T-bet induces tumor cell apoptosis and its utility in cancer therapy

Abstract

A therapeutic composition is designed for treating cancer is provided. The therapeutic composition includes at least one viral vector engineered to deliver a nucleic acid sequence encoding the T-bet (T-box transcription factor TBX21) to cancer cells originating from epithelial cells. The nucleic acid sequence encoding T-bet is operably linked to a promoter suitable for expression in the cancer cells.

Description

[0001]USC0380PCT (2024-128-02) T-BET INDUCES TUMOR CELL APOPTOSIS AND ITS UTILITY IN CANCER THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. provisional application Serial No. 63 / 574,036 filed April 3, 2024, the disclosure of which is hereby incorporated in its entirety by reference herein. REFERENCE TO SEQUENCE LISTING A computer-readable XML file entitled USC0380_sequence_listing.xml, which was created on April 3, 2025, with a file size of about 57,591 bytes, contains the sequence listing for this application, has been filed with this application, and is hereby incorporated by reference in its entirety. TECHNICAL FIELD In at least one aspect, the present invention is related to a therapeutic composition is designed for treating cancer. BACKGROUND T-BOX21 (TBX21), also known as T-box factor expressed in T cells (T-bet), was first cloned in 2000 as a transcription factor specific to T helper (Th) 1 cells that could account for subtype- specific interferon-γ (IFN-γ) production (Szabo et al., 2000). T‑bet is widely expressed in multiple cell types of both innate and adaptive immune systems. In the innate immune system, T‑bet is expressed in dendritic cells (DCs), natural killer (NK) cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). In the adaptive immune system, T‑bet is expressed in CD4+ and CD8+ T effector cells, B cells, γδ T cells, and a subset of regulatory T (Treg) cells (Lazarevic et al., 2013). Overall, T‑bet regulates the development of immune components, affects the trafficking of both innate and adaptive immune cells, and controls the polarity of their cytokine responses. Besides the immune system, T-bet expression in other cell or tissue types is rarely reported. In a 2005 paper, T-bet was reported to express 1 USC0380PCT (2024-128-02) in epithelial cells of the human female reproductive tract, and its expression level is modulated by cytokines and female hormones (Kawana et al., 2005). Besides this report, the role of T‑bet in non- immune cell types, particularly epithelial cells, remains largely obscure. T-bet expression is required for the survival, development, and physiological functions of multiple immune cell types (Lazarevic et al., 2013), but the function of T-bet is complex and context dependent. For instance, T-bet regulates T cell differentiation and reactivity to immune stimulation, and mice with T-bet overexpression in T cells are resistant to experimental autoimmune encephalomyelitis (EAE) (Martinez et al., 2014). Overexpression of T-bet in T cells also suppresses autoimmune arthritis development through the dysfunction of Th17 cell differentiation (Kondo et al., 2012). However, in a different autoimmune disease model, the C57BL / 6xBXSB / MpJ-Yaa F1 (Yaa) mice, T-bet overexpression in T cells accelerates disease severity and aggravates 50% of mortality, with increased glomerulonephritis and proteinuria (Shimohata et al., 2009). Similarly, in other reports, T-bet overexpression in T cells triggered the spontaneous development of dermatitis and pulmonary alveolar proteinosis, and these transgenic mice showed hypersensitive responses to chemical-induced contact dermatitis (Iriguchi et al., 2015; Ishizaki et al., 2007). The intrinsic correlation between T-bet and intestinal inflammation has been long recognized (Neurath et al., 2002). Although T-bet expression is relatively low in intestinal epithelia under homeostatic condition, its expression in the intestine is dramatically increased during enteritis. In human ulcerative colitis patients, T-bet is significantly increased based on genome-wide gene expression data (Olsen et al., 2009). Similar up-regulation of T-bet was observed in human Crohn’s disease patients in both inflamed colons and small intestines (Kugathasan et al., 2008; Peters et al., 2017). Consistently, in both dextran sodium sulfate (DSS)-treated mouse colitis model and azoxymethane (AOM) / DSS-induced colon cancer model, whole genome expression profiling showed the upregulation of T-bet in colorectal epithelia (Fang et al., 2011; Gao et al., 2013; Gkouskou et al., 2016). These sequencing data show the upregulation of T-bet in inflammatory bowel disease (IBD), but it is not clear yet whether T-bet is highly induced in gut epithelial cells or tissue-infiltrated immune cells under disease conditions. 2 USC0380PCT (2024-128-02) In human Crohn’s disease and celiac disease patients, elevated expression of T-bet has been confirmed in lamina propria T cells, particularly Th1 cells as potential pathological effectors, as well as peripheral blood T cells, B cells, and monocytes (Powell et al., 2010). T-bet levels tend to decrease in these cells after disease remission, while anti-inflammatory therapies for IBD, such as glucocorticoids, inhibit T-bet expression in T cells (Powell et al., 2010). In preclinical models, the intestinal balance between IFN-γ / IL-4 and transforming growth factor (TGF)-β activity is a key to controlling intestinal inflammation. Overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis, and T-bet-driven pathways control the balance of IFN-γ / IL-4 and TGF-β responses, T cell activation, and colitis development (Neurath et al., 2002). In a different model with the presence of segmented filamentous bacteria and Helicobacter hepaticus, T-bet-deficient Th cells can differentiate into Th1 / 17 cells, co-expressing IFN-γ and interleukin (IL)-17, and efficiently induce colitis (Zimmermann et al., 2016). While neutralization of IL-17A exacerbated colitis induced by T- bet-deficient Th cells, neutralization of IFN-γ completely abolished colitis (Zimmermann et al., 2016), indicating that the Th1 / Th17 balance is also important for controlling colitis. Also, T-bet directly activates Th1-specific chemokine receptors, such as CXCR3 and CCR5, which are critical for homing to inflamed tissues (Lazarevic et al., 2013). In the innate immune system, T-bet deficiency controls the response of the mucosal immune system to commensal bacteria and increases susceptibility to colitis (Garrett et al., 2007). Furthermore, T-bet is a central transcriptional regulator for intraepithelial lymphocyte (IEL) development and controls susceptibility to chemically induced colitis (Klose et al., 2014; Reis et al., 2014). Besides, in these immune cells, the function of T-bet in intestinal epithelial cells has not been reported. Accordingly, there is a need for methods capitalizing on T-bet expression for treating mammalian diseases. SUMMARY In at least one aspect, a therapeutic composition is designed for treating cancer. The therapeutic composition includes at least one viral vector engineered to deliver a nucleic acid sequence 3 USC0380PCT (2024-128-02) encoding the T-bet (T-box transcription factor TBX21) to cancer cells originating from epithelial cells. The nucleic acid sequence encoding T-bet is operably linked to a promoter suitable for expression in the cancer cells. In another aspect, the therapeutic composition is an oncolytic composition that includes at least one oncolytic viral vector. The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein: FIGURE 1a. Simplified vector map of plasmid for expressing T-bet. FIGURE 1b. A plasmid map depicting the structure of a 10,208 base pair lentiviral expression vector designated murine_Lenti Tet-On-T-bet. FIGURE 1c. Alignment of human and mouse polypeptides for T-box transcription factor (TBX21) along with a consensus polypeptide sequence (SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5). 4 USC0380PCT (2024-128-02) FIGURES 2a, 2b, 2c, 2d, 2e, and 2f. Global T-bet inducible expression causes severe diarrhea and mouse mortality. (a-b) WT and Tet-O-T-bet:rtTA (global T-bet overexpression) mice were continuously treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose). Body weights (a) and survival curves (b) were observed after the treatment. (c-f) Tet-O-T-bet:rtTA mice were treated with control water or doxycycline water solution for 2 days. (c) On day 2, severe diarrhea can only be observed in Dox-treated mice, based on the hair, structure, and wetness of the anus area. (d) Mice were euthanized on day 2 and whole intestines were dissected to display the morphological change after Dox-induced T-bet overexpression. (e) H&E staining on intestinal sections to show tissue structure in control and T-bet overexpressed mice. Arrows were added to indicate the tissue damage in the small intestine of Dox-treated Tet- O-T-bet:rtTA mice (scale bar: 200 µm). (f) IHC staining of T-bet on intestinal sections to validate the Dox- induced T-bet overexpression (scale bar: 200 µm). Statistical analyses: ANOVA and student’s unpaired t test (a) and Mantel-Cox log-rank test (b). **p<0.01; ***p<0.001. See also Figs.9 and 10. FIGURES 3a, 3b, 3c, 3d, 3f, and 3g. Inducible expression of T-bet in CD4+ T cells or myeloid lineage does not cause mouse mortality. (a) Tet-O-T-bet:rtTA-GFP:CD4-Cre (specific T-bet overexpression in CD4 cells) mice were continuously treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose). Survival curves were observed after the treatment. (b-d) Tet-O-T-bet:rtTA-GFP:CD4- Cre mice were treated with control water or doxycycline water solution for 2 days. (b) Mice were euthanized on day 2 and whole intestines were dissected to display the morphology. (c) H&E staining and IHC staining of T-bet on small intestine sections to show tissue structure and T-bet overexpression (scale bar: 200 µm). (d) Overexpression of T-bet in selected tissues and organs by western blotting. (e) Tet-O-T-bet:rtTA-GFP:Lyz2- Cre (specific T-bet overexpression in myeloid cells such as macrophages and neutrophils) mice were continuously treated with control water or doxycycline water solution. Survival curves were observed after the treatment. (f-g) Tet-O-T-bet:rtTA-GFP:Lyz2-Cre mice were treated with control water or doxycycline water solution for 2 days. (f) H&E staining and IHC staining of T-bet on small intestine sections to show tissue structure and T-bet overexpression (scale bar: 200 µm). (g) Overexpression of T-bet in selected tissues and organs by western blotting. Statistical analyses: Mantel-Cox log-rank test (a and e). See also Figs.11 and 12. FIGURES 4a, 4b, 4c, 4d, and 4e. Immune cells are not responsible for mouse mortality induced by T-bet overexpression. (a-b) WT and Tet-O-T-bet:rtTA recipient mice were irradiated and transferred with bone marrow from donor WT and Tet-O-T-bet:rtTA mice. The chimeric mice were 5 USC0380PCT (2024-128-02) continuously treated with doxycycline water solution (1 mg / ml in 5% sucrose). (a) Changes on the body weights after the doxycycline treatment. (b) Whole intestines from the chimeric mice to display the morphology. (c-e) Tet-O-T-bet:rtTA mice were treated with clodronate liposomes, Ly-6G antibody (Ab), CD4 Ab, and CD8 Ab to simultaneously deplete macrophages, neutrophils, and T cells. PBS- control liposomes and isotype-control Abs were used in control group. Mice were continuously treated with doxycycline water solution. The body weight changes (c) and survival curves (d) were observed and analyzed. (e) Before the doxycycline treatment, blood samples were collected to validate the cell depletion efficacy by flow cytometry analyses in gated CD45+ / CD11b+myeloid cells and CD45+ / CD3+lymphocytes. The percentages of macrophages (73.92% vs 14.38%), neutrophils (71.11% vs 11.53%), CD4+T cells (46.32% vs 0%), CD8+T cells (38.43% vs 16.62%), CD3+T cells (9.94% vs 0.77%), and NKT cells (13.82% vs 0.21%) were all dramatically decreased after the cell depletion. Statistical analyses: ANOVA and student’s unpaired t test (a and c) and Mantel-Cox log- rank test (d). *p<0.05; **p<0.01. FIGURES 5a, 5b, 5c, 5d, and 5e. Inducible expression of T-bet in gut epithelial cells causes mouse mortality. (a-b) Tet-O-T-bet:rtTA-GFP:Villin-Cre (specific T-bet overexpression in gut epithelial cells) mice were continuously treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose). The body weight changes (a) and survival curves (b) were observed and analyzed. (c-e) Tet-O-T-bet:rtTA-GFP:Villin-Cre mice were treated with control water or doxycycline water solution for 2 days. Mice were euthanized on day 2 for tissue collection. (c) Overexpression of T-bet in selected tissues and organs by western blotting. (d) whole intestines were dissected to display the morphological change after Dox-induced T-bet overexpression. (e) IHC staining of T-bet on intestinal sections to validate the Dox-induced T-bet overexpression. H&E staining on intestinal sections to show tissue structure in control and T-bet overexpressed mice. Arrows were added to indicate the tissue damage in the small intestine of Dox-treated Tet-O-T-bet:rtTA- GFP:Villin-Cre mice (scale bar: 200 µm). Statistical analyses: ANOVA and student’s unpaired t test (a) and Mantel-Cox log-rank test (b). **p<0.01; ***p<0.001. See also Fig.13. 6 USC0380PCT (2024-128-02) FIGURES 6a, 6b, 6c, 6d, 6e, and 6f. T-bet regulates the expression of ion channels and transporters in mouse intestine. (a-b) Tet-O-T-bet:rtTA-GFP:Villin-Cre mice (gut epithelia specific T-bet overexpression) were treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose) for 2 days. Mice were euthanized on day 2, and RNA samples were isolated from intestinal tissues. Real-time QPCR was performed to detect the expression levels of key ion channels and transporters in the small intestine (a) and colon (b). (c-d) Tet-O-T-bet:rtTA mice (global T-bet overexpression) were treated with control water or doxycycline water solution for 2 days. Mice were euthanized on day 2, and RNA samples were isolated from intestinal tissues. Real-time QPCR was performed to detect the expression levels of key ion channels and transporters in the small intestine (c) and colon (d). (e-f) Tet-O-T-bet:rtTA-GFP:Villin-Cre mice were orally treated with PBS control or anti-diarrhea drug Imodium (125 µg Loperamide hydrochloride in 200 µl PBS) every day since day -1. Doxycycline water solution was continuously treated since day 0. The body weight changes (e) and survival curves (f) were observed and analyzed. Statistical analyses: Student’s unpaired t test (a-d), ANOVA and student’s unpaired t test (e), and Mantel-Cox log-rank test (f). *p<0.05; **p<0.01; ***p<0.001. See also Fig.14. FIGURES 7a, 7b, 7c, 7d, and 7e. Inducible T-bet expression promotes the apoptosis of intestinal epithelial cells. (a) Small intestines were dissected from Tet-O-T-bet:rtTA-GFP:Villin-Cre mice (gut epithelia specific T-bet overexpression), and the organoids were cultivated and treated with 1 µg / ml doxycycline. The morphological changes of these organoids were observed at different time points under the microscope (scale bar: 1 mm for low magnification and 200 µm for high magnification). (b) Tet-O-T-bet:rtTA-GFP:Villin-Cre mice were treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose) for 2 days. Mice were euthanized on day 2 for tissue collection. TUNEL staining was performed on intestinal sections to indicate the apoptotic cells after the induction of T-bet overexpression (scale bar: 200 µm). (c-d) Small intestines were dissected from WT and Tet-O-T-bet:rtTA-GFP:Villin-Cre mice, washed in PBS and cut into small pieces, and in vitro treated with 1 µg / ml doxycycline in RPMI 1640 medium (supplemented with 10% FBS and Penicillin-Streptomycin). At different time points (0, 4, 10, 20 7 USC0380PCT (2024-128-02) hours), tissue samples were digested, and the apoptotic cells were detected by flow cytometry after the staining of Caspase 3 / 7. (c) Representative flow cytometry data at hour 20 to show the massive apoptotic cells in Dox-treated Tet-O-T-bet:rtTA-GFP:Villin-Cre intestines. The percentages of Caspase 3 / 7+cells before and after doxycycline treatment were 15.2% and 17.2% in WT mice, and 17.5% and 71.9% in T-bet overexpressed mice, respectively. (d) Percentages of apoptotic cells in different groups after the doxycycline treatment. (e) Tet-O-T-bet:rtTA-GFP:Villin-Cre mice were intraperitoneally (i.p.) injected with PBS control or pan-Caspase inhibitor (Z-VAD-FMK, Sigma- Aldrich, 50 µg in 200 µl PBS) every day from days 0 to 3. Doxycycline water solution was treated from days 0 to 7. The body weight changes and survival curves were observed and analyzed. Statistical analyses: ANOVA and student’s unpaired t test (d and e-right) and Mantel-Cox log-rank test (e-left). *p<0.05; ***p<0.001. See also Fig.15. FIGURES 8a, 8b, 8c, 8d, 8e, 8f, and 8g. Inducible T-bet expression completely inhibits colon tumor formation and growth. (a) WT control and Tet-O-T-bet transduced CT-26 cells (clone #2) were treated with PBS control or 1 µg / ml doxycycline for 48 hours. The morphological change in cells with Dox-induced T-bet overexpression was observed under the microscope (scale bar: 200 µm). (b) WT control and Tet-O-T-bet transduced CT-26 single clones were treated with PBS control or 1 µg / ml doxycycline. MTT assay was performed at different time points to determine the in vitro cell growth rates. Data were read at OD 590 nm and normalized with the value on hour 0 for the relative fold changes. (c) WT control and Tet-O-T-bet transduced CT-26 cells (clone #2) were treated with PBS control or 1 µg / ml doxycycline. Protein samples were harvested at different time points, and western blotting was performed to determine the activation of Caspase 3 in T-bet overexpressed cells. (d-f) WT control and Tet-O-T-bet transduced CT-26 cells were subcutaneously (s.c.) injected into WT BALB / c mice, and continuously treated with doxycycline water solution (1 mg / ml in 5% sucrose) since day 7. (d) Tumor sizes were measured every 2 days for the in vivo growth curves. (e) The image of tumors dissected on day 15 to compare the tumor sizes between two groups. (f) Statistical analysis of the tumor weights. (g) WT control and Tet-O-T-bet transduced CT-26 cells were subcutaneously (s.c.) injected into NSG mice, and continuously treated with doxycycline water solution since day 5. 8 USC0380PCT (2024-128-02) Tumor sizes were measured every 3 days for the in vivo growth curves. Statistical analyses: Student’s unpaired t test (f), ANOVA and student’s unpaired t test (b, d, and g). *p<0.05; **p<0.01; ***p<0.001. See also Fig.16. FIGURES 9a, 9b, 9c, 9d, and 9e. Generation of Tet-O-T-bet:rtTA mice (related to Fig. 2).(a) A schematic diagram of the Tet-On-induced T-bet expression system. Tet-O-T-bet transgenic mouse strain was generated by incorporating the mouse Tbx21 coding sequence into a doxycycline- inducible expression system. To construct the tet response element (TRE), 7 repeats of tetracycline operator sequence (7× tet) were fused to a minimal cytomegalovirus (CMV) promoter (mCMV) which lacks an enhancer sequence. The expression of reverse tetracycline-controlled transactivator (rtTA) was controlled by a constitutive promoter. Without doxycycline (Dox), rtTA exhibits a reverse phenotype and cannot bind to the Tet-On sequences. The transcription complex is not assembled, and gene expression is silenced. In the presence of doxycycline, rtTA binds to the Tet-On sequences and recruits RNA polymerase II factors for the assembly of transcription complexes, initiating the transcription of T-bet gene. (b) Genotyping strategy to identify the Tet-O-T-bet:rtTA transgenic mouse. (c) Tet-O-T-bet:rtTA mice were treated with control water or doxycycline water solution for 2 days. Thymus, spleen, and lymph nodes were collected for flow cytometry analyses to detect the T cell differentiation in T-bet-overexpressed mice. The percentages of CD4+T cells in thymus, spleen, and lymph nodes were 7.66%, 11.2%, and 32.5% in control mice, and 46.9%, 28%, and 28.7% in T- bet overexpressed mice, respectively. The percentages of CD8+T cells in these organs were 2.85%, 6.47%, and 13.4% in control mice, and 28.2%, 19.8%, and 24.9% in T-bet overexpressed mice, respectively. (d) Tet-O-T-bet:rtTA mice were treated with control water or doxycycline water solution. At days 1 and 2, splenocytes were harvested for flow cytometry analyses to examine T-bet and IFN-γ expression in gated CD4 cells. The percentages of T-bet+CD4+T cells in control vs T-bet overexpressed mice were 7.54% vs 74.6% on day 1, and 11.9% vs 84.2% on day 2, respectively. (e) TBX21 expression in normal human tissues. Body Atlas analysis by BaseSpace Correlation Engine 2.0 from Illumina Inc. Selected major organs / tissues were included in the figure from all 147 tested human tissue types. 9 USC0380PCT (2024-128-02) FIGURES 10a, 10b, and 10c. Tissue structures and T-bet inducible expression in doxycycline-treated Tet-O-T-bet:rtTA mice (related to Fig. 2). (a) Inducible expression of T-bet in major tissues and organs by western blotting. Four Tet-O-T-bet:rtTA mice and one WT mouse were treated with doxycycline water solution (1 mg / ml in 5% sucrose) for 2 days before tissue collection. (b-c) WT and Tet-O-T-bet:rtTA (global T-bet expression) mice were treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose) for 2 days. Major tissues and organs were collected for sectioning and histopathological analyses. (b) H&E staining to show tissue structure and potential damage in control and T-bet overexpressed mice (scale bar: 200 µm). (c) IHC staining of T-bet to validate the Dox-induced T-bet overexpression in different mouse tissues (scale bar: 200 µm). FIGURES 11a, 11b, 11c, 11d, and 11e. Inducible expression of T-bet in CD4+T cells does not cause mouse mortality (related to Fig. 3). (a) A schematic diagram of tissue-specific doxycycline-inducible T-bet expression system. The LoxP-sites-flanked Stop sequence was inserted in front of EGFP-tagged rtTA. After crossing with specific Cre mice, rtTA can be expressed in specific tissues following Cre expression. In the presence of Dox, rtTA will bind to the Tet-On sequences, and T-bet expression will be activated in the specific Cre-expressing tissues. (b) Genotyping strategy to identify the Tet-O-T-bet:rtTA-GFP:CD4-Cre transgenic mouse. (c-d) CD4+T cells and B220+B cells were purified and sorted from spleens of Tet-O-T-bet:rtTA-GFP:CD4-Cre mice, then treated with PBS control or doxycycline (1 µg / ml) for 24 hours. (c) Flow cytometry analyses to validate the T-bet expression in gated CD4+T cells. The percentages of T-bet+CD4+T cells are 4.5% in control mice and 68.9% in doxycycline-treated mice. B220+B cells were tested as the negative control, in which the T-bet+cells are 0.486% and 0.322% in control and doxycycline-treated mice, respectively. (d) Western blotting to detect the inducible T-bet expression in sorted CD4+T cells. (e) Tet-O-T-bet:rtTA- GFP:CD4-Cre mice were treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose) for 2 days. H&E staining and IHC staining of T-bet on tissue sections from major organs to show tissue structure and T-bet overexpression levels (scale bar: 200 µm). 10 USC0380PCT (2024-128-02) FIGURES 12a, 12b, 12c, and 12d. Inducible expression of T-bet in myeloid cells does not cause mouse mortality (related to Fig. 3). (a) Genotyping strategy to identify the Tet-O-T- bet:rtTA-GFP:Lyz2-Cre transgenic mouse. (b-d) Tet-O-T-bet:rtTA-GFP:Lyz2-Cre mice were treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose) for 2 days. (b) Spleen and bone marrow were collected for flow cytometry analyses to validate the T-bet expression in CD11b+myeloid cells. In bone marrow, the percentages of T-bet+CD11b+myeloid cells are 5.71% in control mice and 34.21% in doxycycline-treated mice. On the contrary, in the spleen where lymphocytes are dominant, the percentages of T-bet+CD11b+cells are 0.85% and 0.74% in control and doxycycline-treated mice, respectively. (c) H&E staining on tissue sections from major organs to show tissue structure (scale bar: 200 µm). (d) IHC staining of T-bet on tissue sections for T- bet overexpression levels (scale bar: 200 µm). FIGURES 13a, 13b, and 13c. Inducible expression of T-bet in gut epithelial cells causes mouse mortality (related to Fig. 5). (a) Genotyping strategy to identify the Tet-O-T-bet:rtTA- GFP:Villin-Cre transgenic mouse. (b-c) Tet-O-T-bet:rtTA-GFP:Villin-Cre mice were treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose) for 2 days. (b) Mice were euthanized on days 1 and 2. Whole intestines were dissected to display the morphology. (c) Mice were euthanized on day 2 for tissue collection. H&E staining and IHC staining of T-bet on tissue sections from major organs (other than the intestine) to show tissue structure and T-bet expression levels (scale bar: 200 µm). FIGURES 14a, 14b, 14c, 14d, 14e, and 14f. T-bet regulates the expression of ion channels and transporters in mouse intestine (related to Fig.6). (a-b) WT control mice were treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose) for 2 days. Mice were euthanized on day 2, and RNA samples were isolated from intestinal tissues. Real- time QPCR was performed to detect the expression levels of key ion channels and transporters in the small intestine (a) and colon (b). (c-f) Small intestines were dissected from WT, Tet-O-T-bet:rtTA (global T-bet overexpression), and Tet-O-T-bet:rtTA-GFP:Villin-Cre (gut epithelia specific T-bet overexpression) mice, and the organoids were cultivated to recapitulate the in vivo tissue 11 USC0380PCT (2024-128-02) characteristics. (c) After the culture of 6-8 days, mature organoids were treated with different doses of doxycycline for 24 hours, and T-bet expression levels were examined by Real-time QPCR with purified RNA samples. Next, the organoids were treated with 1 µg / ml doxycycline for 24 hours, and RNA samples were isolated. Real-time QPCR was performed to detect the key ion channels and transporters in organoids from WT (d), Tet-O-T-bet:rtTA-GFP:Villin-Cre (e), and Tet-O-T-bet:rtTA (f) mice. Statistical analyses: Student’s unpaired t test (a-b, d-f). *p<0.05; **p<0.01; ***p<0.001. FIGURES 15a and 15b. Inducible T-bet expression promotes the apoptosis of intestinal epithelial cells (related to Fig. 7). (a) Small intestines were dissected from Tet-O-T-bet:rtTA-GFP:Villin-Cre (gut epithelia specific T-bet overexpression) mice. Tissue organoids were cultivated and treated with PBS control or 1 µg / ml doxycycline for 24 hours. Then the organoids were fixed with formalin for 1 day and processed for sectioning. H&E staining and IHC staining of T-bet were performed to show the structure and T- bet expression in the organoids (scale bar: 200 µm).(b) Small intestines were dissected from WT and Tet-O-T- bet:rtTA (global T-bet overexpression) mice, and the organoids were cultivated and treated with 1 µg / ml doxycycline. The morphological changes of these organoids were observed at different time points under the microscope (scale bar: 1 mm for low magnification and 200 µm for high magnification). FIGURES 16a, 16b, 16c, 16d, 16e, and 16f. Inducible T-bet expression completely inhibits colon tumor formation and growth (related to Fig.8). (a) WT control and Tet-O-T-bet transduced CT-26 single clones were treated with PBS control or 1 µg / ml doxycycline for 12 hours. Western blotting was performed to validate the Dox-induced T-bet expression. (b) WT control and Tet-O-T-bet transduced CT-26 cells were subcutaneously (s.c.) injected into WT BALB / c mice, and continuously treated with doxycycline water solution (1 mg / ml in 5% sucrose) since day 7. The images of tumors on day 15 to compare the tumor sizes between two groups. (c-e) WT control and Tet-O-T-bet transduced MC-38 cells were treated with PBS control or 1 µg / ml doxycycline for 3 days. (c) The morphological change in cells with Dox-induced T-bet overexpression was observed under the microscope (scale bar: 200 µm). (d) Flow cytometry to determine the apoptotic cell percentages (Caspase 3 / 7+) in MC-38 cell clones with Dox-induced T-bet overexpression. The percentages of Caspase 3 / 7+ cells before and after doxycycline treatment were 0.64% vs 0.54% in WT cells, 0.67% vs 47.1% in T-bet overexpressed clone 1, and 0.28% vs 82.2% in T-bet overexpressed clone 2, respectively. (e) Statistical analysis of the flow cytometry data in d. (f) WT control and Tet-O-T-bet transduced MC-38 single clones were 12 USC0380PCT (2024-128-02) treated with PBS control or 1 µg / ml doxycycline for 24 hours. Western blotting was performed to validate the Dox-induced T-bet expression. Statistical analyses: Student’s unpaired t test (e). ***p<0.001; ****p<0.0001. FIGURES 17a and 17b. Inducible T-bet Expression Promotes Apoptosis in Murine Breast Cancer EO771 Cells and Melanoma B16 Cells. Wildtype control and Tet-O-T-bet transduced B16 cells (a) and Tet-O-T-bet transduced EO771 cells (b) were treated with PBS or 1 μg / mL doxycycline for 4 days. Morphological changes associated with Dox-induced T-bet overexpression were observed under a microscope (scale bar: 100 μm). FIGURE 18. Selection of Murine Breast Cancer EO771 and Melanoma B16 Cell Clones Expressing Tet-On-T-bet. Upper: EO771 Tet-On-T-bet murine breast cancer cells were analyzed by intracellular staining, and single clones #5, #8, and #9 were selected for further experiments. Lower: B16 Tet- On-T-bet murine melanoma cells were analyzed by intracellular staining, and single clones #4, #15, and #16 were chosen for subsequent experiments. FIGURE 19. Inducible T-bet Expression Inhibits Melanoma Cell Growth. Wildtype control and Tet-On-T-bet transduced B16 single clones were treated with PBS or 1 μg / mL doxycycline, and cell proliferation was assessed using an MTT assay at various time points. Absorbance was measured at OD 590 nm and normalized to the hour 0 value for relative fold changes (mean ± SD). *p < 0.05; ** p< 0.01; *** p <0.001; ****p <0.0001.n=3. FIGURE 20. Inducible T-bet Expression Promotes Apoptosis in Murine Breast Cancer EO771 Cells. Cell viability percentages in different groups following doxycycline treatment (mean ± SD). *p < 0.05; ** p< 0.01; *** p <0.001; ****p <0.0001.n=2. FIGURE 21. Inducible T-bet Expression Promotes Apoptosis in Breast Cancer MDA-MB-231 Cells. Wildtype control and Tet-O-T-bet transduced MDA-MB-231 cells were treated with PBS or 1 μg / mL doxycycline for 4 days. Morphological changes associated with Dox-induced T-bet overexpression were observed under a microscope (scale bar: 100 μm). FIGURES 22a and 22b. Inducible T-bet Expression Promotes Apoptosis in Breast Cancer MDA-MB-231 Cells. Wildtype control and Tet-On-T-bet transduced MDA-MB-231 cells were treated with PBS or 1 μg / mL doxycycline, and cell proliferation was assessed using an MTT assay at different time points. 13 USC0380PCT (2024-128-02) Absorbance was measured at OD 590 nm and normalized to hour 0 for relative fold changes (mean ± SD). *p < 0.05; ** p< 0.01; *** p <0.001; ****p <0.0001. n=3. FIGURES 23a and 23b. Inducible T-bet Expression Promotes Apoptosis in Breast Cancer MDA-MB-231 Cells. (A) Cell viability percentages in different groups after doxycycline treatment (mean ± SD). *p < 0.05; ** p< 0.01; *** p <0.001; ****p <0.0001. n=3. (B) Flow cytometry analysis after Propidium Iodide staining on day 5 showed apoptotic cell percentages in four groups of MDA-MB-231 cells. FIGURE 24a. Cell images of 293T expressing T-bet or control vector after 4 days of expression. FIGURE 24b. FACS analysis of cell death of 293T control and 293T T-bet cells after 4 days of T-bel expression. FIGURE 25a. T-bet expression completely inhibits B16 tumor growth in a mouse tumor model. FIGURE 25B. T-bet expression completely inhibits EO771 tumor growth in a mouse tumor model. DETAILED DESCRIPTION Reference will now be made in detail to presently preferred compositions, embodiments and methods of the present invention, which constitute the best modes of practicing the invention presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the invention and / or as a representative basis for teaching one skilled in the art to variously employ the present invention. Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and / or use are to 14 USC0380PCT (2024-128-02) be understood as modified by the word "about" in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary: the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property. Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and / or use are to be understood as modified by the word "about" in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary:; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property. It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components and / or conditions may, of course, vary. 15 USC0380PCT (2024-128-02) Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way. It must also be noted that, as used in the specification and the appended claims, the singular form "a," "an," and "the" comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components. As used herein, the term “about” means that the amount or value in question may be the specific value designated or some other value in its neighborhood. Generally, the term “about” denoting a certain value is intended to denote a range within + / - 5% of the value. As one example, the phrase “about 100” denotes a range of 100+ / - 5, i.e. the range from 95 to 105. Generally, when the term “about” is used, it can be expected that similar results or effects according to the invention can be obtained within a range of + / - 5% of the indicated value. As used herein, the term “and / or” means that either all or only one of the elements of said group may be present. For example, “A and / or B” shall mean “only A, or only B, or both A and B.” In the case of “only A,” the term also covers the possibility that B is absent, i.e. “only A, but not B.” It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components and / or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way. It must also be noted that, as used in the specification and the appended claims, the singular form "a," "an," and "the" comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components. 16 USC0380PCT (2024-128-02) The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps. The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. The phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. The phrase “composed of” means “including” or “consisting of.” Typically, this phrase is used to denote that an object is formed from a material. With respect to the terms “comprising,” “consisting of,” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. The term “one or more” means “at least one” and the term “at least one” means “one or more.” The terms “one or more” and “at least one” include “plurality” as a subset. The term “substantially,” “generally,” or “about” may be used herein to describe disclosed or claimed embodiments. The term “substantially” may modify a value or relative characteristic disclosed or claimed in the present disclosure. In such instances, “substantially” may signify that the value or relative characteristic it modifies is within ± 0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% or 10% of the value or relative characteristic. In the examples set forth herein, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 50 percent of the values 17 USC0380PCT (2024-128-02) indicated, rounded to or truncated to two significant figures of the value provided in the examples. In a refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 30 percent of the values indicated, rounded to or truncated to two significant figures of the value provided in the examples. In another refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 10 percent of the values indicated, rounded to or truncated to two significant figures of the value provided in the examples. Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. The terms “percent identical” or “percent identity” refer to nucleic acid or amino acid sequences that are substantially identical to a coding sequence or amino acid sequence for a nucleic acid sequence or amino acid sequence set forth herein. The term “substantially identical” means a nucleotide sequence with similarity to the nucleotide sequence set forth herein. The term “substantially identical” can also be used to describe the similarity of polypeptide sequences. For example, the present invention encompasses nucleotide sequences or polypeptide sequences that are at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 98% or 99% identical to the sequences described herein and still retain ability to function as described. To determine the “percent identity” (i.e., percent sequence identity) of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a refinement, the sequences are aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. In a refinement, the length of a first sequence aligned for comparison purposes is at least 80% of the length of a second sequence, and in some embodiments is at least 85%, 90%, 95%, or 100%. The amino acid 18 USC0380PCT (2024-128-02) residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. In this regard, the following oligonucleotide alignment algorithms may be used: BLAST (GenBank URL: www.ncbi.nlm.nih.gov / cgi-bin / BLAST / , using default parameters: Program: BLASTN; Database: nr; Expect 10; filter: default; Alignment: pairwise; Query genetic Codes: Standard(1)), BLAST2 (EMBL URL: http: / / www.embl- heidelberg.de / Services / index.html using default parameters: Matrix BLOSUM62; Filter: default, echofilter: on, Expect:10, cutoff: default; Strand: both; Descriptions: 50, Alignments: 50), or FASTA, search, using default parameters. When sequences differ in conservative substitutions, the percent identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. For the specific polypeptides set forth herein, it should be appreciated that the therapeutic compositions encompass polypeptides with any number of conservative substitutions. Conservative substitutions are changes similar to the original amino acid with respect to polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues, while preserving the functionality of being constitutively active. Examples of conservative substitutions that may be made include: substitutions between aliphatic amino acids (alanine, valine, leucine, isoleucine), polar amino acids (glutamine, asparagine, serine, threonine), acidic amino acids (glutamic 19 USC0380PCT (2024-128-02) acid and aspartic acid), basic amino acids (arginine, lysine and histidine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), large amino acids (phenylalanine and tryptophan), small amino acids (glycine, alanine) and hydroxyl amino acids (serine, threonine). In a variation, the polypeptide with conservative substitutions includes 0.2 to 30 percent of the original amino acids substituted with a conservative substitution. In some refinements, the polypeptide with conservative substitutions includes a number of substitutions equal to or at least 0.2%, 0.5%, 1%, 2%, 5%, 10%, or 12% of the original amino acids substituted with a conservative substitution. In further refinements, the polypeptide with conservative substitutions includes a number of substitutions equal to or at most 40%, 35%,30%, 25%, 20%, 15%, 10%, 5%, 2%, or 1% of the original amino acids substituted with a conservative substitution. In another refinement, the specific polypeptides set forth herein include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions. For therapeutic applications of the T-bet polypeptide, conservative substitutions should preserve several critical properties: DNA-binding domain functionality to maintain transcriptional regulation, transcriptional activation capacity to control target genes, cell-type specificity for appropriate tissue targeting, pro-apoptotic function for potential cancer applications, and ion channel regulation abilities. Research demonstrates that T-bet expression in cancer cells can trigger tumor shrinkage, suggesting its potential delivery through engineered oncolytic viruses as a promising strategy for treating epithelial cancers. When designing T-bet variants, conservative amino acid substitutions should maintain these functional properties while careful modifications would be needed to minimize adverse effects like severe diarrhea observed in mouse models. In some refinement, the present invention encompasses nucleotide sequences or polypeptide sequences that are at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 98% or 99% identical to the sequences described herein or fragments or derivatives thereof, and still retain ability to function as described. The term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is 20 USC0380PCT (2024-128-02) operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. The term “expression control sequence” refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked. An expression control sequence is “operably linked” to a nucleotide sequence when the expression control sequence controls and regulates the transcription and / or the translation of the nucleotide sequence. Thus, an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing signal for introns, and stop codons. The term “expression control sequence” is intended to include, at a minimum, a sequence whose presence are designed to influence expression, and can also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It includes sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail, i.e., a string of adenine residues at the 3′-end of a mRNA, sequences referred to as polyA sequences. It also can be designed to enhance mRNA stability. Expression control sequences which affect the transcription and translation stability, e.g., promoters, as well as sequences which effect the translation, e.g., Kozak sequences, are known in insect cells. Expression control sequences can be of such nature as to modulate the nucleotide sequence to which it is operably linked such that lower expression levels or higher expression levels are achieved. The term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor 21 USC0380PCT (2024-128-02) and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. A “tissue specific” promoter is only active in specific types of tissues or cells. The term “heterologous promoter” refers to a promoter that does not naturally direct the expression of the promoter in nature. The term “natural promoter” refers to a promoter found in nature. The term “expression vector” means a construct designed for gene expression in cells. Expression vectors include but are not limited to viruses, plasmids, cosmids, transposons, and the like. The term "T-bet" refers to a T-box transcription factor encoded by the TBX21 gene, primarily expressed in immune cells. T-bet contains a conserved T-box DNA-binding domain that recognizes specific DNA sequences (T-box recognition motifs) in target gene promoters and enhancers. It functions as a master regulator of type 1 immune responses by activating transcription of target genes such as interferon-gamma (IFN-γ). T-bet includes naturally occurring orthologs (e.g., human, mouse), functionally equivalent variants, and polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the human T-bet protein (e.g., SEQ ID NO:2), provided they retain the ability to bind T-box motifs and modulate gene expression. Beyond immune cells, T-bet regulates ion channels, apoptosis, and cancer development in other cell types, including intestinal epithelial cells. The term “response element (TRE)” refers to a synthetic or engineered DNA regulatory sequence recognized by a tetracycline-controlled transactivator (e.g., tTA or rtTA) in a tetracycline- or doxycycline-dependent manner. TRE sequences typically consist of tandem tet operator (tetO) repeats upstream of a minimal promoter (such as CMV or TK minimal promoter) and are used to 22 USC0380PCT (2024-128-02) control inducible gene expression in Tet-On or Tet-Off regulatory systems. In the presence (or absence) of tetracycline or its analogs, the TRE can regulate the transcription of a downstream gene, such as a nucleic acid encoding T-bet. There is a Tet-On 3G system available with more restricted expression under TRE3G promoter. Abbreviation "AAV" means Adeno-Associated Virus. "Ab" means Antibody. "ANO1" means Anoctamin-1 (also known as TMEM16A). "AOM" means Azoxymethane. "AMP" means Ampicillin. "bGH" means Bovine Growth Hormone. "BGN" means Biglycan. "BM" means Bone Marrow. "CFTR" means Cystic Fibrosis Transmembrane Conductance Regulator. "CMV" means Cytomegalovirus. "cPPT" means Central Polypurine Tract. "CTS" means Central Termination Sequence. "CXCR3" means C-X-C Motif Chemokine Receptor 3. "CCR5" means C-C Motif Chemokine Receptor 5. 23 USC0380PCT (2024-128-02) "DC" means Dendritic Cell. "DMSO" means Dimethyl Sulfoxide. "DNA" means Deoxyribonucleic Acid. "Dox" means Doxycycline. "DRA" means Downregulated in Adenoma (also known as SLC26A3). "DSS" means Dextran Sodium Sulfate. "EAE" means Experimental Autoimmune Encephalomyelitis. "EDTA" means Ethylenediaminetetraacetic Acid. "EF1α" means Elongation Factor 1-Alpha. "EGFP" means Enhanced Green Fluorescent Protein. "FBS" means Fetal Bovine Serum. "FDA" means Food and Drug Administration. "GFP" means Green Fluorescent Protein. "H&E" means Hematoxylin and Eosin. "HSV" means Herpes Simplex Virus. "IBD" means Inflammatory Bowel Disease. "IEL" means Intraepithelial Lymphocyte. "IFN-γ" means Interferon-gamma. 24 USC0380PCT (2024-128-02) "IHC" means Immunohistochemistry. "IL" means Interleukin. "ILC" means Innate Lymphoid Cell. "IRES" means Internal Ribosome Entry Site. "LN" means Lymph Node. "LTR" means Long Terminal Repeat. "Lyz2" means Lysozyme 2. "MCS" means Multiple Cloning Site. "mCMV" means minimal Cytomegalovirus. "MTT" means 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide. "MUC1" means Mucin 1. "NHE3" means Sodium / Hydrogen Exchanger 3 (also known as SLC9A3). "NK" means Natural Killer. "NKT" means Natural Killer T. "NSG" means NOD scid gamma. "PBS" means Phosphate-Buffered Saline. "PCR" means Polymerase Chain Reaction. "PGK" means Phosphoglycerate Kinase. 25 USC0380PCT (2024-128-02) "PMA" means Phorbol-12-myristate 13-acetate. "PSA" means Prostate-Specific Antigen. "PVDF" means Polyvinylidene Difluoride. "qPCR" means quantitative Polymerase Chain Reaction. "RIPA" means Radioimmunoprecipitation Assay. "RNA" means Ribonucleic Acid. "rtTA" means reverse tetracycline-controlled transactivator. "SDS" means Sodium Dodecyl Sulfate. "SDS-PAGE" means Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. "SEM" means Standard Error of the Mean. "SGLT1" means Sodium-Glucose Linked Transporter 1 (also known as SLC5A1). "SLC" means Solute Carrier. "SLC5A1" means Solute Carrier Family 5 Member 1 (also known as SGLT1). "SLC9A3" means Solute Carrier Family 9 Member 3 (also known as NHE3). "SLC26A3" means Solute Carrier Family 26 Member 3 (also known as DRA). "SLC26A6" means Solute Carrier Family 26 Member 6. "SV40" means Simian Virus 40. "T-bet" means T-box expressed in T cells (also known as TBX21). 26 USC0380PCT (2024-128-02) "TBST" means Tris-Buffered Saline with Tween 20. "TBX21" means T-Box Transcription Factor 21 (also known as T-bet). "TGF-β" means Transforming Growth Factor-beta. "Th" means T helper. "TMEM16A" means Transmembrane Member 16A (also known as ANO1). "TRE" means tetracycline response element. "Treg" means regulatory T cell. "TUNEL" means Terminal deoxynucleotidyl transferase dUTP nick end labeling. "VSV" means Vesicular Stomatitis Virus. "VP16" means Viral Protein 16. "WPRE" means Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. "WT" means Wild Type. In at least one aspect, a therapeutic composition is designed for treating cancer. Referring to Figure 1a, the therapeutic composition includes at least one viral vector 10 engineered to deliver a nucleic acid sequence encoding T-bet 12 (T-box transcription factor TBX21), specifically targeting cancer cells originating from epithelial cells. In some refinements, the T-bet protein can be expressed from an inducible system or a non-inducible system as set forth below in more detail. Figure 1a depicts explicitly a lentiviral vector. The nucleic acid sequence encoding T- bet is operably linked to an inducible promoter 14 designed for precise, controlled expression. An expression cassette 18 includes a central polypurine tract (Flap) 20, inducible promoter 14 (TRE with TetO and minimal CMV promoter), T-bet coding sequence 12, and WPRE 22. The expression cassette 27 USC0380PCT (2024-128-02) 18 is flanked by long terminal repeats (LTRs) 26 and 28, which facilitating integration into the host genome. The central polypurine tract (Flap) 20 enhances nuclear import and is downstream of the 5′ LTR 26. The tetracycline response element (TRE) 14 (i.e., the inducible promoter) includes the tetracycline operator (TetO) 30 and minimal CMV promoter (mCMV) 32, enabling inducible transcription of the T-bet gene. T-bet expression is further enhanced by a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). In a refinement, for bacterial propagation, the plasmid optionally includes an ampicillin resistance gene (AMP) and bacterial origin of replication (Origin). These latter two elements are not need for a human therapeutic composition. SEQ ID NO: 1 provides an example of a polynucleotide encoding human TBX21 (SEQ ID NO: 2), and SEQ ID NO: 3 provides a polynucleotide encoding mouse TBX21 (Mus musculus, SEQ ID NO: 4). Sequences provides as mRNA are directly transcribable to DNA for incorporation in the vector. In particular, mRNA sequence is converted to its DNA equivalent by replacing uracil (U) with thymine (T), and this DNA is then inserted into the viral vector. It is noteworthy that the sequence listing already has T instead of U, so they correspond directly to the DNA sequences (see, SEQ ID Nos: 43-44). Thus, the viral vector can include polynucleotides sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3. In SEQ ID NO: 2, the DNA-binding domain of T-box transcription factor 21 and related T-box proteins is from positions 136 to 326. In a refinement, the viral vector delivers a polynucleotide encoding a polypeptide with a T-box domain, having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 (a consensus sequence between human and mouse). SEQ ID NO: 6 provides the amino acid sequence for the DNA-binding domain of T-box transcription factor 21 in humans. The box in Figure 1C identifies these domains. In a refinement, T-bet encodes a T-box transcription factor that includes a polypeptide having SEQ ID NO 6. In a refinement, T-bet encodes a T-box transcription factor that includes a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO 6. In some variation, these polypeptides retain the ability to bind a T-box recognition motif and / or activate interferon-gamma (IFN-γ) transcription in T cells. Several properties should be preserved for 28 USC0380PCT (2024-128-02) therapeutic applications using T-bet variants: DNA-binding domain functionality, transcriptional activation capacity, cell-type specificity, pro-apoptotic function (particularly valuable for cancer applications), and ion channel regulation abilities. The experiments set forth below demonstrate that T-bet expression in cancer cells can trigger tumor shrinkage, suggesting its potential delivery through engineered viral vectors. Still referring to Figure 1a, the vector employs a tetracycline-inducible TRE-minimal CMV promoter for precise temporal control of T-bet expression in cancer cells. This Tet-On system utilizes a reverse tetracycline-controlled transactivator (rtTA) that is constitutively expressed from a separate plasmid or a distinct cassette within the same viral vector. rtTA becomes activated upon binding doxycycline or another tetracycline analog, allowing it to bind the TRE upstream of the T-bet gene and recruit transcriptional machinery. Consequently, T-bet expression occurs exclusively in the presence of doxycycline, ensuring minimal background expression and tight temporal control, distinguishing it from Tet-Off systems activated by doxycycline removal. In another aspect, the tetracycline response element includes a plurality of repeats of Tet operator sequences (e.g., SEQ ID NO: 7 - TCCCTATCAGTGATAGAGA) with an optional intervening sequence (i.e., spacers) interposed between each Tet operator sequence. In a refinement, the tetracycline response element includes 1 to 15 repeats of the Tet operator sequence. Typically, the tetracycline response element includes 7 repeats of the Tet operator sequence. The intervening sequences can be 1 to 50 nucleotides in length. The spacers interposed between TetO sequences in the tetracycline response element are designed to optimize DNA spacing and orientation for efficient binding of tetracycline-controlled transactivators such as rtTA. These spacer sequences typically range from 4 to 30 base pairs in length and may include AT-rich or GC-rich regions to modulate DNA flexibility and accessibility. The spacers can be non-repetitive or degenerate in sequence to minimize homologous recombination and reduce epigenetic silencing. Functionally, they serve to enhance transcriptional responsiveness, reduce basal (leaky) expression in the absence of doxycycline, and support cooperative binding of multiple transactivator molecules. In some embodiments, the spacer 29 USC0380PCT (2024-128-02) sequences may resemble eukaryotic enhancer elements or include insulator-like features to further refine transcriptional control. In a refinement, the tetracycline response element includes a plurality of repeats of SEQ ID NO: 8 (TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAG), which includes the spacers. These can be repeated 1 to 15 times (preferably 7) as above. In a further refinement, the tetracycline response element includes a polynucleotide having SEQ ID NO: 9 (TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCA GTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTG AAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCAC TCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGA GAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGA G) Figure 1b is a plasmid map (SEQ ID NO: 10) depicting the structure of a 10,208 base pair lentiviral expression vector designated murine_Lenti Tet-On-T-bet. This plasmid is designed for tetracycline-inducible expression of the murine T-bet (Tbx21) transcription factor in mammalian cells. The plasmid incorporates several regulatory elements to enable high-level, tightly regulated gene expression and efficient packaging into lentiviral particles. The vector includes a CMV promoter and CMV enhancer for strong constitutive expression in mammalian cells, and a tetracycline response element (TRE) enabling inducible gene expression in Tet-On systems. The T-bet coding sequence is positioned downstream of these regulatory elements and is followed by a bovine growth hormone (bGH) polyadenylation signal to facilitate transcript stability and termination. To support lentiviral packaging and integration, the plasmid includes a Ψ (psi) packaging signal, a Rev response element, fragments of the HIV-1 gag and nef genes, and truncated 5’ and 3’ LTRs. A central polypurine tract / central termination sequence (cPPT / CTS) is included to enhance nuclear import of the viral genome following transduction. The plasmid also incorporates attB1 and attB2 recombination sites for Gateway® cloning, as well as multiple cloning site (MCS) fragments that provide flexibility for further genetic modification. The backbone includes a SV40 promoter and polyadenylation signal, an 30 USC0380PCT (2024-128-02) f1 origin of replication, and a colE1 origin (ori) for plasmid propagation in E. coli. Selection in bacterial systems is enabled by an ampicillin resistance gene (AmpR) and a bleomycin resistance gene (Ble), with associated regulatory elements including a lac operator and CAP binding site. Numerous restriction sites are annotated along the plasmid sequence, including but not limited to NdeI, SnaBI, EcoNI, AleI, BbvCI, XbaI, PmeI, AvrII, MluI, ScaI, and SgrDI, enabling straightforward cloning and linearization. The full-length vector is optimized for use in gene function studies, immunological modulation, and therapeutic delivery using lentiviral systems. As set forth above, the nucleic acid sequence encoding T-bet (T-box transcription factor TBX21) is operably linked to a promoter that facilitates expression in cancer cells. A useful promoter is the cytomegalovirus (CMV) immediate-early promoter, a strong, constitutive promoter commonly used in gene therapy applications due to its ability to drive high-level, sustained expression across a wide variety of mammalian cell types, including tumor cells. The CMV promoter is advantageous for ensuring the robust expression of T-bet in heterogeneous tumor microenvironments. However, other promoters may be used depending on the desired specificity, expression kinetics, or regulatory requirements. Examples include EF1α (elongation factor 1-alpha), PGK (phosphoglycerate kinase), and SV40 promoters for broad expression, as well as tumor- or tissue-specific promoters such as the telomerase reverse transcriptase (hTERT) promoter, the prostate-specific antigen (PSA) promoter, or the MUC1 promoter for more targeted delivery in specific cancer types. Inducible promoters, such as those responsive to tetracycline (Tet-On / Tet-Off systems), may also be employed to provide temporal control over T-bet expression. The choice of promoter can be optimized based on the target cancer type, the delivery system, and the desired therapeutic profile. In some aspects, the T-bet protein is expressed from a non-inducible system to ensure consistent and robust expression regardless of the immunologic status of the tumor microenvironment. For example, T-bet may be operably linked to a constitutive promoter such as the cytomegalovirus (CMV) immediate-early promoter, elongation factor 1-alpha (EF1α) promoter, phosphoglycerate kinase (PGK) promoter, or ubiquitin C (UbC) promoter. These promoters drive continuous expression in a broad range of cell types and enable T-bet production independent of interferon-gamma or other 31 USC0380PCT (2024-128-02) cytokine-mediated signals. Such constitutive expression may be advantageous in tumors that are poorly immunogenic or immunosuppressed, as it allows for early and sustained reprogramming of the tumor microenvironment toward a Th1-dominant immune response. In some embodiments, the expression cassette may further include elements such as an internal ribosomal entry site (IRES), 2A peptide sequence, or woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to facilitate co-expression of additional genes or to enhance transcript stability and translational efficiency. In refinements utilizing a retroviral or lentiviral delivery platform, the vector may include 5' and 3' Long Terminal Repeats (LTRs) that flank the expression cassette. These LTRs are critical for integration into the host genome and can also influence transcriptional regulation. In self-inactivating (SIN) lentiviral vectors, the 3' LTR is modified to reduce promoter activity after integration, enhancing safety. The internal expression cassette comprising the constitutive promoter (e.g., CMV), the T-bet coding sequence, and any posttranscriptional regulatory elements (e.g., WPRE) is typically inserted between these LTRs. The vector may also include a packaging signal (Ψ) for efficient encapsidation and, optionally, Rev Response Elements (RRE) or central polypurine tracts (cPPT) to enhance nuclear import and transgene expression. In another aspect, the therapeutic composition includes at least one viral vector. In a refinement, the at least one viral vector is an oncolytic virus. In some refinements, the viral vector can be a retroviral or lentiviral system. Examples of the viral vector include, but are not limited to, adenovirus, lentivirus, adeno-associated virus (AAV), herpes simplex virus (HSV), vaccinia virus, vesicular stomatitis virus (VSV), reovirus, myxoma virus, maraba virus, measles virus, Newcastle disease virus, picornavirus, reovirus, vesicular stomatitis virus, and combinations thereof. In some aspects, the oncolytic virus is a Newcastle Disease Virus (NDV) or an attenuated strain thereof. NDV is a paramyxovirus known for its ability to selectively replicate in tumor cells due to impaired interferon signaling in such cells, while exhibiting limited replication in non-transformed cells. Attenuated strains, such as lentogenic NDV isolates, are particularly suitable for therapeutic applications due to their reduced pathogenicity and retained tumor-selective replication properties. 32 USC0380PCT (2024-128-02) In certain aspects, the oncolytic virus is engineered to express one or more xenoantigens that are not endogenously present in humans. The expression of such xenoantigens on the surface of infected tumor cells is configured to promote a hyperacute rejection response in a host organism, characterized by complement activation, natural antibody binding, and rapid immune- mediated clearance of virus-infected cells. This immune activation mechanism enhances the overall antitumor effect. In some aspects, the xenoantigen is an α-galactosyl epitope. The α-galactosyl epitope is a carbohydrate structure not naturally expressed on human cells and is recognized by pre-existing anti-αGal antibodies in most humans. In certain configurations, the α-galactosyl epitope is produced by expression of an α1,3-galactosyltransferase gene that has been inserted into the genome of the oncolytic virus. The α1,3-galactosyltransferase enzyme catalyzes the transfer of galactose in an α1,3 linkage to glycoproteins or glycolipids on the surface of virus-infected cells, thereby generating α- galactosyl epitopes in situ. When the virus infects tumor cells, the α1,3-galactosyltransferase gene is expressed, resulting in cell surface display of αGal epitopes, which then elicit a hyperacute immune response through recognition by circulating anti-αGal antibodies, activation of complement, and immune cell recruitment. This engineered mechanism amplifies tumor-specific clearance while sparing non-infected normal cells, which do not express the αGal antigen or the α1,3- galactosyltransferase enzyme. In some aspects, the oncolytic virus is further engineered to express a transgene encoding one or more immune-activating proteins, including but not limited to a checkpoint inhibitor, a cytokine, or a T-cell engaging molecule. These immune-enhancing payloads are intended to boost local and systemic antitumor immunity when expressed in the tumor microenvironment. The combination of virus-mediated oncolysis and local immunostimulation is intended to induce more durable tumor regression and systemic immune memory. In another aspect, the transgene encodes one or more of: (a) a programmed death-ligand 1 (PD-L1) inhibitor, such as an anti-PD-L1 single-chain antibody fragment; (b) granulocyte- 33 USC0380PCT (2024-128-02) macrophage colony-stimulating factor (GM-CSF), a cytokine that recruits antigen-presenting cells to the tumor; or (c) a single-chain variable fragment (scFv) that binds CD3, to facilitate T-cell engagement. Expression of these immune payloads by the virus enhances T-cell activation, dendritic cell maturation, and immune checkpoint blockade within the tumor microenvironment. In certain aspects, the virus selectively induces tumor cell apoptosis via interferon- stimulated expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and Noxa. Upon infection, tumor cells exhibit heightened interferon responses that upregulate apoptotic mediators such as TRAIL and Noxa. These molecules engage both the extrinsic and intrinsic apoptosis pathways. Normal cells, which maintain tighter regulation of interferon signaling and apoptosis, remain largely unaffected. In addition to apoptosis, the virus may further induce at least one non-apoptotic cell death pathway, selected from autophagy, ferroptosis, necroptosis, or macropinocytosis in tumor cells. NDV has been shown to disrupt intracellular signaling pathways and redox homeostasis, leading to multiple regulated cell death responses depending on the tumor type and microenvironment. In some implementations, the virus exhibits restricted replication in non-tumor cells by virtue of one or more tumor-specific promoter elements or viral gene deletions. For example, promoters responsive to p53 dysfunction or Ras activation can confine gene expression to tumor cells. Alternatively, deletion of viral genes essential for replication in healthy cells—but dispensable in tumor cells—can further restrict tropism and enhance biosafety (Ring et al., Gene Therapy, 2001; the entire disclosure of which is hereby incorporated by reference). Advantageously, the oncolytic virus may be administered intravenously, intratumorally, or intraperitoneally in a therapeutically effective amount sufficient to induce tumor- selective viral replication, immune activation, and / or transgene expression. Dosing and route of administration may vary based on tumor location, immune status, and viral vector design. A therapeutically effective amount is defined as a dose that produces measurable tumor regression or immune response without systemic toxicity. 34 USC0380PCT (2024-128-02) In another aspect, the oncolytic virus is administered in combination with an immune checkpoint inhibitor, such as an anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody. This combination may potentiate systemic immune responses by relieving T cell inhibition while the virus primes or expands tumor-reactive T cells. Studies have demonstrated synergy between intratumoral OV administration and systemic checkpoint blockade in various models, including NDV with CTLA-4 blockade in poorly immunogenic melanoma. In another aspect, the oncolytic virus further comprises a nucleic acid encoding an immunostimulatory cytokine, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-12 (IL-12), or interleukin-15 (IL-15). Expression of these cytokines in the tumor microenvironment can enhance the recruitment, maturation, and activation of dendritic cells and cytotoxic T lymphocytes. For example, GM-CSF expression in T-VEC, a herpes-based oncolytic virus, was shown to contribute to enhanced intratumoral antigen presentation and systemic immunity. In another aspect, the oncolytic virus or a composition comprising the virus includes a STING agonist or a nucleic acid encoding a RIG-I ligand. These innate immune agonists activate pattern recognition receptors in phagocytic antigen-presenting cells, leading to type I interferon production and efficient cross-priming of tumor antigens. STING and RIG-I pathway activation is critical for overcoming immune suppression in the tumor microenvironment and for eliciting robust T cell responses. In another aspect, the virus is configured to induce tumor antigen-agnostic immunization by promoting in situ immunogenic cell death and presentation of endogenous tumor antigens. Upon infection, the virus elicits release of PAMPs and DAMPs, converting the tumor into a site of immune activation. As described by Russell and Barber, this leads to the generation of cytotoxic T lymphocytes reactive to patient-specific neoantigens without prior identification of the antigen repertoire, thereby functioning as an "antigen-agnostic vaccine.” In another aspect, the virus selectively replicates in tumor cells due to the presence of a tumor-specific microRNA response element (MRE) or via deletion of a viral gene required for 35 USC0380PCT (2024-128-02) replication in non-transformed cells. MREs targeting miRNAs that are abundant in normal cells but downregulated in tumors can be inserted into essential viral genes to restrict replication. Alternatively, deletion of viral genes that are complemented by tumor-specific mutations (e.g., p53 or Rb pathway defects) provides an additional layer of tumor selectivity. In another aspect, several strategic options for translating a Tet-On system into viral vector-based therapeutic applications can be implemented. A single vector system incorporates both the rtTA regulator and TRE-therapeutic gene components within one viral construct using different promoters, ensuring all transduced cells receive the complete system but potentially facing size limitations depending on the vector type. Alternatively, a dual vector approach utilizes two separate viral vectors - one delivering the rtTA component and another carrying the TRE-regulated therapeutic gene - offering flexibility in dosing and design but requiring successful co-transduction of the same target cells to function properly. In a refinement, this packaging strategy involves the first plasmid being encapsulated within a first viral vector, and the second plasmid within a second viral vector. For enhanced specificity, cell-type targeting can be achieved by placing the rtTA component under a tissue-specific promoter that activates only in desired cell populations, providing spatial control of gene expression without requiring the Cre-loxP system used in transgenic animal models. Each approach presents distinct advantages depending on the therapeutic application, target tissue, and safety considerations. In another aspect, the viral vector includes a first plasmid configured for the expression of T-bet (T-box transcription factor TBX21). This provides a more precise and controlled approach in cancer treatment, ensuring that the therapeutic genes are delivered efficiently and effectively. In a refinement, the viral vector includes a second plasmid configured for expression of a reverse tetracycline-controlled transactivator (rtTA). In such configurations, the first plasmid may include a promoter, a tetracycline response element (TRE) operably linked to the promoter, and a coding sequence for T-bet positioned downstream of the TRE, such that T-bet expression is inducible by doxycycline in the presence of rtTA. The TRE may include Tet operator (tetO) sequences that are responsive to rtTA. In related refinements, the promoter of the first plasmid may itself contain tetO 36 USC0380PCT (2024-128-02) sequences responsive to rtTA, with the T-bet coding sequence positioned downstream of this inducible promoter. These arrangements allows for the inducible expression of T-bet through doxycycline interaction with the TRE, presenting a targeted and controlled therapeutic strategy. In another aspect, the first plasmid may further comprise a plasmid backbone that includes an origin of replication and regulatory sequences ensuring replication and maintenance in the host cells of the viral vector. Similarly, the second plasmid for rtTA expression may include a constitutive promoter driving expression of rtTA, and in some embodiments, a coding sequence for a rtTA-VP16 fusion protein downstream of the constitutive promoter. The second plasmid may also include a plasmid backbone comprising an origin of replication and associated regulatory elements for stability and replication in host cells. In some implementations, the first and second plasmids are packaged separately, such that the first plasmid is carried by a first viral vector and the second plasmid is carried by a second viral vector, enabling modular or co-delivery strategies. Additionally, the therapeutic composition may further comprise a pharmaceutically acceptable carrier suitable for administration to a subject. In another aspect, the complexity and effectiveness of the therapeutic composition are enhanced by incorporating a second plasmid within the viral vector for the expression of a reverse tetracycline-controlled transactivator (rtTA). This system introduces a layer of control over the expression of therapeutic genes, offering a sophisticated means to regulate the treatment process. In another aspect, the first plasmid for T-bet expression is designed with a promoter that harbors Tet operator sequences (tetO) responsive to rtTA, alongside a coding sequence for the T- bet protein downstream of the promoter. This design ensures a regulated expression of T-bet, tailored to exert a therapeutic effect precisely when needed. In another aspect, the second plasmid within the therapeutic composition, dedicated to rtTA expression, includes a constitutive promoter and a coding sequence for rtTA. This configuration ensures the constant availability of rtTA, which is essential for the inducible expression system's functionality. 37 USC0380PCT (2024-128-02) In another aspect, the therapeutic composition is made suitable for clinical application by including a pharmaceutically acceptable carrier. This carrier facilitates the administration of the composition to subjects, ensuring the treatment is delivered effectively and safely. In another aspect, the therapeutic composition targets a range of cancers. In a refinement, the cancer cells are epithelial in origin. Examples of cancers that can be treated include, but are not limited to, colon cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, and melanoma. This broad applicability highlights the composition's potential to make a significant impact across various cancer types. In another aspect, the therapeutic composition is further diversified to include at least one oncolytic viral vector. This addition leverages the natural cancer-killing properties of certain viruses, thereby enhancing the treatment's effectiveness against cancer cells. In another aspect, an oncolytic virus—such as an adenovirus, herpesvirus (oHSV), vaccinia virus, or other tumor-selective replicating virus—is engineered to carry the T-bet (TBX21) gene under the control of a tetracycline response element (TRE). This design mirrors the function of the first plasmid described in the system, allowing T-bet to remain transcriptionally silent until induced. The virus is further engineered to selectively replicate in tumor cells, ensuring that both viral replication and eventual T-bet expression are localized to the tumor microenvironment. Separately, a second vector—either viral or non-viral—is delivered to provide constitutive expression of rtTA, the reverse tetracycline-controlled transactivator. This second vector may take the form of a non- replicating virus, a second oncolytic virus, or transfected cells, and serves as the regulatory component that activates TRE-driven transcription only when doxycycline is present. The oncolytic virus is administered systemically or locally to the tumor site, where it infects and replicates within tumor cells, delivering the TRE-controlled T-bet gene and simultaneously lysing cancer cells. Upon administration of doxycycline, rtTA binds to the TRE and induces T-bet expression, triggering a robust, localized Th1-type immune response that enhances antitumor immunity without widespread immune activation. This dual-action approach—combining direct oncolysis with spatially and 38 USC0380PCT (2024-128-02) temporally controlled immune stimulation—produces a synergistic therapeutic effect. Moreover, the dosing schedule of doxycycline can be adjusted to fine-tune the level and duration of T-bet expression, enhancing both safety and efficacy of the treatment. The use of a single vector offers several advantages over dual-vector systems. It simplifies manufacturing, regulatory compliance, and clinical delivery, as only one virus needs to be produced and administered. Moreover, it ensures that both genetic components—rtTA and the TRE- controlled transgene—are delivered to the same cell, increasing consistency and reducing variability in transgene expression. This is particularly advantageous in therapeutic settings, where reliable and localized immune activation is critical. Oncolytic viruses such as modified herpes simplex virus (HSV), vaccinia virus, or adenovirus are particularly suitable for this embodiment due to their large genome capacity, allowing the incorporation of both expression cassettes without compromising replication competency. The use of an oncolytic virus also enhances tumor targeting, as these viruses preferentially replicate in and lyse tumor cells, further concentrating T-bet expression and its immunomodulatory effects within the tumor microenvironment. By combining both cassettes into a single oncolytic vector, the invention enables inducible, spatially restricted, and temporally controlled expression of T-bet, facilitating safer and more effective cancer immunotherapy. In another aspect, a single oncolytic virus is engineered to carry both the reverse tetracycline-controlled transactivator (rtTA) and the T-bet (TBX21) gene under the control of a tetracycline response element (TRE) within the same viral genome. The vector includes two expression cassettes: a constitutive promoter, such as CMV or EF1α, driving continuous expression of rtTA, and a second cassette in which the TRE controls inducible expression of T-bet. This unified design ensures that every infected cell receives both the regulatory and effector components necessary for doxycycline-inducible expression. The oncolytic virus selectively infects and replicates within tumor cells, concentrating both viral lysis and T-bet-mediated immunomodulation within the tumor microenvironment. T-bet expression remains off until doxycycline is administered systemically, at which point rtTA binds the TRE and activates T-bet transcription. This induces a Th1-polarized immune response in the tumor site, enhancing antitumor activity without inducing systemic immune 39 USC0380PCT (2024-128-02) toxicity. By combining both expression systems into a single vector, this approach simplifies delivery, ensures co-expression, and improves the uniformity of response. Additionally, the use of an oncolytic virus enables direct tumor killing while simultaneously delivering an immune-activating payload that can be turned on or off with doxycycline, offering precise control over therapeutic timing and intensity for enhanced safety and efficacy. Several commercially available plasmids and kits support the Tet-On gene expression system and provide constitutive expression of the reverse tetracycline-controlled transactivator (rtTA). Addgene, a nonprofit plasmid repository widely used by academic and commercial researchers, offers a number of popular rtTA-expressing plasmids. These include pLVX-Tet-On Advanced, a lentiviral vector originally developed by Clontech (now Takara Bio), which expresses rtTA under the control of either the CMV or EF1α promoter; pLenti CMV rtTA3 Blast (Addgene #26429), a lentiviral plasmid expressing the rtTA3 variant from the CMV promoter and carrying a blasticidin resistance gene for selection; and pTet-On Advanced (Addgene #2010), which encodes an improved rtTA variant with reduced basal expression. Takara Bio, the developer of the original Tet-On and Tet-Off systems, offers additional commercial kits, including the Tet-On 3G System and pLVX-Tet-On 3G, a lentiviral backbone vector that drives rtTA3 expression from a CMV promoter. These vectors are commonly available in formats compatible with stable transduction, including lentiviral-ready constructs, and are widely used to enable doxycycline-inducible expression of target genes in mammalian cells. In addition to viral and oncolytic viral vector approaches, T-bet can be delivered to the tumor microenvironment through a variety of alternative modalities. These include direct injection of T-bet-encoding plasmid DNA or synthetic mRNA into the tumor site, enabling localized expression in tumor cells. mRNA constructs may be formulated with lipid nanoparticles or other delivery systems to enhance cellular uptake and transient protein expression. T-bet protein itself may also be delivered directly, optionally conjugated to cell-penetrating peptides or encapsulated within nanoparticles to facilitate intracellular delivery and nuclear localization. Another potential strategy involves the use of engineered microbial vectors, such as probiotic or tumor-colonizing bacterial strains, designed to selectively home to tumors and transfer T-bet via secretion systems or through direct bacterial uptake 40 USC0380PCT (2024-128-02) by tumor cells. These delivery routes offer modular, flexible platforms for achieving spatially targeted, cell-specific, and temporally controlled modulation of the tumor immune microenvironment via T-bet expression. In another aspect, a method for treating cancer involves administering an effective amount of the therapeutic composition set forth above to a subject in need. This administering results in the expression of T-bet within cancer cells, which induces apoptosis and alters the expression of ion channels and transporters, leading to cell death. This method signifies a strategic approach to combat cancer by directly targeting the disease's cellular mechanisms. Moreover, the method of administering the therapeutic composition is versatile, allowing for intravenous, intratumoral, or oral routes. This flexibility ensures that the treatment can be tailored to the specific needs and circumstances of the patient, enhancing the potential for successful outcomes. Advantageously, tumor regression is facilitated by a combination of T-bet-mediated apoptosis, tumor-specific viral replication, and immune activation through tumor antigen release. As set forth above, the cancer can be colon cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, or melanoma, and treatment results in inhibition of tumor growth or reduction of tumor volume. In another aspect, the viral vector is an oncolytic viral vector that selectively infects tumor cells, replicates, and facilitates tumor cell lysis in combination with T-bet expression to enhance therapeutic efficacy. As set forth above in more detail, T-bet expression is inducible and regulated by doxycycline administration through a Tet-On system. In another aspect, when the therapeutic method is applied to colon cancer, it results in the inhibition of tumor growth or the reduction of tumor volume. This specific outcome underscores the therapeutic composition's efficacy in not only treating but potentially reversing the progression of cancer, offering hope for those affected by this particular disease. 41 USC0380PCT (2024-128-02) In another aspect, the therapeutic composition comprising T-bet demonstrates remarkable cancer-specific cytotoxicity, wherein T-bet expression selectively induces apoptosis and cell death in multiple cancer cell types while showing minimal to no cytotoxic effects on non- cancerous cells. This selective targeting is evidenced by the unaffected viability of normal transformed cells following T-bet overexpression, in stark contrast to the significant cytotoxicity observed in cancer cells of various lineages. Furthermore, the composition demonstrates a predictable and reproducible timeframe of T-bet-induced cancer cell death, with initial morphological changes and decreased cell viability detectable within 24-48 hours after induction, progressive decline in cancer cell viability to approximately 30% by day 5, and further reduction to less than 10% viability by day 7 post-induction. This temporal pattern of T-bet-mediated cancer cell destruction provides a valuable therapeutic window that can be precisely controlled through the tetracycline-inducible system, allowing for optimized treatment protocols that maximize tumor-specific cytotoxicity while minimizing potential adverse effects on surrounding healthy tissues. The following examples illustrate the various embodiments of the present invention. Those skilled in the art will recognize many variations that are within the spirit of the present invention and scope of the claims. 1. Ion Channels and Transporters In Intestinal Epithelial Cells In the experiments set forth below, Tet-O-T-bet transgenic mice for doxycycline- inducible T-bet expression are generated to investigate the in vivo function of T-bet in the established immune system. Unexpectedly, the global T-bet overexpression causes severe diarrhea, intestinal tissue damage, and rapid mouse mortality in 2-4 days. Using transgenic mice with cell-type-specific T-bet induction, it was found that T-bet expression in CD4+ T cells or myeloid cells did not cause mouse death. Instead, T-bet expression in intestinal epithelial cells was responsible for the lethality. Mechanistically, it was showed that inducible T-bet expression in gut epithelial cells altered the expression of ion channels and transporters, which, in turn, triggered fluid secretion into the small intestine and leads to fatal dehydration in mice. Furthermore, ectopic T-bet expression directly induced 42 USC0380PCT (2024-128-02) the apoptosis of gut epithelial cells and was involved in tissue damage. Overall, our findings provide novel insight into the mechanism by which T-bet expression in gut epithelial cells causes lethal phenotype through altered expression of ion channels and transporters and induction of apoptosis, thus identifying the potential therapeutic target for the treatment of cancer and inflammatory diseases. RESULTS Global T-bet inducible expression causes severe diarrhea and mouse mortality T-bet is a key transcription factor in multiple types of immune cell populations, however, the function of T-bet in non-immune cells is rarely reported. To evaluate the expression levels of T-bet (encoded by TBX21) in normal human tissues, the “Body Atlas” analysis was conducted using BaseSpace Correlation Engine 2.0 from Illumina Inc. Among the 147 human tissue types analyzed from the database, T-bet expression was found to be high in the spleen, heart, and kidney; intermediate in the liver, lung, and digestive tissues; and low in the nervous system and glands (Fig. 9e). Within the immune system, T-bet was highly expressed in peripheral CD4 and CD8 T cells, but exhibited low expression in Treg cells and macrophages. (Fig.9e). To study the in vivo function of T-bet, the Tet-O-T-bet transgenic mouse strain was generated by incorporating the mouse Tbx21 coding sequence into a doxycycline-inducible expression system (Fig.9a). The Tet-O-T-bet mouse strain was then crossed with the R26-M2rtTA (global rtTA expression) strain, and double-positive Tet-O-T-bet:rtTA mice were generated for further experiments (Fig.9b). The inducible expression levels of T-bet in different tissues were evaluated in 4 mice at early generation, after doxycycline (Dox) treatment. Although the expression levels varied, T-bet was induced in all the tested organs and tissues from the transgenic mice, including lymph nodes (LNs), thymus, skin, spleen, lung, liver, and purified splenocytes (Fig.9a). Since T-bet is a key regulator in CD8+ and Th1 CD4+ T cells, the T cell populations in lymphoid organs from Tet-O-T-bet:rtTA mice, were evaluated and found that doxycycline-induced T-bet overexpression enhanced the differentiation of both CD4+ and CD8+ T cells in thymus and spleen (Fig. 9c). CD8+ T cell population was also increased in the LNs (Fig.9c). These observations are consistent with previous reports that the T-bet 43 USC0380PCT (2024-128-02) promotes the differentiation of both CD4+ and CD8+ T cells (Yeo and Fearon, 2011; Zhu et al., 2010). The inducible expression of T-bet was further evaluated by flow cytometry, and observed a dramatic increase of T-bet+ cells even after the first day of doxycycline treatment in gated CD4+ lymphocytes (Fig.9d). Next, it was observed the animal condition and unexpectedly found that Dox-treated Tet-O-T-bet:rtTA mice had significantly decreased body weights and died within 4 days (Figs.1a-b). On the contrary, Dox-treated wild-type (WT) mice and control water-treated Tet-O-T-bet:rtTA mice all survived with stable body weights (Figs. 1a-b). Furthermore, after doxycycline-induced overexpression of T-bet, severe diarrhea was observed in the Tet-O-T-bet:rtTA mice (Fig. 2c). The potentially dehydrated status of the mice was consistent with the decreased body weight after doxycycline treatment. The intestines were dissected for morphological analysis, and it was found that the intestines of the Dox-treated Tet-O-T-bet:rtTA mice were swollen and empty, without solid fecal materials but full of yellowish fluids, indicating diarrhea and gut inflammation (Fig.2d). Major organs and tissues were collected from control and T-bet-expressing mice for histopathological analyses. Hematoxylin and eosin (H&E) staining on tissue sections showed that the epithelial layer of the Dox- treated Tet-O-T-bet:rtTA mice was severely destroyed in the small intestine (Fig.2e), while most of the other major organs did not show any difference between control and T-bet-overexpressing mice (Fig.10b). The overexpression of T-bet was also evaluated by Immunohistochemistry (IHC) staining, and observed the successful induction of T-bet expression in the liver, spleen, lung, kidney, thymus, LNs, stomach, and intestine from Dox-treated Tet-O-T-bet:rtTA mice (Figs. 1f and 10c). Overall, these data suggest that the global expression of T-bet is lethal to mice, probably due to severe diarrhea and intestinal tissue damage. Inducible expression of T-bet in CD4+ T cells or myeloid lineage does not cause mouse mortality To identify which cell subset is responsible for T-bet-induced mouse mortality, we generated a cell-type specific T-bet expression system by crossing Tet-O-T-bet mice with the rtTA- 44 USC0380PCT (2024-128-02) EGFP strain in which the LoxP-flanked stop sequence was inserted in front of EGFP-tagged rtTA. After crossing with specific Cre mice, the stop sequence was removed, and rtTA was expressed in Cre-expressing cells to induce cell-specific T-bet expression (Fig.11a). We first generated the Tet-O-T-bet:rtTA-GFP:CD4-Cre mice (Fig. 11b), in which T- bet expression could be induced by doxycycline in CD4+ T cells. The inducible expression of T-bet in T cells, but not in B cells (as a negative control), was validated by flow cytometry and western blotting in sorted splenocytes from Tet-O-T-bet:rtTA-GFP:CD4-Cre mice (Figs. 11c-d). Next, we treated the Tet-O-T-bet:rtTA-GFP:CD4-Cre mice with doxycycline, and surprisingly found that these mice remained alive (Fig. 3a) and were all under a healthy condition (data not shown). The whole intestines were collected for morphological analysis, and we did not observe any differences between the control and T-bet overexpressed mice (Fig.3b). H&E staining was also performed on the intestine sections and other major tissue types, and we did not find any obvious damage in these organs (Figs. 2c and 11e). T-bet expression levels in different organs were also evaluated by IHC staining and western blotting. The expression of T-bet was highly induced in lymphoid organs (such as thymus, spleen, and LNs), and could also be detected in tissues with frequent T cell infiltration (such as lung and intestine), but not others (Figs.2c-d and 11e). Similarly, we generated the Tet-O-T-bet:rtTA-GFP:Lyz2-Cre mice (Fig. 12a) to specifically induce T-bet expression in myeloid lineages, such as macrophages and neutrophils. The Dox-induced expression of T-bet in CD11b+ myeloid population was validated in myeloid cell enriched bone marrow samples (Fig. 12b). After the treatment with doxycycline, the Tet-O-T- bet:rtTA-GFP:Lyz2-Cre mice still survived (Fig. 3e) and were under a healthy condition (data not shown). H&E staining further showed that there was no damage in the small intestine or other major organs from the Dox-treated Tet-O-T-bet:rtTA-GFP:Lyz2-Cre mice (Figs.2f and 12c). Tissue-specific T-bet overexpression in Dox-treated Tet-O-T-bet:rtTA-GFP:Lyz2-Cre mice was also evaluated by western blotting and IHC staining (Figs.2g and 12d). Together, these data rule out that the diarrhea and mortality observed in globally T-bet-expressing mice are due to the enhanced T-bet expression in CD4+ T cells or myeloid lineage. 45 USC0380PCT (2024-128-02) Immune cells are not responsible for mouse mortality induced by T-bet overexpression To determine whether immune cells or other cell types (such as epithelial cells) play the critical role in T-bet-induced mouse mortality, we performed bone marrow adoptive transfer and generated chimeric mice with altered immune cell genotypes. The chimeric mice were treated with doxycycline, and their body weights were recorded. We found that, regardless of the donor mouse’s genotype, the WT recipient mice all maintained healthy with no change in body weights after doxycycline treatment (Fig. 4a). In contrast, Tet-O-T-bet:rtTA recipient mice with either WT or T- bet+ immune cells showed significantly declined body weights and conditions compared to the WT recipients (Fig.4a). The morphology of the whole intestine also revealed severe gut inflammation in the Tet-O-T-bet:rtTA recipient mice, as they were swollen and lacked solid fecal materials (Fig.4b), consistent with the phenotypes observed in the Dox-treated Tet-O-T-bet:rtTA mice. Furthermore, we simultaneously depleted T cells and myeloid cells in Tet-O-T- bet:rtTA mice to determine whether the loss of these key T-bet-expressing immune cell populations may alter the mouse phenotype upon doxycycline treatment. We found that the simultaneous depletion of multiple immune cell types could not improve body weight loss and mouse survival in Dox-treated Tet-O-T-bet:rtTA mice (Figs.3c-d). Successful depletion of these immune cells was validated by flow cytometry analysis of the mouse blood samples (Fig. 4e). Taken together, these data suggest that immune cells are not the key reason for the diarrhea and mortality observed in Dox-treated Tet-O-T- bet:rtTA mice, and that other cell types (such as epithelial cells) may play an important role. Inducible expression of T-bet in gut epithelial cells causes mouse mortality Since the Tet-O-T-bet:rtTA mice had diarrhea, gut inflammation, and tissue damage in the small intestine, we therefore propose a hypothesis that the epithelial cells in the intestine may play an important role in T-bet-induced mouse mortality. Therefore, we crossed Tet-O-T-bet:rtTA-GFP mice with Villin-Cre mice, and generated the Tet-O-T-bet:rtTA-GFP:Villin-Cre strain (Fig. 13a) to specifically induce T-bet expression in gut epithelial cells. 46 USC0380PCT (2024-128-02) After treatment with doxycycline, the body weights of the Tet-O-T-bet:rtTA- GFP:Villin-Cre mice decreased significantly compared to the control group (Fig.5a). Similar to what we observed in Tet-O-T-bet:rtTA mice (Fig. 2b), the Tet-O-T-bet:rtTA-GFP:Villin-Cre mice died within 4 days after the induced expression of T-bet (Fig. 5b). Western blotting data validated the specific expression of T-bet in the intestine but not in other organs in the Tet-O-T-bet:rtTA- GFP:Villin-Cre mice (Fig.5c). Consistently, we observed morphological differences in the intestine between the control and Dox-treated Tet-O-T-bet:rtTA-GFP:Villin-Cre mice, which showed gut inflammation and signs of diarrhea after the induction of T-bet (Fig.5d). Notably, most of the intestinal damage was observed after day 2, and on the first day of doxycycline treatment, no significant damage was found in the intestine (Fig.13b). The H&E staining confirmed gut epithelial damage in the small intestine of Dox-treated Tet-O-T-bet:rtTA-GFP:Villin-Cre mice (Fig. 5e), in which the inducible expression of T-bet was further validated by IHC staining in all the small intestine sections and the colon (Fig.5e). As expected, we did not observe either T-bet expression or tissue abnormality in other major organs from Dox-treated Tet-O-T-bet:rtTA-GFP:Villin-Cre mice (Fig. 12c). Therefore, these data suggest that the specific T-bet overexpression in gut epithelial cells is the key event in inducing diarrhea and mortality in Dox-treated transgenic mice. T-bet regulates the expression of ion channels and transporters in mouse intestine The ion channels and transporters in the intestinal epithelial cells are key regulators in maintaining the water-electrolyte balance and acid / base homeostasis, thus controlling water secretion and absorption in the gut (Deng et al., 2021). Since we observed severe mouse diarrhea and dehydration after the induction of T-bet in gut epithelial cells, we further examined the according levels of ion channels and transporters. In all the tested candidates, cystic fibrosis transmembrane conductance regulator (CFTR) and Anoctamin-1 (ANO1, also known as Transmembrane member 16A, or TMEM16A) are the most important chloride channels that mediate chloride efflux, thus promoting water secretion from the cells and tissue (Das et al., 2018; Dulin, 2020). In contrast, SLC5A1 (or SGLT1), SLC9A3 (or NHE3), SLC26A3 (or DRA), and SLC26A6 are all ion transporters 47 USC0380PCT (2024-128-02) from the solute carriers (SLCs) family, which mediate the uptake of chloride and sodium ions, thus promoting water absorption into the cells (Das et al., 2018; Wang et al., 2020). We treated the Tet-O-T-bet:rtTA-GFP:Villin-Cre mice with control water or doxycycline water solution, and euthanized the mice on day 2 for RNA samples from intestinal tissues. With real-time qPCR, we found that the expression levels of Cftr and Ano1 were significantly increased in both the small intestine and colon tissues after the induction of T-bet expression (Figs. 5a-b). Meanwhile, the expression levels of Slc5a1, Slc9a3, Slc26a3, and Slc26a6 were all significantly decreased after doxycycline treatment (Figs.5a-b). Similar expression patterns were observed in Dox- treated Tet-O-T-bet:rtTA mice (Figs. 5c-d), consistent with their sensitive phenotype after T-bet inducible expression. On the other hand, the expression of these ion channels and transporters did not change in the WT control mice, with or without doxycycline treatment (Figs.14a-b). Besides the in vivo study, we also cultured the intestinal organoids, which are superior 3D in vitro models that better recapitulate tissue development and morphology, while retaining parental genomic and transcriptomic characteristics. The organoids were treated with different doses of doxycycline, and the induced T-bet expression was clearly observed at 1 μg / ml dosage in organoids from both Tet-O-T-bet:rtTA and Tet-O-T-bet:rtTA-GFP:Villin-Cre mice (Fig. 14c). We selected 1 μg / ml as the working dose, and observed increased Cftr and Ano1, but decreased Slc5a1, Slc9a3, Slc26a3, and Slc26a6, in Dox-treated Tet-O-T-bet:rtTA-GFP:Villin-Cre organoids (Fig. 14e), consistent with their expression levels in the in vivo study (Fig.6a). Similar expression patterns were observed in Dox-treated Tet-O-T-bet:rtTA organoids (Fig. 14f), although statistically significant changes occurred only in some of the candidates, probably because the T-bet expression in Tet-O-T- bet:rtTA organoids was not as high as it was in Tet-O-T-bet:rtTA-GFP:Villin-Cre organoids (Fig. 14c). As a negative control group, the WT organoids did not show any dramatic changes in the expression of ion channels and transporters after doxycycline treatment (Fig. 14d). Overall, the data from tissue organoid system confirmed our observations in the in vivo studies. 48 USC0380PCT (2024-128-02) Next, we aimed to investigate whether the therapeutic intervention of mouse diarrhea may ameliorate dehydration and survival in T-bet overexpressed mice. The Tet-O-T-bet:rtTA- GFP:Villin-Cre mice were orally treated with the anti-diarrhea drug Imodium (Loperamide hydrochloride), and then fed with doxycycline water solution for T-bet induction. We found that the anti-diarrhea drug could dramatically improve the health condition of Dox-treated mice, with a significantly milder body weight loss (Fig. 6e). The survival of Tet-O-T-bet:rtTA-GFP:Villin-Cre mice was also significantly extended in the Imodium-treated group, with 80% of the mice surviving on day 8 after doxycycline treatment (Fig. 6f). Taken together, these data suggest that T-bet overexpression in gut epithelial cells could alter the expression of ion channels and transporters and induce fatal diarrhea and dehydration, while the treatment of anti-diarrhea drugs could ameliorate the symptoms. Inducible T-bet expression promotes the apoptosis of intestinal epithelial cells. The intestinal epithelium is the most highly regenerative tissue in the human body and is self-renewed every 4-7 days from the Lgr5+ stem cells at the crypt base (Barker et al., 2007; Liu and Chen, 2020), allowing it to recover from constant damage during food breakdown, nutrient absorption, and waste elimination. The apoptosis of epithelial cells is a frequent event in the intestine and is essential for maintaining cellular balance (Ramachandran et al., 2000). Under pathological conditions, such as inflammation and infection, an aberrant increase in apoptotic cells in the gut is commonly observed, accompanied by extensive epithelial erosion (Patankar and Becker, 2020). When treating the Tet-O-T-bet:rtTA-GFP:Villin-Cre organoids with doxycycline, we surprisingly found that the cells were mostly dying after 2 days, and the 3D structure of the organoids was severely damaged after the induction of T-bet expression (Figs.6a and 15a). Similar results were observed in Dox-treated Tet-O-T-bet:rtTA organoids, but not WT organoids (Fig.15b), indicating that T-bet overexpression not only induces animal mortality but also triggers the death of gut epithelial cells in the in vitro model. We performed a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay on intestinal sections to detect apoptotic cells. Indeed, we observed an 49 USC0380PCT (2024-128-02) increased amount of dark-staining apoptotic cells in both the small intestines and colons of T-bet overexpressed mice (Fig. 7b). However, the number of apoptotic cells detected is still too small for such a severe phenotype. We hypothesize that apoptotic cells may have detached from the intestinal epithelia and thus cannot be detected in tissue sections. To overcome this technical issue, we cultured the small intestine pieces in vitro, added doxycycline to the medium for T-bet induction, and harvested all the cells in the plates. We found the caspase 3 / 7+ apoptotic cells were dramatically increased in Dox-treated intestinal tissues from Tet-O-T-bet:rtTA-GFP:Villin-Cre mice, but not WT control mice (Fig. 7c). The percentages of apoptotic cells at different time points were statistically analyzed, and we observed that T-bet overexpression could induce massive cell apoptosis in over 70% of intestinal epithelial cells in 2 days (Fig.7d). To determine whether the blockage of cell apoptosis may rescue the mortality of T-bet overexpressed mice, we injected a pan-caspase inhibitor Z-VAD-FMK into Dox-treated Tet-O-T- bet:rtTA-GFP:Villin-Cre mice. We found that the caspase inhibitor-treated mice had significantly extended survival and ameliorated body weight loss (Fig. 7e). About 20% of the mice survived for over 12 days, with fully recovered body weights, after the stop of Dox treatment on day 7 (Fig.7e). These data suggest that T-bet inducible expression triggers the apoptosis of intestinal epithelial cells, and the intervention of apoptotic pathways could partially but significantly protect mice from T-bet- induced mortality. Inducible T-bet expression completely inhibits colon tumor formation and growth After we found that T-bet promoted apoptosis in gut epithelial cells, we next investigated whether this mechanism could be utilized to control the growth and progression of epithelial-type cancers in the intestine, such as colon cancer. For this purpose, we generated Tet-O-T- bet-transduced CT-26 cells, in which T-bet overexpression could be induced by doxycycline treatment. Two clones were picked, and inducible T-bet expression was validated by western blotting (Fig. 16a). After doxycycline treatment, we found that the Tet-O-T-bet CT-26 cells died massively, with dramatic morphological changes (Fig. 8a), while WT cells and untreated control cells grew 50 USC0380PCT (2024-128-02) vigorously. From the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, we found that the in vitro growth rates of the untreated WT and Tet-O-T-bet CT-26 cells were comparable (Fig. 8b). However, after doxycycline treatment, the growth rates of the Tet-O-T-bet CT-26 clones significantly declined (Fig. 8b). Consistently, we observed the enhanced activation of caspase 3 in Tet-O-T-bet CT-26 cells after 1 day of doxycycline treatment (Fig.8c), thus proving the induction of cell apoptosis. Furthermore, we injected WT and Tet-O-T-bet CT-26 cells into BALB / c mice for in vivo tumor growth and found the two groups had similar levels of tumor growth before the doxycycline treatment (Fig.8d). After Dox treatment in both groups at day 7, we found that WT tumors continued to grow, while the sizes of T-bet-expressing tumors were decreased, with significantly smaller volumes than WT tumors at all following time points (Fig.8d). On day 15, tumors were harvested for size comparison between the two groups (Figs. 16b and 7e), and the T-bet-expressing tumors had significantly reduced weights compared to WT controls (Fig. 8f). Similar experiments were also performed in NSG mice, and consistent results were observed (Fig.8g). Notably, the Tet-O-T-bet CT- 26 tumors totally disappeared at the final time point (Fig.8g), so we showed only the growth curves in the figure. Besides CT-26 cells, we also investigated the role of T-bet in another colon cancer model, MC-38 cells. Consistent with the data in CT-26 cells, we found that inducible T-bet expression triggered cell apoptosis, with dramatic morphological changes, in two Tet-O-T-bet MC-38 clones (Fig. 16c). The cells were then harvested and stained for flow cytometry analysis, and the percentage of caspase 3 / 7+ cells was significantly increased in the Dox-treated Tet-O-T-bet MC-38 clones compared to WT cells (Figs.16d-e). Notably, the percentage of caspase 3 / 7+ cells tended to be higher in MC-38 clone #2, in which the T-bet expression was higher after Dox treatment (Fig.16f). Similar results were also observed in Tet-O-T-bet CT-26 cells, in which clone #2 had relatively higher T-bet expression and lower cell growth rate than clone #1 (Fig. 16a and 8b). Altogether, these data suggest that the ectopic expression of T-bet in colon cancer cells could induce apoptosis and tumor shrinkage, thus may be utilized as a novel approach for treating the patients with established tumors. 51 USC0380PCT (2024-128-02) Summary of the Experiments The experiments set forth above reveal a novel function of the T-bet transcription factor in intestinal epithelial cells. While T-bet has been implicated in inflammatory bowel disease (IBD), previous research focused only on its role in immune cells. The researchers discovered that inducible T-bet expression in intestinal epithelial cells causes severe diarrhea, tissue damage, and mortality in mice through two main mechanisms: (1) altered expression of ion channels and transporters (increased CFTR and ANO1; decreased SLC family transporters) leading to secretory diarrhea and dehydration, and (2) promotion of epithelial cell apoptosis. Treatment with anti-diarrhea medication (Imodium) or anti-apoptosis drugs (Z-VAD-FMK) significantly extended survival in affected mice. Importantly, induced T-bet expression in cancer cells triggered apoptosis and tumor shrinkage, suggesting potential therapeutic applications using oncolytic viruses to selectively express T-bet in tumors. This research provides new insights into T-bet's role in gut epithelial homeostasis and its potential as a cancer treatment. MATERIALS AND METHODS Animals and in vivo procedures Tet-O-T-bet mice were generated in our lab. Other strains were purchased from The Jackson Laboratory, including the wild-type (WT) C57BL / 6 control mice (Cat#: 000664), rtTA mice (whole body, Cat#: 006965), rtTA-EGFP mice (tissue specific, Cat#: 005572), Lyz2-Cre mice (Cat#: 004781), CD4-Cre mice (Cat#: 017336), and Villin-Cre mice (Cat#: 004586). Mice were crossed in our lab to generate different strains for experiments, including: Tet-O-T-bet:rtTA mice (global T-bet inducible expression), Tet-O-T-bet:rtTA-EGFP:CD4-Cre mice (CD4 T cell specific T-bet inducible expression), Tet-O-T-bet:rtTA-EGFP:Lyz2-Cre mice (myeloid cell specific T-bet inducible expression), and Tet-O-T-bet:rtTA-EGFP:Villin-Cre mice (gut epithelia specific T-bet inducible expression). For tumor studies, WT BALB / c mice (Cat#: 000651) and NSG mice (Cat#: 005557) were ordered from The Jackson Laboratory. In all experiments, 6 to 8 weeks old mice were used unless specifically described. All the mice were housed under a 12:12 light:dark cycle, and maintained in 52 USC0380PCT (2024-128-02) specific pathogen-free facilities that are accredited by Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Houston Methodist Research Institute (#IS00004400) and University of Southern California (#21097). Tet-O-T-bet transgenic mouse strain was generated by incorporating the mouse Tbx21 coding sequence into a doxycycline-inducible expression system (Fig. 9a). To construct the tet response element (TRE), 7 repeats of tetracycline operator sequence (7× tet) were fused to a minimal cytomegalovirus (CMV) promoter (mCMV) which lacks an enhancer sequence. The constructed DNA fragments, containing TRE-Tbx21 and homologous arm, were linearized, purified, and microscopically injected into the fertilized eggs and transferred into the pseudo-pregnant female mice for generating the Tet-O-T-bet transgenic pups. The expression of reverse tetracycline-controlled transactivator (rtTA) from either rtTA or rtTA-EGFP mouse strains was controlled by a constitutive or tissue-specific promoter, respectively. Without doxycycline (Dox), rtTA exhibits a reverse phenotype and cannot bind to the Tet-On sequences. In the presence of doxycycline, rtTA binds to the Tet-On sequences and recruits RNA polymerase II factors for the assembly of transcription complexes, initiating the transcription of T-bet gene. In mouse survival assay, mice were continuously treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose) through the drinking water. Body weight of each mouse was measured daily, and the survival was observed until the sensitive groups were all euthanized, while the resistant groups remained healthy. To generate bone marrow chimeric mice, recipient WT and Tet-O-T-bet:rtTA mice were γ-irradiated at 1000 rad (or 10 Gy), and intravenously injected with 5 million fresh bone marrow cells from donor mice after 24 hours. Recipient mice were treated with antibiotic water (0.5 mg / ml Baytril, Bayer) for two weeks starting from 2 days before irradiation. Regular drinking water was replaced to the mice after antibiotic water treatment. Thirty-five days after the bone marrow transfer, 53 USC0380PCT (2024-128-02) chimeric mice were treated with doxycycline water solution (1 mg / ml in 5% sucrose) for future experiments. For immune cell depletion, Tet-O-T-bet:rtTA mice were intraperitoneally (i.p.) injected with clodronate liposomes (150 μl / injection, C-020, Liposoma BV) on days -2 / 0 / +2, and a combination of Ly-6G antibody (Ab) (250 µg / injection, BE0075-1, BioXCell), CD4 Ab (300 µg / injection, TIB-207, ATCC), and CD8 Ab (300 µg / injection, TIB-210, ATCC) on days -1 / +1 / +3, to simultaneously deplete macrophages, neutrophils, and T cells. PBS-control liposomes and isotype- control Abs were used in the control group. For each injection, 200 µl volume was used (supplemented with PBS). On day 0, blood samples were collected to validate the depletion efficacy by flow cytometry. Mice were continuously treated with doxycycline water solution (1 mg / ml in 5% sucrose) from day 0. The body weights and survival curves were observed. For testing the changes in the ion channels and transporters, different groups of mice were treated with control water (5% sucrose alone) or doxycycline water solution (1 mg / ml in 5% sucrose) for 2 days. Mice were euthanized on day 2, and RNA samples were isolated from PBS- cleaned small intestine and colon tissues for real-time QPCR. For anti-diarrhea drug treatment, Tet-O-T-bet:rtTA-GFP:Villin-Cre mice were orally treated with PBS control or Imodium (125 µg Loperamide hydrochloride in 200 µl PBS) every day since day -1. Drinking water with doxycycline (1 mg / ml in 5% sucrose) was continuously treated since day 0. The body weights and survival curves were observed. For anti-apoptosis drug treatment, Tet-O-T-bet:rtTA-GFP:Villin-Cre mice were intraperitoneally (i.p.) injected with PBS control or pan-Caspase inhibitor (Z-VAD-FMK, Sigma- Aldrich, 50 µg in 200 µl PBS) every day from days 0 to 3. Doxycycline water solution (1 mg / ml in 5% sucrose) was treated from days 0 to 7. The body weights and survival curves were observed and analyzed. 54 USC0380PCT (2024-128-02) For in vivo tumor studies, WT and inducible T-bet expressing CT-26 cells (0.5 million cells in 50 µl PBS + 50 µl Matrigel) were subcutaneously (s.c.) injected into WT BALB / c or NSG mice. Doxycycline water solution (1 mg / ml in 5% sucrose) were continuously treated since day 7 in BALB / c mice, or day 5 in NSG mice. Tumor sizes were observed every 2-3 days. For BALB / c mice, tumors were dissected on day 15 to compare the tumor sizes between two groups. For NSG mice, the tumors in T-bet overexpression group disappeared after day 11, therefore, only the growth curves were showed. Histology and tissues staining Fresh organs and tissues were fixed with 3.7% formalin for 24 hours, and then sent to the histology laboratory at Baylor Breast Care Center or USC School of Pharmacy for further processing and H&E staining. Images were acquired using the Olympus BX61 microscope along with DP71 digital camera (Olympus). IHC staining was performed following previously published protocol (Xing et al., 2021). Briefly, unstained tissue sections were deparaffinized in xylene, rehydrated in graded ethanol solutions, and washed in tap water. Antigen retrieval was achieved by boiling the slides in a pressure cooker for 3 min in a citrated buffer (10 mM trisodium citrate, pH 6.0). After 10 min treatment with 3% H2O2, tissue sections were blocked with 5% normal goat serum in TBST for 1 hour at room temperature, incubated with primary antibodies at 4°C overnight, and with EnVision Polymer-HRP secondary antibodies (Dako) at room temperature for 30 min. After the application of DAB chromogen (Vector), tissue sections were stained with hematoxylin, dehydrated, and mounted. The antibodies used for IHC staining are listed in Table 1. TUNEL staining was performed on intestinal sections to indicate the apoptotic cells, using the TumorTACS In Situ Apoptosis Detection Kit (Cat#: 4815-30-K, R&D Systems), following the manufacturers’ instructions. Western blotting 55 USC0380PCT (2024-128-02) For both mouse tissues and cultured cells, RIPA buffer (150 mM NaCl, 50 mM Tris- HCl pH 7.4, 2 mM EDTA, 0.1% SDS, and 1% NP-40; supplemented with Roche protease inhibitor cocktail) was used for cell lysis and protein extraction. Tissues were homogenized in RIPA buffer on ice for thorough lysis. After shaking the samples at 4 °C for 30 min, tissue / cell debris was removed by centrifugation for 3 min at 12,000 g at 4 ℃ in a microcentrifuge. Lysate was mixed with 5X SDS loading buffer and heated for 5 min at 100 °C. Prepared samples were resolved by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad). The membrane was blocked for 1 h at room temperature using blocking buffer (5% nonfat dried milk in TBST). Blots were incubated with appropriate primary antibodies (diluted at 1:1000) and HRP-conjugated secondary antibodies (diluted at 1:3000). Protein expression was normalized to β-actin antibody (diluted at 1:3000). HRP was detected using chemiluminescent HRP substrate (Millipore). Digital images were acquired with the ChemiDoc XRS+ System and analyzed by Image Lab v.5.1 (Bio-Rad). The antibodies for Western blotting are listed in Table 1. Flow cytometry and sorting For surface marker staining, cells were directly stained in a cocktail of fluorochrome- conjugated antibodies for 30 min on ice, washed twice in 2% FBS / PBS, and resuspended in 2% FBS / PBS for flow cytometry. For intracellular staining, cells were activated with PMA (phorbol-12- myristate 13-acetate), Ionomycin, and protein transport inhibitor for 4 hours at 37°C. Then the cells were resuspended in Fix / Perm buffer (BD) and treated for 20 min on ice, washed with Perm / Wash buffer (BD), and stained with the antibody cocktail for 30 min on ice. After washing twice, cells were re-suspended in 2% FBS / PBS for flow cytometry. BD LSR II analyzer was used to distinguish different subsets. For the sorting of T and B cells, mouse splenocytes were purified from control water or doxycycline water solution treated Tet-O-T-bet:rtTA-GFP:CD4-Cre mice on day 2. Cells were stained with fluorochrome-conjugated antibodies for 10 min at room temperature. After washing twice with 2% FBS / RPMI-1640, cells were filtered with 40 µm strainer, and resuspended in 2% FBS / RPMI- 56 USC0380PCT (2024-128-02) 1640 for sorting. BD FACSAria II sorter was used to acquire the CD45+ / B220+ / CD3- B cells and CD45+ / B220- / CD3+ T cells. The antibodies for flow cytometry are listed in Table 1. Table 1. Antibodies used in this study Antibody Purpose Source Identifier AtiTb tIHC staining / WesternTh Fi h C t# 14582582.7 Mouse genotyping 57 USC0380PCT (2024-128-02) For genotyping, 0.5 cm length of mouse tail tip were collected for DNA purification. The tail tips were digested in 0.1 ml DNA digestion buffer (Viagen, #102-T) with Proteinase K (0.5 mg / ml final concentration), and incubated overnight at 56 ℃. The lysates were heated at 100 ℃ for 3 min to inactivate the Proteinase K, then directly used for genotyping PCR. Red Master Mix (2×, VWR) was used for genotyping PCR. The samples were run at “95℃ 30 seconds, 57℃ 30 seconds, 72℃ 1 minute” for 35 cycles. The primers for genotyping PCR are listed in Table 2. Real-time qPCR analysis Total RNA was isolated from mouse tissues or cultured cells by TRIzol Reagent (Invitrogen), and the first-strand cDNA was generated from total RNA using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific), following the manufacturers’ instructions. Real-time qPCR was performed using iTaq Universal SYBR Green Supermix (Bio- Rad) and specific primers on the Applied Biosystems QuantStudio 6 Flex Real-Time PCR system (Thermo Fisher Scientific). The results were analyzed using QuantStudio Software v.1.3 (Thermo Fisher Scientific). Relative expression of target genes was normalized with mouse Gapdh primers. The primers for real-time qPCR are listed in Table 2. Table 2. Primers used in this study Primer Purpose Sequence (5’-3’) IDDDDDO: USC0380PCT (2024-128-02) R: EGFP GenotypingTGAAGTCGATGCCCTTCAG (SEQ ID NO:17)QQQDDDO:O:O:O:DO:DDDDDD USC0380PCT (2024-128-02) F: Slc26a3 Real-time qPCRGCTTTAGCAGGTCCAGGGAA (SEQ IDNO: 37)DDDDQ Small intestine organoid culture Using previously reported method (Sato et al., 2009), small intestinal crypts were purified from untreated mice in different genotypes for the 3D culture of organoids. Briefly, small intestines were dissected, opened longitudinally, and cleaned with cold PBS. Villi were scraped with a hemacytometer coverslip, leaving only the crypts. The intestine tissues were cut into 5 mm pieces, washed with cold PBS for 10-20 times, and incubated in 2 mM EDTA in PBS for 30 minutes on ice. After the removal of EDTA solution, gut pieces were vigorously suspended in 10% FBS / PBS with a 10-ml pipette, and the supernatant was collected as one fraction. The wash steps were repeated, and more fractions were collected. Each fraction was filtered with a 70 μm strainer, centrifuged and resuspended for enrichment, and examined under microscope for the number and purity of crypts. Proper fractions consisting of essentially pure crypts were used for culture. Crypts were mixed with cold Matrigel (Cat#: 356234, Corning) and seeded in pre-cooled 24-well plates (100-1000 crypts in 50 μl Matrigel per well). After 5-10 minutes’ incubation at 37 °C, 500 μl IntestiCult™ mouse organoid growth medium (Cat#: 06005, Stemcell) was added into each well. The organoids were maintained at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was replaced every 2-3 days. Generation of Tet-O-T-bet cancer cells 60 USC0380PCT (2024-128-02) To generate doxycycline-inducible expression of T-bet (Tet-O-T-bet) in CT-26 and MC-38 cell lines, two types of lentiviral particles were produced by transfecting HEK293T cells with pl-TRE-T-bet, VSV-G, and ∆8.9; or pl-CMV-rtTA, VSV-G, and ∆8.9. The lentiviruses were further concentrated by ultra-centrifugation. Colon cancer cells were transduced by incubating with mixed lentivirus for 24 hours and then replaced with fresh medium. Three days after transduction, the cells were treated with doxycycline (1 µg / ml) for 24 hours, and T-bet expression was verified by western blotting. The T-bet-positive polyclonal cells were subsequently seeded into 96-well plates for monoclonal selection. The T-bet-positive monoclonal cells were picked and identified by western blotting. Monoclonal cells expressing high levels of T-bet were selected for further functional studies. Cell viability assay Using previously reported technique (Ma et al., 2020), the MTT assay was used to determine the in vitro growth rate of cancer cells with inducible T-bet expression. Briefly, cells were cultured in 96-well plates at 1000 cells / well and incubated at 37 °C with 5% CO2. MTT solution was prepared in PBS at 5 mg / ml. PBS control or 1 µg / ml doxycycline was added into the cells at hour 0. At each time points (12, 24, 36 hours), 20 μl MTT solution was added into the 200 μl cell culture medium. After 4-hour incubation at 37°C, the medium was discarded and 100 μl DMSO was added into each well. The 96-well plates were then covered with aluminum foil and shaken for 10 min (360 rpm) at room temperature. The absorbance was read at OD=590 nm on Biotek Synergy 2 microplate reader. Data were normalized with the value on hour 0 for the relative fold changes. Statistical analysis Descriptive statistics, including means, standard deviations, medians, and ranges, were computed for each group and analyzed with Student’s t-test or for multiple comparisons, with ANOVA. Data are presented as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM), as described in figure legends. Differences in mice survival were evaluated with Mantel-Cox log-rank test. The sample size for each experiment is included in the Results section and the associated 61 USC0380PCT (2024-128-02) figure legends. All analyses were performed with GraphPad Prism 5 (GraphPad Software, La Jolla, CA). P-values < 0.05 were considered significant. 2. T-bet-mediated tumor cell killing The transcription factor T-bet, encoded by TBX21 gene, is a key lineage-defining transcription factor primarily expressed in many different cell types of the innate and adaptive immune system. T-bet is highly homologous between human and mouse and plays an essential role in T helper 1 (TH1) cell differentiation, the development of CD8⁺ cytotoxic T lymphocytes, and the cytotoxic activity of NK cells, making it a central factor in TH1-mediated immune responses[1]. However, recent studies show that increased TBX21 expression has been linked to cancer development[2,3,4], yet its precise role in cancer cell growth, tumor regulation, and interactions with immune mechanisms remains unclear. Our recent study demonstrates that T-bet overexpression in colon cancer cells result in tumor cell death. We hypothesize that T-bet-mediated tumor cell death occurs in other types of cancer and T-bet may play a tumor suppressive role in cancer therapy. To address our hypothesis, we first engineered murine melanoma cell line B16 and murine breast cancer line EO771 with a lentiviral Tet-On system and generated stable cell lines with induciable expression of T-bet. Both wildtype and engineered tumor cells were treated with culture medium containing 1μg / mL of doxycycline for up to 4 days. We observed that either B16 or EO771 cells engineered with inducible T-bet expression showed visible slow cell growth, cell death or apoptosis. The T-bet-B16 cells (Fig. 17a) and T-bet-EO771 (Fig. 17b) cells appeared dramatic morphological changes respectively after doxycycline treatment, while wildtype (control) cells without T-bet engineering or those engineered cells without doxycycline treatment remained similar cell growth density and normal morphology, indicating the inducible overexpression of T-bet triggered significant cell apoptosis and death in these two types of tumor. Next, we generated single cell clones of the engineered EO771 and B16 cells by serial dilution for high level of T-bet expression. When each tumor cell clone was expaned, they were treated with 1μg / mL doxycycline for one day and the T-bet expression level was examined by intracellular 62 USC0380PCT (2024-128-02) staining with T-bet antibody and flow cytometry. Three T-bet-EO771 clones (#5, #8, and #9) and three T-bet-B16 clones (#4, #15, and #16) displayed nearly 100% of T-bet expression level respectively and were selected for further studies (Fig.18). With the selected tumor clones, we demonstrated that T-bet overexpression resulted in the inhibition of cell growth. MTT assay was applied to determine the cellular viability when T-bet- B16 clones were treated with or without 1μg / mL of doxycycline. As shown in Fig. 19, wildtype (control) B16 cells and the three T-bet-B16 clones without doxycycline treatment exhibited similar and rapid cell proliferation during 4 days. In contrast, the same tumor clones showed significantly and consistently suppressed cell proliferation after doxycycline treatment. Tet-On-T-bet transduced B16 single clones were treated with PBS or 1 μg / mL doxycycline, and cell proliferation was assessed using an MTT assay at various time points. Absorbance was measured at OD 590 nm and normalized to the hour 0 value for relative fold changes (mean ± SD). *p < 0.05; ** p< 0.01; *** p <0.001; ****p <0.0001.n=3. Another analysis of cell viability was conducted by flow cytometry after cells were stained with Propidium Iodide. Both wildtype and T-bet engineered EO771 cells were treated with 1μg / mL of doxycycline for seven days. As shown in Fig. 20, The viability of T-bet-EO771 cells decreased to 29.5% on day 5, 14.4% on day 6, and 9.0% on day 7 after doxycycline treatment. Meanwhile, wildtype (control) EO771 cells or T-bet-EO771 cells without doxycycline treatment maintained similar cellular viability of over 85% up to day 6 and showed a slight decline on day 7, potentially due to the saturated cell density. These results further confirmed the T-bet induced cell death in multiple tumor types. Besides murine cell lines, we also engineered human breast cancer line MDA-MB-231 with inducible overexpression of T-bet to validate its role in the human cells. Similarly, inducible overexpression of T-bet led to dramatic morphological changes in engineered MDA-MB-231 cells, compared to wildtype (control) cells and the engineered cells without doxycycline treatment (Fig.21). 63 USC0380PCT (2024-128-02) Similar MTT assay and flow cytometry analysis as murine cells were performed simultaneously to evaluate the cellular viability of T-bet engineered MDA-MB-231 cells and wildtype (control) cells during treatment with 1 μg / mL of doxycycline. The expected cell death induced by overexpression of T-bet in human cells was observed. As shown in Fig. 22, the suppression of cell growth was only observed in T-bet engineered MDA-MB-231 cells after doxycycline treatment, while its counterparts and control cells maintained normal cell growth. Meanwhile, Propidium Iodide staining showed a continuous decrease in cell viability of T-bet engineered cells after doxycycline treatment (Fig. 23a). Based on the flow cytometry on day 5, the doxycycline treated and T-bet engineered cells showed a dramatic increase in apoptosis, with 75.4% of cells undergoing cell death (Fig.23b). These results suggested that the inducible overexpression of T-bet was triggering apoptosis in MDA-MB-231 cells. To determine whether T-bet overexpression induces normal cell death, we selected 293T cell line (a normal and transformed cell line), and did not observe cell death after T-bet expression (Fig.24a, 24b), suggesting that T-bet overexpression specifically induces tumor cell death and destruction. Altogether, these data suggest that the ectopic expression of T-bet in melanoma cancer cells, breast cancer cells, and colorectal carcinoma cells[5]could induce apoptosis and tumor regression. These findings highlight T-bet as a potential therapeutic target for cancer treatment and inflammatory disease management. 3. In vivo function data of T-bet in mouse tumor models Test in vivo function of T-bet-mediated tumor inhibition and destruction, we transduced murine melanoma B16 and breast cancer EO771 cells with Tet-O-T-bet lentiviral particles. We then subcutaneously inject 0.1 x 106B16 / Tet-O-T-bet cells and 0.5 x 106EO771 / Tet-O-T-bet cells into C57BL / 6 mice, respectively. Tumor-bearing mice were treated with control water (5% sucrose) or water containing 1 mg / ml in 5% sucrose. Tumor growth was monitored every 2-3 days. We found that T-bet expression in the Dox treated groups completely inhibited tumor growth of B16 / Tet-O-T- 64 USC0380PCT (2024-128-02) bet cells (Fig. 25a) and EO771 / Tet-O-T-bet cells (Fig. 25B). By contrast, in water-treated control groups, both B16 / Tet-O-T-bet cells (Fig.25A) and EO771 / Tet-O-T-bet cells (Fig.25B) grew rapidly. These results suggest that T-bet overexpression induces tumor cell death and growth inhibition. 4. Examples for Oncolytic viral vector applications. Example 1: Cloning T-bet into a Newcastle Disease Virus (NDV) Vector A cDNA encoding human T-bet (TBX21) will be synthesized with mammalian codon optimization and flanked by NDV-specific gene start (GS) and gene end (GE) sequences. The gene cassette will be inserted into a full-length infectious clone of an attenuated lentogenic NDV strain (e.g., LaSota) using reverse genetics techniques. The T-bet transgene will be inserted between the P and M genes to preserve optimal gene expression hierarchy. The recombinant plasmid will be transfected into Vero cells along with support plasmids encoding the NDV NP, P, and L proteins under T7 polymerase control. After 72 hours, cytopathic effect will be monitored and supernatants will be harvested for passage into embryonated chicken eggs to amplify recombinant virus stocks. Expression of T-bet protein will be confirmed by Western blot, qRT-PCR, and immunofluorescence in infected tumor cell lines (e.g., A549, B16). Viral titers and replication kinetics will be compared to wild-type NDV to assess whether T-bet expression impacts viral fitness. Example 2: Engineering an HSV Vector Expressing T-bet A recombinant herpes simplex virus (HSV-1) oncolytic backbone (e.g., Δγ34.5 and ΔICP47) will be modified to express human T-bet under the control of the CMV promoter. The transgene cassette will be inserted into the ICP6 locus, which is dispensable for replication in tumor cells. 65 USC0380PCT (2024-128-02) Recombinant viruses will be generated using BAC recombineering or homologous recombination in complementing Vero cells. After plaque purification, viral clones will be screened for correct insertion using PCR, Southern blotting, and sequencing. T-bet expression in HSV-infected tumor cells (e.g., CT26, Panc02) will be verified by Western blotting, and localization will be confirmed by confocal microscopy to ensure nuclear translocation. Cytokine profiling will be performed to assess IFN-γ production and downstream immune signaling activation. Example 3: T-bet Expression Validation in Tumor Cells Infected with Recombinant Virus Recombinant NDV-T-bet virus will be used to infect various human and mouse tumor cell lines (e.g., A549, MC38, B16) at a multiplicity of infection (MOI) of 0.5–5.0. At 24–48 hours post-infection, T-bet expression will be measured by qPCR and immunoblotting. RNA-seq or NanoString profiling will be performed on infected cells to assess upregulation of canonical T-bet–regulated genes, including IFNG, CXCL9, CXCL10, and TBX21. Functional assays will include ELISA for IFN-γ and flow cytometry to detect MHC class I / II upregulation. These findings will confirm that T-bet expression from the viral vector is biologically active in infected tumor cells. Example 4: Combination Therapy with Immune Checkpoint Inhibitor (Claim 11) An oncolytic Newcastle Disease Virus (NDV) vector expressing human T-bet will be administered intratumorally in a C57BL / 6 mouse model bearing subcutaneous B16-F10 melanoma tumors. Mice will receive concurrent systemic administration of an anti-CTLA-4 antibody at 100 μg per dose on days 1, 4, and 7 post-virus injection. It is anticipated that tumors will exhibit increased infiltration of CD8+ T cells and IFN-γ+ CD4+ T cells, particularly in virus-injected lesions. Example 5: Virus Encoding GM-CSF and T-bet (Claim 12) 66 USC0380PCT (2024-128-02) An oncolytic virus vector will be engineered to co-express T-bet and granulocyte- macrophage colony-stimulating factor (GM-CSF). Following intratumoral injection into CT26 colon carcinoma-bearing mice, it is expected that tumor-draining lymph nodes will show increased activation of antigen-presenting cells, and the frequency of IFN-γ–producing CD8+ T cells will be enhanced. Example 6: STING Agonist Formulation (Claim 13) A recombinant T-bet–expressing NDV vector will be co-administered intratumorally with a STING agonist (e.g., cGAMP) in BALB / c mice with established tumors. It is anticipated that co-treatment will enhance IFN-β production and upregulate CXCL10 and MHC class I expression, leading to increased T cell infiltration and tumor regression. Example 7: Antigen-Agnostic In Situ Vaccination (Claim 14) An NDV-based vector expressing T-bet will be injected into pancreatic tumors in immunocompetent mice. Splenic T cells harvested two weeks post-treatment will respond to autologous tumor lysate but not to irrelevant cell lines. This is expected to demonstrate in situ priming of tumor-specific T cells without prior antigen selection. Example 8: Tumor-Specific Replication via miR-122 Response Element (Claim 15) A T-bet–expressing oncolytic virus will be engineered to include a microRNA-122 (miR-122) response element within the viral polymerase gene. Virus replication will be restricted in miR-122–high normal liver cells but will proceed in miR-122–low tumor cells. Selective replication and tumor lysis without hepatotoxicity is expected. Example 9: α-Gal Xenoantigen for Hyperacute Rejection (Claims 3–4) 67 USC0380PCT (2024-128-02) An NDV-based oncolytic virus will be engineered to express an α1,3- galactosyltransferase gene, resulting in the production of α-galactosyl epitopes on the surface of infected tumor cells. Upon intratumoral injection in mice, pre-existing natural anti-αGal antibodies will bind the epitopes, triggering complement activation and immune-mediated tumor destruction. Example 10: Induction of TRAIL and Noxa (Claim 7) A T-bet–expressing NDV vector will be used to infect A549 tumor cells in vitro and in vivo. The virus is expected to upregulate expression of TRAIL and Noxa in tumor cells via type I interferon signaling. Apoptosis will be confirmed via caspase activation assays and qPCR for TRAIL and Noxa transcripts. Example 11: Ferroptosis and Autophagy Induction (Claim 8) Tumor-bearing mice will be treated with NDV expressing T-bet. It is anticipated that tumor cells will undergo ferroptosis and autophagy as evidenced by lipid ROS accumulation, LC3B staining, and partial reversal with ferrostatin or autophagy inhibitors. Example 12: Tumor-Specific Promoter or Viral Gene Deletion (Claim 9) A modified T-bet NDV will be engineered with a tumor-specific promoter driving an essential viral gene, or a deletion of a viral replication gene that is rescued in p53-deficient tumor cells. The virus will replicate in tumor xenografts but not in matched normal tissue, demonstrating tumor- restricted replication. Example 13: Routes of Administration (Claim 10) The T-bet–expressing oncolytic virus will be administered intravenously, intratumorally, or intraperitoneally in mice bearing orthotopic and subcutaneous tumors. All delivery routes are expected to result in tumor infection and regression, with the most robust effects seen with intratumoral and intravenous delivery. 68 USC0380PCT (2024-128-02) Example 14: OV Expressing T-bet for the Treatment of Pancreatic Cancer A recombinant oncolytic Newcastle Disease Virus (NDV), engineered to express a human T-bet transgene under the control of a viral early promoter, will be administered intratumorally to immunocompetent C57BL / 6 mice bearing orthotopically implanted Panc02 pancreatic tumors. The virus will be designed to selectively replicate in tumor cells and to induce immunogenic cell death. Mice will receive intratumoral injections of NDV-T-bet (1 × 10⁷ PFU / dose) on days 7, 10, and 13 following tumor implantation. Control groups will receive either the parental NDV lacking the T-bet transgene or PBS. Treatment with NDV-T-bet is expected to result in a significant reduction in tumor volume relative to both control groups, as assessed by bioluminescence imaging and caliper measurements. Tumors harvested on day 15 will be evaluated by flow cytometry, and the NDV-T-bet group is expected to exhibit enhanced infiltration of CD8⁺ T cells co-expressing IFNγ and granzyme B, along with a reduced proportion of regulatory T cells (CD4⁺CD25⁺FoxP3⁺). Macrophage polarization toward the M1 phenotype is also expected to be observed, as indicated by increased iNOS and decreased arginase-1 expression. Rechallenge of cured mice with Panc02 cells injected contralaterally on day 30 will demonstrate protection from tumor growth, supporting the establishment of systemic antitumor immunity. Example 15: OV Encoding a Tumor Neoantigen-Xenoantigen Fusion for Triple- Negative Breast Cancer An NDV-based oncolytic virus encoding a fusion construct comprising a tumor neoantigen (mutant p53 peptide) and a xenoantigen (porcine α-Gal epitope), separated by a P2A self- cleaving sequence, will be administered systemically to immunocompetent Balb / c mice bearing orthotopic 4T1 triple-negative breast tumors that express the corresponding mutant p53. 69 USC0380PCT (2024-128-02) Mice will receive intravenous injections of NDV-p53-xeno at a dose of 1 × 10⁸ PFU on days 5, 8, and 12 after tumor implantation. Comparative groups will include mice treated with NDV encoding p53 alone, NDV encoding α-Gal alone, and wild-type NDV. Mice treated with NDV-p53-xeno are expected to exhibit a marked reduction in primary tumor volume (>70%) by day 18, and a near-complete abrogation of spontaneous lung metastases by day 25, as determined by histopathological analysis. Immunohistochemical staining will show robust infiltration of CD4⁺ T-bet⁺ helper T cells and CD8⁺ cytotoxic T lymphocytes expressing IFNγ, as well as a decrease in tumor-associated macrophages expressing arginase-1. Bulk RNA sequencing of tumor tissue will reveal an upregulation of genes involved in antigen presentation (H2-K1, Tap1, Tapbp), Th1 chemokine signaling (CXCL9, CXCL10), and type I interferon-stimulated genes (Ifit1, Mx1), consistent with activation of innate and adaptive antitumor responses. Treated animals will not exhibit significant weight loss or other signs of systemic toxicity. Additional details of the invention are found in Chen L, Yi H, Li Q, Duan T, Liu X, Li L, Wang HY, Xing C, Wang RF. T-bet Regulates Ion Channels and Transporters and Induces Apoptosis in Intestinal Epithelial Cells. Adv Sci (Weinh). 2024 Jul;11(28):e2401654. doi: 10.1002 / advs.202401654. Epub 2024 Apr 22. 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Nature Reviews Drug Discovery, 14(9), 642–662. https: / / doi.org / 10.1038 / nrd4663 Sequences 1 to 10 >SEQ ID NO: 1 (NM_013351.2:220-1827 Homo sapiens T-box transcription factor 21 (TBX21), mRNA) ATGGGCATCGTGGAGCCGGGTTGCGGAGACATGCTGACGGGCACCGAGC CGATGCCGGGGAGCGACGAGGGCCGGGCGCCTGGCGCCGACCCGCAGCACCGCTACTT CTACCCGGAGCCGGGCGCGCAGGACGCGGACGAGCGTCGCGGGGGCGGCAGCCTGGG GTCTCCCTACCCGGGGGGCGCCTTGGTGCCCGCCCCGCCGAGCCGCTTCCTTGGAGCCT ACGCCTACCCGCCGCGACCCCAGGCGGCCGGCTTCCCCGGCGCGGGCGAGTCCTTCCCG CCGCCCGCGGACGCCGAGGGCTACCAGCCGGGCGAGGGCTACGCCGCCCCGGACCCGC GCGCCGGGCTCTACCCGGGGCCGCGTGAGGACTACGCGCTACCCGCGGGACTGGAGGT GTCGGGGAAACTGAGGGTCGCGCTCAACAACCACCTGTTGTGGTCCAAGTTTAATCAGC ACCAGACAGAGATGATCATCACCAAGCAGGGACGGCGGATGTTCCCATTCCTGTCATTT ACTGTGGCCGGGCTGGAGCCCACCAGCCACTACAGGATGTTTGTGGACGTGGTCTTGGT GGACCAGCACCACTGGCGGTACCAGAGCGGCAAGTGGGTGCAGTGTGGAAAGGCCGAG GGCAGCATGCCAGGAAACCGCCTGTACGTCCACCCGGACTCCCCCAACACAGGAGCGC USC0380PCT (2024-128-02) ACTGGATGCGCCAGGAAGTTTCATTTGGGAAACTAAAGCTCACAAACAACAAGGGGGC GTCCAACAATGTGACCCAGATGATTGTGCTCCAGTCCCTCCATAAGTACCAGCCCCGGC TGCATATCGTTGAGGTGAACGACGGAGAGCCAGAGGCAGCCTGCAACGCTTCCAACAC GCATATCTTTACTTTCCAAGAAACCCAGTTCATTGCCGTGACTGCCTACCAGAATGCCG AGATTACTCAGCTGAAAATTGATAATAACCCCTTTGCCAAAGGATTCCGGGAGAACTTT GAGTCCATGTACACATCTGTTGACACCAGCATCCCCTCCCCGCCTGGACCCAACTGTCA ATTCCTTGGGGGAGATCACTACTCTCCTCTCCTACCCAACCAGTATCCTGTTCCCAGCCG CTTCTACCCCGACCTTCCTGGCCAGGCGAAGGATGTGGTTCCCCAGGCTTACTGGCTGG GGGCCCCCCGGGACCACAGCTATGAGGCTGAGTTTCGAGCAGTCAGCATGAAGCCTGC ATTCTTGCCCTCTGCCCCTGGGCCCACCATGTCCTACTACCGAGGCCAGGAGGTCCTGG CACCTGGAGCTGGCTGGCCTGTGGCACCCCAGTACCCTCCCAAGATGGGCCCGGCCAGC TGGTTCCGCCCTATGCGGACTCTGCCCATGGAACCCGGCCCTGGAGGCTCAGAGGGACG GGGACCAGAGGACCAGGGTCCCCCCTTGGTGTGGACTGAGATTGCCCCCATCCGGCCG GAATCCAGTGATTCAGGACTGGGCGAAGGAGACTCTAAGAGGAGGCGCGTGTCCCCCT ATCCTTCCAGTGGTGACAGCTCCTCCCCTGCTGGGGCCCCTTCTCCTTTTGATAAGGAAG CTGAAGGACAGTTTTATAACTATTTTCCCAACTGA > SEQ ID NO: 2 (NP_037483.1 T-Bet human) MGIVEPGCGDMLTGTEPMPGSDEGRAPGADPQHRYFYPEPGAQDADERRGG GSLGSPYPGGALVPAPPSRFLGAYAYPPRPQAAGFPGAGESFPPPADAEGYQPGEGYAAPDP RAGLYPGPREDYALPAGLEVSGKLRVALNNHLLWSKFNQHQTEMIITKQGRRMFPFLSFTV AGLEPTSHYRMFVDVVLVDQHHWRYQSGKWVQCGKAEGSMPGNRLYVHPDSPNTGAHW MRQEVSFGKLKLTNNKGASNNVTQMIVLQSLHKYQPRLHIVEVNDGEPEAACNASNTHIFT FQETQFIAVTAYQNAEITQLKIDNNPFAKGFRENFESMYTSVDTSIPSPPGPNCQFLGGDHYS PLLPNQYPVPSRFYPDLPGQAKDVVPQAYWLGAPRDHSYEAEFRAVSMKPAFLPSAPGPTM SYYRGQEVLAPGAGWPVAPQYPPKMGPASWFRPMRTLPMEPGPGGSEGRGPEDQGPPLV WTEIAPIRPESSDSGLGEGDSKRRRVSPYPSSGDSSSPAGAPSPFDKEAEGQFYNYFPN 81 USC0380PCT (2024-128-02) > SEQ ID NO: 3 (NM_019507.2:195-1787 Mus musculus T-box 21 (Tbx21), mRNA) ATGGGCATCGTGGAGCCGGGCTGCGGAGACATGCTGACCGGCACCGAGC CGATGCCGAGTGACGAGGGCCGGGGGCCCGGAGCGGACCAACAGCATCGTTTCTTCTA TCCCGAGCCGGGCGCACAGGACCCGACCGATCGCCGCGCAGGTAGCAGCCTGGGGACG CCCTACTCTGGGGGCGCCCTGGTGCCTGCCGCGCCGGGTCGCTTCCTTGGATCCTTCGCC TACCCGCCCCGGGCTCAGGTGGCTGGCTTTCCCGGGCCTGGCGAGTTCTTCCCGCCGCC CGCGGGTGCGGAGGGCTACCCGCCCGTGGATGGCTACCCTGCCCCTGACCCGCGCGCG GGGCTCTACCCAGGGCCGCGCGAGGACTACGCATTGCCCGCGGGGTTGGAGGTGTCTG GGAAGCTGAGAGTCGCGCTCAGCAACCACCTGTTGTGGTCCAAGTTCAACCAGCACCA GACAGAGATGATCATCACTAAGCAAGGACGGCGAATGTTCCCATTCCTGTCCTTCACCG TGGCCGGGCTGGAGCCCACAAGCCATTACAGGATGTTTGTGGATGTGGTCTTGGTGGAC CAGCACCACTGGCGGTACCAGAGCGGCAAGTGGGTGCAGTGTGGAAAGGCAGAAGGC AGCATGCCAGGGAACCGCTTATATGTCCACCCAGACTCCCCCAACACCGGAGCCCACTG GATGCGCCAGGAAGTTTCATTTGGGAAGCTAAAGCTCACCAACAACAAGGGGGCTTCC AACAATGTGACCCAGATGATCGTCCTGCAGTCTCTCCACAAGTACCAGCCCCGGCTGCA CATCGTGGAGGTGAATGATGGAGAGCCAGAGGCTGCCTGCAGTGCTTCTAACACACAC GTCTTTACTTTCCAAGAGACCCAGTTCATTGCAGTGACTGCCTACCAGAACGCAGAGAT CACTCAGCTGAAAATCGACAACAACCCCTTTGCCAAAGGATTCCGGGAGAACTTTGAGT CCATGTACGCATCTGTTGATACGAGTGTCCCCTCGCCACCTGGACCCAACTGTCAACTG CTTGGGGGAGACCCCTTCTCACCTCTTCTATCCAACCAGTATCCTGTTCCCAGCCGTTTC TACCCCGACCTTCCAGGCCAGCCCAAGGATATGATCTCACAGCCTTACTGGCTGGGGAC ACCTCGGGAACACAGTTATGAAGCGGAGTTCCGAGCTGTGAGCATGAAGCCCACACTC CTACCCTCTGCCCCGGGGCCCACTGTGCCCTACTACCGGGGCCAAGACGTCCTGGCGCC TGGAGCTGGTTGGCCCGTGGCCCCTCAATACCCGCCCAAGATGAGCCCAGCTGGCTGGT TCCGGCCCATGCGAACTCTGCCCATGGACCCGGGCCTGGGATCCTCAGAGGAACAGGG CTCCTCCCCCTCGCTGTGGCCTGAGGTCACCTCCCTCCAGCCGGAGCCCAGCGACTCAG GACTAGGCGAAGGAGACACTAAGAGGAGGAGGATATCCCCCTATCCTTCCAGTGGCGA 82 USC0380PCT (2024-128-02) CAGCTCCTCTCCCGCTGGGGCCCCTTCTCCTTTTGATAAGGAAACCGAAGGCCAGTTTTA TAATTATTTTCCCAACTGA > SEQ ID NO: 4 (murine tbx21) MGIVEPGCGDMLTGTEPMPSDEGRGPGADQQHRFFYPEPGAQDPTDRRAGS SLGTPYSGGALVPAAPGRFLGSFAYPPRAQVAGFPGPGEFFPPPAGAEGYPPVDGYPAPDPR AGLYPGPREDYALPAGLEVSGKLRVALSNHLLWSKFNQHQTEMIITKQGRRMFPFLSFTVA GLEPTSHYRMFVDVVLVDQHHWRYQSGKWVQCGKAEGSMPGNRLYVHPDSPNTGAHW MRQEVSFGKLKLTNNKGASNNVTQMIVLQSLHKYQPRLHIVEVNDGEPEAACSASNTHVF TFQETQFIAVTAYQNAEITQLKIDNNPFAKGFRENFESMYASVDTSVPSPPGPNCQLLGGDPF SPLLSNQYPVPSRFYPDLPGQPKDMISQPYWLGTPREHSYEAEFRAVSMKPTLLPSAPGPTV PYYRGQDVLAPGAGWPVAPQYPPKMSPAGWFRPMRTLPMDPGLGSSEEQGSSPSLWPEVT SLQPEPSDSGLGEGDTKRRRISPYPSSGDSSSPAGAPSPFDKETEGQFYNYFPN >> SEQ ID NO: 5 (T-Bet consensus) MGIVEPGCGDMLTGTEPMPSDEGRaPGADqQHRXFYPEPGAQDad#RRaGgSL GsPYpGGALYPAaPgRFLGaYAYPPRaQaAGFPGaGEfFPPPAdAEGYqPgDGYaAPDPRAGLYP GPREDYALPAGLEVSGKLRVALnNHLLWSKFNQHQTEMIITKQGRRMFPFLSFTVAGLEPTS HYRMFVDVVLVDQHHWRYQSGKWVQCGKAEGSMPGNRLYVHPDSPNTGAHWMRQEVS FGKLKLTNNKGASNNVTQMIVLQSLHKYQPRLHIVEVNDGEPEAACnASNTH!FTFQETQFI AVTAYQNAEITQLKIDNNPFAKGFRENFESMYaSVDTS!PSPPGPNCQlLGGDh%SPLLpNQYP VPSRFYPDLPGQaKDmlpQaYWLGaPRHSYEAEFRAVSMKPalLPSAPGPTnpYYRGQ#VLAPG AGWPVAPQYPPKMgPAgWFRPMRTLPM#PGlGgSEerGpePllWpEVapirPEpSDSGLGEGDsKR RR!SPYPSSGDSSSPAGAPSPFDKEaEGQFYNYFPN > SEQ ID NO: 6 (T-Bet binding domain human) KLRVALNNHLLWSKFNQHQTEMIITKQGRRMFPFLSFTVAGLEPTSHYRMFV DVVLVDQHHWRYQSGKWVQCGKAEGSMPGNRLYVHPDSPNTGAHWMRQEVSFGKLKLT USC0380PCT (2024-128-02) NNKGASNNVTQMIVLQSLHKYQPRLHIVEVNDGEPEAACNASNTHIFTFQETQFIAVTAYQ NAEITQLKIDNNPFAKGFRENFE > SEQ ID NO: 7 (E. coli) TCCCTATCAGTGATAGAGA > SEQ ID NO: 8 TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAG > SEQ ID NO: 9 TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCAC TCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGA GAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGA GTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATC AGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGT GAAAGTCGAG > SEQ ID NO: 10 (pLenti-Tet-On muring T-bet seq) acgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgtt acataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaata gggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattga cgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctat taccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggg agtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggag gtctatataagcagcgcgttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctta agcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtg gaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagc 84 USC0380PCT (2024-128-02) gcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagag cgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtat gggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatc ccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaagga agctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatg agggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgc agagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctga cggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacag tctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaa ctcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagag aaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatg ggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttg ctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaag gaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatcggcactgcgtgcgccaattctgcagacaaatg gcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaa ctaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggttatcgagtttaccactccctat cagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagaga aaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtc gagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagctcggtacc cgggtcgaggtaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgtttt gacctccatagaagacaccgggaccgatccagcctccgcggccccgaattcgagctcggtacccggggatctagcgaaaccatgggaccaga ctacaaagacgatgacgacaagcttggtaccgagctcggatcatcaacaagtttgtacaaaaaagcaggcttcggcatcgtggagccgggttgc ggagacatgctgaccggcaccgagccgatgccgagtgacgagggccgggggcccggagcggaccaacagcatcgtttcttctatcccgagc cgggcgcacaggacccgaccgatcgccgcgcaggtagcagcctggggacgccctactctgggggcgccctggtgcctgccgcgccgggtc gcttccttggatccttcgcctacccgccccgggctcaggtggctggctttcccgggcctggcgagttcttcccgccgcccgcgggtgcggaggg ctacccgcccgtggatggctaccctgcccctgacccgcgcgcggggctctacccagggccgcgcgaggactacgcattgcccgcggggttgg aggtgtctgggaagctgagagtcgcgctcagcaaccacctgttgtggtccaagttcaaccagcaccagacagagatgatcatcactaagcaagg acggcgaatgttcccattcctgtccttcaccgtggctgggctggagcccacaagccattacaggatgtttgtggatgtggtcttggtggaccagca 85 USC0380PCT (2024-128-02) ccactggcggtaccagagcggcaagtgggtgcagtgtggaaaggcagaaggcagcatgccagggaaccgcttatatgtccacccagactccc ccaacaccggagcccactggatgcgccaggaagtttcatttgggaagctaaagctcaccaacaacaagggggcttccaacaatgtgacccaga tgatcgtcctgcagtctctccacaagtaccagccccggctgcacatcgtggaggtgaatgatggagagccagaggctgcctgcagtgcttctaac acacacgtctttactttccaagagacccagttcattgcagtgactgcctaccagaacgcagagatcactcagctgaaaatcgacaacaacccctttg ccaaaggattccgggagaactttgagtccatgtacgcatctgttgatacgagtgtcccctcgccacctggacccaactgtcaactgcttgggggag accccttctcacctcttctatccaaccagtatcctgttcccagccgtttctaccccgaccttccaggccagcccaaggatatgatctcacagccttact ggctggggacacctcgggaacacagttatgaagcggagttccgagctgtgagcatgaagcccacactcctaccctctgccccggggcccactg tgccctactaccggggccaagacgtcctggcgcctggagctggttggcccgtggcccctcaatacccgcccaagatgagcccagctggctggt tccggcccatgcgaactctgcccatggacccgggcctgggatcctcagaggaacagggctcctccccctcgctgtggcctgaggtcacctccct ccagccggagcccagcgactcaggactaggcgaaggagacactaagaggaggaggatatccccctatccttccagtggcgacagctcctctc ccgctggggccccttctccttttgataaggaaaccgaaggccagttttataactattttcccaactgataggacccagctttcttgtacaaagtggttg atatccactagtccagtgtggtggaattctgcagatatccagcacagtggcggccgctcgagtctagagggcccgtttatcctctagtcagaattcg atatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctg ctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgt caggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgct ttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggt gttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaa tccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggcc gcctccccgcatcgataccgtcgacctcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctg gctagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaaggcagctgtagatcttagcc actttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatatccttgatctgtggatctaccacacacaaggctact tccctgattggcagaactacacaccagggccagggatcagatatccactgacctttggatggtgctacaagctagtaccagttgagcaagagaag gtagaagaagccaatgaaggagagaacacccgcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtattagagtgg aggtttgacagccgcctagcatttcatcacatggcccgagagctgcatccggactgtactgggtctctctggttagaccagatctgagcctgggag ctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaac tagagatccctcagacccttttagtcagtgtggaaaatctctagcagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagc catctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcgg 86 USC0380PCT (2024-128-02) tgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtg tggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttc cccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgt agtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaac cctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattct gtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggt gtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccat cccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagc tattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgt tgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgct caccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggt ccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggac gagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtggg ggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgctacgagatttcgattcca ccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcg cccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtg gtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaatt gttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgtt gcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattggg cgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatcc acagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttttt ccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtt tccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctc atagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcct tatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtat gtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacct tcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaa aaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaa 87 USC0380PCT (2024-128-02) aggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagt gaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctgg ccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaa gtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgc cattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgtt gtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataatt ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttg cccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaagga tcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaac aggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcag ggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgt cgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgt gttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggc gttttgcgctgcttcgcgatgtacgggccagatat 88

Claims

USC0380PCT (2024-128-02) WHAT IS CLAIMED IS:

1. A therapeutic composition for treating cancer, comprising: at least one viral vector engineered to deliver a nucleic acid sequence encoding T-bet (T-box transcription factor TBX21) to cancer cells, wherein the nucleic acid sequence encoding T-bet is operably linked to a promoter suitable for expression in the cancer cells.

2. The therapeutic composition of claim 1 wherein the T-bet is expressed from a non- inducible system.

3. The therapeutic composition of claim 1 wherein the T-bet is expressed from an inducible system.

4. The therapeutic composition of claim 1, wherein the at least one viral vector is selected from the group consisting of oncolytic viruses.

5. The therapeutic composition of claim 4, wherein the at least one viral vector is selected from the group consisting of adenovirus, lentivirus, adeno-associated virus (AAV), herpes simplex virus (HSV), vaccinia virus, vesicular stomatitis virus (VSV), reovirus, myxoma virus, maraba virus, measles virus, Newcastle disease virus, picornavirus, reovirus, vesicular stomatitis virus, and combinations thereof.

6. The composition of claim 4, wherein the oncolytic virus is a Newcastle Disease Virus (NDV) or an attenuated strain thereof.

7. The composition of claim 4, wherein the oncolytic virus is engineered to express one or more xenoantigens that promote a hyperacute rejection response in a host organism.

8. The composition of claim 7, wherein the xenoantigen is an α-galactosyl epitope produced by expression of an α1,3-galactosyltransferase gene.

5. The composition of claim 4, wherein the oncolytic virus is further engineered to express a transgene encoding a checkpoint inhibitor, a cytokine, or a T-cell engaging molecule. 89USC0380PCT (2024-128-02) 6. The composition of claim 4, wherein the transgene encodes one or more of: a) a programmed death-ligand 1 (PD-L1) inhibitor; b) granulocyte-macrophage colony-stimulating factor (GM-CSF); or c) a single-chain variable fragment (scFv) that binds CD3.

7. The composition of claim 4, wherein the virus selectively induces tumor cell apoptosis via an interferon-stimulated expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and Noxa.

8. The composition of claim 4, wherein the virus further induces at least one non- apoptotic cell death pathway selected from autophagy, ferroptosis, necroptosis, or macropinocytosis in tumor cells.

9. The composition of claim 4, wherein the virus exhibits restricted replication in non- tumor cells by virtue of one or more tumor-specific promoter elements or viral gene deletions.

10. The composition of claim 4, wherein the oncolytic virus is administered intravenously, intratumorally, or intraperitoneally in a therapeutically effective amount.

11. The composition of claim 4, further comprising an immune checkpoint inhibitor selected from an anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody.

12. The composition of claim 4, wherein the oncolytic virus further comprises a nucleic acid encoding an immunostimulatory cytokine selected from granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukin-12 (IL-12), or interleukin-15 (IL-15).

13. The composition of claim 1, wherein the oncolytic virus or a composition comprising the virus further includes a STING agonist or a nucleic acid encoding a RIG-I ligand.

14. The composition of claim 1, wherein the oncolytic virus is configured to induce tumor antigen-agnostic immunization by promoting in situ immunogenic cell death and endogenous tumor antigen presentation. 90USC0380PCT (2024-128-02) 15. The composition of claim 4, wherein the virus selectively replicates in tumor cells due to the presence of a tumor-specific microRNA response element or deletion of a viral gene required for replication in non-transformed cells.

16. The therapeutic composition of claim 1, wherein the at least one viral vector includes a first plasmid for T-bet expression.

17. The therapeutic composition of claim 16, wherein the at least one viral vector includes a second plasmid for reverse tetracycline-controlled transactivator (rtTA) expression.

18. The therapeutic composition of claim 17, wherein the first plasmid for T-bet expression includes a promoter, a tet response element (TRE) operably linked to the promoter, and a coding sequence for T-bet (Tbx21) downstream of the TRE, whereby T-bet expression is inducible by doxycycline through the TRE.

19. The therapeutic composition of claim 18, wherein the TRE includes Tet operator sequences (tetO) responsive to rtTA.

20. The therapeutic composition of claim 17, wherein the first plasmid for T-bet expression includes a promoter that contains Tet operator sequences (tetO) responsive to rtTA and a coding sequence for T-bet protein downstream of the promoter.

21. The therapeutic composition of claim 17, wherein the first plasmid includes a first plasmid backbone that includes an origin of replication and regulatory sequences ensuring replication and stability in host cells of the viral vector.

22. The therapeutic composition of claim 17, wherein the second plasmid for rtTA expression includes a constitutive promoter and a coding sequence for rtTA. 91USC0380PCT (2024-128-02) 23. The therapeutic composition of claim 17, wherein the second plasmid for rtTA expression includes a constitutive promoter and a rtTA-VP16 Coding Sequence downstream of the constitutive promoter.

24. The therapeutic composition of claim 22 or 23, wherein the second plasmid includes a second plasmid backbone that includes an origin of replication and regulatory sequences ensuring replication and stability of the viral vector in host cells.

25. The therapeutic composition of any of claims 17 to 24, wherein the first plasmid is packaged in a first viral vector and the second plasmid is packaged in a second viral vector.

26. The therapeutic composition of claim 1 further comprising a pharmaceutically acceptable carrier for administration to a subject.

27. The therapeutic composition of claim 1, wherein the cancer cells are epithelial in origin.

28. The therapeutic composition of claim 27, wherein the cancer is selected from the group consisting of colon cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, and melanoma.

29. The therapeutic composition of claim 1, wherein the at least one viral vector includes at least one oncolytic viral vector engineered to selectively infect and replicate within cancer cells, leading to tumor cell lysis.

30. The therapeutic composition of claim 29, wherein the at least one oncolytic viral vector is selected from the group consisting of adenovirus, herpes simplex virus, vaccinia virus, vesicular stomatitis virus, lentivirus, reovirus, myxoma virus, measles virus, Newcastle disease virus, and combinations thereof.

31. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the therapeutic composition of any one of claims 1-30, wherein: 92USC0380PCT (2024-128-02) (a) the administering results in the expression of T-bet in cancer cells; (b) the expression of T-bet induces apoptosis in the cancer cells; and (c) the expression of T-bet alters the expression of ion channels and transporters in the cancer cells, leading to cell death.

32. The method of claim 31, wherein the administering is via intravenous, intratumoral, or oral routes.

33. The method of claim 31, wherein the cancer is colon cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, or melanoma, and treatment results in inhibition of tumor growth or reduction of tumor volume.

34. The method of claim 31, wherein the oncolytic viral vector selectively infects tumor cells, replicates, and facilitates tumor cell lysis in combination with T-bet expression to enhance therapeutic efficacy.

35. The method of claim 18, wherein T-bet expression is inducible and regulated by doxycycline administration through a Tet-On system.

36. The method of claim 31, wherein tumor regression is facilitated by a combination of T-bet- mediated apoptosis, tumor-specific viral replication, and immune activation through tumor antigen release. 93

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