acid form including cDNA, genomic DNA or synthetic DNA. In some embodiments, the recombinant polynucleotide comprising a sequence encoding truncated RFX5 polypeptide can be DNA. In some embodiments, the recombinant polynucleotide comprising a sequence encoding truncated RFX5 polypeptide can be a singlestranded DNA or double -stranded DNA.

[0110] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can be exogenous to a cell in which it is expressed. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise sequences from the same species as the cell in which it is expressed. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise sequences from a different species as the cell in which it is expressed. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of a recombinant polypeptide can be endogenous to a cell in which it is expressed. For instance, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise sequences from the same species as the cell in which it is expressed.

[oni] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence in Table 1. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 1. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 1. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 1. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 1. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 1. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 1. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs: 1. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 1.

[0112] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 2. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 2. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 2. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 2. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 2. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 2. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs: 2. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 2.

[0113] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 3. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 3. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 3. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 3. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 3. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 3. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs:

3. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 3.

[0114] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 4. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 4. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 4. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 4. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 4. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 4. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs:

4. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 4.

[0115] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a DNA binding domain (DBD), a NK decoy and a nuclear localization signal (nls) sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 5. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a DNA binding domain (DBD), a NK decoy and a nuclear localization signal (nls) sequence with at least about 80% sequence identity to a sequence selected from SEQ ID NO: 5. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a DNA binding domain (DBD), a NK decoy and a nuclear localization signal (nls) sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NO: 5. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a DNA binding domain (DBD), a NK decoy and a nuclear localization signal (nls) sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NO: 5. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a DNA binding domain (DBD), a NK decoy and a nuclear localization signal (nls) sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NO: 5. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a DNA binding domain (DBD), a NK decoy and a nuclear localization signal (nls) sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NO: 5. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a DNA binding domain (DBD), a NK decoy and a nuclear localization signal (nls) sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NO: 5. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a DNA binding domain (DBD), a NK decoy and a nuclear localization signal (nls) sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NO: 5.

[0116] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 6. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 6. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 6. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 6. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 6. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 6. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs:

6. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 6.

[0117] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 7. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 7. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 7. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 7. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 7. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 7. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs:

7. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 7.

[0118] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 8. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 8. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 8. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 8. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 8. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 8. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs:

8. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 8.

[0119] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 9. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 9. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 9. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 9. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 9. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 9. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs:

9. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 9.

[0120] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 10. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 10. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 10. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 10. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 10. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 10. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs:

10. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 10.

[0121] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 11. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 11. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 11. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 11. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 11. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 11. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs:

11. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 11.

[0122] In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 13. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NOs: 13. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 13. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 13. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 13. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 13. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NOs: 13. In some embodiments, a truncated RFX5 polypeptide, encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NOs: 13.

[0123] In some embodiments, a truncated RFX5 polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a simian virus (SV40) nuclear localization signal (nls) and a porcine teschovirus- 1 2A (P2A) sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NO: 14. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a simian virus (SV40) nuclear localization signal (nls) and a porcine teschovirus-1 2A (P2A) sequence with at least about 80%, sequence identity to a sequence selected from SEQ ID NO: 14. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a simian virus (SV40) nuclear localization signal (nls) and a porcine teschovirus-1 2A (P2A) sequence with at least about 85% sequence identity to a sequence selected from SEQ ID NO: 14. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a simian virus (SV40) nuclear localization signal (nls) and a porcine teschovirus-1 2A (P2A) sequence with at least about 90%, 95% sequence identity to a sequence selected from SEQ ID NO: 14. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a simian virus (SV40) nuclear localization signal (nls) and a porcine teschovirus-1 2A (P2A) sequence with at least about 98% sequence identity to a sequence selected from SEQ ID NO: 14. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a simian virus (SV40) nuclear localization signal (nls) and a porcine teschovirus- 1 2A (P2A) sequence with at least about 99% sequence identity to a sequence selected from SEQ ID NO: 14. In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a simian virus (SV40) nuclear localization signal (nls) and a porcine teschovirus- 1 2A (P2A) sequence with at least about 100% sequence identity to a sequence selected from SEQ ID NO: 14. [0124] In some embodiments, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFX5 polypeptide sequence from SEQ ID NO: 20.

[0125] In some embodiments, a truncated or mutant RFXANK polypeptide, encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence in Table 2.

[0126] In some embodiments, a truncated RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFXANK polypeptide sequence from SEQ ID NO: 15. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFXANK polypeptide sequence from SEQ ID NO: 16. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFXANK polypeptide sequence from SEQ ID NO: 17. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFXANK polypeptide sequence from SEQ ID NO: 18. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a truncated RFXANK polypeptide sequence from SEQ ID NO: 19.

[0127] In some embodiments, a recombinant polynucleotide comprising a sequence encoding truncated RFXANK polypeptide can be a DNA sequence. In some embodiments, the recombinant polynucleotide comprising a sequence encoding truncated RFXANK polypeptide can be an mRNA sequence. For instance, the recombinant polynucleotide comprising a sequence encoding truncated RFXANK polypeptide can be a nucleic acid form including cDNA, genomic DNA or synthetic DNA. In some embodiments, the recombinant polynucleotide comprising a sequence encoding truncated RFXANK polypeptide can be DNA. In some embodiments, the recombinant polynucleotide comprising a sequence encoding truncated RFXANK polypeptide can be a single-stranded DNA or double-stranded DNA.

[0128] In some embodiments, a truncated RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can be exogenous to a cell in which it is expressed. For example, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise sequences from the same species as the cell in which it is expressed. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise sequences from a different species as the cell in which it is expressed. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of a recombinant polypeptide can be endogenous to a cell in which it is expressed. For instance, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise sequences from the same species as the cell in which it is expressed.

[0129] In some embodiments, a truncated RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to sequence selected from SEQ ID NO: 15. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80% sequence identity to sequence selected from SEQ ID NO: 15. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to sequence selected from SEQ ID NO: 15. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to sequence selected from SEQ ID NO: 15. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to sequence selected from SEQ ID NO: 15. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to sequence selected from SEQ ID NO: 15. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to sequence selected from SEQ ID NO: 15.

[0130] In some embodiments, a truncated RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to sequence selected from SEQ ID NO: 16. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80% sequence identity to sequence selected from SEQ ID NO: 16. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to sequence selected from SEQ ID NO: 16. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to sequence selected from SEQ ID NO: 16. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to sequence selected from SEQ ID NO: 16. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to sequence selected from SEQ ID NO: 16. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to sequence selected from SEQ ID NO: 16. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to sequence selected from SEQ ID NO: 16.

[0131] In some embodiments, a truncated RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to sequence selected from SEQ ID NO: 17. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80% sequence identity to sequence selected from SEQ ID NO: 17. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to sequence selected from SEQ ID NO: 17. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to sequence selected from SEQ ID NO: 17. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to sequence selected from SEQ ID NO: 17. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to sequence selected from SEQ ID NO: 17. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to sequence selected from SEQ ID NO: 17. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to sequence selected from SEQ ID NO: 17.

[0132] In some embodiments, a truncated RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to sequence selected from SEQ ID NO: 18. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80% sequence identity to sequence selected from SEQ ID NO: 18. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to sequence selected from SEQ ID NO: 18. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to sequence selected from SEQ ID NO: 18. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to sequence selected from SEQ ID NO: 18. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to sequence selected from SEQ ID NO: 18. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to sequence selected from SEQ ID NO: 18. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to sequence selected from SEQ ID NO: 18.

[0133] In some embodiments, a truncated RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to sequence selected from SEQ ID NO: 19. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 80% sequence identity to sequence selected from SEQ ID NO: 19. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to sequence selected from SEQ ID NO: 19. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to sequence selected from SEQ ID NO: 19. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to sequence selected from SEQ ID NO: 19. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to sequence selected from SEQ ID NO: 19. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to sequence selected from SEQ ID NO: 19. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to sequence selected from SEQ ID NO: 19. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to sequence selected from SEQ ID NO: 19.

[0134] In some embodiments, a truncated RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can comprise a sequence with at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to sequence selected from SEQ ID NO: 20. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 85% sequence identity to sequence selected from SEQ ID NO: 20. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 90% sequence identity to sequence selected from SEQ ID NO: 20. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 95% sequence identity to sequence selected from SEQ ID NO: 20. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 98% sequence identity to sequence selected from SEQ ID NO: 20. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 99% sequence identity to sequence selected from SEQ ID NO: 20. In some embodiments, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can comprise a sequence with at least about 100% sequence identity to sequence selected from SEQ ID NO: 20.

[0135] The present disclosure provides compositions, kits, methods of use, pharmaceutical comprising a truncated RFX polypeptide, e.g. a truncated RFX5 polypeptide encoded by a sequence comprising a recombinant polynucleotide. In some embodiments provided herein is a composition comprising a cell comprising a recombinant polynucleotide (can be used interchangeably with e.g. nucleic acid, polynucleic acid, nucleotide, etc.) comprising a sequence encoding a mutant RFX5 polypeptide, and wherein the cell expresses endogenous RFX5. In some embodiments provided herein is a composition comprising a cell comprising a recombinant polynucleotide comprising a sequence encoding a mutant RFX5 polypeptide, and wherein the cell expresses endogenous RFXANK.

[0136] In some embodiments provided herein is a composition comprising a cell comprising a recombinant polynucleotide comprising a sequence encoding a mutant RFX5 polypeptide wherein the cell expresses endogenous RFXANK and endogenous RFXAP or a combination thereof. In some embodiments the cell comprising the mutant RFX5 polypeptide encoded by a sequence of a recombinant polynucleotide expresses endogenous RFXANK. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide does not express endogenous RFXANK. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide does not express endogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide does not express endogenous RFXANK and/or endogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide expresses endogenous RFXANK but not RFXAP. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide expresses endogenous RFXAP but not endogenous RFXANK. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide expresses exogenous RFXANK and exogenous RFXAP, or a combination thereof. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide expresses exogenous RFXANK or exogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide expresses exogenous RFXANK but not exogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide expresses exogenous RFXAP but not exogenous RFXANK. In some embodiments, the cell comprising truncated or mutant RFX5 encoded by a sequence of the recombinant polynucleotide expresses exogenous RFXANK or exogenous RFXAP. In some embodiments, the cell comprising the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide expresses truncated RFXANK, or truncated RFXAP or a combination thereof. In some embodiments, the cell comprising the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide expresses truncated RFXANK, or truncated RFXAP or a combination thereof.

[0137] In some embodiments, the cell comprising an exogenous truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide expresses an exogenous truncated RFXANK, or an exogenous truncated RFXAP or a combination thereof. In some embodiments, the cell comprising the exogenous truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide expresses exogenous truncated RFXANK, and/or endogenous RFXAP. In some embodiments, the cell comprising the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide expresses an endogenous RFXANK, and/or an exogenous truncated RFXAP or a combination thereof.

[0138] The present disclosure provides compositions, kits, methods of use, pharmaceutical comprising a truncated RFX polypeptide, e.g. a truncated RFXANK polypeptide encoded by a sequence comprising a recombinant polynucleotide. In some embodiments provided herein is a composition comprising a cell comprising a recombinant polynucleotide (can be used interchangeably with e.g. nucleic acid, polynucleic acid, nucleotide, etc.) comprising a sequence encoding a mutant RFXANK polypeptide, and wherein the cell expresses endogenous RFXANK. In some embodiments provided herein is a composition comprising a cell comprising a recombinant polynucleotide comprising a sequence encoding a mutant RFXANK polypeptide, and wherein the cell expresses endogenous RFX5.

[0139] In some embodiments provided herein is a composition comprising a cell comprising a recombinant polynucleotide comprising a sequence encoding a mutant RFXANK polypeptide wherein the cell expresses endogenous RFX5 and endogenous RFXAP or a combination thereof. In some embodiments the cell comprising the mutant RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide expresses endogenous RFX5. In some embodiments, the cell comprising truncated or mutant RFXANK encoded by a sequence of the recombinant polynucleotide does not express endogenous RFX5. In some embodiments, the cell comprising truncated or mutant RFXANK encoded by a sequence of the recombinant polynucleotide does not express endogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFXANK encoded by a sequence of the recombinant polynucleotide does not express endogenous RFX5 and/or endogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFXANK encoded by a sequence of the recombinant polynucleotide expresses endogenous RFX5 but not RFXAP. In some embodiments, the cell comprising truncated or mutant RFXANK (truncated and mutant are used interchangeably) encoded by a sequence of the recombinant polynucleotide expresses endogenous RFX5 but not endogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFXANK encoded by a sequence of the recombinant polynucleotide expresses exogenous RFX5 and exogenous RFXAP, or a combination thereof. In some embodiments, the cell comprising truncated or mutant RFXANK encoded by a sequence of the recombinant polynucleotide expresses exogenous RFX5 or exogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFXANK encoded by a sequence of the recombinant polynucleotide expresses exogenous RFX5 but not exogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFXANK encoded by a sequence of the recombinant polynucleotide expresses exogenous RFX5 but not exogenous RFXAP. In some embodiments, the cell comprising truncated or mutant RFXANK encoded by a sequence of the recombinant polynucleotide expresses exogenous RFX5 or exogenous RFXAP. In some embodiments, the cell comprising the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide expresses truncated RFX5, or truncated RFXAP or a combination thereof. In some embodiments, the cell comprising the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide expresses truncated RFX5, or truncated RFXAP or a combination thereof.

[0140] In some embodiments, a cell comprising an exogenous truncated RFXANK polypeptide encoded by a sequence of a recombinant polynucleotide can express an exogenous truncated RFX5, or an exogenous truncated RFXAP or a combination thereof. In some embodiments, the cell comprising the exogenous truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide expresses exogenous truncated RFX5, and/or endogenous RFXAP. In some embodiments, the cell comprising the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide expresses an endogenous RFX5, and/or an exogenous truncated RFXAP or a combination thereof.

RFX complex within MHC promoters

[0141] An RFX complex can contain two molecules of RFX5 and one molecule each of RFX- B/RFXANK/RFXAP polypeptides. Formation of the RFX complex can utilize RFX5 to bind and interact with RFXAP/ANK/B to form the RFX complex. In some embodiments, formation of the RFX complex and binding in the SXY-module may be essential for the enhancement and activation of gene transcription and therefore, the function of the promoter. Wild-type or endogenous RFP polypeptides can interact with each other to form a heterotetrameric RFX complex.

[0142] The present disclosure provides for compositions comprising a cell comprising a truncated RFX5 polypeptide (a non-functional molecule that retains DNA-binding activity) encoded by a sequence of a recombinant polynucleotide, can compete with endogenous RFX5 polypeptide to interact with other RFX polypeptides to form a non-functional RFX complex. For example, the truncated RFX5 polypeptide encoded by a sequence of the recombinant polynucleotide can interact with endogenous RFXB/AP/ANK/B to form the non-functional RFX complex in the cell. In some embodiments, expression of the truncated RFX5 polypeptide or truncated RFXANK in a cell can inhibit or interfere with MHC expression in the cell. In some embodiments, the cell comprising truncated RFX5 polypeptide disclosed herein comprises specificity to bind to an endogenous site in the MHC promoter. In some embodiments, the truncated RFX5 polypeptide can interrupt the activity of the MHC promoter. In some embodiments, the truncated RFX5 polypeptide is a dominant negative RFX5 polypeptide (DN RFX5).

[0143] The present disclosure provides for compositions comprising a cell comprising a truncated RFXANK polypeptide (a non-functional molecule that can interact with transcriptional factor via the ankyrin repeats) encoded by a sequence of a recombinant polynucleotide, can compete with endogenous RFXANK polypeptide to interact with RFX-B/RFX5/RFXAP polypeptides to form a non-functional RFX complex. For example, the truncated RFXANK polypeptide encoded by a sequence of the recombinant polynucleotide can interact with endogenous RFXB/RFX5/AP to form the non-functional RFX complex in the cell. In some embodiments, expression of the truncated RFXANK in a cell can inhibit or interfere with MHC expression in the cell. In some embodiments, the cell comprising truncated RFXANK polypeptide disclosed herein comprises specificity to bind to an endogenous site in the MHC promoter. In some embodiments, the truncated RFXANK polypeptide can interrupt the activity of the MHC promoter. In some embodiments, the truncated RFXANK polypeptide is a dominant negative RFXANK polypeptide (DN RFXANK).

[0144] An MHC promoter is a primary regulator of an HLA/MHC class I/II gene transcription. An MHC promoter region comprises various regulatory factors/elements which interact to drive and/or control the expression of genes and gene products; such as the MHC class I/II genes.

[0145] The present disclosure comprises a truncated RFX polypeptide encoded by a sequence comprising a recombinant polynucleotide wherein the truncated RFX polypeptide can be expressed from a cell and can bind to a cognate site in the MHC promoter. In some embodiments, the truncated RFX polypeptide can be truncated RFX5 polypeptide or truncated RFXANK polypeptide encoded by a sequence comprising the recombinant polynucleotide.

[0146] In some embodiments, the truncated RFX5 polypeptide encoded by the sequence of the recombinant polynucleotide can bind to the ISRE and SXY-module elements and disrupt their activity. In some embodiments, the binding of the truncated RFX polypeptide to the ISRE and SXY-module elements can modulate an MHC promoter region. In some embodiments, modulation of the MHC promoter by the truncated RFX5 can interrupt MHC promoter activity e.g. inhibit transcription of MHC class I gene. Activation of MHC- class 1 genes, can be mediated by several conserved cis-acting regulatory elements in the promoter e.g. enhancer a, interferon-stimulated response element (ISRE) and the SXY-module, comprising the S/W, XI, X2, and Y- regulatory boxes - these regulator factors can be critical in the induction and constitutive expression of MHC-I genes (Van den Eisen et al., 2004). In some embodiments, the present disclosure can disrupt interaction of the enhancer A with nuclear factor ( F)-KB, and subsequently interrupt the ISRE from interacting with the interferon regulatory factor (IRF) family members (Gobin et al., 1998, 1999). For example, expression of the present composition(s) can hinder the transcription factor NF-KB and IRF-1 which can block facilitation of TNFA and IFNY (Janus-family kinase/signal transducer and activator of transcription, Jak/STAT) routes of activation thereby blocking an MHC promoter. In some embodiments, expression of the present compositions can hinder binding within the MHC promoter region and subsequently disrupt interaction of regulatory elements thereby hindering interaction or promoter activity. In some instances, the binding of the present compositions within the MHC promoter can interrupt promoter activity so that nucleotide binding domain, leucine-rich repeat containing (NLR) family member NLRC5 do not properly associate with and can activate the promoters of MHC class I genes as is required in vivo (Meissner et.al., 2010). For example, the binding of truncated RFX5 polypeptide disrupts formation of an RFX complex in the promoter region which can prohibit interaction of wild-type or endogenous RFX complex/NLRC5 or can prohibit interaction of endogenous RFX complex with other regulatory elements.

[0147] In some embodiments, the SXY-module can be present also in the MHC class II promoter region and the binding of truncated RFX5 polypeptide to the SXY -module can block activity of the MHC class II promoter. MHC class II promoters lack the enhancer A and the ISRE factors. In some embodiments, the class II transactivator (CIITA) can act as a co-activator and may be essential for MHC II transcription (Steimle et al., 1993). In some embodiments, truncated RFX5 of the present disclosure when expressed can bind to the SXY- module which may block the constitutive expression of CIITA to potentially interrupt the constitutive expression of MHC II molecule in APCs. For example, the binding of truncated RFX5 polypeptide can disrupt formation of an RFX complex in the promoter region which may prohibit interaction of endogenous RFX complex/CIITA heterocomplex or hinder interaction of endogenous RFX complex with other regulatory elements.

[0148] The present disclosure includes compositions comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFX5 polypeptide wherein the mutant or truncated RFX5 polypeptide comprises a DNA binding domain, a C-terminal truncation, an RFXAP binding site, an RFXANK binding site and wherein the truncated RFX5 lacks an NF-Y binding domain. The sequence and stereo-specific alignment of various other boxes in the SXY-module can be very conserved because of a critical function in constitutive and inducible-transcriptional activation of MHC I/II genes (Gobin et al., 2001, Ting and Trowsdale, 2002). The SXY-module can be bound by a multimer protein complex comprising regulator factor X complex of factors that consist of RFX5, RFXB/ANK, RFXAP and nuclear factor-Y (NF-Y) (Mastemak et al., 2000; Gobin et al, 2001; Choi et al., 2011). In some embodiments, expression of the truncated RFX5 polypeptide lacking the NF-Y binding domain can lead to disruption of the MHC promoter. In some embodiments, truncated RFX5 polypeptide disclosed herein functions as a negative dominant to disrupt the expression from the MHC promoter.

[0149] Presentation of foreign peptides by MHC molecules e.g. by antigen presenting cells can be critical for stimulating an appropriate immune response. In some embodiments, expression of the present compositions in a cell can block the MHC promoters which may interrupt expression of MHC-bound foreign peptides and thereby block presentation of MHC-bound peptides from being presented to the T cell receptors. In some embodiments, the present compositions disclosed herein can block presentation of antigens by APC on the cell surface which interrupts antigen presentation and subsequently can block immune stimulation in a cell.

[0150] The present disclosure provides an exogenous mutant or truncated RFX polypeptide, for example, an exogenous truncated RFX5 polypeptide or exogenous truncated RFXANK wherein the modification modulates or blocks HLA/MHC class I/II expression in a dominant negative manner. For example, expression of the truncated RFX5 polypeptide or truncated RFXANK polypeptide as a dominant negative can disrupt expression of HLA/MHC genes or gene products, for example, expression can disrupt expression of MHC-bound antigens. [0151] In humans, HLA is the MHC molecule. MHC glycoproteins are found in the membranes of cells of vertebrate animals and are required in order to recognize and differentiate self and non-self-antigens, position antigenic epitopes in the MHC grooves and present antigens to T cells receptors to activate immune response. In some embodiments, the present compositions disclosed can affect how MHC molecules participate in the humoral and cell-mediated immunity. In some embodiments, the present compositions disclosed can affect the promiscuous MHC binding of peptides or fragments of antigens derived from degraded foreign molecules present inside the cell. In some embodiments, expression of the present compositions disclosed may affect MHC molecules presentation of fragments of the foreign antigens to stimulate a defense against the infection or diseased cell to neutralize the antigen. For example, expression of a truncated RFX polypeptide such as for example, without limitation, a truncated RFX5 polypeptide or truncated RFXANK polypeptide may inhibit MHC molecules from recognizing self-antigen and can instead recognize self as a foreign e.g. in autoimmune disorders in for instance, a recombinant cell of a recipient of immunotherapies. MHC molecules expressed on the surface of cells are required for T cells to recognize antigens in the vertebrate organism.

[0152] In some embodiments, the present compositions disclosed herein bind to an MHC promoter which can inhibit expression of MHC class I glycoproteins.

[0153] In some embodiments, the present compositions disclosed herein can block MHC promoter which can lead to inhibition of expression of HLA/MHC class I expression, which can block class I activation by cytokine. MHC class I protein are found on membrane surfaces of all nucleated cells with exception of red blood cells, platelets and nerve tissue cells. In some embodiments, the present compositions disclosed herein can block the MHC class I recognition of endogenously produced antigens. For example, because MHC class I may be blocked by the compositions disclosed herein, the recognition, binding and presentation of the antigens which can be accomplished by binding with CD8 adhesion molecules on T cell receptors (TCR) of cytotoxic (Tc) CD8+ lymphocyte, is also blocked. In some embodiments, the present compositions disclosed herein can block binding of MHC class I to CD8+ T cells; binding of MHC I to CD8+ T cells may be required to trigger a cell- mediated immune response.

[0154] MHC class I proteins consist of two polypeptide chains; the a chain and 2 microglobulin. The a chain comprise a transmembrane glycoprotein encoded by the HLA class I molecules including an HLA-A, HLA-B and HLA-C gene. The a chain is organized by three domains including al, a2 and a3 with each domain comprising 90 amino acid sequences. When the 0 macroglobulin does not have a trans membrane protein it resembles a3. In some embodiments, the present composition comprising for example, a truncated RFX5 polypeptide or a truncated RFXANK polypeptide, can hinder MHC class I molecules by hindering their activation by cytokines e.g. interferons (a, 0, y) and tumor necrosis factor (TNF). In some embodiments, the present compositions disclosed herein can hinder constitutive expression of MHC class I gene; MHC I is constitutively expressed on all nucleated cells. MHC class I are essential in the detection of and elimination of virus-infected cells, tumor cells and transplanted allogeneic cells. In some embodiments, the present compositions disclosed herein can hinderance of MHC class I can block detection in a recombinant cell when expressed in the cell which can block elimination of transplant/graft cells or infused allogeneic cells.

[0155] Expression of MHC class II gene products may be essential in the initiation of cellular and humoral immune response, which may be critical for the specific recognition of antigens by the immune system. Class II have also been implicated in contributing to a variety of autoimmune disorders. In some embodiments, MHC class II, heterodimeric glycoproteins, can be found on membrane surfaces of antigen presenting cells (APCs e.g. dendritic cells, macrophages and B cells), in thymus epithelial cells and by activated T cells that engulf foreign antigens. In some instances, MHC class II molecules can be constitutively expressed in these specific T cells. In some embodiments, the present disclosures disclosed herein inhibit MHC -bound antigens which can disrupt antigen presenting cells (APC) from e.g. encountering and digesting foreign molecules into antigens or peptides. Antigens digested by APC are bound and displayed on the MHC proteins. In some embodiments, the compositions provided herein can inhibit recognition of an MHC protein on the surface of an APC thereby disrupting the ability of the cell to display antigen to T cell receptor (TCR). In some embodiments, the present compositions disclosed herein can block recipient/host T cell activation of MHC class II molecules by cytokines e.g. interferon gamma. In some embodiments, prohibiting MHC glycoproteins from attaching on the membrane of APC cells can block the immune cell from recognizing self and/or non-self or disrupt the presentation of antigens to the T cell receptor on a0 T cells (TCRa0). In some embodiments, the present compositions disclosed herein can block class II molecules from being recognized and bound by exogenous antigens (endocytic degraded) which hinders their interaction with CD4 adhesion molecules on TCRs of T helper (Th) CD4+ T lymphocytes. MHC class II consists of two polypeptide chains; al and a2 chain and 01 and 02 chain. The a2 and 02 are transmembrane domains that anchor the MHC to the plasma membrane while the a 1 and 0 1 domains jointly comprise a peptide binding groove. In some embodiments, the present compositions disclosed herein can block Th cells activation, which can occur when Th cells encounter an antigen presenting cell (APC) that has been degraded and may be displaying the peptized antigen on its cell surface via its MHC polypeptide. In some embodiments, the present compositions disclosed herein can hinder MHC polypeptides or molecules from recognizing Th lymphocytes and hinder them from being primed to distinguish between the self and non-self. In some embodiments, the present compositions disclosed herein can block MHC proteins bound to foreign molecules/peptides or antigens from being recognized by Th/CD4+ cells. Recognition of MHC proteins bound to antigens may be required for stimulation of T cell receptor on Th/CD4+ cells; this allows secretion of cytokines to activate naive CD4+ cells to differentiate into specific immune cell subtypes depending on the cytokine induced (e.g. pro-inflammatory or anti-inflammatory); the plasticity of T cell subset can be under the influence of specific cytokines.

[0156] In some embodiments, the present compositions disclosed herein by blocking MHC promoter can block expression of MHC class II molecules. Three classical class II molecules are encoded by the HLA loci - HLA- DP, HLA-DR and HLA-DQ.

[0157] The ability to disrupt or block an MHC promoter using the disclosures presented herein, which subsequently may block or hinder expression of HLA/MHC class I/II genes and gene products, can be an attractive model for use in designing ready-made allogeneic or allograft therapeutics; blocking MHC promoters or class I/II genes can be achieved using the present disclosure in which an exogenous truncated RFX polypeptide (e.g. truncated RFX5 polypeptide or truncated or mutant RFXANK polypeptide) can inhibit endogenous MHC expression when the truncated protein is expressed in a cell. In some embodiments, the cell may comprise any recombinant cell comprising a sequence encoding the disclosures presented herein and which recombinant cell can express any of the disclosures presented herein.

[0158] Provided herein are compositions that can be employed simultaneously in a recombinant cell comprising a non-gene editing mechanism of the present disclosure to achieve MHC modulation. In some embodiments, the non-gene editing mechanism comprises a truncated or mutant transcription factor e.g. truncated RFX5 polypeptide or truncated RFXANK which can impede MHC expression in a dominant negative manner. For example, a composition comprising a cell comprising truncated RFX5 polypeptide encoded by a sequence of a recombinant polynucleotide can modulate the MHC promoter region in a negative dominant manner when expressed in the cell. In some embodiment, a truncated RFX5 dominant negative (DN) expression occurs when truncated RFX5 polypeptide disrupts, in a dominant fashion, the function of simultaneously expressed wild-type (or normal) RFX (complex) proteins in vivo. In the present disclosure, the use of dominant negative mutations is an alternative approach wherein mutations or truncations in a RFX polypeptide e.g. truncated RFX5 polypeptide abolish the function of a wild-type RFX protein and also because the truncated RFX5 polypeptide is inactive, the recombinant cell comprising truncated RFX5 hinders the function of RFX5/B/RFXANK/RFXAP complex in the MHC promoter. In some aspect, a mutant RFX5 polypeptide when expressed in a cell comprising wild-type RFX polypeptides will block some regulatory feature, such as for example, transcription of wild-type RFX gene expressed in the same cell. In some embodiments, the truncated RFX5 DN polypeptide or mutant RFXANK DN polypeptide can interact with the same features e.g. regulatory elements, or transcription factor in the MHC promoter or elsewhere in the cell, as would the wild-type RFX complex gene product. In some embodiments, the expression or presence of the dominant negative compositions disclosed herein e.g. truncated RFX5 DN polypeptide (RFX5 DN) or mutant or truncated RFXANK DN polypeptide (RFXANK DN), when expressed in a cell can inhibit a regulatory element in the MHC promoter, the promoter activity, transcription, or translation activity. For example, expression of a dominant negative such as truncated RFX polypeptides can modulate MHC translation when they bind to and block an MHC promoter because assembly of an RFX multimer complex is essential to its function in the promoter region. [0159] The present disclosure provides a composition comprising a cell comprising a truncated RFX5 polypeptide or truncated RFXANK polypeptide wherein the truncation reduces or eliminates a level of expression or activity of a wild-type MHC promoter in a cell. The present disclosure provides a composition comprising a cell comprising a truncated RFX5 polypeptide or truncated RFXANK polypeptide wherein the truncation reduces or eliminates a level of expression or activity of a wild-type HLA/MHC genes in a cell. The present disclosure provides a composition comprising a cell comprising a truncated RFX5 polypeptide or truncated RFXANK polypeptide wherein the truncation reduces or eliminates a level of expression or activity of a wild-type HLA/MHC gene product in a cell. In some embodiments, the composition provided herein comprising can provide a rapid, non-gene editing method that can be used to modulate or disrupt, (or block, impede, inhibit, downregulate, attenuate, inactivate) HLA/MHC gene transcription via a dominant negative expression mechanism.

[0160] The present disclosure provides an exogenous mutant or truncated RFX polypeptide, for example, an exogenous truncated RFX5 polypeptide or an exogenous truncated RFXANK wherein an expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is less than an expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide.

[0161] In some embodiments, the exogenous truncated RFX polypeptide for example and without limitations, an exogenous truncated or mutant RFX5 polypeptide, or an exogenous truncated or mutant RFX5 polypeptide may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 5%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 10%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 15%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 20%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 25%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 30%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription at least about 35%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription at least about 40%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 45%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 50%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 55%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 60%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 65%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 70%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 75%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 80%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 85%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 90%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 95%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression may decrease and/or modulate expression of an MHC promoter activity or transcription by at least about 100%.

[0162] In some embodiments, the administration of an exogenous truncated or mutant RFX5/RFXANK polypeptide to a first cell may inhibit expression of a T-cell receptor on the cell. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 5%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 10%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 15%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 20%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 25%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 30%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 35%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 40%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 45%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 50%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 55%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 60%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 65%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 70%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 75%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 80%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 85%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 90%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 95%. In some embodiments, the mutant RFX5/RFXANK polypeptide may decrease and/or modulate expression of the T-cell receptor by at least about 100%.

[0163] In some embodiments, the administration a composition to a cell comprising a recombinant polynucleotide comprising a sequence encoding comprising a truncated or mutant RFX5 or a truncated or RFXANK polypeptide (used interchangeably as RFX5/RFXANK polypeptide) may inhibit expression the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the MHC in a cell not expressing the mutant RFX5/RFXANK polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 5% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 15% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 25% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 30% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 35% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 45% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 50% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 50% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 60% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 65% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 70% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 75% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 80% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 85% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 90% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 95% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. In some embodiments, the truncated or mutant RFX5/RFXANK may inhibit the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide by at least 100% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide.

[0164] In some embodiments, the administration of a composition to a cell comprising a recombinant polynucleotide comprising a sequence encoding comprising a truncated or mutant RFX5 or a truncated or RFXANK polypeptide (used interchangeably as RFX5/RFXANK polypeptide) may inhibit expression the expression level of an MHC in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the MHC in a cell not expressing the mutant RFX5/RFXANK polypeptide. In some embodiments, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an MHC class I in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the MHC class I in a cell not expressing the mutant RFX5/RFXANK polypeptide. For example, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an HLA/MHC class I gene such as inhibit an HLA-A, HLA-B, HLA-C gene in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the HLA/MHC class I an HLA-A, HLA-B, HLA-C gene in a cell not expressing the mutant RFX5/RFXANK polypeptide. For example, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an HLA/MHC class I gene such as inhibit an HLA-A gene in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the HLA/MHC class I an HLA-A gene in a cell not expressing the mutant RFX5 /RFXANK polypeptide. For example, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an HLA/MHC class I gene such as inhibit an HLA-B gene in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the HLA/MHC class I an HLA-B, gene in a cell not expressing the mutant RFX5/RFXANK polypeptide. For example, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an HLA/MHC class I gene such as inhibit an HLA-C gene in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the HLA/MHC class I an HLA-C gene in a cell not expressing the mutant RFX5/RFXANK polypeptide.

[0165] In some embodiments, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an MHC class II in the cell expressing the mutant RFX5 /RFXANK polypeptide that is less than the expression level of the MHC class II in a cell not expressing the mutant RFX5/RFXANK polypeptide. For example, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an HLA/MHC class II gene such as inhibit an HLA-DP, HLA-DR, HLA-DQ gene in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the HLA/MHC class II an HLA- DP, HLA-DR, HLA-DQ gene in a cell not expressing the mutant RFX5 /RFXANK polypeptide. For example, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an HLA/MHC class II gene such as inhibit an HLA-DP gene in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the HLA/MHC class II HLA-DP gene in a cell not expressing the mutant RFX5/RFXANK polypeptide. For example, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an HLA/MHC class II gene such as inhibit an HLA-DR gene in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the HLA/MHC class II an HLA-DR, gene in a cell not expressing the mutant RFX5/RFXANK polypeptide. For example, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an HLA/MHC class II gene such as inhibit an HLA-DQ gene in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the HLA/MHC class II an HLA-DQ gene in a cell not expressing the mutant RFX5 /RFXANK polypeptide.

[0166] In some embodiments, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an MHC class I and II in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the MHC class I and II in a cell not expressing the mutant RFX5/RFXANK polypeptide.

[0167] In some embodiments, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an MHC class I and class II in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the MHC class I and class II in a cell not expressing the mutant RFX5/RFXANK polypeptide. For example, the truncated or mutant RFX5 or a truncated or RFXANK polypeptide may inhibit expression of an HLA/MHC class I and class II gene such as inhibit an HLA-A, HLA- B, HLA-C and HLA-DP, HLA-DR, HLA-DQ gene in the cell expressing the mutant RFX5/RFXANK polypeptide that is less than the expression level of the HLA/MHC class I and II HLA-A, HLA-B, HLA-C and HLA-DP, HLA-DR, HLA-DQ gene in a cell not expressing the mutant RFX5/RFXANK polypeptide.

[0168] In some embodiments, disclosed herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX polypeptide, such as for example, a truncated or mutant RFX5 wherein the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is substantially the same as the expression level of the MHC in a RFX5 knockout cell. In some embodiments, the MHC is an endogenous MHC molecule. In some embodiments, the MHC is a class I protein. In some embodiments, the MHC class I is encoded by an HLA-A, HLA-B, HLA-C genes. In some embodiments, the MHC class I is encoded by an HLA-A gene. In some embodiments, the MHC class I is encoded by an HLA-B gene. In some embodiments, the MHC class I is encoded by an HLA-C gene. In some embodiments, the MHC is a class II molecule. In some embodiments, the MHC class II is encoded by an HLA-DP, HLA-DR, HLA-DQ genes. In some embodiments, the MHC class II is encoded by an HLA-DP gene. In some embodiments, the MHC class II is encoded by an HLA-DR gene. In some embodiments, the MHC class II is encoded by an HLA- DQ gene. In some embodiments, the MHC comprises both MHC class I and II molecules. In some embodiments, mutant RFX polypeptide comprises a truncated or mutant RFXANK encoded by a sequence comprising a recombinant nucleic acid wherein the expression level of an MHC in the cell expressing the mutant RFXANK polypeptide is substantially the same as the expression level of the MHC in a RFXANK knockout cell.

[0169] In some embodiments, disclosed herein is a composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide wherein the truncated RFX5 polypeptide comprises a PX2LPX5X6 motif; wherein X2 is any amino acid, X5 is any amino acid, and X₍, is isoleucine (I) or leucine (L).

[0170] In some embodiments, use of suitable vectors for expression of a truncated RFX protein, such as for example, a truncated RFX5 polypeptide or a truncated RFXANK polypeptide in eukaryotic cells such as those listed herein and any additional vectors known in the art and are commercially available, for example, in Ausubel, F. M., et al, Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al, “ Molecular Cloning: A Laboratory Manual,” 2nd Ed. (1989), may be utilized.

[0171] Accordingly, in some embodiments, any of the truncated RFX5 proteins of the present disclosure can be expressed from these expression vectors. The vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors direct the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant nucleic acid technologies may be used e.g. plasmids or viral vectors as listed herein or as utilized in recombinant nucleic acid technologies listed above.

[0172] In some embodiments, host cells can be genetically engineered (e.g., transduced, transformed, or transfected) with, for example, a vector construct of the present disclosure that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule. In some embodiments, the host cell is an immune cell, a mammalian cell, a primate cell, or a human cell, a chimeric antigen cell (e.g. chimeric antigen receptor T cell (CAR-T), a stem cell such as a hematopoietic stem cell, or an induced pluripotent stem cell (iPSC) a human cell, an insect cell, an animal cell or any living cell comprising the disclosure.

[0173] In some embodiment, host cells can be transduced with a nucleic acid encoding any of the truncated RFX protein herein. In some embodiments, the host cell can be an autologous cell. In some embodiments, the host cell can be an allogeneic cell or used in an allogeneic therapy. In some embodiments, the host cell is a T cell, a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, a helper T cell, or a tumor infiltrating lymphocyte, a hematopoietic stem cell (HSC), an induced pluripotent stem cell-derived cell (iPSC), placental-derived cell, or fetal-derived cell.

[0174] In some embodiment, host cells can be transduced with a nucleic acid encoding any of the truncated RFX protein herein, such as for example, a truncated or mutant RFX5 protein (polypeptide) or a truncated or mutant RFXANK protein, comprising vectors that are constructed for a monocistronic, bicistronic or multicistronic expression system. For example, without limitations, a host cell can be transduced with a vector to express (i) a polynucleotide encoding the truncated RFX5 protein comprising regulatory tags to direct expression of the truncated RFX5 protein in vivo. The regulatory tags may comprise a sequence encoding the protease, which acts on cleavage site cloned in the truncated RFX5 protein. A signal retention or protein localization tag are cloned or incorporated and embedded in the recombinant polypeptide sequence. The embedded protein localization tag or intracellular retention tag arrests the truncated RFX5 protein in the host cell on the endoplasmic reticulum. The combination of the protease cleavage site and protein localization tag on which a protease can act is central to the controlled expression of the present truncated RFX5 protein. Regulatory control sequence P2A (a ribose skipping site (P2A from porcine teschovirus) is engineered into the construct together with an epitope-based selection marker (e.g. EGFRt, CD34, Myc Tag) fused to the protease cleavage site in the intracellular retention tag to allow selection of desired cells. For example, without limitation, a host cell can be transduced with a nucleic acid encoding the truncated RFX5 protein, and an additional nucleic acid encoding PA2 and TM protease and any additional nucleic acid encoding one or more additional therapeutic agents such as, a protein therapeutic capable of stimulating any signaling of the domains.

[0175] In some embodiments, disclosed herein are methods for preparing a cell culture including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any one of suitable culture media for the cell cultures described herein. In some embodiments, the recombinant cell expresses the truncated RFX5 protein and/or a CAR described herein.

[0176] In some embodiments, the composition comprising the truncated RFX5 protein disclosed herein may be introduced into a cell. In some embodiments, the composition comprising the truncated RFX5 protein disclosed herein may be introduced into a lymphocyte cell. In some embodiments, the composition comprising the truncated RFX5 protein disclosed herein may be introduced into a T cell or any recombinant cell e.g. a CAR T cell, a HSC, an iPSC cell, a tumor infiltrating T lymphocyte, a CAR T cell, a CD8+ lymphocyte, a CD4+ lymphocyte etcetera . In some embodiments, the composition comprising the truncated RFX5 protein disclosed herein may be introduced into a cytotoxic T lymphocyte cell. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into a natural killer (NK) cell. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into a killer cell. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into a stem cell such as a hematopoietic stem cell. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a mammalian-associated iPSC, e.g. a human-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as beta islet cell-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as natural killer (NK) -associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a chimeric antigen NK-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a retinal cell-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as or neural cell-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as cardiomyocytes-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as or CAR T cell-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a macrophage-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a B cells-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a T cell or T lymphocyte-associated iPSC (comprising for example, T cell receptor -T, T-regulatory cells). In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as an innate lymphoid cells (ILCs)-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a mesenchymal stem cells (MSC)-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a placenta cells-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a fetal cells- associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as any modified, mutant, variant, recombinant and non-modified, non-mutant, wild-type or non-recombinant cell that is an iPSC-derived cell comprising a mammalian cell e.g. comprising a human cell, an insect cell, an animal cell or any other living cell comprising the disclosure.

[0177] In some embodiment, host cells can be transduced with a nucleic acid encoding any of the truncated RFX protein herein, such as for example, a truncated or mutant RFX5 protein (polypeptide) or a truncated or mutant RFXANK protein, comprising vectors that are constructed for a monocistronic, bicistronic or multicistronic expression system. For example, without limitations, a host cell can be transduced with a vector to express (i) a polynucleotide encoding the truncated RFXANK protein comprising regulatory tags to direct expression of the truncated RFXANK protein in vivo. The regulatory tags may comprise a sequence encoding the protease, which acts on cleavage site cloned in the truncated RFXANK protein. A signal retention or protein localization tag are cloned or incorporated and embedded in the recombinant polypeptide sequence. The embedded protein localization tag or intracellular retention tag arrests the truncated RFXANK protein in the host cell on the endoplasmic reticulum. The combination of the protease cleavage site and protein localization tag on which a protease can act is central to the controlled expression of the present truncated RFXANK protein. Regulatory control sequence PA2 (a ribose skipping site (P2A from porcine teschovirus) is engineered into the construct together with an epitope-based selection marker (e.g. EGFRt, CD34, Myc Tag) fused to the protease cleavage site in the intracellular retention tag to allow selection of desired cells. For example, without limitation, a host cell can be transduced with a nucleic acid encoding the truncated RFXANK protein, and an additional nucleic acid encoding PA2 and TM protease and any additional nucleic acid encoding one or more additional therapeutic agents such as, a protein therapeutic capable of stimulating any signaling of the domains.

[0178] In some embodiments, disclosed herein are methods for preparing a cell culture including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any one of suitable culture media for the cell cultures described herein. In some embodiments, the recombinant cell expresses the truncated RFXANK protein and/or a CAR described herein.

[0179] In some embodiments, the composition comprising the truncated RFXANK protein disclosed herein may be introduced into a cell. In some embodiments, the composition comprising the truncated RFXANK protein disclosed herein may be introduced into a lymphocyte cell. In some embodiments, the composition comprising the truncated RFXANK protein disclosed herein may be introduced into a T cell or any recombinant cell e.g. a CAR T cell, a HSC, an iPSC cell, a tumor infiltrating T lymphocyte, a CAR T cell, a CD8+ lymphocyte, a CD4+ lymphocyte etcetera . In some embodiments, the composition comprising the truncated RFXANK protein disclosed herein may be introduced into a cytotoxic T lymphocyte cell. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into a natural killer (NK) cell. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into a killer cell. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into a stem cell such as a hematopoietic stem cell. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a mammalian-associated iPSC, e.g. a human- associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as beta islet cell-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as natural killer (NK) -associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a chimeric antigen NK-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a retinal cell-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as or neural cell-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as cardiomyocytes-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as or CAR T cell-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a macrophage-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a B cells- associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a T cell or T lymphocyte- associated iPSC (comprising for example, T cell receptor -T, T-regulatory cells). In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as an innate lymphoid cells (ILCs)-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a mesenchymal stem cells (MSC)-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a placenta cells-associated iPSC. In some embodiments, the composition comprising the truncated RFXANK protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as a fetal cells-associated iPSC. In some embodiments, the composition comprising the truncated RFX5 protein introduced herein may be introduced into an induced pluripotent stem cell (iPSC) such as any recombinant and non-recombinant cell that is an iPSC-derived cell comprising a mammalian cell e.g. comprising a human cell, an insect cell, an animal cell or any other living cell comprising the disclosure.

Cells

[0180] In some embodiments, the cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a mouse cell. In some embodiments, the animal cell is a human cell . In some embodiments, the composition comprising a truncated RFX polypeptide (such as a truncated RFX5 protein or truncated RFXANK polypeptide) encoded by a sequence of a recombinant polynucleotide or a chimeric antigen receptor (CAR or recombinant cell) (CAR derived from recombinant polynucleotide) may be introduced into a host cell. In some embodiments, the recombinant comprising truncated RFX5 protein cells may be introduced into the host is a population of at least IxlO⁵ T cells (or any other cells used to generate recombinant cells). In some embodiments, the recombinant cells may be introduced into the host is a population of greater than IxlO⁵ T cells (or any other cells used to generate CARs). In some embodiments, the recombinant cells may be introduced into the host is a population of greater than IxlO¹⁰ T cells. In some embodiments, the recombinant cell is an immune system cell, for example without limitation, a T lymphocyte, natural killer cell or NK cell, natural killer T cell or NKT cell, a B cell, a plasma cell, tumor-infiltrating lymphocyte (TIL), a monocyte or macrophage, or a dendritic cell, cytotoxic T cell, cytotoxic -T lymphocytes, plasma cells, tumorinfiltrating lymphocytes, monocytes, macrophages, dendritic cells, CD4+ T cells, epithelial cells or precursor non-differentiated immune cells. The immune cell can also be a precursor cell, i.e., a cell that is capable of differentiating into an immune cell. In some embodiments, the cell is a stem cell such as a hematopoietic stem cell, or an induced pluripotent stem cell (iPSC). For example, an iPSC cell can include for example without limitations to any iPSC cells e.g. a mammalian-associated iPSC, e.g. a human-associated iPSC), e.g. beta islet cell iPSC, natural killer (NK) iPSC or chimeric antigen NK iPSC, or retinal cell iPSC, or neural cell iPSC, cardiomyocytes-associated iPSC, or CAR T cell-associated iPSC, or macrophage-associated iPSC, or B cells- associated iPSC, or T cell or T lymphocyte-associated iPSC or innate lymphoid cells (ILCs)-associated iPSC, or mesenchymal stem cells (MSC)-associated iPSC; or placenta cells-associated iPSC or fetal cells-associated iPSC or any recombinant and non-recombinant cell that is an iPSC-derived cell comprising a mammalian cell e.g. comprising a human cell, an insect cell, an animal cell or any living cell comprising the disclosure.

[0181] In some embodiments, the truncated RFX protein comprising CARs may be autologous/allogeneic (“self’) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells derived from the same individual to which they are subsequently administered. “Allogeneic” as used herein refers to cells of the same species that differ genetically from the cell in comparison. “Syngeneic,” as used herein, refers to cells of a different individual that are genetically identical to the cell in comparison. In some embodiments, the cells are T cells obtained from a mammal. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human.

[0182] T cells may be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from an individual using any number of known techniques such as sedimentation, e.g., FICOLL™ separation.

[0183] In some embodiments, an isolated or purified population of T cells is used. In some embodiments, TCTL and TH lymphocytes are purified from PBMCs. In some embodiments, the TCTL and TH lymphocytes are sorted into naive (TN), memory (TMEM), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and effector (TEFF) T cell subpopulations either before or after activation, expansion, and/or genetic modification. Suitable approaches for such sorting are known and include, e.g., magnetic-activated cell sorting (MACS), where TN are CD45RA+ CD62L+ CD95-; TSCM are CD45RA+ CD62L+ CD95+; TCM are CD45RO+ CD62L+ CD95+; and TEM are CD45RO+ CD62L- CD95+. An exemplary approach for such sorting is described in Wang et al. (2016) Blood 127(24):2980- 90.

[0184] In some embodiments, a specific subpopulation of T cells expressing truncated RFX protein can be isolated by positive selection. In some embodiments, a specific subpopulation of cells (example of cell types are disclosed herein) expressing truncated RFX protein can be isolated by negative selection.

[0185] In order to achieve therapeutically effective doses of T cell compositions, the T cells may be subjected to one or more rounds of stimulation, activation and/or expansion. T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, T cells are activated and expanded for about 1 to 21 days, e.g., about 5 to 21 days. In some embodiments, T cells are activated and expanded for about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 3 days to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to introduction of a nucleic acid (e.g., expression vector) encoding the polypeptide into the T cells.

[0186] In some embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours or about 24 hours prior to introduction of a nucleic acid (e.g., expression vector) encoding the cell surface receptor the into the T cells. In some embodiments, T cells are activated at the same time that a nucleic acid (e.g., an expression vector) encoding the cell surface receptor is introduced into the T cells.

[0187] In some embodiments, conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary for proliferation and viability including, but not limited to serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL- 4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFb, and TNF-a or any other additives suitable for the growth of cells known to the skilled artisan. Further illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Clicks, AEVI-V, DMEM, MEM, a- MEM, F-12, X-Vivo 15, and/or X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. In some embodiments, the nucleic acid (e.g., an expression vector) encoding the cell surface receptor is introduced into the cell (e.g., a T cell) by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, microinjection, DEAE- dextran-mediated transfer, and the like. In some embodiments, the recombinant polynucleotide comprising the sequence encoding the truncated RFX protein may be introduced into the cell (e.g., a T cell) by any gene delivery methods disclosed herein or known in the art and may be driven by viral vector or non-viral vectors bearing the recombinant polynucleotide comprising the sequence encoding the truncated RFX protein disclosed herein. [0188] In some instances, the recombinant polynucleic acid further comprises a sequence encoding a chimeric antigen receptor (CAR). The CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain. In some instances, the recombinant polynucleic acid comprises the sequences in the following order (from 5’ end to 3’ end): the sequence encoding the CAR and the sequence encoding the DN RFX polypeptide. The sequence encoding the CAR can be linked to the sequence encoding the DN RFX polypeptide by a linker comprising or consisting of a sequence encoding a self-cleaving peptide. In some instances, the linker is a P2A cleavable linker.

[0189] The extracellular domain of the CAR comprises an antigen binding domain. The antigen binding domain can be any domain that specifically binds to an antigen. In some instances, the antigen is an antigen expressed by a tumor cell. In some instances, the antigen binding domain comprises a scFv, a nanobody, a ligand, or a receptor.

[0190] The antigen binding domain can be any molecule that binds to the selected antigen with sufficient affinity and specificity, and is often an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab')2 fragment, nanobody, diabody, or the like. Alternatively, the antigen binding domain can be a receptor or a receptor fragment that binds specifically to the target antigen. The antigen binding domain can be attached to the rest of the receptor directly (covalently) or indirectly (for example, through the noncovalent binding of two or more binding partners). Antibody derivatives are molecules that resemble antibodies in their mechanism of ligand binding, and include, for example, nanobodies, duobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, Fab fragments, single chain variable fragments (scFv), single domain antibodies (sdAb), and functional fragments thereof. See for example, D.L. Porter et al., N Engl J Med ( 2011) 365(8):725-33 (scFv); E.L. Smith et al, Mol Ther (2018)26(6): 1447-56 (scFv); S.R. Banihashemi et al., Iran J Basic Med Sci (2018) 21(5):455-64 (CD19 nanobody); F. Rahbarizadeh et al Adv Drug Deliv Rev (2019) 141:41-46 (sdAb);S.M. Kipriyanov et al., Int J Cancer (1998) 77(5):763-72 (diabody); F. Le Gall et al., FEBS Lett (1999) 453(1-2): 164-68 (triabody); M.A. Ghetie et al., Blood (1994) 83(5): 1329-36 (F(ab')2); and M.A. Ghetie et al., Clin Cancer Res (1999) 5(12):3920-27 (F(ab')2 and Fab'). Antibody derivatives can also be prepared from therapeutic antibodies, for example without limitation, by preparing a nanobody, duobody, diabody, triabody, minibody, F(ab')2 fragment, Fab fragment, single chain variable fragment (scFv), or single domain antibody (sdAb) based on a therapeutic antibody. Antibody derivatives can also be designed using phage display techniques (see, e.g., E. Romao et al., Curr Pharm Des (2016) 22(43):6500-18).

[0191] In some instances, the antigen binding domain specifically binds to CD 19. In some instances, the antigen binding domain is an anti-CD19 binding domain. In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity to

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT. In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having at least about 80% sequence identity to

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTS VTVS S .

[0192] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPS QSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

[0193] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGG GGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGS GSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT.

[0194] In some instances, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 60. In some instances, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 60. In some instances, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 60. In some instances, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 60. In some instances, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 60. In some instances, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 60. In some instances, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 60. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 60. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 60. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 60. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 60. [0195] In some instances, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 61. In some instances, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 61. In some instances, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 61. In some instances, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 61. In some instances, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 61. In some instances, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 61 . In some instances, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 61. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 61. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 61. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 61. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 61. [0196] In some instances, the antigen binding domain specifically binds to CD22. In some instances, the antigen binding domain is an anti-CD22 binding domain.

[0197] In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY. AAS and QQSYSIPQT. respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity to DIOMTOSPSSLSASVGDRVTITCRASQTIWSYLNWYOQRPGKAPNLLIYAASSLOSGVPSRFSGRGSG TDFTLTISSLOAEDFATYYCQQSYSIPQTFGOGTKLEI. [0198] In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 ofGDSVSSNSAA. TYYRSKWYN and AREVTGDLEDAFDI. respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having at least about 80% sequence identity to OVOLOQSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRSKWYNDYAVS VKSRITINPDTSKNOFSLOLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVTVSS.

[0199] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

OVOLOQSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRSKWYNDYAVS VKSRITINPDTSKNOFSLOLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVTVSSGGGGSDIOM TOSPSSLSASVGDRVTITCRASQTIWSYLNWYOQRPGKAPNLLIYAASSLOSGVPSRFSGRGSGTDFTL TISSLOAEDFATYYCOOSYSIPOTFGOGTKLEIK.

[0200] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSG TDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTI<LEII<GGGGSQVQLQQSGPGLVI<PSQTLSLTCAIS GDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPE DTA VYY CAREVTGDLED AFDIW GQGTMVTV S S . In some embodiments, the antigen binding domain comprises an scFv with at least about 85, 90, 95, 97, 98, or 99% sequence identity to OVOLOQSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRSKWYNDYAVS VKSRITINPDTSKNOFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVTVSSGGGGSDIOM TOSPSSLSASVGDRVTITCRASQTIWSYLNWYOQRPGKAPNLLIYAASSLOSGVPSRFSGRGSGTDFTL TISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK. In some embodiments, the antigen binding domain comprises an scFv with 100% sequence identity

OVOLOQSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRSKWYNDYAVS VKSRITINPDTSKNOFSLOLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVTVSSGGGGSDIOM TQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTL TISSLOAEDFATYYCOOSYSIPOTFGOGTKLEIK.

[0201] In some instances, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 62. In some instances, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 62. In some instances, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 62. In some instances, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 62. In some instances, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 62. In some instances, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 62. In some instances, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 62. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 62. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 62. In some instances, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 62. In some instances, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 62. [0202] In some instances, the antigen binding domain binds to an antigen that is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate -carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF- II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothelin, MART-1, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR- ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a- fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, ROR1, and GD2, or amixture thereof. [0203] In some embodiments, the antigen binding domain of a CAR provided herein is operatively linked to a transmembrane domain by a hinge domain. In some embodiments, the antigen binding domain of a CAR provided herein is directly linked to a transmembrane domain by a hinge domain. In some embodiments, the hinge domain of a CAR provided herein is from CD28. In some embodiments, the hinge domain of a CAR provided herein has the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP. In some embodiments, the hinge domain of a CAR or provided herein is from CD8. In some embodiments, the hinge domain of a CAR provided herein has the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY. [0204] In certain embodiments, a hinge or spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain includes the CH2 and/or CH3 of IgG 1, lgG4, or IgD. Illustrative spacer domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a and CD28, which may be wild-type hinge regions from these molecules or variants thereof. In certain aspects, the hinge domain includes a CD8a or CD28 hinge region. In some embodiments, the hinge is a PD-1 hinge or CD152 hinge.

[0205] In some embodiments, the CAR further includes an extracellular spacer domain, which may include a hinge domain. The hinge domain is generally a flexible polypeptide connector region disposed between the targeting moiety and the transmembrane domain. Exemplary hinge domain sequences include those from IgG subclasses (such as IgGl and IgG4), IgD, CD28, and CD8 domains. In some embodiments, the hinge domain provides structural flexibility to flanking polypeptide regions. The hinge domain may consist of natural or synthetic polypeptides. It will be appreciated by those skilled in the art that hinge domains may improve the function of the CAR by promoting optimal positioning of the antigen binding domain in relationship to the portion of the antigen recognized by it. In some embodiments, a hinge domain may not be required for optimal CAR activity. In some embodiments, a hinge domain comprising a short sequence of amino acids promotes CAR activity by facilitating antigen-binding by, for example, relieving steric constraints that could otherwise alter antibody binding kinetics. In some embodiments, the hinge domain is linked downstream of the antigenbinding domain of a CAR and upstream of the transmembrane domain of a CAR.

[0206] Non-limiting examples of suitable hinge domains include those derived from CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4. In some embodiments, the hinge domain can include regions derived from a human CD8a (also known as CD8a) molecule, a CD28 molecule, and any other receptors that provide a similar function in providing flexibility to flanking regions. In some embodiments, the CAR disclosed herein includes a hinge domain derived from a CD8a hinge domain. In some embodiments, the CAR disclosed herein includes a hinge domain derived from a CD28 or CD8 hinge domain. In some embodiments, the hinge domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to a CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4 hinge domain.

[0207] In some embodiments, the spacer domain further comprises a linker including one or more intervening amino acid residues that are positioned between the antigen binding domain and the extracellular hinge domain. In some embodiments, the linker is positioned downstream from the antigen binding domain and upstream from the hinge domain. In principle, there are no particular limitations to the length and/or amino acid composition of the linker. In some embodiments, any arbitrary single-chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker. In some embodiments, the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues. In some embodiments, the length and amino acid composition of the extracellular spacer can be optimized to vary the orientation and/or proximity of the antigen binding domain and the extracellular hinge domain to one another to achieve a desired activity of the CAR. In some embodiments, the orientation and/or proximity of the antigen binding domain and the extracellular hinge domain to one another can be varied and/or optimized as a “tuning” tool or effect to enhance or reduce the efficacy of the CAR. In some embodiments, the orientation and/or proximity of the antigen binding domain and the hinge domain to one another can be varied and/or optimized to create a partially functional version of the CAR. In some embodiments, the extracellular spacer domain includes an amino acid sequence corresponding to an IgG4 hinge domain and an IgG4 CH2-CH3 domain.

[0208] Alternatively, the spacer domain can be a synthetic polypeptide spacer, such as a spacer having a random sequence, a (gly-gly-ser)n (“GGSn”) sequence, or a variation thereof such as (SGG)n, (GGGS)n, (SGGG)n, (GSGGG)n, and the like, where n can range from about 1 to about 15. The synthetic polypeptide spacer domain can also include a naturally occurring sequence, such as a hinge domain derived from CD8a, IgG, and the like.

[0209] The extracellular domain of the CAR is operably connected to the transmembrane domain. In some instances, the extracellular domain is connected to the transmembrane domain by a spacer. The transmembrane domain of the CAR serves to transduce the external signal received by the extracellular domain to the intracellular domain. The transmembrane domain can be any proper transmembrane domain known in the art, including but not limited to, CD3^ transmembrane domain, CD28 transmembrane domain, CD8 transmembrane domain, CD8H transmembrane domain, and transmembrane and immunoglobulin domain containing 2 protein (CD28H). The transmembrane domain can be selected from a transmembrane region of a transmembrane protein such as, for example, Type I transmembrane proteins, an artificial hydrophobic sequence or a combination thereof. Examples of the transmembrane domain include the transmembrane regions of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Synthetic transmembrane domains may comprise a triplet of phenylalanine, tryptophan and valine. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the intracellular signaling domain of the CAR. A glycine -serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

[0210] In some embodiments, the CAR comprises a transmembrane domain from a polypeptide selected from the group consisting of: CD4, CD8a, CD28, CD 154, and PD-1; and one or more intracellular costimulatory signaling domains from a polypeptide selected from the group consisting of: 4-1BB, CD28, CD134, and CD137; and an intracellular signaling domain from a polypeptide selected from the group consisting of: FcyRI, FcRy, FcR, CD3y, CD33, CD3e, CD3zeta, CD35, CD22, CD79a, CD79, and CD665. Such a CAR may further include a spacer domain between the antigen-binding portion and the transmembrane domain, e.g., a CD8a hinge. In some embodiments, the CAR comprises a transmembrane domain from CD28. In some embodiments, the CAR comprises a transmembrane domain with the sequence FWVLVVVGGVLACYSLLVTVAFIIFWV. In some embodiments, the CAR comprises a transmembrane domain from CD8. In some embodiments, the CAR comprises a transmembrane domain with the sequence IWAPLAGTCGVLLLSLVITLYC.

[0211] The transmembrane domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, the TM domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of the T-cell receptor, CD3y, CD38, CD3e, CD35, CD3zeta, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and/or PD-1.

[0212] The transmembrane domain may include, for example without limitation, all or part of the transmembrane domain of the CD3zeta chain), CD28, CD2, CD4, 0X40, 4-1BB (CD137), ICOS (CD278), ILRB (CD122), IL-2RG (CD132), CTLA-4, PD-1, or CD40, or a sequence derived from such a transmembrane domain. The cytoplasmic signaling domain in general comprises a domain that transduces the event of ligand binding into an intracellular signal that activates the T cell. The CD3z intracellular domain/activating domain is frequently used, although others such as MyD88 can be used. In an embodiment, the transmembrane domain is the transmembrane domain from CD3eta, CD2, CD8, or CD28. In an embodiment, the transmembrane domain is derived from the transmembrane domain from CD2 or CD28. In some embodiments, the transmembrane domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to a CD3zeta, CD28, CD2, CD4, 0X40, 4-1BB (CD137), FcERIy, ICOS (CD278), ILRB (CD122), IL-2RG (CD132), or CD40 transmembrane domain.

[0213] According to some embodiments, a CAR includes a transmembrane domain derived from CD8a or CD28 and a short polypeptide linker, e.g., between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the transmembrane domain and the intracellular signaling domain of the CAR. A glycine-serine linker may be employed as such a linker, for example.

[0214] The transmembrane domain of the CAR is operably connected to the intracellular domain. The intracellular domain serves to transduce the received external signal to kick-start the downstream signaling cascade. The intracellular domain comprises an intracellular signaling domain. In some instances, the intracellular domain comprises an intracellular signaling domain from CD3^, 4-1BB (CD137) CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3e, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70.

[0215] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from 4-1BB (CD137). In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0216] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0217] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta and an intracellular signaling domain from 4-1BB (CD137).

[0218] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD2. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence

KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPAT. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence PATSQHPPPPPGHRSQAPSHRPPPPGHRVQH.

[0219] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3epsilon. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RPPPVPNPDYEPIRKGQRDLYSGLNQRRI. In some embodiments, the CAR comprises an intracellular domain comprising a truncated CD3epsilon intracellular domain.

[0220] Signals generated through the T cell receptor (TCR) alone may be insufficient for full activation of the T cell and a secondary or costimulatory signal may also be required. Thus, T cell activation can be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigendependentprimary activation through the TCR (e g., aTCR/CD3 complex) and costimulatory signaling domains that act in an antigen- independent manner to provide a secondary or costimulatory signal. As such, the CAR may include an intracellular signaling domain that includes one or more costimulatory signaling domains and a primary signaling domain.

[0221] Primary signaling domains can regulate primary activation of the TCR complex either in a stimulatory manner, or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”). Nonlimiting examples of ITAM-containing primary signaling domains suitable for use in a CAR include those derived from FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD3zeta, CD35, CD22, CD79a, CD79b, and CD665. In certain embodiments, a CAR includes a CD3zeta primary signaling domain and one or more costimulatory signaling domains. In certain embodiments, a CAR includes a 4-1BB costimulatory signaling domain. The intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain. In certain embodiments, a CAR lacks a CD2 intracellular signaling domain.

[0222] In some embodiments, the CAR includes one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR. Exemplary costimulatory molecules suitable for use in CARs contemplated in particular embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD3O, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70. In some embodiments, the costimulatory signaling domain has at least about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to a costimulatory signaling domain from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70 domain. In some embodiments, a CAR includes one or more costimulatory signaling domains selected from the group consisting of CD2, 4-1BB, CD28, CD137, and CD134, and a CD3zeta primary signaling domain. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like. In certain embodiments, the CAR comprises two or more intracellular signaling domains. For example, the CAR may comprise a first signaling domain and a second signaling domain or fragments thereof independently selected from a CD3zeta intracellular signaling domain, a CD28 intracellular signaling domain, a 4- IBB intracellular signaling domain, an OX-40 intracellular signaling domain, an inducible co-stimulator (ICOS) intracellular signaling domain, a CD27 intracellular signaling domain, and a MyD88/CD40 intracellular signaling domain. By way of example, a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof that is a CD28 intracellular signaling domain. Also, by way of example, a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof that is a 4- IBB intracellular signaling domain. Also, by way of example, a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain, a second intracellular signaling domain or fragment thereof that is a 4- IBB intracellular signaling domain, and a third intracellular signaling domain or fragment thereof that is a CD3 epsilon intracellular signaling domain.

[0223] CARs of the disclosure may comprise a CD3^, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4- 1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70 cytoplasmic signaling domain. In some embodiments, the cytoplasmic signaling domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to an CD3 , 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3e, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4- 1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70 cytoplasmic signaling domain. CARs of the disclosure may comprise a CD2 co-stimulatory domain, and one or more additional co-stimulatory domains to increase cytokine production or sensitivity, reduce or prevent anergy, and/or to increase proliferation and cytotoxic activity. These additional co-stimulatory domains can be derived from co-stimulatory proteins such as B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7-H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 41BB (CD137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, 0X40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F-10, NTB-A, CD48, SLAM (CD150), CD58, ikaros, CD53, integrin a4, CD82, integrin a4bl, CD90, integrin a4b7, CD96, LAG-3, CD160, LMIR, CRTAM, TCL1A, DAP12; TIM-1, Dectin-1, TIM-4, TSLP, EphB6, TSLP-R, and/or HLA-DR. In some embodiments, the cytoplasmic signaling domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to an B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7-H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 41BB (CD137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, 0X40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F-10, NTB-A, CD48, SLAM (CD150), CD58, ikaros, CD53, integrin a4, CD82, integrin a4bl, CD90, integrin a4b7, CD96, LAG-3, CD160, LMIR, CRTAM, TCL1A, DAP12; TIM-1, Dectin-1, TIM-4, TSLP, EphB6, TSLP-R, and/or HLA-DR domains.

[0224] In some instances, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some instances, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0225] In some instances, the CAR comprises an amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65. In some instances, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 63. In some instances, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 64. In some instances, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 65. In some instances, the CAR consists of an amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65. In some instances, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 63. In some instances, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 64. In some instances, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 65.

[0226] In some instances, the CAR further comprises a protein localization tag. The protein localization tag can be operably linked to the intracellular domain of the CAR. The protein localization tag can be operably linked to the extracellular domain of the CAR. The protein localization tag can be an ER localization tag, a Golgi apparatus (Golgi) localization tag, a lysosome localization tag, a plasma membrane localization tag, a mitochondria localization tag, a peroxisome localization tag, a cytosolic localization tag, or a nuclear localization tag. In some instances, the protein localization tag is an ER localization tag. In some instances, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 52. In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 52. In some instances, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 53. In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 53. In some instances, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 54. In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 54. In some instances, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 55. In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 55. In some instances, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 56. In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 56. In some instances, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 57. In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 57. In some instances, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 58. In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 58. In some instances, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 59. In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 59. In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 52. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 52. In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 53. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 53. In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 54. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 54. In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 54. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 54. In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 55. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 55. In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 56. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 56. In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 57. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 57. In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 58. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 58. In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 59. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 59.

[0227] In some instances, the ER localization tag comprising an amino acid sequence LYKYKSRRSFIDEKKMP (SEQ ID NO: 66). In some instances, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 66. In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 66. In some instances, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 40. In some instances, the ER localization tag comprises the amino acid sequence KKMP (SEQ ID NO: 67). In some instances, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 67.

[0228] In some instances, the protein localization tag is a Golgi localization tag. In some instances, the Golgi localization tag comprises the amino acid sequence YQRL (SEQ ID NO: 68). In some instances, the Golgi localization tag consists of the amino acid sequence YQRL (SEQ ID NO: 68). In some instances, the protein localization tag is a lysosome localization tag. In some instances, the lysosome localization tag comprises the amino acid sequence KFERQ (SEQ ID NO: 69). In some instances, the lysosome localization tag consists of the amino acid sequence KFERQ (SEQ ID NO: 69).

[0229] In some instances, a protease cleavage site is disposed between the protein localization tag and the CAR. In some instances, the protease cleavage site is disposed between the protein localization tag and the intracellular domain of the CAR. In some instances, the protease cleavage site is disposed between the protein localization tag and the extracellular domain of the CAR. Protease cleavage sites are to be understood as amino acid residues that are recognized by proteases and/or amino acid residues whose peptide bond is cleaved by proteases. In some instances, a protease cleavage site can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. Optionally, additional amino acids can be present at the N-terminus and/or C-terminus of the cleavage site. A protease cleavage site also can be a variant of a cleavage site of a known protease as long as it is recognized/cleaved by the protease.

[0230] Various protease cleavage sites include, but are not limited to protease cleavage sites for proteases from the serine protease family, or for metalloproteases, or for a protease from the cysteine protease family, and/or the aspartic acid protease family, and/or the glutamic acid protease family. In some embodiments, serine proteases cleavage sites include, but are not limited to, cleavage sites for chymotrypsin-like proteases, and/or subtilisin-like proteases, and/or alpha/beta hydrolases, and/or signal peptidases. In some embodiments, metalloprotease recognition sites include, but are not limited to, cleavage sites for metallocarboxypeptidases or metalloendopeptidases. In some instances, the protease cleavage site is TEV protease cleavage site.

[0231] The sequence encoding the DN RFX polypeptide disclosed herein and the sequence encoding the CAR disclosed herein can be a single polynucleotide sequence. In some instances, the single polynucleotide sequence comprises a self-cleaving site (e.g., 2A peptides) separating the sequence encoding the DN RFX polypeptide and the sequence encoding the CAR. In some instances, the sequence encoding the DN RFX polypeptide and the sequence encoding the CAR are separated sequences. The sequence encoding the DN RFX polypeptide or the sequence encoding the CAR can be inserted to a vector for expression in a cell. In some instances, the sequence encoding the DN RFX polypeptide and the sequence encoding the CAR are inserted into the same vector. In some instances, the sequence encoding the DN RFX polypeptide and the sequence encoding the CAR are inserted into different vectors. In some instances, the vector comprising the sequence encoding the DN RFX polypeptide is different from the vector comprising the sequence encoding the CAR. In some instances, the vector comprising the sequence encoding the DN RFX polypeptide and the vector comprising the sequence encoding the CAR are the same. [0232] The compositions of the present disclosure can be vector-delivered e.g. truncated RFX polypeptides can be delivered by a vector which removes the need for gene editing. Because of the delivery method, there is no need to use complicated manufacturing e.g. use of a vector to introduce a CAR or a recombinant construct and electroporation to introduce Cas/sgRNA complex not necessary and avoids potential safety concerns of dsDNA breaks by Cas. In some embodiments, the polynucleotide comprising the sequences encoding the truncated RFX protein e.g. truncated RFX5 polypeptide or truncated RFXANK polypeptide may be introduced into a cell using any conventional methods in the art or using a gene delivery system. In some instances, the recombinant polynucleotide may be introduced into a cell using one or more viruses or other delivery vehicles as outlined above in some non -limiting examples, including for example delivery via a liposome.

[0233] In some embodiments, the polynucleotide comprising the sequences encoding the truncated RFX protein may be introduced into a cell using biological methods. For example, biological methods may employ delivery methods such as vectors or synthetic liposomes, non-limiting examples described above; other examples may include non-viral biological agents, such as bacteria, bacteriophage, virus-like particles, erythrocyte ghosts, exosomes among others known in the art. In some embodiments, the polynucleotide may be introduced into a cell via any other delivery system. In some embodiments, the polynucleotide may be introduced into a cell using a cell transformation method. In some embodiments, the polynucleotide may be introduced into a cell using a cell transduction method. In some embodiments, the polynucleotide may be introduced into a cell by any transfection method wherein the transfection leads to uptake of any artificial introduction of foreign cargo e.g. nucleic acid into a cell. In some embodiments, the polynucleotide comprising the sequences encoding the truncated RFX protein may be introduced into a cell using physical methods in which physical energy is applied for intracellular delivery. Such physical methods use application of force to generate transient pores in the cell membrane. Some non-limiting examples of physical methods of delivering the polynucleotide or a cell bearing polynucleotide include, microfluidic electroporation, nanochannel electroporation, nanostraw electroporation, laser-induced photoporation, optical transfection, mechanoporation, ballistic gene delivery, cell squeezing, microinjection, nanofountain probe electroporation, particle bombardment, field-induced membrane disruption, sonoporation, optoporation, magnetoporation, constriction channel based intracellular delivery, thermoporation and any other electroporation-based cell delivery technique or device. Various physical delivery methods have long demonstrated the ability to deliver cargo molecules directly to the cell intracellular environment such as for example, the cytoplasm or nucleus of the cell. The methods of delivery may depend on if the introduction may be for a single-cell intracellular delivery or not. art. In some embodiments, the polynucleotide may be introduced into a cell using chemical methods, such as chemical vector-based non-viral cargo delivery which may require modifying cell-penetrating peptides or proteins or endosomal escape to transfect cargo molecules into the cytoplasm directly. The chemical transfection methods are techniques that catalyze DNA cross-membrane transport. In some embodiments, chemical methods may use Ca2+phosphate, polycations or dendrimers including for example, without limitations, such methods as, use of cationic polymers e.g. diethylaminoethyl -dextran (DEAE-dextran). Chemical methods of cell delivery may apply cell transfection with cationic lipids (non-viral vectors), also known as lipofection or lipid- mediated/liposome transfection are used in cargo or gene transfection.

[0234] In some embodiments, the recombinant polynucleotide comprising the sequences encoding the truncated RFX protein maybe integrated to the genome of the cell. In some embodiments, the polynucleotide integrating into the cell may be single stranded. In some embodiments, the polynucleotide integrating may be double stranded DNA. In some embodiments, the polynucleotide may be short nucleotide sequences. In some embodiments, the polynucleotide may be long nucleotide sequences. In some embodiments, the integration into the genome of the cell may be transient integration in the cell. In some embodiments, the integration into the genome of the cell may be stable and integrate into the genome of the recipient cell. In some embodiments, the polynucleotide may integrate into the cell genome within a random locus. In some embodiments, the polynucleotide may integrate into the cell genome within a directed or targeted locus. In instances where the polynucleotide may be integrated into the cell genome, the polynucleotide may replicate when the cell genome replicates.

[0235] In some embodiments, the cells bearing the polynucleotide comprising the sequences encoding the truncated RFX protein having integrated or transduced or transformed into the cell may be characterized using various methods.

[0236] In some embodiments, the recombinant polynucleotide comprising the sequences encoding the truncated RFX protein may be encoded by a vector as described above. In some embodiments, the vector or cell comprising the recombinant polynucleotide is a recombinant vector or cell. In some embodiments, the recombinant cell or recombinant vector may comprise a selectable biomarker. In some embodiments, the selectable marker that is expressed by the recombinant vector or a cell may be used to select and characterized the recombinant polynucleotide.

[0237] In some embodiments, the selectable biomarker in the vector comprising the recombinant polynucleotide comprising the sequences encoding the truncated RFX protein may be a fluorescent biomarker. In some embodiments, the selectable biomarker may be an antibiotic cassette. In some embodiments, the selectable marker may be a vector or molecule that produces a morphological change, wherein the morphological change denotes integration of the recombinant polynucleotide or cell or vector bearing the polynucleotide. In some embodiments, the selectable biomarker may be any selectable biomarker used in recombinant nucleic acid cloning technology or in the selection of recombinant molecules. Examples, of selectable markers without limitations, include, a transgene, a suicide gene, an activation biomarker, an antibiotic resistance cassette, a morphological change marker or a fluorescent marker. Non-limiting examples of protein genes that may be used to encode fluorescent biomarker proteins include, green fluorescent protein (GFP) gene, enhanced green fluorescent protein (eGFP) gene, mScarlet fluorescent protein gene, red fluorescent protein (RFP) gene, infrared fluorescent protein (iRFP) gene, cyan fluorescent protein (CFP) gene, yellow fluorescent protein (YFP) gene, mCherry/texasRed gene, Cy5.5 fluorescent protein gene and many other fluorescent protein gene in the art. Non-limiting examples of antibiotic selectable resistance marker gene include, kanamycin gene, ampicillin gene, streptomycin gene, neomycin gene, puromycin gene gentamycin gene, erythromycin gene, Blasticidin S gene, hygromycin B gene among many others known in the art. In some embodiments, the polynucleotide integrating may be small interfering RNA or miRNA wherein the siRNA or miRNA may be short hairpin transcripts, or the short hairpins may be made from a selectable DNA vector.

[0238] In some embodiments, the recombinant polynucleotide comprising the sequences encoding the truncated RFX protein may comprise sequences wherein at least portions of the sequences encoding the truncated RFX protein are endogenous to a cell. In some embodiments, the recombinant polynucleotide may comprise sequences, wherein at least portions of the sequences encoding the truncated RFX protein are exogenous/heterologous to a cell. In some embodiments, the polynucleotide may comprise sequences encoding the truncated RFX protein wherein all the sequences are exogenous to a cell. In some embodiments, the polynucleotide may comprise sequences encoding the truncated RFX protein that are partially endogenous to a cell and partially exogenous to a cell.

[0239] In some embodiments, the polynucleotide comprising the sequences encoding the truncated RFX protein may comprise oligonucleotides. In some embodiments, the polynucleotide may comprise nucleotides. In some embodiments, the polynucleotide may comprise nucleic acids such as DNA sequences of any length or synthetic or artificial nucleotide analogues thereof. In some embodiments, the polynucleotide may comprise nucleic acids, such as RNA sequences or synthetic or artificial nucleotide analogues thereof. Nucleotide analogues may comprise peptide nucleic acids (PNA) or locked nucleic acids (LNA). The polynucleotide may comprise amino acid sequences with the ability to fold to protein with a bioactive form, including ability to form a polypeptide similar in function, tertiary or functional structure to the native polypeptide. In some embodiments, the recombinant polypeptide may not retain the native confirmation and function.

[0240] In some embodiments, the polynucleotide (where polynucleotide hereinafter refers to polynucleotide comprising the sequence encoding the truncated RFX protein) may comprise a polymeric form of nucleotides of any length that are single stranded or double stranded or multi-stranded or nucleotide analogues, or combinations of these.

[0241] In some embodiments, the polynucleotide comprising the sequences encoding the truncated RFX protein may comprise modified bases. In some embodiments, the polynucleotide may comprise mutated bases. In some instances, the synthetic or artificial nucleotide analogues or bases can comprise chemical modifications at one or more of ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof In some embodiments, the polynucleotide may comprise modified bases, but wherein the modification to the bases does not change the amino acids or protein encoded. Chemical modifications increase stability. For example, a modification in nucleotide base may be any naturally occurring, unmodified nucleotide base such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified base that is sufficiently similar to an unmodified nucleotide base such that it is capable of hydrogen bonding with a base present on a target pre- mRNA. Examples of modified nucleotide bases include, without limitation, hypoxanthine, xanthine, 7- methylguanine, 5,6-dihydrouracil, 5 -methylcytosine, and 5-hydroxymethoylcytosine. In some embodiments, the polynucleotide may comprise modified bases that alter the original amino acid codon as wherein the modification results in a different protein/peptide than a previous protein/peptide. In some embodiments, the polynucleotide may comprise modified bases that do not alter the original amino acid codon as wherein the modification results in a different protein/peptide than a previous protein/peptide. In some embodiments, the polynucleotide may comprise a sequence encoding a gene or a fragment thereof. In some embodiments, the polynucleotide may comprise sequences encoding any three-dimensional structure, and may perform functions that are new, known or unknown. In some embodiments, the polynucleotide may comprise sequences encoding one or more analogues (e.g. altered backbone, sugar or nucleobase). In some embodiments, the polynucleotide structure, if present, may comprise modifications that may not alter the nucleotide structures. In some embodiments, the polynucleotide structure, if present, may comprise modifications that may alter the nucleotide structures. In some embodiments, the said modifications on the nucleotide structures of the polynucleotide may be imparted before or after assembly of the polymer.

[0242] In some embodiments, the recombinant polypeptide comprising the sequences encoding the truncated RFX protein may be encoded by a naked DNA or RNA expression vector. Non limiting examples of recombinant vectors include, naked DNA or RNA expression vectors or expression vectors associated with cationic condensing agents.

[0243] In some embodiments, the recombinant polynucleotide comprising the sequences encoding the truncated RFX protein maybe encoded by a vector. In some embodiments, the vector encoding the recombinant polynucleotide may be a conventionally available vector. In some embodiments, the vector may be a commercially available vector; examples of such vectors include but are not limited to, for example, a shuttle vector, a plasmid vector, or a viral vector such as, without limitation, a lentiviral vector, a poxviral vector, an adenoviral vector, a herpes simplex viral vector, retroviral vector, an adeno-associated vector (AAV), an influenza vector, a measles virus vector, a CRISPR-Cas based vector, a vesicular stomatitis virus vector, a hybrid vector, a nanoparticle-associated vector, a nanoparticle vector, simian virus 40 (SV40), and bovine papilloma virus vectors (or any other vector known in the art see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0244] In some embodiments, the recombinant polynucleotide comprising the sequences encoding the truncated RFX protein may be encoded by a suicide vector, a shuttle vector, an expression vector, a viral vector, a phage vector, a cosmid vector, a plasmid vector, a cloning vector or any other vectors in the art. In some embodiments, the polynucleotide may be encoded by a suicide vector. In some embodiments, the polynucleotide may be encoded by a shuttle vector. In some embodiments, the polynucleotide may be encoded by a cloning vector. In some embodiments, the polynucleotide may be encoded by a bacteriophage lambda vector. In some embodiments, the polynucleotide may be encoded by a cosmid vector. In some embodiments, the polynucleotide may be encoded by a phage vector. In some embodiments, the polynucleotide may be encoded by an expression vector. In some embodiments, the polynucleotide may be encoded by a hybrid vector comprising combinations of any vector(s) disclosed or any vector(s) known in the art. In some embodiments, the polynucleotide may be encoded by a plasmid vector. In some embodiments, the polynucleotide may be encoded by a viral vector. In some embodiments, the polynucleotide may be encoded by an AAV. In some embodiments, the polynucleotide may be encoded by a CRISPR-Cas-based vector. In some embodiments, the polynucleotide may be encoded by a viral vector such as a lentivirus. In some embodiments, the polynucleotide may be encoded by a viral vector such as a retrovirus. In some embodiments, the polynucleotide may be encoded by a viral vector such as an adenovirus. In some embodiments, the polynucleotide may be encoded by a viral vector such as an adeno- associated virus. In some embodiments, the polynucleotide may be encoded by a viral vector such as a herpes simplex virus. In some embodiments, the polynucleotide may be encoded by a viral vector such as a measles virus. In some embodiments, the polynucleotide may be encoded by a viral vector such as an influenza virus. In some embodiments, the polynucleotide may be encoded by a viral vector such as a vesicular stomatitis virus. In some embodiments, the polynucleotide may be encoded by an RNA vector. In some embodiments, the polynucleotide may be encoded by a DNA vector.

[0245] In some embodiments, the polynucleotide comprising the sequences encoding the truncated RFX protein may be encoded by a non-viral vector or template. In some embodiments, the polynucleotide may be encoded from a single-stranded template. In some embodiments, the polynucleotide may be encoded from a double -stranded template. In some embodiments, the polynucleotide may be encoded from a sample comprising an RNA template. In some embodiments, the polynucleotide may be encoded from a sample comprising a DNA template. In some embodiments the polypeptide may be encoded from a sample comprising any combinations of nucleic acids.

[0246] Pharmaceutical compositions are also provided. The pharmaceutical compositions may include any of the cells comprising the compositions of the present disclosure, such as for example, truncated RFX polypeptides (e.g. a truncated/mutant RFX5 polypeptide or a truncated/mutant RFXANK polypeptide) and a pharmaceutically acceptable carrier. The pharmaceutical compositions generally include a therapeutically effective amount of the cells. By “therapeutically effective amount” is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease, condition, symptom, or disorder. The term “therapeutically effective amount is defined in more detail elsewhere in this application.

[0247] The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents. Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0248] The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.

[0249] Pharmaceutical compositions that include the cells of the present disclosure may be prepared by mixing the cells having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate,

-n- citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m- cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0250] An aqueous formulation of the recombinant polypeptides, proteases, nucleic acids, expression vectors, and/or cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate -buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0251] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0252] A surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylenepolypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w/v.

[0253] In some embodiments, the pharmaceutical composition includes cells of the present disclosure, and one or more of the above -identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v). [0254] In some embodiments, provided is a pharmaceutical composition that includes a therapeutically effective amount of cells (e.g., any T cells, such as CAR T cells, hematopoietic stem cells-derived cells, induced pluripotent stem cells or any iPSC-derived cells or any cells disclosed herein e.g. a living cell) and comprises compositions of the present disclosure. Examples of compositions as disclosed herein comprise a truncated RFX polypeptides e.g. a truncated RFX5 or a truncated/mutant RFXANK polypeptide. A “therapeutically effective amount” of such cells may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” an individual, e.g., a patient. When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual).

[0255] Additionally, the term “therapeutically effective amount” is defined elsewhere throughout this application. In some embodiments, a pharmaceutical composition of the present disclosure includes from IxlO⁶ to 5xlO¹⁰ of the cells disclosed herein comprising (expressing) the present disclosure. In some embodiments, a pharmaceutical composition of the present disclosure includes from about IxlO⁶ to about 5xlO¹⁰ of the cells of the present disclosure. In some embodiments, a pharmaceutical composition of the present disclosure includes less than IxlO⁶ cells of the present disclosure. In some embodiments, a pharmaceutical composition of the present disclosure includes from 5xlO¹⁰ or more than 5xlO¹⁰ cells of the present disclosure.

[0256] In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutically acceptable carrier or excipient comprises pharmaceutically acceptable diluent, excipient, vehicle, or carrier; where the composition is suitable for injection to a mammal or animal such as for example, to a human subject in need thereof. The pharmaceutically acceptable carrier or excipient can include but not limited to, inert solid diluents and fillers, diluents, sterile aqueous solution and various organic solvents, permeation enhancers, and solubilizers. These compositions can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science (Martin E.W., Easton Pennsylvania, Mack Publishing Company, 19^th ed., 1995) describes formulations which can be used in connection with the subject invention.

[0257] In some embodiments, a composition comprising cells expressing the DN RFX protein and chimeric antigen receptors (CARs) may be administered to a subject in need thereof. In some embodiments, the subject in need thereof may comprise a subject with a disease. In some embodiments, the subject may comprise an individual with a health disorder. In some embodiments, the subject is an individual with a medical condition. In some embodiments, the subject may comprise an individual with a disorder deemed treatable by administration of the truncated RFX protein disclosed herein. In some embodiments, the disorder is any disorder that may receive palliative care by provision of the truncated RFX protein. In some embodiments, the disorder may be any disorder that may experience relief from pain, for example, from inflammatory disorder. In some embodiments, the disorder may be any disorder that may experience prevention of the disorder, for example, from inflammatory disorder. In some embodiments, the disorder may be any disorder that may experience curative treatment, for example, from a disorder, a disease, condition, infection, tumor, inflammation. The disorder may be due to an autoimmune disease, cancer, infectious disease symptoms or disorder or condition. [0258] In some embodiments, the therapeutic benefits of recombinant cells e.g. T cells, iPSC-derived cells (human induced PSC), embryonic or adult hematopoietic stem cell-derived cells, or any cells disclosed herein which express the truncated RFX protein (e.g. truncated RFX5 protein or truncated RFXANK protein) or chimeric antigen receptors (CARs) disclosed herein may benefit any inflammatory disorder. In some embodiments, a cell comprising the present disclosure may provide therapeutic benefit of treating an inflammatory disease or condition in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of the present disclosure. For instance, without limitations, the inflammatory disorder (disease, condition, symptom) may be multiple sclerosis. In other cases, the inflammatory disorder may be an autoimmune disease. Examples of the diseases or conditions that the subject recombinant cell (for example, CAR T cells, CARs, hematopoietic stem cell-derived cells, iPSC-derived cells, T lymphocytes, NK cell, gamma delta T cells, allogeneic cells, autologous cells, CD8+ T cells, CD4+ T cells) expressing the present disclosure can be administered to as a treatment or prevention include but are not limited to acute disseminated encephalomyelitis (ADEM), Addison's disease, allergy, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-phospholipid syndrome, asthma (including allergic asthma), autoimmune haemolytic anaemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, Behcet’s disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, coeliac disease, Crohn's disease, Cushing's Syndrome, dermatomyositis, diabetes mellitus type 1, eosinophilic granulomatosis with polyangiitis, graft versus host disease, Graves' disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, Hidradenitis Suppurativa, inflammatory fibrosis (e.g., scleroderma, lung fibrosis, and cirrhosis), juvenile arthritis, genetic diseases or disorders (including for example, monogenic diseases such as sickle cell disease and any other monogenic disease or any genetic disease or condition for which therapy would benefit from administration of the disclosure), Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjogren's syndrome, systemic lupus erythematosus, Takayasu’s arteritis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo and Vogt-Koyanagi- Harada Disease. In some cases, the disease or condition comprises rheumatoid arthritis. In some cases, the disease or condition comprises multiple sclerosis.

[0259] In some embodiments, the treatment may comprise administering to a subject a plurality of treatments comprising the truncated RFX protein or CAR T described herein. In some embodiments, treatment of a disease or condition in a subject in need thereof, may comprise administering to the subject a therapeutically effective amount of the composition comprising cells comprising the truncated RFX protein disclosed herein by administering to the subject the pharmaceutical composition comprising therapeutically effective amount of the cells comprising the truncated RFX protein and at least one pharmaceutically acceptable excipient, in the presence of a multimer inducing agent. In some embodiments, the cells comprising the truncated RFX protein may be administered for the treatment of any condition known to man, such as for example, without limitation, a disease or condition further comprising an infection such as, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, Alzheimer's Disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's Disease, coeliac disease, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme arthritis, meningoencephalitis, immune mediated inflammatory disorders of the central and peripheral nervous system, autoimmune disorders, pancreatitis, trauma from surgery, graft-versus-host disease, transplant rejection, heart disease, bone resorption, bums patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, liver/lung fibrosis, periodontitis, hypochlorhydia, solid tumors (renal cell carcinoma), liver cancer, multiple myeloma, prostatic cancer, bladder cancer, pancreatic cancer, neurological cancers, and B-cell malignancies (e.g., Casteleman's disease, certain lymphomas, chronic lymphocytic leukemia, and multiple myeloma). In some cases, the disease or condition comprises Sjogren’s syndrome. In some cases, the disease or condition comprises inflammatory bowel disease (IBD). In some cases, the disease or condition comprises systemic lupus erythematosus (SLE). In some cases, the disease or condition comprises vasculitis, such as anti-neutrophil cytoplasmic antibody (ANCA) Associated (ANCA) vasculitis. In some cases, the disease or condition comprises graft-versus-host disease (GvHD). In some cases, the disease or condition comprises type 1 diabetes. In some cases, the disease or condition comprises Behcet’s syndrome. In some cases, the disease or condition comprises sepsis.

[0260] In some embodiments, the present disclosure provides a therapeutic composition comprising administering the CAR for the treatment of a cancer in a mammal (animal, human) in need thereof, comprising administering to the mammal a therapeutically effective amount the composition of the present disclosure.

[0261] Engineered cells of the disclosure may be used to aid in the therapy of a hyperproliferative disorder, for example a cancer. Administration of engineered cells (or nucleic acids for generating engineered cells in situ), alone or in combination with other therapeutic agents or a molecule (administered, secreted, or surface expressed) in response to a tumor e.g. in the tumor microenvironment), aids in the treatment or therapy by reducing the number and/or severity of symptoms experienced by a subject, increasing overall or long term survival, killing pathological cells such as tumor cells or other hyperproliferative cells, reducing the tumor burden, inhibiting the growth of tumor cells or other hyperproliferative cells, inhibiting the spread or proliferation of tumor cells or other hyperproliferative cells, and the like.

[0262] In some embodiments, in addition to the composition comprising the truncated RFX protein or CARs of the present disclosure, the additional therapeutic agent may include for example, an antibody, vaccine, any anti-cancer plant-based therapeutics, an oncoloytic virus, a checkpoint inhibitor, a T cell agonist antibody, any anti -cancer chemotherapy treatments in the market that is an anti -cancer agent or anti-cancer therapy, and/or a bispecific antibody, any anti-cancer radiation therapy administered at therapeutically effective dosages and administered in combination with CAR T cells. Administration of the truncated RFX protein or CARs with any of the anti-cancer agents or therapies may be provided in any order, any combinations, any dosages, at any frequency of treatment deemed suitable. Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration or in any order. In some embodiments, the one or more additional therapeutic agents, chemotherapeutics, anti-cancer agents, or anticancer therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. “Chemotherapy” and “anti-cancer agent” are used interchangeably herein. Various classes of anti-cancer agents can be used. Non-limiting examples include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), checkpoint inhibitors, immunomodulators, cytokines, nanoparticles, radiation therapy, tyrosine kinase inhibitors (for example, imatinib mesylate), hormone treatments, soluble receptors and/or other antineoplastics.

[0263] Hyperproliferative disorders include cancers and hyperplasia characterized by the unregulated overgrowth of cells. Hyperproliferative disorders frequently display loss of genetic regulatory mechanisms and may express native proteins inappropriately (including expression of proteins from other cell types or developmental stages, expression of mutated proteins, and expression of proteins at levels higher or lower than normal).

[0264] B-cell hyperproliferative disorders include B-cell leukemias and lymphomas such as, but not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, MALT lymphoma, Waldenstrom’s macroglobulinemia, and/or other disorders characterized by the overgrowth of B-lineage cells. [0265] Hyperproliferative disorders include diseases such as, but not limited to, bladder cancer, including upper tract tumors and urothelial carcinoma of the prostate; bone cancer, including chondrosarcoma, Ewing's sarcoma, and osteosarcoma; breast cancer, including noninvasive, invasive, phyllodes tumor, Paget's disease, and breast cancer during pregnancy; central nervous system cancers, adult low-grade infiltrative supratentorial astrocytoma/oligodendroglioma, adult intracranial ependymoma, anaplastic astrocytoma/anaplastic oligodendroglioma/glioblastoma multiforme, limited (1-3) metastatic lesions, multiple (>3) metastatic lesions, carcinomatous lymphomatous meningitis, non-immunosuppressed primary CNS lymphoma, and metastatic spine tumors; cervical cancer; colon cancer, rectal cancer, anal carcinoma; esophageal cancer; gastric (stomach) cancer; head and neck cancers, including ethmoid sinus tumors, maxillary sinus tumors, salivary gland tumors, cancer of the lip, cancer of the oral cavity, cancer of the oropharynx, cancer of the hypopharynx, occult primary, cancer of the glottic larynx, cancer of the supraglottic larynx, cancer of the nasopharynx, and advanced head and neck cancer; hepatobiliary cancers, including hepatocellular carcinoma, gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma; Hodgkin disease/lymphoma; kidney cancer; melanoma; multiple myeloma, systemic light chain amyloidosis, Waldenstrom's macroglobulinemia; myelodysplastic syndromes; neuroendocrine tumors, including multiple endocrine neoplasia, type 1, multiple endocrine neoplasia, type 2, carcinoid tumors, islet cell tumors, pheochromocytoma, poorly differentiated/small cell/atypical lung carcinoids; Non-Hodgkin's Lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B-Cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, AIDS-Related B-Cell lymphoma, peripheral T-Cell lymphoma, and mycosis fungoides/Sezary Syndrome; non-melanoma skin cancers, including basal and squamous cell skin cancers, dermatofibrosarcoma protuberans, Merkel cell carcinoma; non-small cell lung cancer (NSCLC), including thymic malignancies; occult primary; ovarian cancer, including epithelial ovarian cancer, borderline epithelial ovarian cancer (Low Malignant Potential), and less common ovarian histologies; pancreatic adenocarcinoma; prostate cancer; small cell lung cancer and lung neuroendocrine tumors; soft tissue sarcoma, including soft-tissue extremity, retroperitoneal, intra-abdominal sarcoma, and desmoid; testicular cancer; thymic malignancies, including thyroid carcinoma, nodule evaluation, papillary carcinoma, follicular carcinoma, Hurthle cell neoplasm, medullary carcinoma, and anaplastic carcinoma; uterine neoplasms, including endometrial cancer and/or uterine sarcoma.

[0266] Methods for administering CAR-comprising immune cells for therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described in US 2003/0170238; US 4690915; S.A. Rosenberg, Nat Rev Clin Oncol (2011) 8(10):577-85. See also M. Themeli et al., Nat Biotechnol (2013) 31(10):928-33; and T. Tsukahara et al., Biochem Biophys Res Commun (2013) 438(1): 84-89. In an aspect of the disclosure, the method comprises administering a CAR-T, an iPSC, an HSC, NK cell, gamma delta T cell, T cell or any immunotherapies or grafts comprising the present disclosure.

[0267] In some embodiments, the subject to be treated is a mammal, such as a human being. In other cases, the mammal is a mouse, a rat, a cat, a dog, a rabbit, a pig, a sheep, a horse, a bovine, a goat, a gerbil, a hamster, a guinea pig, a monkey or any other mammal. Many such mammals may be subjects that are known to the art as preclinical models for certain diseases or disorders, including inflammatory diseases, solid tumors and/or other cancers (e.g., Talmadge et al., 2007 Am. J. Pathol. 170:793; Kerbel, 2003 Cane. Biol. Therap. 2(4 Suppl 1): S 134; Man et al., 2007 Cane. Met. Rev. 26:737; Cespedes et al., 2006 Clin. TransL Oncol. 8:318).

[0268] In some embodiments, the aspects of the disclosure comprise a method for increasing an immune response in a subject, comprising administering to the subject a cell comprising the recombinant polynucleotide encoding the truncated RFX protein or CAR as described herein. In some embodiments, the disclosure relates to a method for treating disease, infection, symptom, disorder, or condition in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition comprising cells comprising the truncated RFX protein disclosed herein to a subject for the treatment of a disease, a disorder, cancer, condition, inflammatory disorder, tumor or an infection.

[0269] In some embodiments, the disease or disorder or condition may be a cancer. In some embodiments, the cancer may be a lymphoma. In some embodiments, the cancer may be a leukemia. Some non-limiting examples of leukemia include chronic lymphocytic leukemia ("CLL"), acute lymphoblastic leukemia ("ALL"), chronic lymphocytic leukemia, myelogenous leukemia, acute myelogenous leukemia (AML) and chronic myeloid leukemia (CML).

[0270] In other embodiments, the tumor is a solid tumor cancer. In some embodiments, the solid tumor cell is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, or esophageal cancer. In some embodiments, the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell. In some embodiments, the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC). In some embodiments, the administered first therapeutic agent inhibits tumor growth or metastasis of the cancer in the subject. In some embodiments, the cancer includes a metastatic cancer cell, a multiply drug resistant cancer cell, or a recurrent cancer cell. In some embodiments, the administered first therapeutic agent confers increased production of interferon gamma (IFNy) and/or interleukin-2 (IL-2) in the subject. In some embodiments, the cancer has reduced expression of CD58. In some embodiments, the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM), or skin cutaneous melanoma (SKCM).

[0271] In some embodiments, an effective amount of the engineered cells described herein is determined based on the intended goal, for example tumor regression. For example, where existing cancer is being treated, the amount of a therapeutic agent disclosed herein to be administered may be greater than where administration of the therapeutic agent is for prevention of cancer. One of ordinary skill in the art will be able to determine the amount of a therapeutic agent to be administered and the frequency of administration in view of this disclosure. The quantity to be administered, both according to number of treatments and dose, also depends on the individual to be treated, the state of the individual, and the protection desired. Precise amounts of the therapeutic agent also depend on the judgment of the practitioner and can be peculiar to each individual. Frequency of administration could range from 1-2 days, to 2-6 hours, to 6-10 hours, to 1-2 weeks or longer depending on the judgment of the practitioner.

[0272] In some embodiments of the present disclosure, the therapeutic agents will be an aqueous composition that includes the engineered cells described herein. Aqueous compositions of the present disclosure contain an effective amount of a therapeutic agent disclosed herein in a pharmaceutically acceptable carrier or aqueous medium. Thus, the “pharmaceutical preparation” or “pharmaceutical composition” of the disclosure can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the recombinant cells disclosed herein, its use in the manufacture of the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Center for Biologies.

[0273] The engineered cells described herein can be used to cure established tumors, inhibit tumor growth or metastasis of cancer in the treated subject relative to the tumor growth or metastasis in subjects who have not been administered one of the therapeutic compositions disclosed herein. In some embodiments, the engineered cells can be used to stimulate immune responses against the tumor via inducing the production of interferon gamma (IFNy) and/or interleukin-2 (IL-2), and other pro-inflammatory cytokines. The production of interferon gamma (IFNy) and/or interleukin-2 (IL-2) can be stimulated to produce up to about 20 fold, such as any of about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold 16 fold, 17 fold, 18 fold, 19 fold, or 20 fold or higher compared to the production of interferon gamma (IFNy) and/or interleukin-2 (IL-2) in subjects who have not been administered one of the therapeutic compositions disclosed herein.

[0274] Also provided by the present disclosure are kits. In certain embodiments, provided are kits that include any of the nucleic acids and/or expression vectors of the present disclosure, and instructions for introducing the nucleic acid or expression vector into a cell. According to some embodiments, when the expression vector encodes a recombinant polypeptide that does not comprise the protease (trans configuration), the expression vector further encodes the protease. In certain embodiments, the expression vector is configured to express the recombinant polypeptide and the protease from the same promoter. For example, the expression vector may be a bicistronic expression vector for expression of separate recombinant polypeptides and protease molecules under the same promoter in the cell.

[0275] The kits of the present disclosure may further include any other reagents useful for regulatable signaling of the cell surface receptor, such as transfection/transduction reagents useful for introducing the nucleic acid or expression vector into cells of interest, e.g., immune cells (e.g., T cells) or other cells of interest.

[0276] Components of the kits may be present in separate containers, or multiple components may be present in a single container. A suitable container includes a single tube (e.g., vial), one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), or the like.

[0277] The instructions of the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub packaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, DVD, CD- ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0278] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0279] List of Numbered Embodiments

1. A composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFX5 polypeptide, wherein the truncated RFX5 polypeptide has a length of at most 359 amino acids.

2. A composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFX5 polypeptide, wherein the truncated RFX5 polypeptide comprises a truncation of at least 257 amino acids.

3. A composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFX5 polypeptide, wherein the truncated RFX5 polypeptide comprises an N-terminal truncation.

4. A composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFX5 polypeptide, wherein the truncated RFX5 polypeptide comprises an amino acid sequence consisting of amino acids 1-198 of SEQ ID NO: 1.

5. A composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFX5 polypeptide, and wherein the cell expresses endogenous RFX5.

6. The composition of any one of embodiments 1-3 and 5, wherein the mutant or truncated RFX5 polypeptide comprises from 150-250 amino acids, from 175-225 amino acids, from 190-210 amino acids, or about 200 amino acids.

7. The composition of embodiment 5, wherein the mutant RFX5 polypeptide is a truncated RFX5 polypeptide.

8. The composition of embodiment 5, wherein the cell expresses endogenous RFXANK and endogenous RFXAP.

9. The composition of embodiment 5, wherein the mutant RFX5 polypeptide interacts with endogenous RFXANK, endogenous RFXAP or a combination thereof.

10. The composition of embodiment 5, wherein the mutant RFX5 polypeptide interacts with endogenous RFXANK and endogenous RFXAP in the cell to form a complex.

11. The composition of embodiment 5, wherein expression of the mutant RFX5 polypeptide inhibits or interferes with MHC expression in the cell. The composition of embodiment 11, wherein an expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is less than an expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. The composition of embodiment 12, wherein the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is as least 25% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. The composition of embodiment 12, wherein the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is at least 50% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide. The composition of any one of embodiments 11-14, wherein the MHC is an endogenous MHC. The composition of any one of embodiments 11-15, wherein the MHC is an MHC Class I protein. The composition of any one of embodiments 11-15, wherein the MHC is an MHC Class II protein. The composition of any one of embodiments 11-15, wherein the MHC comprises an MHC Class I protein and an MHC Class II protein. The composition of embodiment 16 or 18, wherein the MHC Class I protein is encoded by an HLA-A, HLA-B or HLA-C gene. The composition of embodiment 17 or 18, wherein the MHC Class II protein is encoded by an HLA- DR, HLA-DP, or HLA-DQ gene. The composition of any one of embodiments 11-20, wherein the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is substantially the same as the expression level of the MHC in a RFX5 knockout cell. The composition of any one of embodiments 1-21, wherein the truncated RFX5 polypeptide comprises a PX2LPX5X6 motif; wherein X2 is any amino acid, X5 is any amino acid, and X₍, is isoleucine (I) or leucine (L). The composition of any one of embodiments 1-22, wherein the mutant or truncated RFX5 lacks an NFY binding domain. The composition of any one of embodiments 1-23, wherein the mutant or truncated RFX5 polypeptide comprises:

(i) a DNA binding domain;

(ii) a C-terminal truncation;

(iii) an RFXAP binding site;

(iv) an RFXANK binding site; and wherein the truncated RFX5 lacks an NFY binding domain. The composition of any one of embodiments 1-24, wherein the truncated RFX5 polypeptide is a dominant negative RFX5 polypeptide (RFX5 DN). The composition of any one of embodiments 5-25, wherein the truncated RFX5 polypeptide lacks a nuclear localization signal (NLS). The composition of any one of embodiments 5-26, wherein the cell is an allogeneic cell. The composition of any one of embodiments 5-27, wherein the cell is a lymphocyte. The composition of any one of embodiments 5-28, wherein the cell is a T cell, an NK cell, an NKT cell,

T reg cell, a monocyte, a myeloid cell, a macrophage, a hematopoietic stem cell or an iPSC. The composition of embodiment 29, wherein the T cell is a gamma-delta T cell, a CD8+ T cell or a CD4+ T cell. The composition of embodiment 29 or 30, wherein the T cell is a CAR-T cell. The composition of any one of embodiments 5-31, wherein the cell is a host cell. The composition of any one of embodiments 5-32, wherein the cell is a population of cells. The composition of embodiment 33, wherein the population of cells comprises at least IxlO⁵ cells. The composition of any one of embodiments 1-34, wherein the recombinant nucleic acid comprises a sequence encoding a chimeric antigen receptor (CAR). The composition of embodiment 35, wherein the CAR comprises: (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain. The composition of embodiment 36, wherein the antigen binding domain is an anti-CD19 binding domain. The composition of embodiment 37, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. The composition of embodiment 36, wherein the antigen binding domain is an anti-CD22 binding domain. The composition of embodiment 39, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QHWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. The composition of embodiment 36, wherein the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothelin, MART-1, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A- PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, R0R1, and GD2. The composition of any one of embodiments 36-41, wherein the intracellular domain of the CAR comprises an intracellular signaling domain from CD3zeta, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD35, CD3c, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70. The composition of any one of embodiments 36-42, wherein the transmembrane domain of the CAR comprises a transmembrane domain from CD 8 or CD28. The composition of any one of embodiments 36-43, wherein the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28. The composition of any one of embodiments 1-44, wherein the recombinant nucleic acid is a vector. The composition of any one of embodiments 1-45, wherein the recombinant nucleic acid is a viral vector or a plasmid. The composition of any one of embodiments 1-44, wherein the recombinant nucleic acid is an RNA. A pharmaceutical composition comprising the composition of any one of embodiment 1-47, and a pharmaceutically acceptable excipient, diluent or carrier. A method of modulating MHC expression comprising expressing the mutant or truncated RFX5 polypeptide encoded by the nucleic acid sequence of the recombinant nucleic acid of the composition of any one of embodiments 1-47 in a cell. The method of embodiment 49, wherein the modulating MHC expression comprises inhibiting MHC expression. The method of embodiment 49 or 50, wherein the mutant or truncated RFX5 polypeptide binds to a promoter for an MHC in the cell. The method of any one of embodiments 49-51, wherein the cell is a population of T cells, and wherein a higher percentage of T cells in the population of T cell survive when administered to a subject compared to the percentage of T cells that survive in a population of T cells that do not express the mutant or truncated RFX5 polypeptide when administered to the subject. The method of any one of embodiments 49-52, wherein the cell is a population of T cells, and wherein a percentage of T cells in the population of T cell that survive when administered to a subject is about 50%- 150% of the T cells that survive in a population of RFX5 knockout T cells when administered to the subject. The method of embodiment 52 or 53, wherein the subject comprises alloreactive T cells. The method of any one of embodiments 52-54, wherein the number of T cells in the population of T cell that survive when administered to a subject is at least 1.5 fold higher than the number of T cells T cells that survive in a population of T cells that do not express the mutant or truncated RFX5 polypeptide when administered to the subject. The method of any one of embodiments 52-54, wherein the number of T cells in the population of T cell that survive when administered to a subject is at least 2 or 3 or 4 or 5 fold higher than the number of T cells T cells that survive in a population of T cells that do not express the mutant or truncated RFX5 polypeptide when administered to the subject. The method of any one of embodiments 49-56, wherein the mutant or truncated RFX5 polypeptide inhibits binding or recruitment of RFXANK and/or RFXAP to a promoter for an MHC in the cell. The method of any one of embodiments 49-57, wherein the mutant or truncated RFX5 polypeptide inhibits binding or recruitment of endogenous RFX5 to a promoter for an MHC in the cell. The method of any one of embodiments 49-58, wherein the mutant or truncated RFX5 polypeptide competes with endogenous RFX5 for a promoter for an MHC in the cell. A method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of embodiment 48 to the subject. The method of embodiment 60, wherein the subject is a human subject. The method of embodiment 60 or 61, wherein the disease or condition is cancer or an auto-immune disease or condition. The method of embodiment 62, wherein the cancer is lymphoma or leukemia. The method of embodiment 62, wherein the cancer is a solid tumor cancer. The method of embodiment 62, wherein the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and/or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LU AD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC). The method of embodiment 62, wherein the autoimmune disease is an inflammatory condition. A vector comprising a sequence encoding a recombinant nucleic acid comprising a mutant RFX5 polypeptide, wherein the vector further comprising a sequence encoding a chimeric antigen receptor (CAR). A composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFXANK polypeptide, wherein the truncated RFXANK polypeptide has a length of less than 260 amino acids. The composition of embodiment 68, wherein the truncated RFXANK polypeptide further comprises a mutation. The composition of embodiment 69, wherein the mutation is a mutation at a position corresponding to position 121 and/or 224 of SEQ ID NO: 15. A composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFXANK polypeptide, and wherein the cell expresses endogenous RFXANK. The composition of embodiment 71, wherein the mutant RFXANK polypeptide is a truncated RFXANK polypeptide. The composition of any one of embodiments 68-72, wherein the mutant or truncated RFXANK polypeptide lacks one or more ankyrin repeat domains and/or lacks one or more domains that bind to CIITA and/or NLRC5. The composition of embodiment 73, wherein the truncated RFXANK polypeptide comprises 3 or fewer ankyrin repeat domains. The composition of embodiment 73, wherein the truncated RFXANK polypeptide comprises 2 or fewer ankyrin repeat domains. The composition of embodiment 73, wherein the truncated RFXANK polypeptide comprises 1 ankyrin repeat domain. The composition of embodiment 73, wherein the truncated RFXANK polypeptide lacks an ankyrin repeat domain. The composition of any one of embodiments 71-77, wherein the mutant RFXANK polypeptide comprises a mutation at a position corresponding to position 121 and/or 224 of SEQ ID NO: 15. The composition of any one of embodiments 69-76, wherein the mutation is selected from the group consisting of a D 12 IV mutation, a Y224A mutation and a combination thereof. The composition of any one of embodiments 71-78, wherein expression of the mutant RFXANK polypeptide inhibits or interferes with expression of an MHC in the cell. The composition of embodiment 80, wherein an expression level of an MHC in the cell expressing the mutant RFXANK polypeptide is less than an expression level of the MHC in a cell not expressing the mutant RFXANK polypeptide. 82. The composition of embodiment 80, wherein the MHC is an endogenous MHC.

83. The composition of embodiment 80 or 81, wherein the MHC is an MHC Class II protein or an MHC Class I protein.

84. The composition of embodiment 83, wherein the MHC Class II protein is encoded by an HLA-DR, HLA-DP, or HLA-DQ gene; or wherein the MHC Class I protein is encoded by an HLA-A, HLA-B or HLA-C gene.

85. The composition of any one of embodiments 80-84, wherein the expression level of an MHC in the cell expressing the mutant RFXANK polypeptide is substantially the same as the expression level of the MHC in a RFXANK knockout cell.

86. The composition of any one of embodiments 71-85, wherein the mutant RFXANK polypeptide is a dominant negative RFXANK polypeptide (RFXANK DN).

87. The composition of any one of embodiments 71-86, wherein the truncated RFXANK polypeptide has a length of less than 260 amino acids.

88. The composition of any one of embodiments 68-87, wherein the mutant or truncated RFXANK polypeptide has a length of less than 150, 125 or 100 amino acids.

89. The composition of any one of embodiments 68-87, wherein the mutant or truncated RFXANK polypeptide has a length of about 122 amino acids.

EXAMPLES

[0280] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature cited herein.

[0281] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

Example 1: Cloning of various constructs

[0282] The native protein gene sequences encoding components of the truncated RFX5 protein and truncated or mutant RFXANK comprise the sequences from Table 1 and/or Table 2. Exemplary plasmids were designed to express various RFX5 and RFXANK constructs. To generate plasmids encoding truncated RFX5 proteins or RFXANK proteins the sequences in Table 3 were used for cloning.

Example 2: Generation of DN RFX constructs and testing of their effects on MHC expression and survival of graft T cells

Synthesis of RFX DN Constructs

[0283] Genes encoding RFX5 or RFXANK dominant negative constructs were human codon optimized, synthesized and cloned into a MSGV1 retroviral vector by Gene Art (Life Technologies). The effects of DN RFX constructs on MHC expression are shown in FIG. 5 and FIG. 6.

Retroviral vector production and primary T cell transduction

[0284] Retroviral supernatant was produced via transient transfection of 293 GP producer cells. 293 GP cells were seeded onto poly-D-lysine (R&D Systems) coated plates the day prior to transfection. After the cells reached 70-90% confluence, plasmids encoding the genes of interest were co-transfected with Lipofectamine 2000 (Life Technologies). Media is replaced 24 h after transfection. Viral supernatant was harvested 24 to 48 h after media replacement and filtered through a 0.45 pm filter to remove cell debris. Viral supernatant was stored long term at -80°C or used immediately for transduction.

[0285] Primary human T cells were isolated from fresh or frozen PBMCs obtained from healthy donor leukopaks from Stem Cell Technologies using negative MACS selection (Miltenyi). Isolated T cells were cryopreserved in Bambanker (GC Lymphotec Inc.). After thawing, T cells were activated with TransACT (Miltenyi) and 100 lU/mL IL-2 (Miltenyi) for 48 h and cultured in RPMI-1640 + 10% fetal bovine serum + non-essential amino acids (R10). T cells were transduced with retroviral vector on days 2 or 3 post activation. Gene editing of primary T cells by CRISPR/Cas9

[0286] T cells were activated with TransACT and 100 lU/mL IL-2 for 48-72 hr. T cells were gene edited using 1: 1 molar ratio of sgRNA:Cas9 on days 2 or 3 post activation via CRISPR/Cas9 using the Neon electroporator (Life Technologies). After 24 h, fresh RPMI-1640 + 10% fetal bovine serum + non-essential amino acids + 100 lU/mL IL-2 was added.

T Cell Mixed Lymphocyte Reaction

[0287] Engineered graft T cells were purified by negative MACS selection for TCRa[3 negativity (Stem Cell Technologies). Purity was confirmed via flow cytometry. Graft T cells were mixed at a 1: 1 E:T ratio with primed, allogeneic host T cells in R10 + 20 lU/mL IL-2 and co-incubated for 48 h at 37 °C, 5% CO2. % Graft survival was determined by flow cytometry, gating on absolute event counts of graft T cells and normalizing in the absence of effector cells, competitive inhibition approach but the overexpressed competitor (truncated RFX5) is competing for only two sites for each of the MHCI and MHCII loci (binding site upstream of each locus on two copies of the relevant chromosome, 6) . One therefore expects minimal dependence of the dominant negative effect of truncated RFX5 on the expression level achieved, FIG. 7. Flow plots of MHC-I expression vs BFP (transduction marker) Dominant negative RFX5 (AA1-198) showed decreased MHC-I expression, even at lower BFP MFI. If SV40 NLS (PKKKRKV) was C-terminally fused, higher expression was required for MHC-I reduction.

NK Cell Mixed Lymphocyte Reaction

[0288] Engineered graft T cells were purified by negative MACS selection for TCRa|3 negativity (Stem Cell Technologies). Purity was confirmed via flow cytometry. Graft T cells were mixed at a 1: 1 E:T ratio with activated, allogeneic host NK cells in R10 + 1000 lU/mL IL-2 and co-incubated for 48 h at 37 °C, 5% CO2. %Graft survival was determined by flow cytometry, gating on absolute event counts of graft T cells and normalizing in the absence of effector cells.

[0289] Survival of graft T cells in comparison to control cells comprising RFX knockout cell or NTD are shown in FIG. 8. Truncated RFX5 (AA 1-198), 1371. that retained RFXANK, RFXAP and DNA binding domain but lacks C-terminal NF-Y interacting domain functioned as a dominant negative, phenotypically equivalent to RFX5 KO. Inclusion of SV40 NLS (1372) or RFX5 NLS (1373) also worked but was unnecessary. RFXANK Y224A (1484) functions like a class II KD, class I unaffected. Allogeneic T cells co-cultured with graft T cells for 48 h in 1-way T cell mixed lymphocyte reaction.

[0290] Percent survival of graft cells varied by HLA-specific gene, however, overall, percent survival of graft cells clones 1371-1373 graft T cells maintained similar levels to those of the cells expressing an RFX knockout (RFX5 KO) construct; RFX5 DN had similar levels of protection as RFX5 KO (FIG. 8). Addition of NLS to RFX5 DN decreased efficacy.

[0291] FIG. 9 shows the percentage of graft survival when allogeneic NK cells were co-cultured with graft T cells for 48 hours in a 1-way NK cell mixed lymphocyte reaction. Percent survival of cells expressing RFX5 dominant negative (DN) was comparable to that for RFX5 knockout (KO) cells and was significantly higher than survival of |32m KO cells.

Example 3 - Exemplary sequences of the disclosure

Table 1. Sequences of Exemplary RFX5 polypeptides

Table 2. Sequences of Exemplary RFXANK polypeptides

Table 3. Other exemplary sequences

Table 4: Exemplary Antigen Binding Domain for CARs

[0292] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

6. The composition of any one of claims 1-3 and 5, wherein the mutant or truncated RFX5 polypeptide comprises from 150-250 amino acids, from 175-225 amino acids, from 190-210 amino acids, or about 200 amino acids.

7. The composition of claim 5, wherein the mutant RFX5 polypeptide is a truncated RFX5 polypeptide.

8. The composition of claim 5, wherein the cell expresses endogenous RFXANK and endogenous RFXAP.

9. The composition of claim 5, wherein the mutant RFX5 polypeptide interacts with endogenous RFXANK, endogenous RFXAP or a combination thereof.

10. The composition of claim 5, wherein the mutant RFX5 polypeptide interacts with endogenous RFXANK and endogenous RFXAP in the cell to form a complex.

11. The composition of claim 5, wherein expression of the mutant RFX5 polypeptide inhibits or interferes with MHC expression in the cell.

12. The composition of claim 11, wherein an expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is less than an expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide.

13. The composition of claim 12, wherein the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is as least 25% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide.

14. The composition of claim 12, wherein the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is at least 50% less than the expression level of the MHC in a cell not expressing the mutant RFX5 polypeptide.

15. The composition of any one of claims 11-14, wherein the MHC is an endogenous MHC.

16. The composition of any one of claims 11-15, wherein the MHC is an MHC Class I protein. The composition of any one of claims 11-15, wherein the MHC is an MHC Class II protein. The composition of any one of claims 11-15, wherein the MHC comprises an MHC Class I protein and an MHC Class II protein. The composition of claim 16 or 18, wherein the MHC Class I protein is encoded by an HLA-A, HLA-B or HLA-C gene. The composition of claim 17 or 18, wherein the MHC Class II protein is encoded by an HLA-DR, HLA-DP, or HLA-DQ gene. The composition of any one of claims 11-20, wherein the expression level of an MHC in the cell expressing the mutant RFX5 polypeptide is substantially the same as the expression level of the MHC in a RFX5 knockout cell. The composition of any one of claims 1-21, wherein the truncated RFX5 polypeptide comprises a PX2LPX5X6 motif; wherein X2 is any amino acid, X5 is any amino acid, and X₍, is isoleucine (I) or leucine (L). The composition of any one of claims 1-22, wherein the mutant or truncated RFX5 lacks an NFY binding domain. The composition of any one of claims 1-23, wherein the mutant or truncated RFX5 polypeptide comprises:

(v) a DNA binding domain;

(vi) a C-terminal truncation;

(vii) an RFXAP binding site;

(viii)an RFXANK binding site; and wherein the truncated RFX5 lacks an NFY binding domain. The composition of any one of claims 1-24, wherein the truncated RFX5 polypeptide is a dominant negative RFX5 polypeptide (RFX5 DN). The composition of any one of claims 5-25, wherein the truncated RFX5 polypeptide lacks a nuclear localization signal (NLS). The composition of any one of claims 5-26, wherein the cell is an allogeneic cell. The composition of any one of claims 5-27, wherein the cell is a lymphocyte. The composition of any one of claims 5-28, wherein the cell is a T cell, an NK cell, an NKT cell, a monocyte, a myeloid cell, a macrophage, a dendritic cell, a hematopoietic stem cell or an iPSC. The composition of claim 29, wherein the T cell is a gamma-delta T cell, a CD8+ T cell or a CD4+ T cell. The composition of claim 29 or 30, wherein the T cell is a CAR-T cell. The composition of any one of claims 5-31, wherein the cell is a host cell. The composition of any one of claims 5-32, wherein the cell is a population of cells. The composition of claim 33, wherein the population of cells comprises at least IxlO⁵ cells. The composition of any one of claims 1-34, wherein the recombinant nucleic acid comprises a sequence encoding a chimeric antigen receptor (CAR). The composition of claim 35, wherein the CAR comprises: (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain. The composition of claim 36, wherein the antigen binding domain is an anti-CD19 binding domain. The composition of claim 37, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. The composition of claim 36, wherein the antigen binding domain is an anti-CD22 binding domain. The composition of claim 39, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. The composition of claim 36, wherein the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothelin, MART-1, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE- 3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD20, CD22, ROR1, and GD2. The composition of any one of claims 36-41, wherein the intracellular domain of the CAR comprises an intracellular signaling domain from CD3zeta, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3e, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70. The composition of any one of claims 36-42, wherein the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28. The composition of any one of claims 36-43, wherein the extracellular domain of the CAR comprises a hinge domain from CD 8 or CD28. The composition of any one of claims 1-44, wherein the recombinant nucleic acid is a vector. The composition of any one of claims 1-45, wherein the recombinant nucleic acid is a viral vector or a plasmid. The composition of any one of claims 1-44, wherein the recombinant nucleic acid is an RNA. A pharmaceutical composition comprising the composition of any one of claim 1-47, and a pharmaceutically acceptable excipient, diluent or carrier. A method of modulating MHC expression comprising expressing the mutant or truncated RFX5 polypeptide encoded by the nucleic acid sequence of the recombinant nucleic acid of the composition of any one of claims 1-47 in a cell. The method of claim 49, wherein the modulating MHC expression comprises inhibiting MHC expression. The method of claim 49 or 50, wherein the mutant or truncated RFX5 polypeptide binds to a promoter for an MHC in the cell. The method of any one of claims 49-51, wherein the cell is a population of T cells, and wherein a higher percentage of T cells in the population of T cell survive when administered to a subject compared to the percentage of T cells that survive in a population of T cells that do not express the mutant or truncated RFX5 polypeptide when administered to the subject. The method of any one of claims 49-52, wherein the cell is a population of T cells, and wherein a percentage of T cells in the population of T cell that survive when administered to a subject is about 50%- 150% of the T cells that survive in a population of RFX5 knockout T cells when administered to the subject. The method of claim 52 or 53, wherein the subject comprises alloreactive T cells. The method of any one of claims 52-54, wherein the number of T cells in the population of T cell that survive when administered to a subject is at least 1.5 fold higher than the number of T cells T cells that survive in a population of T cells that do not express the mutant or truncated RFX5 polypeptide when administered to the subject. The method of any one of claims 52-54, wherein the number of T cells in the population of T cell that survive when administered to a subject is at least 2 or 3 or 4 or 5 fold higher than the number of T cells T cells that survive in a population of T cells that do not express the mutant or truncated RFX5 polypeptide when administered to the subject. The method of any one of claims 49-56, wherein the mutant or truncated RFX5 polypeptide inhibits binding or recruitment of RFXANK and/or RFXAP to a promoter for an MHC in the cell. The method of any one of claims 49-57, wherein the mutant or truncated RFX5 polypeptide inhibits binding or recruitment of endogenous RFX5 to a promoter for an MHC in the cell. The method of any one of claims 49-58, wherein the mutant or truncated RFX5 polypeptide competes with endogenous RFX5 for a promoter for an MHC in the cell. A method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 48 to the subject. The method of claim 60, wherein the subject is a human subject. The method of claim 60 or 61, wherein the disease or condition is cancer or an auto-immune disease or condition. The method of claim 62, wherein the cancer is lymphoma or leukemia. The method of claim 62, wherein the cancer is a solid tumor cancer. The method of claim 62, wherein the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and/or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LU AD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC). The method of claim 62, wherein the autoimmune disease is an inflammatory condition. A vector comprising a sequence encoding a recombinant nucleic acid comprising a mutant RFX5 polypeptide, wherein the vector further comprising a sequence encoding a chimeric antigen receptor (CAR). A composition comprising a recombinant nucleic acid comprising a sequence encoding a truncated RFXANK polypeptide, wherein the truncated RFXANK polypeptide has a length of less than 260 amino acids. The composition of claim 68, wherein the truncated RFXANK polypeptide further comprises a mutation. The composition of claim 69, wherein the mutation is a mutation at a position corresponding to position 121 and/or 224 of SEQ ID NO: 15.

-HO- A composition comprising a cell comprising a recombinant nucleic acid comprising a sequence encoding a mutant RFXANK polypeptide, and wherein the cell expresses endogenous RFXANK. The composition of claim 71, wherein the mutant RFXANK polypeptide is atruncated RFXANK polypeptide. The composition of any one of claims 68-72, wherein the mutant or truncated RFXANK polypeptide lacks one or more ankyrin repeat domains and/or lacks one or more domains that bind to CIITA and/or NLRC5. The composition of claim 73, wherein the truncated RFXANK polypeptide comprises 3 or fewer ankyrin repeat domains. The composition of claim 73, wherein the truncated RFXANK polypeptide comprises 2 or fewer ankyrin repeat domains. The composition of claim 73, wherein the truncated RFXANK polypeptide comprises 1 ankyrin repeat domain. The composition of claim 73, wherein the truncated RFXANK polypeptide lacks an ankyrin repeat domain. The composition of any one of claims 71-77, wherein the mutant RFXANK polypeptide comprises a mutation at a position corresponding to position 121 and/or 224 of SEQ ID NO: 15. The composition of any one of claims 69-76, wherein the mutation is selected from the group consisting of a D121V mutation, a Y224A mutation and a combination thereof. The composition of any one of claims 71-78, wherein expression of the mutant RFXANK polypeptide inhibits or interferes with expression of an MHC in the cell. The composition of claim 80, wherein an expression level of an MHC in the cell expressing the mutant RFXANK polypeptide is less than an expression level of the MHC in a cell not expressing the mutant RFXANK polypeptide. The composition of claim 80, wherein the MHC is an endogenous MHC. The composition of claim 80 or 81, wherein the MHC is an MHC Class II protein or an MHC Class I protein. The composition of claim 83, wherein the MHC Class II protein is encoded by an HLA-DR, HLA-DP, or HLA-DQ gene; or wherein the MHC Class I protein is encoded by an HLA-A, HLA-B or HLA-C gene. The composition of any one of claims 80-84, wherein the expression level of an MHC in the cell expressing the mutant RFXANK polypeptide is substantially the same as the expression level of the MHC in a RFXANK knockout cell. The composition of any one of claims 71-85, wherein the mutant RFXANK polypeptide is a dominant negative RFXANK polypeptide (RFXANK DN). The composition of any one of claims 71-86, wherein the truncated RFXANK polypeptide has a length of less than 260 amino acids. The composition of any one of claims 68-87, wherein the mutant or truncated RFXANK polypeptide has a length of less than 150, 125 or 100 amino acids. The composition of any one of claims 68-87, wherein the mutant or truncated RFXANK polypeptide has a length of about 122 amino acids.