Drug-inducible gene expression system

Abstract

Provided herein is a method for user controlled gene expression. In one embodiment, provided is a method for the activation of a T cell, comprising the steps of ectopically expressing farnesoid X receptor (FXR) in said T cell; activating said FXR; and transcribing a gene that contains a farnesoid response element (FRE) in a promoter region.

Description

[0001] DRUG-INDUCIBLE GENE EXPRESSION SYSTEM

[0002] FIELD OF THE INVENTION

[0003] The invention relates generally to cells that are engineered in a way that allows for drug inducible control of gene expression.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This work was supported by the U.S. Department of Veterans Affairs, and the Federal Government has certain rights in this invention.

[0006] BACKGROUND OF THE INVENTION

[0007] User controlled gene expression has been a highly sought after technology in the context of gene and cell therapy. Several approaches have been deployed leveraging transgenic expression of drug receptors coupled with transcriptional activation elements. These include doxy cycline / Tetracy cline inducible systems, cumate inducible systems (1), and rapamycin inducible systems which have been mainstays for controlling gene expression in pre-clinical systems (2).

[0008] Each of these systems relies on the expression of chimeric fusion proteins containing components of non-human proteins. As such, these systems are inherently immunogenic and this has precluded application of these tools to control gene expression in the clinical setting. A need exists in the art for user controlled gene expression that is non-immunogenic. SUMMARY OF THE INVENTION

[0009] In one embodiment, provided is a method for the activation of a T cell, comprising the steps of ectopically expressing farnesoid X receptor (FXR) in said T cell; activating said FXR; and transcribing a gene that contains a farnesoid response element (FRE) in a promoter region.

[0010] In another embodiment, provided is a method for drug-inducible control of gene expression in a T-cell, comprising the steps of ectopically expressing farnesoid X receptor (FXR) in said T cell; activating said FXR by administering a drug; and transcribing a gene that contains a farnesoid response element (FRE) in a promoter region.

[0011] In a further embodiment, provided is an engineered T-cell, comprisnig a transgene comprising a promoter region comprising a FRE.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows a schematic diagram depicting an embodiment of the invention: FXR controllable gene expression. Cells are modified to ectopically express a FXR which can be activated by administering an FXR agonist, like obeticholic acid.

[0014] Figure 2 shows the respective constructs used to explore the utility of the hFXR inducible gene expression.

[0015] Figure 3 shows the function of the FXR inducible system in human cells and its effects on proliferation in human T cells.

[0016] Figure 4 shows schematic diagrams of tandem transposon constructs for reprograming CAR-T cells to express the cytokine interleukin 12 under control of FXR activating ligands.

[0017] Figure 5 provides specific examples of promoter sequences that are inducible by FXR activation.

[0018] Figure 6 shows the results of a study examining the relative induction efficiency of two different FRE elements in a human Jurkat T cell line.

[0019] Figure 7 is a schematic of the respective constructs used to explore the utility of the hFXR inducible gene expression system. Figure 8 show proliferation of human T cells that have been stably modified to express GFP or a hFXR expression construct after exposure to lum activating ligand Fexaramine (Fex) for 1 week under stimulating conditions.

[0020] Figure 9 shows quantification of luminescence in cells transfected with each hFXR expression construct and exposed to varying concentrations of Fex and Obeti cholic Acid (OA) as indicated at (A) 48 hours and (B) 7 days post transfection.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] The ability to control gene expression using a drug is a long sought after feature that will be of great utility in the context of gene and cell therapy. Conventional gene therapies rely on the inherent cell-specific properties of gene promoters that are built into the gene modifying vectors. This limits user control over gene expression that could be used to improve function and safety depending on the application. Several methods have been developed previously to control gene expression using drugs (1) (cite), light (4), or thermal pulses (5). Many of these systems, including all the drug inducible systems, rely on synthetic constructs that are non-human in origin. This has precluded adoption of these systems into the clinical setting.

[0023] This invention circumvents the immune system by using a fully human system based on ectopically expressing FXR in cells of interest which could include: Embryonic Stem Cells (ESCs), Adult Stem Cells, Induced Pluripotent Stem Cells (iPSCs), Hematopoietic Stem Cells (HSCs), Peripheral Blood Mononuclear Cells (PBMCs), CAR-T Cells, T Regulatory Cells, Dendritic Cells, Fibroblasts, Endothelial Cells, Neuronal Cells, Epithelial Cells, Skeletal Muscle Cells, Adipose-Derived Stem Cells (ADSCs). When these cells are also engineered with a gene of interest (GOI) flanked by a FRE containing promoter the GOI expression can be controlled by activation of the FXR. Since natural FXR ligands normally only exist in the gut, the receptor will remain inactive unless a ligand as administered. This enables pharmacological control of gene expression. Some agents that could be used to activate FXR include: Ursodeoxycholic Acid (UDCA), Obeticholic Acid (OCA), GW4064, INT-747 (6-ECDCA), Chenodeoxycholic Acid (CDCA), and Fexaramine (Fex). The same system could be deployed in the context of gene therapy, where genes are delivered directly to a patient using a vector intended to modify cells in vivo.

[0024] Thus, in one embodiment, the system disclosed herein relies on ectopic expression of a nuclear receptor for bile acids, the farnesoid X receptor (FXR), in cells of interest. The FXR is activated upon ligand binding, and it moves into the nucleus where it activates transcriptions of specific genes that contain farnesoid response elements (FREs) in the promoter region. This allows for control of gene expression by engineering cells with transgenes containing FREs in promoter sequence (Fig 1). Normally, farnesoid receptors are primarily expressed in cells of the small intestine where they function to respond to bile acids. Bile acids and other FXR agonists are not generally found outside of the small intestine, so it is reasonable to assume that unwanted activation of FXR in T cells or other non-gut cells would not be likely due to the lack of natural agonist in most tissues (3). Similarly, FXR can be activated with a variety of safe, clinically approved small molecules with high bioavailability and few side effects. The invention paired this designer ectopic receptor system with FXR responsive promoter sequences as a method to control gene expression in T cells.

[0025] Some specific examples of clinical applications for this technology that are enabled by the invention disclosed here include controlling expression of secreted cytokines or chemokines, controlling gene expression of synthetic receptors like chimeric antigen receptors, or genes that drive cell fate (Fig 1).

[0026] Examples of cytokines that could be delivered include: Interleukin-2 (IL-2), Interleukin- 10 (IL- 10), Interferon-alpha (IFN-a), Interferon-beta (IFN-P), Interleukin- 12 (IL- 12), Tumor Necrosis Factor-alpha (TNF-a) inhibitors, Transforming Growth Factor-beta (TGF- ).

[0027] Examples of chemokines that could be delivered include: Interleukin-8 (IL-8), RANTES, MCP-1 (Monocyte Chemoattractant Protein- 1), MIP-la (Macrophage Inflammatory Protein- 1 alpha), MIP-ip (Macrophage Inflammatory Protein- 1 beta), CXCL10 (Interferon-gamma Inducible Protein-10), CXCL12 (Stromal Cell-Derived Factor- 1), CX3CL1 (Fractalkine), CCL5 (Chemokine (C-C motif) ligand 5), CXCL13 (B-cell Attracting Chemokine 1).

[0028] Examples of genes that modify cell fate include: Oct4 (POU5F1), Sox2, Klf4, c-Myc, Nanog, GATA4, TBX5, Runxl, YAP (Yes-associated protein), c-Jun, TNF (Tumor Necrosis Factor), IL1B (Interleukin- 1 beta), IL6 (Interleukin-6), IL12B (Interleukin- 12 subunit beta), N0S2 (Nitric Oxide Synthase 2), CCL2 (Chemokine (C-C motif) ligand 2), CXCL9 (C-X-C Motif Chemokine Ligand 9), CXCL10 (C-X-C Motif Chemokine Ligand 10), IRF5 (Interferon Regulatory Factor 5), STAT1 (Signal Transducer and Activator of Transcription 1).

[0029] To avoid the immune complications associated with xenogenic gene expression, a system was developed that leverages a human nuclear receptor that has a highly restricted gene expression profde in adults. This restricted gene expression profde provides an opportunity to ectopically express this receptor in therapeutic transgene constructs used in cell and gene therapy. The invention demonstrate efficient engineering of several cell types including human T cells to constitutively express the FXR, thus enabling activation of genes harboring FREs in those cells. The invention demonstrates that by engineering FXR expressing cells with FRE driven reporter constructs like luciferase, the invention can use activating ligands to control gene expression.

[0030] Specifically, cells can be modified to express FXR using viral transduction, transposon based engineering, or targeted integration strategies to introduce a transgene encoding FXR under control of a constitutive promoters for example: CMV (Cytomegalovirus) Promoter, SV40 (Simian Virus 40) Promoter, EFla (Elongation Factor 1 Alpha) Promoter, UBC (Ubiquitin C) Promoter, RPL13a (Ribosomal Protein LI 3a) Promoter, PGK (Phosphoglycerate Kinase) Promoter, Actin Promoter, T7 Promoter.

[0031] Cells are modified either in parallel, or sequentially with another transgene encoding a GOI flanked by promoter containing FRE sequence suitable for controlling gene expression in that cell type. Some examples of minimal FRE sequences are included in Figure 6. Some other gene promoters that could be used to control gene expression in the FXR system include components of the promotors of the genes CYP7A1, CYP8B1, BSEP (ABCB11), SHP (NR0B2), ABC Al, PLTP, G6Pase, PEPCK, FGF19.

[0032] EXAMPLES

[0033] A gene expression system was designed that relies on the ectopic expression of a nuclear receptor for bile acids, the farnesoid x receptor (FXR), FXR is activated upon ligand binding, translocates to the nucleus and activates the transcription of genes that contain farnesoid response elements (FREs) in the promotor region. This allows for the control of gene expression by engineering cells with transgenes containing FREs in the promotor sequence. Farnesoid receptors (FXR) are primarily expressed in the small intestine, where they respond to bile acids. Since bile acids and other FXR agonists are generally not present outside of the small intestine, the likelihood of unwanted FXR activation in T cells or other non-gut cells is low due to the absence of natural agonists in most tissues. Additionally, FXR can be activated by a variety of safe, clinically approved small molecules that have high bioavailability and few side effects. The invention combined this designer ectopic receptor system with FXR-responsive promoter sequences to control gene expression in T cells.

[0034] Figure 1 shows FXR controllable gene expression. Cells are modified to ectopically express a FXR which can be activated by administering an FXR agonist, like obeticholic acid. The activated FXR then translocates to the nucleus and dimerizes with the retinoid x receptor (RXR) and promotes transcription of loci with upstream FXR response elements (FRE). In the examples depicted, cells can be engineered with transgenes expressing cytokines, CAR constructs, or transcription factors driving phenotypic polarization or differentiation of cell types.

[0035] The respective constructs used to explore the utility of the hFXR inducible gene expression are shown in Figure 2: (A) is a piggyback transposon containing EFl A driving the hFXR and a GFP reporter for enriching / identifying stably modified cells. (B) is a FRE response element luciferase reporter comprised of a component of the BSEP gene promoter driving expression if enhanced firefly luciferase. Panel C shows an experimental schematic for generating Jurkat T cell lines stably expressing FXR and enrichment of stable cells using GFP sorting. After sorting the cells are transiently transfected with a luciferase reporter construct and assessed for luciferase induction after exposure to FXR agonist. Panel D shows the results of an experiment evaluating FXR inducible luciferase expression in the Jurkat cell line.

[0036] The function of the FXR inducible system in human cells and its effects on proliferation in human T cells is shown in Figure 3. Panel A shows the drug dependent induction of luminescence in human T cells co-transfected with pTpB-hFXR and BSEP eFFluc reporter (defined in Fig 2). Panel B shows the proliferation of human T cells that have been stably modified to express GFP or a hFXR expression construct and exposed to luM activating ligand fexaramine for 1 week under stimulating conditions (IL2+aCD3 / aCD28). Expression and activation of FXR does not adversely affect T cell proliferation in vitro.

[0037] Figure 4 shows schematic diagrams of tandem transposon constructs for reprograming CAR-T cells to express the cytokine interleukin 12 under control of FXR activating ligands.

[0038] Figure 5 provides specific examples of promoter sequences that are inducible by FXR activation.

[0039] The results of a study examining the relative induction efficiency of two different FRE elements in a human Jurkat T cell line is shown in Figure 6. “Long” refers to The full length BSEP promoter and “short” refers to the minimal BSEP promoter (Fig 5). the respective constructs used to explore the utility of the hFXR inducible gene expression system is shown in Figure 7. PiggyBac transposons containing (A) CMV or (B, C) EFl A driving the hFXR and a GFP reporter for enriching / identifying stably modified cells. The farnesoid response element (FRE) luciferase reporter is comprised of a component of the BSEP gene promoter driving expression of an enhanced version of firefly luciferase.

[0040] Proliferation of human T cells that have been stably modified to express GFP or a hFXR expression construct after exposure to lum activating ligand fexaramine for 1 week under stimulating conditions is shown in Figure 8. And quantification of luminescence in cells transfected with each hFXR expression construct and exposed to varying concentrations of Fexaramine (Fex) and Obeti cholic Acid (OA) as indicated at (A) 48 hours and (B) 7 days post transfection is presented in Figure 9.

[0041] The data presented here demonstrates that that by ectopically expressing FXR in T cells, the invention can control the activation of the T cells via FDA approved drugs. Such drugs include, for example, Obeticholic acid and Fexeramine. These drugs selectively bind and activate FXR, generating a drug-inducible gene system. Based on these data, the FXR system can be utilized in a variety of therapeutic indications where cells can be engineered to express transgenes such as CAR constructs, cytokines, or transcription factors that drive phenotypic polarization or differentiation of different cell types, as depicted in Figure 1.

[0042] Jurkats: Cultures were maintained in RPMI 1640 supplemented with final concentrations of 10% FBS, 2mM GlutaMAX, and 0.2% PenStrep. Cells were maintained in 37°C in 5% CO2. Human T Cells: Cultures were maintained in RPMI supplemented with 10% FBS, 300 mg / L L- Glutamine, 2 mmol / L GlutaMAX, 100 U / mL penicillin, 100 pg / mL streptomycin, 50 pg / mL gentamycin, 25 mmol / L HEPES and 55 pmol / L 2-mercaptoethanol. After transfection, human T cells were activated with Immunocult CD3 / CD28 T-cell activators from STEMCELL Technologies and expanded in rhIL2. Cultures were maintained in an incubator set at 37°C with 5% CO2.

[0043] Molecular Biology

[0044] Plasmid vectors, indicated in Figure 7, were synthesized by Vector Builder Biosciences. All plasmid vectors were confirmed via sequencing. The plasmids were prepared according to the manufacturer’s protocol from PureLink HiPure Plasmid DNA Purification Maxiprep Kit.

[0045] CAR-T Cell Generation

[0046] Jurkats: Jurkats were transfected using the Neon NxT Electroporation System using the following parameters: 1350 volts, a pulse width of 10ms, and a pulse number of 3. The cells were transfected with one of the following plasmid vectors: Full BSEP nLuc-EFS, half BSEP nLuc-EFS, and Tandem hFXR CMV promoter. After transfection, the cells were cultured in the aforementioned conditions.

[0047] Human T cells: Leukopaks were purchased from STEMCELL Technologies which collects and distributes healthy non-identified donor blood with the appropriate consent forms in accordance with the Institutional Review Board (IRB). PBMCs were transfected with one of the following plasmids: Full BSEP nLuc-EFS, half BSEP nLuc-EFS, and Tandem hFXR CMV promoter at the following electroporation parameters: 2150 volts, a pulse width of 20ms, and a pulse number of 1. After transfection, T cells were activated from the PMBCs using Immunocult CD3 / CD28 T- cell activators from STEMCELL Technologies and cultured in 300 IU / mL rhIL-2 (NCI) for 7 days.

[0048] Luminescence Quantification

[0049] Jurkats: After electroporation, cells were left to recover in the aforementioned culture conditions for 48 hours or 7 days. After this time, electroporated and control jurkats were plated in a clear bottom 96-well plate (Coming) at le5 GFP+cells per well. Cells were treated with one of the following conditions: no drug control, I pM Fexeramine (Sigma Aldrich), 5 pM Fexeramine, IpM Obeticholic Acid (Sigma Aldrich) or 5pM Obeticholic Acid. After treatment, the cells were cultured in the incubator overnight. The following morning, luminescence was measured using a SpectraMax plate reader.

[0050] Human T cells: After PBMCs were transfected and T cells were activated for 7 days, the cells were plated in a clear bottom 96 well plate. Cells were treated with no drug control, IpM Fexeramine, 5pM Fexeramine, IpM Obeticholic Acid or 5pM Obeticholic Acid for 24 hours at 37°C in 5% CO2. After incubation, the plate was loaded into the SpectraMax plate reader and luminescence was measured.

[0051] To determine the function of the FR inducible system in human cells, the invention first examined the proliferation of human T cells that had been stably modified to express GFP or a hFXR expression construct and exposed to lum activating ligand fexaramine for 1 week of T cell stimulation (IL2 +aCD3 / aCD28). These data suggest that FXR does not adversely affect T cell proliferation in vitro.

[0052] After confirming no differences in proliferation of the stably modified human T cells, the invention examined the drug dependent induction of luminescence in jurkat cells transfected with each of the plasmid vectors depicted in Figure 7. Luminescence was observed 48 hours (Figure 9a) and 7 days (Figure 9b) post transfection. These results suggest that the luciferase reporter which serves as the farnesoid response element (FRE), is activated regardless of drug treatment.

[0053] To avoid the immune complications associated with xenogeneic gene expression, a system was developed that leverages a human nuclear receptor with a highly restricted gene expression profile in adults. This restricted profile allows for ectopic expression of this receptor in therapeutic transgene constructs used in cell and gene therapy. The preliminary data suggest that even without the administration of the drug, the constructs appear to have nonspecific activation as shown by the luminescence in the transfection control group in Figure 9. This suggests that the constructs depicted in Figure 7 are constitutively activated. 1 . Kallunki T, Barisic M, Jaattela M, Liu B. How to Choose the Right Inducible Gene Expression System for Mammalian Studies? Cells. 2019;8(8). doi: 10.3390 / cells8080796. PubMed PMID: 31366153; PMCID: PMC6721553.

[0054] 2. Tri stan -Manzano M, Justicia-Lirio P, Maldonado-Perez N, Cortijo-Gutierrez M, Benabdellah K, Martin F. Externally-Controlled Systems for Immunotherapy: From Bench to Bedside. Front Immunol. 2020;ll:2044. doi: 10.3389 / fimmu.2020.02044. PubMed PMID: 33013864; PMCID: PMC7498544.

[0055] 3. Sun L, Cai J, Gonzalez FJ. The role of farnesoid X receptor in metabolic diseases, and gastrointestinal and liver cancer. Nat Rev Gastroenterol Hepatol. 2021 ; 18(5):335-47. doi: 10.1038 / s41575-020-00404-2. PubMed PMID: 33568795.

[0056] 4. Hartmann D, Smith JM, Mazzotti G, Chowdhry R, Booth MJ. Controlling gene expression with light: a multidisciplinary endeavour. Biochem Soc Trans. 2020;48(4): 1645-59. doi: 10.1042 / BST20200014. PubMed PMID: 32657338; PMCID: PMC7458398.

[0057] 5. Miller IC, Gamboa Castro M, Maenza J, Weis JP, Kwong GA. Remote Control of Mammalian Cells with Heat-Triggered Gene Switches and Photothermal Pulse Trains. ACS Synth Biol. 2018;7(4):1167-73. doi: 10.1021 / acssynbio.7b00455. PubMed PMID: 29579381; PMCID: PMC5929470.

Claims

WHAT IS CLAIMED IS1. A method for the activation of a T cell, comprising the steps of ectopically expressing farnesoid X receptor (FXR) in said T cell; activating said FXR; and transcribing a gene that contains a farnesoid response element (FRE) in a promoter region.

2. The method according to claim 1, wherein the FXR is activated by administering a FXR agonist.

3. The method according to claim 1, wherein the method is non-immunogenic.

4. A method for drug-inducible control of gene expression in a T-cell, comprising the steps of ectopically expressing farnesoid X receptor (FXR) in said T cell; activating said FXR by administering a drug; and transcribing a gene that contains a farnesoid response element (FRE) in a promoter region.

5. The method according to claim 4, wherein the drug is Obeticholic acid or Fexeramine.

6. The method according to claim 4, wherein the method is non-immunogenic.

7. An engineered T-cell, comprisnig a transgene comprising a promoter region comprising a FRE.

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