Compositions and methods for tunable regulation of transcription

Abstract

To provide compositions and methods for tunable regulation of transcription.SOLUTION: The present disclosure provides compositions and methods related to transcription factor systems. Such systems provide for modular and tunable protein expression driven by regulated transcriptional activity. The present disclosure relates to systems, compositions and methods for tunable protein expression driven by regulated transcriptional activity. Provided in the present disclosure are modular transcription factor systems, polynucleotides of transcription factor systems, polypeptides, vectors, cells, compositions and methods for use in regulation of transcription and regulated protein expression driven by regulated transcriptional activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 958,693, filed January 8, 2020, and U.S. Provisional Patent Application No. 62 / 959,859, filed January 10, 2020. The entire contents of the aforementioned applications are incorporated herein by reference.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on January 8, 2021, is named 268052_483267_SL.txt and is 241,815 bytes in size.

[0003] Field The present disclosure relates to systems, compositions, and methods for regulatable protein expression driven by controlled transcriptional activity. Provided in the present disclosure are modular transcription factor systems, transcription factor system polynucleotides, polypeptides, vectors, cells, compositions, and methods for use in regulating transcription and controlled protein expression driven by controlled transcriptional activity. [Background technology]

[0004] background Gene therapy and cell therapy have revolutionized medicine, offering new possibilities for the treatment of previously intractable conditions. However, most current technologies do not allow for titration of the timing or level of target protein induction. This makes many potential gene therapy and cell therapy applications difficult or impossible to use safely and effectively.

[0005] Insufficient exogenous and / or endogenous gene regulation is a significant problem in many gene and cell therapy settings. This lack of regulatability also makes it difficult to safely express proteins that have narrow or uncertain therapeutic windows or that require more titrated or transient expression.

[0006] One approach to controlled protein expression or function is the use of drug-responsive domains (DRDs). Drug-responsive domains are small protein domains that can be attached to target proteins of interest. In the absence of a DRD-binding ligand, the DRD destabilizes the attached protein of interest, causing it to be rapidly degraded by the cellular ubiquitin-proteasome system. However, when a specific small molecule DRD-binding ligand binds to the DRD, the attached protein of interest is stabilized and protein function is achieved.

[0007] DRD technology forms the basis of a new class of cell and gene therapies that can regulate and temporally control gene expression and function, expanding the scope of protein therapeutics that can be safely and effectively incorporated into cell and gene therapy modalities. However, current DRD technologies generate fusion proteins in which a protein of interest is linked to a DRD, which may be unsuitable for some indications. Therefore, there remains a need to develop cell and gene therapies in which a native protein of interest can be expressed in a controlled manner. Summary of the Invention

[0008] overview The present invention provides cell and gene therapies in which the timing or levels of engineered cells, nucleic acid molecules, vectors, and native therapeutic proteins can be controlled by the administration of oral small molecule drugs.

[0009] Additionally, the present disclosure provides compositions, systems, and methods for tunable control of transcription. The compositions relate to transcription factor systems and agents that induce transcriptional activity of a polynucleotide encoding a protein of interest. The compositions provided by the present disclosure include nucleic acid molecules, polypeptides, and cells associated with the transcription factor systems. Methods related to the transcription factor systems provided by the present disclosure include methods for generating modified cells and methods for treating or preventing disease.

[0010] Provided herein is a transcription factor system. The transcription factor system of the present disclosure is a combination of one or more polynucleotides including: (1) one or more nucleic acid sequences encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; (2) a nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor, or a portion thereof, is operably linked to the DRD; and (3) a nucleic acid sequence encoding a payload, the nucleic acid sequence being operably linked to an inducible promoter that includes the specific polynucleotide binding site.

[0011] The present disclosure provides modified cells related to transcription factor systems.

[0012] In some aspects, the present disclosure provides engineered cells that may regulate the expression or transcription of a payload. The engineered cells comprise a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug-responsive domain (DRD). At least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD. The transcription factor activation domain and the transcription factor DNA-binding domain interact to form a transcription factor that can activate transcription of a fourth nucleic acid sequence upon binding to the specific polynucleotide binding site, and the fourth nucleic acid sequence encodes a protein of interest and is operably linked to either the specific polynucleotide binding site, an exogenous inducible promoter containing the specific polynucleotide binding site, or both. In some embodiments, the protein of interest is a heterologous protein. In some embodiments, the fourth nucleic acid sequence is located on the first polynucleotide. In some embodiments, the engineered cells further comprise a second polynucleotide comprising the fourth nucleic acid sequence.

[0013] In some aspects, the present disclosure provides modified cells comprising a polynucleotide comprising a first nucleic acid sequence encoding a drug response domain (DRD) and a second nucleic acid sequence encoding a transcription factor. The transcription factor is operably linked to the DRD and is capable of binding to a specific polynucleotide binding site and activating transcription of a third nucleic acid sequence encoding a protein of interest, the third nucleic acid sequence being operably linked to either the specific polynucleotide binding site, an exogenous inducible promoter comprising the specific polynucleotide binding site, or both. In some embodiments, the protein of interest is a heterologous protein. In some embodiments, the third nucleic acid sequence is located on a polynucleotide comprising the first nucleic acid sequence and the second nucleic acid sequence. In some embodiments, the modified cell further comprises a second polynucleotide comprising the third nucleic acid sequence.

[0014] In another aspect, the disclosure provides a modified cell comprising: (a) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription, and a second nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor, or a portion thereof, is operably linked to the DRD; and (b) a second polynucleotide comprising a third nucleic acid sequence encoding a protein of interest, wherein the third nucleic acid sequence is operably linked to an exogenous inducible promoter that comprises the specific polynucleotide binding site.

[0015] In another aspect, the disclosure provides a modified cell comprising: (a) a polynucleotide comprising a first nucleic acid sequence encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription of a second nucleic acid sequence encoding a protein of interest, wherein the second nucleic acid sequence is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; and (b) a third nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor is operably linked to the DRD.

[0016] In another aspect, the disclosure provides a modified cell comprising: (a) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD; and (b) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter that comprises the specific polynucleotide binding site, wherein the transcription factor activation domain and the transcription factor DNA-binding domain interact to form a transcription factor that can activate transcription upon binding to the specific polynucleotide binding site.

[0017] In another aspect, the disclosure provides a modified cell comprising: (a) a first polynucleotide comprising a nucleic acid sequence encoding a transcription factor activation domain; (b) a second polynucleotide comprising a nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide-binding site located on an exogenous inducible promoter; and (c) a third polynucleotide comprising a nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD. In one aspect, the transcription factor activation domain and the transcription factor DNA-binding domain interact to form a transcription factor that can bind to the specific polynucleotide-binding site and activate transcription of a nucleic acid sequence encoding a protein of interest, which nucleic acid sequence is operably linked to an exogenous inducible promoter.

[0018] In various embodiments, one or more of the transcription factor DNA-binding domain, the transcription factor activation domain, and the DRD are derived from a parent protein. In some embodiments, the transcription factor DNA-binding domain is derived from a parent protein selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL. In some embodiments, the transcription factor activation domain is derived from a parent protein, and the parent protein is p65. In some embodiments, the DRD is derived from a parent protein selected from the group including human carbonic anhydrase 2 (CA2), human DHFR, E. coli DHFR (ecDHFR), human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5.

[0019] In some embodiments, the DRD is stabilized in the presence of a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). In some embodiments, the DRD responds to or interacts with a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP).

[0020] In some embodiments, the protein of interest is a wild-type protein.

[0021] In some embodiments, the protein of interest is a therapeutic protein.

[0022] In some embodiments, the protein of interest is selected from the group consisting of a cytokine, an antibody, or an antigen-binding fragment thereof, a clotting factor, an enzyme, a gene-editing protein, a T-cell receptor (TCR), and a chimeric antigen receptor (CAR).

[0023] In some embodiments, the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

[0024] In some embodiments, the protein of interest is a secreted protein.

[0025] In some embodiments, the cell is a T cell, a natural killer cell (NK cell), or a tumor-infiltrating lymphocyte (TIL).

[0026] In some embodiments, the cell is a stem cell, a liver cell, a blood cell, a pancreatic cell, a nerve cell, an eye cell, a muscle cell, or a bone cell.

[0027] Also provided by the present disclosure are nucleic acid molecules related to transcription factor systems.

[0028] In one aspect, the present disclosure provides a nucleic acid molecule comprising: (a) a first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and (b) a second nucleic acid sequence encoding a drug-responsive domain (DRD). In some embodiments, the nucleic acid molecule further comprises (c) a third nucleic acid sequence encoding a transcription factor activation domain, wherein either (i) the transcription factor DNA-binding domain is operably linked to the DRD, (ii) the transcription factor activation domain is operably linked to the DRD, or (iii) a combination of the transcription factor DNA-binding domain and the transcription factor activation domain is operably linked to the DRD. In some embodiments, the transcription factor DNA-binding domain is derived from a parent protein selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL. In some embodiments, the transcription factor activation domain is derived from a parent protein, and the parent protein is p65.

[0029] In one aspect, the present disclosure provides a nucleic acid molecule comprising: (a) a first nucleic acid sequence encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; and (b) a second nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor is operably linked to the DRD. In some embodiments, the nucleic acid molecule further comprises (c) a third nucleic acid sequence encoding a protein of interest, wherein the third nucleic acid sequence is operably linked to either the specific polynucleotide binding site, an exogenous inducible promoter comprising the specific polynucleotide binding site, or both.

[0030] In some embodiments, the specific polynucleotide binding site is located on an exogenous inducible promoter.

[0031] In some embodiments, the DRD is derived from a parent protein selected from the group including human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5.

[0032] In some embodiments, the DRD is stabilized in the presence of a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). In some embodiments, the DRD responds to or interacts with a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP).

[0033] In some embodiments, the protein of interest is a wild-type protein.

[0034] In some embodiments, the protein of interest is a therapeutic protein.

[0035] In some embodiments, the protein of interest is selected from the group consisting of a cytokine, an antibody, a clotting factor, an enzyme, a gene-editing protein, a T cell receptor (TCR), and a chimeric antigen receptor (CAR).

[0036] In some embodiments, the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

[0037] In some embodiments, the protein of interest is a secreted protein.

[0038] Also provided herein is a vector comprising the nucleic acid molecule described herein.Vector provided by the present disclosure includes plasmid or viral vector.In some aspects, viral vector is derived from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus and picornavirus.In some aspects, viral vector is selected from the group consisting of lentivirus vector, gamma retrovirus vector, adeno-associated virus (AAV) vector, adenovirus vector and herpesvirus vector.

[0039] Also provided by the present disclosure are first and second polynucleotides comprising nucleic acid sequences encoding one or more components of a transcription factor system.

[0040] In one aspect, the disclosure provides a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD; the second polynucleotide comprises a fourth nucleic acid sequence encoding a protein of interest, wherein the fourth nucleic acid sequence is operably linked to an inducible promoter comprising the specific polynucleotide-binding site, wherein the transcription factor activation domain and the transcription factor DNA-binding domain interact to form a transcription factor that can activate transcription upon binding to the specific polynucleotide-binding site; and wherein the first polynucleotide and the second polynucleotide are each carried on a single vector, or the first polynucleotide and the second polynucleotide are carried on separate vectors.

[0041] In one aspect, the present disclosure provides a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a first nucleic acid sequence encoding a transcription factor and a second nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor is operably linked to the DRD, and the transcription factor is capable of activating transcription upon binding to a specific polynucleotide binding site; the second polynucleotide comprises a third nucleic acid sequence encoding a protein of interest, wherein the third nucleic acid sequence is operably linked to an inducible promoter comprising the specific polynucleotide binding site; and wherein the first polynucleotide and the second polynucleotide are each carried on a single vector, or the first polynucleotide and the second polynucleotide are carried on separate vectors.

[0042] In some embodiments, the DRD is derived from a parent protein selected from the group including human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. In some embodiments, the DRD is stabilized in the presence of a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP).

[0043] In some embodiments, the protein of interest is a wild-type protein. In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the protein of interest is selected from the group consisting of a cytokine, an antibody, a clotting factor, an enzyme, a gene editing protein, a T cell receptor (TCR), and a chimeric antigen receptor (CAR). In some embodiments, the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre. In some embodiments, the protein of interest is a secreted protein.

[0044] Also provided by the present disclosure are methods related to transcription factor systems.

[0045] In one aspect, the disclosure provides a method of producing a modified cell, the method comprising introducing into the cell a nucleic acid molecule comprising: (a) a first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and (b) a second nucleic acid sequence encoding a drug-responsive domain (DRD). In one embodiment, the nucleic acid molecule further comprises a third nucleic acid sequence encoding a transcription factor activation domain. In some embodiments, either (i) the transcription factor DNA-binding domain is operably linked to the DRD, (ii) the transcription factor activation domain is operably linked to the DRD, or (iii) a combination of the transcription factor DNA-binding domain and the transcription factor activation domain is operably linked to the DRD.

[0046] In some embodiments, the method further includes introducing into the cell a fourth nucleic acid sequence encoding a protein of interest, the fourth nucleic acid sequence being operably linked to an inducible promoter comprising a specific polynucleotide binding site. In some embodiments, the protein of interest is a heterologous protein. In one embodiment, the fourth nucleic acid sequence is on the same nucleic acid molecule as the first, second, and third nucleic acid sequences. In one embodiment, the fourth nucleic acid sequence is on a different nucleic acid molecule from the first, second, and third nucleic acid sequences.

[0047] In some embodiments, the protein of interest is selected from the group consisting of a cytokine, an antibody, or an antigen-binding fragment thereof, a clotting factor, an enzyme, a gene-editing protein, a T-cell receptor (TCR), and a chimeric antigen receptor (CAR).

[0048] In some embodiments, the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

[0049] In some embodiments, the protein of interest is a secreted protein.

[0050] In some embodiments, the nucleic acid molecule is introduced into cells by a plasmid or a viral vector.In one embodiment, the viral vector is derived from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus.In one embodiment, the viral vector is selected from the group consisting of lentivirus vector, gamma retrovirus vector, adeno-associated virus (AAV) vector, adenovirus vector, and herpesvirus vector.

[0051] In some embodiments, the nucleic acid molecule is introduced into the cell by a non-viral delivery method.

[0052] In some embodiments, the cell is a T cell, a natural killer cell (NK cell), or a tumor-infiltrating lymphocyte (TIL). In some embodiments, the cell is a stem cell, a liver cell, a blood cell, a pancreatic cell, a neuron, an eye cell, a muscle cell, or a bone cell.

[0053] Also provided by the present disclosure are methods related to treating or preventing disease.

[0054] In one aspect, the disclosure provides a method of treating or preventing a disease in a subject in need thereof, the method comprising: (a) providing a cell population; (b) introducing into at least one cell within the cell population at least one nucleic acid molecule, the at least one nucleic acid molecule comprising: (i) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; and (ii) a protein of interest that prevents or treats the disease, or a symptom thereof. wherein the fourth nucleic acid sequence comprises the second polynucleotide operably linked to an exogenous inducible promoter comprising a specific polynucleotide binding site; (c) delivering the cell to the subject; and (d) administering to the subject a ligand that stabilizes the DRD sufficiently to allow expression of at least one of a transcription factor activation domain and a transcription factor DNA binding domain, in an amount sufficient to bind to the specific polynucleotide binding site and form a transcription factor that allows expression of a protein of interest in the cell, wherein expression of the protein of interest is controlled by the presence of the ligand in the subject, and wherein the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0055] In one aspect, the disclosure provides a method of introducing modified cells into a subject in need of disease treatment or prevention, the method comprising: (a) providing a cell population; (b) introducing at least one nucleic acid molecule into at least one cell within the cell population, the at least one nucleic acid molecule comprising: (i) a first polynucleotide comprising: a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD; and (ii) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter that comprises the specific polynucleotide binding site; and (c) delivering the cell to the subject.

[0056] In one aspect, the present disclosure provides a method of introducing modified cells into a subject in need of disease treatment or prevention, the method comprising: (a) providing a cell population; (b) introducing at least one nucleic acid molecule or a first polynucleotide and a second polynucleotide of any of the above-listed aspects into at least one cell within the cell population; and delivering the cell to the subject.

[0057] In one embodiment, the disclosure provides a method of genetically modifying one or more cells in a subject in need of disease treatment or prevention, the method comprising: (a) introducing at least one nucleic acid molecule into at least one cell of the subject, the at least one nucleic acid molecule comprising: (i) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug response domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD upon expression in the cell; and (ii) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter that comprises the specific polynucleotide binding site.

[0058] In one aspect, the disclosure provides a method of genetically modifying one or more cells in a subject in need of disease treatment or prevention, the method comprising: (a) introducing into at least one cell of the subject at least one nucleic acid molecule, the at least one nucleic acid molecule comprising: (i) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD upon expression in the cell; and (ii) a protein of interest that treats the disease. (b) administering to a subject a ligand that stabilizes the DRD sufficiently to permit expression of at least one of a transcription factor activation domain and a transcription factor DNA binding domain, in an amount sufficient to bind to the specific polynucleotide binding site and form a transcription factor that permits expression of the protein of interest in the cell, wherein expression of the protein of interest is controlled by the presence of the ligand in the subject, and wherein the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0059] In one aspect, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising: (a) providing a cell population; (b) introducing at least one first nucleic acid molecule and at least one second nucleic acid molecule into at least one cell within the cell population, wherein (i) the first nucleic acid molecule comprises a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD upon expression in the cell; and (ii) the second nucleic acid molecule induces a disease. (c) delivering the cells to the subject; and (d) administering to the subject a ligand that stabilizes the DRD sufficiently to allow expression of a transcription factor activation domain and a transcription factor DNA binding domain, in an amount sufficient to bind to the specific polynucleotide binding site and form a transcription factor that allows expression of the protein of interest in the cells, wherein expression of the protein of interest is controlled by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0060] In one aspect, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising: (a) providing a cell population; (b) introducing at least one first nucleic acid molecule and at least one second nucleic acid molecule into at least one cell within the cell population, wherein (i) the first nucleic acid molecule comprises a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD upon expression in the cell; (ii) the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein of interest that prevents and / or treats the disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter that comprises the specific polynucleotide binding site; and (c) delivering the cell to the subject.

[0061] In a related embodiment, the present disclosure provides a method for preventing and / or treating a disease in a subject in need thereof. The method includes: (a) providing a cell population; and (b) introducing at least one first nucleic acid molecule and at least one second nucleic acid molecule into at least one cell within the cell population. In this example method, the first nucleic acid molecule includes a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug-responsive domain (DRD). At least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD when expressed in the cell, and the second nucleic acid molecule includes a fourth nucleic acid sequence encoding a protein of interest that prevents and / or treats the disease in the subject in need thereof. The fourth nucleic acid sequence is operably linked to an exogenous inducible promoter that includes the specific polynucleotide binding site. The method also includes (c) delivering the cells to a subject, and (d) administering to the subject a ligand that stabilizes the DRD sufficiently to allow expression of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to bind to the specific polynucleotide binding site and form a transcription factor that allows expression of a protein of interest in the cell. In this example method, expression of the protein of interest is controlled by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0062] In a related embodiment, the disclosed methods of treatment and prevention may be achieved by introducing a single vector into a cell, wherein the vector carries a first nucleic acid molecule and a second nucleic acid molecule, wherein (i) the first nucleic acid molecule comprises a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor activation domain and / or the transcription factor DNA-binding domain is operably linked to the DRD upon expression in the cell, and the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein of interest that treats or prevents the disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter that comprises the specific polynucleotide binding site.

[0063] In some alternative embodiments, the disclosed methods of treatment and prevention may be achieved by introducing a first vector and a second vector into a cell, wherein the first vector comprises a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor activation domain and / or the transcription factor DNA-binding domain are operably linked to the DRD when expressed in the cell, and the second vector comprises a fourth nucleic acid sequence encoding a protein of interest that prevents and / or treats a disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter that comprises the specific polynucleotide binding site.

[0064] In some embodiments, nucleic acid molecule is introduced into cell by plasmid or viral vector.In some aspects, viral vector is derived from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus and picornavirus.In some aspects, viral vector is selected from the group consisting of lentivirus vector, gamma retrovirus vector, adeno-associated virus (AAV) vector, adenovirus vector and herpesvirus vector.

[0065] In some embodiments, the nucleic acid molecule is introduced into the cell by a non-viral delivery method.

[0066] Also provided by the present disclosure is a system for regulatable expression of a protein of interest in a cell, the system comprising: (a) a first polynucleotide encoding a transcription factor linked to a drug response domain (DRD), wherein the transcription factor selectively transcribes a polynucleotide sequence encoding the protein of interest; (b) a second polynucleotide comprising an exogenous transcription factor binding site positioned upstream and adjacent to the nucleic acid sequence encoding the protein of interest; (c) introducing the first polynucleotide and the second polynucleotide into the cell under conditions such that the first polynucleotide and the second polynucleotide are stably integrated into the genome of the cell; and (d) modulating expression of the transcription factor by adding a ligand that stabilizes the DRD, wherein the transcription factor specifically binds to the transcription factor binding site positioned upstream and adjacent to the polynucleotide sequence encoding the protein of interest, and wherein expression of the protein of interest is controlled by the amount of transcription factor present in the cell.

[0067] The present disclosure also provides a pharmaceutical composition comprising a composition described herein and a pharmaceutically acceptable excipient. In certain embodiments, for example, the following are provided: (Item 1) 1. A modified cell comprising a first polynucleotide, the first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD); at least one of the transcription factor activation domain, the transcription factor DNA binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor capable of activating transcription of a fourth nucleic acid sequence upon binding to the specific polynucleotide binding site; The modified cell, wherein the fourth nucleic acid sequence encodes a protein of interest and is operably linked to an exogenous inducible promoter containing the specific polynucleotide binding site. (Item 2) 1. A modified cell comprising a first polynucleotide, the first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD); at least one of the transcription factor activation domain, the transcription factor DNA binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor capable of activating transcription of a fourth nucleic acid sequence upon binding to the specific polynucleotide binding site; The modified cell, wherein the fourth nucleic acid sequence encodes a protein of interest and is operably linked to the specific polynucleotide binding site. (Item 3) 3. The modified cell of item 2, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site. (Item 4) 4. The modified cell according to any one of items 1 to 3, wherein the protein of interest is a heterologous protein. (Item 5) 5. The modified cell of any one of items 1 to 4, wherein the fourth nucleic acid sequence is located on the first polynucleotide. (Item 6) 5. The modified cell of any one of items 1 to 4, further comprising a second polynucleotide, wherein the second polynucleotide comprises the fourth nucleic acid sequence. (Item 7) 7. The modified cell of any one of items 1 to 6, wherein the transcription factor DNA-binding domain is derived from a parent protein selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL. (Item 8) 8. The modified cell according to any one of items 1 to 7, wherein the transcription factor activation domain is derived from a parent protein, and the parent protein is p65. (Item 9) 1. A modified cell comprising a polynucleotide comprising a first nucleic acid sequence encoding a drug response domain (DRD) and a second nucleic acid sequence encoding a transcription factor, wherein the transcription factor is operably linked to the DRD; The modified cell, wherein the transcription factor is capable of binding to a specific polynucleotide binding site and activating transcription of a third nucleic acid sequence encoding a protein of interest, and the third nucleic acid sequence is operably linked to an exogenous inducible promoter that contains the specific polynucleotide binding site. (Item 10) 1. A modified cell comprising a polynucleotide comprising a first nucleic acid sequence encoding a drug response domain (DRD) and a second nucleic acid sequence encoding a transcription factor, wherein the transcription factor is operably linked to the DRD; The modified cell, wherein the transcription factor is capable of binding to a specific polynucleotide binding site and activating transcription of a third nucleic acid sequence encoding a protein of interest, and the third nucleic acid sequence is operably linked to the specific polynucleotide binding site. (Item 11) 11. The modified cell of claim 10, wherein the third nucleic acid sequence is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site. (Item 12) 12. The modified cell according to any one of items 9 to 11, wherein the protein of interest is a heterologous protein. (Item 13) 13. The modified cell according to any one of items 9 to 12, wherein the third nucleic acid sequence is located on the polynucleotide comprising the first nucleic acid sequence and the second nucleic acid sequence. (Item 14) 13. The modified cell of any one of items 9 to 12, further comprising a second polynucleotide comprising the third nucleic acid sequence. (Item 15) 15. The modified cell of any one of items 1 to 14, wherein the DRD is derived from a parent protein selected from the group comprising human carbonic anhydrase 2 (CA2), human DHFR, E. coli DHFR (ecDHFR), human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. (Item 16) Item 1 to 1, wherein the DRD is stabilized in the presence of a ligand selected from the group consisting of acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). 6. The modified cell of any one of claims 5. (Item 17) 17. The modified cell according to any one of items 1 to 16, wherein the protein of interest is a wild-type protein. (Item 18) 18. The modified cell according to any one of items 1 to 17, wherein the protein of interest is a therapeutic protein. (Item 19) 19. The modified cell of item 18, wherein the protein of interest is selected from the group consisting of a cytokine, an antibody, a clotting factor, an enzyme, a gene-editing protein, a T cell receptor (TCR), and a chimeric antigen receptor (CAR). (Item 20) 18. The modified cell according to any one of items 1 to 17, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre. (Item 21) 18. The modified cell according to any one of items 1 to 17, wherein the protein of interest is a secreted protein. (Item 22) 22. The modified cell of any one of items 1 to 21, wherein the cell is a T cell, a natural killer cell (NK cell), or a tumor-infiltrating lymphocyte (TIL). (Item 23) 22. The modified cell of any one of items 1 to 21, wherein the cell is a stem cell, a liver cell, a blood cell, a pancreatic cell, a nerve cell, an eye cell, a muscle cell, or a bone cell. (Item 24) A nucleic acid molecule comprising: a. a first nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; b. a second nucleic acid sequence encoding a drug responsive domain (DRD); The nucleic acid molecule comprising: (Item 25) c. The nucleic acid molecule of item 24, further comprising a third nucleic acid sequence encoding a transcription factor activation domain, wherein either (i) the transcription factor DNA binding domain is operably linked to the DRD, (ii) the transcription factor activation domain is operably linked to the DRD, or (iii) a combination of the transcription factor DNA binding domain and the transcription factor activation domain is operably linked to the DRD. (Item 26) d. The nucleic acid molecule of item 24 or 25, further comprising a fourth nucleic acid sequence encoding a protein of interest, wherein the fourth nucleic acid sequence is operably linked to an inducible promoter comprising the specific polynucleotide binding site. (Item 27) 27. The nucleic acid molecule of any one of items 24 to 26, wherein the transcription factor DNA-binding domain is derived from a parent protein selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL. (Item 28) 28. The nucleic acid molecule according to any one of items 24 to 27, wherein the transcription factor activation domain is derived from a parent protein, and the parent protein is p65. (Item 29) A nucleic acid molecule comprising: a. a first nucleic acid sequence encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; b. a second nucleic acid sequence encoding a drug responsive domain (DRD); wherein the transcription factor is operably linked to the DRD. (Item 30) c. The nucleic acid molecule of Item 29, further comprising a third nucleic acid sequence encoding a protein of interest, wherein the third nucleic acid sequence is operably linked to an inducible promoter comprising the specific polynucleotide binding site. (Item 31) 31. The nucleic acid molecule of any one of items 24 to 30, wherein the DRD is derived from a parent protein selected from the group comprising human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. (Item 32) 32. The nucleic acid molecule of any one of items 24 to 31, wherein the DRD is stabilized in the presence of a ligand selected from the group consisting of acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). (Item 33) 33. The nucleic acid molecule of any one of items 26 to 28 or 30 to 32, wherein the protein of interest is a wild-type protein. (Item 34) 33. The nucleic acid molecule of any one of items 26 to 28 or 30 to 32, wherein the protein of interest is a therapeutic protein. (Item 35) 35. The nucleic acid molecule of item 34, wherein the protein of interest is selected from the group consisting of a cytokine, an antibody, a coagulation factor, an enzyme, a gene-editing protein, a T cell receptor (TCR), and a chimeric antigen receptor (CAR). (Item 36) 33. The nucleic acid molecule of any one of items 26 to 28 or 30 to 32, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre. (Item 37) 33. The nucleic acid molecule according to items 26 to 28 or 30 to 32, wherein the protein of interest is a secreted protein. (Item 38) A vector comprising the nucleic acid molecule according to any one of Items 24 to 37. (Item 39) 39. The vector according to item 38, wherein the vector is a plasmid or a viral vector. (Item 40) 40. The vector of item 39, wherein the viral vector is derived from an adenovirus, an adeno-associated virus (AAV), an alphavirus, a flavivirus, a herpesvirus, a measles virus, a rhabdovirus, a retrovirus, a lentivirus, a Newcastle disease virus (NDV), a poxvirus, or a picornavirus. (Item 41) 40. The vector of item 39, wherein the viral vector is selected from the group consisting of a lentiviral vector, a gammaretroviral vector, an adeno-associated viral (AAV) vector, an adenoviral vector, and a herpes viral vector. (Item 42) A first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises: a first nucleic acid sequence encoding a transcription factor activation domain, a specific polynucleotide binding site; a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds; and a third nucleic acid sequence encoding a drug response domain (DRD), wherein at least one of the transcription factor activation domain, the transcription factor DNA binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; the second polynucleotide is a fourth nucleic acid sequence encoding a protein of interest, said fourth nucleic acid sequence operably linked to an inducible promoter comprising said specific polynucleotide binding site; the first polynucleotide and the second polynucleotide, wherein the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that can activate transcription upon binding to the specific polynucleotide binding site, and wherein the first polynucleotide and the second polynucleotide are each carried on a single vector, or the first polynucleotide and the second polynucleotide are carried on separate vectors. (Item 43) A first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises: a first nucleic acid sequence encoding a transcription factor and a second nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor is operably linked to the DRD, and the transcription factor is capable of activating transcription upon binding to a specific polynucleotide binding site; the second polynucleotide is a third nucleic acid sequence encoding a protein of interest, said third nucleic acid sequence operably linked to an inducible promoter comprising said specific polynucleotide binding site; The first polynucleotide and the second polynucleotide are each carried on a single vector, or the first polynucleotide and the second polynucleotide are carried on separate vectors. (Item 44) 44. The first and second polynucleotides according to item 42 or 43, wherein the DRD is derived from a parent protein selected from the group comprising human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. (Item 45) 45. The first polynucleotide and the second polynucleotide according to any one of items 42 to 44, wherein the DRD is stabilized in the presence of a ligand selected from the group comprising acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). (Item 46) 46. The first polynucleotide and the second polynucleotide according to any one of Items 42 to 45, wherein the protein of interest is a wild-type protein. (Item 47) 46. The first polynucleotide and the second polynucleotide according to any one of Items 42 to 45, wherein the protein of interest is a therapeutic protein. (Item 48) 48. The first polynucleotide and the second polynucleotide according to Item 47, wherein the protein of interest is selected from the group consisting of a cytokine, an antibody, a coagulation factor, an enzyme, a gene-editing protein, a T cell receptor (TCR), and a chimeric antigen receptor (CAR). (Item 49) 46. The first polynucleotide and the second polynucleotide according to any one of Items 42 to 45, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre. (Item 50) 46. The method according to any one of items 42 to 45, wherein the protein of interest is a secreted protein. The first polynucleotide and the second polynucleotide. (Item 51) 1. A method for producing a modified cell, said method comprising: a. a first nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; b. a second nucleic acid sequence encoding a drug responsive domain (DRD); The method comprises introducing into a cell a nucleic acid molecule comprising: (Item 52) 52. The method of claim 51, wherein the nucleic acid molecule further comprises a third nucleic acid sequence encoding a transcription factor activation domain. (Item 53) 53. The method of claim 52, wherein either (i) the transcription factor DNA binding domain is operably linked to the DRD, (ii) the transcription factor activation domain is operably linked to the DRD, or (iii) a combination of the transcription factor DNA binding domain and the transcription factor activation domain is operably linked to the DRD. (Item 54) 54. The method of claim 53, further comprising introducing into the cell a fourth nucleic acid sequence encoding a protein of interest, wherein the fourth nucleic acid sequence is operably linked to an inducible promoter comprising the specific polynucleotide binding site. (Item 55) 55. The method of item 54, wherein the protein of interest is a heterologous protein. (Item 56) 56. The method of item 54 or 55, wherein the fourth nucleic acid sequence is on the same nucleic acid molecule as the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence. (Item 57) 56. The method of item 54 or 55, wherein the fourth nucleic acid sequence is on a different nucleic acid molecule than the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence. (Item 58) 58. The method according to any one of items 54 to 57, wherein the protein of interest is selected from the group consisting of a cytokine, an antibody, a coagulation factor, an enzyme, a gene-editing protein, a T cell receptor (TCR), and a chimeric antigen receptor (CAR). (Item 59) 58. The method according to any one of items 54 to 57, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre. (Item 60) 58. The method according to any one of items 54 to 57, wherein the protein of interest is a secreted protein. (Item 61) 61. The method of any one of items 51 to 60, wherein the nucleic acid molecule is introduced into the cell by a plasmid or viral vector. (Item 62) 62. The method of claim 61, wherein the viral vector is derived from an adenovirus, an adeno-associated virus (AAV), an alphavirus, a flavivirus, a herpesvirus, a measles virus, a rhabdovirus, a retrovirus, a lentivirus, a Newcastle disease virus (NDV), a poxvirus, or a picornavirus. (Item 63) 62. The method of claim 61, wherein the viral vector is selected from the group consisting of a lentiviral vector, a gammaretroviral vector, an adeno-associated viral (AAV) vector, an adenoviral vector, and a herpes viral vector. (Item 64) 61. The method of any one of items 51 to 60, wherein the nucleic acid molecule is introduced into the cell by a non-viral delivery method. (Item 65) 65. The method of any one of items 51 to 64, wherein the cells are T cells, natural killer cells (NK cells), or tumor-infiltrating lymphocytes (TILs). (Item 66) 65. The method according to any one of items 51 to 64, wherein the cells are stem cells, liver cells, blood cells, pancreatic cells, nerve cells, eye cells, muscle cells, or bone cells. (Item 67) 1. A method of treating or preventing a disease in a subject in need thereof, said method comprising: a. providing a cell population; b. introducing at least one nucleic acid molecule into at least one cell within said cell population, said at least one nucleic acid molecule comprising: i. a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; ii. a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest for preventing or treating said disease or a symptom thereof, said fourth nucleic acid sequence being operably linked to an exogenous inducible promoter comprising said specific polynucleotide binding site; c. delivering the cells to the subject; and d. administering to the subject a ligand that stabilizes the DRD sufficiently to permit expression of at least one of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to bind to the specific polynucleotide binding site and form a transcription factor that permits expression of the protein of interest in the cell; The method, wherein expression of the protein of interest is controlled in the subject by the presence of the ligand, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest. (Item 68) 1. A method for introducing modified cells into a subject in need of disease treatment or prevention, said method comprising: a. providing a cell population; b. introducing at least one nucleic acid molecule into at least one cell within said cell population, said at least one nucleic acid molecule comprising: i. a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; ii. a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; and c. delivering said cells to said subject. (Item 69) 1. A method for introducing modified cells into a subject in need of disease treatment or prevention, said method comprising: a. providing a cell population; b. at least one nucleic acid molecule according to any one of items 24 to 37 or items 42 to 49 introducing the first and second polynucleotides of any one of claims 50 into at least one cell within said cell population; and c. delivering said cells to said subject. (Item 70) 1. A method for genetically modifying one or more cells in a subject in need of treatment or prevention of a disease, said method comprising: a. introducing at least one nucleic acid molecule into at least one cell of said subject, said at least one nucleic acid molecule comprising: i. a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD upon expression in the cell; ii. a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site. (Item 71) 1. A method for genetically modifying one or more cells in a subject in need of treatment or prevention of a disease, said method comprising: a. introducing at least one nucleic acid molecule into at least one cell of said subject, said at least one nucleic acid molecule comprising: i. a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD upon expression in the cell; ii. a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; and b. administering to the subject a ligand that stabilizes the DRD sufficiently to permit expression of at least one of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to bind to the specific polynucleotide binding site and form a transcription factor that permits expression of the protein of interest in the cell; The method, wherein expression of the protein of interest is controlled in the subject by the presence of the ligand, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest. (Item 72) 1. A method of treating a disease in a subject in need thereof, said method comprising: a. providing a cell population; b. introducing at least one of the first nucleic acid molecules and at least one of the second nucleic acid molecules into at least one cell within the cell population; i. the first nucleic acid molecule comprises a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD upon expression in the cell; ii. the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein of interest for treating the disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; c. delivering the cells to the subject; and d. administering to the subject a ligand that stabilizes the DRD sufficiently to permit expression of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to bind to the specific polynucleotide binding site and form a transcription factor that permits expression of the protein of interest in the cell; The method, wherein expression of the protein of interest is controlled in the subject by the presence of the ligand, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest. (Item 73) 1. A method of treating a disease in a subject in need thereof, said method comprising: a. providing a cell population; b. introducing at least one of the first nucleic acid molecules and at least one of the second nucleic acid molecules into at least one cell within the cell population; i. the first nucleic acid molecule comprises a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD upon expression in the cell; ii. the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein of interest that treats the disease, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter that comprises the specific polynucleotide binding site; and c. delivering said cells to said subject. (Item 74) 74. The method of any one of items 67 to 73, wherein the nucleic acid molecule is introduced into the cell by a plasmid or viral vector. (Item 75) 75. The method of item 74, wherein the viral vector is derived from an adenovirus, an adeno-associated virus (AAV), an alphavirus, a flavivirus, a herpesvirus, a measles virus, a rhabdovirus, a retrovirus, a lentivirus, a Newcastle disease virus (NDV), a poxvirus, or a picornavirus. (Item 76) 75. The method of claim 74, wherein the viral vector is selected from the group consisting of a lentiviral vector, a gammaretroviral vector, an adeno-associated viral (AAV) vector, an adenoviral vector, and a herpes viral vector. (Item 77) 74. The method of any one of items 67 to 73, wherein the nucleic acid molecule is introduced into the cell by a non-viral delivery method. (Item 78) A system for regulatable expression of a protein of interest in a cell, said system comprising: a. a first polynucleotide encoding a transcription factor linked to a drug response domain (DRD), wherein the transcription factor selectively transcribes a polynucleotide sequence encoding the protein of interest; b. a second polynucleotide comprising an exogenous transcription factor binding site positioned upstream and adjacent to the nucleic acid sequence encoding the protein of interest; c. introducing the first polynucleotide and the second polynucleotide into the cell under conditions such that the first polynucleotide and the second polynucleotide are stably integrated into the genome of the cell; d. Regulating expression of the transcription factor by adding a ligand that stabilizes the DRD. Including, The transcription factor is located upstream of the polynucleotide sequence encoding the protein of interest. The system wherein the target protein specifically binds to adjacently arranged transcription factor binding sites, and the expression of the target protein is controlled by the amount of the transcription factor present in the cell. [Brief explanation of the drawings]

[0068] [Figure 1] Figure 1 shows a schematic diagram of the transcription factor system design scheme. A shows a schematic diagram of a transcription factor construct, referred to as a "DRD-TF construct," which contains nucleic acid sequences encoding a transcription factor DNA-binding domain, a transcription factor activation domain, and a drug-responsive domain (DRD). B shows a schematic diagram of a payload construct, which contains an inducible promoter containing a binding site for the transcription factor DNA-binding domain. [Figure 2] Ligand-dependent activity of a transcription factor system containing DRD-regulated transcription factors with different DRDs is shown. (A) Western blots of lysates from untransfected ("mock") HEK293T cells and HEK293T cells transfected with a construct encoding a constitutive transcription factor (construct ZFHD-055, "Cons.") or a construct encoding a transcription factor operably linked to a DRD derived from the CA2, ecDHFR, ER, or hDHFR parent protein are shown. Details of each construct and ligand treatment conditions are provided in Tables 4 and 6. The top panel of the Western blot shows bands for endogenous p65, the transcription factor and DRD polypeptides encoded by each of the DRD-TF constructs, and the transcription factor polypeptide encoded by the constitutive construct ZFHD-055. (B) Quantification of the Western blot in Figure 2A, normalized to 1.0 for the constitutive condition. [Figure 3]Figure 1 shows the ligand-dependent activity of a transcription factor system containing the ecDHFR DRD-regulated transcription factor. A shows a schematic diagram of transcription factor construct ZFHD-005. B shows a schematic diagram of payload construct ZFHD-007. C shows a schematic diagram of constitutive transcription factor construct ZFHD-004. D shows a Western blot of lysates from U2OS cells stably integrated with the indicated constructs and treated with 10 μM TMP or 0.1% DMSO. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 44.3 kDa represents the transcription factor and DRD polypeptides encoded by transcription factor construct ZFHD-005. The band appearing at approximately 26.5 kDa represents the transcription factor polypeptide encoded by construct ZFHD-004. E shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry on U2OS cells stably integrated with the indicated constructs treated with 10 μM TMP or 0.1% DMSO. [Figure 4] Figure 4 shows the dose response of the transcription factor system, including the ecDHFR DRD-regulated transcription factor, to ligands. Figure 4A shows a Western blot of lysates from U2OS cells stably integrating constructs ZFHD-005 and ZFHD-007 treated with DMSO or the indicated concentrations of TMP. The lane labeled "U2OS" represents untransduced U2OS cells treated with TMP. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 44.3 kDa represents the transcription factor and DRD polypeptide encoded by transcription factor construct ZFHD-005. Figure 4B shows quantification of the "ZFHD-005 polypeptide" band shown in the Western blot in Figure 4A. Fluorescence was normalized to endogenous p65. (C) GFP median fluorescence intensity (MFI) as assessed by flow cytometry on U2OS cells harboring stably integrated constructs ZFHD-005 and ZFHD-007 treated with the indicated concentrations of TMP. The highest concentration of TMP used in Figure 4C is 33 μM. Data shown are from triplicates. Error bars represent standard deviation. [Figure 5]Figure 1 shows the ligand-dependent activity of the transcription factor system, including the ecDHFR DRD-regulated transcription factor, in T cells. (A) Western blot of lysates from untransduced T cells or T cells transduced with virus (OTLV-ZFHD-005 or OTLV-ZFHD-007) treated with TMP or DMSO. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 44.3 kDa (indicated by an arrow) represents the transcription factor and DRD polypeptide encoded by the transcription factor construct ZFHD-005. (B) GFP median fluorescence intensity (MFI) as assessed by flow cytometry of untransduced T cells or T cells transduced with virus generated from the indicated constructs treated with TMP or DMSO. Data shown are from triplicates. Error bars represent standard deviation from the mean. [Figure 6] Figure 6 shows the ligand-dependent activity of a transcription factor system containing the CA2 DRD-regulating transcription factor in ARPE-19 cells. Figure 6A shows a schematic diagram of the transcription factor construct ZFHD-019. Figure 6B shows Western blots of lysates from untransduced ARPE-19 cells or ARPE-19 cells stably harboring the ZFHD-019 and ZFHD-007 constructs treated with 10 μM ACZ or 1% DMSO. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 55.8 kDa represents the transcription factor and DRD polypeptide encoded by the transcription factor construct ZFHD-019. Figure 6C shows quantification of the "ZFHD-019 polypeptide" band shown in the Western blot in Figure 6B. Fluorescence was normalized to endogenous p65. D shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry of non-transduced ARPE-19 cells or ARPE-19 cells that have stably integrated the indicated constructs, either untreated or treated with 10 μM ACZ or 1% DMSO. Data shown are from triplicates. Error bars represent standard deviation from the mean. Non-transduced ARPE-19 cells and ARPE-19 cells that have stably integrated the construct ZFHD-007, as indicated on the graph, were treated with DMSO. [Figure 7] Figure 7 shows the dose response of the transcription factor system, including the CA2 DRD-regulating transcription factor, to ligands. Figure 7A shows a Western blot of lysates from ARPE-19 cells harboring stably integrated constructs ZFHD-007 and ZFHD-019 treated with the indicated concentrations of ACZ. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 55.8 kDa represents the transcription factor and DRD polypeptide encoded by the transcription factor construct ZFHD-019. Figure 7B shows quantification of the "ZFHD-019 polypeptide" band shown in the Western blot in Figure 7A. Fluorescence was normalized to the endogenous p65 band. [Figure 8] Figure 1 shows the dose response of the transcription factor system, including the CA2 DRD-regulating transcription factor, to ligands. The graph shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry in U2OS cells stably integrated with constructs ZFHD-007 and ZFHD-019, treated with the indicated concentrations of ACZ. Data shown are from two replicates. Error bars represent standard deviation. [Figure 9] Figure 1 shows the ligand-dependent activity of a transcription factor system containing the CA2 DRD-regulating transcription factor in Jurkat cells. A shows a schematic diagram of the transcription factor construct ZFHD-048. B shows a schematic diagram of the payload construct ZFHD-022. C shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry on Jurkat cells stably integrated with constructs ZFHD-048 and ZFHD-022 and treated with DMSO (0.1%) or ACZ (10 μM final concentration). Data presented are for cells that were positive for the transduction marker. [Figure 10A] Figure 1 shows the ligand-dependent activity of a single vector transcription factor system containing the ecDHFR DRD-regulated transcription factor. A schematic diagram of construct ZFHD-012 is shown. [Figure 10B] Figure 1 shows the ligand-dependent activity of a single vector transcription factor system containing the ecDHFR DRD-regulated transcription factor. A schematic diagram of construct ZFHD-018 is shown. [Figure 10C]Figure 1 shows the ligand-dependent activity of a single-vector transcription factor system containing the ecDHFR DRD-regulated transcription factor. Western blots of lysates from U2OS cells transduced with lentivirus prepared from the indicated constructs and treated with 10 μM TMP or 0.1% DMSO are shown. The band appearing at approximately 44.3 kDa represents the transcription factor and DRD polypeptide encoded by the indicated construct. In the single-vector construct, the presence of a stop codon at the end of the EGFP sequence and a stop codon at the end of the transcription factor-DRD sequence results in the approximately 44.3 kDa band representing the transcription factor and DRD polypeptide. [Figure 10D] Figure 1 shows the ligand-dependent activity of a single-vector transcription factor system containing the ecDHFR DRD-regulated transcription factor. Western blots of lysates from U2OS cells transduced with lentivirus prepared from the indicated constructs and treated with 10 μM TMP or 0.1% DMSO are shown. The band appearing at approximately 44.3 kDa represents the transcription factor and DRD polypeptide encoded by the indicated construct. In the single-vector construct, the presence of a stop codon at the end of the EGFP sequence and a stop codon at the end of the transcription factor-DRD sequence results in the approximately 44.3 kDa band representing the transcription factor and DRD polypeptide. [Figure 10E] Ligand-dependent activity of the single-vector transcription factor system containing the ecDHFR DRD-regulated transcription factor is shown. GFP median fluorescence intensity (MFI) as assessed by flow cytometry is shown for U2OS cells transduced with lentiviruses generated from the indicated constructs and treated with 10 μM TMP or 0.1% DMSO. [Figure 10F] Ligand-dependent activity of the single-vector transcription factor system containing the ecDHFR DRD-regulated transcription factor is shown. GFP median fluorescence intensity (MFI) as assessed by flow cytometry is shown for U2OS cells transduced with lentiviruses generated from the indicated constructs and treated with 10 μM TMP or 0.1% DMSO. [Figure 11]Figure 1 shows the ligand-dependent activity of a single-vector transcription factor system containing a CA2 DRD-regulating transcription factor. A shows a schematic diagram of the single-vector system, designated as construct ZFHD-036. B shows the GFP median fluorescence intensity (MFI) as assessed by flow cytometry in Jurkat cells transduced with lentivirus generated from the indicated constructs and treated with 10 μM ACZ or 0.1% DMSO. ZFHD-036.1 and ZFHD-036.2 on the graph represent two cell lines, each transduced with lentivirus generated from construct ZFHD-036. [Figure 12] Figure 1 shows the ligand-dependent activity of transcription factor systems containing variants of transcription factor constructs. A shows a schematic diagram of the transcription factor construct variants. B shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry on U2OS cells stably integrated with the indicated constructs and treated with either 0.1% DMSO or 10 μM TMP. [Figure 13] Figure 1 shows a ligand response time course analysis of a transcription factor system containing variants of the transcription factor construct. The graph shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry of U2OS cells stably integrated with the indicated constructs and treated with either 0.1% DMSO or 10 µM TMP for the indicated times. [Figure 14] Figure 1 shows the ligand-dependent activity of transcription factor systems containing variants of the payload constructs. A shows a schematic diagram of payload construct ZFHD-007. B shows a schematic diagram of payload construct ZFHD-017. C-D show GFP median fluorescence intensity (MFI) as assessed by flow cytometry on U2OS cells stably integrated with the indicated constructs and treated with either 0.1% DMSO or 10 μM TMP. [Figure 15] 1 shows the ligand-dependent activity of a transcription factor system containing a payload construct encoding a secreted IL12 payload. The graph shows the concentration of secreted IL12 in supernatants collected from U2OS cells that have stably integrated the indicated constructs and are treated with either 0.1% DMSO or 10 μM TMP. [Figure 16A] Ligand-dependent regulation of different transcription factors operably linked to the DRD derived from the parent CA2 protein is shown. Western blots of lysates from untransfected ("mock") HEK293T cells and HEK293T cells transfected with the following constructs: (1) cjun-001 ("001"), (2) cjun-002 ("002"), or (3) cjun-003 ("003"). Each construct-transfected cell population is shown after treatment with DMSO or ACZ (indicated by a "+" symbol). The band labeled "c-Jun-001 and -002 polypeptides" identifies the CA2-linker-C-Jun polypeptides encoded by the cjun-001 and cjun-002 constructs. The band labeled "c-Jun-003 polypeptide" identifies the c-Jun polypeptide encoded by the cjun-003 construct. [Figure 16B] Figure 16A shows the ligand-dependent regulation of different transcription factors functionally linked to the DRD from the parent CA2 protein. Quantification of the Western blot in Figure 16A is shown. [Figure 16C] Figure 1 shows ligand-dependent regulation of different transcription factors operably linked to the DRD derived from the parent CA2 protein. Western blots of lysates from untransfected ("mock") HEK293T cells and HEK293T cells transfected with the following constructs: (1) FOXP3-013 ("013"), (2) FOXP3-014 ("014"), or (3) FOXP3-015 ("015"). Each construct-transfected cell population is shown after treatment with DMSO or ACZ (indicated by a "+" symbol). The band labeled "FOXP3-013 and -014 polypeptides" identifies the CA2-FOXP3 polypeptides encoded by the FOXP3-013 and FOXP3-014 constructs. The band labeled "FOXP3-015 polypeptide" identifies the FOXP3 polypeptide encoded by the construct FOXP3-015. [Figure 16D]Figure 16C shows the ligand-dependent regulation of different transcription factors functionally linked to the DRD from the parent CA2 protein. Quantification of the Western blot in Figure 16C is shown. [Figure 17] Ligand-dependent regulation of a c-Jun transcription factor construct stably integrated into Jurkat cells is shown. (A) Western blots of lysates from untransduced ("mock") Jurkat cells and Jurkat cells transduced with lentiviruses generated from constructs cjun-001 ("001") and cjun-002 ("002"). Cell lines transduced with each construct are shown after treatment with DMSO or ACZ (indicated by a "+" symbol). Bands for c-Jun polypeptide and phosphorylated c-Jun polypeptide are indicated. (B) Quantification of the Western blot in Figure 17A. [Figure 18-1] FIG. 18 shows the nucleotide sequence of the pELDS-puro transfer vector (SEQ ID NO: 68). [Figure 18-2] FIG. 18 shows the nucleotide sequence of the pELDS-puro transfer vector (SEQ ID NO: 68). [Figure 19-1] FIG. 19 shows the nucleotide sequence of the pELNS-puro transfer vector (SEQ ID NO: 69). [Figure 19-2] FIG. 19 shows the nucleotide sequence of the pELNS-puro transfer vector (SEQ ID NO: 69). DETAILED DESCRIPTION OF THE INVENTION

[0069] Detailed Description Transcription factor system According to the present disclosure, a transcription factor system is a combination of one or more polynucleotides including: (1) one or more nucleic acid sequences encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; (2) a nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor is operably linked to the DRD; and (3) a nucleic acid sequence encoding a payload, the nucleic acid sequence being operably linked to an inducible promoter comprising the specific polynucleotide binding site.

[0070] In some embodiments, a combination of one or more polynucleotides of a transcription factor system may be used to create a system for modifying cells, e.g., immune cells, useful for treating disease, to allow regulatable expression of a protein of interest by controlling the presence of a transcription factor acting on a polynucleotide(s) encoding the payload or protein of interest.

[0071] In some embodiments, the combination of one or more polynucleotides of a transcription factor system comprises a polynucleotide comprising a first nucleic acid sequence encoding a transcription factor and a second nucleic acid sequence encoding a DRD.

[0072] The present disclosure also provides a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug-responsive domain (DRD). In this example, at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD exemplified herein. The second polynucleotide comprises a fourth nucleic acid sequence encoding a protein of interest, which is operably linked to an inducible promoter that comprises a specific polynucleotide-binding site. In this example, the transcription factor activation domain and the transcription factor DNA-binding domain interact to form a transcription factor that can activate transcription upon binding to the specific polynucleotide-binding site, and the first polynucleotide and the second polynucleotide are each carried on a single vector, or the first polynucleotide and the second polynucleotide are carried on separate vectors.

[0073] In a related example, the disclosure provides compositions and nucleic acids operable to regulate transcription. For example, the disclosure provides a first polynucleotide and a second polynucleotide of a regulatable transcription factor system. The first polynucleotide comprises a first nucleic acid sequence encoding a transcription factor and a second nucleic acid sequence encoding a drug response domain (DRD), where the transcription factor is operably linked to the DRD and the transcription factor is capable of activating transcription upon binding to a specific polynucleotide binding site. The second polynucleotide comprises a third nucleic acid sequence encoding a protein of interest, where the third nucleic acid sequence is operably linked to an inducible promoter comprising the specific polynucleotide binding site. The first polynucleotide and the second polynucleotide can each be carried on a single vector, or the first polynucleotide and the second polynucleotide can be carried on separate vectors.

[0074] In some embodiments, the combination of one or more polynucleotides of a transcription factor system comprises a first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, a second nucleic acid sequence encoding a transcription factor activation domain, and a third nucleic acid sequence encoding a DRD. In some aspects, the combination of one or more polynucleotides of a transcription factor system comprises a polynucleotide comprising the first, second, and third nucleic acid sequences. In some aspects, the combination of one or more polynucleotides of a transcription factor system comprises a polynucleotide comprising two of the first, second, and third nucleic acid sequences. In some aspects, the combination of one or more polynucleotides of a transcription factor system comprises a first polynucleotide comprising the first nucleic acid sequence, a second polynucleotide comprising the second nucleic acid sequence, and a third polynucleotide comprising the third nucleic acid sequence. In one aspect, the transcription factor DNA-binding domain is operably linked to the DRD. In another aspect, the transcription factor activation domain is operably linked to the DRD. In another aspect, both the transcription factor DNA-binding domain and the transcription factor activation domain are operably linked to the DRD. In some aspects, the transcription factor DNA-binding domain and the transcription factor activation domain are expressed as a transcription factor fusion protein.

[0075] According to the present disclosure, the transcription factor system encodes a transcription factor capable of driving expression of a payload. In some embodiments, the transcription factor is encoded by a first nucleic acid sequence encoding a transcription factor activation domain and a second nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site. The transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that activates transcription of the nucleic acid sequence encoding the payload upon binding to the specific polynucleotide binding site.

[0076] In some embodiments, the specific polynucleotide binding site comprises at least one nucleic acid site having a specific sequence recognized and bound by a transcription factor DNA binding domain. In some embodiments, the specific polynucleotide binding site comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten nucleic acid sites recognized by a DNA binding domain of the present disclosure. In some embodiments, the specific polynucleotide binding site comprises eight nucleic acid sites recognized by a DNA binding domain. In some embodiments, the specific polynucleotide binding site comprises two or more tandem nucleic acid sites, each having a specific sequence recognized and bound by a transcription factor DNA binding domain. In some aspects, the tandem nucleic acid sites comprise the same nucleic acid sequence. In some embodiments, the specific polynucleotide binding site comprises tandem repeat nucleic acid sites recognized by a DNA binding domain of the present disclosure.

[0077] As described herein, the transcription factor or a portion thereof is functionally linked to the DRD in the transcription factor system of the present disclosure. The presence, absence, or amount of a ligand that binds to or interacts with the DRD can regulate the stability of the transcription factor upon such binding or interaction, and thus the function of the transcription factor. Thus, the transcription factor system can exhibit ligand-dependent activity.

[0078] In some embodiments, the transcription factor system is present in a cell or cell population. In some embodiments, one or more polynucleotides of the transcription factor system are introduced into a cell or cell population.

[0079] Transcription factor system constructs A combination of one or more polynucleotides of a transcription factor system may also be referred to herein as a combination of one or more nucleic acid constructs. The polynucleotides or nucleic acid constructs may comprise different arrangements of nucleic acid sequences and / or may be uniquely combined as part of a transcription factor system, so long as the resulting combination of polynucleotides or nucleic acid constructs comprises (1) one or more nucleic acid sequences encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription, (2) a nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor is operably linked to the DRD, and (3) a nucleic acid sequence encoding a payload and operably linked to an inducible promoter comprising the specific polynucleotide binding site.

[0080] In some embodiments, the transcription factor system comprises multiple constructs. In some embodiments, the transcription factor system comprises a transcription factor construct and a payload construct. In one aspect, the transcription factor construct comprises a nucleic acid sequence encoding a transcription factor. In one aspect, the transcription factor construct comprises a nucleic acid sequence encoding a transcription factor activation domain and a nucleic acid sequence encoding a transcription factor DNA binding domain.

[0081] In some embodiments, the transcription factor system comprises a single construct. The single construct comprises nucleic acid sequences encoding the transcription factor, DRD, and payload of the transcription factor system. In some embodiments, such a single construct transcription factor system may be introduced into a cell on a single nucleic acid molecule, such as a plasmid or vector. A transcription factor system comprising a single construct may be referred to herein as a single vector transcription factor system.

[0082] In addition to including the nucleic acid sequences described herein for the transcription factor systems, the nucleic acid constructs of the present disclosure may include additional nucleic acid sequences, including, but not limited to, regulatory elements, polyadenylation sequences, linkers, and cleavage sites.

[0083] In some embodiments, the transcription factor construct may include a promoter, a transcription factor DNA-binding domain, a transcription factor activation domain, and a nucleic acid sequence encoding a DRD. In some embodiments, the nucleic acid sequence encoding the DRD is adjacent to a nucleic acid sequence encoding at least one of the transcription factor domains. In some embodiments, the nucleic acid sequence encoding the DRD is located between the nucleic acid sequence encoding the transcription factor DNA-binding domain and the nucleic acid sequence encoding the transcription factor activation domain.

[0084] In some embodiments, the transcription factor construct may comprise a promoter, a transcription factor DNA-binding domain, a transcription factor activation domain, a linker, and a nucleic acid sequence encoding a DRD. In some aspects, the linker is disposed between the nucleic acid sequence encoding the transcription factor domain and the nucleic acid sequence encoding the DRD.

[0085] In some embodiments, the promoter in the transcription factor construct is EF1a. In some embodiments, the transcription factor DNA binding domain encoded in the transcription factor construct is ZFHD1. In some embodiments, the transcription factor activation domain encoded in the transcription factor construct is p65.

[0086] In some embodiments, the payload construct may comprise a specific polynucleotide binding site comprising at least one nucleic acid site having a specific sequence recognized and bound by a transcription factor DNA binding domain, a promoter, and a nucleic acid sequence encoding the payload. Exemplary binding sites include the eight nucleic acid sites recognized by the ZFHD1 DNA binding domain.

[0087] In some embodiments, a construct of the present disclosure, such as a transcription factor construct or a payload construct, is incorporated into a plasmid or viral vector. In some embodiments, the plasmid or viral vector comprises one or more regulatory elements that become operably linked to one or more components of the construct incorporated into the plasmid or viral vector. In some embodiments, the plasmid or viral vector comprises regulatory elements well known in the art, including, for example, promoters, introns, spacers, stuffer sequences, etc. In some embodiments, a transcription factor construct is incorporated into a plasmid or viral vector such that the components of the transcription factor construct are operably linked to the regulatory elements of the plasmid or viral vector. In some embodiments, such a transcription factor construct comprises a nucleic acid sequence encoding a transcription factor DNA-binding domain, a transcription factor activation domain, and a DRD, and is incorporated into the plasmid or viral vector such that a promoter sequence in the plasmid or viral vector drives expression of the transcription factor DNA-binding domain, the transcription factor activation domain, and the DRD. Such a promoter may be selected from a constitutive promoter, a tissue-specific promoter, a cell-specific promoter, a cell differentiation-specific promoter, and / or a disease-specific promoter. Optionally, the promoter may be selected from EF1a, CMV, EFS, RSV, SFFV, PGK, CAG, and SV40.

[0088] Components of transcription factor systems As described above, the polynucleotides or nucleic acid constructs of a transcription factor system may contain different arrangements of nucleic acid sequences and / or may be uniquely combined as part of a transcription factor system, so long as the resulting combination of polynucleotides or nucleic acid constructs contains (1) one or more nucleic acid sequences encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription, (2) a nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor is operably linked to the DRD, and (3) a nucleic acid sequence encoding a payload and operably linked to an inducible promoter comprising the specific polynucleotide binding site. Thus, a transcription factor system is a modular system, and each component of the transcription factor system can be selected independently.

[0089] The nucleic acid sequence encoding the drug responsive domain (DRD) may be selected from the sequences of DRDs described in more detail in the "Drug responsive domain (DRD)" section below.

[0090] The one or more nucleic acid sequences encoding a transcription factor may be selected from an existing transcription factor, an engineered transcription factor derived from an existing transcription factor, or one or more sequences encoding an engineered transcription factor containing a DNA-binding domain and an activation domain. As used herein, an "engineered transcription factor derived from an existing transcription factor" refers to an engineered transcription factor that is derived at least in part from a parent (natural) transcription factor molecule or sequence and retains the ability to bind to a specific polynucleotide binding site and activate transcription. For example, an engineered transcription factor may be derived from a parent transcription factor containing one or more zinc finger domains capable of sequence-specific contact with DNA. An engineered TAL effector transcription factor may be designed to contain a TAL effector repeat region that recognizes a specific DNA-binding site, a mammalian nuclear localization signal (NLS), and a synthetic transcription activation domain. When the transcription factor is an engineered transcription factor containing a DNA-binding domain and an activation domain, both the DNA-binding domain and the activation domain may be selected separately and combined to form the complete transcription factor.

[0091] The transcription factor DNA-binding domain may be derived from an existing nucleic acid-binding protein. For example, the DNA-binding sequence or domain of an existing DNA-binding protein may be used as, or further modified to generate, the transcription factor DNA-binding domain of the present disclosure.

[0092] In some embodiments, the transcription factor DNA binding domain is derived from a parent protein selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL.

[0093] In some embodiments, the transcription factor DNA-binding domain is derived from the ZFHD1 parent protein. ZFHD1 is a zinc finger-homeodomain fusion protein designed by Pomerantz, JL, et al. (Pomerantz, JL, et al. "Structure-Based Design of Transcription Factors," Science, vol. 267, no. 5194, 1995). ZFHD1 contains fingers 1 and 2 of Zif268, a gly-gly-arg-arg linker, and an OCT-1 homeodomain. ZFHD1 can bind to a nucleic acid sequence containing the sequence TAATGATGGGCG (SEQ ID NO: 70). In some embodiments, the transcription factor DNA-binding domain consists of or comprises the amino acid sequence of ZFHD1.

[0094] In some embodiments, the present disclosure provides methods for regulating target genes and their corresponding functional proteins (e.g., payloads or proteins of interest) using a Cas / guide RNA system. It should be understood that one skilled in the art will be able to design suitable guide RNAs to form co-localized complexes with target nucleic acids, including target genes as described herein.

[0095] Various Cas proteins are known to those skilled in the art, including CasI (Cas3), CasIA (Cas8a), CasIB (Cas8b), CasIC (Cas8c), CasID (Cas10d), CasIE (Cse1), CasIF (Csy1), CasIU, CasII (Cas9), CasIIA (Csn2), CasIIB (Cas4), CasIIC, CasIII (Ca10), CasIIIA (Csm2), CasIIIB (Cmr5), CasIIIC, CasIIBD, CasIV (Csf1), CasIVA, CasIVB, CasV (Cpf1), C2c2, and C2c1.

[0096] In some embodiments, the transcription factor DNA binding domain is selected from the group consisting of C2C1, C2C3, Cpf1 (also referred to as Cas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, C The Cas protein is derived from a Cas protein selected from the group consisting of se1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

[0097] In one embodiment, the Cas9 protein comprises a sequence as described for naturally occurring Cas9 from S. aureus, S. thermophiles, S. pyogenes, or Neisseria meningitidis Cas9, and a protein sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology thereto, and which is a DNA-binding protein, such as an RNA-guided DNA-binding protein.

[0098] In one embodiment, the Cas12 protein comprises a sequence as described for naturally occurring Cas12 from Francisella novicida, Acidaminococcus species, Lachnospiraceae species, or Prevotella species, and a protein sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto, and which is a DNA-binding protein, such as an RNA-guided DNA-binding protein.

[0099] In some embodiments, the transcription factor DNA binding domain is derived from a parent Cas protein, such as a parent Cas9 or Cas12 protein. In some embodiments, the transcription factor DNA binding domain is or comprises a Cas9 that has been modified to lack nuclease activity. In some embodiments, the transcription factor DNA binding domain is or comprises a Cas12 that has been modified to lack nuclease activity.

[0100] Naturally occurring Cas9 contains two nuclease domains: an HNH-like nuclease domain that cleaves the DNA strand complementary to the guide RNA sequence (the target strand), and a RuvC-like nuclease domain that cleaves the DNA strand opposite the complementary strand (the non-target strand). By mutating both the HNH and RuvC nuclease domains (resulting in a so-called "dead Cas9" or "dCas9"), the resulting dCas9 retains its RNA-guided DNA targeting ability but loses its endonuclease activity. In some embodiments, the transcription factor DNA binding domain is a dCas9 containing mutated HNH and RuvC nuclease domains. In some embodiments, the transcription factor DNA binding domain is a dCas9 containing mutated HNH and RuvC nuclease domains, derived from the parent S. aureus, S. thermophiles, S. pyogenes, or Neisseria meningitidis Cas9.

[0101] Naturally occurring Cas12 (e.g., Cas12a and Cas12b) contain a RuvC-like domain that cleaves DNA. By mutating the RuvC nuclease domain, a catalytically dead Cas12 (with inactivated DNase activity, also referred to herein as "dCas12") can be derived from the parent Cas12 protein. In some embodiments, the transcription factor DNA-binding domain is or comprises a catalytically dead Cas12 (dCas12).

[0102] In some embodiments, the transcription factor DNA-binding domain is derived from a parent protein that is a type II Cas homolog. Cas9 is an example of a type II Cas protein. In some embodiments, the transcription factor DNA-binding domain is or comprises a type II Cas homolog that lacks nuclease activity or has been modified to lack nuclease activity. In some embodiments, the transcription factor DNA-binding domain is or comprises a type II Cas homolog that comprises a mutated HNH and RuvC nuclease domain.

[0103] In exemplary embodiments, Cas9 is altered or otherwise modified to inactivate nuclease activity. Such alterations or modifications include altering one or more amino acids to inactivate nuclease activity or the nuclease domain. Such modifications include removing the polypeptide sequence(s) exhibiting nuclease activity, i.e., the nuclease domain, such that the polypeptide sequence(s) exhibiting nuclease activity, i.e., the nuclease domain, are absent from the Cas9 DNA-binding protein. Other modifications for inactivating nuclease activity will be readily apparent to those skilled in the art. Thus, a nuclease-null DNA-binding protein includes a polypeptide sequence altered to inactivate nuclease activity or the removal of a polypeptide sequence(s) to inactivate nuclease activity. A nuclease-null DNA-binding protein retains the ability to bind to DNA even when the nuclease activity is inactivated. Thus, a DNA-binding protein may contain the polypeptide sequence(s) necessary for DNA binding but lack one or more or all of the nuclease sequences that exhibit nuclease activity. See Jinek et al., (2012) Science 337, 816-821. Cas9 proteins lacking nuclease activity are referred to as nuclease-null Cas9 ("Cas9Nuc," "dead Cas9," or "dCas9") and exhibit reduced or eliminated nuclease activity, or nuclease activity that is absent or substantially absent within the detection level. According to this embodiment, the nuclease activity of Cas9Nuc may be undetectable using known assays, i.e., below the detection level of known assays.

[0104] In some embodiments, the transcription factor DNA binding domain is derived from the Cas9 parent protein. In some embodiments, the transcription factor DNA binding domain comprises a Cas9 with a mutated nuclease domain (referred to as "dead Cas9" or "dCas9"). The resulting dCas9 retains its RNA-guided DNA targeting ability but loses its endonuclease activity. In some embodiments, the transcription factor DNA binding domain is dCas9.

[0105] The present disclosure provides the use of guide RNAs to target a polynucleotide binding sequence, such as those described herein, to a Cas protein, e.g., a nuclease null-Cas9 operably linked to a DRD. Such guide RNAs can be easily designed by those skilled in the art given the specific polynucleotide binding sequence. The guide RNA may include one or more of a spacer sequence, a tracr mate sequence, and a tracr sequence. The term spacer sequence is understood by those skilled in the art and may include any polynucleotide that has sufficient complementarity with a polynucleotide binding sequence to hybridize with the polynucleotide binding sequence and allow sequence-specific binding of a CRISPR complex to the polynucleotide binding sequence. A guide RNA may be formed from a spacer sequence covalently linked to a tracr mate sequence (which may be referred to as a crRNA) and a separate tracr sequence, where the tracr mate sequence hybridizes to a portion of the tracr sequence. According to certain embodiments, the tracr mate sequence and the tracr sequence are linked or connected, such as by a covalent bond via a linker sequence, and this construct may be referred to as a fusion of the tracr mate sequence and the tracr sequence. A linker sequence, as referred to herein, is a sequence of nucleotides, referred to herein as a nucleic acid sequence, that joins the tracr mate sequence and the tracr sequence. Thus, the guide RNA can be a two-component species (i.e., separate crRNA and tracr RNA that hybridize to each other) or a single species (i.e., a crRNA-tracr RNA fusion, often referred to as an sgRNA).

[0106] In some embodiments, guide RNAs may be delivered directly into cells as native species by methods known to those of skill in the art, including injection or lipofection, or may be delivered into cells to be transcribed from their cognate DNA, with the cognate DNA introduced into the cell by electroporation, transient and stable transfection (including lipofection), and viral transduction.

[0107] In some embodiments, the transcription factor system comprises one or more polynucleotides encoding a DRD-regulating transcription factor, where the transcription factor comprises a DNA-binding domain that is or includes a nuclease-null Cas9. When a DRD-stabilizing ligand is added, the DRD and transcription factor are stabilized, and the nuclease-null Cas9 is expressed and becomes capable of binding to the guide RNA. Upon binding to the guide RNA, the Cas9-gRNA system binds to a polynucleotide binding sequence operably linked to a protein of interest. When the Cas9-gRNA system is bound to the polynucleotide binding sequence, the protein of interest gene is transcribed due to the presence of the transcription factor activation domain. Thus, when a regulatable transcription factor expression construct comprises a Cas9-gRNA system, RNA-guided DNA regulation is achieved in cells, such as human cells, by tethering or binding the DRD to either the nuclease-null Cas9 or the transcription factor activation domain. Accordingly, embodiments of the present disclosure include methods and materials for localizing a transcriptional regulatory domain to a target locus by fusing, conjugating, or linking a DRD to either a Cas9Nuc or a transcription factor activation domain, or both.

[0108] In some embodiments, the transcription factor DNA-binding domain is derived from a TAL parent protein. TAL (Transcription Activator-Like) effectors (also referred to as "TALEs") are proteins secreted by Xanthomonas bacteria that regulate gene expression in host plants and aid in bacterial infection. TAL effectors have a repeat region consisting primarily of tandem repeats of 33 or 34 amino acid residues. The repeat monomers differ from each other primarily at amino acid positions 12 and 13, with a strong correlation between the unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotides in the TALE binding site. The transcription factor DNA-binding domain of the present disclosure may comprise all or part of the repeat region of a TAL effector that can bind to a specific DNA-binding site. In some embodiments, the DNA-binding domain comprises a synthetic TAL effector that can recognize a desired nucleic acid sequence. Methods for assembling custom TAL effectors are readily available to those skilled in the art. "Engineered TAL effector" as used herein refers to a polypeptide derived from a parent TAL effector protein, a TAL effector, and / or a polypeptide comprising a repeat region of a synthetic TAL effector or region thereof. In some embodiments, the transcription factor DNA-binding domain is an engineered TAL effector capable of binding to a specific nucleic acid site. In some embodiments, the transcription factor DNA-binding domain is derived from a parent protein of a zinc finger protein. In some embodiments, the parent zinc finger protein may be a C2H2 zinc finger protein. In some embodiments, the transcription factor DNA-binding domain may comprise one or more zinc finger domains that make sequence-specific contacts with DNA. In some embodiments, the transcription factor DNA-binding domain may comprise at least two zinc finger domains, at least three zinc finger domains, at least four zinc finger domains, or at least five zinc finger domains that form a zinc finger array capable of specifically recognizing a DNA site. In some embodiments, the transcription factor DNA-binding domain comprises a three-finger array.An engineered DNA binding domain that comprises one or more zinc finger domains is referred to herein as an "engineered zinc finger binding protein."

[0109] In some embodiments, the transcription factor DNA binding domain may be selected from an engineered zinc finger binding protein, an engineered TAL effector, or other natural or engineered DNA binding domain.

[0110] Zinc finger and TALE DNA-binding domains can be "engineered" to bind to a given nucleotide sequence, for example, by manipulating the recognition region of a naturally occurring zinc finger or TALE protein (changing one or more amino acids). Thus, engineered DNA-binding proteins (zinc fingers or TALEs) are proteins that do not occur in nature. A non-limiting example of a method for engineering DNA-binding proteins is design and selection. Engineered DNA-binding proteins are proteins that do not occur in nature whose design / composition is driven primarily by rational criteria. Rational criteria for design include the application of substitution rules and the application of computerized algorithms to process information in databases that store information on existing ZFP and / or TALE designs and binding data. See, e.g., U.S. Patent Nos. 8,586,526, 6,140,081, 6,453,242, 6,534,261, and 8,586,526, and also WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016496, the disclosures of these references, insofar as they relate to the design and selection of DNA-binding proteins derived from existing ZFP and / or TALE proteins and their associated binding data, are incorporated herein by reference in their entireties.

[0111] The activation domain of an engineered transcription factor according to the present disclosure may be derived from a region or domain of an existing transcription factor. In some embodiments, the activation domain is a region of an existing transcription factor that is capable of transcriptional activation. In some embodiments, the transcription factor activation domain may be selected from the activation domains of p65, VP64, p300, SAM, VPR, or other activation domains. In some embodiments, the activation domain is derived from the carboxy-terminal region of the human transcription factor NF-κβ p65 protein (referred to herein as "p65"). In some embodiments, the activation domain comprises the carboxy-terminal region of the human transcription factor NF-κβ p65 protein.

[0112] Considerations in the design of the transcription factor systems provided herein are that the encoded transcription factor be capable of binding to a specific polynucleotide binding site, and that the nucleic acid sequence encoding the payload be operably linked to an inducible promoter that contains the specific polynucleotide binding site. In various embodiments, the inducible promoter is an exogenous inducible promoter. Pairs of transcription factors (including engineered transcription factors) and their corresponding polynucleotide binding sites are known in the art. DNA-binding domains of DNA-binding proteins are also known, along with their corresponding polynucleotide binding sites, and methods for identifying novel DNA-binding domain sequences and corresponding polynucleotide binding sites that can be used to design synthetic transcription factors and corresponding synthetic promoters are also known. See, for example, Khalil AS, et al. provide zinc finger arrays that can be used as core components for constructing synthetic transcription factors, and further provide corresponding nucleic acid binding sequences that can be inserted into synthetic promoters and recognized by the zinc finger array (Khalil AS, et al. Cell 2012, 150, 647-658, the entire contents of which are incorporated by reference). Khalil AS, et al. also identify synthetic transcription factor-promoter pairs and provide design strategies for modifying the promoter (e.g., multimerizing zinc finger binding sequences to create promoters with repeat operators) and the synthetic transcription factor (e.g., by creating variants) to alter transcriptional output. Any of the transcription factor-promoter pairs or engineered zinc finger arrays and their corresponding nucleic acid binding sites disclosed by Khalil AS, et al. can be used in the transcription factor system of the present disclosure. As an example, Figure 3A of Khalil, AS, et al. provides a library of amino acid residues of recognition helices of zinc finger arrays and corresponding DNA-binding sequences that can be used to design transcription factor DNA-binding domains and specific polynucleotide-binding sites of the present disclosure. One skilled in the art can modify the transcription factor or zinc finger array sequences provided by Khalil, AS, et al. by cloning these transcription factor or array sequences into the transcription factor system constructs provided herein. As another example, Zhang, F., et al. describe methods for designing and producing engineered TAL effectors with corresponding nucleic acid-binding sites. These can be used to prepare engineered transcription factors and their specific polynucleotide-binding sites. Any of the TAL effectors provided by Zhang, F., et al. may be used to prepare transcription factor DNA-binding domains in the transcription factor systems of the present disclosure. For example, Zhang, F., et al. disclose the construction of 17 artificial TAL effectors that target specific DNA binding sites, and also provide the sequences of the TAL effector repeat regions and corresponding nucleic acid binding sequences in Figure 2a.The TAL effectors or DNA-binding portions thereof disclosed in Zhang, F., et al. can be used in addition to the DNA-binding domain to construct the corresponding nucleic acid-binding sequence of the inducible promoter of the present disclosure. Those skilled in the art will recognize that there are several options for selecting and designing the DNA-binding domain of the present disclosure. In addition to selecting recognized DNA-binding proteins and domains known in the art, the DNA-binding domain of the present disclosure can be designed based on the framework of an existing DNA-binding protein. For example, methods for selecting DNA-binding domains based on the Cys2His2 zinc finger protein framework are available to those skilled in the art (Pabo, CO, et al. Annu. Rev. Biochem. 2001. 70:313-40).

[0113] In some embodiments, the inducible promoter operably linked to the nucleic acid sequence encoding the payload comprises a minimal promoter (also referred to as a "min promoter" or "core promoter") and a specific polynucleotide binding site. In this scenario, both the minimal promoter and the specific polynucleotide binding site are operably linked to the nucleic acid sequence encoding the payload. The term "minimal promoter" refers to the minimal structure that allows for the formation of an initiation complex. A minimal promoter may include an RNA polymerase binding site, a TATA box, and a transcription start site. A minimal promoter may be associated with one or more response elements (such as enhancers or transcription factor binding sites) to generate an inducible promoter. Additional details regarding minimal promoters and the association of minimal promoters with response elements are provided by Ede and colleagues (Ede et al., ACS Synth Biol. 2016 May 20;5(5):395-404). In some embodiments, an inducible promoter for a transcription factor system or component thereof of the present disclosure is selected from the following minimal promoters: minCMV, CMV53 (minCMV with the addition of an upstream GC box), minSV40 (minimal simian virus 40 promoter), miniTK (-33 to +32 region of the herpes simplex thymidine kinase promoter), MLP (-38 to +6 region of the adenovirus major late promoter), pJB42CAT5 (minimal promoter from the human junB gene), YB_TATA (Benenson and colleagues (Hansen, J. et al. Proc Natl Acad Sci USA. 2014;111:15705-15710), as well as minimal promoters selected from TATA boxes alone.

[0114] As described above, a specific polynucleotide binding site may comprise at least one nucleic acid site having a specific sequence that is recognized and bound by a transcription factor DNA binding domain. In some embodiments, a specific polynucleotide binding site comprises two or more nucleic acid sites, each having a specific sequence that is recognized and bound by a transcription factor DNA binding domain. The pairing of DNA binding domains with their corresponding polynucleotide binding sites is described above.

[0115] The nucleic acid sequence encoding the payload may be selected to encode any payload or protein of interest. Additional details regarding payloads are provided in the "Payload" section below.

[0116] Exemplary nucleic acid constructs that may be used individually (as a single construct) or in combination as part of a transcription factor system are set forth in Table 1. An asterisk ( 「*」 ) indicates translation of the stop codon. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17]

[0117] Additional exemplary constructs containing structurally distinct transcription factor components are provided in Table 2. An asterisk ( 「*」 ) indicates translation of the stop codon. Separate construct components are also provided, along with corresponding control constructs that do not contain regulated transcription factors. As shown in the descriptions of constructs cjun-001 and cjun-002, a peptide linker is placed between the CA2 and c-Jun components of each construct. Additionally, all constructs contain the P2A peptide. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0118] Characterization of ligand-dependent activity of transcription factor systems The ligand-dependent activity of a transcription factor system may be characterized by a variety of methods.

[0119] In some embodiments, the ligand-dependent activity of a transcription factor system is characterized by ligand-dependent regulation of a transcription factor polypeptide (e.g., a transcription factor DNA-binding domain, a transcription factor activation domain, or both a transcription factor DNA-binding domain and a transcription factor activation domain) encoded by the transcription factor system. In some embodiments, the ligand-dependent activity of a transcription factor system is characterized by ligand-dose-dependent regulation of a transcription factor polypeptide encoded by the transcription factor system. In one aspect, the transcription factor polypeptide is a polypeptide comprising a transcription factor activation domain. In another aspect, the transcription factor polypeptide is a polypeptide comprising a transcription factor DNA-binding domain. In another aspect, the transcription factor polypeptide is a polypeptide comprising both a transcription factor activation domain and a transcription factor DNA-binding domain. Ligand-dependent regulation of a transcription factor polypeptide may be characterized by various methods. In some aspects, ligand-dependent regulation of a transcription factor polypeptide may be assessed by measuring levels of the transcription factor polypeptide or a domain thereof, such as by immunoassay.

[0120] In some embodiments, the ligand-dependent activity of the transcription factor system is characterized by ligand-dependent expression of a payload encoded by the transcription factor system. Payload expression may be assessed by various methods. In some aspects, payload expression is assessed by measuring payload mRNA levels. In some aspects, payload expression is assessed by measuring payload polypeptide levels.

[0121] In some embodiments, the transcription factor system may be compared to a control transcription factor system lacking the DRD. In some embodiments, the ligand-dependent activity of the transcription factor system may be analyzed or characterized in comparison to the activity of a transcription factor system comprising a control transcription factor construct lacking the DRD. One example of a control transcription factor construct is construct ZFHD-004, which is described by the present disclosure (as shown in Table 1).

[0122] transcription factors Transcription factors are proteins that bind to sequence-specific sites (transcription factor polynucleotide binding sites) on DNA, preferably located within or near a promoter, thereby facilitating the binding of the transcription machinery to the promoter, thereby activating transcription of the DNA sequence. Such entities are also known as transcriptional regulatory proteins.

[0123] In various embodiments, transcription factors for use in the transcription factor systems, compositions, and methods described herein comprise a transcription factor DNA-binding domain and a transcription factor activation domain. In some embodiments, the combination of the transcription factor DNA-binding domain and the transcription factor activation domain results in a functional transcription factor. In various embodiments, the transcription factor DNA-binding domain and / or the transcription factor activation domain may interact with other transcriptional regulatory elements.

[0124] In some embodiments, transcription factors are exemplified as proteins that recognize and bind to specific short DNA sequences, thereby inevitably affecting gene expression. The recognition of DNA sequences by transcription factors is achieved through chemical interactions between the amino acid side chains of the transcription factor protein and the base-pairing residues of DNA that function as regulatory sequences. Thus, transcription factors "read" genomic sequences, and this mechanism provides the sequence recognition function on which the informational aspects of the regulatory process that controls gene expression depend.

[0125] Transcription factors typically consist of a DNA-binding domain and an effector or activation domain that mediate interactions with other proteins required for transcription, including other transcription factors. Transcription factors perform many functions, including gene activation. They are transcribed in the nucleus, translated in the cytoplasm, and find their target sites within genomic DNA upon re-entry into the nucleus, mediated by a nuclear localization site contained in all transcription factor protein sequences. Transcription factors contain basic domains that nonspecifically concentrate in the vicinity of DNA, facilitating diffusion-limited discovery of their target sites.

[0126] In various embodiments of the present disclosure, transcription factor systems utilize transcription factors that are composed of and / or include a transcription factor DNA-binding domain and a transcription factor effector or activation domain or protein (used interchangeably herein). The transcription factor activation domain, the transcription factor DNA-binding domain, and / or a combination of a transcription factor activation domain and a transcription factor DNA-binding domain may be operably linked to a DRD (any of which is a DRD-TF). Upon stabilization of the linked DRD by binding of an exogenous stabilizing ligand, the stabilized DRD-TF can transcribe a protein of interest.

[0127] The DNA sequence to which the transcription factor DNA binding domain binds is called a transcription factor binding site or response element, or, as used interchangeably herein, a specific polynucleotide binding site, and these binding sites are found in or near the promoter of the regulated DNA sequence. The promoter containing the specific polynucleotide binding site can be an exogenous promoter. In some embodiments, the promoter can be an exogenous inducible promoter. When incorporated into the transcription factor system containing the target protein or payload, the transcription factor binding site or the specific polynucleotide binding site is an exogenous nucleic acid sequence.

[0128] In various embodiments of the present disclosure, suitable transcription factors useful in synthesizing transcription factor systems can include any known transcription factor for which a transcription factor binding site is known. Some examples of such transcription factors include the STAT family (STAT1, 2, 3, 4, 5a, 5b, and 6), c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD, fos / jun, NF-kappaB, HIV-TAT, E2F family, T-Box gene family, helix-loop-helix transcription factors, zinc finger transcription factors such as ZFHD1, Oct4, and Zif268, engineered zinc finger transcription factors, and the following families: bHLH, bZIP, forkhead, nuclear receptor, HMG / Sox, Ets, T-box, AT hook, homeodomain+POU, Myb / SANT, THAP finger, CENPB, E2F, BED Examples of transcription factor DNA-binding domains include, but are not limited to, ZF, GATA, Rel, CxxC, IRF, SAND, SMAD, HSF, MBD, RFX, CUT+homeodomain, DM, STAT, ARID / BRIGHT, Grainyhead, MADS box, AP-2, CSD, and homeodomain+PAX transcription factors. Exemplary transcription factor DNA-binding domains may comprise one or more DNA-binding domains derived from a parent protein selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL.

[0129] In various embodiments, the transcription factor system provides regulatable transcription of a protein of interest or payload (used interchangeably herein). In various embodiments, the nucleic acid sequence encoding the protein of interest is operably linked to an exogenous inducible promoter that contains a specific polynucleotide binding site, i.e., a defined DNA polynucleotide sequence, that specifically binds to the transcription factor DNA binding domain. The transcription factor binding domain can then be combined with the transcription factor DNA activation domain to regulate the transcription of the protein of interest.

[0130] When a cell or organism containing a DRD-TF is exposed to an exogenous stabilizing ligand, the DRD-TF becomes stabilized. The stabilized DRD-TF then binds to the specific polynucleotide binding site to which the DRD-TF binds, and can therefore control the transcription of a polynucleotide encoding a protein of interest. In some embodiments, binding of the stabilized DRD-TF activates the transcription of the polynucleotide encoding the protein of interest, resulting in protein expression in the cell or organism. In the absence of an exogenous stabilizing ligand, the DRD-TF is degraded and cannot activate transcription. Thus, both the amount and timing of protein expression can be controlled by administering an exogenous stabilizing ligand to a cell or organism.

[0131] In various embodiments, the transcription factor DNA-binding domain and the transcription factor activation domain are typically operably linked or may be separated by one or more intervening sequences, e.g., a linker or cleavage site. In various embodiments, the first polynucleotide may comprise a first nucleic acid sequence encoding the transcription factor DNA-binding domain, a second nucleic acid sequence encoding the transcription factor activation domain, and a third nucleic acid sequence encoding a drug-responsive domain (DRD). In such embodiments, the transcription factor activation domain and / or the transcription factor DNA-binding domain is operably linked to the DRD upon expression in the cell. In addition, the cell will also comprise a second polynucleotide comprising a fourth nucleic acid sequence capable of being specifically bound by the transcription factor DNA-binding domain and a fifth nucleic acid sequence encoding a protein of interest or payload as described herein.

[0132] The transcription factor DNA binding domain, the transcription factor activation domain, and the protein of interest or payload may be provided for the methods of the present disclosure on the same vector or in separate vectors.

[0133] In some embodiments, a vector comprises a polynucleotide described herein. In some embodiments, the vector comprises at least a first nucleic acid sequence encoding at least one of a transcription factor DNA-binding domain and a transcription factor activation domain, and a second nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor DNA-binding domain and / or the transcription factor activation domain is operably linked to the DRD. Optionally, in some embodiments, the first vector comprises a transcription factor linked to the DRD, and the second vector comprises a protein of interest or payload operably linked to the transcription factor polynucleotide binding site. In further embodiments, a single vector comprises a first nucleic acid sequence encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription, a second nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is operably linked to the DRD, and optionally a third nucleic acid sequence encoding a protein of interest operably linked to an inducible promoter comprising the transcription factor polynucleotide binding site. In some embodiments, the first vector comprises at least a first nucleic acid sequence encoding at least one of a transcription factor DNA-binding domain and a transcription factor activation domain, and a second nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor DNA-binding domain and / or the transcription factor activation domain is operably linked to the DRD, and the second vector comprises a third nucleic acid sequence capable of being specifically bound by the transcription factor DNA-binding domain and a fourth nucleic acid sequence encoding a protein of interest or payload as described herein.

[0134] In some embodiments, the vector also comprises an origin of replication (ori), which allows for the vector to be amplified, for example, in bacteria. Additionally or alternatively, the vector comprises a selectable marker, such as an antibiotic resistance gene, a gene for a color marker, or a suicide gene.

[0135] Drug-responsive domain (DRD) A drug-responsive domain (DRD) is a protein domain that is unstable and degraded in the absence of a ligand, but its stability is restored by binding to a corresponding DRD-binding ligand. The term drug-responsive domain (DRD) is interchangeable with the term destabilization domain (DD). A drug-responsive domain (DRD) can be added to a polypeptide or protein and can destabilize the added polypeptide or protein in the absence of a DRD-binding ligand. DRDs confer their destabilizing properties to the added polypeptide or protein by proteolysis. Without wishing to be bound by any theory, in the absence of a DRD-binding ligand, the added polypeptide or protein is rapidly degraded by the cellular ubiquitin-proteasome system. Ligands that bind to or interact with the DRD can modulate the stability of the added polypeptide or protein upon such binding or interaction. When a ligand binds to its intended DRD, the instability can be reversed and the function of the added polypeptide or protein can be restored. The conditional stability of the DRD allows for a rapid and unimpeded switch from a stable protein to an unstable substrate for degradation, and its dependence on ligand concentration further provides tunable control of the degradation rate.

[0136] In some embodiments, the DRDs of the present disclosure may be derived from known polypeptides capable of post-translational regulation of proteins. In some embodiments, the DRDs of the present disclosure may be developed or derived from known proteins. Regions, portions, or domains of wild-type proteins may be used as DRDs, in whole or in part. They may be combined or rearranged to create new peptides, proteins, regions, or domains, any of which may be used as a DRD or as a starting point for the design of additional DRDs.

[0137] In some embodiments, the DRD may be derived from a parent protein or a mutant protein having one, two, three, or more amino acid mutations compared to the parent protein. In some embodiments, the parent protein may be selected from, but is not limited to, FKBP, human protein FKBP, human DHFR (hDHFR), E. coli DHFR (ecDHFR), PDE5 (phosphodiesterase 5), CA2 (carbonic anhydrase II), and ER (estrogen receptor). Examples of proteins that may be used to develop DRDs and their ligands are listed in Table 3. [Table 3-1] [Table 3-2]

[0138] In some embodiments, the sequence of a protein used to develop a DRD may include all, a portion, or a region of a protein sequence in Table 3. In some embodiments, proteins that may be used to develop a DRD include isoforms of proteins listed in Table 3.

[0139] hPDE5 DRD In some embodiments, the DRD of the present disclosure is derived from hPDE5. In some embodiments, the DRD of the present disclosure is derived from hPDE5 isoform 2. In some embodiments, the DRD of the present disclosure is derived from hPDE5 isoform 3. In some embodiments, the DRD of the present disclosure is derived from hPDE5 isoform X1.

[0140] In some embodiments, the DRD of the present disclosure is derived from a cGMP-specific 3',5'-cyclic phosphodiesterase (hPDE5) comprising the amino acid sequence of SEQ ID NO:71.

[0141] In some embodiments, the DRD of the present disclosure may comprise the entire hPDE5 (SEQ ID NO: 71). In some embodiments, the DRD derived from hPDE5 may comprise the catalytic domain of hPDE5 (e.g., amino acids 535-860 of SEQ ID NO: 71). In some embodiments, the hPDE5 DRD of the present disclosure may comprise a methionine at the N-terminus of the catalytic domain of hPDE5, i.e., amino acids 535-860 of wild-type (WT) hPDE5.

[0142] In some embodiments, a DRD of the disclosure comprises, in whole or in part, a cGMP-specific 3',5'-cyclic phosphodiesterase (hPDE5, SEQ ID NO: 71) and further comprises an amino acid mutation at position 732 (R732) of SEQ ID NO: 71. In some embodiments, the amino acid mutation at position 732 (R732) is selected from the group consisting of R732L, R732A, R732G, R732V, R732I, R732P, R732F, R732W, R732Y, R732H, R732S, R732T, R732D, R732E, R732Q, R732N, R732M, R732C, and R732K.

[0143] In some embodiments, the hPDE5 DRD of the disclosure is selected from the group consisting of H653A, F736A, D764A, D764N, Y612F, Y612W, Y612A, W853F, I821A, Y829A, F787A, D656L, Y728L, M625I, E535D, E536G, Q541R, K555R, F559L, F561L, F564L, F564S, K591E, N587S, K604E, K608E, N609H, K630R, K633E, N636S, N661S, Y676D, Y676N, C677R, H678R, D687A , T712S, D724N, D724G, L738H, N742S, A762S, D764G, D764V, S766F, K795E, L797F, I799T, T802P, S815C, M816A, I824T, C839S, K852E, S560G, V585A, I599V, I648V, S663P, L675P, T711A, F744L, L746S, F755L, L804P, M816T, and F840S.

[0144] In some embodiments, the DRD of the disclosure comprises, in whole or in part, a cGMP-specific 3',5'-cyclic phosphodiesterase (hPDE5, SEQ ID NO: 71) and further comprises an amino acid mutation at position 732 (R732) of SEQ ID NO: 71. In some such embodiments, the DRD further comprises (i) an amino acid mutation at position 764 (D764) of SEQ ID NO: 71, wherein the D764 mutation is selected from D764N and D764A, (ii) an amino acid mutation at position 612 (Y612) of SEQ ID NO: 71, wherein the Y612 mutation is selected from the group consisting of Y612A, Y612F, and Y612W, (iii) an amino acid F736A mutation at position 736 (F736) of SEQ ID NO: 71, or (iv) an amino acid H653A mutation at position 653 (H653) of SEQ ID NO: 71.

[0145] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, a cGMP-specific 3',5'-cyclic phosphodiesterase (hPDE5, SEQ ID NO: 71) and further comprises an amino acid mutation at a position compared to SEQ ID NO: 71, the mutation being selected from the group consisting of W853F, I821A, Y829A, F787A, F736A, D656L, Y728L, M625I, and H653A.

[0146] In some embodiments, the hPDE5 DRD of the disclosure comprises any of the following amino acids: T537A, E539G, V548E, D558G, F559S, E565G, C574N, R577Q, R577W, N583S, Q586R, Q589L, K591R, K591R, L595P, C596R, W615R, F619S, Q623R, K633I, Q635R, N636S, T639S, D640N, E642G, I643 T, L646S, A649V, A650T, S652G, H653A, D654G, V660A, V660A, L672P, A673T, C677Y, M681T, E682G , H685R, F686S, Q688R, M691T, S695G, G697D, S702I, I706T, E707K, Y709H, Y709C, I715V, I720V, A 722V, D724G, Y728C, K730E, R732L, L738I, I739M, K741N, K741R, F744L, D748N, K752E, K752E, K7 52E, E753K, L756V, M758T, M760T, A762V, C763R, D764N, D764N, I774V, L781F, L781P, E785K, R794 and / or E858G, M805T, R807G, K812R, I813T, I813T, M816R, Q817R, V818A, F820S, I821V, C825R, Y829C, E830K, L832P, S836L, C846Y, C846S, L856P, L856P, A857T, or E858G.

[0147] In some embodiments, the hPDE5 DRD of the disclosure comprises E536K, I739W; H678F, S702F; E669G, I700T; G632S, I648T; T639S, M816R; Q586R, D724G; E539G, L738I; L672P, S836L; M691T, D764N; I720V, F820S; E682G, D748N; S652G, Q688R; Y728C, Q817R; H653, R732L; L595P, K741R; R732D, F736S; R732E, F736D; R732V, F736G; R732 It may comprise two mutations independently selected from W, F736G; R732W, F736V; R732L, F736W; R732P, F736Q; R732A, F736A; R732S, F736G; R732T, F736P; R732M, F736H; R732Y, F736M; R732P, F736D; R732P, F736G; R732W, F736L; R732L, F736S; R732D, F736T; R732L, F736V; R732G, F736V; and R732W, F736A.

[0148] In some embodiments, the hPDE5 DRD of the present disclosure may comprise two mutations independently selected from Q623R, D654G, K741N; A673T, L756V, C846Y; E642G, G697D, I813T; C677Y, H685R, A722V; Q635R, E753K, I813T; Y709H, K812R, L832P; N583S, K752E, C846S; K591R, I643T, L856P; F619S, V818A, Y829C; and F559S, Y709C, M760T. In some embodiments, the hPDE5 DRD of the present disclosure may comprise two mutations independently selected from S695G, E707K, I739M, C763R; A649V, A650T, K730E, E830K; and R577W, W615R, M805T, I821V.

[0149] In some embodiments, the hPDE5 DRD of the present disclosure may comprise multiple mutations independently selected from V660A, L781F, R794G, C825R, E858G; T537A, D558G, I706T, F744L, D764N; R577Q, C596R, V660A, I715V, E785K, L856P; and V548E, Q589L, K633I, M681T, S702I, K752E, L781P, A857T.

[0150] hDHFR DRD In some embodiments, the DRD of the present disclosure is derived from a human dihydrofolate reductase (hDHFR) protein, such as, but not limited to, human dihydrofolate reductase 1 (hDHFR1), human dihydrofolate reductase 2 (hDHFR2), or a fragment or variant thereof.

[0151] In some embodiments, the DRD is derived from an hDHFR protein and may contain at least one mutation. In some embodiments, the DRD is derived from an hDHFR protein and may contain two or more mutations. In some embodiments, the DRD is derived from an hDHFR protein and may contain two, three, four, or five mutations.

[0152] In some embodiments, a DRD of the present disclosure may comprise the entire hDHFR (SEQ ID NO: 2). In some embodiments, a DRD derived from hDHFR may comprise amino acids 2-187 of the parent hDHFR sequence (e.g., amino acids 2-187 of SEQ ID NO: 2). This is referred to herein as the hDHFR M1del mutation.

[0153] In some embodiments, the DRD of the present disclosure comprises a region of hDHFR (SEQ ID NO: 2) or its entirety, and further comprises a mutation selected from I17V, F59S, N65D, K81R, Y122I, N127Y, M140I, K185E, N186D, and M140I compared to SEQ ID NO: 2.

[0154] In some embodiments, the DRD of the present disclosure comprises a region of hDHFR (SEQ ID NO:2), or all of it, and further comprises two or more mutations compared to SEQ ID NO:2.

[0155] In some embodiments, the hDHFR DRD of the disclosure comprises (A10V,H88Y), (C7R / Y163C), (I17V,Y122I), (Q36H,Y122I), (Q36K,Y122I), (Q36R,Y122I), (Q36S,Y122I), (Q36T,Y122I), (N65H,Y122I), (N65L,Y122I), (N65R,Y122I), (N65W,Y122I), (Q103E,Y122I), (Q103S,Y122I), (N108D ... 22I), (V121A, Y122I), (Y122I, K174N), (Y122I, E162G), (A125F, Y122I), (N127Y, Y122I), (H131R / E144G), (E162G / I176F), (K55R, N65K, Y122I), (Q36E, Q103H, Y122I), (Q36F, N65F, Y122I), and (V110A / V136M / K177R).

[0156] In some embodiments, the hDHFR DRD of the disclosure comprises the amino acids (I17V, Y122I), (G21T, Y122N), (Q36H, Y122I), (Q36K, Y122I), (Q36R, Y122I), (Q36S, Y122I), (Q36T, Y122I), (N65H, Y122I), (N65L, Y122I), (N65R, Y122I), (N65W, Y122I), (L74N, Y122I), (Q103E, Y122I), ), (Q103S, Y122I), (N108D, Y122I), (V121A, Y122I), (Y122I, K174N), (Y122I, E162G), (A125F, Y122I), (N127Y, Y122I), (K55R, N65K, Y122I), (Q36E, Q103H, Y122I), and (Q36F, N65F, Y122I).

[0157] In some embodiments, the DRD of the disclosure comprises, in whole or in part, human dihydrofolate reductase (hDHFR, SEQ ID NO:2) and further comprises an amino acid Y122I mutation at position 122 (Y122) of SEQ ID NO:2. In some such embodiments, the DRD further comprises (i) an amino acid Q36K mutation at position 36 (Q36) of SEQ ID NO:2, (ii) an amino acid A125F mutation at position 125 (A125) of SEQ ID NO:2, or (iii) an amino acid N65F mutation at position 65 (N65) of SEQ ID NO:2 and a substitution of F or K at amino acid position 36 (Q36) of SEQ ID NO:2.

[0158] In some embodiments, the hDHFR of the present disclosure DRD is M1del, V2A, C7R, I8V, V9A, A10T, A10V, Q13R, N14S, G16S, I17N, I17 V, K19E, N20D, G21T, G21E, D22S, L23S, P24S, L28P, N30D, N30H, N30S, E31G , E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E, K56R, T57A, F59S, I61T, K 64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N7 3G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F89L, R92G, S93G, S93R, L94 A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107 T, A107V, N108D, K109E, K109R, V110A, D111N, M112T, M112V, V113A, W114R , I115V, I115L, V116I, G117D, V121A, Y122C, Y122D, Y122I, K123R, K123E , A125F, M126I, N127R, N127S, N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, T137R, R138G, R138I, I139T, I 139V, M140I, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, T147A, F1 48S, F148L, F149L, P150L, E151G, I152V, D153A, D153G, E155G, K156R, Y15 7R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, V166A, S168C, D169 G, V170A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, I176T, K177E , K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G,It may contain one or more mutations independently selected from the group consisting of K185R, K185del, K185E, N186S, N186D, D187G, and D187N.

[0159] In some embodiments, the DRD of the disclosure is selected from the group consisting of hDHFR(C7R, Y163C), hDHFR(E162G, I176F), hDHFR(G21T, Y122I), hDHFR(H131R, E144G), hDHFR(I17V, Y122I), hDHFR(L74N, Y122I), hDHFR(L94A, T147A), hDHFR(M53T, R138I), hDHFR(N127Y, Y122I), hDHFR(Q36K, Y122I), hDHFR(T137R, F143L), hDHFR(T57A, I72A), hDHFR(V121A , Y122I), hDHFR(V75F, Y122I), hDHFR(Y122I, A125F), hDHFR(Y122I, M140I), hDHFR(Y178H, E181G), hDHFR(Y183H, K185E), hDHFR(amino acids 2 to 187 of WT)(G21T, Y122I), hDHFR(amino acids 2 to 187 of WT)(I17V, Y122I), hDHFR(amino acids 2 to 187 of WT)(L74N, Y122I), hDHFR(amino acids 2 to 187 of WT)(L94A, T147A), hDHFR(amino acids 2 to 187 of WT) (M53T, R138I), hDHFR (amino acids 2 to 187 of WT) (N127Y, Y122I), hDHFR (amino acids 2 to 187 of WT) (Q36K, Y122I), hDHFR (amino acids 2 to 187 of WT) (V121A, Y122I), hDHFR (amino acids 2 to 187 of WT) (V75F, Y122I), hDHFR (amino acids 2 to 187 of WT) (Y122I, A125F), hDHFR (amino acids 2 to 187 of WT) (Y122I, M140I), hDHFR(E31D, F32M, V116I), hDHFR(G21E, I72V, I176T), hDHFR(I8V, K133E, Y163C), hDHFR(K19E, F89L, E181G), hDHFR(L23S, V121A, Y157C), hDHFR(N49D, F59S, D153G), hDHFR(Q36F, N65F, Y122I), h DHFR(Q36F, Y122I, A125F), hDHFR(V110A, V136M, K177R), hDHFR(V9A, S93R, P150L), hDHFR(Y122I, H131R, E144G), hDHFR(G54R, I115L, M140V, S168C),hDHFR (amino acids 2-187 of WT) (E31D, F32M, V116I), hDHFR (amino acids 2-187 of WT) (Q36F, N65F, Y122I), hDHFR (amino acids 2-187 of WT) (Q36F, Y122I, A125F), hDHFR (amino acids 2-187 of WT) (Y122I, H131R, E144G), hDHFR (V2A, R33G, Q36R, L100P, K185R), hDHFR (D22S, F32M, R33S, Q36S, N65S), hDHFR (amino acids 2-187 of WT) (D22S, F32M, R33S) , Q36S, N65S), hDHFR(I17N, L98S, K99R, M112T, E151G, E162G, E172G), hDHF R(G16S, I17V, F89L, D96G, K123E, M140V, D146G, K156R), hDHFR(K81R, K99R, L100P, E102G, N108D, K123R, H128R, D142G, F180L, K185E), hDHFR(R138G, D 142G, F143S, K156R, K158E, E162G, V166A, K177E, Y178C, K185E, N186S), hDH FR(N14S, P24S, F35L, M53T, K56E, R92G, S93G, N127S, H128Y, F135L, F143S, L159P, L160P, E173A, F180L), hDHFR(F35L, R37G, N65A, L68S, K69E, R71G, L8 0P, K99G, G117D, L132P, I139V, M140I, D142G, D146G, E173G, D187G), hDHFR (L28P, N30H, M38V, V44A, L68S, N73G, R78G, A97T, K99R, A107T, K109R, D111N , L134P, F135V, T147A, I152V, K158R, E172G, V182A, E184R), hDHFR(V2A, I1 7V, N30D, E31G, Q36R, F59S, K69E, I72T, H88Y, F89L, N108D, K109E, V110A, I1 15V, Y122D, L132P, F135S, M140V, E144G, T147A, Y157C, V170A, K174R, N186 S), hDHFR(L100P, E102G, Q103R, P104S, E105G, N108D, V113A, W114R, Y122C,M126I, N127R, H128Y, L132P, F135P, I139T, F148S, F149L, I152V, D153A, D169G, V170A, I176A, K177R, V182A, K185R, N186S), and hDHFR (A10T, Q13R, N14S, N20D, P24S, N30S, M38T, T40A, K47R, N 49S, K56R, I61T, K64R, K69R, I72A, R78G, E82G, F89L, D96G, N108D, M112V, W114R, Y122D, K123E, I139V, Q141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y183C, E184G, K185del, D187N).

[0160] ecDHFR DRD In some embodiments, the DRD of the present disclosure is derived from E. coli dihydrofolate reductase (ecDHFR). In some embodiments, the DRD is derived from an ecDHFR protein and may comprise at least one mutation. In some embodiments, the DRD is derived from an ecDHFR protein and may comprise two or more mutations. In some embodiments, the DRD is derived from an ecDHFR protein and may comprise two, three, four, or five mutations. In some embodiments, the DRD is derived from an ecDHFR protein and may comprise at least one mutation selected from Y100I, F103L, and G121V. In some embodiments, the DRD is derived from an ecDHFR protein and may comprise at least two mutations selected from R12Y, Y100I; R12H, E129K; H12Y, Y100I; H12L, Y100I; R98H, F103S; M42T, H114R; N18T, A19V; and I61F, T68S.

[0161] FKBP DRD In some embodiments, the DRD of the present disclosure is derived from an FK506 binding protein (FKBP) protein or a fragment or variant thereof. In some embodiments, the DRD is derived from an FKBP protein and may contain at least one mutation. In some embodiments, the DRD is derived from an FKBP protein and may contain two or more mutations. In some embodiments, the DRD is derived from an FKBP protein and may contain two, three, four, or five mutations.

[0162] In some embodiments, the DRD of the disclosure is derived, in whole or in part, from the human FKBP protein (SEQ ID NO: 3) and comprises at least one mutation selected from F36V, F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K105I. In some embodiments, the FKBP DRD of the disclosure comprises two or more mutations selected from F36P, L106P; and E31G, F36V, R71G, K105E.

[0163] ER DRD In some embodiments, the DRD of the present disclosure is derived from an estrogen receptor (ER) protein or a fragment or variant thereof. In some embodiments, the DRD is derived from an ER protein and may contain at least one mutation. In some embodiments, the DRD is derived from an ER protein and may contain two or more mutations. In some embodiments, the DRD is derived from an ER protein and may contain two, three, four, or five mutations.

[0164] In some embodiments, the DRD of the present disclosure comprises the ligand binding domain of ER (amino acids 305-509 of SEQ ID NO: 6). In some embodiments, the DRD may comprise at least one mutation compared to the ligand binding domain of ER, wherein the mutation occurs at position 413 (N413) and / or position 502 (Q502). In some embodiments, the mutation is at position N413 and is N413D, N413T, N413H, N413A, N413Q, N413V, N413C, N413K, N413M, N413R, N413S, N413W, N413I, N413E, N413L, N413P, N413F, N413Y, or N413G. In some embodiments, the mutation is at position Q502 and is Q502H, Q502D, Q502E, Q502V, Q502A, Q502T, Q502N, Q502K, Q502S, Q502L, Q502Y, Q502W, Q502F, Q502I, Q502G, Q502P, Q502M, or Q502C. In some embodiments, the DRD comprises mutations at positions N413 and Q502, and the mutations at position N413 are N413D, N413T, N413H, N413A, N413Q, N413V, N413C, N413K, N413M, N413R, N413S, N413W, N413I, N413E, N413L, N413P, N413F, The mutation at position Q502 is selected from Q502H, Q502D, Q502E, Q502V, Q502A, Q502T, Q502N, Q502K, Q502S, Q502L, Q502Y, Q502W, Q502F, Q502I, Q502G, Q502P, Q502M, or Q502C.

[0165] In some embodiments, at least one mutation is N413D. In some embodiments, at least one mutation is N413T. In some embodiments, at least one mutation is Q502H. In some embodiments, the ER DRD comprises at least two mutations, N413T, Q502H or N413D, Q502H.

[0166] In some embodiments, the ER DRD may further comprise one or more mutations independently selected from L384M, M421G, G521R, or Y537S.

[0167] In some embodiments, a DRD of the disclosure comprises: ER(aa 305-549 of WT, L384M, N413F, M421G, G521R, Y537S), ER(aa 305-549 of WT, L384M, N413L, M421G, G521R, Y537S), ER(aa 305-549 of WT, L384M, N413Y, M421G, G521R, Y537S), ER(aa 305-549 of WT, L384M, N413H, M421G, G521R, Y537S), ER(aa 305-549 of WT, L384M, N413Q, M421G, G 521R, Y537S), ER(WT aa305~549, L384M, N413I, M421G, G521R, Y537S), ER( WT aa305~549, L384M, N413M, M421G, G521R, Y537S), ER (WT aa305~549, L38 4M, N413K, M421G, G521R, Y537S), ER(WT aa305~549, L384M, N413V, M421G, G521R, Y537S), ER(WT aa305~549, L384M, N413S, M421G, G521R, Y537S), ER( WT aa 305-549, L384M, N413C, M421G, G521R, Y537S), ER (WT aa 305-549, L384M, N413W, M421G, G521R, Y537S), ER (WT aa 305-549, L384M, N413P, M421G, G521R, Y537S), ER (WT aa 305-549, L384M, N413R, M421G, G521R, Y537S), ER (WT aa 305-549, L384M, N413T, M421G, G521R, Y537S), ER (WT aa 305-549, L3 84M, N413A, M421G, G521R, Y537S), ER(WT aa305~549, L384M, N413E, M421G , G521R, Y537S), ER (WT aa305~549, L384M, N413G, M421G, G521R, Y537S), ER (WT aa305~549, L384M, M421G, Q502F, G521R, Y537S), ER (WT aa305~549, L3 84M, M421G, Q502L, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502Y,G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502H, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502I, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502M, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502M, G521R, Y537S) G, Q502N, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502K, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502V, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502S, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502S, G521R, Y537S) 84M, M421G, Q502C, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502W, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502P, G521R, Y537S), ER(WT aa305~549, L384M, M421G, Q502T, G521R, Y537S), ER(WT aa30 5~549、L384M、M421G、Q502A、G521R、Y537S)、ER(WTのaa305~549、L384M、M421G、Q502D、G521R、Y537S)、ER(WTのaa305~549、L384M、M421G、Q502E、G521R、Y537S)、&ER(WTのaa305~549、L384M、M421G、Q502G、G521R、Y537S)。、

[0168] CA2 DRD In some embodiments, the DRD of the present disclosure may be derived from human carbonic anhydrase 2 (hCA2), which is a member of the carbonic anhydrase superfamily of metalloenzymes. In some embodiments, the DRD may be derived from the hCA2 protein and include at least one mutation. In some embodiments, the DRD may be derived from the hCA2 protein and include two or more mutations. In some embodiments, the DRD may be derived from the hCA2 protein and include two, three, four, or five mutations.

[0169] In some embodiments, the DRD of the present disclosure may be derived from amino acids 1-260 of CA2 (SEQ ID NO: 5). In some embodiments, the DRD is derived from a CA2 that includes amino acids 2-260 of the parent CA2 sequence (e.g., amino acids 2-260 of SEQ ID NO: 5). This is referred to herein as the CA2 M1del mutation. In one embodiment, the DRD derived from CA2 may include amino acids 2-237 of the parent CA2 sequence (e.g., amino acids 2-237 of SEQ ID NO: 5).

[0170] In some embodiments, the DRD of the present disclosure comprises a region of, or the entirety of, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises a mutation selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T compared to SEQ ID NO: 5.

[0171] In some embodiments, the DRD of the disclosure comprises a region of human carbonic anhydrase 2 (CA2, SEQ ID NO: 5), or the entirety thereof, and includes any of the following amino acids as compared to SEQ ID NO: 5: A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C205R, C205V, C205R ... 5W, C205Y, D101G, D101M, D110I, D129I, D138G, D138M, D138N, D161*, D161M , D161V, D164G, D164I, D174*, D174T, D179E, D179I, D179R, D189G, D189I, D1 9T, D19V, D242G, D242T, D32T, D34T, D41T, D52I, D52L, D71F, D71G, D71K, D7 1M, D71S, D71Y, D72I, D72S, D72T, D72X, D75T, D75V, D85M, E106D, E106G, E10 6S, E117*, E117N, E14N, E186*, E186N, E204A, E204D, E204G, E204N, E213*, E213G, E213N, E220K, E220R, E220S, E233D, E233G, E233R, E235*, E235G, E23 5N, E237K, E237R, E238*, E238N, E238R, E26S, E69D, E69K, E69S, F130L, F14 6V, F175I, F175L, F175S, F178L, F178S, F20L, F20S, F225I, F225L, F225S, F2 25Y, F230I, F230L, F230S, F259L, F259S, F66S, F70I, F70L, F95Y, G102D, G1 04R, G104V, G128R, G12D, G12E, G131E, G131R, G131W, G139D, G144D, G144V, G 150A, G150S, G150W, G155A, G155C, G155D, G155S, G170A, G170D, G182A, G182 W, G195A, G195R, G232R, G232W, G234L, G234V, G25E, G63D, G63V, G81E, G81V,G82D、G86A、G86D、G98V、H107I、H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I215S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153、 * 、K153N、K158E、K158N、K167 * 、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227 * 、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143 * 、L147 * 、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197 * 、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211 * 、L211A、L211S、L223 * 、L223I、L223V、L228F、L228H、L228T、L239 * 、L239F、L239T、L250 * 、L250P、L250T、L44 * 、L44M、L47C、L47V、L57 * 、L57X、L60S、L79F、L79S、L84W、L90 * 、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177 *、N177T、N229 * 、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、P13A、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、R181S、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206 *, V206C, V206M, V210C, V217L, V217R, V217S, V222A, V222C, V222G, V241 G, V241W, V241X, V31L, V49F, V68L, V68W, V78C, W123G, W123R, W16G, W191 * , W191G, W191L, W208G, W208L, W208S, W244 * , W244G, W244L, W97C, W97G, Y114H, Y114M, Y127M, Y190 * , Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, and S29A. 「*」 indicates the translation of a stop codon, and X indicates any amino acid.

[0172] In some embodiments, a DRD of the present disclosure comprises a region of or all of human carbonic anhydrase 2 (CA2, SEQ ID NO:5) and further comprises two or more mutations compared to SEQ ID NO:5.

[0173] In some embodiments, the DRD of the disclosure is selected from the group consisting of CA2(aa2-260 of WT, R27L, H122Y), CA2(aa2-260 of WT, T87I, H122Y), CA2(aa2-260 of WT, H122Y, N252D), CA2(aa2-260 of WT, D72F, V241F), CA2(aa2-260 of WT, V241F, P249L), CA2(aa2-260 of WT, D72F, P249L), CA2(aa2-260 of WT, D71L, L250R), CA2(aa2-260 of WT, D72F, P249F), C A2 (aa 2-260 of WT, T55K, G63N, Q248N), CA2 (aa 2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa 2-260 of WT, W4Y, L156H), CA2 (aa 2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 of WT, L156H, F225L), CA2 (aa 2-260 of WT, L156H, CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R), CA2 (aa 2-260 of WT, G63D, E69V, N231I), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D), CA2 (aa 2-260 of WT, D72F, V241F, P249L), CA2 (aa 2-260 of WT, D71L, T87N, L250R), CA2 (aa 2-260 of WT, L156H, S172C, F178Y, E186D), CA2 (aa 2-260 of WT, CA2 (aa2-260 of WT, A77I, P249F), CA2 (aa2-260 of WT, E106D, C205S), CA2 (aa2-260 of WT, C205S, W208S), CA2 (aa2-260 of WT, S73N, R89Y), CA2 (aa2-260 of WT, D71K, T192F), CA2 (aa2-260 of WT, S73N, R89F), CA2 (aa2-260 of WT, G63D, M240L), CA2 (aa2-260 of WT, V134F, L228F), or CA2 (aa2-260 of WT, S56F, D71S).

[0174] In some embodiments, the DRD of the disclosure is CA2(aa2-260 of WT, R27L, H122Y), CA2(aa2-260 of WT, T87I, H122Y), CA2(aa2-260 of WT, H122Y, N252D), CA2(aa2-260 of WT, D72F, V241F), CA2(aa2-260 of WT, V241F, P249L), CA2(aa2-260 of WT, D72F, P249L), CA2(aa2-260 of WT, D71L, L250R), CA2(aa2-260 of WT, D72F, P249F ...2F, P249F), CA2(aa2-260 of WT, D72F, P249 ~260, T55K, G63N, Q248N), CA2 (WT aa2~260, L156H, A257del, S258del, F259del, K260del), CA2 (WT aa2~260, L156H, S2del, H3del, H4del, W5del), CA2 (WT aa2~260, W4Y, L156H), CA2 (WT aa2~260, L156H, G234del, E235del, P236del), CA2 (WT aa2~260, L156H, F225L), CA2 (WT aa2~260, D70N, D74N, D10 0N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R), CA2 (aa 2-260 of WT, G63D, E69V, N231I), CA2 (aa 2-260 of WT, R27L, T87I, H122Y, N252D), CA2 (aa 2-260 of WT, D72F, V241F, P249L), CA2 (aa 2-260 of WT, D71L, T87N, L250R), CA2 (aa 2-260 of WT, L156H, S172C, F178Y, E186D), CA2 (aa 2-260 of WT, D71F, N231F), CA2 (aa 2-260 of WT) CA2(aa2-260 of WT, D71K, P249H), CA2(aa2-260 of WT, D72F, P249H), CA2(aa2-260 of WT, Q53N, N61Y), CA2(aa2-260 of WT, E106D, C205S), CA2(aa2-260 of WT, C205S, W208S), CA2(aa2-260 of WT, S73N, R89Y), CA2(aa2-260 of WT, D71K, T192F), CA2(aa2-260 of WT, Y193L, K260L), CA2(aa2-260 of WT,D71F, V241F, P249L), CA2 (aa 2-260 of WT, L147F, Q248F), CA2 (aa 2-260 of WT, D52I, S258P), CA2 (aa 2-260 of WT, D72S, T192N), CA2 (aa 2-260 of WT, D179E, T192I), CA2 (aa 2-260 of WT, S56N, Q103K), CA2 (aa 2-260 of WT, D71Y, Q248L), CA2 (aa 2-260 of WT, S73N, R89F), CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F), CA CA2 (aa2-260 of WT, D72F, P249I), CA2 (aa2-260 of WT, D72X, V241X, P249X), CA2 (aa2-260 of WT, A54X, S56X, L57X, T192X), CA2 (aa2-260 of WT, Y193V, K260F), CA2 (aa2-260 of WT, G63D, M240L), CA2 (aa2-260 of WT, V134F, L228F), CA2 (aa2-260 of WT, D71G, N231K), CA2 (aa2-260 of WT, S56F, D71S), CA2 (aa2-260 of WT, D52 L, G128R, Q248F), CA2 (aa 2-260 of WT, S73X, R89X), CA2 (aa 2-260 of WT, Y51X, D72X, V241X, P249X), CA2 (aa 2-260 of WT, D72I, W97C), CA2 (aa 2-260 of WT, D71K, T192F, N231F), CA2 (aa 2-260 of WT, H36Q, S43T, Y51F, N67D, G131W, R226H), CA2 (aa 2-260 of WT, F70I, F146V), CA2 (aa 2-260 of WT, K45N, V68L, H119Y, K169R , D179E), CA2 (aa 2-260 of WT, H15L, A54V, K111E, E220K, F225I), CA2 (aa 2-260 of WT, P13S, P83A, D101G, K111N, F230I), CA2 (aa 2-260 of WT, G63D, W123R, E220K), CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S), CA2 (aa 2-260 of WT, I59N, G102R, A173T), CA2 (aa 2-260 of WT, L79F, P180S),CA2 (aa 2-260 of WT, A77P, G102R, D138N), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I), CA2 (aa 2-260 of WT, T199N, L202P, L228F), CA2 (aa 2-260 of WT, K9N, H122Y, T168K), CA2 (aa 2 of WT) ~260, Q53H, L90V, Q92H, G131E), CA2 (aa2~260 of WT, L44M, L47V, N62K, E69D), CA2 (aa2~260 of WT, D75V, K169N, F259L), CA2 (aa2~260 of WT, T207S, V222A, N231D), CA2 (aa2~260 of WT, I59F, V 206M, G232R), CA2 (aa 2-260 of WT, P13A, A133T), CA2 (aa 2-260 of WT, I59N, R89I), CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L), CA2 (aa 2-260 of WT, G12R, H15Y , D19V), CA2 (aa 2 to 260 of WT, A65V, F95Y, E106G, H107Q, I145M, F175I), CA2 (aa 2 to 260 of WT, G63D, E69V, N231I), CA2 (aa 2 to 260 of WT, S29A, C205S) and / or CA2 (aa 2 to 260 of WT, S29C, C205S).

[0175] In some embodiments, the DRD of the disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises an H122Y amino acid mutation at position 122 (H122) of SEQ ID NO: 5. In some such embodiments, the DRD further comprises (i) an R27L amino acid mutation at position 27 (R27) of SEQ ID NO: 5, (ii) a T87I amino acid mutation at position 87 (T87) of SEQ ID NO: 5, (iii) an N252D amino acid mutation at position 252 (N252) of SEQ ID NO: 5, or a combination of (i), (ii), and / or (iii).

[0176] In some embodiments, the DRD of the disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises an amino acid E106D mutation at position 106 (E106) of SEQ ID NO: 5. In some such embodiments, the DRD further comprises an amino acid C205S mutation at position 205 (C205) of SEQ ID NO: 5.

[0177] In some embodiments, the DRD of the disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises an amino acid W208S mutation at position 208 (W208) of SEQ ID NO: 5. In some such embodiments, the DRD further comprises an amino acid C205S mutation at position 205 (C205) of SEQ ID NO: 5.

[0178] In some embodiments, the DRD of the disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises an amino acid I59N mutation at position 59 (I59) of SEQ ID NO: 5. In some such embodiments, the DRD further comprises an amino acid G102R mutation at position 102 (G102) of SEQ ID NO: 5.

[0179] In some embodiments, the DRD of the disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises an amino acid L156H mutation at position 156 (L156) of SEQ ID NO: 5. In some such embodiments, the DRD further comprises (i) an amino acid W4Y mutation at position 4 (W4) of SEQ ID NO: 5, (ii) an amino acid F225L mutation at position 225 (F225) of SEQ ID NO: 5, (iii) a deletion of amino acids at positions 257-260 of SEQ ID NO: 5, (iv) a deletion of amino acids at positions 1-5 of SEQ ID NO: 5, or (v) a deletion of amino acids G234, E235, and P236 of SEQ ID NO: 5.

[0180] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises four mutations compared to SEQ ID NO: 5, the mutations corresponding to (i) L156H, S172C, F178Y, and E186D, or (ii) D70N, D74N, D100N, and L156H.

[0181] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises a first mutation and a second mutation compared to SEQ ID NO: 5, wherein (i) the first mutation is an S73N mutation of amino acid at position 73 (S73) of SEQ ID NO: 5, and (ii) the second mutation is a substitution of F or Y at amino acid position 89 (R89) of SEQ ID NO: 5.

[0182] In some embodiments, the DRD of the disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises an N or F substitution at amino acid position 56 (S56) of SEQ ID NO: 5. In some such embodiments, the DRD comprises two substitutions relative to SEQ ID NO: 5 corresponding to S56F and D71S.

[0183] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises one or more substitutions compared to SEQ ID NO: 5, wherein at least one substitution is a D or N substitution at amino acid position 63 (G63) of SEQ ID NO: 5, and the one or more substitutions correspond to: (i) G63D, (ii) G63D and M240L, (iii) G63D, E69V and N231I, or (iv) T55K, G63N and Q248N.

[0184] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises two or more substitutions compared to SEQ ID NO: 5, one of the two or more substitutions being a substitution of L or K at amino acid position 71 (D71) of SEQ ID NO: 5, the two or more substitutions corresponding to (i) D71L and T87N, (ii) D71L and L250R, (iii) D71L, T87N and L250R, or (iv) D71K and T192F.

[0185] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises two or more substitutions compared to SEQ ID NO: 5, wherein at least one of the two or more substitutions is (i) a substitution of F at amino acid position 241 (V241) of SEQ ID NO: 5, or (ii) a substitution of F or L at amino acid position 249 (P249) of SEQ ID NO: 5, and the two or more substitutions correspond to: (i) D72F and V241F; (ii) D72F and P249L; (iii) D72F and P249F; (iv) D72F, V241F and P249L; (v) A77I and P249F; or (vi) V241F and P249L.

[0186] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5) and further comprises one or more substitutions selected from Y51T, L183S, Y193I, L197P, and a combination of V134F and L228F compared to SEQ ID NO: 5.

[0187] The amino acid sequence of a DRD encompassed by the present disclosure has at least about 70% identity, preferably at least about 75% or 80% identity, more preferably at least about 85%, 86%, 87%, 88%, 89% or 90% identity, and even more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the parent protein from which it is derived. In some embodiments, the amino acid sequence of a DRD encompassed by the present disclosure has at least about 70% identity, preferably at least about 75% or 80% identity, more preferably at least about 85%, 86%, 87%, 88%, 89% or 90% identity, and even more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the parent protein from which it is derived (e.g., a parent protein having the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 71).

[0188] Examples of DRDs of the present disclosure include those derived from human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. Suitable DRDs may also be referred to as destabilizing domains or ligand-binding domains, which are also known in the art. See, for example, WO2018 / 161000, WO2018 / 231759, WO2019 / 241315, US8,173,792, US8,530,636, WO2018 / 237323, WO2017 / 181119, US2017 / 0114346, US2019 / 0300864, WO2017 / 156238, Miyazaki et al., J Am Chem Soc,134:3942(2012), Banaszynski et al.(2006)Cell 126:995-1004, Stankunas,K.et al.(2003)Mol.Cell 12:1615-1624, Banaszynski et al. al. (2008) Nat. Med.14:1123-1127, Iwamoto et al. (2010) Chem. Biol.17:981-988, Armstrong et al. (2007) Nat. Methods 4:1007-1009, Madeira da Silva et al. (2009) Proc. Natl. Acad. Sci. USA 106:7583-7588, Pruett-Miller et al. (2009) PLoS See Genet. 5:e1000376, and Feng et al. (2015) Elife 4:e10606.

[0189] As provided above in the "Transcription Factor Systems" section, one or more polynucleotide combinations of a transcription factor system include a nucleic acid sequence encoding a drug response domain (DRD), wherein the transcription factor (e.g., a transcription factor DNA binding domain, a transcription factor activation domain, or both) is operably linked to the DRD. The nucleic acid sequence encoding the DRD may be selected from the sequences of the DRDs described herein. Constructs comprising DRD sequences are provided in Table 1 above. Additional constructs comprising different DRDs are provided in Table 4. The asterisks ( 「*」 ) indicates translation of the stop codon. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0190] Stimulation of transcription factor systems The transcription factor system of the present disclosure can be responsive to a stimulus.

[0191] In some embodiments, the stimulus is a ligand. The ligand may be nucleic acid-based, protein-based, lipid-based, organic, inorganic, or any combination of the foregoing. In some embodiments, the ligand may be a synthetic molecule. In some embodiments, the ligand may be a small molecule therapeutic compound. In some embodiments, the ligand may be a small molecule drug previously approved by a regulatory agency, such as the U.S. Food and Drug Administration (FDA).

[0192] As described in this disclosure, the transcription factor system can exhibit ligand-dependent activity. A ligand can bind to the DRD and stabilize the transcription factor or domain of the transcription factor encoded by the transcription factor system. Ligands known to bind to candidate DRDs can be tested for their effect on the activity of the transcription factor system.

[0193] In some embodiments, the ligand is cell-permeable. In some embodiments, the ligand may be designed to be lipophilic to improve cell permeability.

[0194] In some embodiments, the ligand is a small molecule. Small molecule ligands may be clinically approved to be safe and have suitable pharmacokinetics and distribution.

[0195] In some embodiments, the ligand may be conjugated or bound to one or more other molecules, such as, but not limited to, another ligand, a protein, a peptide, a nucleic acid, a lipid, a lipid derivative, a sterol, a steroid, a metabolite, a metabolite derivative, or a small molecule. In some embodiments, the ligand stimulus is conjugated or bound to one or more different types and / or numbers of other molecules. In some embodiments, the ligand stimulus is a multimer of the same type of ligand. In some embodiments, the ligand stimulus multimer includes two, three, four, five, six, or more monomers.

[0196] CA2 ligand In some embodiments, the ligands of the present disclosure bind to carbonic anhydrase, hi some embodiments, the ligands bind to carbonic anhydrase and inhibit its function, and are referred to herein as carbonic anhydrase inhibitors.

[0197] In some embodiments, the ligand is a small molecule that binds to carbonic anhydrase 2. In one embodiment, the small molecule is a CA2 inhibitor. Examples of CA2 inhibitors include, but are not limited to, celecoxib (also known as Celebrex), valdecoxib, rofecoxib, acetazolamide, methazolamide, dorzolamide, brinzolamide, diclophenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide.

[0198] In some embodiments, the ligand may comprise a portion of a small molecule known to mediate binding to CA2. The ligand may also be modified to reduce off-target binding to carbonic anhydrases other than CA2 and increase specific binding to CA2.

[0199] In some embodiments, the stimulus can be a ligand that binds to more than one carbonic anhydrase, hi one embodiment, the stimulus is a pan-carbonic anhydrase inhibitor that may bind to more than one carbonic anhydrase.

[0200] DHFR ligand In some embodiments, the ligands of the present disclosure bind to dihydrofolate reductase, hi some embodiments, the ligands bind to and inhibit the function of dihydrofolate reductase and are referred to herein as dihydrofolate inhibitors.

[0201] In some embodiments, the ligands may be selective inhibitors of human DHFR. The ligands of the present disclosure may also be selective inhibitors of dihydrofolate reductase from bacteria and parasites, such as Pneumocystis species, Toxoplasma species, Trypanosoma species, Mycobacterium species, and Streptococcus species. Ligands specific for other DHFR may be modified to improve binding to human dihydrofolate reductase.

[0202] Examples of dihydrofolate inhibitors include, but are not limited to, trimethoprim (TMP), methotrexate (MTX), pralatrexate, piritrexim, pyrimethamine, tarotrexin, chlorguanide, pentamidine, trimetrexate, aminopterin, C1 898 trihydrochloride, pemetrexed disodium, raltitrexed, sulfaguanidine, Folotyn, iclaprim, and diaveridine.

[0203] In some embodiments, the ligands of the present disclosure may include dihydrofolic acid or any of its derivatives that may bind to human DHFR. In some embodiments, the ligands of the present disclosure may be 2,4,diaminoheterocyclic compounds. In some embodiments, the 4-oxo group in dihydrofolic acid may be modified to generate a DHFR inhibitor. In one example, the 4-oxo group may be replaced with a 4-amino group. Various diaminoheterocycles, including pteridines, quinazolines, pyridopyrimidines, pyrimidines, and triazines, may also be used as scaffolds for developing DHFR inhibitors and may be used in accordance with the present disclosure.

[0204] In some embodiments, the ligand comprises a TMP-derived ligand containing a portion of the ligand known to mediate binding to DHFR. The ligand may also be modified to reduce off-target binding to other folate metabolic enzymes and increase specific binding to DHFR.

[0205] ER ligand In some embodiments, the ligands of the present disclosure bind to ER. The ligands may be agonists or antagonists. In some embodiments, the ligands bind to ER and inhibit its function, and are referred to herein as ER inhibitors. In some embodiments, the ligands may be selective inhibitors of human ER. The ligands of the present disclosure may also be selective inhibitors of ER of other species. Ligands specific for other ERs may be modified to improve binding to human ER.

[0206] The ligand may be an ER agonist, such as, but not limited to, the endogenous estrogen 17b-estradiol (E2) and the synthetic nonsteroidal estrogen diethylstilbestrol (DES). In some embodiments, the ligand may be an ER antagonist, such as, for example, ICI-164,384, RU486, tamoxifen, 4-hydroxytamoxifen (4-OHT), fulvestrant, toremifene, lasofoxifene, clomiphene, femarelle, and ormeloxifene and raloxifene (RAL).

[0207] In some embodiments, the stimulus of the present disclosure can be an ER antagonist, such as, but not limited to, bazedoxifene and / or raloxifene.

[0208] In some embodiments, the ligand comprises a bazedoxifene-derived ligand containing a portion of the ligand known to mediate binding to the ER. The ligand may also be modified to reduce off-target binding to other folate-metabolizing enzymes and increase specific binding to the DRD from the ER.

[0209] Phosphodiesterase Ligands In some embodiments, the ligands of the present disclosure bind to phosphodiesterases, hi some embodiments, the ligands bind to phosphodiesterases and inhibit their function, and are referred to herein as phosphodiesterase inhibitors.

[0210] In some embodiments, the ligand is a small molecule that binds to phosphodiesterase 5. In one embodiment, the small molecule is a hPDE5 inhibitor. Examples of hPDE5 inhibitors include, but are not limited to, sildenafil, vardenafil, tadalafil, avanafil, lodenafil, mirodenafil, udenafil, benzamidenafil, dasantafil, beminafil, SLx-2101, LAS34179, UK-343,664, UK-357903, UK-371800, and BMS-341400.

[0211] In some embodiments, the ligand comprises a sildenafil-derived ligand containing a portion of the ligand known to mediate binding to hPDE5. The ligand may also be modified to reduce off-target binding to phosphodiesterase and increase specific binding to hPDE5.

[0212] In some embodiments, the stimulus can be a ligand that binds to more than one phosphodiesterase, hi one embodiment, the stimulus is a pan-phosphodiesterase inhibitor that may bind to more than one hPDE, such as aminophylline, paraxanthine, pentoxifylline, theobromine, dipyridamole, theophylline, zaprinast, icariin, CDP-840, etazolate, and glaucine.

[0213] In some embodiments, the ligand is an hPDE1 inhibitor. In some embodiments, the ligand is an hPDE2 inhibitor. In some embodiments, the ligand is an hPDE3 inhibitor. In some embodiments, the ligand is an hPDE4 inhibitor. In some embodiments, the ligand is an hPDE6 inhibitor. In some embodiments, the ligand is an hPDE7 inhibitor. In some embodiments, the ligand is an hPDE8 inhibitor. In some embodiments, the ligand is an hPDE9 inhibitor. In some embodiments, the ligand is an hPDE10 inhibitor.

[0214] FKBP ligand In some embodiments, the ligand of the present disclosure binds to FKBP, including human FKBP, hi some embodiments, the ligand is SLF or Shield-1.

[0215] payload The payload may comprise any polypeptide or protein or fragment thereof. The payload may be a wild-type sequence, a fragment of a wild-type sequence, and / or may contain one or more mutations. The payload may be a naturally occurring protein derived from the genome of an organism, or a variant, mutant, or derivative thereof. Naturally occurring proteins may be derived from, for example, mammalian organisms, bacteria, and viruses. The payload may be a protein or polypeptide encoded by a recombinant nucleic acid molecule, a fusion or chimeric polypeptide, or a polypeptide that functions as part of a protein complex.

[0216] In one example, the payload can be a polypeptide encoded by a nucleic acid sequence in the human genome.

[0217] In some embodiments, the payload may be a variant sequence of a parent polypeptide. In some aspects, the variant sequence may have the same or similar activity as the reference sequence. Alternatively, the variant may have altered activity (e.g., increased or decreased) compared to the reference sequence. Generally, a variant of a particular polypeptide of the present disclosure will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, but less than 100%, with the particular reference polypeptide, as determined by sequence alignment programs known to those skilled in the art.

[0218] Therapeutic agents as payloads In some embodiments, the payload of the present disclosure can be a therapeutic agent. For example, the payload can be a cancer therapeutic agent, an autoimmune disease therapeutic agent, an immunotherapeutic agent, an anti-inflammatory agent, an anti-pathogen agent, or a gene therapy agent. In some aspects, the immunotherapeutic agent can be an antibody and fragments and variants thereof, a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric switch receptor, an antagonist of a co-inhibitory molecule, an agonist of a co-stimulatory molecule, a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a metabolic factor, a coagulation factor, an enzyme, a homing receptor, and a safety switch.

[0219] In some embodiments, the payload of the present disclosure may be an immunotherapeutic agent that induces an immune response in an organism. The immunotherapeutic agent may be, but is not limited to, an antibody and fragments and variants thereof, a TCR, a chimeric antigen receptor (CAR), a chimeric switch receptor, a cytokine, a chemokine, a cytokine receptor, a chemokine receptor, a cytokine-cytokine receptor fusion polypeptide, or any agent that induces an immune response. In one embodiment, the immunotherapeutic agent induces an anti-cancer immune response in a cell or a subject.

[0220] Cytokines, chemokines and other soluble factors as payloads In some embodiments, the payloads of the present disclosure may be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells, and other cell types, which act as chemical messengers between cells and tissues in the body. These proteins mediate a wide range of physiological functions, from effects on cell growth, differentiation, migration, and survival to numerous effector activities. For example, activated T cells produce various cytokines for cytotoxic function to eliminate tumor cells.

[0221] In some embodiments, the payload of the present disclosure may be a cytokine, as well as fragments, variants, analogs, and derivatives thereof, including, but not limited to, interleukins, tumor necrosis factors (TNFs), interferons (IFNs), TGF-beta, and chemokines. In some embodiments, the payload of the present disclosure may be a cytokine that stimulates an immune response. In other embodiments, the payload of the present disclosure may be an antagonist of a cytokine that negatively impacts anti-cancer immune responses.

[0222] In some embodiments, the payload of the present disclosure may be a cytokine receptor, a recombinant receptor thereof, a variant, analog, or derivative thereof, or a signaling component of a cytokine. In various embodiments, the payload of the present disclosure may include a secreted cytokine or a membrane-bound form of a cytokine. Illustrative examples of membrane cytokines may include cytokines (e.g., immunostimulatory cytokines, e.g., IL12, IL2, IL15, and IL18) operably fused, linked, or attached to a transmembrane domain, e.g., a CD8α transmembrane domain, a B7-1 transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, or a human IgG4 Fc region. In various embodiments, the cytokine may be fused or attached to the transmembrane domain by an intervening peptide or protein sequence, such as a linker, hinge, or transmembrane tail.

[0223] In one embodiment, the payload of the present disclosure may be a cytokine fused to a TNF alpha ectodomain. Such a payload is produced as a membrane-associated cytokine fused to a TNF ectodomain. In one embodiment, the cytokine may be shed from the cell surface by the action of membrane-associated proteases and / or proteases in the extracellular space, such as MMP9.

[0224] In some embodiments, the payload of the present disclosure may be an interleukin (IL) cytokine. Interleukins (IL) are a class of glycoproteins produced by white blood cells to regulate immune responses. As used herein, the term "interleukin (IL)" refers to an interleukin polypeptide from any species or source, including full-length proteins as well as fragments or portions of proteins.

[0225] In some embodiments, the payload of the present disclosure may comprise IL12. IL12 is a heterodimeric protein of two subunits (p35, p40) secreted by antigen-presenting cells such as macrophages and dendritic cells. Expression of IL12 requires coexpression of the two subunits to produce a biologically active heterodimer. In some embodiments, the payload of the present disclosure may be the p35 subunit or the p40 subunit.

[0226] In some embodiments, the payload of the present disclosure may comprise all or a portion of IL12.

[0227] In some embodiments, the IL12 may be Flexi IL12, in which both the p35 and p40 subunits are encoded by a single cDNA that produces a single polypeptide chain. The single polypeptide chain may be generated by placing the p35 subunit at the N-terminus or C-terminus of the single polypeptide chain. Similarly, the p40 subunit may be at the N-terminus or C-terminus of the single polypeptide chain.

[0228] The format of the IL12 payload of the present disclosure may be optimized. In one embodiment, the payload may be a bicistronic IL12 containing p40 and p35 subunits separated by an internal ribosome entry site or a cleavage site such as P2A or furin to allow independent expression of both subunits from a single vector. In another embodiment, the payload may be the p40 subunit of IL12 or the p35 subunit of IL12.

[0229] In some embodiments, the payload may be membrane-bound IL12. IL12 may be membrane-bound by a transmembrane domain. The transmembrane domain may also include an optional hinge domain. In some aspects, the IL12 molecule is extracellular and tethered to the cell by the transmembrane domain. In some aspects, membrane-bound IL12 may be shed or cleaved from the cell surface by the action of a protease. In some embodiments, the transmembrane domain of the present disclosure may be derived from either a natural source or a synthetic source. The transmembrane domain may be derived from any naturally occurring membrane-associated or transmembrane protein. Alternatively, the transmembrane domain of the present disclosure may be synthetic. In some aspects, synthetic sequences may include primarily hydrophobic residues such as leucine and valine. In some aspects, transmembrane and / or hinge domains may be selected that are resistant to protease activity.

[0230] In some embodiments, the payload of the present disclosure may comprise IL15, a potent immunostimulatory cytokine and essential survival factor for T cells and natural killer cells.

[0231] In some embodiments, the payload of the present disclosure may comprise all or a portion of IL15. Any portion of IL15 that retains one or more functions of full-length or mature IL15 may be useful in the present disclosure. Such functions include promoting NK cell survival, regulating NK cell and T cell activation and proliferation, as well as supporting NK cell development from hematopoietic stem cells.

[0232] In some cases, all or a portion of IL15 is linked to all or a portion of one or more transmembrane proteins.

[0233] The IL15 payload may be designed to be secreted (eg, using an IL2 signal sequence) or membrane-bound (eg, using an IgE or CD8a signal sequence).

[0234] A unique feature of IL15-mediated activation is the trans-presentation mechanism, in which IL15 is presented as a complex with the alpha subunit of the IL15 receptor (IL15Ra), which binds to and activates membrane-bound IL15 beta / gamma receptors, either on the same cell or on different cells. In some embodiments, the payload of the present disclosure is membrane-bound IL15. In some embodiments, the payload of the present disclosure may comprise an IL15 / IL15Ra fusion polypeptide. In some embodiments, the payload may be all or a portion of IL15 fused to all or a portion of IL15Ra. Any portion of IL15 and IL15Ra that retains one or more functions of full-length or mature IL15 or IL15Ra, respectively, may be used.

[0235] In some embodiments, the IL15 molecule is extracellular and tethered to the cell by a transmembrane domain, hi some embodiments, membrane-bound IL15 may be shed or cleaved from the cell surface by the action of a protease.

[0236] The entire membrane-bound IL15 or IL15 / IL15Ra fusion polypeptide of the present disclosure, or a portion thereof, may be shed into the extracellular space. As used herein, shedding refers to the release of membrane-associated biomolecules from the membrane to which they are tethered. In some cases, shedding may be induced by proteolytic cleavage.

[0237] The payload of the present disclosure may comprise an amino acid sequence similar to the amino acid sequence of human IL15, for example, UniProtKB-P40933 (IL15_HUMAN).

[0238] In some embodiments, the payloads of the present disclosure may be utilized to improve the proliferation, survival, persistence, and efficacy of immune cells, such as CD8+ TEM, natural killer cells, and tumor-infiltrating lymphocyte (TIL) cells, as well as CAR T cells used in immunotherapy. In one aspect, the present disclosure provides a payload to minimize toxicity associated with cytokine therapy. In some embodiments, the payload of the present disclosure may comprise all or a portion of IL2. Any portion of IL2 that retains one or more functions of full-length or mature IL2 may be useful in the present disclosure.

[0239] It is understood in the art that a particular gene and / or protein nomenclature for the same gene or protein may or may not include punctuation marks such as a dash "-" or symbols such as Greek letters. Whether they are included or excluded herein is not intended to change the meaning as understood by those of skill in the art. For example, IL2, IL-2, and IL 2 refer to the same interleukin. Similarly, IL15, IL 15, and IL-15 refer to the same interleukin. Similarly, TNF alpha, TNFα, TNF-alpha, TNF-α, TNF alpha, and TNFα all refer to the same protein.

[0240] Antibodies and antibody fragments as payloads In some embodiments, the payloads of the present disclosure may be antibodies, antibody fragments, and variants thereof.

[0241] The antibody may be an intact antibody, an antibody light chain, an antibody heavy chain, an antibody fragment, an antibody variant, or an antibody derivative.

[0242] For purposes herein, an "antibody" may include an Fc region in addition to heavy and light chain variable domains.

[0243] In some embodiments, the payload may be a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., each individual antibody in the population binds to the same and / or identical epitope, excluding possible variants that may arise during the production of the monoclonal antibody, such as variants generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0244] In one embodiment, the payload of the present disclosure may be a humanized antibody. As used herein, the term "humanized antibody" refers to a chimeric antibody that contains minimal portions from one or more non-human (e.g., murine) antibody source(s), the remainder being derived from one or more human immunoglobulin sources. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient antibody are replaced by hypervariable region residues of an antibody of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capacity. In one embodiment, the antibody may be a humanized full-length antibody.

[0245] As used herein, the term "antibody variant" refers to an engineered antibody (related to a natural or starting antibody) or a biomolecule (e.g., an antibody mimetic) that is similar in structure and / or function to a natural or starting antibody. Antibody variants may have altered amino acid sequence, composition, or structure when compared to a natural antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.

[0246] In some embodiments, antibody fragments and variants may comprise the antigen-binding region of an intact antibody. Examples of antibody fragments and variants may include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules such as single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site. A remaining "Fc" fragment is also produced, the name reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. Payloads of the present disclosure may comprise one or more of these fragments.

[0247] In some embodiments, the antibody payload of the present disclosure may be a therapeutic antibody.

[0248] Chimeric Antigen Receptor Payloads In some embodiments, the payload of the present disclosure may be a chimeric antigen receptor (CAR). As used herein, the term "chimeric antigen receptor (CAR)" refers to a synthetic receptor that mimics the T cell receptor (TCR) on the surface of a T cell. Generally, a CAR is composed of an extracellular targeting domain, a transmembrane domain / region, and an intracellular signaling / activation domain. Cells, such as T cells, engineered to express a CAR can be redirected to attack target cells expressing a molecule that can be recognized by the targeting portion of the CAR. In a standard CAR receptor, the components: the extracellular targeting domain, the transmembrane domain, and the intracellular signaling / activation domain are linearly assembled into a single fusion protein. The extracellular region includes a targeting domain / moiety (e.g., an scFv) that recognizes a specific tumor antigen or other tumor cell surface molecule. The intracellular region may contain a signaling domain of the TCR complex (e.g., the signaling region of CD3ζ) and / or one or more costimulatory signaling domains, such as those derived from CD28, 4-1BB (CD137), and OX-40 (CD134). For example, while "first-generation CARs" only have a CD3ζ signaling domain, a costimulatory intracellular domain is added to enhance T cell persistence and proliferation, forming second-generation CARs with a CD3ζ signaling domain and one costimulatory signaling domain, and third-generation CARs with a CD3ζ signaling domain and two or more costimulatory signaling domains. When expressed by a T cell, the CAR confers antigen specificity to the T cell as determined by the extracellular targeting portion of the CAR. Fourth-generation CARs include the addition of one or more elements, such as a homing gene and a suicide gene, to develop a more effective and safer CAR structure.

[0249] In some embodiments, the CAR payload, when transduced into immune cells (e.g., T cells and NK cells), can redirect the immune cells to targets (e.g., tumor cells) that express a molecule recognized by the extracellular targeting portion of the CAR.

[0250] Nucleic acid modifiers as payloads In some embodiments, the payload of the present disclosure may be a nucleic acid modifying agent.

[0251] In some embodiments, the payload of the present disclosure may be a component of a gene editing system. In some embodiments, the payload of the present disclosure may be a Cas protein (CRISPR-associated protein), including Cas9 and Cas12. The Cas protein may be altered or otherwise modified. For example, the Cas protein may be a dead Cas9. In some embodiments, the Cas9 protein is an enzymatically active Cas9 protein, a wild-type Cas9 protein, a nickase Cas9 protein, or a nuclease-null or nuclease-deficient Cas9 protein. In some embodiments, the payload of the present disclosure may be a zinc finger nuclease, a TALEN (transcription activator-like effector-based nuclease), or a meganuclease.

[0252] In some embodiments, the payload of the present disclosure may be a recombinase, such as Cre recombinase.

[0253] Drugs for treating autoimmune disorders as payloads In some embodiments, the payload of the present disclosure may be a drug for treating, ameliorating, or preventing an autoimmune disorder.

[0254] In some embodiments, payloads of the present disclosure include anti-cytokines, such as neutralizing antibodies against tumor necrosis factor (TNF)-α, IL-1, and IL-6. In some embodiments, payloads of the present disclosure target B cell depletion, such as neutralizing antibodies against CD20, CD22, CD28, CTLA-4, and B lymphocyte stimulatory factor (BLyS).

[0255] Pharmaceutical Compositions and Formulations The present teachings further include pharmaceutical compositions comprising one or more of the disclosed transcription factor systems, nucleic acids, polynucleotides, modified cells, or payloads, and optionally at least one pharmaceutically acceptable excipient or inactive ingredient.

[0256] As used herein, the term "pharmaceutical composition" refers to a preparation of one or more of the transcription factor systems, nucleic acids, polynucleotides, modified cells, payloads or transcription factor system components described herein, or pharmaceutically acceptable salts thereof, optionally including other chemical components such as physiologically suitable carriers and excipients.

[0257] The term "excipient" or "inactive ingredient" refers to an inert or inactive substance added to a pharmaceutical composition to further facilitate administration of a compound.

[0258] In some embodiments, the composition is administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to any one or more transcription factor system components delivered as described herein.

[0259] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, non-human mammals, including agricultural animals such as cows, horses, chickens, and pigs, domestic animals such as cats and dogs, or research animals such as mice, rats, rabbits, dogs, and non-human primates.

[0260] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and / or an appropriate fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0261] The relative amounts of active ingredient, pharmaceutically acceptable excipient or inactive ingredient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may comprise 0.1% to 100%, e.g., 0.5 to 50%, 1 to 30%, 5 to 80%, at least 80% (w / w) active ingredient.

[0262] The effectiveness of disease treatment or amelioration can be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dose of drug required to maintain the treatment effect, the level of disease markers, or any other measurable parameter appropriate for the given disease being treated or targeted for prevention. A medical professional skilled in the art may monitor the effectiveness of treatment or prevention by measuring any one of such parameters or any combination of parameters. In relation to the administration of the compositions of the present disclosure, for example, "effective against" cancer indicates that administration in a clinically relevant manner will result in a beneficial effect in at least a significant proportion of patients, such as symptom improvement, cure, reduction in disease burden, reduction in tumor mass or cell count, prolonged lifespan, improved quality of life, or other effect generally recognized as positive by physicians familiar with the treatment of a particular type of cancer.

[0263] Treatment or prevention effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when symptoms do not worsen or develop, which would otherwise be expected.As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in measurable parameters of the disease can indicate effective treatment.The effectiveness of a given composition or formulation of the present disclosure can also be determined using an experimental animal model of a given disease, as known in the art.When using an experimental animal model, the effectiveness of treatment is proven when a statistically significant change is observed.

[0264] formulation The polynucleotide and vector compositions of the present disclosure may be formulated in any manner suitable for delivery, including, but not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, and combinations thereof.

[0265] In one embodiment, the polynucleotide and vector formulations are nanoparticles that may contain at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, and PEGylated lipids. In another embodiment, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, and DODMA.

[0266] For polynucleotides of the present disclosure, formulations may be selected from, for example, any of those taught in International Application PCT / US2012 / 069610.

[0267] inactive ingredients In some embodiments, a pharmaceutical or other formulation may include at least one excipient that is an inactive ingredient. As used herein, the term "inactive ingredient" refers to one or more inactive agents included in a formulation. In some embodiments, all, some, or none of the inactive ingredients that may be used in the formulations of the present disclosure may be approved by the U.S. Food and Drug Administration (FDA).

[0268] Dosage, Delivery and Administration The compositions of the present disclosure may be delivered to cells or subjects by one or more routes and modalities. Viral vectors containing one or more of the transcription factor systems, nucleic acids, polynucleotides, payloads, and other components described herein may be used to deliver them to cells and / or subjects. Other modalities, such as mRNA and plasmids, may also be used, and may also be used as recombinant proteins.

[0269] Delivery Naked Delivery The pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads of the present disclosure may be delivered to cells, tissues, organs, and / or organisms in a naked form. As used herein, the term "naked" refers to a pharmaceutical composition, transcription factor system, nucleic acid, polynucleotide, or payload that is delivered without agents or modifications that promote transfection or permeability. Naked pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads may be delivered to cells, tissues, organs, and / or organisms using routes of administration known in the art and described herein. In some embodiments, naked delivery may involve formulation in a simple buffer, such as saline or PBS.

[0270] Formulation Delivery In some embodiments, the pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads of the present disclosure may be formulated using the methods described herein. The formulations may include pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads, which may be modified and / or unmodified. The formulations may further include, but are not limited to, cell-penetrating agents, pharmaceutically acceptable carriers, delivery agents, bioerodible or biocompatible polymers, solvents, and / or sustained-release delivery depots. The formulations of the present disclosure may be delivered to cells using routes of administration known in the art and described herein.

[0271] The pharmaceutical composition, transcription factor system, nucleic acid, polynucleotide, or payload may also be formulated for delivery directly to an organ or tissue by any of several methods known in the art, including, but not limited to, direct immersion or bath, via catheter, gel, powder, ointment, cream, gel, lotion, and / or infusion, by means of a substrate such as a fabric or biodegradable material coated or impregnated with the composition, and the like.

[0272] Cellular delivery In another aspect of the present disclosure, polynucleotides of a transcription factor system or components thereof and compositions and vectors comprising polynucleotides of the present disclosure may be introduced into cells, such as immune effector cells.

[0273] In one aspect of the present disclosure, the polynucleotides of the transcription factor system or its components and the compositions of the present disclosure may be packaged into a plasmid, a viral vector, or integrated into a viral genome, allowing transient or stable expression of the polynucleotide. Preferred viral vectors are retroviral vectors, including lentiviral vectors and gammaretroviral vectors. To construct a retroviral vector, a polynucleotide molecule of the transcription factor system is inserted into the viral genome in place of a specific viral sequence to create a replication-deficient virus. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol, and env genes but lacking the LTR and packaging components. The recombinant retroviral particles are secreted into the culture medium and then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are particularly preferred because they can infect both dividing and non-dividing cells.

[0274] Vectors may also be transferred into cells by non-viral methods, such as physical methods, such as needles, electroporation, sonoporation, hydroporation, chemical carriers, such as inorganic particles (e.g., calcium phosphate, silica, gold), and / or chemical methods. In some embodiments, synthetic or natural biodegradable agents may be used for delivery, such as cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based vectors, or polymer-based vectors. In some embodiments, vectors may be transferred into cells by transient membrane disruption, e.g., by rapid cell deformation.

[0275] In some embodiments, polypeptides of the present disclosure may be delivered directly to cells. In one embodiment, polypeptides of the present disclosure may be delivered using a synthetic peptide containing an endosomal leakage domain (ELD) fused to a cell penetration domain (CLD). The polypeptides of the present disclosure are co-transfected into cells with an ELD-CLD-synthetic peptide. ELDs facilitate the escape of endosome-trapped proteins into the cytosol. Such domains are obtained from proteins of microbial and viral origin and have been described in the art. CPDs enable transport of proteins across the plasma membrane and have also been described in the art. ELD-CLD fusion proteins synergistically increase transduction efficiency compared to co-transduction with either domain alone. In some embodiments, a histidine-rich domain may optionally be added to the shuttle construct as an additional method to enable escape of cargo from endosomes to the cytosol. The shuttle may also contain cysteine residues at the N- or C-terminus to generate multimers of the fusion peptide. Multimers of ELD-CLD fusion peptides, generated by the addition of cysteine residues to the peptide termini, exhibit even higher transduction efficiency compared to single fusion peptide constructs. Polypeptides of the present disclosure may also be appended to appropriate localization signals to target the cargo to the appropriate subcellular location, e.g., the nucleus. In some embodiments, any of the ELD, CLD, or fusion ELD-CLD synthetic peptides taught in International Patent Publications WO2016161516 and WO2017175072 may be useful in the present disclosure (the entire contents of each of which are incorporated herein by reference).

[0276] Delivery Modality and / or Vector The transcription factor system of the present disclosure, or components thereof, may be delivered using one or more modalities. The present disclosure also provides vectors that package the polynucleotides of the present disclosure encoding transcription factors and portions thereof, DRDs, or payload constructs, as well as combinations thereof. The vectors of the present disclosure may also be used to deliver the packaged polynucleotides to cells, local tissue sites, or subjects. These vectors may be of any type, including DNA vectors, RNA vectors, plasmids, viral vectors, and particles. Viral vector technology is well known and is described in detail in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, NY, USA).

[0003] Viruses useful as vectors include, but are not limited to, adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus. In some embodiments, the virus is selected from a lentivirus vector, a gammaretrovirus vector, an adeno-associated virus (AAV) vector, an adenovirus vector, and a herpesvirus vector.

[0277] Generally, a vector contains an origin of replication functional in at least one organism, a promoter sequence and convenient restriction endonuclease sites, and one or more selectable markers, such as drug resistance genes.

[0278] In some embodiments, recombinant expression vectors may contain regulatory sequences, such as transcriptional and translational initiation and termination codons, that are specific to the host cell type into which the vector is introduced.

[0279] In some embodiments, a vector of the present disclosure may include one or more payloads taught herein, and two or more payloads may be involved in a single ligand response, where the two or more payloads are simultaneously regulated by the same ligand or response agent.

[0280] Lentiviral vehicles / particles In some embodiments, lentiviral vehicles / particles may be used as a delivery modality. Lentiviruses are a subgroup of viruses in the Retroviridae family, named after the need for reverse transcription of the viral RNA genome into DNA before integration into the host genome. Therefore, the most important feature of lentiviral vehicles / particles is that they integrate their genetic material into the genome of target / host cells. Some examples of lentiviruses include human immunodeficiency viruses: HIV-1 and HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi virus, and caprine arthritis-encephalitis virus (CAEV).

[0281] Typically, lentiviral particles constituting gene delivery vehicles are replication-deficient by themselves (also referred to as "self-inactivating"). Lentiviruses can infect both dividing and non-dividing cells by an entry mechanism through an intact host nuclear membrane. Recombinant lentiviral vehicles / particles have been generated by attenuating HIV pathogenic genes many times over; for example, genes Env, Vif, Vpr, Vpu, Nef, and Tat have been deleted, making the vector biologically safe. Correspondingly, for example, lentiviral vehicles derived from HIV-1 / HIV-2 can mediate efficient delivery, integration, and long-term expression of transgenes in non-dividing cells.

[0282] Lentiviral particles can be generated by co-expressing viral packaging elements and the vector genome itself in producer cells, such as human HEK293T cells. These elements are typically provided on three or four separate plasmids. Producer cells are co-transfected with plasmids encoding lentiviral components, including the viral core (i.e., structural proteins) and enzymatic components, and envelope protein(s) (referred to as the packaging system), as well as a plasmid containing the exogenous transgene to be transferred to target cells, the vehicle itself (also referred to as the transfer vector). Generally, the plasmid or vector is contained in a producer cell line. The plasmid / vector is introduced into the producer cell line via transfection, transduction, or infection. Methods for transfection, transduction, or infection are well known to those skilled in the art. By way of non-limiting example, packaging and transfer constructs can be introduced into a producer cell line by calcium phosphate transfection, lipofection, or electroporation, generally along with a dominant selectable marker such as neo, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.

[0283] The producer cells produce recombinant viral particles containing a foreign gene, such as a transcription factor system component of the present disclosure or a polynucleotide thereof. The recombinant viral particles are recovered from the culture medium and titrated by standard methods used by those skilled in the art. The recombinant lentiviral vehicle can be used to infect target cells.

[0284] Cells that can be used to produce high-titer lentiviral particles include HEK293T cells, 293G cells, STAR cells (Relander et al., Mol. Ther., 2005, 11:452-459), the FreeStyle™ 293 Expression System (ThermoFisher, Waltham, MA), and other HEK293T-based producer cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 22(3):357-369; Lee et al. al., Biotechnol Bioeng, 2012, 10996):1551-1560; Throm et al., Blood. 2009, 113(21):5104-5110, the contents of each of which are incorporated herein by reference in their entirety.

[0285] In some embodiments, the envelope protein may be a heterologous envelope protein of another virus, such as the G protein of vesicular stomatitis virus (VSV G) or the baculovirus gp64 envelope protein. VSV-G glycoprotein is particularly found in the vesiculovirus genus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PV), and the like. Species classified as Vesicular stomatitis virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV), and Vesicular stomatitis New Jersey virus (VSNJV) and / or grass carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Gray Lodge virus (GLOV), Jurona virus (JURY), Klamath virus (KLAV), Kwatta virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike virus (Pike), and others. The virus may be selected from strains tentatively classified in the genus vesiculovirus as fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), spring viremia of carp virus (SVCV), Tupaia virus (TUPV), ulcerative disease rhabdovirus (UDRV) and Yug Bogdanovac virus (YBV). The gp64 or other baculovirus env protein can be derived from Autographa californica nucleopolyhedrovirus (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fumiferana nuclear polyhedrosis virus, Orgyia pseudotsugata single-capsid nuclear polyhedrosis virus, Epiphyas postvittana nuclear polyhedrosis virus, Hyphantria cunea nuclear polyhedrosis virus, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nuclear polyhedrosis virus, or Batken virus.In some embodiments, the envelope protein may be RD114, RD115, or may be derived from gibbon ape leukemia virus (GaLV) or baboon retrovirus envelope glycoprotein (BaEV).

[0286] Other elements provided in the lentiviral particle may include a retroviral LTR (long terminal repeat) at either the 5' or 3' end, a retroviral transport element, optionally a lentiviral reverse response element (RRE), a promoter or an active portion thereof, and a locus control region (LCR) or an active portion thereof.

[0287] Methods for producing recombinant lentiviral particles have been described in the art, for example, in U.S. Patent Nos. 8,846,385, 7,745,179, 7,629,153, 7,575,924, 7,179,903, and 6,808,905.

[0288] The lentiviral vector used may be selected from, but is not limited to, pLVX, pLenti, pLenti6, pLJM1, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJM1-EGFP, pULTRA, pInducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionII.

[0289] Adeno-associated virus particles Delivery of any polynucleotide of the disclosed transcription factor system, transcription factor construct, or payload construct may be achieved using recombinant adeno-associated virus (rAAV) vectors. Such vectors or viral particles may be designed to utilize any of the known serotype capsids or combinations of serotype capsids.

[0290] AAV vectors include not only single-stranded vectors but also self-complementary AAV vectors (scAAV). scAAV vectors contain DNA that anneals together to form a double-stranded vector genome. By skipping double-strand synthesis, scAAV allows for rapid expression in cells.

[0291] rAAV vectors may be produced by standard methods in the art, such as by triple transfection, in sf9 insect cells, or in suspension cell culture of human cells such as HEK293 cells.

[0292] The transcription factor construct and the payload construct may be encoded in one or more viral genomes that are packaged in the AAV capsids taught herein.

[0293] Such vectors or viral genomes may also contain, in addition to at least one or two ITRs (inverted terminal repeats), certain regulatory elements required for expression from the vector or viral genome, such regulatory elements being well known in the art and including, for example, promoters, introns, spacers, stuffer sequences, etc.

[0294] The transcription factor constructs or payload constructs of the present disclosure may be administered in one or more or separate AAV particles.

[0295] In some embodiments, a transcription factor system construct may be administered in one or more AAV particles, hi some embodiments, two or more transcription factor system constructs may be encoded in the viral genome.

[0296] Retroviral vehicles / particles (γ-retroviral vectors) In some embodiments, retroviral vehicles / particles may be used to deliver the transcription factor systems, transcription factor constructs, or payload constructs of the present disclosure. Retroviral vectors (RVs) allow for permanent integration of transgenes into target cells. In addition to complex HIV-1 / 2-based lentiviral vectors, simple gamma-retrovirus-based retroviral vectors have been widely used to deliver therapeutic genes and have been clinically proven as one of the most efficient and powerful gene delivery systems capable of transducing a wide range of cell types. Exemplary species of gammaretroviruses include murine leukemia virus (MLV) and feline leukemia virus (FeLV).

[0297] In some embodiments, gamma-retroviral vectors derived from mammalian gamma-retroviruses, such as murine leukemia viruses (MLV), are recombinant. The MLV family of gammaretroviruses includes ecotropic, amphotropic, xenotropic, and polytropic subfamilies. Ecotropic viruses can infect only mouse cells using the mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect mice, humans, and other species via the Pit-2 receptor. An example of an amphotropic virus is the 4070A virus. Xenotropic and polytropic viruses use the same (Xpr1) receptor but have different species tropisms. Xenotropic viruses, such as NZB-9-1, can infect humans and other species but not mouse species, whereas polytropic viruses, such as focus-forming virus (MCF), can infect mice, humans, and other species.

[0298] Gamma-retroviral vectors may be produced in packaging cells by co-transfecting the cells with several plasmids, including a plasmid encoding the retroviral structural and enzymatic (gag-pol) polyprotein, a plasmid encoding the envelope (env) protein, and a plasmid encoding a vector mRNA comprising a polynucleotide encoding a composition of the present disclosure, which is packaged into newly formed viral particles.

[0299] In some embodiments, recombinant gamma-retroviral vectors are pseudotyped with envelope proteins of other viruses. Envelope glycoproteins are incorporated into the outer lipid layer of the viral particle, which can increase / alter cell tropism. In some embodiments, the envelope protein may be RD114, RD115, or may be derived from gibbon ape leukemia virus (GaLV) or baboon retrovirus envelope glycoprotein (BaEV).

[0300] In some embodiments, the recombinant gamma-retroviral vector is a self-inactivating (SIN) gamma-retroviral vector. The vector is replication-deficient. SIN vectors may have a deletion in the 3'U3 region, which initially contains enhancer / promoter activity. Additionally, the 5'U3 region may be replaced with a strong promoter from cytomegalovirus or RSV (required for packaging cell lines), or a suitable internal promoter and / or enhancer element. The selection of the internal promoter may be made according to the specific requirements of gene expression required for the particular purpose of the present disclosure.

[0301] In some embodiments, the polynucleotide of the transcription factor system, transcription factor construct, or payload construct is inserted into the recombinant viral genome. Other components of the viral mRNA of the recombinant gamma-retroviral vector may be modified by inserting or removing naturally occurring sequences (e.g., inserting an IRES, inserting a heterologous polynucleotide encoding a polypeptide of interest or an inhibitory nucleic acid, shuffling a more effective promoter from a different retrovirus or virus in place of the wild-type promoter, etc.). In some examples, the recombinant gamma-retroviral vector may contain a modified packaging signal, and / or a primer binding site (PBS), and / or a 5'-enhancer / promoter element in the U3 region of the 5'-long terminal repeat (LTR), and / or a modified 3'-SIN element in the U3 region of the 3'-LTR. These modifications may increase the titer and capacity of infection.

[0302] Oncolytic viral vectors In some embodiments, the polynucleotides of the present disclosure may be packaged into an oncolytic virus. As used herein, the term "oncolytic virus" refers to a virus that selectively infects and kills cancer cells, such as a vaccine virus. Oncolytic viruses can be naturally occurring or genetically engineered viruses, such as oncolytic adenoviruses and oncolytic herpes viruses.

[0303] In some embodiments, the oncolytic vaccinia virus may comprise viral particles of a thymidine kinase (TK)-deficient, granulocyte-macrophage (GM)-colony stimulating factor (CSF)-expressing, replication-competent vaccinia virus vector sufficient to induce cellular oncolysis within a tumor; see, e.g., U.S. Patent No. 9,226,977.

[0304] messenger RNA (mRNA) In some embodiments, the transcription factor system, transcription factor construct, or payload construct of the present disclosure may be designed as a messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. Such mRNA molecules may have any of the structural components or characteristics taught in International Application No. PCT / US2013 / 030062.

[0305] In some embodiments, a transcription factor system or its components may be designed as a self-amplifying RNA. "Self-amplifying RNA," as used herein, refers to an RNA molecule that can replicate within a host and increase the amount of RNA and the protein encoded by the RNA. Such self-amplifying RNA may have any of the structural features or components taught in International Patent Application Publication No. WO2011005799.

[0306] dosage The present disclosure provides methods that include administering any one or more components or compositions of a transcription factor system to a subject in need thereof. They may be administered to a subject in any amount and using any route of administration effective to prevent or treat or image a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition associated with cancer or an autoimmune disease). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its method of administration, its method of activity, etc.

[0307] The compositions of the present disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage.However, it will be understood that the total daily amount of the compositions of the present disclosure can be determined by attending physician within the scope of sound medical judgment.The specific therapeutically effective, prophylactically effective or suitable imaging dose level for any specific patient will depend on various factors, including the disorder being treated and the severity of the disorder, the activity of the specific compound used, the specific composition used, the age, weight, general health, sex and diet of the patient, the administration time, administration route and excretion rate of the specific compound used, treatment duration, the drugs used in combination with or simultaneously with the specific compound used, and similar factors well known in the medical field.

[0308] In some embodiments, compositions of the present disclosure may be used in cancer immunotherapy at various doses to avoid T cell exhaustion, prevent cytokine release syndrome, and minimize immunotherapy-related toxicity. For example, a low dose of a composition of the present disclosure may be used initially to treat patients with high tumor burden, while patients with low tumor burden may be treated with high and repeated doses of a composition of the present disclosure to ensure minimal tumor antigen recognition. In another example, compositions of the present disclosure may be delivered in a pulsed manner to reduce strong T cell signaling and enhance in vivo persistence. In some aspects, toxicity may be minimized by initially using a low dose of a composition of the present disclosure before administering a higher dose. Dosing may be modified if serum markers such as ferritin, serum C-reactive protein, IL6, IFN-γ, and TNF-α are elevated.

[0309] In some embodiments, neurotoxicity may be associated with CAR or TIL therapy. Such neurotoxicity may be associated with CD19-CAR. The toxicity may be due to excessive T cell infiltration into the brain. In some embodiments, neurotoxicity may be mitigated by preventing T cells from passing through the blood-brain barrier. This may be achieved by targeted gene deletion of endogenous alpha-4 integrin inhibitors, such as tysabri / natalizumab, which may also be useful in the present disclosure.

[0310] Also provided herein are methods of administering a ligand or DRD ligand according to the present disclosure to a subject in need thereof. In some embodiments, the ligand is selected from acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). The ligand may be administered to a subject or cell in any amount and using any route of administration effective to modulate the transcription factor system, DRD, or payload of the present disclosure. In some embodiments, ACZ may be used with an hCA2 DRD, methotrexate may be used with an hDHFR DRD, and trimethoprim may be used with an ecDHFR DRD. The exact amount required will vary from subject to subject depending on the subject's species, age, and general condition, the severity of the disease, the particular composition, its method of administration, its method of activity, and the like. The subject may be a human, mammal, or animal. Compositions according to the present disclosure are typically formulated in unit dosage form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present disclosure may be determined by the attending physician within the scope of sound medical judgment. In certain embodiments, the ligands of the present disclosure may be administered in an amount of from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, or about 0.1 mg / kg per day to achieve the desired effect. The compound may be administered at a dosage level sufficient to deliver about 100 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg, about 10 mg / kg to about 100 mg / kg, about 50 mg / kg to about 500 mg / kg, or about 100 mg / kg to about 1000 mg / kg of subject body weight once or more per day.In some embodiments, dosage levels can be 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg, 150 mg / kg, 160 mg / kg, 170 mg / kg, 180 mg / kg, 190 mg / kg or mg / kg of subject body weight per day, or more than once per day, to achieve the desired effect.

[0311] The present disclosure provides methods for delivering any of the ligands described herein to cells or tissues, the methods comprising contacting cells or tissues with the ligand, which may be accomplished in vitro, ex vivo, or in vivo. In certain embodiments, a ligand according to the present disclosure may be administered to cells at a dosage level sufficient to deliver about 1 nM to about 10 nM, about 5 nM to about 50 nM, about 10 nM to about 100 nM, about 50 nM to about 500 nM, about 100 nM to about 1000 nM, about 1 μM to about 10 μM, about 5 μM to about 50 μM, about 10 μM to about 100 μM, about 25 μM to about 250 μM, or about 50 μM to about 500 μM. In some embodiments, the ligand may be administered to the cells at a dose selected from, but not limited to, 0.00064 μM, 0.0032 μM, 0.016 μM, 0.08 μM, 0.4 μM, 1 μM, 2 μM, 10 μM, 50 μM, 75 μM, 100 μM, 150 μM, 175 μM, 200 μM, 250 μM.

[0312] The desired dosage of the ligand of the present disclosure may be delivered only once, three times a day, twice a day, once a day, every other day, every other day, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are used, a split-dosing regimen, such as those described herein, may be used. As used herein, a "split dose" refers to the division of a "single unit dose" or total daily dose into two or more doses, e.g., two or more administrations of a "single unit dose." As used herein, a "single unit dose" refers to the dose of any therapeutic agent administered in one dose / once / single route / single contact point, i.e., in a single administration event. The desired dosage of the ligand of the present disclosure may be administered as a "pulse dose" or "continuous flow." As used herein, a "pulse dose" is a series of single unit doses of any therapeutic agent administered at a set frequency over a period of time. As used herein, a "continuous flow" is a dose of a therapeutic agent administered continuously over a period of time by a single route / single point of contact, i.e., continuous administration events. The total daily dose, a predetermined or prescribed amount in a 24-hour period, may be administered by any of these methods, or as a combination of these methods, or by any other method suitable for pharmaceutical administration.

[0313] Administration In some embodiments, compositions for cancer immunotherapy or autoimmune disease treatment may be administered to cells ex vivo and then administered to a subject. In further embodiments, the cells are selected from B cells, T cells, natural killer cells (NK cells), or tumor-infiltrating lymphocytes (TILs). Immune cells can be isolated and expanded ex vivo using various methods known in the art. For example, methods for isolating cytotoxic T cells are described in U.S. Patent Nos. 6,805,861 and 6,531,451. Isolation of NK cells is described in U.S. Patent No. 7,435,596.

[0314] In some embodiments, depending on the nature of the cells, the cells may be introduced into a host organism, e.g., a mammal, in a variety of ways, including injection, transfusion, infusion, local instillation, or transplantation. In some aspects, the cells of the present disclosure may be introduced into the site of a tumor. The number of cells used will depend on a number of circumstances, the purpose of the introduction, the lifespan of the cells, the protocol used, e.g., the number of administrations, the ability of the cells to proliferate, etc. The cells may be present in a physiologically acceptable medium.

[0315] In some embodiments, the cells of the present disclosure may be administered in multiple doses to a subject with a disease or condition, generally resulting in amelioration of one or more symptoms of the cancer or clinical condition and / or treating or preventing the cancer or its clinical condition or symptoms.

[0316] In some embodiments, compositions for immunotherapy or treatment of autoimmune diseases may be administered in vivo, hi some embodiments, polynucleotides of the present disclosure, payloads and compositions of the present disclosure comprising transcription factor systems may be delivered to a subject in vivo via gene therapy.

[0317] Delivery route The pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, payloads, vectors, and cells of the present disclosure may be administered by any route to achieve a therapeutically effective result, including enteral (into the gut), gastrointestinal, epidural (to the dura mater), oral (through the mouth), transdermal, peridural, intracerebral (to the brain), intracerebroventricular (into the brain), epicutaneous (applied on the skin), intradermal (to the skin itself), subcutaneous (under the skin), intranasal (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (injected or poured into the peritoneum), intravesical, intravitreal (through the eye), and intracavity injection. (into a diseased cavity), intracavernosal (to the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), transvaginal, insufflation (inhaled through the nose), sublingual, sublabial, enema, ophthalmic (on the conjunctiva), eardrop, auricular (in or through the ear), buccal (towards the cheek), conjunctival, cutaneous, dental (into tooth(s)), electroosmosis, intracervical, intrasinus, intratracheal, extracorporeal, hemodialysis, infiltration, intrainterstitial, intraperitoneal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intracapsular, intrachondral (into cartilage), intrasacral (into the cauda equina), intracisternal (into the cisterna magna cerebellomedularis), intracorneal (into the cornea), intracoronalintracornal), intracoronary (into a coronary artery), intracavernosal (into the expansion space of the corpus cavernosum of the penis), intraspinal (into an intervertebral disc), intraductal (into the duct of a gland), intraduodenal (into the duodenum), intradural (into or under the dura mater), intraepidermal (into the epidermis), intraesophageal (into the esophagus), intragastric (into the stomach), intragingival (into the gums), intraileal (into the distal part of the small intestine), intralesional (into or introduced directly into a focal lesion), intraluminal (into the lumen of a duct), intralymphatic (into the lymph), intramedullary (into the medullary cavity of a bone), intrameningeal (inside the meninges), intramyocardial (inside the heart muscle), intraocular (inside the eyeball), intraovarian (inside the ovaries), intrapericardial (inside the pericardium), intrapleural (inside the pleura), intraprostatic (inside the prostate), intrapulmonary (inside the lungs or their bronchi), intrasinus (inside the nasal or periorbital spaces), intraspinal (inside the spinal column), intrasynovial (inside the synovial spaces of a joint), intratendinous (inside tendons), intratesticular (inside the testes), intrathecal (into the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (inside the thorax), intraductal (inside the tubules of an organ), intratumoral (inside a tumor), intratympanic ( Into the middle ear), intravascular (into one or more blood vessels), intravenous (into a room), iontophoresis (by electric current transferring ions of soluble salts into body tissues), irrigation (bathing or flushing an open wound or body cavity), laryngeal (directly onto the larynx), nasogastric (through the nose into the stomach), occlusive dressing (administered topically followed by covering the area with an occlusive dressing), ocular (to the outside of the eye), oropharyngeal (directly into the mouth and pharynx), parenteral, transdermal, periarticular, epidural, perineural, periodontal, rectal, respiratory (administered intravenously for local or systemic effect) These include, but are not limited to, oral or nasal insufflation into the airways), retrobulbar (behind the pons or behind the eye), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (across or across the placenta), transtracheal (through the tracheal wall), transtympanic (across or through the tympanic cavity), ureteral (into the ureter), urethral (into the urethra), intravaginal, sacral block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis, or spinal.

[0318] Parenteral and parenteral administration In some embodiments, the pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, payloads, vectors, and cells of the present disclosure may be administered parenterally. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, the compositions are mixed with solubilizing agents, such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof. In other embodiments, surfactants such as hydroxypropyl cellulose are included.

[0319] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to the known art. Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, for example, solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic saline. Sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0320] Injectable preparations may be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0321] Detectable Agents and Labels The transcription factor systems, nucleic acids, polynucleotides, payloads, vectors and cells of the present disclosure may be associated with or bound to one or more radioactive or detectable agents.

[0322] These agents include various small organic molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent substances, luminescent substances (e.g., luminol), bioluminescent substances (e.g., luciferase, luciferin, and aequorin), chemiluminescent substances, radioactive substances (e.g., 18 F, 67 Ga, 81m Kr, 82 Rb, 111 In, 123 I, 133 Xe, 201 Tl, 125 I, 35 S, 14 C. 3 H, or 99m Tc (e.g., pertechnetate (technetate(VII), TcO4- )), and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), single crystal iron oxide nanoparticles (MION), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast agents (iohexol), microbubbles, or perfluorocarbons).

[0323] In some embodiments, the detectable agent may be an undetectable precursor that becomes detectable upon activation (e.g., a fluorogenic tetrazine-fluorophore construct (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or an enzyme-activatable fluorogenic agent (e.g., PROSENSE® (VisEn Medical))). In vitro assays in which the enzyme-labeled composition can be used include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation assay, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.

[0324] Use and application The transcription factor systems, constructs, ligands, or compositions of the present disclosure may be utilized in a wide variety of applications, including but not limited to, therapeutics, diagnostics and prognostics, bioengineering, bioprocessing, biomanufacturing, research medicine, metabolomics, gene expression, enzyme replacement, and the like.

[0325] The present disclosure provides methods that include administering a composition, eg, a pharmaceutical composition, that includes one or more components of a transcription factor system to a subject in need thereof.

[0326] While there may be some uses that do not involve medical treatment, for example, to generate cell lines and reagents for scientific research, one use involves administering the compositions of the present disclosure to generate modified cells for in vivo gene therapy or adoptive cell therapy, including, for example, the treatment of cancer, autoimmune diseases, and other diseases. An exemplary method of medically treating or preventing a disease, condition, or disorder in a subject in need thereof may include the following steps: (a) providing a cell population (either human, animal, primary, or cell culture, including autologous, allogeneic, or syngeneic); (b) introducing at least one nucleic acid molecule into at least one cell within the cell population, wherein the at least one nucleic acid molecule comprises: (i) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug responsive domain (DRD), wherein the transcription factor activation domain and / or the transcription factor DNA-binding domain is operably linked to the DRD; and (ii) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, wherein the fourth nucleic acid sequence comprises the second polynucleotide operably linked to an exogenous inducible promoter comprising a specific polynucleotide binding site; (c) delivering the cell to the subject; and (d) administering to the subject a ligand that stabilizes the DRD sufficiently to allow expression of a transcription factor activation domain and a transcription factor DNA binding domain, in an amount sufficient to bind to the specific polynucleotide binding site and form a transcription factor that allows expression of the protein of interest in the cell, wherein expression of the protein of interest is controlled by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0327] In the above methods, the protein of interest may be used to ameliorate, cure, prevent or reduce one or more symptoms of a disease, condition or disorder.

[0328] The compositions of the present disclosure may be administered to a subject in any amount and using any route of administration effective to prevent or treat or image a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition associated with cancer, autoimmune disease, and other diseases). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its method of administration, its method of activity, etc.

[0329] The compositions of the present disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage.However, it will be understood that the total daily amount of the compositions of the present disclosure can be determined by attending physician within the scope of sound medical judgment.The specific therapeutically effective, prophylactically effective or suitable imaging dose level for any specific patient will depend on various factors, including the disorder being treated and the severity of the disorder, the activity of the specific compound used, the specific composition used, the age, weight, general health, sex and diet of the patient, the administration time, administration route and excretion rate of the specific compound used, treatment duration, the drugs used in combination with or simultaneously with the specific compound used, and similar factors well known in the medical field.

[0330] Also provided herein are methods of administering one or more stabilizing ligands (as used herein, a ligand that stabilizes a DRD may be referred to as a stabilizing ligand or simply a ligand, with the understanding that the ligand is effective to stabilize a DRD used in a transcription factor system according to the present disclosure) to a subject in need thereof. The ligand may be administered to a subject or cell in any amount and using any route of administration effective to modulate the amount of expression of a transcription factor of the present disclosure in a cell comprising the transcription factor system. The exact amount of stabilizing ligand required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its method of administration, its method of activity, etc. The subject may be a human, mammal, or animal.

[0331] therapeutic use Cancer immunotherapy Cancer immunotherapy aims to induce or restore immune system responsiveness against cancer. Significant advances in immunotherapy research have led to the development of various strategies, which can be broadly classified as active and passive immunotherapy. Generally, these strategies may be utilized to directly kill cancer cells or counteract the immunosuppressive tumor microenvironment. Active immunotherapy aims to induce an endogenous, long-lasting tumor antigen-specific immune response. The response can be further enhanced by nonspecific stimulation of immune response regulators such as cytokines. In contrast, passive immunotherapy involves the administration of effector immune molecules, such as tumor antigen-specific cytotoxic T cells or antibodies, to the host. This approach is temporary and requires multiple applications.

[0332] Despite significant advances, the effectiveness of current immunotherapy strategies is limited by associated toxicities. They are often associated with a narrow therapeutic window associated with immunotherapy, which arises in part from the need to approach the limit of potentially lethal toxicity to achieve clinically meaningful treatment effects. Furthermore, doses are escalated in vivo as adoptively transferred immune cells continue to expand within the patient, often unpredictably.

[0333] The major risk associated with immunotherapy is on-target but off-tumor side effects resulting from T cell activation in response to normal tissue expression of tumor-associated antigens (TAA). In clinical trials utilizing T cells expressing T cell receptors against specific TAAs, skin rash, colitis, and hearing loss have been reported in response to immunotherapy.

[0334] Immunotherapy may also cause on-target tumor toxicity, which occurs when tumor cells die in response to immunotherapy.Side effects include tumor lysis syndrome, cytokine release syndrome, and related macrophage activation syndrome.Importantly, these adverse effects may occur during tumor destruction, and therefore may cause toxicity even if tumor immunotherapy is successful.Therefore, the approach of controlling immunotherapy by controlling immunotherapeutic agents is highly desirable, as they have the potential to reduce toxicity and maximize efficacy.

[0335] The present disclosure provides systems, compositions, immunotherapeutic agents, and methods for immunotherapy, which provide tunable control of gene expression and function in immunotherapy, for example, for the prevention and treatment of cancer.

[0336] In one aspect, the systems, compositions, immunotherapeutic agents, and other components of the present disclosure can be controlled by separately added stabilizing ligands, providing significant flexibility for controlling cancer immunotherapy. Additionally, the systems, compositions, and methods of the present disclosure may also be combined with therapeutic agents, such as chemotherapeutic agents, small molecules, gene therapies, and antibodies, to prevent and / or treat diseases, e.g., cancer.

[0337] The tunable nature of the disclosed systems and compositions has the potential to improve the potency and duration of immunotherapy effectiveness. By using the disclosed compositions to reversibly silence the biological activity of adoptively transferred cells, it is possible to maximize the potential of cell therapy without irreversibly killing them and terminating the therapy.

[0338] The present disclosure provides methods aimed at fine-tuning immunotherapies after administration to patients, thereby improving their safety and efficacy and expanding the target population that may benefit from immunotherapy.

[0339] In some embodiments, the immune cells of the present disclosure can be T cells engineered to express a payload or protein of interest, e.g., an antigen-specific T cell receptor (TCR) or antigen-specific chimeric antigen receptor (CAR) (known as a CAR T cell) as taught herein. Thus, at least one polynucleotide, or a vector comprising a polynucleotide, encoding the protein of interest, e.g., a CAR system (or TCR) described herein, is introduced into the T cell. T cells expressing the CAR or TCR bind to a specific antigen via the extracellular targeting portion of the CAR or TCR, thereby transmitting a signal to the T cell via the intracellular signaling domain(s), resulting in activation of the T cell. Activated CAR T cells change their behavior, including the release of cytotoxic cytokines (e.g., tumor necrosis factor and lymphotoxin), improved cell proliferation rate, and changes in cell surface molecules. Such changes result in the destruction of target cells expressing the antigen recognized by the CAR or TCR. In addition, the release of cytokines or changes in cell surface molecules stimulate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0340] The CAR introduced into the T cell may be a first-generation CAR containing only the TCR CD3 zeta intracellular signaling domain, a second-generation CAR containing the TCR CD3 zeta intracellular signaling domain and a costimulatory signaling domain, a third-generation CAR containing the TCR CD3 zeta intracellular signaling domain and two or more costimulatory signaling domains, a split CAR system, or an on / off switch CAR system. In one example, CAR or TCR expression is controlled by a transcription factor, and if the transcription factor or a component thereof is operably linked to a DRD, this results in little or no transcription factor accumulation in the absence of a stabilizing ligand. The payload contains a polynucleotide binding sequence specific to the transcription factor or a component thereof, and therefore, in the absence of a stabilizing ligand, little or no protein of interest is produced. When a stabilizing ligand is administered to cells containing a transcription factor system, the transcription factor is rescued from degradation when bound to the DRD, and then the transcription factor binds to its cognate polynucleotide binding sequence directly adjacent to the protein of interest and is then transcribed. The transcribed mRNA is then translated to produce the polypeptide / protein of interest. In some exemplary embodiments, the presence or absence of a DRD-stabilizing ligand is used to modulate CAR or TCR expression in transduced T cells or NK cells.

[0341] In some embodiments, the CAR T cells of the present disclosure may be further engineered to express one, two, three, or more additional immunotherapeutic agents. The immunotherapeutic agents may be another CAR or TCR specific for a different target molecule; a cytokine such as IL2, IL12, IL15, and IL18, or a cytokine receptor such as IL15Ra; a chimeric switch receptor that converts an inhibitory signal into a stimulatory signal; a homing receptor that directs the adoptively transferred cells to a target site such as tumor tissue; a drug that optimizes immune cell metabolism; or a safety switch gene (e.g., a suicide gene) that kills activated T cells if a severe event is observed after adoptive cell transfer or if the transferred immune cells are no longer needed. These molecules may be included in the same construct or in separate constructs.

[0342] In one embodiment, CAR T cells (including TCR T cells) of the present disclosure may be "armed" CAR T cells that are transfected or transduced with one or more components of either the same or different transcription factor systems, including the CAR payload, and cytokine-encoding transcription factor systems, under the control of the same or different transcription factors operably linked to the same or different DRDs. Inducible cytokines or secreted constitutively activating cytokines further arm the CAR T cells to improve efficacy and persistence. In this regard, such CAR T cells are also referred to as "armored CAR T cells." The "armoring" molecules may be selected based on the tumor microenvironment and other elements of the innate and adaptive immune systems. In some embodiments, the molecules may be stimulatory factors such as IL2, IL12, IL15, IL18, type I IFN, CD40L, and 4-1BBL, which have been shown to further enhance CAR T cell efficacy and persistence through different mechanisms, even in the face of a hostile tumor microenvironment.

[0343] Chimeric antigen receptor-engineered T cell (CAR-T) therapy has not yet been successfully applied to solid tumors. Enhancing CAR-T cell function and selectively delivering cargo to the site of solid tumors are key strategies for achieving effective CAR-T therapy against solid tumors. In one embodiment, the payload or protein of interest may include interleukin-12 (IL12), which may be utilized to enhance the efficacy of CAR-T cells, particularly due to its potential to remodel the tumor microenvironment. IL12 has previously been shown to be effective in enhancing the efficacy of tumor-infiltrating lymphocytes (TILs) in addition to CAR- or TCR-modified T cells in preclinical and clinical models. However, constitutive production of IL12 may compromise safety and / or efficacy, so local delivery of the cytokine may be a preferred approach, if needed. In some embodiments, the disclosed transcription factor system, or components thereof, may be utilized to exogenously control IL12 expression, enabling the use of IL12 in adoptive cell therapy.

[0344] In some embodiments, the transcription factor regulation system of the present disclosure may be used to control payload expression such as Flexi IL12 (or other IL12 constructs, such as membrane-bound IL12) in transformed immune cells by providing controlled local signals for tumor microenvironment remodeling and epitope spreading to improve CAR efficacy, particularly in solid tumor settings. Transcription factor regulation as described herein also provides rapid, dose-dependent, local production of IL12 upon addition of a DRD-specific stabilized ligand.

[0345] In some embodiments, the armed CAR T cells of the present disclosure are engineered to express a CD19 CAR and a payload such as IL12, which is regulated using a transcription factor system or composition of the present disclosure. After CAR-mediated activation in tumors, such T cells release inducible IL12 to enhance T cell activation, attract and activate innate immune cells, and eliminate CD19-positive cancer cells.

[0346] In one embodiment, the T cells of the present disclosure may be modified to incorporate a nucleic acid sequence encoding a suicide gene and a transcription factor system comprising a CAR payload encoded by the transcription factor system or a component thereof.

[0347] In one embodiment, CAR T cells (including TCR T cells) of the present disclosure may be transfected or transduced with one or more components of a transcription factor system, including a cytokine and a safety switch gene (e.g., a suicide gene). The suicide gene may be an inducible caspase, such as caspase 9, that induces apoptosis when activated by an extracellular stabilized ligand of the DRD encoded by the transcription factor system. Such induced apoptosis eliminates the transplanted cells as needed to reduce the risk of direct toxicity and uncontrolled cell proliferation.

[0348] In one embodiment, transcription factor systems and components thereof that regulate the expression levels and activity of any of the described payloads or proteins of interest (used interchangeably) may be used in immunotherapy. By way of non-limiting example, immunotherapeutic agents may be antibodies and fragments and variants thereof, cancer-specific T cell receptors (TCRs) and variants thereof, anti-tumor-specific chimeric antigen receptors (CARs), chimeric switch receptors, inhibitors of co-inhibitory receptors or ligands, agonists of co-stimulatory receptors and ligands, cytokines, chemokines, cytokine receptors, chemokine receptors, soluble growth factors, metabolic factors, suicide genes, homing receptors, or any agent that induces an immune response in cells and subjects.

[0349] In some embodiments, a composition for inducing or suppressing an immune response may comprise one or more components of a transcription factor system or one or more polypeptides encoded by a transcription factor system. In some embodiments, the transcription factor system may comprise the following polynucleotides: a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, and a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operably linked to the DRD; the second polynucleotide comprises a fourth nucleic acid sequence encoding a protein of interest, wherein the fourth nucleic acid sequence is operably linked to an inducible promoter comprising the specific polynucleotide binding site; the transcription factor activation domain and the transcription factor DNA-binding domain interact to form a transcription factor; and binding of the transcription factor to the specific polynucleotide binding site is required for the transcription factor to regulate transcription of the fourth nucleic acid sequence.

[0350] In one embodiment, the payload can be an immunotherapeutic agent.

[0351] In some embodiments, the transcription factor systems and compositions of the present disclosure relate to the transcriptional regulation of protein (protein of interest or payload) function, including, for example, immunotherapeutic anti-tumor immune responses. In some embodiments, immunotherapeutic agents may include cytokines, chemokines, antibodies, integrins, integral proteins, membrane proteins, and extracellular proteins that may be used to upregulate or improve the function of one or more immune cell types or downregulate the activity of one or more immune cell types. In various embodiments, immunotherapeutic agents useful for treating diseases, conditions, or disorders may include cytokines, such as interleukins. In various embodiments, the transcription factor systems provide proteins of interest or payloads that include interleukins, e.g., IL-2, IL-6, IL12, IL15, IL18, and other immunotherapeutic agents that promote or upregulate the lifespan and activity of one or more immune cell types useful for treating diseases, conditions, or disorders, or symptoms associated with any of them.

[0352] In some embodiments, cells that have been genetically modified to encode and express at least one transcription factor operable to enable transcription of a protein of interest (immunotherapeutic agent) linked to a transcription factor polynucleotide binding site may be used in adoptive cell therapy (ACT, also referred to as "adoptive cell transfer"). As used herein, adoptive cell transfer refers to the administration of immune cells (autologous, allogeneic, or derived from a genetically modified host) that have direct anti-cancer activity. ACT has shown promise in clinical applications against malignant diseases and infectious diseases. For example, T cells genetically engineered to recognize CD19 have been used to treat follicular B-cell lymphoma (Kochenderfer et al., Blood, 2010, 116:4099-4102, and Kochenderfer and Rosenberg, Nat Rev Clin Oncol., 2013, 10(5):267-276), and ACT using autologous lymphocytes genetically modified to express anti-tumor T cell receptors has been used to treat metastatic melanoma (Rosenberg and Dudley, Curr. Opin. Immunol. 2009, 21:233-240).

[0353] According to the present disclosure, one or more components of a transcription factor system may be used in the development and implementation of cell therapies, such as adoptive cell therapy. In some embodiments, one or more components of a transcription factor system may be used to encode an engineered or modified TCR, or to enhance T cells other than TCRs (e.g., by introducing cytokine genes, genes for checkpoint inhibitors PD1, CTLA4), in cell therapy to implement CAR therapy, in the manipulation or regulation of TILs, in allogeneic cell therapy, or in the combination of T cell therapy with other lines of treatment (e.g., radiation, cytokines).

[0354] Provided herein is a method for use in adoptive cell therapy, comprising preconditioning a subject in need thereof, modulating immune cells with one or more components of a transcription factor system and / or a composition of the present disclosure, administering engineered immune cells expressing the composition of the present disclosure to the subject, and successfully engrafting the engineered cells in the subject.

[0355] In some embodiments, the regulatable transcription factor expression constructs and compositions of the present disclosure may be used to minimize preconditioning regimens associated with adoptive cell therapy. As used herein, "preconditioning" refers to any therapeutic regimen administered to a subject to improve the outcome of adoptive cell therapy. Preconditioning strategies include, but are not limited to, total body irradiation and / or lymphodepleting chemotherapy. Clinical trials of adoptive therapy without preconditioning have failed to demonstrate any clinical benefit, demonstrating its importance in ACT. Furthermore, preconditioning is associated with significant toxicity, limiting the subject cohort suitable for ACT. In some cases, immune cells in ACT may be engineered to express cytokines such as IL-2, IL-6, IL12, and IL15 as payloads using the transcription factors described herein to allow for selective expression of proteins of interest that may be regulated using the stabilized ligands of the present disclosure to reduce the need for preconditioning (Pengram et al. (2012) Blood 119(18):4133-41, the entire contents of which are incorporated by reference).

[0356] In some embodiments, the immune cells in ACT may be dendritic cells, T cells such as CD8+ T cells and CD4+ T cells, natural killer (NK) cells, NK T cells, cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer (LAK) cells, memory T cells, regulatory T cells (Tregs), helper T cells, cytokine-induced killer (CIK) cells, and any combination thereof. In other embodiments, the immune stimulatory cells in ACT may be generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In some embodiments, autologous or allogeneic immune cells are used in ACT.

[0357] In some embodiments, the cells used for ACT may be T cells engineered to express a CAR containing an antigen-binding domain specific for an antigen on tumor cells of interest. In other embodiments, the cells used for ACT may be NK cells engineered to express a CAR containing an antigen-binding domain specific for an antigen on tumor cells of interest. In addition to adoptive transfer of genetically modified T cells (e.g., CAR T cells) for immunotherapy, alternative types of CAR-expressing leukocytes may be used for adoptive immunotherapy, either alone or in combination with CAR T cells. In one example, a mixture of T cells and NK cells may be used for ACT. According to the present disclosure, the expression level of CAR in T cells and NK cells is regulated and controlled by a small molecule that binds to a DRD(s) operably linked to a transcription factor or its component, thereby allowing selective transcription of the CAR in transfected or transduced T cells and NK cells. In this scenario, the CAR is encoded by a nucleic acid sequence operably linked to an inducible promoter containing a specific polynucleotide binding site for the transcription factor.

[0358] In some embodiments, NK cells engineered to express one or more components of a transcription factor system may be used for ACT. NK cell activation induces perforin / granzyme-dependent apoptosis in target cells. NK cell activation also induces cytokine secretion, such as IFNγ, TNF-α, and GM-CSF. These cytokines enhance the phagocytic function of macrophages and their antibacterial activity, and enhance adaptive immune responses through upregulation of antigen presentation by antigen-presenting cells such as dendritic cells (DCs) (reviewed by Vivier et al., Nat. Immunol., 2008, 9(5):503-510).

[0359] Other examples of genetic modifications may include the introduction of chimeric antigen receptors (CARs) and downregulation of inhibitory NK cell receptors such as NKG2A.

[0360] NK cells may also be genetically reprogrammed to circumvent NK cell inhibitory signals upon interaction with tumor cells. For example, genetically modifying NK cells using CRISPR, ZFN, or TALEN to silence their inhibitory receptors may enhance the anti-tumor potential of NK cells.

[0361] Immune cells can be isolated and expanded ex vivo using various methods known in the art. For example, methods for isolating and expanding cytotoxic T cells are described in U.S. Patent Nos. 6,805,861 and 6,531,451, U.S. Patent Publication No. US20160348072A1, and International Patent Publication No. WO2016168595A1, the entire contents of each of which are incorporated herein by reference. NK cell isolation and expansion are described in U.S. Patent Publication No. US20150152387A1, U.S. Patent No. 7,435,596, and Oyer, JL (2016). Cytotherapy. 18(5):653-63, the entire contents of each of which are incorporated herein by reference. In particular, human primary NK cells can be expanded in the presence of feeder cells, such as bone marrow cell lines that have been genetically modified to express membrane-bound IL15, IL21, IL12, and 4-1BBL.

[0362] In some cases, a subpopulation of immune cells may be enriched for ACT. Methods for immune cell enrichment are taught in International Patent Publication No. WO2015039100A1. In another example, T cells positive for B and T lymphocyte attenuator marker (BTLA) may be used to enrich for anti-cancer reactive T cells, as described in U.S. Patent No. 9,512,401 (the entire contents of each of which are incorporated herein by reference).

[0363] In some embodiments, immune cells for ACT may be depleted of selected subpopulations to enhance T cell proliferation. For example, immune cells may be depleted of Foxp3+ T lymphocytes to minimize anti-tumor immune responses using the methods taught in U.S. Patent Publication No. US20160298081A1, the contents of which are incorporated herein by reference in their entirety.

[0364] In some embodiments, activation and proliferation of T cells for ACT is achieved by antigen stimulation of a chimeric antigen receptor (CAR) transiently expressed on the cell surface. Such activation methods are taught in International Patent Publication No. WO2017015427, the entire contents of which are incorporated herein by reference.

[0365] In some embodiments, immune cells may be activated by antigens associated with antigen-presenting cells (APCs). In some embodiments, APCs may be dendritic cells, macrophages, or B cells, which may be antigen-specific or non-specific. APCs may be autologous or allogeneic within their organs. In some embodiments, APCs may be artificial antigen-presenting cells (aAPCs), such as cell-based aAPCs or acellular aAPCs. Cell-based aAPCs may be selected from either genetically modified allogeneic cells, such as human erythroleukemia cells, or xenogeneic cells, such as mouse fibroblasts and Drosophila cells. Alternatively, APCs may be acellular, with antigens or costimulatory domains displayed on synthetic surfaces, such as latex beads, polystyrene beads, lipid vesicles, or exosomes.

[0366] In some embodiments, cells of the present disclosure, particularly T cells, may be expanded using an artificial cell platform. In one embodiment, mature T cells may be generated using artificial thymic organoids (ATOs) as described by Seet CS et al. 2017. Nat Methods. 14, 521-530 (the entire contents of which are incorporated herein by reference). ATOs are based on a stromal cell line that expresses the delta-like canonical Notch ligand (DLL1). In this method, stromal cells are aggregated with hematopoietic stem and progenitor cells by centrifugation and placed at an air-liquid interface on a cell culture insert to generate organoid cultures. ATO-derived T cells exhibit a naive phenotype, a diverse T cell receptor (TCR) repertoire, and TCR-dependent functions.

[0367] In some embodiments, adoptive cell therapy is performed by autologous transfer, where cells are derived from a subject in need of treatment and the isolated and processed cells are administered to the same subject. In other cases, ACT may involve allogeneic transfer, where cells are isolated and / or prepared from a donor subject other than the recipient subject who will ultimately receive the cell therapy. The donor and recipient subjects may be genetically identical or similar, or may express the same HLA class or subtype.

[0368] In some embodiments, multiple immunotherapeutic agents introduced into immune cells (e.g., T cells and NK cells) for ACT may be controlled by the same or different transcription factor systems. In one example, two payloads, e.g., a cytokine such as IL12 and a CAR construct such as a CD19 CAR, are each transcribed by one or more transcription factor(s) on the same or different transcription factor systems, and the transcription factor(s) are linked to the same or different DRDs. The payloads are transcribed and translated when the DRD(s) are stabilized with a stabilizing ligand specific for the DRD(s). Expression of the IL12 and CD19 CAR is regulated using one or more stabilizing ligands. In other embodiments, multiple immunotherapeutic agents introduced into immune cells (e.g., T cells and NK cells) for ACT may be controlled by different transcription factor systems. In one example, a cytokine such as IL12 and a CAR construct such as a CD19 CAR are each transcribed by one of two different transcription factors, and each transcription factor is operably linked to a different DRD, thereby allowing them to be separately regulated using different stimuli. In another example, the suicide gene and the CAR construct may be transcriptionally activated by two different transcription factors.

[0369] Following gene regulation using one or more components and compositions of the transcription factor system disclosed herein, the cells are administered to a subject in need thereof. Methods for administering cells for adoptive cell therapy are known and may be used in conjunction with the provided methods and compositions. For example, methods for adoptive T cell therapy are described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al., U.S. Patent No. 4,690,915 to Rosenberg, and Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85. See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933, Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9, and Davila et al. (2013) PLoS ONE 8(4):e61338, the entire contents of each of which are incorporated herein by reference.

[0370] In some embodiments, the immune cells of the ACT may be engineered to express one or more immunotherapeutic agents (proteins of interest) that promote immune cell activation, infiltration, proliferation, survival, and anti-tumor function. The immunotherapeutic agent may be a second CAR or TCR specific for a different target molecule, a cytokine or cytokine receptor, a chimeric switch receptor that converts inhibitory signals into stimulatory signals, a homing receptor that directs adoptively transferred cells to a target site such as tumor tissue, a drug that optimizes immune cell metabolism, or a safety switch gene (e.g., a suicide gene) that kills activated T cells if a severe event is observed after adoptive cell transfer or if the transferred immune cells are no longer needed.

[0371] In some embodiments, immune cells used in adoptive cell transfer can be genetically engineered to improve their persistence, cytotoxicity, tumor targeting ability, and ability to home to disease sites in vivo, with the overall goal of further improving their ability to kill tumors in cancer patients. One example is introducing one or more components of the disclosed transcription factor system encoding cytokines, such as gamma-cytokines (e.g., IL2 and IL15), into immune cells to promote their proliferation and survival. Transduction of immune cells with cytokine genes encoded by the transcription factor system (e.g., gamma-cytokines IL2 and IL15) allows for the proliferation of immune cells, e.g., NK cells, without the addition of exogenous cytokines, resulting in enhanced tumor cytotoxicity of cytokine-expressing NK cells.

[0372] In some embodiments, one or more components of the transcription factor system may be utilized to prevent T cell exhaustion. As used herein, "T cell exhaustion" refers to the gradual, progressive loss of T cell function caused by chronic T cell activation. T cell exhaustion is a major factor limiting the effectiveness of antiviral and antitumor immunotherapy. Exhausted T cells have a high rate of apoptosis and high surface expression of multiple inhibitory receptors, as well as reduced proliferative and cytokine-producing capabilities. T cell activation leading to exhaustion can occur either in the presence or absence of antigen.

[0373] In some embodiments, one or more components of the transcription factor system may be utilized to prevent T cell exhaustion in the context of chimeric antigen receptor T cell therapy (CAR-T). In this context, exhaustion may in some cases be caused by oligomerization of the CAR's scFv on the cell surface, resulting in continuous activation of the CAR's intracellular domain. As a non-limiting example, a CAR of the present disclosure may include an scFv that cannot oligomerize. As another non-limiting example, a CAR that is rapidly internalized and re-expressed after antigen exposure may also be selected to prevent chronic scFv oligomerization on the cell surface. In one embodiment, the framework region of the scFv may be modified to prevent constitutive CAR signaling (Long et al. 2014. Cancer Research. 74(19)S1, the entire contents of which are incorporated by reference). One or more components of the transcription factor system of the present disclosure may also be used to control the surface expression of a CAR on the T cell surface to prevent chronic T cell activation. The CAR of the present disclosure may also be designed to minimize exhaustion. As a non-limiting example, a 41-BB signaling domain may be incorporated into the CAR design to ameliorate T cell exhaustion. In some embodiments, any of the strategies disclosed by Long HA et al. may be utilized to prevent exhaustion (Long AH et al. (2015) Nature Medicine 21, 581-590, the entire contents of which are incorporated herein by reference).

[0374] In some embodiments, the regulatable nature of the transcription factor system of the present disclosure can be utilized to reverse the human T cell exhaustion observed with strong CAR signaling. Reversible silencing of the biological activity of adoptively transferred cells using the compositions of the present disclosure can be used to reverse strong signaling, thereby reactivating T cells. Reversal of exhaustion can be measured by downregulation of multiple inhibitory receptors associated with exhaustion.

[0375] In some embodiments, T cell metabolic pathways may be modified to reduce the susceptibility of T cells to exhaustion. Metabolic pathways may include, but are not limited to, glycolysis, the urea cycle, the citric acid cycle, beta-oxidation, fatty acid biosynthesis, the pentose phosphate pathway, nucleotide biosynthesis, and glycogen metabolism. As a non-limiting example, a payload that reduces the rate of glycolysis may be utilized to limit or prevent T cell exhaustion (Long et al. Journal for Immunotherapy of Cancer 2013, 1(Suppl 1):P21, the entire contents of which are incorporated by reference). In one embodiment, the T cells of the present disclosure may be used in combination with inhibitors of glycolysis, such as 2-deoxyglucose and rapamycin.

[0376] In some embodiments, the payload or protein of interest of the present disclosure may be used in combination with an antibody or fragment that targets a T cell surface marker associated with T cell exhaustion. T cell surface markers associated with T cell exhaustion that may be used include, but are not limited to, CTLA-1, PD-1, TGIT, LAG-3, 2B4, BTLA, TIM3, VISTA, and CD96. In some embodiments, one or more components of the transcription factor system may be utilized to prevent T cell exhaustion.

[0377] In some embodiments, compositions of the present disclosure may be utilized to alter TIL (tumor infiltrating lymphocyte) populations in a subject. In one embodiment, any of the payloads described herein may be utilized to alter the ratio of CD4-positive cells to CD8-positive populations. In some embodiments, TILs may be selected ex vivo and engineered to express any of the cytokines described herein. Payloads of the present disclosure may be used to expand the CD4 and / or CD8 populations of TILs to enhance TIL-mediated immune responses. Parameters for improving CAR-T therapy outcomes are described in Finney et al. JCI. 2019;129(5):2123-2132 (the entire contents of which are incorporated herein by reference). Levels of the biomarker LAG3(high) / TNF-α(low) in peripheral blood CD8+ T cells at the time of apheresis may also predict subsequent dysfunctional responses in subjects with high antigen burden who do not achieve a complete response lasting more than several weeks. The convergence of T cell intrinsic characteristics, resulting from the starting T cell repertoire and the effects of the manufacturing process, on CD19 antigen-induced activation after adoptive transfer may also play a role in the outcome of CAR-T therapy. The starting T cell repertoire may be partially influenced by the timing of apheresis. In one embodiment, apheresis may be performed before chemotherapy. The cumulative burden of CD19-expressing leukemia and normal B cells, as assessed in bone marrow before lymphodepleting chemotherapy, may be important in determining the outcome of CAR-T therapy. According to Finney et al., increasing antigen burden improves the outcome of CAR-T therapy. To increase the CD19 antigen load in vivo, subjects may also be infused with subject-derived T cells (also referred to as T-APCs) that have been genetically modified to express CD19 and expanded.

[0378] In some embodiments, the regulatable transcription factor expression constructs, payloads of interest (e.g., immunotherapeutics), vectors, cells and compositions of the present disclosure may be used in combination with a cancer vaccine.

[0379] In some embodiments, cancer vaccines may include peptides and / or proteins derived from tumor-associated antigens (TAAs). Such strategies may be used to induce an immune response in a subject, which in some cases may be a cytotoxic T lymphocyte (CTL) response. Peptides used in cancer vaccines may be modified to match the mutation profile of the subject. For example, EGFR-derived peptides with mutations consistent with those found in subjects in need of therapy have been successfully used in patients with lung cancer (Li F et al. (2016) Oncoimmunology. Oct 7;5(12):e1238539, the entire contents of which are incorporated herein by reference).

[0380] In one embodiment, the cancer vaccine of the present disclosure may include superagonist modified peptide ligands (APLs) derived from tumor-associated antigens (TAAs). These are mutant peptide ligands that deviate from the native peptide sequence by one or more amino acids, which activate specific CTL clones more effectively than the native epitope. These changes may enable the peptide to better bind to restricted class I MHC molecules or to interact more favorably with the TCR of a given tumor-specific CTL subset. APLs may be selected using the methods taught in U.S. Patent Publication No. US20160317633A1, the entire contents of which are incorporated herein by reference.

[0381] In some embodiments, effector immune cells genetically modified to encode components of the disclosed transcription factor system and a payload may be combined with a biological adjuvant as described herein. Dual control of the CAR and cytokines and ligands separates kinetic control of target-mediated activation from endogenous T cell proliferation. Such dual control also minimizes the need for patient preconditioning regimens. As a non-limiting example, a DRD-regulated transcription factor that transcribes a payload, e.g., a CAR, e.g., a CD19 CAR, may be combined with a cytokine, e.g., IL12, to enhance the antitumor effect of the CAR (Pegram HJ, et al. Tumor-targeted T cells modified to secrete IL12 eradicate systemic tumors without need for prior conditioning. Blood. 2012;119:4133-41, the entire contents of each of which are incorporated herein by reference). As another non-limiting example, Merchant et al. combined dendritic cell-based vaccination with recombinant human IL7 to improve outcomes in high-risk pediatric sarcoma patients (Merchant, MSet. al. Adjuvant immunotherapy to Improve Outcome in High-Risk Pediatric Sarcomas. Clin Cancer Res. 2016. 22(13):3182-91, the contents of each of which are incorporated herein by reference in their entirety).

[0382] In some embodiments, effector immune cells engineered to express one or more antigen-specific TCRs or CARs may be combined with compositions of the present disclosure comprising immunotherapeutic agents that alter the immunosuppressive tumor microenvironment.

[0383] In one embodiment, effector immune cells engineered to express CARs specific for different target molecules on the same cell may be combined. In another embodiment, different immune cells engineered to express the same CAR construct, such as NK cells and T cells, may be used in combination for tumor treatment, for example, T cells engineered to express a CD19 CAR may be combined with NK cells engineered to express the same CD19 CAR to treat B-cell malignancies.

[0384] In other embodiments, immune cells engineered to express a CAR may be combined with a checkpoint blockade agent.

[0385] In some embodiments, one or more components of the transcription factor system of the present disclosure, e.g., effector immune cells genetically engineered to express a payload, may be combined with the cancer vaccines of the present disclosure and other immunotherapies and adjuvant treatments.

[0386] In some embodiments, the methods of the present disclosure may include a combination of a composition of the present disclosure with other agents effective in treating cancer, infectious diseases, and other immunodeficiency disorders, such as, for example, anti-cancer agents. As used herein, the term "anti-cancer agent" refers to any agent that can adversely affect cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, decreasing the rate of proliferation of cancer cells, decreasing the incidence or number of metastases, shrinking tumor size, inhibiting tumor growth, decreasing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[0387] In some embodiments, the anti-cancer agent or therapy may be a chemotherapeutic agent, or radiation therapy, immunotherapy, surgery, or any other therapeutic agent that improves the therapeutic efficacy of the treatment in combination with the present disclosure.

[0388] In one embodiment, one or more components of a transcription factor system, including a CD19 CAR, may be used in combination with an aminopyrimidine derivative, such as a Burkitt tyrosine receptor kinase (BTK) inhibitor, using the methods taught in International Patent Application No. WO2016164580, the contents of which are incorporated herein by reference in their entirety.

[0389] In some embodiments, the compositions of the present disclosure may be used in combination with immunotherapies other than the therapies of the present invention described herein, such as antibodies specific for some target molecule on the surface of tumor cells.

[0390] Exemplary chemotherapeutics include acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, amsacrine, anastrozole, anthramycin, asparaginase, asperin, sulindac, curcumin, alkylating agents including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; nitrosoureas, such as carmustine (BCU), lomustine (CCNU), and semustine (methyl-CC U), ethyleneimines / methylmelamines, for example, triethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine), alkyl sulfonates such as busulfan, triazines such as dacarbazine (DTIC), antimetabolites, including: folic acid analogs, for example, methotrexate and trimetrexate, pyrrolidine analogs, for example, 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs, for example, 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine ( natural products, including: antimitotics such as paclitaxel; vinca alkaloids, including vinblastine (VLB), vincristine, and vinorelbine; taxotere, estramustine, and estramustine phosphate; epipodophyllotoxins, such as etoposide and teniposide; antibiotics, such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes, such as L-asparaginase; cytokines, such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha;antiangiogenic factors such as TNF-beta and GM-CSF, for example, angiostatin and endostatin, inhibitors of FGF or VEGF, for example, soluble receptors for angiogenic factors, including soluble VGF / VEGF receptors, platinum coordination complexes, for example, cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIFf) and procarbazine, adrenocortical suppressants such as mitotane (ο,ρ'-DDD) and aminoglutethimide Hormones and antagonists, including: corticosteroid antagonists, such as prednisone and equivalents, dexamethasone and aminoglutethimide, progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate, estrogens, such as diethylstilbestrol and ethinyl estradiol equivalents, antiestrogens such as tamoxifen, androgens, antiandrogens, e.g. testosterone propionate and fluoxymesterone / equivalents nonsteroidal antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide, kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, antioxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosine kinase inhibitors, such as imatinib mesylate (sold as Gleevec or Glivec) and erlotinib (EGF receptor inhibitor currently sold as Tarveca) inhibitors), antiviral drugs, such as oseltamivir phosphate, amphotericin B, and palivizumab, Sdi1 mimetics, semustine, senescence induction inhibitor 1, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stipiamide, stromelysin inhibitors, sulfinosine, superactive vasoactive intestinal peptide antagonists, veraresol, veramine, verudine, verteporfin, vinorelbine, vinxartin, Vitaxin, vorozole, zanoteron, zeniplatin, zilascorub,and zinostatin stimalamer, the PI3Kβ small molecule inhibitor, GSK2636771, the pan-PI3K inhibitor (BKM120), the BRAF inhibitors, vemurafenib (Zelboraf) and dabrafenib (Tafinlar), or analogs or derivatives and variants of any of the foregoing.

[0391] Radiotherapeutic agents and factors include radiation and waves that induce DNA damage, such as gamma irradiation, X-rays, UV irradiation, microwaves, electronic emissions, and radioisotopes. Therapy may be achieved by delivering these forms of radiation to the localized tumor site. All of these factors most likely affect widespread damaged DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0392] In some embodiments, the chemotherapeutic agent may be an immunomodulatory agent such as lenalidomide (LEN). Recent studies have demonstrated that lenalidomide can enhance the anti-tumor function of CAR-modified T cells (Otahal et al., Oncoimmunology, 2015, 5(4): e1115940). Some examples of anti-tumor antibodies include tocilizumab and siltuximab.

[0393] Other agents that may be used in combination with the compositions of the present disclosure may also include, but are not limited to, agents that affect the upregulation of cell surface receptors and their ligands, such as Fas / Fas ligand, DR4 or DR5 / TRAIL and gap junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, such as focal adhesion kinase (FAK) inhibitors and lovastatin, or agents that increase t...

Claims

[Claim 1] The invention described in the specification.

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