Compositions and methods for regulated control of transcription

Abstract

The present disclosure provides compositions and methods related to transcription factor systems. Such systems provide modular and regulatable protein expression driven by controlled transcriptional activity. 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.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 958,693 filed on 8 January 2020 and U.S. Provisional Patent Application No. 62 / 959,859 filed on 10 January 2020. The entirety of the above-mentioned applications is incorporated herein by reference.

[0002] Sequence listing reference This application includes an array listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. Created on 8 January 2021, its ASCII copy is named 268052_483267_SL.txt and is 241,815 bytes in size.

[0003] field This disclosure relates to systems, compositions, and methods for regulated protein expression driven by controlled transcriptional activity. Provided in this disclosure are modular transcription factor systems, polynucleotides, polypeptides, vectors, cells, compositions, and methods for use in regulated protein expression driven by transcriptional control and controlled transcriptional activity. [Background technology]

[0004] background Gene therapy and cell therapy have revolutionized medicine, offering new possibilities for treating previously intractable conditions. However, most current technologies lack the ability to precisely time or control the dose level of targeted protein induction. This makes it difficult or impossible to safely and effectively utilize many potential gene and cell therapies.

[0005] Inadequate exogenous and / or endogenous gene regulation is a significant problem in many gene therapy and cell therapy scenarios. This lack of regulatory capability also makes it difficult to safely express proteins with narrow or uncertain therapeutic windows, or proteins requiring more dose-specific or transient expression.

[0006] One approach to controlled protein expression or function is the use of drug-responsive domains (DRDs). A drug-responsive domain is a small protein domain that can be attached to a target protein of interest. In the absence of a DRD-binding ligand, the attached target protein becomes unstable and is rapidly degraded by the cell's ubiquitin-proteasome system. However, when a specific small molecule DRD-binding ligand binds to the DRD, the attached target protein is stabilized, and protein function is achieved.

[0007] DRD technology forms the basis for a new class of cell and gene therapies that can regulated and transiently control gene expression and function, expanding the realm of protein therapy that can be safely and effectively incorporated into cell and gene therapy modalities. However, current DRD technologies produce fusion proteins in which the target protein is bound to the DRD, which may make them unsuitable for some indications. Therefore, there remains a need to develop cell and gene therapies in which the target native protein can be expressed in a controlled manner. [Overview of the Initiative]

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

[0009] Furthermore, this disclosure provides compositions, systems, and methods for regulated control of transcription. The compositions relate to transcription factor systems and agents that induce the transcriptional activity of polynucleotides encoding a protein of interest. The compositions provided by this disclosure include nucleic acid molecules, polypeptides, and cells related to transcription factor systems. The methods related to transcription factor systems provided by this disclosure include methods for producing modified cells and methods for treating or preventing diseases.

[0010] Provided herein are transcription factor systems. The transcription factor systems of this disclosure are combinations of one or more polynucleotides, comprising: (1) one or more nucleic acid sequences encoding a transcription factor that can bind to a specific polynucleotide binding site to activate transcription; (2) a nucleic acid sequence encoding a drug-responsive domain (DRD), wherein a transcription factor, or a portion thereof, is functionally linked to the DRD; and (3) a nucleic acid sequence encoding a payload and functionally linked to an inducible promoter containing a specific polynucleotide binding site.

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

[0012] In some embodiments, the present disclosure provides modified cells that may be capable of controlling the expression or transcription of a payload. The modified cells include 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 the transcription of a fourth nucleic acid sequence when bound to a 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 comprising 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 modified cells further comprise a second polynucleotide comprising the fourth nucleic acid sequence.

[0013] In some embodiments, the present disclosure provides modified cells comprising a polynucleotide comprising a first nucleic acid sequence encoding a drug-responsive domain (DRD) and a second nucleic acid sequence encoding a transcription factor. The transcription factor is operably linked to the DRD, binds to a specific polynucleotide binding site, and can activate the transcription of a third nucleic acid sequence encoding a protein of interest, and 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. In some embodiments, the protein of interest is a heterologous protein. In some embodiments, the third nucleic acid sequence is located on the polynucleotide comprising the first nucleic acid sequence and the second nucleic acid sequence. In some embodiments, the modified cells further comprise a second polynucleotide comprising the third nucleic acid sequence.

[0014] In another aspect, the present 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-responsive 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 comprising the specific polynucleotide binding site.

[0015] In another aspect, the present 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-responsive domain (DRD), wherein the transcription factor is operably linked to the DRD.

[0016] In another aspect, the present 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 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 capable of activating transcription upon binding to the specific polynucleotide binding site.

[0017] In another embodiment, 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 functionally linked to the DRD. In one embodiment, the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that can bind to a specific polynucleotide binding site and activate the transcription of a nucleic acid sequence encoding a protein of interest, the nucleic acid sequence being functionally linked to an exogenous inducible promoter.

[0018] In various embodiments, one or more of the transcription factor DNA-binding domain, transcription factor activation domain, and DRD are derived from the 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 the parent protein, which is p65. In some embodiments, the DRD is derived from a parent protein selected from the group consisting of 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, DRD is stabilized in the presence of a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). In some embodiments, 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 target protein is a wild-type protein.

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

[0022] In some embodiments, the protein of interest is selected from the group consisting of cytokines, antibodies or their antigen-binding fragments, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs).

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

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

[0025] In some embodiments, the cells are T cells, natural killer cells (NK cells), or tumor-infiltrating lymphocytes (TILs).

[0026] In some embodiments, the cells are stem cells, liver cells, blood cells, pancreatic cells, nerve cells, eye cells, muscle cells, or bone cells.

[0027] Furthermore, this disclosure provides nucleic acid molecules related to transcription factor systems.

[0028] In one embodiment, the 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, where (i) the transcription factor DNA-binding domain is functionally linked to the DRD, (ii) the transcription factor activation domain is functionally linked to the DRD, or (iii) a combination of the transcription factor DNA-binding domain and the transcription factor activation domain is functionally 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, which is p65.

[0029] In one embodiment, the 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 to activate transcription, and (b) a second nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is functionally 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 functionally linked to either a specific polynucleotide binding site, an exogenous inducible promoter containing a specific polynucleotide binding site, or both.

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

[0031] In some embodiments, 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, DRD is stabilized in the presence of a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). In some embodiments, 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 target protein is a wild-type protein.

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

[0035] In some embodiments, the protein of interest is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs).

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

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

[0038] Also provided herein are vectors comprising nucleic acid molecules as described herein. Vectors provided by this disclosure include plasmids or viral vectors. In some embodiments, viral vectors are derived from adenoviruses, adeno-associated viruses (AAVs), alphaviruses, flaviviruses, herpesviruses, measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease virus (NDV), poxviruses, and picornaviruses. In some embodiments, viral vectors are selected from the group consisting of lentiviral vectors, gamma-retrovirus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

[0039] Also provided by this disclosure are a first polynucleotide and a second polynucleotide, each containing a nucleic acid sequence encoding one or more components of a transcription factor system.

[0040] In one embodiment, the Disclosure provides a first polynucleotide and a second 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), 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 functionally linked to the DRD; the second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest, the fourth nucleic acid sequence being functionally linked to an inducible promoter comprising a specific polynucleotide binding site, where the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that can activate transcription when bound to the specific polynucleotide binding site; the first polynucleotide and the second polynucleotide are each supported on a single vector, or the first polynucleotide and the second polynucleotide are supported on separate vectors.

[0041] In one embodiment, the Disclosure provides a first polynucleotide and a second polynucleotide, the first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor and a second nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is functionally linked to the DRD and the transcription factor can activate transcription upon binding to a specific polynucleotide binding site; the second polynucleotide comprising a third nucleic acid sequence encoding a protein of interest, wherein the third nucleic acid sequence is functionally linked to an inducible promoter comprising a specific polynucleotide binding site; the first polynucleotide and the second polynucleotide are each supported on a single vector, or the first polynucleotide and the second polynucleotide are supported 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 target protein is a wild-type protein. In some embodiments, the target protein is a therapeutic protein. In some embodiments, the target protein is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs). In some embodiments, the target protein is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFNs, and Cre. In some embodiments, the target protein is a secreted protein.

[0044] Furthermore, this disclosure provides methods related to transcription factor systems.

[0045] In one embodiment, the disclosure provides a method for producing modified cells, the method comprising introducing a nucleic acid molecule into cells 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, (i) the transcription factor DNA-binding domain is functionally linked to the DRD, (ii) the transcription factor activation domain is functionally linked to the DRD, or (iii) a combination of the transcription factor DNA-binding domain and the transcription factor activation domain is functionally linked to the DRD.

[0046] In some embodiments, the method further includes introducing a fourth nucleic acid sequence encoding the protein of interest into a cell, wherein the fourth nucleic acid sequence is functionally linked to an inducible promoter containing 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 located on the same nucleic acid molecule as the first, second, and third nucleic acid sequences. In one embodiment, the fourth nucleic acid sequence is located on a different nucleic acid molecule than the first, second, and third nucleic acid sequences.

[0047] In some embodiments, the protein of interest is selected from the group consisting of cytokines, antibodies or their antigen-binding fragments, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs).

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

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

[0050] In some embodiments, nucleic acid molecules are introduced into cells by plasmids or viral vectors. 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 lentiviral vectors, gamma-retrovirus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

[0051] In some embodiments, nucleic acid molecules are introduced into cells by non-viral delivery methods.

[0052] In some embodiments, the cells are T cells, natural killer cells (NK cells), or tumor-infiltrating lymphocytes (TILs). In some embodiments, the cells are stem cells, liver cells, blood cells, pancreatic cells, nerve cells, eye cells, muscle cells, or bone cells.

[0053] Furthermore, this disclosure provides methods related to treating or preventing diseases.

[0054] In one embodiment, the disclosure provides a method for treating or preventing a disease in a person in need thereof, the method comprising: (a) providing a cell population; (b) introducing at least one nucleic acid molecule into at least one cell in 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 functionally linked to the DRD; and (ii) a protein intended for the prevention or treatment of a disease or its symptoms. (c) a second polynucleotide comprising a fourth nucleic acid sequence encoding a specific polynucleotide, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising a specific polynucleotide binding site; (c) delivered to a target cell; and (d) administered to the target in an amount sufficient to bind a ligand to the specific polynucleotide binding site and form a transcription factor that enables the expression of the protein of interest in the cell, wherein the expression of the protein of interest is controlled in the target 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.

[0055] In one embodiment, the Disclosure provides a method for introducing modified cells into a subject requiring treatment or prevention of a disease, the method comprising (a) providing a cell population, (b) introducing at least one nucleic acid molecule into at least one cell in 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 functionally linked to the DRD, and (ii) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest for treating the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising a specific polynucleotide binding site, and (c) delivering the cells to the subject.

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

[0057] In one embodiment, the Disclosure provides a method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, 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-responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during intracellular expression, and (ii) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest for treating the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising a specific polynucleotide binding site.

[0058] In one embodiment, the Disclosure provides a method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, the method comprising (a) introducing at least one nucleic acid molecule into at least one cell of the subject, wherein the at least one nucleic acid molecule is a first polynucleotide comprising (i) 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 functionally linked to the DRD when expressed in the cell, and (ii) a protein intended to treat the disease. (b) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising a specific polynucleotide binding site, and (b) administering to a subject in an amount sufficient to bind a ligand that stabilizes the DRD to enable the expression of at least one of a transcription factor activating domain and a transcription factor DNA binding domain to the specific polynucleotide binding site and form a transcription factor that enables the expression of the protein of interest in the cell, wherein the 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.

[0059] In one embodiment, the present disclosure provides a method for treating a disease in a subject in need thereof, the method comprising (a) providing a cell population, and (b) introducing at least one of a first nucleic acid molecule and at least one of a second nucleic acid molecule into at least one cell in 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 functionally linked to the DRD when expressed in the cell, and (ii) the second nucleic acid molecule treats the disease The present invention comprises (c) a fourth nucleic acid sequence encoding a protein of interest to be treated, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing a specific polynucleotide binding site; (c) delivery to a target cell; and (d) administration to the target in an amount sufficient to bind a ligand to the specific polynucleotide binding site and form a transcription factor that enables the expression of the protein of interest in the cell, wherein the expression of the protein of interest is controlled in the target 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.

[0060] In one embodiment, the Disclosure provides a method for treating a disease in a target that requires such treatment, the method comprising (a) providing a cell population, (b) introducing at least one of a first nucleic acid molecule and at least one of a second nucleic acid molecule into at least one cell in the cell population, (i) the first nucleic acid molecule 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 functionally linked to the DRD when expressed in the cell, (ii) the second nucleic acid molecule comprising a fourth nucleic acid sequence encoding a protein intended to prevent and / or treat a disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing a specific polynucleotide binding site, and (c) delivering the cell to the target.

[0061] In related embodiments, the disclosure provides a method for preventing and / or treating a disease in a target population where it is needed. The method comprises (a) providing a cell population, and (b) introducing at least one of a first nucleic acid molecule and at least one of a second nucleic acid molecule into at least one cell in the cell population. In this example method, 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). At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during intracellular expression, and the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a target protein for preventing and / or treating a disease in a target population where it is needed. The fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing a specific polynucleotide binding site. The method also includes the steps of (c) delivering cells to a target, and (d) administering to the target a ligand that stabilizes the DRD sufficiently to enable the expression of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to bind to a specific polynucleotide binding site and form a transcription factor that enables the expression of the target protein in the cell. In this example method, the expression of the target protein is controlled in the target 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 target protein.

[0062] In related embodiments, the treatment and prevention methods of the present disclosure may be achieved by introducing a single vector into a cell, the vector carrying a first nucleic acid molecule and a second nucleic acid molecule, (i) the first nucleic acid molecule 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 are functionally linked to the DRD upon intracellular expression; and the second nucleic acid molecule comprising a fourth nucleic acid sequence encoding a protein intended to treat or prevent a disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing a specific polynucleotide binding site.

[0063] In some alternative embodiments, the treatment and prevention methods of the present disclosure may be achieved by introducing a first vector and a second vector into cells, the first vector 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 transcription factor DNA binding domain are functionally linked to the DRD upon intracellular expression; the second vector comprising a fourth nucleic acid sequence encoding a protein intended to prevent and / or treat a disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing a specific polynucleotide binding site.

[0064] In some embodiments, nucleic acid molecules are introduced into cells by plasmids or viral vectors. In some embodiments, the viral vector is derived from adenoviruses, adeno-associated viruses (AAVs), alphaviruses, flaviviruses, herpesviruses, measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease viruses (NDVs), poxviruses, and picornaviruses. In some embodiments, the viral vector is selected from the group consisting of lentiviral vectors, gamma-retrovirus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

[0065] In some embodiments, nucleic acid molecules are introduced into cells by non-viral delivery methods.

[0066] Also provided by this disclosure is a system for the regulated expression of a protein of interest in a cell, the system comprising: (a) a first polynucleotide encoding a transcription factor linked to a drug-responsive domain (DRD), wherein the transcription factor selectively transcribes the polynucleotide sequence encoding the protein of interest; (b) a second polynucleotide containing an exogenous transcription factor binding site located upstream of the nucleic acid sequence encoding the protein of interest; (c) introducing the first and second polynucleotides into a cell under conditions that allow for stable integration of the first and second polynucleotides into the cell's genome; and (d) regulating the 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 located upstream of the polynucleotide sequence encoding the protein of interest, and the expression of the protein of interest is controlled by the amount of transcription factor present in the cell.

[0067] This disclosure also provides pharmaceutical compositions comprising the compositions described herein and pharmaceutically acceptable excipients. In certain embodiments, for example, the following are provided: (Item 1) A modified cell comprising a first 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), 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 functionally linked to the DRD. The transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that, upon binding to the specific polynucleotide binding site, can activate the transcription of the fourth nucleic acid sequence. The modified cell wherein the fourth nucleic acid sequence encodes a target protein and is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site. (Item 2) A modified cell comprising a first 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), 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 functionally linked to the DRD. The transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that, upon binding to the specific polynucleotide binding site, can activate the transcription of the fourth nucleic acid sequence. The modified cell wherein the fourth nucleic acid sequence encodes a target protein and is functionally linked to the specific polynucleotide binding site. (Item 3) The modified cell according to item 2, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site. (Item 4) A modified cell as described in any one of items 1 to 3, wherein the target protein is a heterologous protein. (Item 5) A modified cell according to any one of items 1 to 4, wherein the fourth nucleic acid sequence is located on the first polynucleotide. (Item 6) A modified cell according to any one of items 1 to 4, further comprising a second polynucleotide, wherein the second polynucleotide comprises the fourth nucleic acid sequence. (Item 7) A modified cell according to 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) A 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) A modified cell comprising a polynucleotide containing a first nucleic acid sequence encoding a drug-responsive domain (DRD) and a second nucleic acid sequence encoding a transcription factor, wherein the transcription factor is functionally linked to the DRD. The modified cell wherein the transcription factor can bind to a specific polynucleotide binding site and activate the transcription of a third nucleic acid sequence encoding a target protein, and the third nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site. (Item 10) A modified cell comprising a polynucleotide containing a first nucleic acid sequence encoding a drug-responsive domain (DRD) and a second nucleic acid sequence encoding a transcription factor, wherein the transcription factor is functionally linked to the DRD. The modified cell wherein the transcription factor can bind to a specific polynucleotide binding site and activate the transcription of a third nucleic acid sequence encoding a target protein, and the third nucleic acid sequence is functionally linked to the specific polynucleotide binding site. (Item 11) The modified cell according to item 10, wherein the third nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site. (Item 12) A modified cell as described in any one of items 9 to 11, wherein the target protein is a heterologous protein. (Item 13) A 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) A modified cell according to any one of items 9 to 12, further comprising a second polynucleotide containing the third nucleic acid sequence. (Item 15) Modified cells as described in any one of items 1 to 14, wherein 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. (Item 16) Modified cells according to any one of items 1 to 15, wherein the DRD is stabilized in the presence of a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). (Item 17) Modified cells as described in any one of items 1 to 16, wherein the target protein is a wild-type protein. (Item 18) Modified cells as described in any one of items 1 to 17, wherein the target protein is a therapeutic protein. (Item 19) The modified cell described in item 18, wherein the target protein is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs). (Item 20) A modified cell according to any one of items 1 to 17, wherein the target protein is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre. (Item 21) A modified cell according to any one of items 1 to 17, wherein the target protein is a secreted protein. (Item 22) A modified cell according to 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) Modified cells as described in any one of items 1 to 21, wherein the cells are stem cells, liver cells, blood cells, pancreatic cells, nerve cells, eye cells, muscle cells, or bone cells. (Item 24) nucleic acid molecules, a. A first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, b. The second nucleic acid sequence encoding the drug response domain (DRD) and The nucleic acid molecule, including the above. (Item 25) c. A nucleic acid molecule according to item 24, further comprising a third nucleic acid sequence encoding a transcription factor activation domain, wherein (i) the transcription factor DNA binding domain is functionally linked to the DRD, (ii) the transcription factor activation domain is functionally linked to the DRD, or (iii) a combination of the transcription factor DNA binding domain and the transcription factor activation domain is functionally linked to the DRD. (Item 26) d. A nucleic acid molecule according to item 24 or 25, further comprising a fourth nucleic acid sequence encoding a protein of interest, wherein the fourth nucleic acid sequence is functionally linked to an inducible promoter containing the specific polynucleotide binding site. (Item 27) A nucleic acid molecule according to 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) A 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) nucleic acid molecules, a. A first nucleic acid sequence encoding a transcription factor that can bind to a specific polynucleotide binding site and activate transcription, b. The second nucleic acid sequence encoding the drug response domain (DRD) and The nucleic acid molecule comprising the transcription factor functionally linked to the DRD. (Item 30) c. A nucleic acid molecule according to item 29, further comprising a third nucleic acid sequence encoding a target protein, wherein the third nucleic acid sequence is functionally linked to an inducible promoter containing the specific polynucleotide binding site. (Item 31) The nucleic acid molecule described in any one of items 24 to 30, wherein 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. (Item 32) The nucleic acid molecule described in any one of items 24 to 31, wherein the DRD is stabilized in the presence of a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). (Item 33) A nucleic acid molecule as described in any one of items 26-28 or 30-32, wherein the target protein is a wild-type protein. (Item 34) A nucleic acid molecule as described in any one of items 26-28 or 30-32, wherein the target protein is a therapeutic protein. (Item 35) The nucleic acid molecule described in item 34, wherein the target protein is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs). (Item 36) The target protein is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre, and is a nucleic acid molecule as described in any one of items 26-28 or 30-32. (Item 37) A nucleic acid molecule as described in items 26-28 or 30-32, wherein the target protein is a secreted protein. (Item 38) A vector containing a nucleic acid molecule as described in any one of items 24-37. (Item 39) The vector described in item 38, wherein the vector is a plasmid or a viral vector. (Item 40) The vector described in item 39, wherein 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. (Item 41) The vector described in item 39, wherein the viral vector is selected from the group consisting of lentiviral vectors, gamma retrovirus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors. (Item 42) A first polynucleotide and a second polynucleotide, wherein the first polynucleotide is 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 The third nucleic acid sequence comprises a drug-responsive domain (DRD), wherein at least one of the following is functionally linked to the DRD: 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. The second polynucleotide is The fourth nucleic acid sequence comprises a fourth nucleic acid sequence encoding the target protein, wherein the fourth nucleic acid sequence is functionally linked to an inducible promoter containing the specific polynucleotide binding site. The transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that can activate transcription when bound to a specific polynucleotide binding site, wherein the first polynucleotide and the second polynucleotide are each supported on a single vector, or the first polynucleotide and the second polynucleotide are supported on separate vectors. (Item 43) A first polynucleotide and a second polynucleotide, wherein the first polynucleotide is The material comprises a first nucleic acid sequence encoding a transcription factor and a second nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is functionally linked to the DRD, and the transcription factor can activate transcription when it binds to a specific polynucleotide binding site. The second polynucleotide is The third nucleic acid sequence comprises a third nucleic acid sequence encoding the target protein, wherein the third nucleic acid sequence is functionally linked to an inducible promoter containing the specific polynucleotide binding site. The first polynucleotide and the second polynucleotide, wherein each of the first polynucleotide and the second polynucleotide is supported on a single vector, or the first polynucleotide and the second polynucleotide are supported on separate vectors. (Item 44) The first and second polynucleotides described in item 42 or 43, wherein 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. (Item 45) The first and second polynucleotides according to any one of items 42 to 44, wherein the DRD is stabilized in the presence of a ligand selected from the group including acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). (Item 46) The first polynucleotide and the second polynucleotide as described in any one of items 42 to 45, wherein the target protein is a wild-type protein. (Item 47) The first polynucleotide and the second polynucleotide described in any one of items 42 to 45, wherein the target protein is a therapeutic protein. (Item 48) The first polynucleotide and the second polynucleotide described in item 47, wherein the target protein is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs). (Item 49) The first polynucleotide and the second polynucleotide described in any one of items 42 to 45, wherein the target protein is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre. (Item 50) The first polynucleotide and the second polynucleotide described in any one of items 42 to 45, wherein the target protein is a secreted protein. (Item 51) A method for producing modified cells, wherein the method is a. A first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, b. The second nucleic acid sequence encoding the drug response domain (DRD) and The method comprising introducing nucleic acid molecules containing into cells. (Item 52) The method according to item 51, wherein the nucleic acid molecule further comprises a third nucleic acid sequence encoding a transcription factor activation domain. (Item 53) The method according to item 52, wherein (i) the transcription factor DNA binding domain is functionally linked to the DRD, (ii) the transcription factor activation domain is functionally linked to the DRD, or (iii) a combination of the transcription factor DNA binding domain and the transcription factor activation domain is functionally linked to the DRD. (Item 54) The method according to item 53, further comprising introducing a fourth nucleic acid sequence encoding a target protein into the cell, wherein the fourth nucleic acid sequence is functionally linked to an inducible promoter containing the specific polynucleotide binding site. (Item 55) The method according to item 54, wherein the target protein is a heterologous protein. (Item 56) The method according to item 54 or 55, wherein the fourth nucleic acid sequence is located 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) The method according to item 54 or 55, wherein the fourth nucleic acid sequence is located on a nucleic acid molecule different from the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence. (Item 58) The method according to any one of items 54 to 57, wherein the target protein is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs). (Item 59) The method according to any one of items 54 to 57, wherein the target protein is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre. (Item 60) The method according to any one of items 54 to 57, wherein the target protein is a secreted protein. (Item 61) The method according to 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) The method according to item 61, wherein 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. (Item 63) The method according to item 61, wherein the viral vector is selected from the group consisting of lentiviral vectors, gamma retroviral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors. (Item 64) The method according to 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) The method according to 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) 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) A method for treating or preventing a disease in a person in need thereof, wherein the method is a. To provide a cell population, 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 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 functionally linked to the DRD. ii. A second polynucleotide comprising a fourth nucleic acid sequence encoding a protein intended to prevent or treat the disease or its symptoms, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site. c. Delivering the cells to the target, and d. Administer to the subject an amount sufficient to form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the target protein in the cell, thereby stabilizing the DRD to a degree sufficient to allow expression of at least one of the transcription factor activation domain and the transcription factor DNA binding domain. The method comprising the following: the expression of the target protein 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 target protein. (Item 68) A method for introducing modified cells into a subject requiring treatment or prevention of a disease, wherein the method is a. To provide a cell population, 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 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 functionally linked to the DRD. ii. comprising a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein intended to treat the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising the specific polynucleotide binding site, and c. The method comprising delivering the cells to the target. (Item 69) A method for introducing modified cells into a subject requiring treatment or prevention of a disease, wherein the method is a. To provide a cell population, b. Introducing at least one nucleic acid molecule described in any one of items 24-37 or a first polynucleotide and a second polynucleotide described in any one of items 42-50 into at least one cell in the cell population, c. The method comprising delivering the cells to the target. (Item 70) A method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, wherein the method is a. Introduce at least one nucleic acid molecule into at least one cell of the subject, and the at least one nucleic acid molecule 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 functionally linked to the DRD during expression in the cell. ii. The method comprising a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein for the purpose of treating the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising the specific polynucleotide binding site. (Item 71) A method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, wherein the method is a. Introduce at least one nucleic acid molecule into at least one cell of the subject, and the at least one nucleic acid molecule 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 functionally linked to the DRD during expression in the cell. ii. comprising a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein intended to treat the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising the specific polynucleotide binding site, and b. A ligand that stabilizes the DRD to a sufficient degree to enable the expression of at least one of the transcription factor activation domain and the transcription factor DNA binding domain is administered to the subject in an amount sufficient to bind to the specific polynucleotide binding site and form a transcription factor that enables the expression of the target protein in the cell. The method comprising the following: the expression of the target protein 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 target protein. (Item 72) A method for treating a disease in a person who requires such treatment, wherein the method is a. To provide a cell population, b. Introduce 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 functionally linked to the DRD during expression in the cell. ii. The second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein intended to treat the disease, and the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site. c. Delivering the cells to the target, and d. A ligand that stabilizes the DRD to a sufficient degree to enable the expression of the transcription factor activation domain and the transcription factor DNA binding domain is administered to the subject in an amount sufficient to form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the target protein in the cell. The method comprising the following: the expression of the target protein 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 target protein. (Item 73) A method for treating a disease in a person who requires such treatment, wherein the method is a. To provide a cell population, b. Introduce 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 functionally linked to the DRD during expression in the cell. ii. The second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein intended to treat the disease, and the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site, and c. The method comprising delivering the cells to the target. (Item 74) The method according to 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) The method according to item 74, wherein 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. (Item 76) The method according to item 74, wherein the viral vector is selected from the group consisting of lentiviral vectors, gamma retroviral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors. (Item 77) The method according to 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 the regulated expression of a target protein in a cell, wherein the system is a. A first polynucleotide encoding a transcription factor linked to a drug-responsive domain (DRD), wherein the transcription factor selectively transcribes the polynucleotide sequence encoding the target protein, b. A second polynucleotide containing an exogenous transcription factor binding site located adjacent to the upstream of the nucleic acid sequence encoding the target protein, c. Introducing the first polynucleotide and the second polynucleotide into the cell under conditions that allow for stable integration of the first polynucleotide and the second polynucleotide into the cell's genome. d. Regulating the expression of the transcription factor by adding a ligand that stabilizes the DRD. Includes, The system wherein the transcription factor specifically binds to a transcription factor binding site located adjacent to the upstream of the polynucleotide sequence encoding the target protein, and the expression of the target protein is controlled by the amount of the transcription factor present in the cell. [Brief explanation of the drawing]

[0068] [Figure 1]A schematic diagram of the transcription factor system design scheme is shown. A shows a schematic diagram of the transcription factor construct, referred to as the "DRD-TF construct," which includes 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 the payload construct, which includes an inducible promoter containing a binding site for the transcription factor DNA binding domain. [Figure 2] This shows the ligand-dependent activity of transcription factor systems, including DRD-regulating transcription factors, with different DRDs. A shows 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 functionally linked to a DRD derived from CA2, ecDHFR, ER, or hDHFR parental proteins. 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 transcription factors and DRD polypeptides encoded by endogenous p65, each of the DRD-TF constructs, and transcription factor polypeptides encoded by the constitutive construct ZFHD-055. B shows the quantification of the Western blot from Figure 2A, normalized with the constitutive condition set to 1.0. [Figure 3]This shows the ligand-dependent activity of a transcription factor system, including the ecDHFR DRD regulatory transcription factor. A shows a schematic diagram of the transcription factor construct ZFHD-005. B shows a schematic diagram of the payload construct ZFHD-007. C shows a schematic diagram of the constitutive transcription factor construct ZFHD-004. D shows a Western blot of lysates from U2OS cells stably incorporating the shown constructs, 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 polypeptide encoded by the transcription factor construct ZFHD-005. The band appearing at approximately 26.5 kDa represents the transcription factor polypeptide encoded by construct ZFHD-004. E exhibits GFP central fluorescence intensity (MFI) as assessed by flow cytometry on U2OS cells stably incorporating the indicated construct, treated with 10 μM TMP or 0.1% DMSO. [Figure 4] This shows the dose-response to ligands in a transcription factor system including the ecDHFR DRD regulatory transcription factor. A shows a Western blot of lysates from U2OS cells stably incorporating constructs ZFHD-005 and ZFHD-007, treated with DMSO or TMP at the indicated concentrations. 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 the transcription factor construct ZFHD-005. B shows the quantification of the "ZFHD-005 polypeptide" band shown in the Western blot of Figure 4A. Fluorescence is normalized to endogenous p65. C represents the GFP central fluorescence intensity (MFI) as evaluated by flow cytometry on U2OS cells containing stably incorporated constructs ZFHD-005 and ZFHD-007, treated with TMP at the indicated concentrations. The maximum TMP concentration used in Figure 4C was 33 μM. The data shown are repeated three times. Error bars represent the standard deviation. [Figure 5]This shows the ligand-dependent activity of the transcription factor system, including the ecDHFR DRD regulatory transcription factor, in T cells. A shows Western blots of lysates from untransduced T cells or T cells transduced with a 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 the arrow) represents the transcription factor and DRD polypeptide encoded by the transcription factor construct ZFHD-005. B shows the GFP central fluorescence intensity (MFI) as assessed by flow cytometry of untransduced T cells or T cells transduced with a virus prepared from the shown construct, treated with TMP or DMSO. The data shown are repeated three times. Error bars represent the standard deviation from the mean. [Figure 6] This shows the ligand-dependent activity of a transcription factor system, including a CA2 DRD regulatory transcription factor, in ARPE-19 cells. A shows a schematic diagram of the transcription factor construct ZFHD-019. B shows Western blots of lysates from untransduced ARPE-19 cells or ARPE-19 cells containing stably incorporated constructs ZFHD-019 and ZFHD-007, 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. C shows the quantification of the "ZFHD-019 polypeptide" band shown in the Western blot of Figure 6B. Fluorescence is normalized to endogenous p65. D represents the GFP central fluorescence intensity (MFI) as assessed by flow cytometry of ARPE-19 cells that stably incorporate the indicated construct, either untransduced or untreated or treated with 10 μM ACZ or 1% DMSO. The data shown are repeated three times. Error bars represent the standard deviation from the mean. Untransduced ARPE-19 cells and ARPE-19 cells that stably incorporate the construct ZFHD-007, as shown on the graph, were treated with DMSO. [Figure 7] This shows the dose-response relationship to ligands in a transcription factor system, including the CA2 DRD regulatory transcription factor. Figure A shows a Western blot of lysates from ARPE-19 cells containing stably incorporated constructs ZFHD-007 and ZFHD-019, treated with ACZ at the indicated concentrations. 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 B shows the quantification of the "ZFHD-019 polypeptide" band shown in the Western blot of Figure 7A. Fluorescence is normalized relative to the endogenous p65 band. [Figure 8] This graph shows the dose-response relationship to ligands in transcription factor systems, including CA2 DRD regulatory transcription factors. The graph displays GFP central fluorescence intensity (MFI) as assessed by flow cytometry in U2OS cells stably incorporating constructs ZFHD-007 and ZFHD-019, treated with ACZ at the indicated concentrations. The data shown are repeated twice. Error bars represent the standard deviation. [Figure 9] This shows the ligand-dependent activity of transcription factor systems, including CA2 DRD regulatory transcription factors, 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 the GFP central fluorescence intensity (MFI) as evaluated by flow cytometry on Jurkat cells stably incorporating constructs ZFHD-048 and ZFHD-022, treated with DMSO (0.1%) or ACZ (final concentration of 10 μM). The data presented pertain to cells that were positive for the transduction marker. [Figure 10A] This shows the ligand-dependent activity of a single-vector transcription factor system containing the ecDHFR DRD regulatory transcription factor. A schematic diagram of construct ZFHD-012 is shown. [Figure 10B] This shows the ligand-dependent activity of a single-vector transcription factor system containing the ecDHFR DRD regulatory transcription factor. A schematic diagram of construct ZFHD-018 is shown. [Figure 10C]This shows the ligand-dependent activity of a single-vector transcription system containing the ecDHFR DRD regulatory transcription factor. Western blots of lysates from U2OS cells transduced with a lentivirus prepared from the shown construct 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 shown construct. In the single-vector construct, the presence of stop codons at the end of the EGFP sequence and the transcription factor-DRD sequence results in the approximately 44.3 kDa band representing the transcription factor and DRD polypeptide. [Figure 10D] This shows the ligand-dependent activity of a single-vector transcription system containing the ecDHFR DRD regulatory transcription factor. Western blots of lysates from U2OS cells transduced with a lentivirus prepared from the shown construct 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 shown construct. In the single-vector construct, the presence of stop codons at the end of the EGFP sequence and the transcription factor-DRD sequence results in the approximately 44.3 kDa band representing the transcription factor and DRD polypeptide. [Figure 10E] This exhibits ligand-dependent activity of a single-vector transcription factor system containing the ecDHFR DRD regulatory transcription factor. Transduced with lentiviruses prepared from the shown constructs, U2OS cells treated with 10 μM TMP or 0.1% DMSO exhibit GFP central fluorescence intensity (MFI) as assessed by flow cytometry. [Figure 10F] This exhibits ligand-dependent activity of a single-vector transcription factor system containing the ecDHFR DRD regulatory transcription factor. Transduced with lentiviruses prepared from the shown constructs, U2OS cells treated with 10 μM TMP or 0.1% DMSO exhibit GFP central fluorescence intensity (MFI) as assessed by flow cytometry. [Figure 11]This shows the ligand-dependent activity of a single-vector transcription factor system containing a CA2 DRD regulatory transcription factor. A shows a schematic diagram of the single-vector system, shown as construct ZFHD-036. B shows the GFP central fluorescence intensity (MFI) as evaluated by flow cytometry of Jurkat cells transduced with a lentivirus prepared from the shown construct 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 a lentivirus prepared from construct ZFHD-036. [Figure 12] This shows the ligand-dependent activity of transcription factor systems, including variants of transcription factor constructs. A shows a schematic diagram of the transcription factor construct variant. B shows the GFP central fluorescence intensity (MFI) as assessed by flow cytometry on U2OS cells stably incorporating the shown constructs, treated with either 0.1% DMSO or 10 μM TMP. [Figure 13] This shows a ligand response time course analysis of transcription factor systems, including variants of the transcription factor construct. The graphs show the GFP central fluorescence intensity (MFI) as assessed by flow cytometry of U2OS cells stably incorporating the indicated construct, treated with either 0.1% DMSO or 10 μM TMP at the indicated time. [Figure 14] This shows the ligand-dependent activity of transcription factor systems, including 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 the GFP central fluorescence intensity (MFI) as assessed by flow cytometry on U2OS cells stably incorporating the shown constructs, treated with either 0.1% DMSO or 10 μM TMP. [Figure 15] This graph shows the ligand-dependent activity of a transcription factor system containing a payload construct encoding the secreted IL-12 payload. The graph shows the concentration of secreted IL-12 in the supernatant collected from U2OS cells stably incorporating the shown construct, treated with either 0.1% DMSO or 10 μM TMP. [Figure 16A] This shows ligand-dependent regulation of different transcription factors functionally linked to the DRD derived from the parental CA2 protein. 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") are shown. Cell populations transfected with each construct after treatment with DMSO or ACZ (indicated by the "+" symbol) are shown. The labeled band "c-Jun-001 and -002 polypeptides" identifies the CA2-linker-C-jun polypeptide encoded by the cjun-001 and cjun-002 constructs. The labeled band "c-Jun-003 polypeptide" identifies the c-Jun polypeptide encoded by the construct cjun-003. [Figure 16B] This shows ligand-dependent regulation of different transcription factors functionally linked to DRD derived from the parental CA2 protein. Figure 16A shows the quantification of the Western blot. [Figure 16C] This shows ligand-dependent regulation of different transcription factors functionally linked to the DRD derived from the parental 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") are shown. Cell populations transfected with each construct after treatment with DMSO or ACZ (indicated by the "+" symbol) are shown. The labeled band "FOXP3-013 and -014 polypeptides" identifies the CA2-FOXP3 polypeptides encoded by the FOXP3-013 and FOXP3-014 constructs. The labeled band "FOXP3-015 polypeptide" identifies the FOXP3 polypeptide encoded by the construct FOXP3-015. [Figure 16D]This shows ligand-dependent regulation of different transcription factors functionally linked to DRD derived from the parental CA2 protein. Figure 16C shows the quantification of the Western blot. [Figure 17] Figure 17A shows ligand-dependent regulation of c-Jun transcription factor constructs stably incorporated into Jurkat cells. Figure A shows Western blots of lysates from non-transduced ("mock") Jurkat cells and Jurkat cells transduced with lentiviruses prepared from constructs cjun-001 ("001") and cjun-002 ("002"). Transduced cell lines of each construct after treatment with DMSO or ACZ (indicated by the "+" symbol) are shown. Bands for c-Jun polypeptide and phosphorylated c-Jun polypeptide are shown. Figure B shows quantification of the Western blots in Figure 17A. [Figure 18-1] Figure 18 shows the nucleotide sequence of the pELDS-puro introduction vector (SEQ ID NO: 68). [Figure 18-2] Figure 18 shows the nucleotide sequence of the pELDS-puro introduction vector (SEQ ID NO: 68). [Figure 19-1] Figure 19 shows the nucleotide sequence of the pELNS-puro introduction vector (SEQ ID NO: 69). [Figure 19-2] Figure 19 shows the nucleotide sequence of the pELNS-puro introduction vector (SEQ ID NO: 69). [Modes for carrying out the invention]

[0069] Detailed explanation Transcription factor system According to this disclosure, a transcription factor system is a combination of one or more polynucleotides comprising: (1) one or more nucleic acid sequences encoding a transcription factor that can bind to a specific polynucleotide binding site and activate transcription; (2) a nucleic acid sequence encoding a drug-responsive domain (DRD) to which the transcription factor is functionally linked; and (3) a nucleic acid sequence encoding a payload and functionally linked to an inducible promoter containing a specific polynucleotide binding site.

[0070] In some embodiments, a combination of one or more polynucleotides in a transcription factor system may be used to create a system that modifies cells, such as immune cells useful for treating a disease, and modifies the expression of a target protein by controlling the presence of a transcription factor that acts on a polynucleotide(s) encoding a target payload or protein.

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

[0072] This disclosure also provides a first polynucleotide and a second 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). 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 functionally ligated to the DRD as illustrated herein. The second polynucleotide comprises a fourth nucleic acid sequence encoding the protein of interest, the fourth nucleic acid sequence is functionally ligated to an inducible promoter containing 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 when bound to a specific polynucleotide binding site, the first polynucleotide and the second polynucleotide each being supported on a single vector, or the first polynucleotide and the second polynucleotide being supported on separate vectors.

[0073] In related examples, the Disclosure provides compositions and nucleic acids that are operable to control transcription. For example, the Disclosure provides a first polynucleotide and a second polynucleotide of a tunable 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-responsive domain (DRD), the transcription factor being functionally linked to the DRD, and the transcription factor being able to activate transcription upon binding to a specific polynucleotide binding site. The second polynucleotide comprises a third nucleic acid sequence encoding a protein of interest, the third nucleic acid sequence being functionally linked to an inducible promoter containing a specific polynucleotide binding site, the first and second polynucleotides being each supported on a single vector, or the first and second polynucleotides being supported on separate vectors.

[0074] In some embodiments, a combination of one or more polynucleotides in a transcription factor system includes 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 embodiments, a combination of one or more polynucleotides in a transcription factor system includes a polynucleotide comprising the first, second, and third nucleic acid sequences. In some embodiments, a combination of one or more polynucleotides in a transcription factor system includes a polynucleotide comprising two of the first, second, and third nucleic acid sequences. In some embodiments, a combination of one or more polynucleotides in a transcription factor system includes 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 embodiment, the transcription factor DNA-binding domain is functionally linked to the DRD. In another embodiment, the transcription factor activation domain is functionally linked to the DRD. In yet another embodiment, both the transcription factor DNA-binding domain and the transcription factor activation domain are functionally linked to the DRD. In some embodiments, the transcription factor DNA-binding domain and the transcription factor activation domain are expressed as a transcription factor fusion protein.

[0075] According to this disclosure, the transcription factor system encodes a transcription factor that can drive the 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, upon binding to a specific polynucleotide binding site, activates the transcription of the nucleic acid sequence encoding the payload.

[0076] In some embodiments, a particular polynucleotide binding site comprises at least one nucleic acid site having a specific sequence recognized and bound by the transcription factor DNA-binding domain. In some embodiments, a particular 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 the DNA-binding domain of the disclosure. In some embodiments, a particular polynucleotide binding site comprises eight nucleic acid sites recognized by the DNA-binding domain. In some embodiments, a particular polynucleotide binding site comprises two or more tandem nucleic acid sites, each having a specific sequence recognized and bound by the transcription factor DNA-binding domain. In some embodiments, the tandem nucleic acid sites comprise identical nucleic acid sequences. In some embodiments, a particular polynucleotide binding site comprises tandem repeat nucleic acid sites recognized by the DNA-binding domain of the disclosure.

[0077] As described herein, a transcription factor or a portion thereof is functionally linked to a DRD in the transcription factor system of this disclosure. The presence, absence, or amount of ligands that bind to or interact with the DRD may modulate the stability of the transcription factor and consequently its function in the event of such binding or interaction. Thus, the transcription factor system may exhibit ligand-dependent activity.

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

[0079] Transcription factor system constructs A combination of one or more polynucleotides in a transcription factor system may also be referred to herein as a combination of one or more nucleic acid constructs. A polynucleotide or nucleic acid construct may include different arrangements of nucleic acid sequences and / or may be combined in a specific way as part of a transcription factor system, insofar as the resulting combination of polynucleotides or nucleic acid constructs includes: (1) one or more nucleic acid sequences encoding a transcription factor that can bind to a specific polynucleotide binding site and activate transcription; (2) a nucleic acid sequence encoding a drug-responsive domain (DRD) to which the transcription factor is functionally linked to the DRD; and (3) a nucleic acid sequence encoding a payload and functionally linked to an inducible promoter containing a specific polynucleotide binding site.

[0080] In some embodiments, the transcription factor system comprises a plurality of constructs. In some embodiments, the transcription factor system comprises a transcription factor construct and a payload construct. In one embodiment, the transcription factor construct comprises a nucleic acid sequence encoding a transcription factor. In one embodiment, 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 the transcription factor, DRD, and a nucleic acid sequence encoding the payload of the transcription factor system. In some embodiments, such a single construct transcription factor system may be introduced into cells 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 transcription factor systems, the nucleic acid constructs of this disclosure may include additional nucleic acid sequences. These additional nucleic acid sequences include, but are 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 the DRD. In some embodiments, the nucleic acid sequence encoding the DRD is adjacent to the nucleic acid sequence encoding at least one of the transcription factor domains. In some embodiments, the nucleic acid sequence encoding the DRD is positioned 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 include a promoter, a transcription factor DNA-binding domain, a transcription factor activation domain, a linker, and a nucleic acid sequence encoding the DRD. In some embodiments, the linker is positioned 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 encoded transcription factor DNA-binding domain in the transcription factor construct is ZFHD1. In some embodiments, the encoded transcription factor activation domain in the transcription factor construct is p65.

[0086] In some embodiments, the payload construct may include a specific polynucleotide binding site having 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. An exemplary binding site includes eight nucleic acid sites recognized by the ZFHD1 DNA-binding domain.

[0087] In some embodiments, constructs of the present disclosure, such as transcription factor constructs or payload constructs, are incorporated into a plasmid or viral vector. In some embodiments, the plasmid or viral vector includes one or more regulatory elements that are functionally linked to one or more components of the construct incorporated into the plasmid or viral vector. In some embodiments, the plasmid or viral vector includes regulatory elements well known in the art, such as 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 functionally linked to the regulatory elements of the plasmid or viral vector. In some embodiments, such a transcription factor construct includes a transcription factor DNA-binding domain, a transcription factor activation domain, and a nucleic acid sequence encoding a DRD, and the promoter sequence in the plasmid or viral vector is incorporated into the plasmid or viral vector such that it drives the expression of the transcription factor DNA-binding domain, the transcription factor activation domain, and the DRD. Such promoters may be selected from constitutive promoters, tissue-specific promoters, cell-specific promoters, cell differentiation-specific promoters, and / or disease-specific promoters. Depending on the circumstances, the promoter may be selected from EF1a, CMV, EFS, RSV, SFFV, PGK, CAG, and SV40.

[0088] Components of the transcription factor system As described above, the polynucleotide or nucleic acid constructs of a transcription factor system may include different arrangements of nucleic acid sequences and / or be combined in a specific way as part of a transcription factor system, insofar as the resulting combination of polynucleotides or nucleic acid constructs includes: (1) one or more nucleic acid sequences encoding a transcription factor that can bind to a specific polynucleotide binding site and activate transcription; (2) a nucleic acid sequence encoding a drug-responsive domain (DRD) to which the transcription factor is functionally linked; and (3) a nucleic acid sequence encoding a payload and functionally linked to an inducible promoter containing a specific polynucleotide binding site. Thus, the transcription factor system is a modular system, and each component of the transcription factor system can be selected separately.

[0089] The nucleic acid sequence encoding the drug-responsive domain (DRD) may be selected from the DRD sequences described in more detail in the following section, "Drug-Responsive Domain (DRD)".

[0090] One or more nucleic acid sequences encoding a transcription factor may be selected from existing transcription factors, engineered transcription factors derived from existing transcription factors, or one or more sequences encoding an engineered transcription factor containing a DNA-binding domain and an activation domain. As used herein, “engineered transcription factor derived from existing transcription factors” refers to an engineered transcription factor that is at least partially derived 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 include a TAL effector repeat region that recognizes a specific DNA-binding site, a mammalian nuclear localization signal (NLS), and a synthetic transcription activation domain. If 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 a 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 the transcription factor DNA-binding domain of this disclosure, or may be further modified to generate it.

[0092] In some embodiments, the transcription factor DNA-binding domain is derived from a parental 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. ("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 the OCT-1 homeodomain. ZFHD1 can bind to nucleic acid sequences including the sequence TAATGATGGGCG (SEQ ID NO: 70). In some embodiments, the transcription factor DNA-binding domain consists of or includes the amino acid sequence of ZFHD1.

[0094] In some embodiments, the disclosure provides a method for controlling target genes and their corresponding functional proteins (e.g., payloads or proteins of interest) using a Cas / guide RNA system. Those skilled in the art will understand that suitable guide RNAs can be designed to form colocalization complexes with target nucleic acids, including target genes as described herein.

[0095] Various Cas proteins are known to those skilled in the art, and these proteins include CasI (Cas3), CasIA (Cas8a), CasIB (Cas8b), CasIC (Cas8c), CasID (Cas10d), CasIE (Cse1), CasIF (Csy1), CasIU, CasII (Cas9), CasIIA (Csn2), CasIIB (Cas4), CasIIC, CasIII (Cas10), 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 C2C1, C2C3, Cpf1 (also called Cas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, C It 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] According to one embodiment, the Cas9 protein includes a protein sequence that is a DNA-binding protein, such as an RNA-induced DNA-binding protein, and has at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology to naturally occurring Cas9 derived from S. aureus, S. thermophiles, S. pyogenes, or Neisseria meningitidis Cas9.

[0098] According to one embodiment, the Cas12 protein includes a protein sequence that is a DNA-binding protein, such as an RNA-induced DNA-binding protein, and has at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology to naturally occurring Cas12 derived from Francisella novicida, Acidaminococcus, Lachnospiraceae, or Prevotella species.

[0099] In some embodiments, the transcription factor DNA-binding domain is derived from a parental Cas protein, such as a parental Cas9 or Cas12 protein. In some embodiments, the transcription factor DNA-binding domain is or includes Cas9 modified to lack nuclease activity. In some embodiments, the transcription factor DNA-binding domain is or includes Cas12 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 (target strand), and a RuvC-like nuclease domain that cleaves the DNA strand opposite the complementary strand (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-induced DNA targeting ability but loses its endonuclease activity. In some embodiments, the transcription factor DNA-binding domain is a dCas9 containing the mutated HNH and RuvC nuclease domains, derived from 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, catalytically dead Cas12 (having inactivated DNase activity and also referred to herein as "dCas12") may be obtained from the parent Cas12 protein. In some embodiments, the transcription factor DNA-binding domain is or contains 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 a type II Cas homolog that lacks nuclease activity or is modified to lack nuclease activity, or includes such a homolog. In some embodiments, the transcription factor DNA-binding domain is a type II Cas homolog that includes mutated HNH and RuvC nuclease domains, or includes such a homolog.

[0103] In exemplary embodiments, Cas9 is modified or otherwise altered to inactivate its nuclease activity. Such modification or alteration involves altering one or more amino acids to inactivate nuclease activity or the nuclease domain. Such alteration involves removing a polypeptide sequence(s) exhibiting nuclease activity, i.e., the nuclease domain, so that the polypeptide sequence(s) exhibiting nuclease activity, i.e., the nuclease domain, is no longer present in 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 comprises a polypeptide sequence modified to inactivate nuclease activity or the removal of a polypeptide sequence(s) to inactivate nuclease activity. The nuclease-null DNA-binding protein retains its ability to bind to DNA even after its nuclease activity has been inactivated. Therefore, DNA-binding proteins may contain the polypeptide sequence(s) necessary for DNA binding but lack one or more 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 is absent or substantially absent within the detection level. In 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 contains Cas9 having a mutated nuclease domain (referred to as "dead Cas9" or "dCas9"). The resulting dCas9 retains its RNA-induced DNA targeting ability but loses its endonuclease activity. In some embodiments, the transcription factor DNA-binding domain is dCas9.

[0105] This disclosure provides the use of guide RNA for targeting a polynucleotide binding sequence, such as those described herein, to a Cas protein, for example, a nuclease null Cas9 functionally linked to DRD. Such guide RNA can be readily designed by those skilled in the art, given knowledge of a particular polynucleotide binding sequence. The guide RNA may comprise one or more of a spacer sequence, a tracr mate sequence, and a tracr sequence. The term spacer sequence will be understood by those skilled in the art and may comprise any polynucleotide that hybridizes with the polynucleotide binding sequence and has sufficient complementarity with the polynucleotide binding sequence to cause the CRISPR complex to sequence-specifically bind to the polynucleotide binding sequence. The guide RNA may be formed from a spacer sequence and a separate tracr sequence covalently bonded to a tracr mate sequence (which may be referred to as crRNA), and the tracr mate sequence is hybridized to a portion of the tracr sequence. In certain embodiments, the tracr mate sequence and the tracr sequence are joined or linked by covalent bonding, such as by a linker sequence, and this construct may be referred to as a fusion of the tracr mate sequence and the tracr sequence. The linker sequences referred to herein are sequences of nucleotides, which are referred to herein as nucleic acid sequences, and which link the tracr mate sequence and the tracr sequence. Thus, the guide RNA may be a species of two components (i.e., separate crRNA and tracr RNA that hybridize with each other) or a monospecies (i.e., a crRNA-tracr RNA fusion, often referred to as sgRNA).

[0106] In some embodiments, the guide RNA may be delivered directly into the cell as a native species by methods known to those skilled in the art, including injection or lipofection, or it may be delivered into the cell to be transcribed from congenital 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, the transcription factor being either nuclease null Cas9 or comprising a DNA-binding domain containing it. When a DRD-stabilizing ligand is added, the DRD and transcription factor are stabilized, and nuclease null Cas9 is expressed and becomes capable of binding to a guide RNA. Upon binding to the guide RNA, the Cas9-gRNA system binds to a polynucleotide-binding sequence functionally linked to the protein of interest. When the Cas9-gRNA system binds to the polynucleotide-binding sequence, the protein gene of interest is transcribed in the presence of the transcription factor activation domain. Therefore, when a regulatory transcription factor expression construct contains a Cas9-gRNA system, RNA-induced DNA regulation occurs in cells such as human cells by linking or binding the DRD to either nuclease null Cas9 or the transcription factor activation domain. Accordingly, aspects of this disclosure include methods and materials for localizing a transcriptional regulatory domain to a target gene locus by fusing, binding, or ligating a DRD to either Cas9Nuc or a transcription factor activation domain, or both.

[0108] In some embodiments, the transcription factor DNA-binding domain is derived from the parental TAL protein. TAL (transcription activator-like) effectors (also referred to as "TALE") are proteins secreted by Xanthomonas bacteria that regulate gene expression in host plants and assist in bacterial infection. TAL effectors have a repeating region consisting mainly of 33 or 34 amino acid tandem repeats. The repeat monomers differ mainly at amino acid positions 12 and 13, and there is a strong correlation between the specific pairs of amino acids at positions 12 and 13 and the corresponding nucleotides of the TALE binding site. The transcription factor DNA-binding domain of this disclosure may include all or part of the repeating region of a TAL effector that can bind to a specific DNA-binding site. In some embodiments, the DNA-binding domain includes 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 polypeptides derived from parental TAL effector proteins, TAL effectors and / or synthetic TAL effectors, or polypeptides containing repeating regions of such regions. 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 contact 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 three finger arrays.An engineered DNA-binding domain containing 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 native or engineered DNA-binding domains.

[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 (by altering one or more amino acids). Thus, an engineered DNA-binding protein (zinc finger or TALE) is a protein that does not exist in nature. Non-limiting examples of methods for manipulating DNA-binding proteins include design and selection. An engineered DNA-binding protein is a protein that does not exist in nature, whose design / composition is primarily determined by rational criteria. Rational criteria for design include the application of substitution rules and the application of computerized algorithms for processing information in databases storing information on existing ZFP and / or TALE design and binding data. For example, see U.S. Patents 8,586,526, 6,140,081, 6,453,242, 6,534,261 and 8,586,526, and also see WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536 and WO03 / 016496, where the disclosures of these references relate to the design and selection of DNA-binding proteins derived from existing ZFP and / or TALE proteins and associated binding data, the entirety of which is incorporated herein by reference.

[0111] The activation domain of the manipulated transcription factor according to this 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 includes the carboxy-terminal region of the human transcription factor NF-κβ p65 protein.

[0112] Considerations in the design of transcription factor systems provided herein include the ability of the encoded transcription factor to bind to a specific polynucleotide binding site, and the functional linkage of the nucleic acid sequence encoding the payload to an inducible promoter containing a 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 in the design of synthetic transcription factors and corresponding synthetic promoters are also known. For example, Khalil AS, et al. provides a zinc finger array that can be used as a core component for constructing synthetic transcription factors and further provides a corresponding nucleic acid-binding sequence that can be inserted into a synthetic promoter and recognized by the zinc finger array (Khalil AS, et al. Cell 2012, 150, 647-658, the whole of which is incorporated by reference). Khalil AS, et al. also provide design strategies for identifying synthetic transcription factor-promoter pairs, altering promoters (e.g., by polymerizing zinc finger binding sequences to create promoters with repeat operators), and modifying transcriptional output by altering synthetic transcription factors (e.g., by creating variants). Either the transcription factor-promoter pairs or the manipulated zinc finger arrays and their corresponding nucleic acid binding sites disclosed by Khalil AS, et al. can be used in the transcription factor systems of this disclosure. As an example, Figure 3A by Khalil AS, et al. provides a library of amino acid residues of recognition helices of zinc finger arrays and their corresponding DNA binding sequences that can be used to design the transcription factor DNA binding domains and specific polynucleotide binding sites of this disclosure.One of ordinary skill in the art will recognize that the transcription factors or zinc finger array sequences provided by Khalil, A.S., et al. can be modified by cloning the sequences of these transcription factors or arrays into the constructs of the transcription factor system provided herein. As another example, Zhang, F., et al. have described methods for designing and producing engineered TAL effectors having corresponding nucleic acid binding sites. These can be used for the preparation of 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 the 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 the corresponding nucleic acid binding sequences in FIG. 2a. The TAL effectors or their DNA binding portions disclosed by Zhang, F., et al. may be used, in addition to the DNA binding domain, to construct the corresponding nucleic acid binding sequences of the inducible promoters of the present disclosure. One of ordinary skill in the art will recognize that there are several options for the selection and design of the DNA binding domains of the present disclosure. In addition to the selection of recognized DNA binding proteins and domains known in the art, the DNA binding domains of the present disclosure may be designed based on the frameworks of existing DNA binding proteins. For example, methods for selecting DNA binding domains based on the Cys2His2 zinc finger protein framework are available to one of ordinary skill in the art (Pabo, C.O., et al. Annu. Rev. Biochem. 2001. 70:313-40).

[0113] In some embodiments, the inducible promoter functionally ligated to the nucleic acid sequence encoding the payload includes a minimal promoter (also referred to as the “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 functionally ligated to the nucleic acid sequence encoding the payload. The term “minimal promoter” refers to the smallest structure that enables the formation of an initiation complex. The minimal promoter may include an RNA polymerase binding site, a TATA box, and a transcription initiation site. The minimal promoter may be bound to one or more response elements (such as enhancer or transcription factor binding sites) to generate an inducible promoter. Additional details regarding the minimal promoter and the binding of the minimal promoter to response elements are provided by Ede et al. (ACS Synth Biol. 2016 May 20;5(5):395-404). In some embodiments, the inducible promoters of the transcription factor system or its components of the Disclosure include 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 derived from the human junB gene), YB_TATA (synthetic minimal promoter developed by Benenson and colleagues (Hansen, J. et al. Proc Natl Acad Sci USA. 2014;111:15705-15710)), and minimal promoters selected from the TATA box alone.

[0114] As described above, a particular 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 particular 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 has been described above.

[0115] The nucleic acid sequence encoding the payload may be selected to encode any payload or protein of interest. Additional details regarding the payload 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. The asterisk ( ) in Table 1 indicates translation of a 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 that include structurally different transcription factor components are provided in Table 2. The asterisk ( 「*」 ) in Table 2 indicates translation of the stop codon. In addition to the corresponding control constructs that do not contain a regulated transcription factor, separate construct components are also provided. As shown in the description of constructs cjun-001 and cjun-002, the peptide linker is placed between the CA2 component and the c-Jun component of each construct. Further, all constructs contain a P2A peptide.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

[0118] Characterization of ligand-dependent activity of transcription factor systems The ligand-dependent activity of transcription factor systems can be characterized by various methods.

[0119] In some embodiments, the ligand-dependent activity of a transcription factor system is characterized by ligand-dependent control of the transcription factor polypeptide encoded by the transcription factor system (e.g., a transcription factor DNA-binding domain, a transcription factor activation domain, or both the transcription factor DNA-binding domain and the transcription factor activation domain). In some embodiments, the ligand-dependent activity of a transcription factor system is characterized by ligand-dose-dependent control of the transcription factor polypeptide encoded by the transcription factor system. In one embodiment, the transcription factor polypeptide is a polypeptide containing a transcription factor activation domain. In another embodiment, the transcription factor polypeptide is a polypeptide containing a transcription factor DNA-binding domain. In yet another embodiment, the transcription factor polypeptide is a polypeptide containing both a transcription factor activation domain and a transcription factor DNA-binding domain. The ligand-dependent control of the transcription factor polypeptide may be characterized by various methods. In some embodiments, the ligand-dependent control of the transcription factor polypeptide may be evaluated by measuring the level of the transcription factor polypeptide or its domains, for example, by an immunoassay.

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

[0121] In some embodiments, the transcription factor system may be compared to a control transcription factor system lacking 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 including a control transcription factor construct lacking DRD. An example of a control transcription factor construct is construct ZFHD-004, which is described herein (as shown in Table 1).

[0122] Transcription factor Transcription factors are proteins that bind to DNA, preferably to sequence-specific sites on DNA located within or near the promoter (transcription factor polynucleotide binding sites). They activate the transcription of DNA sequences by promoting the binding of the transcription mechanism to the promoter. Such entities are also known as transcription regulatory proteins.

[0123] In various embodiments, the transcription factors for use in the transcription factor systems, compositions, and methods described herein include 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 influencing gene expression. Recognition of DNA sequences by transcription factors occurs through chemical interactions between the amino acid side chains of the transcription factor protein and base pair residues of DNA that function as regulatory sequences. Thus, transcription factors "read" genomic sequences, and this mechanism provides a sequence recognition function on which the informative aspects of regulatory processes controlling 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 necessary 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, upon re-entry into the nucleus, find their target sites within genomic DNA, mediated by nuclear localization sites present in all transcription factor protein sequences. Transcription factors contain basic domains that are non-specifically concentrated near DNA, facilitating the discovery of diffusion-limited target sites.

[0126] In various embodiments of this disclosure, the transcription factor system utilizes a transcription factor comprising and / or containing a transcription factor DNA-binding domain and a transcription factor effector or activating domain or protein (as used interchangeably herein). The transcription factor activating domain, the transcription factor DNA-binding domain, and / or a combination of the transcription factor activating domain and the transcription factor DNA-binding domain may be functionally ligated to a DRD (any of which is a DRD-TF). Once the ligated DRD is stabilized by the binding of an exogenous stabilizing ligand, the stabilized DRD-TF can transcribe the 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. These binding sites are found within or near a promoter of a controlled DNA sequence. The promoter containing the specific polynucleotide binding site may be an exogenous promoter. In some embodiments, the promoter may be an exogenous inducible promoter. When incorporated into a transcription factor system containing the protein or payload of interest, the transcription factor binding site or specific polynucleotide binding site is an exogenous nucleic acid sequence.

[0128] In various embodiments of this disclosure, suitable transcription factors useful for the synthesis of transcription factor systems may include any known transcription factors whose transcription factor binding sites are 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 kappa B, HIV-TAT, E2F family, T-Box gene family, helix-loop-helix transcription factors, zinc finger transcription factors, e.g., 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 factors 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. Exemplary transcription factor DNA-binding domains may include one or more DNA-binding domains derived from parental proteins selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL.

[0129] In various embodiments, the transcription factor system provides regulated transcription of the protein of interest or payload (used interchangeably herein). In various embodiments, the nucleic acid sequence encoding the protein of interest is functionally ligated to a specific polynucleotide binding site that specifically binds to the transcription factor DNA-binding domain, i.e., an exogenous inducible promoter containing a defined DNA polynucleotide sequence. The transcription factor-binding domain is then combined with the transcription factor DNA-activating domain to regulate the transcription of the protein of interest.

[0130] When cells or organisms containing DRD-TF are exposed to an exogenous stabilizing ligand, the DRD-TF is stabilized. The stabilized DRD-TF then binds to a specific polynucleotide binding site to which it binds, and thus can control the transcription of the polynucleotide encoding the protein of interest. In some embodiments, the binding of 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. Therefore, both the amount and timing of protein expression can be controlled by administering an exogenous stabilizing ligand to the cell or organism.

[0131] In various embodiments, the transcription factor DNA-binding domain and the transcription factor activation domain may typically be functionally linked or separated by one or more intervening sequences, such as linkers or cleavage sites. In various embodiments, the first polynucleotide may include 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 are functionally linked to the DRD during intracellular expression. In addition, the cell may also include a second polynucleotide containing a fourth nucleic acid sequence that can be specifically bound by the transcription factor DNA-binding domain and a fifth nucleic acid sequence encoding the protein of interest or payload as described herein.

[0132] The transcription factor DNA-binding domain, the transcription factor activation domain, and the target protein or payload may be supplied for the method of this disclosure on the same vector or in separate vectors.

[0133] In some embodiments, the vector comprises a polynucleotide as 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 are functionally ligated to the DRD. Optionally, in some embodiments, the first vector comprises a transcription factor ligated to the DRD, and the second vector comprises a protein of interest or payload functionally ligated 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 to activate transcription, a second nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is functionally ligated to the DRD, and optionally comprises a third nucleic acid sequence encoding a protein of interest functionally ligated to an inducible promoter containing a 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 are functionally ligated to the DRD, and the second vector comprises a third nucleic acid sequence that can be 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 has, for example, an origin of replication (ori) that allows for amplification of the vector in bacteria. Furthermore, or alternatively, the vector may include selectable markers such as antibiotic resistance genes, color marker genes, and suicide genes.

[0135] Drug-responsive domain (DRD) A drug-responsive domain (DRD) is a protein domain that is unstable and degradable 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 attached to a polypeptide or protein, and in the absence of a DRD-binding ligand, it can destabilize the attached polypeptide or protein. DRDs confer their destabilizing properties to the attached polypeptide or protein by proteolysis. While we do not wish to be bound by any theory, in the absence of a DRD-binding ligand, the attached polypeptide or protein is rapidly degraded by the cellular ubiquitin-proteasome system. Ligands that bind to or interact with a DRD can modulate the stability of the attached 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 attached polypeptide or protein can be restored. The conditional stability of DRDs allows for a rapid and unimpeded switch from a stable protein to an unstable substrate intended for degradation. Furthermore, its dependence on ligand concentration provides further controllable control of the degradation rate.

[0136] In some embodiments, the DRDs of the Disclosure may be derived from known polypeptides that are post-translationally controllable. In some embodiments, the DRDs of the Disclosure may be developed from or derived from known proteins. Regions, parts, or domains of wild-type proteins may be used as DRDs, either 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 DRDs or as starting points for the design of further 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 protein sequences used to develop the DRD may include all, part, or regions of the protein sequences listed in Table 3. In some embodiments, the proteins that may be used to develop the DRD may include isoforms of the proteins listed in Table 3.

[0139] hPDE5 DRD In some embodiments, the DRD of the Disclosure is derived from hPDE5. In some embodiments, the DRD of the Disclosure is derived from hPDE5 isoform 2. In some embodiments, the DRD of the Disclosure is derived from hPDE5 isoform 3. In some embodiments, the DRD of the 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: 0.71.

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

[0142] 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), further comprising 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 this disclosure is 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 The mutants may further include one or more mutations independently selected from the group consisting of 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 present disclosure comprises, in whole or in part, a cGMP-specific 3',5'-cyclic phosphodiesterase (hPDE5, SEQ ID NO: 71), further comprising 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 mutation at D764 is selected from D764N and D764A; (ii) an amino acid mutation at position 612 (Y612) of SEQ ID NO: 71, wherein the mutation at Y612 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), further comprising an amino acid mutation at a certain 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 this disclosure is 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 It may contain one or more mutations independently selected from the group consisting of G, 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 this disclosure is 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 include 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 this disclosure may include 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 this disclosure may include 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 include a plurality of 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 human dihydrofolate reductase (hDHFR) proteins, such as, but not limited to, human dihydrofolate reductase 1 (hDHFR1), human dihydrofolate reductase 2 (hDHFR2), or fragments or variants thereof.

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

[0152] In some embodiments, the DRD of this disclosure may include the entire hDHFR (SEQ ID NO: 2). In some embodiments, the DRD derived from hDHFR may include 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 the region or the whole of hDHFR (SEQ ID NO: 2) and further comprises mutations 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 the region or the whole of hDHFR (SEQ ID NO: 2) and further comprises two or more mutations compared to SEQ ID NO: 2.

[0155] In some embodiments, the hDHFR DRD of the present disclosure is (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, Y1 It includes two or more mutations selected from (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 present disclosure is (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) The variant contains two or more mutations selected from (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 present disclosure comprises, in whole or in part, human dihydrofolate reductase (hDHFR, SEQ ID NO: 2), further comprising a Y122I mutation of the amino acid at position 122 (Y122) of SEQ ID NO: 2. In some such embodiments, the DRD further comprises (i) a Q36K mutation of the amino acid at position 36 (Q36) of SEQ ID NO: 2, (ii) an A125F mutation of the amino acid at position 125 (A125) of SEQ ID NO: 2, or (iii) an N65F mutation of the amino acid at position 65 (N65) of SEQ ID NO: 2 and an F or K substitution at 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 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(WT amino acids 2-187)(G21T, Y122I), hDHFR(WT amino acids 2-187)(I17V, Y122I), hDHFR(WT amino acids 2-187)(L74N, Y122I), hDHFR(WT amino acids 2-187)(L94A, T147A), hDHFR(WT amino acids 2-187) (M53T, R138I), hDHFR(WT amino acids 2-187)(N127Y, Y122I), hDHFR(WT amino acids 2-187)(Q36K, Y122I), hDHFR(WT amino acids 2-187)(V121A, Y122I), hDHFR(WT amino acids 2-187)(V75F, Y122I), hDHFR(WT amino acids 2-187)(Y122I, A125F), hDHFR(WT amino acids 2-187)(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(WT amino acids 2-187)(E31D, F32M, V116I), hDHFR(WT amino acids 2-187)(Q36F, N65F, Y122I), hDHFR(WT amino acids 2-187)(Q36F, Y122I, A125F), hDHFR(WT amino acids 2-187)(Y122I, H131R, E144G), hDHFR(V2A, R33G, Q36R, L100P, K185R), hDHFR(D22S, F32M, R33S, Q36S, N65S), hDHFR(WT amino acids 2-187)(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 Includes 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 this disclosure is derived from E. coli dihydrofolate reductase (ecDHFR). In some embodiments, the DRD is derived from the ecDHFR protein and may contain at least one mutation. In some embodiments, the DRD is derived from the ecDHFR protein and may contain two or more mutations. In some embodiments, the DRD is derived from the ecDHFR protein and may contain two, three, four, or five mutations. In some embodiments, the DRD is derived from the ecDHFR protein and may contain at least one mutation selected from Y100I, F103L, and G121V. In some embodiments, the DRD may be derived from the ecDHFR protein and may contain 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 Disclosure is derived from the FK506-binding protein (FKBP) protein or a fragment or variant thereof. In some embodiments, the DRD is derived from the FKBP protein and may contain at least one mutation. In some embodiments, the DRD is derived from the FKBP protein and may contain two or more mutations. In some embodiments, the DRD is derived from the FKBP protein and may contain two, three, four, or five mutations.

[0162] In some embodiments, the DRD of the Disclosure is derived whole or in part from the human FKBP protein (SEQ ID NO: 3) and includes 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 includes F36P, L106P, and two or more mutations selected from E31G, F36V, R71G, and K105E.

[0163] ER DRD In some embodiments, the DRD of the Disclosure is derived from an estrogen receptor (ER) protein or a fragment or variant thereof. In some embodiments, the DRD is derived from the ER protein and may contain at least one mutation. In some embodiments, the DRD is derived from the ER protein and may contain two or more mutations. In some embodiments, the DRD is derived from the 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 the ER (amino acids 305-509 of SEQ ID NO: 6). In some embodiments, the DRD may contain at least one mutation compared to the ligand-binding domain of the ER, the mutation occurring at position 413 (N413) and / or position 502 (Q502). In some embodiments, the mutation is located 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 located at the Q502 position 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 includes mutations at positions N413 and Q502, where the N413 mutation is 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 either N413Y or N413G, and 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, ER DRD comprises at least two mutations, N413T, Q502H or N413D, Q502H.

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

[0167] In some embodiments, the DRD of this disclosure includes: ER(WT aa305~549, L384M, N413F, M421G, G521R, Y537S), ER(WT aa305~549, L384M, N413L, M421G, G521R, Y537S), ER(WT aa305~549, L384M, N413Y, M421G, G521R, Y537S), ER(WT aa305~549, L384M, N413H, M421G, G521R, Y537S), ER(WT aa305~549, 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 aa305~549, L384M, N413C, M421G, G521R, Y537S), ER (WT aa305~549, L384M, N413W, M421G, G521R, Y537S), ER (WT aa305~549, L384M, N413P, M421G, G521R, Y537S), ER (WT aa305~549, L384M, N413R, M421G, G521R, Y537S), ER (WT aa305~549, L384M, N413T, M421G, G521R, Y537S), ER (WT aa305~549, L384M, N413T, M421G, G521R, Y537S), ER (WT aa305~549, 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 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 contain at least one mutation. In some embodiments, the DRD may be derived from the hCA2 protein and contain two or more mutations. In some embodiments, the DRD may be derived from the hCA2 protein and contain two, three, four, or five mutations.

[0169] In some embodiments, the DRD of this disclosure may be derived from amino acids 1-260 of CA2 (SEQ ID NO: 5). In some embodiments, the DRD is derived from CA2 containing 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 contain 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 or the whole of human carbonic anhydrase 2 (CA2, SEQ ID NO: 0.5) and further comprises mutations selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T compared to SEQ ID NO: 0.5.

[0171] In some embodiments, the DRD of the present disclosure includes the region or the whole of human carbonic anhydrase 2 (CA2, SEQ ID NO: 0.5), compared to SEQ ID NO: 0.5, 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, C20 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 * The following further include mutations selected from Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, and S29A. When used herein, 「*」 The symbol indicates the translation of a stop codon, and X represents any amino acid.

[0172] In some embodiments, the DRD of the present disclosure comprises a region or the whole of human carbonic anhydrase 2 (CA2, SEQ ID NO: 0.5) and further comprises two or more mutations compared to SEQ ID NO: 0.5.

[0173] In some embodiments, the DRD of the present disclosure is CA2(WT aa2~260, R27L, H122Y), CA2(WT aa2~260, T87I, H122Y), CA2(WT aa2~260, H122Y, N252D), CA2(WT aa2~260, D72F, V241F), CA2(WT aa2~260, V241F, P249L), CA2(WT aa2~260, D72F, P249L), CA2(WT aa2~260, D71L, L250R), CA2(WT aa2~260, D72F, P249F), C A2 (WT aa2~260, 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 aa 2~260, D70N, D74N, D100N, L156H), (CA2 (WT aa2~260, I59N, G102R), CA2 (WT aa2~260, G63D, E69V, N231I), CA2 (WT aa2~260, R27L, T87I, H122Y, N252D), CA2 (WT aa2~260, D72F, V241F, P249L), CA2 (WT aa2~260, D71L, T87N, L250R), CA2 (WT aa2~260, L156H, S172C, F178Y, E186D), CA2 (WT a Includes a2~260, A77I, P249F), CA2 (WT aa2~260, E106D, C205S), CA2 (WT aa2~260, C205S, W208S), CA2 (WT aa2~260, S73N, R89Y), CA2 (WT aa2~260, D71K, T192F), CA2 (WT aa2~260, S73N, R89F), CA2 (WT aa2~260, G63D, M240L), CA2 (WT aa2~260, V134F, L228F), or CA2 (WT aa2~260, S56F, D71S).

[0174] In some embodiments, the DRD of the present disclosure is CA2(WT aa2~260, R27L, H122Y), CA2(WT aa2~260, T87I, H122Y), CA2(WT aa2~260, H122Y, N252D), CA2(WT aa2~260, D72F, V241F), CA2(WT aa2~260, V241F, P249L), CA2(WT aa2~260, D72F, P249L), CA2(WT aa2~260, D71L, L250R), CA2(WT aa2~260, D72F, P249F), CA2(WT aa2 ~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(WT aa2~260, I59N, G102R), CA2(WT aa2~260, G63D, E69V, N231I), CA2(WT aa2~260, R27L, T87I, H122Y, N252D), CA2(WT aa2~260, D72F, V241F, P249L), CA2(WT aa2~260, D71L, T87N, L250R), CA2(WT aa2~260, L156H, S172C, F178Y, E186D), CA2(WT aa2~260, D71F, N231F), CA2(WT aa 2~260, A77I, P249F), CA2 (WT aa2~260, D71K, P249H), CA2 (WT aa2~260, D72F, P249H), CA2 (WT aa2~260, Q53N, N61Y), CA2 (WT aa2~260, E106D, C205S), CA2 (WT aa2~260, C205S, W208S), CA2 (WT aa2~260, S73N, R89Y), CA2 (WT aa2~260, D71K, T192F), CA2 (WT aa2~260, Y193L, K260L), CA2 (WT aa2~260,D71F, V241F, P249L), CA2 (WT aa2~260, L147F, Q248F), CA2 (WT aa2~260, D52I, S258P), CA2 (WT aa2~260, D72S, T192N), CA2 (WT aa2~260, D179E, T192I), CA2 (WT aa2~260, S56N, Q103K), CA2 (WT aa2~260, D71Y, Q248L), CA2 (WT aa2~260, S73N, R89F), CA2 (WT aa2~260, D71K, N231L, E235G, L239F), CA 2 (WT aa2~260, D72F, P249I), CA2 (WT aa2~260, D72X, V241X, P249X), CA2 (WT aa2~260, A54X, S56X, L57X, T192X), CA2 (WT aa2~260, Y193V, K260F), CA2 (WT aa2~260, G63D, M240L), CA2 (WT aa2~260, V134F, L228F), CA2 (WT aa2~260, D71G, N231K), CA2 (WT aa2~260, S56F, D71S), CA2 (WT aa2~260, D52 L, G128R, Q248F), CA2 (WT aa2~260, S73X, R89X), CA2 (WT aa2~260, Y51X, D72X, V241X, P249X), CA2 (WT aa2~260, D72I, W97C), CA2 (WT aa2~260, D71K, T192F, N231F), CA2 (WT aa2~260, H36Q, S43T, Y51F, N67D, G131W, R226H), CA2 (WT aa2~260, F70I, F146V), CA2 (WT aa2~260, K45N, V68L, H119Y, K169R , D179E), CA2 (WT aa2~260, H15L, A54V, K111E, E220K, F225I), CA2 (WT aa2~260, P13S, P83A, D101G, K111N, F230I), CA2 (WT aa2~260, G63D, W123R, E220K), CA2 (WT aa2~260, N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S), CA2 (WT aa2~260, I59N, G102R, A173T), CA2 (WT aa2~260, L79F, P180S),CA2 (WT aa2~260, A77P, G102R, D138N), CA2 (WT aa2~260, F20L, K45N, G63D, E69V, N231I), CA2 (WT aa2~260, T199N, L202P, L228F), CA2 (WT aa2~260, K9N, H122Y, T168K), CA2 (WT aa2 ~260, Q53H, L90V, Q92H, G131E), CA2 (WT aa2~260, L44M, L47V, N62K, E69D), CA2 (WT aa2~260, D75V, K169N, F259L), CA2 (WT aa2~260, T207S, V222A, N231D), CA2 (WT aa2~260, I59F, V 206M, G232R), CA2 (WT aa2~260, P13A, A133T), CA2 (WT aa2~260, I59N, R89I), CA2 (WT aa2~260, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L), CA2 (WT aa2~260, G12R, H15Y) Includes D19V), CA2 (WT aa2~260, A65V, F95Y, E106G, H107Q, I145M, F175I), CA2 (WT aa2~260, G63D, E69V, N231I), CA2 (WT aa2~260, S29A, C205S) and / or CA2 (WT aa2~260, S29C, C205S).

[0175] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 0.5), further comprising an H122Y mutation of the amino acid at position 122 (H122) of SEQ ID NO: 0.5. In some such embodiments, the DRD further comprises (i) an R27L mutation of the amino acid at position 27 (R27) of SEQ ID NO: 0.5, (ii) a T87I mutation of the amino acid at position 87 (T87) of SEQ ID NO: 0.5, (iii) an N252D mutation of the amino acid at position 252 (N252) of SEQ ID NO: 0.5, or a combination of (i), (ii), and / or (iii).

[0176] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 0.5), further comprising an E106D mutation of the amino acid at position 106 (E106) of SEQ ID NO: 0.5. In some such embodiments, the DRD further comprises a C205S mutation of the amino acid at position 205 (C205) of SEQ ID NO: 0.5.

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

[0178] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 0.5), further comprising an I59N mutation of the amino acid at position 59 (I59) of SEQ ID NO: 0.5. In some such embodiments, the DRD further comprises a G102R mutation of the amino acid at position 102 (G102) of SEQ ID NO: 0.5.

[0179] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 5), further comprising an L156H mutation of the amino acid at position 156 (L156) of SEQ ID NO: 5. In some such embodiments, the DRD further comprises (i) a W4Y mutation of the amino acid at position 4 (W4) of SEQ ID NO: 5, (ii) an F225L mutation of the amino acid at position 225 (F225) of SEQ ID NO: 5, (iii) a deletion of the amino acid at positions 257-260 of SEQ ID NO: 5, (iv) a deletion of the amino acid at positions 1-5 of SEQ ID NO: 5, or (v) a deletion of the 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: 0.5), further comprising four mutations compared to SEQ ID NO: 0.5, 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), further comprising a first mutation and a second mutation compared to SEQ ID NO: 5, wherein (i) the first mutation is an S73N mutation of the amino acid at position 73 (S73) of SEQ ID NO: 5, and (ii) the second mutation is an F or Y substitution at position 89 (R89) of the amino acid of SEQ ID NO: 5.

[0182] In some embodiments, the DRD of the present disclosure comprises, in whole or in part, human carbonic anhydrase 2 (CA2, SEQ ID NO: 0.5), further comprising an N or F substitution at amino acid position 56 (S56) of SEQ ID NO: 0.5. In some such embodiments, the DRD comprises two substitutions corresponding to S56F and D71S compared to SEQ ID NO: 0.5.

[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), further comprising 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 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: 0.5), further comprising two or more substitutions compared to SEQ ID NO: 0.5, one of which is an L or K substitution at amino acid position 71 (D71) of SEQ ID NO: 0.5, and the two or more substitutions correspond 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), further comprising two or more substitutions compared to SEQ ID NO: 5, where 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, where 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: 0.5) and further comprises one or more substitutions selected from Y51T, L183S, Y193I, L197P and combinations of V134F and L228F compared to SEQ ID NO: 0.5.

[0187] The amino acid sequences of the DRDs included in this disclosure have at least about 70% identity with the amino acid sequences of the parental proteins from which they are derived, 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. In some embodiments, the amino acid sequence of the DRDs included in this 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 of the parent protein from which it is derived (e.g., a parent protein having any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 71).

[0188] Examples of DRDs of this disclosure include those derived from human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. Preferred DRDs may be referred to as destabilizing domains or ligand-binding domains, which are also known in the art. For example, WO2018 / 161000, WO2018 / 231759, WO2019 / 241315, US8,173,792, US8,530,636, WO201 8 / 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 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-responsive domain (DRD), and the transcription factor (e.g., a transcription factor DNA-binding domain, a transcription factor activation domain, or both) is functionally linked to the DRD. The nucleic acid sequence encoding the DRD may be selected from the DRD sequences described herein. Constructs containing DRD sequences are provided in Table 1 above. Additional constructs containing different DRDs are provided in Table 4. (Asterisks in Table 4)「*」 ) indicates the 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 the transcription factor system The transcription factor system of this disclosure is capable of responding to stimuli.

[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 thereof. 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 may exhibit ligand-dependent activity. Ligands may bind to DRDs and stabilize the transcription factor or domain of a transcription factor encoded by the transcription factor system. Ligands known to bind to candidate DRDs may be tested for their effects 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 as being safe and having appropriate pharmacokinetics and distribution.

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

[0196] CA2 ligand In some embodiments, the ligands of this disclosure bind to carbonic anhydrase. In 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, metazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxyzolamide, zonisamide, dansylamide, and dichlorfenamide.

[0198] In some embodiments, the ligand may comprise a subset of small molecules 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 stimulant may be a ligand that binds to two or more carbonic anhydrases. In one embodiment, the stimulant is a pancarbonate inhibitor that may bind to two or more carbonic anhydrases.

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

[0201] In some embodiments, the ligand may be a selective inhibitor of human DHFR. The ligands of this disclosure may also be selective inhibitors of dihydrofolate reductases from bacteria and parasites, such as species Pneumocystis, Toxoplasma, Trypanosoma, Mycobacterium, and Streptococcus. Other DHFR-specific ligands 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, pyritrexime, pyrimethamine, talotrexin, chloroguanide, pentamidine, trimethrexate, aminopterin, C1 898 trihydrochloride, pemetrexed disodium, larcitrexed, sulfaguanidine, Folotyn, ikuraprim, and diaveridine.

[0203] In some embodiments, the ligand of the Disclosure may include any of the dihydrofolate or its derivatives that may bind to human DHFR. In some embodiments, the ligand of the Disclosure may be a 2,4,diaminoheterocyclic compound. In some embodiments, the 4-oxo group in dihydrofolate may be modified to produce a DHFR inhibitor. In one example, the 4-oxo group may be replaced with a 4-amino group. Various diaminoheterocyclic compounds, 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 Disclosure.

[0204] In some embodiments, the ligand includes a TMP-derived ligand that contains a subset of ligands known to mediate binding to DHFR. The ligand may also be modified to reduce off-target binding to other folate metabolizing enzymes and increase specific binding to DHFR.

[0205] ER ligand In some embodiments, the ligands of this disclosure bind to the ER. The ligands may be agonists or antagonists. In some embodiments, the ligands bind to the 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 this disclosure may also be selective inhibitors of other species of ER. Ligands specific to other ERs may be modified to improve binding to human ER.

[0206] The ligand may be an ER agonist, for example, 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, for example, ICI-164,384, RU486, tamoxifen, 4-hydroxytamoxifen (4-OHT), fulvestrant, toremifene, rasofoxifen, clomiphene, femarelle, and olmeroxifen and raloxifen (RAL).

[0207] In some embodiments, the stimulus of this disclosure may be an ER antagonist, for example, but not limited to, bazedoxifene and / or raloxifene.

[0208] In some embodiments, the ligand includes a bazedoxifen-derived ligand that contains a subset of ligands 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 ER-derived DRDs.

[0209] Phosphodiesterase ligand In some embodiments, the ligands of this disclosure bind to phosphodiesterases. In 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 an hPDE5 inhibitor. Examples of hPDE5 inhibitors include, but are not limited to, sildenafil, vardenafil, tadalafil, avanafil, rodenafil, mirodenafil, udenafil, benzamidenafil, dasantafil, beminafil, SLx-2101, LAS34179, UK-343,664, UK-357903, UK-371800, and BMS-341400.

[0211] In some embodiments, the ligand includes a sildenafil-derived ligand that contains a subset of ligands 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 stimulant may be a ligand that binds to two or more phosphodiesterases. In one embodiment, the stimulant is a panphosphodiesterase inhibitor that can bind to two or more hPDEs, such as aminophylline, paraxanthine, pentoxifylline, theobromine, dipyridamole, theophylline, zaprinast, icariin, CDP-840, ethazolate, 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 this disclosure binds to FKBP, including human FKBP. In some embodiments, the ligand is SLF or Shield-1.

[0215] payload The payload may include any polypeptide or any protein or a fragment thereof. The payload may include a wild-type sequence, a fragment of a wild-type sequence, and / or one or more mutations. The payload may be a native protein derived from an organism's genome, or a variant, mutant, and derivative thereof. Native proteins may be derived, for example, from 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 may be a polypeptide encoded by the nucleic acid sequence of the human genome.

[0217] In some embodiments, the payload may be a variant sequence of the parent polypeptide. In some embodiments, 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, variants 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 with that particular reference polypeptide, but less than 100%, as determined by sequence alignment programs known to those skilled in the art.

[0218] Therapeutic agent as payload In some embodiments, the payload of the Disclosure may be a therapeutic agent. For example, the payload may be a cancer treatment agent, a treatment agent for autoimmune diseases, an immunotherapy agent, an anti-inflammatory agent, an anti-pathogen agent, or a gene therapy agent. In some embodiments, the immunotherapy agent may be an antibody and its fragments and variants, 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 costimulatory molecule, a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a metabolic factor, a coagulation factor, an enzyme, a homing receptor, or a safety switch.

[0219] In some embodiments, the payload of the present disclosure may be an immunotherapy agent that induces an immune response in a living organism. The immunotherapy agent may be, but is not limited to, antibodies and their fragments and variants, TCRs, chimeric antigen receptors (CARs), chimeric switch receptors, cytokines, chemokines, cytokine receptors, chemokine receptors, cytokine-cytokine receptor fusion polypeptides, or any agent that induces an immune response. In one embodiment, the immunotherapy agent induces an anti-cancer immune response in cells or subjects.

[0220] Cytokines, chemokines, and other soluble factors as payloads In some embodiments, the payload 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 functions that eliminate tumor cells.

[0221] In some embodiments, the payload of the present disclosure may be cytokines, as well as fragments, variants, analogs, and derivatives thereof, and may include, but are not limited to, interleukins, tumor necrosis factor (TNF), interferons (IFN), TGF-beta, and chemokines. In some embodiments, the payload of the present invention may be cytokines that stimulate an immune response. In other embodiments, the payload of the present invention may be cytokine antagonists that negatively affect the anti-cancer immune response.

[0222] In some embodiments, the payload of the Disclosure may be a cytokine receptor, its recombinant receptor, variant, analogue and derivative, or a signaling component of a cytokine. In various embodiments, the payload of the Disclosure may include a secreted cytokine or a membrane-bound form of a cytokine. Exemplary examples of membrane cytokines include transmembrane domains, e.g., CD8α transmembrane domain, B7-1 transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, or cytokines functionally fused, linked or bound to the human IgG4 Fc region (e.g., immunostimulatory cytokines, e.g., IL12, IL2, IL15 and IL18). In various embodiments, the cytokine may be fused or bound 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 the TNF alpha extradomain. Such a payload is produced as a membrane-associated cytokine fused to the TNF extradomain. In one embodiment, the cytokine may be detached from the cell surface by the action of a membrane-associated protease and / or an extracellular space protease, such as MMP9.

[0224] In some embodiments, the payload of the present disclosure may be an interleukin (IL) cytokine. Interleukins (ILs) are a class of glycoproteins produced by leukocytes to regulate the immune response. 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 Disclosure may include IL12. IL12 is a heterodimer protein consisting of two subunits (p35, p40) secreted by antigen-presenting cells such as macrophages and dendritic cells. IL12 expression requires the simultaneous expression of both subunits to produce a biologically active heterodimer. In some embodiments, the payload of the Disclosure may be the p35 subunit or the p40 subunit.

[0226] In some embodiments, the payload of this disclosure may include the entirety or a portion of IL12.

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

[0228] The format of the IL12 payload in this disclosure may be optimized. In one embodiment, the payload may be bicistronic IL12 containing p40 and p35 subunits separated by an internal ribosome entry site or a cleavage site such as P2A or furin, in order to enable 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 bound to the membrane by a transmembrane domain. The transmembrane domain may also contain an optional hinge domain. In some embodiments, the IL12 molecule is extracellular and ligated to the cell by the transmembrane domain. In some embodiments, the membrane-bound IL12 may be detached or cleaved from the cell surface by the action of a protease. In some embodiments, the transmembrane domain of this disclosure may originate from either a natural or synthetic source. The transmembrane domain may originate from any natural membrane-binding protein or transmembrane protein. Alternatively, the transmembrane domain of this disclosure may be synthetic. In some embodiments, the synthetic sequence may contain primarily hydrophobic residues such as leucine and valine. In some embodiments, transmembrane and / or hinge domains resistant to protease activity may be selected.

[0230] In some embodiments, the payload of the present disclosure may include IL-15. Interleukin-15 is a potent immunostimulatory cytokine and an essential survival factor for T cells and natural killer cells.

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

[0232] In some cases, IL15, either entirely or partially, is ligated to one or more transmembrane proteins, either entirely or partially.

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

[0234] A distinctive 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) that binds to and activates the membrane-bound IL15 beta / gamma receptor, either on the same cell or on different cells. In some embodiments, the payload of the Disclosure is membrane-bound IL15. In some embodiments, the payload of the Disclosure may comprise an IL15 / IL15Ra fusion polypeptide. In some embodiments, the payload may be whole or a portion of IL15 fused to whole or a portion of IL15Ra. Any portion of IL15 and IL15Ra, either full-length or mature, retaining one or more functions of each, may be used.

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

[0236] The membrane-bound IL15 or IL15 / IL15Ra fusion polypeptides of this disclosure, in whole or in part, may be detached into the extracellular space. Detachment, as used herein, refers to the release of membrane-related biomolecules from the membrane to which they are tethered. In some cases, detachment may be induced by proteolytic cleavage.

[0237] The payload of this disclosure may include 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 Disclosure may be used to improve the proliferation, survival, persistence, and efficacy of immune cells, such as CD8+ TEMs, natural killer cells, and tumor-infiltrating lymphocytes (TILs), as well as CAR T cells used in immunotherapy. In one embodiment, the Disclosure provides a payload for minimizing toxicity associated with cytokine therapy. In some embodiments, the payloads of the Disclosure may include all or part of IL2. Any part of IL2 that retains one or more functions of full-length or mature IL2 may be useful in the Disclosure.

[0239] In the art, it is understood that certain gene and / or protein nomenclature for the same gene or protein may or may not include punctuation marks such as dashes "-" or symbols such as Greek letters. Whether they are included or excluded herein, their meaning is not intended to be altered to be understood by those skilled 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 payload of the present disclosure may be an antibody, an antibody fragment, or a variant 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 the purposes of this specification, the “antibody” may include an Fc region in addition to the heavy chain and light chain variable domains.

[0243] In some embodiments, the payload may be a monoclonal antibody. As used herein, the term “monoclonal antibody” means an antibody obtained from a substantially homogeneous population of cells (or clones), i.e., each antibody contained in that population binds to the same and / or same epitope, excluding possible variants that may arise during the production of the monoclonal antibody, such as variants that are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen.

[0244] In one embodiment, the payload of the Disclosure may be a humanized antibody. As used herein, the term “humanized antibody” refers to a chimeric antibody comprising a minimum portion from one or more non-human (e.g., mouse) antibody sources and the remainder derived from one or more human immunoglobulin sources. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues in the hypervariable region of the recipient antibody are replaced by residues in the hypervariable region of an antibody (donor antibody) from a non-human species such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capabilities. In one embodiment, the antibody may be a humanized full-length antibody.

[0245] As used herein, the term “antibody variant” refers to a modified antibody (related to a native or starting antibody) or a biomolecule (e.g., an antibody mimetic) whose structure and / or function are similar to a native or starting antibody. Antibody variants may have altered amino acid sequences, composition, or structure compared to native antibodies. 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 include the antigen-binding region of an intact antibody. Examples of antibody fragments and variants include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments. Papain digestion of the antibody produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site. A residual "Fc" fragment is also produced, its name reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still being crosslinkable with an antigen. The payload of this disclosure may include one or more of these fragments.

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

[0248] Chimeric antigen receptor payload In some embodiments, the payload of this 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 T cells. Generally, a CAR consists 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 molecules that can be recognized by the targeting portion of the CAR. In a typical CAR receptor, the components—extracellular targeting domain, transmembrane domain, and intracellular signaling / activation domain—are constructed linearly as a single fusion protein. The extracellular region includes a targeting domain / region (e.g., scFv) that recognizes a specific tumor antigen or other tumor cell surface molecule. The intracellular region may include the signaling domain of the TCR complex (e.g., the signaling region of CD3ζ), as well as / or one or more co-stimulatory signaling domains, e.g., those derived from CD28, 4-1BB (CD137), and OX-40 (CD134). For example, while "first-generation CARs" only possess a CD3ζ signaling domain, a costimulatory intracellular domain is added to enhance the persistence and proliferation of T cells, resulting in second-generation CARs having a CD3ζ signaling domain and one costimulatory signaling domain, and third-generation CARs having a CD3ζ signaling domain and two or more costimulatory signaling domains. When expressed by T cells, CARs confer antigen specificity to T cells, which is determined by the extracellular targeting portion of the CAR. Fourth-generation CARs involve the addition of one or more elements, such as homing genes and suicide genes, to develop a more effective and safer CAR structure.

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

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

[0251] In some embodiments, the payload of the Disclosure may be a component of a gene editing system. In some embodiments, the payload of the Disclosure may be a Cas protein (CRISPR-related protein) including Cas9 and Cas12. The Cas protein may be altered or otherwise modified. For example, the Cas protein may be dead Cas9. In some embodiments, the Cas9 protein may be an enzymatically active Cas9 protein, a wild-type Cas9 protein, a Cas9 protein nickase, or a nuclease null or nuclease-deficient Cas9 protein. In some embodiments, the payload of the Disclosure may be a zinc finger nuclease, a TALEN (transcription activator-like effector-based nuclease), and 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 Disclosure may be a drug for treating, improving or preventing an autoimmune disorder.

[0254] In some embodiments, the payload of the Disclosure includes anti-cytokines such as neutralizing antibodies against tumor necrosis factor (TNF)-α, IL-1, and IL-6. In some embodiments, the payload of the Disclosure targets B cell depletion and includes neutralizing antibodies against CD20, CD22, CD28, CTLA-4, and B lymphocyte-stimulating factor (BLyS).

[0255] Pharmaceutical compositions and preparations This instruction further includes a pharmaceutical composition comprising one or more of the transcription factor systems, nucleic acids, polynucleotides, modified cells, or payloads of this disclosure, and optionally at least one pharmaceutically acceptable excipient or inactive component.

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

[0257] The terms "excipient" or "inactive ingredient" refer to inert or inactive substances added to a pharmaceutical composition to further facilitate the administration of a compound.

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

[0259] The descriptions of pharmaceutical compositions provided herein primarily concern pharmaceutical compositions suitable for administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animals, such as non-human animals, such as non-human mammals. Targets to which the pharmaceutical compositions are intended include, but are not limited to, agricultural animals such as cattle, horses, chickens, and pigs; domestic animals such as cats and dogs; or research animals such as mice, rats, rabbits, dogs, and non-human primates, as well as non-human mammals.

[0260] The pharmaceutical compositions described herein may be prepared, packaged, and / or sold in bulk as single unit doses and / or as multiple single unit doses. As used herein, “unit dose” refers to a specific amount of a pharmaceutical composition containing a given amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or an appropriate fraction of such a dose, for example, half or one-third of such a dose.

[0261] The relative amounts of the active ingredient, pharmaceutically acceptable excipients or inactive ingredients, and / or any additional ingredients in the pharmaceutical compositions according to this disclosure will vary depending on the identifying information, size, and / or state of the object being treated, and further depending on the route through which the composition is administered. For example, a composition may contain 0.1% to 100%, e.g., 0.5 to 50%, 1 to 30%, 5 to 80%, or at least 80% (w / w) of the active ingredient.

[0262] The effectiveness of a treatment or improvement of a disease may be evaluated, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dose of the drug required to maintain the treatment effect, levels of disease markers, or any other measurable parameters appropriate to a given disease being treated or targeted for prevention. A healthcare professional skilled in the art may monitor the effectiveness of a treatment or prevention by measuring any one of such parameters or any combination of parameters. In relation to the administration of the compositions of this disclosure, for example, "effective against cancer" indicates that administration in a clinically appropriate manner results in beneficial effects in at least a significant proportion of patients, such as improvement of symptoms, cure, reduction of disease burden, reduction of tumor mass or cell number, extension of lifespan, improvement of quality of life, or other effects generally recognized as positive by a physician familiar with the treatment of a particular type of cancer.

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

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

[0265] In one embodiment, the polynucleotide and vector formulation is a nanoparticle which 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 DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, and DODMA, but is not limited to these.

[0266] With respect to the polynucleotides of this disclosure, formulations may be selected from, for example, those taught in international application PCT / US2012 / 069610.

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

[0268] Drug administration, delivery, and administration The compositions of this disclosure may be delivered to cells or subjects by one or more routes and modalities. Viral vectors containing one or more 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 Unpackaged delivery The pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads of this disclosure may be delivered to cells, tissues, organs and / or organisms in a naked form. As used herein, the term “naked” means a pharmaceutical composition, transcription factor system, nucleic acid, polynucleotide, or payload delivered without any agents or modifications that promote transfection or permeability. Naked pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads are known in the art and may be delivered to cells, tissues, organs and / or organisms using the administration routes described herein. In some embodiments, naked delivery may include formulation in a simple buffer such as saline or PBS.

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

[0271] Pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads may also be formulated for direct delivery to organs or tissues by any of several methods in the art, including, but not limited to, direct immersion or bathing, via catheters, by means of using a substrate such as cloth or biodegradable material coated with or impregnated with the composition, by gel, powder, ointment, cream, lotion, and / or infusion.

[0272] Delivery to cells In another aspect of this disclosure, a transcription factor system or its components, and a vector comprising the compositions and polynucleotides of this disclosure, may be introduced into cells such as immune effector cells.

[0273] In one aspect of this disclosure, the polynucleotides of the transcription factor system or its components and the compositions of this disclosure may be packaged in a plasmid, viral vector, or incorporated into a viral genome, enabling transient or stable expression of the polynucleotides. Preferred viral vectors are retroviral vectors, including lentiviral vectors and gamma retroviral vectors. To construct a retroviral vector, polynucleotide molecules of the transcription factor system are inserted into the viral genome in place of certain viral sequences to produce a replication-deficient virus. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components. The recombinant retroviral particles are secreted into a culture medium, then collected and optionally concentrated for gene transfer. Lentiviral vectors are particularly preferred because they can infect both dividing and non-dividing cells.

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

[0275] In some embodiments, the polypeptides of the Disclosure may be delivered directly to cells. In one embodiment, the polypeptides of the Disclosure may be delivered using a synthetic peptide comprising an endosomal leakage domain (ELD) fused to a cell permeable domain (CLD). The polypeptides of the Disclosure are co-introduced into cells together with the ELD-CLD-synthetic peptide. The ELD facilitates the escape of proteins trapped in endosomes into the cytosol. Such domains are derived from proteins of microbial and viral origin and have been described in the art. CPDs enable the transport of proteins across the plasma membrane, and these 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 a shuttle construct as an additional method to enable cargo escape from endosomes to the cytosol. The shuttle may also contain a cysteine ​​residue at the N or C terminus to generate a multimer of the fusion peptide. The polymers of ELD-CLD fusion peptides produced by adding cysteine ​​residues to the peptide terminus exhibit even higher transduction efficiency compared to single fusion peptide constructs. The polypeptides of this disclosure may also be modified with appropriate localization signals to direct the cargo to an appropriate intracellular 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 this disclosure (the entire contents of each of them are incorporated herein by reference).

[0276] Delivery modalities and / or vectors The transcription factor systems or components of the present disclosure may be delivered using one or more modalities. The present disclosure also provides vectors for packaging the polynucleotides of the present disclosure that encode transcription factors and parts thereof, DRDs, or payload constructs, and combinations thereof. The vectors of the present disclosure may also be used to deliver the packaged polynucleotides to cells, local tissue sites, or targets. These vectors may be of any kind, including DNA vectors, RNA vectors, plasmids, viral vectors, and particles. Viral vector technology is well known and is described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Useful viruses as vectors include, but are not limited to, adenoviruses, adeno-associated viruses (AAVs), alphaviruses, flaviviruses, herpesviruses, measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease viruses (NDVs), poxviruses, and picornaviruses. In some embodiments, the virus is selected from lentiviral vectors, gamma retroviral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

[0277] Generally, a vector contains a replication origin that functions in at least one organism, a promoter sequence and a convenient restriction endonuclease site, as well as one or more selectable markers, such as a drug resistance gene.

[0278] In some embodiments, the recombinant expression vector may include regulatory sequences such as transcription and translation start and stop codons, which are specific to the host cell type into which the vector is introduced.

[0279] In some embodiments, the vectors of this disclosure may include one or more payloads taught herein, and two or more payloads may be included in a single ligand response. In this case, the two or more payloads are simultaneously modulated by the same ligand or responsive agent.

[0280] Lentivirus vehicle / particle In some embodiments, lentiviral vehicles / particles may be used as a delivery modality. Lentiviruses are a subgroup of viruses in the family Retroviridae, named for the fact that their viral RNA genome must be reverse-transcribed into DNA before it can be incorporated into the host genome. Therefore, the most important feature of lentiviral vehicles / particles is the integration of 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), Bisna-Maedi virus, and canine arthritis encephalitis virus (CAEV).

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

[0282] Lentiviral particles may be produced by co-expressing the viral packaging elements and the vector genome itself in a producing cell, such as human HEK293T cells. These elements are usually provided in three or four separate plasmids. The producing cell is co-transfected with a plasmid encoding lentiviral components, including the viral core (i.e., structural proteins) and enzymatic components, as well as envelope proteins (plural) (referred to as the packaging system), and a plasmid containing the transgene to be transferred into the target cell, the vehicle itself (also referred to as the transvector). Generally, the plasmid or vector is contained within the producing cell line. The plasmid / vector is introduced into the producing cell line via transfection, transduction, or infection. Methods of transfection, transduction, or infection are well known to those skilled in the art. As a non-limiting example, the packaging and introduction constructs may be introduced into a producing cell line by calcium phosphate transfection, lipofection, or electroporation, generally along with major selectable markers such as neo, DHFR, Gln synthetase, or ADA, followed by selection and isolation of clones in the presence of appropriate drugs.

[0283] The producing cells produce recombinant viral particles containing exogenous genes, such as the transcription factor system components of this disclosure or their polynucleotides. 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 may be used to produce high-titer lentiviral particles include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol.Ther., 2005, 11:452-459), FreeStyle® 293 Expression System (ThermoFisher, Waltham, MA), and other HEK293T-based producing cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 22(3):357-369, Lee et al., Biotechnol Bioeng, 2012, 10996):1551-1560, Throm et al., Blood. 2009, 113(21):5104-5110, the entire contents of each of these are incorporated herein by reference).

[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 effective against species classified as belonging to the genus vesiculovirus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV), and Vesicular stomatitis New Jersey virus (VSNJV), and / or Grass Gorgonirhud virus, 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. The strains may be selected from among those provisionally classified in the vesiculovirus genus as vesiculovirus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Koi spring viremia virus (SVCV), Tupaia virus (TUPV), ulcerative disease rhabdovirus (UDRV), and Yug Bogdanovac virus (YBV).gp64 or other baculovirus env proteins may originate 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 derived from gibbon leukemia virus (GaLV) or baby retrovirus envelope glycoprotein (BaEV).

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

[0287] Methods for generating recombinant lentivirus particles are described in the art, for example, in U.S. Patents 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 polynucleotides of any of the transcription factor systems, transcription factor constructs, or payload constructs of this disclosure may be achieved using recombinant adeno-associated virus (rAAV) vectors. Such vectors or viral particles may be designed to utilize any or a combination of known 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 enables rapid expression within cells.

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

[0292] The transcription factor constructs and payload constructs may be encoded by one or more viral genomes packaged in the AAV capsid as taught herein.

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

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

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

[0296] Retrovirus vehicle / particle (γ-retrovirus vector) In some embodiments, retroviral vehicles / particles may be used to deliver the transcription factor systems, transcription factor constructs, or payload constructs of this disclosure. Retroviral vectors (RVs) enable the permanent integration of transgenes into target cells. In addition to compound HIV-1 / 2-based lentiviral vectors, simple gamma-retrovirus-based retroviral vectors are widely used to deliver therapeutic genes and have been clinically demonstrated as one of the most efficient and potent gene delivery systems capable of transducing a wide range of cell types. Exemplary species of gamma-retroviruses include mouse leukemia virus (MLV) and feline leukemia virus (FeLV).

[0297] In some embodiments, gamma-retroviral vectors derived from mammalian gamma-retroviruses, such as mouse leukemia virus (MLV), are recombinant. The MLV family of gamma-retroviruses includes the ecotropic, amphotropic, xenotropic, and polytropic subfamilies. Ecotropic viruses can infect only mouse cells using the mCAT-1 receptor. Examples of ecotropic viruses are Moloney's 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 utilize the same (Xpr1) receptor, but their species-specification differs. Xenotropic viruses, such as NZB-9-1, infect humans and other species but not mouse species, while polytropic viruses, such as focus-forming viruses (MCF), infect mice, humans, and other species.

[0298] The gamma-retroviral vector may be produced in a packaging cell by co-transfecting the cell with several plasmids, including a plasmid encoding the retroviral structure and enzyme (gag-pol) polyprotein, a plasmid encoding the envelope (env) protein, and a plasmid encoding vector mRNA containing polynucleotides encoding the composition of the Disclosure, which is packaged within a newly formed viral particle.

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

[0300] In some embodiments, the recombinant gamma-retroviral vector is a self-inactivated (SIN) gamma-retroviral vector. The vector is dysfunctional in replication. The SIN vector may initially have a deletion within the 3'U3 region containing enhancer / promoter activity. Furthermore, the 5'U3 region may be replaced with a strong promoter (required for packaging cell lines) derived from cytomegalovirus or RSV, or a suitable internal promoter and / or enhancer element. The selection of the internal promoter may be made in accordance with the specific requirements of gene expression necessary for the particular purposes of this disclosure.

[0301] In some embodiments, polynucleotides of transcription factor systems, transcription factor constructs, or payload constructs are inserted into the recombinant viral genome. Other components of the viral mRNA of the recombinant gamma-retroviral vector may be modified by insertion or removal of naturally occurring sequences (e.g., insertion of IRESs, insertion of heterologous polynucleotides encoding a polypeptide or inhibitory nucleic acid of interest, shuffling a wild-type promoter with a more effective promoter of a different retrovirus or virus). In some examples, the recombinant gamma-retroviral vector may include a modified packaging signal and / or primer-binding site (PBS), and / or a 5'-enhancer / promoter element in the U3 region of a 5'-long-terminal repeat (LTR), and / or a modified 3'-SIN element in the U3 region of a 3'-LTR. These modifications may increase the infectivity titer and capacity.

[0302] Oncolytic viral vectors In some embodiments, the polynucleotides of this disclosure may be packaged in oncolytic viruses. 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 may occur naturally or may be genetically modified viruses such as oncolytic adenoviruses and oncolytic herpesviruses.

[0303] In some embodiments, the oncolytic vaccine virus may comprise viral particles of a replicable vaccine virus vector that is thymidine kinase (TK) deficient, expresses granulocyte-macrophage (GM)-colony-stimulating factor (CSF) sufficiently to induce tumor breakdown of cells within a tumor, see, for example, U.S. Patent No. 9,226,977.

[0304] Messenger RNA (mRNA) In some embodiments, the transcription factor systems, transcription factor constructs, or payload constructs of this disclosure may be designed as 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 structural components or features of those taught in International Application PCT / US2013 / 030062.

[0305] In some embodiments, the transcription factor system or its components may be designed as self-amplifying RNA. “Self-amplifying RNA,” as used herein, refers to an RNA molecule capable of replicating within a host and increasing the amount of RNA and the RNA-encoded protein. Such self-amplifying RNA may have any of the structural features or components taught in International Patent Application Publication WO2011005799.

[0306] dosage This disclosure provides a method comprising administering any one or more components or compositions of a transcription factor system to a subject in need of them. They may be administered to the subject using any amount and any route of administration that is effective in preventing, treating, or imaging a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition associated with cancer or autoimmune disease). The exact amount required will vary from subject to subject depending on the subject's species, age, and overall condition, the severity of the disease, the specific composition, the method of administration, the method of activation, etc.

[0307] The compositions according to this disclosure are typically formulated in dose units to facilitate administration and ensure uniformity of the dose. However, it is understood that the total daily dose of the compositions according to this disclosure may be determined by the attending physician within reasonable medical judgment. Specific therapeutically effective, prophylactically effective, or appropriate imaging dose levels for any particular patient will depend on a variety of factors, including, for example, the disorder being treated and its severity, the activity of the specific compound used, the specific composition used, the patient's age, weight, overall health, sex, and diet, the timing, route of administration, and elimination rate of the specific compound used, the duration of treatment, any drugs used in combination with or concurrently with the specific compound used, and similar factors well known in the medical field.

[0308] In some embodiments, the compositions of the Disclosure may be used in cancer immunotherapy at varying doses to avoid T cell exhaustion, prevent cytokine release syndrome, and minimize immunotherapy-related toxicity. For example, low doses of the compositions of the Disclosure may be used to initially treat patients with a high tumor burden, while patients with a low tumor burden may be treated with high and repeated doses of the compositions of the Disclosure to ensure recognition of minimal tumor antigen burden. In another example, the compositions of the Disclosure may be delivered in a pulsed manner to reduce potent T cell signaling and enhance in vivo persistence. In some embodiments, toxicity may be minimized by using a low dose of the compositions of the Disclosure first before administering a high dose. Dosage may be modified if serum markers such as ferritin, serum C-reactive protein, IL-6, 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 crossing the blood-brain barrier. This may be achieved by target gene deletion of endogenous alpha-4 integrin inhibitors such as tysabri / natalizumab, which may also be useful in this disclosure.

[0310] Also provided herein are methods for administering ligands or DRD ligands to subjects requiring them in accordance with this disclosure. In some embodiments, ligands are selected from acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). The ligands may be administered to subjects or cells using any amount and any route of administration that is effective in modulating the transcription factor system, DRD, or payload of this disclosure. In some embodiments, ACZ may be used with hCA2 DRD, methotrexate with hDHFR DRD, and trimethoprim with ecDHFR DRD. The exact amount required will vary from subject to subject depending on the species, age, and overall condition of the subject, the severity of the disease, the specific composition, the method of administration, and the method of activation. Subjects may be humans, mammals, or animals. The compositions according to this disclosure are typically formulated in unit dosage forms to facilitate administration and ensure uniformity of the dose. However, it is understood that the total daily dose of the compositions of this disclosure may be determined by the attending physician within the bounds of reasonable medical judgment. In certain embodiments, the ligands of this disclosure may be administered per day at doses of approximately 0.0001 mg / kg to approximately 100 mg / kg, approximately 0.001 mg / kg to approximately 0.05 mg / kg, approximately 0.005 mg / kg to approximately 0.05 mg / kg, approximately 0.001 mg / kg to approximately 0.005 mg / kg, approximately 0.05 mg / kg to approximately 0.5 mg / kg, approximately 0.01 mg / kg to approximately 50 mg / kg, and approximately 0.1 mg / kg to approximately 0.1 mg / kg to obtain the desired effect. The drug may be administered at dose levels sufficient to deliver the target body weight at least once a day, ranging from approximately 40 mg / kg to 0.5 mg / kg to 30 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg, or approximately 1 mg / kg to 25 mg / kg, 10 mg / kg to 100 mg / kg, 50 mg / kg to 500 mg / kg, or 100 mg / kg to 1000 mg / kg.In some embodiments, the dosage level may 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 target body weight per day, or once or more times per day, in order to obtain the desired effect.

[0311] This disclosure provides a method for delivering any of the ligands described herein to cells or tissues, the method comprising contacting the cells or tissues with the ligands, and which can be achieved in vitro, ex vivo, or in vivo. In certain embodiments, the ligands according to this disclosure may be administered to cells at dose levels 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, and about 50 μM to about 500 μM. In some embodiments, the ligand may be administered to cells in doses 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, and 250 μM.

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

[0313] Administration In some embodiments, compositions for cancer immunotherapy or treatment of autoimmune diseases may be administered ex vivo to cells and subsequently 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). The immune cells may be isolated and proliferated ex vivo using various methods known in the art. For example, methods for isolating cytotoxic T cells are described in U.S. Patents 6,805,861 and 6,531,451. Isolation of NK cells is described in U.S. Patent 7,435,596.

[0314] In some embodiments, depending on the properties of the cells, the cells may be introduced into a host organism, such as a mammal, by a wide variety of methods, including injection, transfusion, infusion, local intravenous infusion, or transplantation. In some embodiments, the cells of this disclosure may be introduced into a tumor site. The number of cells used will depend on the numerous circumstances, the purpose of introduction, the lifespan of the cells, the protocol used, such as the number of doses and the cell's ability to proliferate. The cells may be present in a physiologically acceptable culture medium.

[0315] In some embodiments, the cells of this disclosure may be administered in multiple doses to subjects having a disease or condition. The administration generally results in improvement of one or more symptoms of cancer or a clinical condition, and / or treats or prevents cancer or its clinical condition or symptoms.

[0316] In some embodiments, compositions for immunotherapy or treatment of autoimmune diseases may be administered in vivo. In some embodiments, the polynucleotides of the Disclosure comprising a transcription factor system, the payloads and compositions of the Disclosure 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 this disclosure may be administered by any route to achieve therapeutically effective results. These include enteral (into the intestines), gastrointestinal, epidural (into the dura mater), oral (by mouth), percutaneous, peridural, intracerebral (into the cerebrum), intraventricular (into the ventricles), topical (applied to the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous infusion, intraarterial (into an artery), intramuscular (into muscle), intracardiac (into the heart), intraosseous (into bone marrow), intrathecal (into the spinal canal), intraperitoneal (injected into the peritoneum), intravesical infusion, intravitreous (through the eye), and intracavitary injection. (Into pathological cavities), intracavernosal (at the base of the penis), intravaginal administration, intrauterine, extraamniotic administration, percutaneous (diffusion from intact skin for systemic distribution), transmucosal (diffusion from mucosa), transvaginal, breech (inhalation through the nose), sublingual, sublabial, enema, eye drops (on the conjunctiva), ear drops, auricle (inside or from the ear), buccal (towards the cheek), conjunctiva, skin, teeth (into teeth(s)), electroosmosis, intracervical, intrasinus, intratracheal, extracorporeal, hemodialysis, infiltration, intrastitium, intraabdomen, intraamniotic, intraarticular, intrabile duct, intrabronchial, intrasacral, intracavitary, intraarticular, intrabile duct, intrabronchial, intrasacral, intracavitary, intracavitary (into the cauda equina), intracisterna magna (into the cisterna magna cerebellomedularis), intracorneal, intradentalIntracornal, within the coronary arteries, within the corpus cavernosum (within the expanded space of the corpus cavernosum of the penis), within the spinal cord (within the intervertebral disc), within the ducts (within the ducts of glands), within the duodenum (within the duodenum), within the dura mater (within or beneath the dura mater), within the epidermis (in the epidermis), within the esophagus (in the esophagus), within the stomach (in the stomach), within the gingiva (in the gingiva), within the ileum (in the distal part of the small intestine), within the lesion (within a localized lesion or directly introduced into a localized lesion), within the lumen (in the lumen of a duct), within the lymphatic vessel (in the lymph), within the medullary cavity (in the medullary cavity of bone), Intrameningeal (within the meninges), intramyocardium (within the myocardium), intraocular (within the eyeball), intraovarian (within the ovary), intrapericardial (within the pericardial), intrapleural (within the pleural), intraprostate (within the prostate), intralungal (within the lung or its bronchi), sinus (within the nasal cavity or periorbital space), intraspinal cord (within the spinal column), synovial bursa (within the synovial space of a joint), intratendinous (within the tendon), testis (within the testis), intraspinal cavity (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thoracic cage), intratubular (within the tubules of organs), intratumoral (within the tumor), intratympanic cavity ( Intra-ear, intravascular, intracavitary, intracavitary, iontophoresis (by an electric current that moves ions of a soluble salt into body tissues), irrigation (by immersing or washing an open wound or body cavity), larynx (directly onto the larynx), transnasogastrostomy (through the nose into the stomach), occlusive dressing (after local administration, the area is covered with a bandage to seal it), transocular (into the external eye), oropharyngeal (directly into the mouth and pharynx), parenteral, transdermal, periarticular, epidural, perineurial, periodontal, rectum, respiratory tract (for local or systemic effects) This includes, but is not limited to, oral or nasal inhalation into the airways, retrobulbar (behind the pons or behind the eyeball), intramyocardium (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosa, local, transplacental (through or beyond the placenta), transtracheal (through the tracheal wall), transtympanic membrane (through or beyond the tympanic cavity), ureter (into the ureter), urethra (into the urethra), intravaginal, sacral block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis, or spinal cord.

[0318] Parenteral administration and injection administration In some embodiments, the pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, payloads, vectors, and cells of the 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 form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly 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, as well as mixtures thereof. In addition to inert diluents, oral compositions may contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or fragrances. In certain embodiments of parenteral administration, the composition is mixed with solubilizers, such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof. In other embodiments, surfactants such as hydroxypropylcellulose are included.

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

[0320] Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0321] Detectable drugs and labels The transcription factor systems, nucleic acids, polynucleotides, payloads, vectors, and cells of this disclosure may be associated with or conjugated to one or more radiopharmaceuticals or detectable drugs.

[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., pertechnetium acid (technetium(VII), TcO4)- Examples include (as a contrast agent), as well as contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelate Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), single-crystal iron oxide nanoparticles (MION), and ultra-small superparamagnetic iron oxide (USPIO)), manganese chelate (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 fluorescent tetrazine-fluorophore construct (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green488, or tetrazine-BODIPY TMR-X)) or an enzyme-activating fluorescent agent (e.g., PROSENSE® (VisEn Medical))). In vitro assays in which the enzyme-labeled composition may be used include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), immunoprecipitation assays, immunofluorescence assays, enzyme immunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.

[0324] Uses and applications The transcription factor systems, constructs, ligands, or compositions of this disclosure may be used in a wide variety of applications, including, but not limited to, therapeutic, diagnostic and prognostic, bioengineering, bioprocessing, biomanufacturing, research agents, metabolomics, gene expression, and enzyme substitution.

[0325] This disclosure provides a method comprising administering a composition, for example, a pharmaceutical composition comprising one or more components of a transcription factor system, to a subject in need of it.

[0326] For example, there may be several non-medical uses for generating cell lines and reagents for scientific research, one of which involves administering the compositions of this 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 medical treatment or prevention of a disease, condition or disorder in a subject in need may include the following steps: (a) providing a cell population (including autologous, allogeneic or syngeneic, human, animal, primary or cell culture); (b) introducing at least one nucleic acid molecule into at least one cell in the cell population, wherein the at least one nucleic acid molecule is a first polynucleotide comprising (i) 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 functionally linked to the DRD, and (ii) a polynucleotide comprising a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest for treating a disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising a specific polynucleotide binding site; (c) delivering cells to a subject; and (d) administering to the subject in an amount sufficient to bind a ligand that stabilizes DRD to such an extent that it enables the expression of a transcription factor activating domain and a transcription factor DNA binding domain, to the subject in an amount sufficient to form a transcription factor that enables the expression of the protein of interest in the cells, wherein the 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.

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

[0328] The compositions of this disclosure may be administered to a subject using any amount and any route of administration that is effective in preventing, treating, or imaging a disease, disorder, and / or condition (e.g., cancer, autoimmune diseases, and other disease-related diseases, disorders, and / or conditions). The exact amount required will vary from subject to subject depending on the species, age, and overall condition of the subject, the severity of the disease, the specific composition, the method of administration, the method of activation, etc.

[0329] The compositions according to this disclosure are typically formulated in dose units to facilitate administration and ensure uniformity of the dose. However, it is understood that the total daily dose of the compositions according to this disclosure may be determined by the attending physician within reasonable medical judgment. Specific therapeutically effective, prophylactically effective, or appropriate imaging dose levels for any particular patient will depend on a variety of factors, including, for example, the disorder being treated and its severity, the activity of the specific compound used, the specific composition used, the patient's age, weight, overall health, sex, and diet, the timing, route of administration, and elimination rate of the specific compound used, the duration of treatment, any drugs used in combination with or concurrently with the specific compound used, and similar factors well known in the medical field.

[0330] Also provided herein are methods for administering one or more stabilizing ligands (wherein used herein, a ligand that stabilizes DRD may be referred to as a stabilizing ligand or simply a ligand, with the understanding that the ligand is effective in stabilizing DRD used in the transcription factor system of this disclosure) to a subject in need of it. The ligand may be administered to the subject or cells in any amount and via any route of administration that is effective in modulating the amount of transcription factor expression of this disclosure in cells containing the transcription factor system. The exact amount of stabilizing ligand required will vary from subject to subject depending on the subject's species, age, and overall condition, the severity of the disease, the specific composition, the method of administration, and the method of activation. The subject may be human, mammal, or animal.

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

[0332] Despite significant advances, the effectiveness of current immunotherapy strategies is limited by associated toxicity. These are often related to the narrow therapeutic window associated with immunotherapy, which in part arises from the need to approach the limits of potential lethal toxicity in order to achieve a clinically meaningful treatment effect. Furthermore, the dose increases in vivo because the adopted immune cells continue to proliferate within the patient, often unpredictably.

[0333] The main risks associated with immunotherapy are on-target but extratumor-related side effects resulting from T cell activation in response to the normal tissue expression of tumor-associated antigens (TAAs). Clinical trials using T cells expressing T cell receptors for specific TAAs have reported skin rashes, colitis, and hearing loss in response to immunotherapy.

[0334] Immunotherapy can also lead to on-target oncotoxicity, which occurs when tumor cells are killed in response to immunotherapy. Side effects include tumor lysis syndrome, cytokine release syndrome, and associated macrophage activation syndrome. Importantly, these adverse effects can occur during tumor destruction and therefore can be toxic even if the tumor immunotherapy is successful. Therefore, approaches that control immunotherapy by controlling the immunotherapeutic agents are highly desirable, as they have the potential to reduce toxicity and maximize efficacy.

[0335] This disclosure provides systems, compositions, immunotherapeutic agents, and methods for immunotherapy. These compositions provide modulotable control of gene expression and function in immunotherapy, for example, for the prevention and treatment of cancer.

[0336] In one embodiment, the systems, compositions, immunotherapeutic agents and other components of the present disclosure may be controlled by separately added stabilizing ligands, which provides significant flexibility for controlling cancer immunotherapy. Furthermore, 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, such as cancer.

[0337] The tunable properties of the systems and compositions of this disclosure have the potential to improve the potency and duration of the efficacy of immunotherapy. By reversibly silencing the bioactivity of adoptive cells using the compositions of this disclosure, it is possible to maximize the potential of cell therapy without irreversibly killing cells and terminating the therapy.

[0338] This disclosure provides a method for fine-tuning immunotherapy after administration to a patient. As a result, the safety and efficacy of immunotherapy are improved, and the target population that may benefit from immunotherapy is increased.

[0339] In some embodiments, the immune cells of this disclosure may be T cells modified to express a payload or protein of interest, such as an antigen-specific T cell receptor (TCR) or an antigen-specific chimeric antigen receptor (CAR) (known as CAR T cells) as taught herein. Thus, a protein of interest, such as at least one polynucleotide encoding the CAR system (or TCR) described herein, or a vector containing a polynucleotide, is introduced into the T cells. T cells expressing a 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 the activation of the T cell. Activated CAR T cells alter their behavior, including the release of cytotoxic cytokines (e.g., tumor necrosis factor and lymphotoxins), improved cell proliferation rate, and alterations of cell surface molecules. Such alterations lead to the destruction of target cells expressing the antigen recognized by the CAR or TCR. In addition, cytokine release 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 T cells may be a first-generation CAR containing only the intracellular signaling domain of the TCR CD3 zeta, a second-generation CAR containing both the intracellular signaling domain and the co-stimulatory signaling domain of the TCR CD3 zeta, a third-generation CAR containing both the intracellular signaling domain and two or more co-stimulatory signaling domains of the TCR CD3 zeta, or a split CAR system, or an on / off switch CAR system. For example, if the expression of a CAR or TCR is controlled by a transcription factor, and the transcription factor or its components are functionally linked to the DRD, then in the absence of a stabilizing ligand, it will result in little to no accumulation of the transcription factor. The payload has a polynucleotide binding sequence specific to the transcription factor or its components, and therefore, in the absence of a stabilizing ligand, little to 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 if bound to the DRD, and then the transcription factor binds to its homologous polynucleotide binding sequence directly adjacent to the protein of interest, and is subsequently transcribed. The transcribed mRNA is then translated to produce the polypeptide / protein of interest. In some exemplary embodiments, the presence or absence of DRD-stabilizing ligands is used to modulate CAR or TCR expression in transduced T cells or NK cells.

[0341] In some embodiments, the CAR T cells of this disclosure may be further modified to express one, two, three or more other immunotherapeutic agents. These immunotherapeutic agents may be other CARs or TCRs specific to different target molecules, cytokines such as IL2, IL12, IL15 and IL18, cytokine receptors such as IL15Ra, chimeric switch receptors that convert inhibitory signals into stimulating signals, homing receptors that guide adoptive cells to target sites such as tumor tissue, agents that optimize the metabolism of immune cells, or safety switch genes (e.g., suicide genes) that kill activated T cells if a serious event is observed after adoptive cell transfer or if the transferred immune cells are no longer needed. These molecules may be contained in the same construct or in separate constructs.

[0342] In one embodiment, the CAR T cells (including TCR T cells) of this disclosure may be “armed” CAR T cells that are transfected or transduced under the control of the same or different transcription factors functionally linked to the same or different DRD, with one or more components of any transcription factor system containing a CAR payload and the same or different transcription factor systems encoding cytokines. Inducible cytokines or secreted constitutive activating cytokines further arm the CAR T cells to improve efficacy and persistence. In this context, such CAR T cells are also referred to as “armored CAR T cells.” The “armor” 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 stimulants such as IL2, IL12, IL15, IL18, type I IFN, CD40L, and 4-1BBL, which have been shown to further enhance the efficacy and persistence of CAR T cells by different mechanisms, even when faced with an unsuitable tumor microenvironment.

[0343] Chimeric antigen receptor-modified 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 a solid tumor are key tactics for achieving effective CAR-T therapy for solid tumors. In one embodiment, the payload or protein of interest may contain interleukin-12 (IL12), which may be used to enhance the efficacy of CAR-T cells, particularly because it may reconstruct the tumor microenvironment. IL12 has 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 impair safety and / or efficacy, so local delivery of cytokines may be a preferred approach as needed. In some embodiments, the transcription factor system or its components of this disclosure may be used to exogenously regulate IL12 expression to enable the use of IL12 in adoptive cell therapy.

[0344] In some embodiments, the transcription factor regulatory systems of this 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 signaling for tumor microenvironment reconstruction and epitope diffusion, particularly in solid tumor settings, in order to improve the effectiveness of CARs. Transcription factor regulation as described herein also provides rapid, dose-dependent local production of IL12 upon addition of DRD-specific stabilizing ligands.

[0345] In some embodiments, the armed CAR T cells of the Disclosure are modified to express a payload such as CD19 CAR and IL12, which is controlled using the transcription factor system or composition of the Disclosure. Such T cells, after CAR-mediated activation in a tumor, 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 transcription factor system containing a CAR payload encoded by the transcription factor system or its components, and a nucleic acid sequence encoding a suicide gene.

[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 cytokines and safety switch genes (e.g., suicide genes). The suicide gene may be an inducible caspase, such as caspase-9, which induces apoptosis when activated by an extracellular stabilizing ligand of the DRD encoded by the transcription factor system. Such induced apoptosis eliminates transplanted cells as needed to reduce the risk of direct toxicity and uncontrolled cell proliferation.

[0348] In one embodiment, a transcription factor system and its components that modulate the expression level and activity of any described payload or protein (used interchangeably) may be used in immunotherapy. In non-limiting examples, immunotherapeutic agents may be antibodies and their fragments and variants, cancer-specific T cell receptors (TCRs) and their variants, antitumor-specific chimeric antigen receptors (CARs), chimeric switch receptors, inhibitors of co-inhibitory receptors or ligands, agonists of costimulatory receptors and ligands, cytokines, chemokines, cytokine receptors, chemokine receptors, soluble growth factors, metabolic factors, suicide genes, homing receptors, or any agents that induce 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: 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 functionally linked to the DRD; the second polynucleotide comprises a fourth nucleic acid sequence encoding the protein of interest, wherein the fourth nucleic acid sequence is functionally linked to an inducible promoter comprising a specific polynucleotide binding site; the transcription factor activation domain and the transcription factor DNA-binding domain interact to form a transcription factor, and the binding of the transcription factor to the specific polynucleotide binding site is required for the transcription of the fourth nucleic acid sequence to be controlled by the transcription factor.

[0350] In one embodiment, the payload may be an immunotherapy agent.

[0351] In some embodiments, the transcription factor systems and compositions of this disclosure relate to the transcriptional regulation of protein (protein of interest or payload) function, including, for example, the antitumor immune response of an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent may include cytokines, chemokines, antibodies, integrins, endogenous proteins, membrane proteins, and extracellular proteins that may be used to upregulate or improve the function of one or more immune cell types, or to downregulate the activity of one or more immune cell types. In various embodiments, the immunotherapeutic agent useful for treating a disease, condition, or disorder may include cytokines, such as interleukins. In various embodiments, the transcription factor system provides a protein of interest or payload containing interleukins, such as IL-2, IL-6, IL-12, IL-15, IL-18, and other immunotherapeutic agents that promote or upregulate the lifespan and activity of one or more immune cell types useful for treating a disease, condition, disorder, or symptoms associated with any of them.

[0352] In some embodiments, cells genetically modified to encode and express at least one transcription factor capable of activating to enable the transcription of a target protein (immunotherapy 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 (derived from the autologous, allogeneic, or genetically modified host) that possess direct anticancer activity. ACT has shown promise in clinical applications for malignant diseases and infectious diseases. For example, T cells genetically engineered to recognize CD19 are 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 antitumor T cell receptors is used to treat metastatic melanoma (Rosenberg and Dudley, Curr. Opin. Immunol. 2009, 21:233-240).

[0353] According to this 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 in cell therapy for implementing CAR therapy, for manipulating or controlling TILs, in allogeneic cell therapy, or in combination with other treatment lines (e.g., radiation, cytokines) to encode an engineered or modified TCR, or to enhance non-TCR T cells (e.g., by introducing cytokine genes, checkpoint inhibitors PD1, CTLA4 genes).

[0354] Provided herein is a method for use in adoptive cell therapy. The method includes preconditioning a subject requiring such therapy, modulating immune cells with one or more components of the transcription factor system and / or composition of the Disclosure, administering engineered immune cells expressing the composition of the Disclosure to the subject, and ensuring successful engraftment of the engineered cells within the subject.

[0355] In some embodiments, the moduloable transcription factor expression constructs and compositions of this 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 lymphocyte depletion chemotherapy. Clinical trials of adoptive therapy without preconditioning have failed to demonstrate any clinical benefit, highlighting its importance in ACT. Furthermore, preconditioning is associated with significant toxicity and limits the suitability of the subject cohort for ACT. In some cases, ACT immune cells may be engineered to express cytokines such as IL-2, IL-6, IL-12, and IL-15 as payloads using transcription factors described herein to enable selective expression of the target protein, which may be regulated using the stabilizing ligands of this disclosure, in order to reduce the need for preconditioning (Pengram et al. (2012) Blood 119(18):4133-41, the entire content of which is incorporated by reference).

[0356] In some embodiments, the immune cells of 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 immunostimulatory cells of 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 in ACT may be T cells engineered to express a CAR containing an antigen-binding domain specific to the antigen on the tumor cells of interest. In other embodiments, the cells used in ACT may be NK cells engineered to express a CAR containing an antigen-binding domain specific to the antigen on the tumor cells of interest. In addition to adoptive transfer of genetically modified T cells (e.g., CAR T cells) for immunotherapy, alternative forms of CAR-expressing leukocytes may be used in 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 in ACT. The expression levels of CAR in T cells and NK cells are regulated and controlled according to this disclosure by small molecules that bind to DRDs (or more) functionally linked to a transcription factor or its components, thereby enabling selective transcription of CAR in transfected or transduced T cells and NK cells. In this scenario, the CAR is encoded by a nucleic acid sequence functionally linked to an inducible promoter containing a specific polynucleotide binding site of the transcription factor.

[0358] In some embodiments, NK cells engineered to express one or more components of a transcription factor system may be used in ACT. Activation of NK cells 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 antimicrobial activity, and enhance the adaptive immune response through upregulation of antigen presentation by antigen-presenting cells such as dendritic cells (DCs) (as outlined by Vivier et al., Nat. Immunol., 2008, 9(5):503-510).

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

[0360] NK cells may also be genetically reprogrammed to evade NK cell inhibitory signals when interacting with tumor cells. For example, genetically modifying NK cells using CRISPR, ZFNs, or TALENs to silence their inhibitory receptors may enhance the antitumor activity of NK cells.

[0361] Immune cells can be isolated and proliferated ex vivo using various methods known in the art. For example, methods for isolating and proliferating 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 these are incorporated herein by reference. Isolation and proliferation of NK cells 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 these are incorporated herein by reference. In particular, human primary NK cells may proliferate in the presence of feeder cells, such as bone marrow cell lines 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. A method for enriching immune cells is taught in International Patent Publication WO2015039100A1. In another example, T cells positive for B and T lymphocyte attenuator markers (BTLA) may be used to enrich T cells that are anti-cancer responsive, as described in U.S. Patent No. 9,512,401 (each of which is incorporated herein by reference in its entirety).

[0363] In some embodiments, immune cells for ACT may deplete a selected subpopulation to enhance T cell proliferation. For example, immune cells may deplete Foxp3+ T lymphocytes to minimize the antitumor immune response using a method taught in U.S. Patent Publication US20160298081A1 (the entire content of which is incorporated herein by reference).

[0364] In some embodiments, T cell activation and proliferation for ACT are achieved by antigenic stimulation of a chimeric antigen receptor (CAR) transiently expressed on the cell surface. Such activation methods are taught in International Patent No. WO2017015427, the entirety of which is 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 that are antigen-specific or nonspecific. APCs may be self or allogeneic within their respective organs. In some embodiments, APCs may be artificial antigen-presenting cells (aAPCs), such as cell-based aAPCs or cell-free aAPCs. Cell-based aAPCs may be selected from either genetically modified allogeneic cells, such as human erythroleukemia cells, or heterogeneic cells, such as mouse fibroblasts and Drosophila cells. Alternatively, APCs may be cell-free, with the antigen or costimulatory domain presented on a synthetic surface such as latex beads, polystyrene beads, lipid vesicles, or exosomes.

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

[0367] In some embodiments, adoptive cell therapy is performed by autotransfer, where cells originate from a subject requiring treatment, and the isolated and processed cells are administered to the same subject. In other cases, ACT may involve allotransfer, where cells are isolated and / or prepared from a donor subject other than the recipient subject ultimately receiving 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, each of two payloads, e.g., a cytokine such as IL12 and a CAR construct such as CD19 CAR, is transcribed by one or more transcription factors on the same or different transcription factor systems, and the transcription factors are ligated to the same or different DRDs. The payloads are transcribed and translated when the DRD(s) are stabilized with DRD(s)-specific stabilizing ligands. The expression of 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, each of a cytokine such as IL12 and a CAR construct such as CD19 CAR is transcribed by one of two different transcription factors, and each transcription factor is functionally ligated to a different DRD, thereby being regulated separately 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 systems of this disclosure, cells are administered to a target requiring them. Methods for administering cells for adoptive cell therapy are known and may be used in connection with the methods and compositions provided. For example, methods for adoptive T cell therapy are described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al., U.S. Patent No. 4,690,915 by 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 Davida et al. (2013) PLoS ONE 8(4):e61338, the entire contents of each of these are incorporated herein by reference.

[0370] In some embodiments, ACT immune cells may be modified to express one or more immunotherapeutic agents (proteins of interest) that promote the activation, invasion, proliferation, survival, and antitumor function of immune cells. The immunotherapeutic agents may be second CARs or TCRs specific to different target molecules, cytokines or cytokine receptors, chimeric switch receptors that convert inhibitory signals into stimulating signals, homing receptors that guide adoptive cells to target sites such as tumor tissue, agents that optimize the metabolism of immune cells, or safety switch genes (e.g., suicide genes) that kill activated T cells if a serious event is observed after adoptive cell transfer or if the transferred immune cells are no longer needed.

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

[0372] In some embodiments, one or more components of a transcription factor system may be used 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 limiting factor in the effectiveness of antiviral and antitumor immunotherapy. Exhausted T cells have a high rate of apoptosis, high surface expression of multiple inhibitory receptors, and reduced proliferative and cytokine production capacity. T cell activation leading to exhaustion may occur in the presence or absence of an antigen.

[0373] In some embodiments, one or more components of a transcription factor system may be used to prevent T cell exhaustion in connection with chimeric antigen receptor T cell therapy (CAR-T). In this regard, exhaustion may in some cases be caused by oligomerization of the scFv of the CAR on the cell surface, resulting in continuous activation of the intracellular domain of the CAR. As a non-limiting example, the CARs of this disclosure may include scFvs that cannot be oligomerized. As another non-limiting example, CARs that are rapidly internalized and reexpressed 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, its entire content incorporated by reference). One or more components of the transcription factor system of this disclosure may also be used to control the surface expression of the CAR on the T cell surface to prevent chronic T cell activation. The CARs of this disclosure may also be designed to minimize exhaustion. As a non-limiting example, the 41-BB signaling domain may be incorporated into the CAR design to mitigate T cell exhaustion. In some embodiments, any of the strategies disclosed by Long HA et al. may be used to prevent exhaustion (Long AH et al. (2015) Nature Medicine 21, 581-590, the entire content of which is incorporated herein by reference).

[0374] In some embodiments, the tunable nature of the transcription factor systems of the present disclosure may be utilized to reverse human T cell exhaustion observed with potent CAR signaling. Reversible silencing of adoptive-transferred cells using compositions of the present disclosure may be used to reverse potent signaling and thereby reactivate T cells. Reversal of exhaustion may be measured by downregulation of several inhibitory receptors associated with exhaustion.

[0375] In some embodiments, T cell metabolic pathways may be modified to reduce the T cell's sensitivity to exhaustion. These 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 the glycogen metabolic pathway. As a non-limiting example, a payload that reduces the rate of glycolysis may be used to limit or prevent T cell exhaustion (Long et al. Journal for Immunotherapy of Cancer 2013, 1(Suppl 1):P21, the entire content of which is incorporated by reference). In one embodiment, the T cells of this disclosure may be used in combination with 2-deoxyglucose and a glycolysis inhibitor such as rapamycin.

[0376] In some embodiments, the payload or protein of interest of this disclosure may be used in combination with an antibody or fragment that targets a T cell surface marker associated with T cell exhaustion. Examples of 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 a transcription factor system may be used to prevent T cell exhaustion.

[0377] In some embodiments, the compositions of this disclosure may be used to alter the TIL (tumor-infiltrating lymphocyte) population in a subject. In one embodiment, any of the payloads described herein may be used to alter the ratio of CD4-positive cells to CD8-positive populations. In some embodiments, TILs may be sorted ex vivo and manipulated to express any of the cytokines described herein. The payloads of this disclosure may be used to increase the CD4 and / or CD8 populations of TILs in order to enhance the TIL-mediated immune response. Parameters for improving the outcomes of CAR-T therapy are described in Finney et al. JCI.2019;129(5):2123-2132 (the entire content of which is incorporated herein by reference). The levels of the biomarker LAG3 (high) / TNF-α (low) in peripheral blood CD8+ T cells at apheresis may also predict subsequent dysfunctional responses in high antigen-loaded subjects that do not achieve a complete response lasting more than several weeks. T-cell-specific characteristics resulting from the originating T-cell repertoire and manufacturing process may also play a role in the outcomes of CAR-T therapy, as they converge on CD19 antigen-induced activation after adoptive transfer. The originating T-cell repertoire may be partially influenced by the timing of apheresis. In one embodiment, apheresis may be performed before chemotherapy. The cumulative load of CD19-expressing leukemia and normal B cells, as assessed in the bone marrow before lymphocyte-depleting chemotherapy, may be important in determining the outcomes of CAR-T therapy. According to Finney et al., increased antigen load improves the outcomes of CAR-T therapy. To increase CD19 antigen loading in vivo, subjects may also be injected with T cells derived from them (also known as T-APCs) that have been genetically modified to express CD19 and proliferated.

[0378] In some embodiments, the moduloable transcription factor expression constructs, the payload of interest (e.g., an immunotherapy agent), the vector, the cells, and the compositions of this disclosure may be used in combination with a cancer vaccine.

[0379] In some embodiments, cancer vaccines may contain 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. The peptides used in cancer vaccines may be modified to match the mutation profile of the subject. For example, EGFR-derived peptides with mutations matching those found in subjects requiring therapy have been successfully used in patients with lung cancer (Li F et al. (2016) Oncoimmunology. Oct 7;5(12):e1238539, the entire content of which is incorporated herein by reference).

[0380] In one embodiment, the cancer vaccine of this 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, thereby activating specific CTL clones more effectively than the native epitopes. Their modifications may allow the peptides to bind better to restriction class I MHC molecules or to interact more favorably with the TCR of a given tumor-specific CTL subset. The APLs may be selected using the methods taught in U.S. Patent Publication US20160317633A1, the entire contents of which are incorporated herein by reference.

[0381] In some embodiments, components of the transcription factor system of this disclosure, and effector immune cells genetically modified to encode the payload, may be combined with the biological adjuvants described herein. Dual regulation of CARs and cytokines and ligands separates the dynamic control of target-mediated activation from endogenous T cell proliferation. Such dual regulation also minimizes the need for patient preconditioning regimens. As a non-limiting example, a payload, e.g., a CAR, e.g., a DRD regulatory transcription factor transcribing 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, each of which is incorporated herein by reference in its entirety). As another non-limiting example, Merchant et al. improved outcomes in high-risk pediatric sarcoma patients by combining dendritic cell-based vaccination with recombinant human IL7 (Merchant, MSet.al. Adjuvant immunotherapy to Improve Outcome in High-Risk Pediatric Sarcomas. Clin Cancer Res. 2016.22(13):3182-91, the entirety of each of these is incorporated herein by reference).

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

[0383] In one embodiment, effector immune cells modified to express CARs specific to different target molecules on the same cell may be combined. In another embodiment, different immune cells modified 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 modified to express the CD19 CAR may be combined with NK cells modified to express the same CD19 CAR to treat B-cell malignancies.

[0384] In other embodiments, immune cells modified to express CAR may be combined with a checkpoint blocker.

[0385] In some embodiments, one or more components of the transcription factor system of the Disclosure, for example, effector immune cells genetically modified to express the payload, may be combined with the cancer vaccine of the Disclosure as well as other immunotherapies and adjuvant treatments.

[0386] In some embodiments, the methods of the Disclosure may include combinations of the compositions of the Disclosure with other agents effective in treating cancer, infections and other immunodeficiency disorders, such as anticancer agents. As used herein, the term “anticancer agent” means any agent that can adversely affect a cancer in question by, for example, killing cancer cells, inducing apoptosis in cancer cells, reducing the rate of growth of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the lifespan of an object having cancer.

[0387] In some embodiments, the anticancer agent or therapy may be a chemotherapeutic agent, or any other therapeutic agent that improves the therapeutic efficacy of the procedure in combination with radiotherapy, immunotherapy, surgery, or any other therapeutic agent.

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

[0389] In some embodiments, the compositions of this disclosure may be used in combination with immunotherapies other than those described herein, such as antibodies specific to certain target molecules on the surface of tumor cells.

[0390] Examples of chemotherapy include asibicin, acralubicin, acodazole hydrochloride, acronin, adzelesin, aldesleukin, altretamine, ambomycin, amethantrone acetate, amsacrin, anastrozole, anthramycin, asparaginase, asperrin, sulindac, curcumin, alkylating agents including: nitrogen mustards such as mechloretamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil, nitrosoureas such as carmustine (BC U), lomustine (CC N U), and semustine (methyl-CC N U). Antimetabolites including: ethyleneimine / methylmelamine, such as triethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altoretamine), alkyl sulfonates such as busulfan, triazines such as dacarbazine (DTIC), and the following: folic acid analogs, such as methotrexate and trimethrexate; pyrrolidine analogs, such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacitidine, 2,2'-difluorodeoxycytidine; purine analogs, such as 6-mercaptopurine, 6-thioguanine, azathiopurine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine ( Natural products including: EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA), antimitotic agents such as paclitaxel, vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and vinca alkaloids including estramustine phosphate, epipodophyllotoxins such as etoposide and teniposide, antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mitramycin), mitomycin C, and actinomycin, enzymes such as L-asparaginase, cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha,Anti-angiogenic factors such as TNF-beta and GM-CSF, such as angiostatin and endostatin, inhibitors of FGF or VEGF, such as soluble VGF / VEGF receptors and other receptors for soluble forms of angiogenic factors, platinum coordination complexes, such as cisplatin and carboplatin, anthracendions such as mitoxantrone, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIFf) and procarbazine, adrenal cortical inhibitors, such as mitotane (ο,ρ'-DDD) and aminoglutethimide Hormones and antagonists, including the following: corticosteroid antagonists, e.g., prednisone and its equivalents, dexamethasone and aminoglutethimide; progestins, e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens, e.g., diethylstilbestrol and ethinylestradiol equivalents; anti-estrogens such as tamoxifen; androgens, anti-androgens, including testosterone propionate and fluoxymesterone / equivalents, e.g., For example, 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 mimetic drugs, ubiquitin ligase inhibitors, stat inhibitors, and receptor tyrosine kinase inhibitors, such as imatinib mesylate (marketed as Gleevec or Glivec) and erlotinib (EGF receptor), currently marketed as Tarveca. Antiviral drugs such as inhibitors, for example, oseltamivir phosphate, amphotericin B, and palivizumab, Sdi1 mimetic drugs, semustine, aging-inducing inhibitors 1, sparfosinic acid, spicamycin D, spiromustine, sprenopentin, spongistatin 1, squalamine, stipamide, stromelicin inhibitors, sulfinosine, hyperactive vasoactive intestinal peptide antagonists, veraresol, veramine, verzin, verteporfin, vinorelbine, vinxaltin, Vitaxin, volozol, zanoterone, zeniplatin, zirascorb,This also includes, but is not limited to, dinostatin stimalamers, PI3Kβ small molecule inhibitors, GSK2636771, pan-PI3K inhibitors (BKM120), BRAF inhibitors, vemurafenib (Zelboraf), and dabrafenib (Tafinlar), or any of the aforementioned analogs, derivatives, and variants.

[0391] Radiotherapy agents and factors include radiation and waves that induce DNA damage, such as gamma rays, X-rays, UV rays, microwaves, electron emission, and radioisotopes. Therapy may be achieved by irradiating localized tumor sites with the above forms of radiation. All of these factors are most likely to affect broad-spectrum damaged DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for X-rays ranges from a daily dose of 50-200 roentgens over a long period (3-4 weeks) to a single dose of 2000-6000 roentgens. The dose range for radioisotopes varies considerably and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by tumor cells.

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

[0393] Other agents that may be used in combination with the compositions of this disclosure 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, cell proliferation inhibitors and differentiation agents, cell adhesion inhibitors, such as adhesion plaque kinase (FAK) inhibitors and lovastatin, or agents that increase the sensitivity of hyperproliferating cells to apoptosis-inducing substances such as the antibody C225.

[0394] The combination may include administering the composition of this disclosure and other agents simultaneously or separately. Alternatively, this immunotherapy may precede or follow other agents / therapies at intervals ranging from minutes, days, weeks to months.

[0395] Provided in this disclosure is a method for reducing tumor volume or load in a subject where it is desired, the method comprising introducing the composition of this disclosure into the subject.

[0396] The disclosure also provides a method for treating a target cancer, the method comprising administering to a target an effective amount of effector immune cells genetically modified to include the transcription factor system of the disclosure.

[0397] cancer Various cancers may be treated with regulated transcr...

Claims

1. A modified cell comprising a first 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. 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 functionally linked to the DRD, The transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that, upon binding to the specific polynucleotide binding site, can activate the transcription of the fourth nucleic acid sequence. The modified cell wherein the fourth nucleic acid sequence encodes a target protein and is functionally linked to or to an exogenous inducible promoter containing the specific polynucleotide binding site.

2. The modified cell according to claim 1, wherein the target protein is a heterologous protein.

3. The modified cell according to claim 1 or claim 2, wherein the fourth nucleic acid sequence is located on the first polynucleotide.

4. A modified cell according to any one of claims 1 to 3, further comprising a second polynucleotide, wherein the second polynucleotide comprises the fourth nucleic acid sequence.

5. The modified cell according to any one of claims 1 to 4, wherein the DRD is stabilized in the presence of ligand: acetazolamide (ACZ).

6. The modified cell according to any one of claims 1 to 5, wherein the target protein is a wild-type protein.

7. The modified cell according to any one of claims 1 to 6, wherein the target protein is a therapeutic protein.

8. The modified cell according to claim 7, wherein the target protein is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs).

9. The modified cell according to any one of claims 1 to 6, wherein the target protein is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

10. The modified cell according to any one of claims 1 to 6, wherein the target protein is a secreted protein.

11. The modified cell according to any one of claims 1 to 10, wherein the cell is a T cell, a natural killer cell (NK cell), or a tumor-infiltrating lymphocyte (TIL).

12. The modified cell according to any one of claims 1 to 10, wherein the cell is a stem cell, liver cell, blood cell, pancreatic cell, nerve cell, eye cell, muscle cell, or bone cell.

13. nucleic acid molecules, a. A first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, b. The second nucleic acid sequence encoding the drug-responsive domain (DRD) and c. A third nucleic acid sequence encoding a transcription factor activation domain and d. The fourth nucleic acid sequence encoding the target protein and Includes, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. The aforementioned transcription factor activation domain is derived from p65, (i) the transcription factor DNA binding domain is functionally linked to the DRD, (ii) the transcription factor activation domain is functionally linked to the DRD, or (iii) a combination of the transcription factor DNA binding domain and the transcription factor activation domain is functionally linked to the DRD. The nucleic acid molecule wherein the fourth nucleic acid sequence is functionally linked to an inducible promoter containing the specific polynucleotide binding site.

14. The nucleic acid molecule according to claim 13, wherein the DRD is stabilized in the presence of ligand: acetazolamide (ACZ).

15. The nucleic acid molecule according to claim 13, wherein the target protein is a wild-type protein.

16. The nucleic acid molecule according to claim 13, wherein the target protein is a therapeutic protein.

17. The nucleic acid molecule according to claim 16, wherein the target protein is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs).

18. The nucleic acid molecule according to claim 13, wherein the target protein is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

19. The nucleic acid molecule according to claim 13, wherein the target protein is a secreted protein.

20. A vector comprising a nucleic acid molecule according to any one of claims 13 to 19.

21. The vector according to claim 20, wherein the vector is a plasmid or a viral vector.

22. The vector according to claim 21, wherein the viral vector is derived from an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picornavirus.

23. The vector according to claim 21, wherein the viral vector is selected from the group consisting of lentiviral vectors, gamma retrovirus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

24. The first polynucleotide and the second polynucleotide, The first polynucleotide is It 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. 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 functionally linked to the DRD, The second polynucleotide is The fourth nucleic acid sequence comprises a fourth nucleic acid sequence encoding the target protein, wherein the fourth nucleic acid sequence is functionally linked to an inducible promoter containing the specific polynucleotide binding site. The transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that can activate transcription when bound to a specific polynucleotide binding site, wherein the first polynucleotide and the second polynucleotide are supported on a single vector, or the first polynucleotide and the second polynucleotide are supported on separate vectors.

25. The first polynucleotide and the second polynucleotide according to claim 24, wherein the DRD is stabilized in the presence of ligand: acetazolamide (ACZ).

26. The first polynucleotide and the second polynucleotide according to any one of claims 24 or 25, wherein the target protein is a wild-type protein.

27. The first polynucleotide and the second polynucleotide according to any one of claims 24 or 25, wherein the target protein is a therapeutic protein.

28. The first polynucleotide and the second polynucleotide according to claim 27, wherein the target protein is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs).

29. The first polynucleotide and the second polynucleotide according to any one of claims 24 or 25, wherein the target protein is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

30. The first polynucleotide and the second polynucleotide according to any one of claims 24 or 25, wherein the target protein is a secreted protein.

31. A composition comprising nucleic acid molecules for use in a method for producing modified cells, wherein the method is a. A first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site, b. The second nucleic acid sequence encoding the drug-responsive domain (DRD) and c. A third nucleic acid sequence encoding a transcription factor activation domain and The process includes introducing the nucleic acid molecule containing into a cell, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. The aforementioned transcription factor activation domain is derived from p65, (i) the transcription factor DNA binding domain is functionally linked to the DRD, (ii) the transcription factor activation domain is functionally linked to the DRD, or (iii) a combination of the transcription factor DNA binding domain and the transcription factor activation domain is functionally linked to the DRD. The composition, wherein the method further comprises introducing a fourth nucleic acid sequence encoding a target protein into the cell, wherein the fourth nucleic acid sequence is functionally linked to an inducible promoter containing the specific polynucleotide binding site.

32. The composition according to claim 31, wherein the target protein is a heterogeneous protein.

33. The composition according to claim 31 or 32, wherein the fourth nucleic acid sequence is located on the same nucleic acid molecule as the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence.

34. The composition according to claim 31 or 32, wherein the fourth nucleic acid sequence is located on a nucleic acid molecule different from the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence.

35. The composition according to any one of claims 31 to 34, wherein the target protein is selected from the group consisting of cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T cell receptors (TCRs), and chimeric antigen receptors (CARs).

36. The composition according to any one of claims 31 to 34, wherein the target protein is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

37. The composition according to any one of claims 31 to 34, wherein the target protein is a secreted protein.

38. The composition according to any one of claims 31 to 37, wherein the nucleic acid molecule is introduced into the cell by a plasmid or viral vector.

39. The composition according to claim 38, wherein the viral vector is derived from an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picornavirus.

40. The composition according to claim 38, wherein the viral vector is selected from the group consisting of lentiviral vectors, gamma retrovirus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

41. The composition according to any one of claims 31 to 37, wherein the nucleic acid molecule is introduced into the cell by a non-viral delivery method.

42. The composition according to any one of claims 31 to 41, wherein the cells are T cells, natural killer cells (NK cells), or tumor-infiltrating lymphocytes (TILs).

43. The composition according to any one of claims 31 to 41, wherein the cells are stem cells, liver cells, blood cells, pancreatic cells, nerve cells, eye cells, muscle cells, or bone cells.

44. A composition for use in a method of treating or preventing a disease in a subject in need thereof, wherein the composition comprises at least one nucleic acid molecule, and the method a. To provide a population of cells. b. Introducing the at least one nucleic acid molecule into at least one cell within the cell population, wherein the at least one nucleic acid molecule 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD, the first polynucleotide and ii. A second polynucleotide comprising a fourth nucleic acid sequence encoding a protein for the purpose of preventing or treating the disease or its symptoms, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising a specific polynucleotide binding site, To introduce the above, c. Delivering the cells to the target, and d. Administering to the subject a ligand that stabilizes the DRD to such an extent that it enables the expression of at least one of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the target protein in the cell, The expression of the target protein 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 target protein. The composition comprising the above.

45. A composition for use in a method for introducing modified cells into a subject requiring treatment or prevention of disease, wherein the composition comprises at least one nucleic acid molecule, and the method a. To provide a population of cells. b. Introducing the at least one nucleic acid molecule into at least one cell within the cell population, wherein the at least one nucleic acid molecule 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD, the first polynucleotide and ii. A second polynucleotide comprising a fourth nucleic acid sequence encoding a protein for the purpose of treating the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising a specific polynucleotide binding site, The aforementioned introduction, and c. Delivering the cells to the target. The composition comprising the above.

46. A composition or combination for use in a method for introducing modified cells into a subject requiring treatment or prevention of disease, wherein the composition comprises at least one nucleic acid molecule as described in any one of claims 13 to 19, and the combination comprises a first polynucleotide and a second polynucleotide as described in any one of claims 24 to 30, wherein the method a. provides a cell population. b. Introducing the at least one nucleic acid molecule or the first polynucleotide and the second polynucleotide into at least one cell within the cell population, and c. Delivering the cells to the target. The composition or combination comprising the above.

47. A composition for use in a method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, wherein the composition comprises at least one nucleic acid molecule, and the method a. Introducing the at least one nucleic acid molecule into the at least one cell of the subject, wherein the at least one nucleic acid molecule 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during intracellular expression, the first polynucleotide ii. A second polynucleotide comprising a fourth nucleic acid sequence encoding a protein for the purpose of treating the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising a specific polynucleotide binding site, The composition, which includes introducing the above.

48. A composition for use in a method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, wherein the composition comprises at least one nucleic acid molecule, and the method a. Introducing the at least one nucleic acid molecule into the at least one cell of the subject, wherein the at least one nucleic acid molecule 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during intracellular expression, the first polynucleotide ii. A second polynucleotide comprising a fourth nucleic acid sequence encoding a protein for the purpose of treating the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter comprising a specific polynucleotide binding site, The aforementioned introduction, and b. Administering to the subject a ligand that stabilizes the DRD to a sufficient degree to enable the expression of at least one of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the target protein in the cell, The composition comprising administering such a substance, wherein the expression of the target protein 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 target protein.

49. A combination for use in a method of treating a disease in a person requiring such treatment, wherein the combination comprises at least one first nucleic acid molecule and at least one second nucleic acid molecule, and the method a. To provide a population of cells. 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during expression within the cell. ii. The introduction wherein the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein intended to treat the disease, and the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site. c. Delivering the cells to the target, and d. Administering to the subject a ligand that stabilizes the DRD to a sufficient degree to enable the expression of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the target protein in the cell, The expression of the target protein 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 target protein. The aforementioned combination, including the above.

50. A combination for use in a method of treating a disease in a person requiring such treatment, wherein the combination comprises at least one first nucleic acid molecule and at least one second nucleic acid molecule, and the method a. To provide a population of cells. 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during expression within the cell. ii. The introduction of the second nucleic acid molecule comprising a fourth nucleic acid sequence encoding a protein intended to treat the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site, and c. Delivering the cells to the target. The aforementioned combination, including the above.

51. The composition according to any one of claims 44 to 48 or the combination according to any one of claims 46, 49, and 50, wherein the nucleic acid molecule is introduced into the cell by a plasmid or viral vector.

52. The composition or combination according to claim 51, wherein the viral vector is derived from an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, or picornavirus.

53. The composition or combination according to claim 51, wherein the viral vector is selected from the group consisting of lentiviral vectors, gamma retroviral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

54. The composition according to any one of claims 44 to 48 or the combination according to any one of claims 46, 49, and 50, wherein the nucleic acid molecule is introduced into the cell by a non-viral delivery method.

55. A composition for use in a method for introducing modified cells into a subject requiring treatment or prevention of disease, wherein the composition comprises a first polynucleotide as described in any one of claims 24 to 30, and the first polynucleotide is introduced into at least one cell in a cell population in combination with a second polynucleotide as described in any one of claims 24 to 30, wherein the method is a. To provide the aforementioned cell population, b. Introducing the first polynucleotide and the second polynucleotide into at least one cell within the cell population, and c. Delivering the cells to the target. The composition comprising the above.

56. A composition for use in a method for introducing modified cells into a subject requiring treatment or prevention of disease, wherein the composition comprises a second polynucleotide as described in any one of claims 24 to 30, and the second polynucleotide is introduced into at least one cell in a cell population in combination with a first polynucleotide as described in any one of claims 24 to 30, wherein the method is a. To provide the aforementioned cell population, b. Introducing the first polynucleotide and the second polynucleotide into at least one cell within the cell population, and c. Delivering the cells to the target. The composition comprising the above.

57. A composition for use in a method of treating a disease in a subject requiring such treatment, wherein the composition comprises at least one of first nucleic acid molecules, and the at least one of the first nucleic acid molecules is introduced into at least one cell in a cell population in combination with at least one of second nucleic acid molecules, wherein the method is a. To provide the aforementioned 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during expression within the cell. ii. The introduction wherein the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein intended to treat the disease, and the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site. c. Delivering the cells to the target, and d. Administering to the subject a ligand that stabilizes the DRD to a sufficient degree to enable the expression of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the target protein in the cell, The expression of the target protein 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 target protein. The composition comprising the above.

58. A composition for use in a method of treating a disease in a subject requiring such treatment, wherein the composition comprises at least one of a second nucleic acid molecule, and the at least one of the second nucleic acid molecules is introduced into at least one cell in a cell population in combination with at least one of a first nucleic acid molecule, and the method is a. To provide the aforementioned 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during expression within the cell. ii. The introduction wherein the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein intended to treat the disease, and the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site. c. Delivering the cells to the target, and d. Administering to the subject a ligand that stabilizes the DRD to a sufficient degree to enable the expression of the transcription factor activation domain and the transcription factor DNA binding domain, in an amount sufficient to form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the target protein in the cell, The expression of the target protein 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 target protein. The composition comprising the above.

59. A composition for use in a method of treating a disease in a subject requiring such treatment, wherein the composition comprises at least one of first nucleic acid molecules, and the at least one of the first nucleic acid molecules is introduced into at least one cell in a cell population in combination with at least one of second nucleic acid molecules, wherein the method is a. To provide the aforementioned 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during expression within the cell. ii. The introduction of the second nucleic acid molecule comprising a fourth nucleic acid sequence encoding a protein intended to treat the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site, and c. Delivering the cells to the target. The composition comprising the above.

60. A composition for use in a method of treating a disease in a subject requiring such treatment, wherein the composition comprises at least one of a second nucleic acid molecule, and the at least one of the second nucleic acid molecules is introduced into at least one cell in a cell population in combination with at least one of a first nucleic acid molecule, and the method is a. To provide the aforementioned 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), The aforementioned transcription factor activation domain is derived from p65, The aforementioned transcription factor DNA-binding domain is derived from ZFHD1, The aforementioned DRD is derived from the human carbonic anhydrase 2 (CA2) parent protein, and An amino acid sequence having at least 90% identity to the amino acid sequence described in SEQ ID NO: 5, and containing one of the following mutations to SEQ ID NO: (i) L156H, (ii) I59N and G102R, (iii) S56N, (iv) L156H, S172C, F178Y and E186D, (v) L197P, (vi) R27L, T87I, H122Y and N252D, (vii) D72F, V241F and P249L, (viiii) D71L, T87N and L250R, (ix) L183S, (x) G63D, E69V and N231I, or (xi) W208S. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is functionally linked to the DRD during expression within the cell. ii. The introduction of the second nucleic acid molecule comprising a fourth nucleic acid sequence encoding a protein intended to treat the disease, wherein the fourth nucleic acid sequence is functionally linked to an exogenous inducible promoter containing the specific polynucleotide binding site, and c. Delivering the cells to the target. The composition comprising the above.

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