Compositions and methods for tunable regulation of transcription

Abstract

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

Description

COMPOSITIONS AND METHODS FOR TUNABLE REGULATION OF TRANSCRIPTION CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Application No. 62 / 958,693, filed January 8, 2020 and U.S. Provisional Application No. 62 / 959,859, filed January 10, 2020. The entire contents of the aforementioned applications are incorporated herein by reference in their entireties. REFERENCE TO THE SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 8, 2021, is named 268052 483267 SL.txt and is 241,815 bytes in size. FIELD

[0003] The present disclosure relates to systems, compositions and methods for tunable protein expression driven by regulated transcriptional activity. Provided in the present disclosure are modular transcription factor systems, polynucleotides of transcription factor systems, polypeptides, vectors, cells, compositions and methods for use in regulation of transcription and regulated protein expression driven by regulated transcriptional activity. BACKGROUND

[0004] Gene and cell therapies are revolutionizing medicine and offering new promise for the treatment of previously intractable conditions. However, most current technologies do not allow titration of the timing or levels of target protein induction. This has rendered many potential gene and cell therapy applications difficult or impossible to safely and effectively deploy.

[0005] Inadequate exogenous and / or endogenous gene control is a critical issue in many gene and cell therapy settings. This lack of tunability also makes it difficult to safely express proteins with narrow or uncertain therapeutic windows or those requiring more titrated or transient expression.

[0006] One approach to regulated protein expression or function is the use of drug responsive domains (DRDs). Drug responsive domains are small protein domains that can be appended to a target protein of interest. DRDs render the attached protein of interest unstable in the absence of a DRD-binding ligand and the protein of interest is rapidly degraded by the ubiquitin-proteasome system of the cell. However, when a specific small molecule DRD-binding ligand binds to the DRD, the attached protein of interest is stabilized, and protein function is achieved.

[0007] DRD technology forms the basis of a new class of cell and gene therapies that can deliver tunable and temporal control of gene expression and function, expanding the universe of protein therapeutics that can be safely and effectively incorporated into cell and gene therapy modalities. However, current DRD technology produces fusion proteins in which the protein of interest is joined to a DRD, which may be unsuitable for some indications. Thus, there remains a need to develop cell and gene therapies in which native proteins of interest can be expressed in a regulated manner. SUMMARY

[0008] The present invention provides modified cells, nucleic acid molecules, vectors, and cell and gene therapies in which the timing or levels of a native therapeutic protein can be regulated through administration of an oral small molecule drug.

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

[0010] Provided herein are transcription factor systems. A transcription factor system of the present disclosure is a combination of one or more polynucleotides that comprise (1) one or more nucleic acid sequences that encode a transcription factor that is able to bind to a specific polynucleotide binding site and activate transcription; (2) a nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor, or part thereof, is operably linked to the DRD; and (3) a nucleic acid sequence that encodes a payload and is operably linked to an inducible promoter comprising the specific polynucleotide binding site.

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

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

[0013] In some aspects, the present disclosure provides a modified cell comprising a polynucleotide that comprises 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 and is able to bind to a specific polynucleotide binding site and activate transcription of a third nucleic acid sequence encoding a protein of interest, the third nucleic acid sequence being operably linked to either the specific polynucleotide binding site, an exogenous inducible promoter comprising the specific polynucleotide binding site, or both. In some embodiments, the protein of interest is a heterologous protein. In some embodiments, the third nucleic acid sequence is located on the polynucleotide that comprises the first nucleic acid sequence and the second nucleic acid sequence. In some embodiments, the modified cell further comprises a second polynucleotide that comprises 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 that is able to bind to a specific polynucleotide binding site and activate transcription, and a second nucleic acid sequence encoding a drug responsive domain (DRD); wherein the transcription factor, or part thereof, is operably linked to the DRD; and (b) a second polynucleotide comprising a third nucleic acid sequence encoding a protein of interest, said third nucleic acid sequence being 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 able to bind to a specific polynucleotide binding site and activate 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 that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD); wherein at least one of the transcription factor activation domain, the transcription factor DNA binding domain, or the 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 that encodes a protein of interest, said fourth nucleic acid sequence being 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 that is able to activate transcription upon binding to the specific polynucleotide binding site.

[0017] In another aspect, the present 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 the combination of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD. In one aspect, the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that is able to bind to the specific polynucleotide binding site and activate transcription of a nucleic acid sequence encoding a protein of interest, said nucleic acid sequence being operably linked to the exogenous inducible promoter.

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

[0019] In some embodiments, the DRD is stabilized in the presence of a ligand selected from the group comprising: Acetazolamide (ACZ), Methotrexate (MTX), and Trimethoprim (TMP). In some embodiments, the DRD is responsive to or interacts with a ligand selected from the group comprising: Acetazolamide (ACZ), Methotrexate (MTX), and Trimethoprim (TMP).

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

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

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

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

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

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

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

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

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

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

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

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

[0032] In some embodiments, the DRD is stabilized in the presence of a ligand selected from the group comprising: Acetazolamide (ACZ), Methotrexate (MTX), and Trimethoprim (TMP). In some embodiments, the DRD is responsive to or interacts with a ligand selected from the group comprising: Acetazolamide (ACZ), Methotrexate (MTX), and Trimethoprim (TMP).

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

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

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

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

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

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

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

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

[0041] In one aspect, the present disclosure provides a first polynucleotide and second polynucleotide, the first polynucleotide comprising: a first nucleic acid sequence that encodes a transcription factor and a second nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor is operably linked to the DRD and wherein the transcription factor is able to activate transcription upon binding to a specific polynucleotide binding site; and a second polynucleotide comprising: a third nucleic acid sequence that encodes a protein of interest, the third nucleic acid sequence being operably linked to an inducible promoter comprising the specific polynucleotide binding site; wherein the first polynucleotide and the second polynucleotide are each carried in a single vector, or the first polynucleotide and the second polynucleotide are carried in separate vectors.

[0042] In some embodiments, the DRD is derived from a parent protein selected from the group comprising: human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDES. In some embodiments, the DRD is stabilized in the presence of a ligand selected from the group comprising: Acetazolamide (ACZ), Methotrexate (MTX), and Trimethoprim (TMP).

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

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

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

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

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

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

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

[0050] In some embodiments, the nucleic acid molecule is introduced into the cell by a plasmid or a viral vector. In one embodiment, the viral vector is derived from an adenovirus, adeno- associated virus (AAV), alphavirus, flavivirus, herpes virus, 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 a lentivirus vector, a gamma retrovirus vector, adeno- associated virus (AAV) vector, adenovirus vector, and a herpes virus vector.

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

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

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

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

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

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

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

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

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

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

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

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

[0063] In some alternate embodiments, methods of treatment and prevention of the present disclosure can be accomplished by introducing a first vector and a second vector into a cell, wherein the first vector comprises a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor activation domain and / or the transcription factor DNA binding domain, upon expression in the cell, is operably linked to the DRD, and the second vector comprises a fourth nucleic acid sequence that encodes a protein of interest that prevents and / or treats the disease, the fourth nucleic acid sequence being operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site.

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

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

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

[0067] The present disclosure also provides pharmaceutical compositions that include the compositions described herein and a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG. 1A-FIG. 1B depict schematic illustrations of a transcription factor system design scheme. FIG. 1A shows a schematic of a transcription factor construct, referred to as “DRD-TF construct”, comprising a nucleic acid sequence encoding a transcription factor DNA binding domain, a transcription factor activation domain, and a drug responsive domain (DRD). FIG. 1B shows a schematic of a payload construct, comprising an inducible promoter that comprises binding sites for the transcription factor DNA binding domain.

[0069] FIG. 2A-FIG. 2B show ligand-dependent activity of transcription factor systems comprising DRD-regulated transcription factors with different DRDs. FIG. 2A shows a western blot of lysates from untransfected (“mock”) HEK293T cells and HEK293T cells transfected with a construct encoding a constitutive transcription factor (construct ZFHD-055; “Cons.”) or a construct encoding a transcription factor operably linked to a DRD derived from CA2, ecDHFR, ER, or hDHFR parent protein. Details on each construct and ligand treatment conditions are provided in Table 4 and Table 6. The top panel of the western blot shows bands for the endogenous p65, the transcription factor and DRD polypeptide encoded by each of the DRD-TF constructs, and the transcription factor polypeptide encoded by the constitutive construct ZFHD-055. FIG. 2B shows quantification of the western blot in FIG. 2A, normalized with the constitutive condition set to 1.0.

[0070] FIG. 3A-FIG. 3E shows ligand-dependent activity of a transcription factor system comprising an ecDHFR DRD-regulated transcription factor. FIG. 3A shows a schematic of the transcription factor construct ZFHD-005. FIG. 3B shows a schematic of the payload construct ZFHD-007. FIG. 3C shows a schematic of a constitutive transcription factor construct ZFHD-004. FIG. 3D shows a western blot of lysates from U20S cells with stable integration of the indicated constructs, treated with 10 uM TMP or 0.1% DMSO. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 44.3kDa 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. FIG. 3E shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry on U20S cells with stable integration of the indicated constructs, treated with 10 uM TMP or 0.1% DMSO.

[0071] FIG. 4A-FIG. 4C shows dose-response to ligand of a transcription factor system comprising an ecDHFR DRD-regulated transcription factor. FIG. 4A shows a western blot of lysates from U20S cells with stably integrated constructs ZFHD-005 and ZFHD-007, treated with DMSO or the indicated concentrations of TMP. The lane labeled “U20S” represents untransduced U20S cells treated with TMP. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 44.3kDa represents the transcription factor and DRD polypeptide encoded by the transcription factor construct ZFHD-005. FIG. 4B shows quantification of the indicated “ZFHD-005 polypeptide” bands in the western blot of FIG. 4A. Fluorescence is normalized to endogenous p65. FIG. 4C shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry on U20S cells with stably integrated constructs ZFHD-005 and ZFHD- 007, treated with the indicated concentrations of TMP. The highest concentration of TMP used in FIG. 4C is 33 uM. Data shown is for 3 replicates. Error bars represent standard deviation.

[0072] FIG. SA- FIG. 5B shows ligand-dependent activity of a transcription factor system comprising an ecDHFR DRD-regulated transcription factor in T cells. FIG. SA shows a western blot of lysates from untransduced T cells or T cells transduced with virus (OTLV-ZFHD-005 or OTLV- ZFHD-007) and 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. FIG. 5B shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry of untransduced T cells or T cells transduced with virus made from the indicated constructs and treated with TMP or DMSO. Data shown is for 3 replicates. Error bars represent standard deviation from the mean.

[0073] FIG. 6A- FIG. 6D shows ligand-dependent activity of a transcription factor system comprising a CA2 DRD-regulated transcription factor in ARPE-19 cells. FIG. 6A shows a schematic of the transcription factor construct ZFHD-019. FIG. 6B shows a western blot of lysates from untransduced ARPE-19 cells or ARPE-19 cells with stably integrated constructs ZFHD-019 and ZFHD-007, treated with 10 pM 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. FIG. 6C shows quantification of the indicated “ZFHD-019 polypeptide” band in the western blot of FIG. 6B. Fluorescence is normalized to endogenous p65. FIG. 6D shows GFP mean fluorescence intensity (MFI) as assessed by flow cytometry of untransduced ARPE-19 cells or ARPE-19 cells with stable integration of the indicated constructs, either untreated or treated with 10 uM ACZ or 1% DMSO. Data shown is for 3 replicates. Error bars represent standard deviation from the mean. The untransduced ARPE-19 cells and ARPE-19 cells with stable integration of construct ZFHD-007 shown on the graph were treated with DMSO.

[0074] FIG. 7A- FIG. 7B shows dose-response to ligand of a transcription factor system comprising a CA2 DRD-regulated transcription factor. FIG. 7A shows a western blot of lysates from ARPE-19 cells with stably integrated constructs ZFHD-007 and ZFHD-019, treated with the indicated concentrations of ACZ. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 55.8 kDa represents the transcription factor and DRD polypeptide encoded by the transcription factor construct ZFHD-019. FIG. 7B shows quantification of the indicated “ZFHD-019 polypeptide” bands in the western blot of FIG. 7A. Fluorescence is normalized to the endogenous P65 band.

[0075] FIG. 8 shows dose-response to ligand of a transcription factor system comprising a CA2 DRD-regulated transcription factor. The graph shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry of U20S cells with stable integration of constructs ZFHD-007 and ZFHD-019, treated with the indicated concentrations of ACZ. Data shown is for 2 replicates. Error bars represent standard deviation.

[0076] FIG. 9A- FIG. 9C shows ligand-dependent activity of a transcription factor system comprising a CA2 DRD-regulated transcription factor in Jurkat cells. FIG. 9A shows a schematic of the transcription factor construct ZFHD-048. FIG. 9B shows a schematic of the payload construct ZFHD-022. FIG. 9C shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry on Jurkat cells with stable integration of constructs ZFHD-048 and ZFHD-022, treated with DMSO (0.1%) or ACZ (10 uM final concentration). The data presented is for cells that were positive for the transduction marker.

[0077] FIG. 10A-FIG. 10F shows ligand-dependent activity of single vector transcription factor systems comprising ecDHFR DRD-regulated transcription factors. FIG. 10A shows a schematic of construct ZFHD-012. FIG. 10B shows a schematic of construct ZFHD-018. FIG. 10C and FIG. 10D show western blots of lysates from U20S cells transduced with lentiviruses made from the indicated constructs and treated with 10 uM TMP or 0.1% DMSO. The band appearing at approximately 44.3 kDa represents the transcription factor and DRD polypeptide encoded by the indicated constructs. In the single vector constructs, there is a stop codon at the end of the EGFP sequence and a stop codon at the end of the transcription factor-DRD sequence, thus resulting in the approximate 44.3 kDa bands representative of the transcription factor and DRD polypeptide. FIG. 10E and FIG. 10F show GFP median fluorescence intensity (MFI) as assessed by flow cytometry for U20S cells transduced with lentiviruses made from the indicated constructs and treated with 10 uM TMP or 0.1% DMSO.

[0078] FIG. 11A-FIG. 11B shows ligand-dependent activity of a single vector transcription factor system comprising a CA2 DRD-regulated transcription factor. FIG. 11A shows a schematic of the single vector system, depicted as construct ZFHD-036. FIG. 11B show GFP median fluorescence intensity (MFI) as assessed by flow cytometry for Jurkat cells transduced with lentiviruses made from the indicated constructs and treated with 10 uM ACZ or 0.1% DMSO. ZFHD-036.1 and ZFHD-036.2 on the graph represent two cell lines, each transduced with lentivirus made from construct ZFHD-036.

[0079] FIG. 12A-FIG. 12B shows ligand-dependent activity of transcription factor systems comprising variants of transcription factor constructs. FIG. 12A shows schematic diagrams of transcription factor construct variants. FIG. 12B shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry on U20S cells with stable integration of the indicated constructs, treated with either 0.1% DMSO or 10 uM TMP.

[0080] FIG. 13 shows ligand-response timecourse analyses of transcription factor systems comprising variants of transcription factor constructs. The graph shows GFP median fluorescence intensity (MFI) as assessed by flow cytometry for U20S cells with stable integration of the indicated constructs, treated for the indicated time periods with either 0.1% DMSO or 10 uM TMP.

[0081] FIG. 14A-FIG. 14D shows ligand-dependent activity of transcription factor systems comprising variants of payload constructs. FIG. 14A shows a schematic diagram of payload construct ZFHD-007. FIG. 14B shows a schematic diagram of payload construct ZFHD-017. FIG. 14C-FIG. 14D show GFP median fluorescence intensity (MFI) as assessed by flow cytometry on U20S cells with stable integration of the indicated constructs, treated with either 0.1% DMSO or 10 uM TMP.

[0082] FIG. 15 shows ligand-dependent activity of a transcription factor system comprising a payload construct encoding a secreted IL12 payload. The graph shows concentration of secreted IL12 in supernatants collected from U20S cells with stable integration of the indicated constructs, treated with either 0.1% DMSO or 10 uM TMP.

[0083] FIG. 16A-FIG. 16D show ligand-dependent regulation of different transcription factors operably linked to a DRD derived from a parent CA2 protein. FIG. 16A shows a western blot of lysates from untransfected (“mock”) HEK293T cells and HEK293T cells transfected with the following constructs: (1) ¢jun-001 (“0017), (2) ¢jun-002 (“002”), or (3) ¢jun-003 (“003”). Each construct-transfected cell population is shown after treatment with DMSO or ACZ (indicated by “+” symbol). The labeled bands “c-Jun -001 and -002 polypeptides” identify the CA2-linker-C-jun polypeptides encoded by the ¢jun-001 and cjun-002 constructs. The labeled bands “c-Jun-003 polypeptide” identify the c-Jun polypeptide encoded by construct ¢jun-003. FIG. 16B shows quantification of the western blot in FIG. 16A. FIG. 16C shows a western blot of lysates from untransfected (“mock”) HEK293T cells and HEK293T cells transfected with the following constructs: (1) FOXP3-013 (“013”), (2) FOXP3-014 (“014”), or (3) FOXP3-015 (“015”). Each construct-transfected cell population is shown after treatment with DMSO or ACZ (indicated by “+” symbol). The labeled bands “FOXP3 -013 and -014 polypeptide” identify the CA2-FOXP3 polypeptides encoded by the FOXP3-013 and FOXP3-014 constructs. The labeled bands “FOXP3- 015 polypeptide” identify the FOXP3 polypeptide encoded by construct FOXP3-015. FIG. 16D shows quantification of the western blot in FIG. 16C.

[0084] FIG. 17A-FIG. 17B show ligand-dependent regulation of c-Jun transcription factor construct stably integrated in Jurkat cells. FIG. 17A shows a western blot of lysates from untransduced (“mock”) Jurkat cells and Jurkat cells transduced with lentivirus made from constructs cjun-001 (“001”) and cjun-002 (“002”). Each construct-transduced cell line is shown after treatment with DMSO or ACZ (indicated by “+” symbol). Bands for a c-Jun polypeptide and a phosphorylated c-Jun polypeptide are shown. FIG. 17B shows quantification of the western blot in FIG. 17A.

[0085] FIG. 18 shows the nucleotide sequence of the pELDS-puro transfer vector (SEQ ID NO: 68).

[0086] FIG. 19 shows the nucleotide sequence of the pELNS-puro transfer vector (SEQ ID NO: 69). DETAILED DESCRIPTION Transcription Factor System

[0087] According to the present disclosure, a transcription factor system is a combination of one or more polynucleotides that comprise (1) one or more nucleic acid sequences that encode a transcription factor that is able to bind to a specific polynucleotide binding site and activate transcription; (2) a nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor is operably linked to the DRD; and (3) a nucleic acid sequence that encodes a payload and is operably linked to an inducible promoter comprising the specific polynucleotide binding site.

[0088] In some embodiments, the combination of one or more polynucleotides of a transcription factor system can be used to modify cells, for example, immune cells useful in the treatment of a disease and create systems for regulatable expression of a protein of interest by regulating the presence of a transcription factor acting on the polynucleotide(s) encoding the payload or protein of interest.

[0089] In some embodiments, the combination of one or more polynucleotides of a transcription factor system comprises a polynucleotide that comprises a first nucleic acid sequence encoding the transcription factor and a second nucleic acid sequence encoding the DRD.

[0090] The present disclosure also provides a first polynucleotide and second polynucleotide, wherein the first polynucleotide comprises: a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD). In this example, at least one of the transcription factor activation domain, the transcription factor DNA binding domain, or the combination of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD exemplified herein. The second polynucleotide comprises: a fourth nucleic acid sequence that encodes a protein of interest, the fourth nucleic acid sequence being operably linked to an inducible promoter comprising the 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 is able to activate transcription upon binding to the specific polynucleotide binding site, and the first polynucleotide and the second polynucleotide are each carried in a single vector, or the first polynucleotide and the second polynucleotide are carried in separate vectors.

[0091] In a related example, the present disclosure provides compositions and nucleic acids that are operable to regulate transcription. For example, the present disclosure provides a first polynucleotide and second polynucleotide of the regulatable transcription factor system. The first polynucleotide comprises a first nucleic acid sequence that encodes a transcription factor and a second nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor is operably linked to the DRD and wherein the transcription factor is able to activate transcription upon binding to a specific polynucleotide binding site. The second polynucleotide comprises: a third nucleic acid sequence that encodes a protein of interest, the third nucleic acid sequence being operably linked to an inducible promoter comprising the specific polynucleotide binding site; such that the first polynucleotide and the second polynucleotide are each carried in a single vector, or the first polynucleotide and the second polynucleotide are carried in separate vectors.

[0092] In some embodiments, the combination of one or more polynucleotides of a transcription factor system comprises a first nucleic acid sequence encoding a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; a second nucleic acid sequence encoding a transcription factor activation domain; and a third nucleic acid sequence encoding the DRD. In some aspects, the combination of one or more polynucleotides of a transcription factor system comprises a polynucleotide that comprises the first, second and third nucleic acid sequences. In some aspects, the combination of one or more polynucleotides of a transcription factor system comprises a polynucleotide that comprises two of the first, second and third nucleic acid sequences. In some aspects, the combination of one or more polynucleotides of a transcription factor system comprises a first polynucleotide comprising the first nucleic acid sequence; a second polynucleotide comprising the second nucleic acid sequence; and a third polynucleotide comprising the third nucleic acid sequence. In one aspect, the transcription factor DNA binding domain is operably linked to the DRD. In another aspect, the transcription factor activation domain is operably linked to the DRD. In another aspect, both the transcription factor DNA binding domain and the transcription factor activation domain are operably linked to the DRD. In some aspects, the transcription factor DNA binding domain and the transcription factor activation domain are expressed as a transcription factor fusion protein.

[0093] According to the present disclosure, a transcription factor system encodes a transcription factor that can drive expression of a payload. In some embodiments, the transcription factor is encoded by a first nucleic acid sequence that encodes a transcription factor activation domain and a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site. The transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that activates transcription of the nucleic acid sequence encoding the payload upon binding to the specific polynucleotide binding site.

[0094] In some embodiments, the specific polynucleotide binding site comprises at least one nucleic acid site with a specific sequence that is recognized and bound by the transcription factor DNA binding domain. In some embodiments, the specific polynucleotide binding site comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten nucleic acid sites that are recognized by the DNA binding domain of the present disclosure. In some embodiments, the specific polynucleotide binding site comprises eight nucleic acid sites that are recognized by the DNA binding domain. In some embodiments, the specific polynucleotide binding site comprises two or more tandem nucleic acid sites, each with a specific sequence that is recognized and bound by the transcription factor DNA binding domain. In some aspects, the tandem nucleic acid sites comprise identical nucleic acid sequences. In some embodiments, the specific polynucleotide binding site comprises tandem repeat nucleic acid sites that are recognized by the DNA binding domain of the present disclosure.

[0095] As described herein, a transcription factor or part thereof, is operably linked to a DRD in a transcription factor system of the present disclosure. The presence, absence or an amount of a ligand that binds to or interacts with the DRD, can, upon such binding or interaction modulate the stability of the transcription factor and consequently the function of the transcription factor. Thus, a transcription factor system can exhibit ligand-dependent activity.

[0096] In some embodiments, a transcription factor system is present in a cell or a population of cells. In some embodiments, one or more polynucleotides of a transcription factor system are introduced into a cell or population of cells. Transcription factor system constructs

[0097] The combination of one or more polynucleotides of a transcription factor system may also be referred to herein as a combination of one or more nucleic acid constructs. The polynucleotides or nucleic acid constructs may comprise different arrangements of nucleic acid sequences, and / or may be uniquely combined as part of a transcription factor system, so long as the resulting combination of polynucleotides or nucleic acid constructs comprises (1) one or more nucleic acid sequences that encode a transcription factor that is able to bind to a specific polynucleotide binding site and activate transcription; (2) a nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor is operably linked to the DRD; and (3) a nucleic acid sequence that encodes a payload and is operably linked to an inducible promoter comprising the specific polynucleotide binding site.

[0098] In some embodiments, a transcription factor system comprises multiple constructs. In some embodiments, a transcription factor system comprises a transcription factor construct and a payload construct. In one aspect, the transcription factor construct comprises a nucleic acid sequence that encodes a transcription factor. In one aspect, the transcription factor construct comprises a nucleic acid sequence that encodes a transcription factor activation domain and a nucleic acid sequence that encodes transcription factor DNA binding domain.

[0099] In some embodiments, a transcription factor system comprises a single construct. The single construct comprises nucleic acid sequences encoding the transcription factor, DRD, and payload of the transcription factor system. In some embodiments, such a single construct transcription factor system may be introduced into a cell on a single nucleic acid molecule, such as a plasmid or vector. A transcription factor system comprising a single construct may be referred to herein as a single vector transcription factor system.

[00100] In addition to comprising the nucleic acid sequences described herein for a transcription factor system, nucleic acid constructs of the present disclosure may comprise additional nucleic acid sequences. Additional nucleic acid sequences of constructs include, but are not limited to, regulatory elements, polyadenylation sequences, linkers, and cleavage sites.

[00101] In some embodiments, a transcription factor construct may comprise nucleic acid sequences encoding: a promoter, a transcription factor DNA binding domain, a transcription factor activation domain, and a DRD. In some embodiments, the nucleic acid sequence encoding the DRD is adjacent to a nucleic acid sequence encoding at least one of the transcription factor domains. In some embodiments, the nucleic acid sequence encoding the DRD is positioned between a nucleic acid sequence encoding the transcription factor DNA binding domain and the transcription factor activation domain.

[00102] In some embodiments, a transcription factor construct may comprise nucleic acid sequences encoding: a promoter, a transcription factor DNA binding domain, a transcription factor activation domain, a linker, and a DRD. In some aspects, the linker is positioned between a nucleic acid sequence encoding a transcription factor domain and the nucleic acid sequence encoding the DRD.

[00103] In some embodiments, a promoter in a transcription factor construct is EFla. In some embodiments, the encoded transcription factor DNA binding domain in a transcription factor construct is ZFHD1. In some embodiments, the encoded transcription factor activation domain in a transcription factor construct is p65.

[00104] In some embodiments, a payload construct may comprise nucleic acid sequences encoding: a specific polynucleotide binding site comprising at least one nucleic acid site with a specific sequence recognized and bound by the transcription factor DNA binding domain, a promoter, and payload. An exemplary binding site comprises eight (8) nucleic acid sites that are recognized by a ZFHD1 DNA binding domain.

[00105] In some embodiments, a construct of the present disclosure, such as a transcription factor construct or a payload construct, is integrated into a plasmid or viral vector. In some embodiments, the plasmid or viral vector comprises one or more regulatory elements that become operably linked to one or more components of the construct that is integrated into the plasmid or viral vector. In some embodiments, the plasmid or viral vector comprises regulatory elements well known in the art, including for example promoters, introns, spacers, stuffer sequences, and the like. In some embodiments, a transcription factor construct is integrated into a plasmid or viral vector such that the components of the transcription factor construct are operably linked to regulatory elements of the plasmid or viral vector. In some embodiments, such a transcription factor construct comprises nucleic acid sequences encoding: a transcription factor DNA binding domain, a transcription factor activation domain, and a DRD, and is integrated into the plasmid or viral vector such that a promoter sequence in the plasmid or viral vector drives expression of the transcription factor DNA binding domain, transcription factor activation domain, and DRD. Such a promoter may be selected from a constitutive promoter, a tissue-specific promoter, a cell-specific promoter, a cell differentiation- specific promoter, and / or a disease-specific promoter. Optionally, the promoter may be selected from EFla, CMV, EFS, RSV, SFFV, PGK, CAG, and SV40. Components of transcription factor systems

[00106] As stated above, the polynucleotides or nucleic acid constructs of a transcription factor system may comprise different arrangements of nucleic acid sequences, and / or may be uniquely combined as part of a transcription factor system, so long as the resulting combination of polynucleotides or nucleic acid constructs comprises (1) one or more nucleic acid sequences that encode a transcription factor that is able to bind to a specific polynucleotide binding site and activate transcription; (2) a nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor is operably linked to the DRD; and (3) a nucleic acid sequence that encodes a payload and is operably linked to an inducible promoter comprising the specific polynucleotide binding site. In this way, the transcription factor system is a modular system and each component of the transcription factor system can be selected separately.

[00107] The nucleic acid sequence that encodes a drug responsive domain (DRD) may be selected from sequences of DRDs described in more detail in the “Drug responsive domains (DRDs)” section below.

[00108] The one or more nucleic acid sequences that encode a transcription factor may be selected from one or more sequences that encode an existing transcription factor, an engineered transcription factor that has been derived from an existing transcription factor, or an engineered transcription factor that comprises a DNA binding domain and an activation domain. As used herein, an “engineered transcription factor that has been derived from an existing transcription factor” refers to an engineered transcription factor that originates at least in part from the parent (native) transcription factor molecule or sequence and retains the ability to bind a specific polynucleotide binding site and activate transcription. For example, an engineered transcription factor may be derived from a parent transcription factor comprising one or more zinc finger domains capable of sequence-specific contacts with DNA. An engineered TAL effector transcription factor may be designed to comprise a TAL effector repetitive regions recognizing 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 that comprises a DNA binding domain and an activation domain, both the DNA binding domain and the activation domain may be separately selected and combined to form the complete transcription factor.

[00109] The transcription factor DNA binding domain may be derived from an existing nucleic acid binding protein. For example, a DNA-binding sequence or domain of an existing DNA binding protein may be used as or further modified to generate the transcription factor DNA binding domain of the present disclosure.

[00110] In some aspects, the transcription factor DNA binding domain is derived from a parent protein selected from the group consisting of: ZFHD1, Cas9, Cas12, and TAL.

[00111] In some embodiments, the transcription factor DNA binding domain is derived from a ZFHD1 parent protein. ZFHD] is a zinc finger-homeodomain fusion protein designed by Pomerantz, J.L., et al. (Pomerantz, J. L., et al. “Structure-Based Design of Transcription Factors.” Science, vol. 267, no. 5194, 1995). ZFHD1 comprises fingers 1 and 2 of Zif268, a gly-gly-arg-arg linker, and the OCT-1 homeodomain. ZFHD1 can bind to a nucleic acid sequence comprising the sequence TAATGATGGGCG (SEQ ID NO: 70). In some embodiments, the transcription factor DNA binding domain consists of or comprises the amino acid sequence of ZFHD1.

[00112] In some embodiments, the present disclosure provides methods of regulating target genes and their corresponding functional proteins (e.g., payload or protein of interest) using a Cas / guide RNA system. It is to be understood that one of skill will be able to design suitable guide RNA for forming a co-localization complex with a target nucleic acid including a target gene as described herein.

[00113] Various Cas proteins are known to those of skill in the art and include CasI (Cas3), Cas IA (Cas8a), CasIB (Cas8b), CasIC (Cas8c), CasID (Cas10d), CasIE (Csel), CasIF (Csyl), CasIU, CasIl (Cas9), CaslIA (Csn2), CaslIB (Cas4), CaslIC, CaslIII (Cas10), CasIIIA (Csm2), CasIIIB (Cmr5), CaslIIC, CasIIBD, CaslV (Csfl), CasIVA, CasIVB, CasV (Cpfl), C2¢c2, and C2cl and the like.

[00114] In some embodiments, the transcription factor DNA binding domain is derived from a Cas protein selected from the group consisting of C2C1, C2C3, Cpf1 (also referred to as Cas12a), Casl12b, Casl2c, Casl12d, Casl2e, Cas13a, Casl13b, Casl3c, Cas13d, Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Casl0, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa$, Csn2, Csm2, Csm3, Csm4, Csm$5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, and Csf4.

[00115] According to one aspect, the Cas9 protein includes the sequence as set forth for naturally occurring Cas9 from S. aureus, S. thermophiles, S. pyogenes or Neisseria meningitidis Cas9 and protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein, such as an RNA guided DNA binding protein.

[00116] According to one aspect, the Cas12 protein includes the sequence as set forth for naturally occurring Cas12 from Francisella novicida, Acidaminococcus sp., Lachnospiraceae sp., or Prevotella sp. and protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein, such as an RNA guided DNA binding protein.

[00117] In some embodiments, the transcription factor DNA binding domain is derived from a parent Cas protein, such as a parent Cas9 or Cas12 protein. In some embodiments, the transcription factor DNA binding domain is or comprises a Cas9 that has been modified to lack nuclease activity. In some embodiments, the transcription factor DNA binding domain is or comprises a Cas12 that has been modified to lack nuclease activity.

[00118] Naturally occurring Cas9 comprises 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 (nontarget strand). By mutating both the HNH and RuvC nuclease domains (resulting in the so-called “dead Cas9” or “dCas9”), the resulting dCas9 retains its RNA-guided DNA targeting ability but loses its endonuclease activity. In some embodiments, the transcription factor DNA binding domain is a dCas9 comprising mutated HNH and RuvC nuclease domains. In some embodiments, the transcription factor DNA binding domain is a dCas9 comprising mutated HNH and RuvC nuclease domains and is derived from a parent S. aureus, S. thermophiles, S. pyogenes or Neisseria meningitidis Cas9.

[00119] Naturally occurring Casl12 (e.g., Cas12a and Cas12b) comprises a RuvC-like domain that cleaves DNA. By mutating the RuvC nuclease domain, a catalytically dead Cas12 (having deactivation of DNase activity, also referred to herein as “dCas12”) may be derived from a parent Casl2 protein. In some embodiments, the transcription factor DNA binding domain is or comprises a catalytically dead Cas12 (dCas12).

[00120] In some embodiments, the transcription factor DNA binding domain is derived from a parent protein that is a Type II Cas homolog. Cas9 is an example of a Type II Cas protein. In some embodiments, the transcription factor DNA binding domain is or comprises a Type II Cas homolog that lacks nuclease activity or has been modified to lack nuclease activity. In some embodiments, the transcription factor DNA binding domain is or comprises a Type II Cas homolog comprising mutated HNH and RuvC nuclease domains.

[00121] In accordance with an exemplary embodiment, Cas is altered or otherwise modified to inactivate the nuclease activity. Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain. Such modification includes removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e., the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from the Cas9 DNA binding protein. Other modifications to inactivate nuclease activity will be readily apparent to one of skill in the art. Accordingly, a nuclease-null DNA binding protein includes polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity. The nuclease-null DNA binding protein retains the ability to bind to DNA even though the nuclease activity has been inactivated. Accordingly, the DNA binding protein includes the polypeptide sequence or sequences required for DNA binding but may lack the one or more or all of the nuclease sequences exhibiting nuclease activity. See Jinek et al., (2012) Science 337, 816-821. A Cas9 protein lacking nuclease activity is referred to as a nuclease-null Cas9 (“Cas9Nuc”, “dead Cas9” or “dCas9”) and exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection. According to this aspect, nuclease activity for a Cas9Nuc may be undetectable using known assays, i.e. below the level of detection of known assays.

[00122] In some embodiments, the transcription factor DNA binding domain is derived from a Cas9 parent protein. In some embodiments, the transcription factor DNA binding domain comprises a Cas9 having mutated nuclease domains (referred to as “dead Cas9” or “dCas9”). The resulting dCas9 retains its RNA-guided DNA targeting ability but loses its endonuclease activity. In some embodiments, the transcription factor DNA binding domain is a dCas9.

[00123] The present disclosure provides for the use of guide RNA to target a Cas protein, for example a nuclease-null Cas9 operably linked to a DRD, to a polynucleotide binding sequence as described herein. Such guide RNA can be readily designed by those of skill in the art when knowing the particular polynucleotide binding sequence. A guide RNA may include one or more of a spacer sequence, a tracr mate sequence and a tracr sequence. The term spacer sequence is understood by those of skill in the art and may include any polynucleotide having sufficient complementarity with a polynucleotide binding sequence to hybridize with the polynucleotide binding sequence and direct sequence-specific binding of a CRISPR complex to the polynucleotide binding sequence. The guide RNA may be formed from a spacer sequence covalently connected to a tracr mate sequence (which may be referred to as a crRNA) and a separate tracr sequence, wherein the tracr mate sequence is hybridized to a portion of the tracr sequence. According to certain aspects, the tracr mate sequence and the tracr sequence are connected or linked such as by covalent bonds by a linker sequence, which construct may be referred to as a fusion of the tracr mate sequence and the tracr sequence. The linker sequence referred to herein is a sequence of nucleotides, referred to herein as a nucleic acid sequence, which connect the tracr mate sequence and the tracr sequence. Accordingly, a guide RNA may be a two component species (i.e., separate crRNA and tracr RNA which hybridize together) or a unimolecular species (i.e., a crRNA-tracr RNA fusion, often termed a sgRNA).

[00124] In some embodiments, the guide RNA may be delivered directly to a cell as a native species by methods known to those of skill in the art, including injection or lipofection, or as transcribed from its cognate DNA, with the cognate DNA introduced into cells through electroporation, transient and stable transfection (including lipofection) and viral transduction.

[00125] In some embodiments, a transcription factor system comprises one or more polynucleotides encoding a DRD-regulated transcription factor, wherein the transcription factor comprises a DNA binding domain that is or comprises a nuclease-null Cas9. When the DRD stabilizing ligand is added, the DRD and transcription factor is stabilized and the nuclease-null Cas9 is expressed and available to bind to the guide RNA. Upon binding with the guide RNA, the Cas9- gRNA system binds to the polynucleotide binding sequence which is operably linked to the protein of interest. When the Cas9-gRNA system is bound to the polynucleotide binding sequence, the protein of interest gene is transcribed due to the presence of the transcription factor activation domain. Thus, when the regulatable transcription factor expression construct comprises the Cas9- gRNA system, RNA-guided DNA regulation is effected in cells such as human cells by tethering or connecting DRDs to either a nuclease-null Cas9 or to transcription factor activation domains. Accordingly, aspects of the present disclosure include methods and materials for localizing transcriptional regulatory domains to targeted loci by fusing, connecting or joining a DRD to either Cas9Nuc or to a transcription factor activation domain, or both.

[00126] In some embodiments, the transcription factor DNA binding domain is derived from a TAL parent protein. TAL (transcription activator-like) effectors (also referred to as “TALEs”) are proteins secreted by Xanthomonas bacteria to modulate gene expression in host plants and aid bacterial infection. TAL effectors have a repetitive region consisting of tandem repeats of mostly 33 or 34 amino acid residues. Repeat monomers differ from each other mainly in amino acid positions 12 and 13, and there is a strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site. The transcription factor DNA binding domain of the present disclosure may comprise all or a portion of the repetitive region of a TAL effector that is capable of binding to a specific DNA binding site. In some embodiments, the DNA binding domain comprises a synthetic TAL effector capable of recognizing a desired nucleic acid sequence. Methods for assembling custom TAL effectors are readily available to one of skill in the art. An “engineered TAL effector” refers herein to a polypeptide derived from a parent TAL effector protein, a polypeptide comprising the repetitive region of a TAL effector and / or a synthetic TAL effector or region thereof. In some embodiments, the transcription factor DNA binding domain is an engineered TAL effector capable of binding to a specific nucleic acid site.In some embodiments, the transcription factor DNA binding domain is derived from a zinc finger protein parent protein. In some embodiments, the parent zinc finger protein may a C2H2 zinc finger protein. In some embodiments, the transcription factor DNA binding domain may comprise one or more zinc finger domains that make sequence-specific contacts with DNA. In some embodiments, the transcription factor DNA binding domain may comprise at least two zinc finger domains, at least three zinc finger domains, at least four zinc finger domains, or at least five zinc finger domains that form a zinc finger array capable of specifically recognizing a DNA site. In some embodiments, the transcription factor DNA binding domain comprises a three-finger array. An engineered DNA binding domain comprising one or more zinc finger domains is referred to herein as an “engineered zinc finger binding protein”.

[00127] In some embodiments, the transcription factor DNA binding domain may be selected from an engineered zinc finger binding protein, engineered TAL effector, or other natural or engineered DNA binding domain.

[00128] Zinc finger and TALE DNA binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition region of a naturally occurring zinc finger or TALE protein. Therefore, engineered DNA binding proteins (zinc fingers or TALES) are proteins that are non-naturally occurring. Non- limiting examples of methods for engineering DNA-binding proteins are design and selection. A designed DNA binding protein is a protein not occurring in nature whose design / composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and / or TALE designs and binding data. See, for example, U.S. Pat. Nos. 8,586,526; 6,140,081; 6,453,242; 6,534,261 and 8,586,526; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060, WO 02 / 016536 and WO 03 / 016496, the disclosures of these references as it pertains to the design and selection of DNA binding proteins derived from existing ZFP and / or TALE proteins and binding data related thereto, is incorporated by reference herein it their entireties.

[00129] The activation domain of an engineered transcription factor according to the present disclosure may be derived from a region or domain of an existing transcription factor. In some embodiments the activation domain is a region of an existing transcription factor that is capable of transcription 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-kf p65 protein (referred to herein as “p65”). In some embodiments, the activation domain comprises the carboxy terminal region of the human transcription factor NF- KP p65 protein.

[00130] A consideration in the design of the transcription factor system provided herein is that the encoded transcription factor is able to bind to a specific polynucleotide binding site and that the nucleic acid sequence that encodes the payload is operably linked to an inducible promoter comprising the specific polynucleotide binding site. In various embodiments, the inducible promoter is an exogenous inducible promoter. Pairs of transcription factors (including engineered transcription factors) and their corresponding polynucleotide binding sites are known in the art. Also known are DNA binding domains of DNA binding proteins along with their corresponding polynucleotide binding sites and method for identifying new DNA binding domain sequences and corresponding polynucleotide binding sites that can be used for the design of synthetic transcription factors and corresponding synthetic promoters. For example, Khalil A.S., et al. provide zinc finger arrays that can be used as core building blocks for constructing synthetic transcription factors and further provide corresponding nucleic acid binding sequences that can be inserted within synthetic promoters and recognized by the zinc finger arrays (Khalil A.S., et al. Ce / / 2012, 150, 647-658, incorporated by reference in its entirety). Khalil A.S., et al. also identify synthetic transcription factor-promoter pairs and provide design strategies to modify transcriptional output by changing the promoters (e.g, multimerizing zinc finger binding sequences to create promoters with repeat operators) and changing the synthetic transcription factors (e.g., by creating variants). Any of the transcription factor-promoter pairs or engineered zinc finger arrays and their corresponding nucleic acid binding sites disclosed by Khalil A.S., et al. can be used for the transcription factor systems of the present disclosure. As an example, Figure 3A of Khalil A.S., et al. provides a library of amino acid residues of the recognitions helices for zinc finger arrays and the corresponding DNA binding sequences which can be used in the design of a transcription factor DNA binding domain and the specific polynucleotide binding site of the present disclosure. One of skill in the art would be able to modify the transcription factors or zinc finger array sequences provided by Khalil, A.S., et al. by cloning the sequences of these transcription factors or arrays into the constructs of a transcription factor system provided herein. As another example, Zhang, F., et al. describe methods for design and production of engineered TAL effectors with corresponding nucleic acid binding sites. These can be used in the preparation of engineered transcription factors and their specific polynucleotide binding site. Any of the TAL effectors provided by Zhang, F., et al. may be used to prepare a transcription factor DNA binding domain in a transcription factor system of the present disclosure. For example, Zhang, F., et al. disclose construction of 17 artificial TAL effectors to target specific DNA binding sites and also provide in Figure 2a the sequences of TAL effector repeat regions and corresponding nucleic acid binding sequences. The TAL effectors or DNA binding parts thereof disclosed in Zhang, F., et al. can be used to construct the DNA binding domains as well as the corresponding nucleic acid binding sequences for the inducible promoters of the present disclosure. One of skill in the art would recognize that there are several options for selection and design of the DNA binding domains of the present disclosure. In addition to selection of recognized DNA binding proteins and domains known in the art, the DNA binding domain 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 Cys;His: zinc finger protein framework is available to the skilled artisan (Pabo, C.O., et al. Annu. Rev. Biochem. 2001. 70:313—40).

[00131] In some embodiments, the inducible promoter that is operably linked to the nucleic acid sequence that encodes the payload comprises a minimal promoter (also referred to as a “min promoter” or “core promoter”) and the specific polynucleotide binding site. In this scenario, both the minimal promoter and the specific polynucleotide binding site are operably linked to the nucleic acid sequence that encodes the payload. The term “minimal promoter” refers to a minimal structure that enables the formation of the initiation complex. A minimal promoter may comprise an RNA polymerase binding site, TATA box and transcription start site. A minimal promoter may be coupled with one or more response elements (such as enhancers or transcription factor binding sites) to generate an inducible promoter. Additional details regarding minimal promoters and coupling of minimal promoters to response elements is provided by Ede and colleagues (Ede et al., ACS Synth Biol. 2016 May 20; 5(5): 395-404). In some embodiments, the inducible promoter of a transcription factor system or component thereof of the present disclosure comprises a minimal promoter selected from the following minimal promoters: minCMV, CMV53 (minCMV with the addition of an upstream GC box), minSV40 (minimal simian virus 40 promoter), miniTK (the —33 to +32 region of the Herpes simplex thymidine kinase promoter), MLP (the —38 to +6 region of the adenovirus major late promoter), pJB42CATS (a minimal promoter derived from the human junB gene), YB_TATA (a synthetic minimal promoter developed by Benenson and colleagues (Hansen, J. et al. Proc Natl Acad Sci USA. 2014; 111:15705-15710)), and the TATA box alone.

[00132] As described above, the specific polynucleotide binding site may comprise at least one nucleic acid site with a specific sequence that is recognized and bound by the transcription factor DNA binding domain. In some embodiments, the specific polynucleotide binding site comprises two or more nucleic acid sites, each with a specific sequence that is recognized and bound by the transcription factor DNA binding domain. Pairing of DNA binding domains with their corresponding polynucleotide binding sites is discussed above.

[00133] The nucleic acid sequence that encodes a payload may be selected to encode any payload or protein of interest. Additional details regarding payloads are provided in the “Payloads” section below.

[00134] Exemplary nucleic acid constructs that may be used individually (as a single construct) or in combination as part of a transcription factor system are described in Table 1. 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[00135] Additional illustrative constructs comprising structurally different transcription factor components are provided in Table 2. An asterisk (“*”) in Table 2 indicates the translation of a stop codon. Corresponding control constructs not comprising a regulated transcription factor as well as separate construct components are also provided. As indicated in the description for constructs cjun- 001 and cjun-002, a peptide linker is positioned between the CA2 component and the c-Jun component of each construct. Additionally, all constructs comprise a P2A peptide. Characterization of ligand-dependent activity of a transcription factor system

[00136] Ligand-dependent activity of a transcription factor system may be characterized by various methods.

[00137] In some embodiments, ligand-dependent activity of a transcription factor system is characterized by ligand-dependent regulation of a transcription factor polypeptide (for example, a transcription factor DNA binding domain, a transcription factor activation domain or both the transcription factor DNA binding domain and transcription factor activation domain), encoded by the transcription factor system. In some embodiments, ligand-dependent activity of a transcription factor system is characterized by ligand dose-dependent regulation of a transcription factor polypeptide encoded by the transcription factor system. In one aspect, a transcription factor polypeptide is a polypeptide comprising a transcription factor activation domain. In another aspect, a transcription factor polypeptide is a polypeptide comprising a transcription factor DNA binding domain. In another aspect, a transcription factor polypeptide is a polypeptide comprising both a transcription factor activation domain and a transcription factor DNA binding domain. Ligand- dependent regulation of a transcription factor polypeptide may be characterized by various methods. In some aspects, ligand-dependent regulation of a transcription factor polypeptide may be assessed by measuring the levels of the transcription factor polypeptide or domain thereof, such as by an immunoassay.

[00138] In some embodiments, ligand-dependent activity of a transcription factor system is characterized by ligand-dependent expression of the payload encoded by the transcription factor system. Expression of the payload may be assessed by various methods. In some aspects, expression of the payload is assessed by measuring payload mRNA levels. In some aspects, expression of the payload is assessed by measuring payload polypeptide levels.

[00139] In some embodiments, a transcription factor system may be compared to a control transcription factor system that lacks a DRD. In some embodiments, ligand-dependent activity of a transcription factor system may be analyzed or characterized relative to the activity of a transcription factor system comprising a control transcription factor construct that lacks a DRD. An example of a control transcription factor construct is construct ZFHD-004, which is described by the present disclosure (as shown in Table 1). Transcription Factors

[00140] A transcription factor is a protein that binds to DNA, preferably to a sequence-specific site on the DNA (a transcription factor polynucleotide binding site) located in or near a promoter, which facilitates the binding of the transcription machinery to the promoter, thus activating transcription of the DNA sequence. Such entities are also known as transcription regulator proteins.

[00141] In various embodiments, a transcription factor for use in the transcription factor system, compositions and methods described herein includes 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 a 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 transcription regulatory elements.

[00142] In some embodiments, transcription factors are exemplified as proteins that recognize and bind to specific short DNA sequences and thereby causally affect gene expression. The recognition of DNA sequences by transcription factors occurs by chemical interactions of the amino acid side chains of a transcription factor protein with base pair residues of the DNA that functions as regulatory sequence. The transcription factors thus “read” the genomic sequence, which mechanism provides the sequence recognition function on which informational aspects of regulatory transactions controlling gene expression depend.

[00143] Transcription factors typically consist of DNA-binding domains and effector or activation domains that mediate interactions with other proteins necessary for transcription, including with other transcription factors. Transcription factors execute many functions, including gene activation. They are transcribed in the nucleus, translated in the cytoplasm, and find their target sites in the genomic DNA on reentry into the nucleus, mediated by nuclear localization sites included in all transcription factor protein sequences. Transcription factors include basic domains which cause them to be concentrated nonspecifically in the vicinity of the DNA, facilitating the diffusion-limited discovery of their target sites.

[00144] In various embodiments of the present disclosure, a transcription factor system utilizes a transcription factor made up of and / or comprising a transcription factor DNA binding domain and a transcription factor effector or activation domain or protein (used interchangeably herein). The transcription factor activation domain, the transcription factor DNA binding domain, and / or the combination of the transcription factor activation domain and the transcription factor DNA binding domain may be operably linked to the DRD (any of which is a DRD-TF). Upon stabilization of the linked DRD through binding of an exogenous stabilizing ligand, the stabilized DRD-TF is able to transcribe a protein of interest.

[00145] The DNA sequence that a transcription factor DNA binding domain binds to is called a transcription factor-binding site or response element, or as used herein interchangeably, a specific polynucleotide binding site; these binding sites are found in or near the promoter of the regulated DNA sequence. A promoter comprising a specific polynucleotide binding site may be an exogenous promoter. In some embodiments, a promoter may be an exogenous inducible promoter. The transcription factor-binding site or specific polynucleotide binding site when incorporated into a transcription factor system containing a protein of interest or payload, is an exogenous nucleic acid sequence.

[00146] In various embodiments of the present disclosure, suitable transcription factors useful in the synthesis of transcription factor system can include any known transcription factor for which the transcription factor-binding site is known. Some examples of such transcription factors include (but are not limited to) the STAT family (STATs 1, 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 transcription factors from the following families: bHLH, bZIP, Forkhead, Nuclear receptor, HMG / Sox, Ets, T-box, AT hook, , Homeodomain + POU, Myb / SANT, THAP finger, CENPB, E2F, BED 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 a parent protein selected from the group consisting of: ZFHD1, Cas9, Cas12, and TAL.

[00147] In various embodiments, the transcription factor system provides for the tunable transcription of a protein of interest or payload (used interchangeably herein). In various embodiments, the nucleic acid sequence encoding the protein of interest is operably linked to an exogenous inducible promoter comprising a specific polynucleotide binding site, that is, a defined DNA polynucleotide sequence, that specifically binds to the transcription factor DNA binding domain. The transcription factor binding domain, in combination with the transcription factor DNA activation domain, is then able to regulate transcription of the protein of interest.

[00148] When a cell or organism comprising a DRD-TF is exposed to an exogenous stabilizing ligand, the DRD-TF is stabilized. The stabilized DRD-TF is then able to bind to the specific polynucleotide binding site to which the DRD-TF binds, and thus regulate transcription of the polynucleotide encoding the protein of interest. In some embodiments, the binding of the stabilized DRD-TF activates transcription of the polynucleotide encoding the protein of interest, which results in protein expression in the cell or organism. In the absence of the exogenous stabilizing ligand, the DRD-TF is degraded and unable to activate transcription. Thus, both the amount and the timing of protein expression can be controlled by administering the exogenous stabilizing ligand to the cell or organism.

[00149] In various embodiments, the transcription factor DNA binding domain, the transcription factor activation domain, are typically operably linked or may be separated by one or more intervening sequences, for example, a linker or a cleavage site. In various embodiments, a first polynucleotide may include a first nucleic acid sequence that encodes a transcription factor DNA binding domain; a second nucleic acid sequence that encodes a transcription factor activation domain; and a third nucleic acid sequence that encodes a drug responsive domain (DRD). In such embodiments, the transcription factor activation domain and / or the transcription factor DNA binding domain, upon expression in the cell, is operably linked to the DRD. In addition, the cell will also include a second polynucleotide that comprises a fourth nucleic acid sequence that can be specifically bound by the transcription factor DNA binding domain and a fifth nucleic acid sequence that encodes a protein of interest or payload as described herein.

[00150] The transcription factor DNA binding domain, the transcription factor activation domain and the protein of interest or payload may be supplied for the methods of the present disclosure on the same vectors or in separate vectors.

[00151] In some embodiments, a vector comprises the polynucleotides described herein. In some embodiments, the vector comprises at least a first nucleic acid sequence that encodes at least one of a transcription factor DNA binding domain and a transcription factor activation domain; and a second nucleic acid sequence that encodes a drug responsive domain (DRD); wherein the transcription factor DNA binding domain and / or the transcription factor activation domain is operably linked to the DRD. Optionally, in some embodiments, a first vector comprises the transcription factor linked to the DRD, and a second vector comprises a protein of interest or payload operably linked to a transcription factor polynucleotide binding site. In a further embodiment, a single vector comprises a first nucleic acid sequence encoding a transcription factor able to bind to a specific polynucleotide binding site and activate transcription; a second nucleic acid sequence encoding a drug responsive domain (DRD); wherein the transcription factor is operably linked to the DRD and optionally a third nucleic acid sequence encoding a protein of interest operably linked to an inducible promoter comprising the transcription factor polynucleotide binding site. In some embodiments, a first vector comprises at least a first nucleic acid sequence that encodes at least one of a transcription factor DNA binding domain and a transcription factor activation domain; and a second nucleic acid sequence that encodes a drug responsive domain (DRD); wherein the transcription factor DNA binding domain and / or the transcription factor activation domain is operably linked to the DRD, and a 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 that encodes a protein of interest or payload as described herein.

[00152] In some embodiments, the vectors also possess an origin of replication (ori) which permits amplification of the vector, for example in bacteria. Additionally, or alternatively, the vector includes selectable markers such as antibiotic resistance genes, genes for colored markers and suicide genes. Drug responsive domains (DRDs)

[00153] Drug responsive domains (DRDs) are protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a corresponding DRD-binding ligand. The term drug responsive domain (DRD) is interchangeable with the term destabilizing domain (DD). Drug responsive domains (DRDs) can be appended to a polypeptide or protein and can render the attached polypeptide or protein unstable in the absence of a DRD-binding ligand. DRDs convey their destabilizing property to the attached polypeptide or protein via protein degradation. Without wishing to be bound by any theory, in the absence of a DRD-binding ligand, the appended polypeptide or protein is rapidly degraded by the ubiquitin-proteasome system of a cell. A ligand that binds to or interacts with a DRD can, upon such binding or interaction, modulate the stability of the appended polypeptide or protein. When a ligand binds its intended DRD, the instability is reversed and function of the appended polypeptide or protein can be restored. The conditional nature of DRD stability allows a rapid and non-perturbing switch from stable protein to unstable substrate for degradation. Moreover, its dependency on the concentration of its ligand further provides tunable control of degradation rates.

[00154] In some embodiments, DRDs of the present disclosure may be derived from known polypeptides that are capable of post-translational regulation of proteins. In some embodiments, DRDs of the present disclosure may be developed or derived from known proteins. Regions or portions or domains of wild type proteins may be utilized as DRDs in whole or in part. They may be combined or rearranged to create new peptides, proteins, regions or domains of which any may be used as DRDs or the starting point for the design of further DRDs.

[00155] In some embodiments, a DRD may be derived from a parent protein or from 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); PDES (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: Proteins and their binding ligands Shield-1 Sildenafil; Vardenafil; Tadalafil Protein Protein Sequence Protein | Ligands SEQ ID NO.: E. coli MISLIAALAVDRVIGMENAMPWNLPADL * Methotrexate Dihydrofolate AWFKRNTLNKPVIMGRHTWESIGRPLPGR MTX) reductase KNIILSSQPGTDDRVTWVKSVDEAIAACG Trimethoprim (ecDHFR) DVPEIMVIGGGRVYEQFLPKAQKLYLTHI (TMP) (Uniprot ID: DAEVEGDTHFPDYEPDDWESVFSEFHDA POABQ4) DAQNSHSYCFEILERR Human MVGSLNCIVAVSQNMGIGKNGDLPWPPL | 2 Methotrexate Dihydrofolate RNEFRYFQRMTTTSSVEGKQNLVIMGKK MTX) reductase TWFSIPEKNRPLKGRINLVLSRELKEPPQG Trimethoprim (hDHFR) AHFLSRSLDDALKLTEQPELANKVDMVW (TMP) (Uniprot ID: IVGGSSVYKEAMNHPGHLKLFVTRIMQDF P00374) ESDTFFPEIDLEKYKLLPEYPGVLSDVQEE KGIKYKFEVYEKND Human FKBP GVQVETISPGDGRTFPKRGQTCVVHYTG | 3 Shield-1 (FK506 binding MLEDGKKFDSSRDRNKPFKFMLGKQEVI protein) (Uniprot | RGWEEGVAQMSVGQRAKLTISPDYAYGA ID: P62942) TGHPGIIPPHATLVFDVELLKLE Phosphodiesteras | MEETRELQSLAAAVVPSAQTLKITDFSFS | 4 Sildenafil; ¢5 (PDE5), DFELSDLETALCTIRMFTDLNLVQNFQMK Vardenafil; ligand binding HEVLCRWILSVKKNYRKNVAYHNWRHA Tadalafil domain (Uniprot | FNTAQCMFAALKAGKIQNKLTDLEILALL ID: Uniprot ID IAALSHDLDHRGVNNSYIQRSEHPLAQLY 076074) CHSIMEHHHFDQCLMILNSPGNQILSGLSI EEYKTTLKIIKQAILATDLALYIKRRGEFFE LIRKNQFNLEDPHQKELFLAMLMTACDLS ATTKPWPIQQRIAELVATEFFDQGDRERKE LNIEPTDLMNREKKNKIPSMQVGFIDAICL QLYEALTHVSEDCFPLLDGCRKNRQKWQ ALAEQQ Phosphodiesteras | MERAGPSFGQ QRQQQQPQQQKQQQR | 71 Sildenafil; e 5 (PDES), full- | DQDSV EAWLDDHWDF TFSYFVRKAT Vardenafil; length (Uniprot REMVNAWFAERVHTIPV CKE GIRGH Tadalafil ID: Uniprot ID TESCS CPLQQSPRAD NSAPGTPTRK 076074) ISASEFDRPL RPIVVKDSEGTVSFLSDSE K | KEQMPLTPPR FDHDEGDQCS RLLELV KDIS SHLDVTALCH KIFLHIHGL ISADR YSLFLV CEDSSNDKFL ISRLFD VAEG STLEEVSNNC IRLEWNKGIV GHVAAL GEPLNIKDAYEDPR FNAEVDQITG YKTQSILCMP IKNHREEVVG VAQAI NKK | SG NGGTFTEKDEKDFAAYLAFC GIVLH NAQLY ETSLLENKRN QVLLDLAS LI FEEQQSLEVI LKKIAATIISFM QVQK CTIF | IVDEDCSDSF SSVFHMECEE LEKSS DTLTR EHDANKINYM YAQYVKN TME PLNIPDVSKD KRFPWTTENT GNVNQQC IRS LLCTPIKNGK KNKVIGVCQL VNK MEENTGKVKPFNRND EQ FLEAFVIFCG LGIQNTQMYE AVERAMAKQM VTLEVLS YHA SAAEEETRELQSLAAAV VPS AQTL KITDFS FSDFELSDLE TALCTIRMFT DLN LVQNFQM KHEVLCRWILSVKKNYR KNV Carbonic anhydrase IT (CA2) (Uniprot ID: P00918) Bazedoxifene Raloxifene AYHNWRHAFN TAQCMFAALK AGKIQ NKLTD LEILALLIAA LSHDLDHRG VNNS | YIQRSEH PLAQLYCHSI MEHHHFDQCL MILNSPGNQI LSGLSIEEYK TTLKIIKQA ILATDLALYIK RRGEFFELIR KNQFNLE DPH QKELFLAMLM TACDLSAITK PWPIQQRIAELVATEFFDQG DRERKE LNIE PTDLMNREKK NKIPSMQVGF ID AICLQLYE ALTHVSED CFPLLDGC RK NR | KWQALAEQQ EKMLINGESG QAKRN Carbonic MSHHWGY GKHNGPEHWHKDFPIAKGER | 5 Celecoxib anhydrase IT QSPVDIDTHTAKYDPSLKPLSVSYDQATS Acetazolamide (CA2) (Uniprot LRILNNGHAFNVEFDDSQDKAVLKGGPL ID: P00918) DGTYRLIQFHFHWGSLDGQGSEHTVDKK KYAAELHLVHWNTKYGDFGKAVQQPDG LAVLGIFLKVGSAKPGLQKVVDVLDSIKT KGKSADFTNFDPRGLLPESLDYWTYPGSL TTPPLLECVTWIVLKEPISVSSEQVLKFRK LNFNGEGEPEELMVDNWRPAQPLKNRQI KASFK (Human estrogen | MTMTLHTKASGMALLHQIQGNELEPLNR | 6 Bazedoxifene receptor (ER) PQLKIPLERPLG EVYLDSSKPA VYNYPEG Raloxifene Uniprot ID: AAYEFNAAAAANA QVYGQTGLPYGP P03372.2) GSEAAAFG SNGLGGFPPLNSVSPSPLML LHPPPQLSPFLQPHGQQVPY YLENEPS GYTVREAGPPAFY RPNSDNRRQGGRERL | ASTND KGSMAMESAKETRYCAVCND YASGYHYGVWSCEGCKAFFK RSIQGHN DYMCPATNQCTID KNRRKSCQACRLRK CYEVGM MKGGIRKDRRGGRMLKHKRQ | RDDGEGRGEVGSAGDMRAAN LWPSPL MIKRSKKNSLALSL TADQMVSALL DAEPPILYSE YDPTRPFSEASMMGLL TNLA DRELVHMINW AKR VPGFVDL TLHDQVHLLE CAWLEILMIGLVWRSM EHPG KLLFAPNLLL DRNQGKCVEG MVEIFDMLLATSSRFRMMNLQGEEFV CLKSIILLNSGVYT FLSSTLKSLEEKDHI HRVLDKITDTLIHLM AKAGLTLQQQ HQRLAQLLLI LSHIRHMSNKGMEH LYSMKC KNVVPLYDLLLEMLDAHRLH APTSRGGASV EETDQSHLATAGSTSS HSLQKYYITGEAEG FPATV

[00156] In some embodiments, the sequence of a protein used to develop DRDs may comprise all, part of, or a region thereof of a protein sequence in Table 3. In some embodiments, proteins that may be used to develop DRDs include isoforms of proteins listed in Table 3. hPDES5 DRDs

[00157] In some embodiments, a DRD of the present disclosure is derived from hPDES. In some embodiments, a DRD of the present disclosure is derived from hPDES isoform 2. In some embodiments, a DRD of the present disclosure is derived from hPDES isoform 3. In some embodiments, a DRD of the present disclosure is derived from hPDES isoform X1.

[00158] In some embodiments, a DRD of the present disclosure is derived from a cGMP-specific 3°,5’-cyclic phosphodiesterase (WPDES) comprising the amino acid sequence of SEQ ID NO. 71.

[00159] In some embodiments, a DRD of the present disclosure may include the whole hPDES (SEQ ID NO. 71). In some embodiments, DRDs derived from hPDES may comprise the catalytic domain of hPDES (e.g., 535-860 of SEQ ID NO. 71). In some embodiments, hPDE5 DRDs of the present disclosure may include a methionine at the N terminal of the catalytic domain of hPDES, i.e. amino acids 535-860 of hPDES wild-type (WT).

[00160] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a cGMP-specific 3°,5’-cyclic phosphodiesterase (WPDES5; SEQ ID NO. 71), and further comprises a mutation in the amino acid at position 732 (R732) of SEQ ID NO. 71. In some embodiments, the mutation in the amino acid at position 732 (R732) is selected from the group consisting of R732L, R732A, R732G, R732V, R732, R732P, R732F, R732W, R732Y, R732H, R732S, R732T, R732D, R732E, R732Q, R732N, R732M, R732C, and R732K.

[00161] In some embodiments, a hPDES DRD of the present disclosure may further comprise one or more mutations independently selected from the group consisting of H653A, F736A, D764A, D764N, Y612F, Y612W, Y612A, W8S3F, I821A, Y829A, F787A, D656L, Y728L, M6251, E535D, E536G, Q541R, KS55R, FS59L, FS61L, F564L, F564S, KS91E, N587S, K604E, K60SE, N609H, K630R, K633E, N636S, N661S, Y676D, Y676N, C677R, H678R, D687A, T7128, D724N, D724G, L738H, N7428, A762S, D764G, D764V, ST66F, K795E, L797F, I799T, T802P, S815C, M816A, I824T, C8398, K852E, S560G, V585A, I599V, 1648V, S663P, L675P, T711A, F744L, L746S, F755L, L804P, M816T, and F840S.

[00162] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a c¢GMP-specific 3°,5’-cyclic phosphodiesterase (lPDE5; SEQ ID NO. 71), and further comprises a mutation in the amino acid at position 732 (R732) of SEQ ID NO. 71. In some such embodiments, the DRD further comprises (i) a mutation in the amino acid at position 764 (D764) of SEQ ID NO. 71, wherein the mutation at D764 is selected from D764N and D764A; (ii) a mutation in the amino acid 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 F736A mutation in the amino acid at position 736 (F736) of SEQ ID NO. 71; or (iv) an H653A mutation in the amino acid at position 653 (H653) of SEQ ID NO. 71.

[00163] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a cGMP-specific 3’,5’-cyclic phosphodiesterase (WPDES; SEQ ID NO. 71), and further comprises a mutation in the amino acid at a position relative to SEQ ID NO. 71, the mutation selected from the group consisting of: W853F, I821A, Y829A, F787A, F736A, D656L, Y728L, M6251, and H653A.

[00164] In some embodiments, a hPDES DRD of the present disclosure may comprise one or more mutations independently selected from the group consisting of T537A, E539G, V548E, D558G, F5598, E565G, C574N, R577Q, R577W, N583S, Q586R, Q589L, K591R, K591R, L595P, CS96R, W615R, F619S, Q623R, K6331, Q635R, N6368S, T639S, D640N, E642G, 1643T, L646S, A649V, A650T, S652G, H653A, D654G, V660A, V660A, L672P, A673T, C677Y, M681T, E682G, H685R, F686S, Q688R, M691T, S695G, G697D, $7021, 1706T, E707K, Y709H, Y709C, 1715V, I1720V, A722V, D724G, Y728C, K730E, R732L, L7381, 1739M, K741N, K741R, F744L, D748N, K752E, K752E, K752E, E753K, L756V, M758T, M760T, A762V, C763R, D764N, D764N, 1774V, L781F, L781P, E785K, R794G, M80ST, R807G, K812R, I813T, I813T, M816R, Q817R, V818A, F820S, 1821V, C825R, Y829C, E830K, L832P, S836L, C846Y, C846S, L856P, L856P, A8S7T, or E8S8G.

[00165] In some embodiments, a hPDES DRD of the present disclosure may comprise two mutations independently selected from ES36K, 1739W; H678F, S702F; E669G, 1700T, G6328, 1648T; T639S, M816R; Q586R, D724G; E5S39G, L7381; L672P, S836L; M691T, D764N; I720V, F820S; E682G, D748N; $652G, Q688R; Y728C, Q817R; H653, R732L; L595P, K741R; R732D, F736S; R732E, F736D; R732V, F736G; R732W, 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.

[00166] In some embodiments, a hPDES DRD of the present disclosure may comprise two mutations independently selected from Q623R, D654G, K741N; A673T, L756V, C846Y; E642G, G697D, I813T; C677Y, H685R, A722V; Q635R, E753K, I813T; Y709H, K812R, L832P; N583S, K752E, C8468; K591R, 1643T, L856P; F619S, V818A, Y829C, and F559S, Y709C, M760T. In some embodiments, a hPDES DRD of the present disclosure may comprise two mutations independently selected from S695G, E707K, 1739M, C763R; A649V, A650T, K730E, E830K; and R577W, W615R, M805T, I821V.

[00167] In some embodiments, a hPDES5 DRD of the present disclosure may comprise multiple mutations independently selected from V660A, L781F, R794G, C825R, E858G; T537A, D558G, I706T, F744L, D764N; R577Q, C596R, V660A, 1715V, E785K, L856P; and V548E, Q3589L, K6331, M681T, $7021, K752E, L781P, A857T. hDHFR DRDs

[00168] In some embodiments, a DRD of the present disclosure is derived from a human dihydrofolate reductase (nDHFR) protein such as, but not limited to, human dihydrofolate reductase 1 (hDHFR1), human dihydrofolate reductase 2 (nDHFR2), or a fragment or variant thereof.

[00169] In some embodiments, the DRD may be derived from a hDHFR protein and include at least one mutation. In some embodiments, the DRD may be derived from a hDHFR protein and include more than one mutation. In some embodiments, the DRD may be derived from a hDHFR protein and include two, three, four or five mutations.

[00170] In some embodiments, a DRD of the present disclosure may include the whole hDHFR (SEQ ID NO. 2). In some embodiments, DRDs derived from hDHFR may comprise amino acids 2- 187 of the parent hDHFR sequence (e.g., amino acids 2-187 of SEQ ID NO. 2). This is referred to herein as an hDHFR M1del mutation.

[00171] In some embodiments, a DRD of the present disclosure comprises a region of or the whole hDHFR (SEQ ID NO. 2), and further comprises a mutation relative to SEQ ID NO. 2 selected from 117V, F598, N65D, K81R, Y122I, N127Y, M1401, K185E, N186D, and M1401.

[00172] In some embodiments, a DRD of the present disclosure comprises a region of or the whole hDHFR (SEQ ID NO. 2), and further comprises two or more mutations relative to SEQ ID NO. 2.

[00173] In some embodiments, a hDHFR DRD of the present disclosure comprises two or more mutations selected from (A10V, H88Y); (C7TR / 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; Y122I); (V121A, Y122I); (Y1221, K174N); (Y122I, E162G); (A125F, Y122I); (N127Y, Y122I); (HI31R / E144G); (E162G / T176F); (K55R, N65K, Y122I); (Q36E, Q103H, Y122I); (Q36F, N65F, Y122I); and (V110A / V136M / K177R).

[00174] In some embodiments, a hDHFR DRD of the present disclosure comprises two or more mutations selected from (I117V, Y122I); (G21T, Y122N); (Q36H, Y122I); (Q36K, Y122I); (Q36R, Y1221I); (Q368, Y122I); (Q36T, Y122I); (N65H, Y122I); (N65L, Y122I); (N65R, Y122I); (N65W, Y1221I); (L74N, Y122I); (Q103E, Y122I); (Q103S, Y122I); (N108D; Y122I); (VI121A, Y122I); (Y1221, K174N); (Y1221, E162G); (A125F, Y122I); (N127Y, Y122I); (K55R, N65K, Y122I); (Q36E, Q103H, Y122I); and (Q36F, N65F, Y1221).

[00175] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human dihydrofolate reductase (nDHFR; SEQ ID NO. 2), and further comprises a Y122I mutation in the amino acid at position 122 (Y122) of SEQ ID NO. 2. In some such embodiments, the DRD further comprises: (i) a Q36K mutation in the amino acid at position 36 (Q36) of SEQ ID NO. 2; (ii) an A125F mutation in the amino acid at position 125 (A125) of SEQ ID NO. 2; or (iii) a N65F mutation in the amino acid at position 65 (N65) of SEQ ID NO. 2 and a substitution of F or K at the amino acid position 36 (Q36) of SEQ ID NO. 2.

[00176] In some embodiments, a hDHFR DRD of the present disclosure may comprise one or more mutations independently selected from the group consisting of M1del, V2A, C7R, I8V, VIA, A10T, A10V, Q13R, N14S, G168S, I17N, 117V, K19E, N20D, G21T, G21E, D228, L238, 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, KS6E, KS6R, T57A, F59S, I61T, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, 172A, 172V, N73G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F89L, R92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, N108D, K109E, K109R, V110A, D111N, M112T, M112V, V113A, W114R, I115V, I115L, V1161, G117D, VI21A, Y122C, Y122D, Y1221, K123R, K123E, A125F, M126], N127R, N127S, N127Y, H128R, H128Y, HI31R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, T137R, R138G, R138, 1139T, 1139V, M1401, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, T147A, F148S, F148L, F149L, P150L, E151G, 1152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, 1176A, 1176F, 1176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.

[00177] In some embodiments, a DRD of the present disclosure comprises hDHFR (C7R, Y163C), hDHFR (E162G, 1176F); 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, 172A), hDHFR (VI21A, Y122I); hDHFR (V75F, Y122I); hDHFR (Y122I, A125F); hDHFR (Y 1221, M1401); hDHFR (Y178H, E181G); hDHFR (Y183H, K185E); hDHFR (Amino acid 2-187 of WT) (G21T, Y122I); hDHFR (Amino acid 2-187 of WT) (I17V, Y122I); hDHFR (Amino acid 2-187 of WT) (L74N, Y122I); hDHFR (Amino acid 2-187 of WT) (L94A, T147A); hDHFR (Amino acid 2-187 of WT) (M53T, R138I); hDHFR (Amino acid 2-187 of WT) (N127Y, Y122I); hDHFR (Amino acid 2-187 of WT) (Q36K, Y122I); hDHFR (Amino acid 2-187 of WT) (V121A, Y122I); hDHFR (Amino acid 2- 187 of WT) (V75F, Y122I); hDHFR (Amino acid 2-187 of WT) (Y122I, A125F); hDHFR (Amino acid 2-187 of WT) (Y 1221, M1401); hDHFR (E31D, F32M, V116I); hDHFR (G21E, 172V, 11767); hDHFR (I8V, K133E, Y163C); hDHFR (K19E, F89L, E181G); hDHFR (L23S, V1214, Y157C); hDHEFR (N49D, F59S, D153G); hDHFR (Q36F, N65F, Y122I); hDHFR (Q36F, Y1221, A125F); hDHEFR (V110A, V136M, K177R); hDHER (V9A, S93R, P150L); hDHFR (Y122I, HI31R, E144G); hDHFR (GS4R, I115L, M140V, S168C); hDHFR (Amino acid 2-187 of WT) (E31D, F32M, V116l); ADHFR (Amino acid 2-187 of WT) (Q36F, N65F, Y122I); hDHFR (Amino acid 2- 187 of WT) (Q36F, Y122I, A125F); hDHFR (Amino acid 2-187 of WT) (Y1221, HI31R, E144G); hDHEFR (V2A, R33G, Q36R, L100P, K185R); hDHFR(D22S, F32M, R338, Q36S, N65S), hDHFR (Amino acid 2-187 of WT) (D228, F32M, R338, Q368, N65S); hDHFR (I17N, L98S, K99R, MI112T, E151G, E162G, E172G); hDHFR (G168, 117V, F89L, D96G, K123E, M140V, D146G, K156R); hDHFR (K81R, K99R, L100P, E102G, N108D, K123R, H128R, D142G, F180L, K185E); hDHEFR (R138G, D142G, F143S, K156R, K158E, E162G, V166A, K177E, Y178C, K185E, N1868S); hDHFR (N14S, P24S, F35L, M53T, K56E, R92G, S93G, N127S, H128Y, F135L, F143S, L159P, L160P, E173A, F180L); hDHFR (F35L, R37G, N65A, L68S, K69E, R71G, L80P, K99G, G117D, L132P, 1139V, M140L 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, 117V, N30D, E31G, Q36R, F598, K69E, I72T, H88Y, F89L, N108D, K109E, V110A, 1115V, Y122D, L132P, F135S, M140V, E144G, T1474, Y157C, V170A, K174R, N186S); hDHFR (L100P, E102G, Q103R, P104S, E105G, N108D, V113A, W114R, Y122C, M1261 N127R, H128Y, L132P, F135P, 1139T, F148S, F149L, I152V, D153A, D169G, V170A, 176A, K177R, V182A, K185R, N186S); and hDHFR (A10T, QI3R, N14S, N20D, P24S, N30S, M38T, T40A, K47R, N49S, K56R, I61T, K64R, K69R, 172A, R78G, E82G, F89L, D96G, N108D, M112V, W114R, Y122D, K123E, 1139V, Q141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y183C, E184G, K185del, D187N). ecDHFR DRDs

[00178] In some embodiments, a DRD of the present disclosure is derived from E. coli dihydrofolate reductase (ecDHFR). In some embodiments, the DRD may be derived from an ecDHEFR protein and include at least one mutation. In some embodiments, the DRD may be derived from an ecDHFR protein and include more than one mutation. In some embodiments, the DRD may be derived from an ecDHFR protein and include two, three, four or five mutations. In some embodiments, the DRD may be derived from an ecDHFR protein and comprise at least one mutation selected from Y100I, F103L, and G121V. In some embodiments, the DRD may be derived from an ecDHFR protein and comprise at least two mutations selected from R12Y,Y100I; R12H,E129K; H12Y,Y100I; H12L,Y100L; R98H,F103S; M42T,H114R; N18T,A19V; and I61F,T68S. FKBP DRDs

[00179] In some embodiments, a DRD of the present disclosure is derived from a FK506 binding protein (FKBP) protein or a fragment or variant thereof. In some embodiments, the DRD may be derived from a FKBP protein and include at least one mutation. In some embodiments, the DRD may be derived from a FKBP protein and include more than one mutation. In some embodiments, the DRD may be derived from an FKBP protein and include two, three, four or five mutations.

[00180] In some embodiments, a DRD of the present disclosure is derived from, in whole or in part, a human FKBP protein (SEQ ID NO. 3) and comprises at least one mutation selected from F36V, F158, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K1051. In some embodiments, a FKBP DRD of the present disclosure comprises more than one mutation selected from F36P, L106P; and E31G, F36V, R71G, K105E. ER DRDs

[00181] In some embodiments, a DRD of the present disclosure is derived from an Estrogen Receptor (ER) protein or a fragment or variant thereof. In some embodiments, the DRD may be derived from an ER protein and include at least one mutation. In some embodiments, the DRD may be derived from an ER protein and include more than one mutation. In some embodiments, the DRD may be derived from an ER protein and include two, three, four or five mutations.

[00182] In some embodiments, a DRD of the present disclosure comprises the ligand binding domain of ER (amino acids 305 to 509 of SEQ ID NO: 6). In some embodiments, a DRD may include at least one mutation relative to the ligand binding domain of ER, wherein the mutation occurs at position 413 (N413) and / or at position 502 (Q502). In some embodiments, the mutation is at position N413 and is N413D, N413T, N413H, N413A, N413Q, N413V, N413C, N413K, N413M, N413R, N413S, N413W, N4131, N413E, N413L, N413P, N413F, N413Y or N413G. In some embodiments, the mutation is at position Q502 and is Q502H, Q502D, QS502E, Q502V, Q502A, Q502T, Q502N, Q502K, Q502S, Q502L, Q502Y, Q502W, Q502F, Q5021, Q502G, Q502P, Q502M, or Q502C. In some embodiments, the DRD comprises mutations at position N413 and at position Q502, wherein the mutation at position N413 is selected from N413D, N413T, N413H, N413A, N413Q, N413V, N413C, N413K, N413M, N413R, N413S, N413W, N4131, N413E, N413L, N413P, N413F, N413Y or N413G and the mutation at position Q502 is selected from Q502H, Q502D, Q502E, Q502V, Q502A, Q502T, Q502N, Q502K, Q502S, Q502L, Q502Y, Q502W, Q502F, Q5021, Q502G, Q502P, Q502M, or Q502C.

[00183] In some embodiments, the at least one mutation is N413D. In some embodiments, the at least one mutation is N413T. In some embodiments, the at least one mutation is Q502H. In some embodiments, the ER DRD comprises at least two mutations and is N413T, Q502H or N413D, Q502H.

[00184] In some embodiments, an ER DRD may further comprise one or more mutations independently selected from L384M, M421G, G521R or Y537S.

[00185] In some embodiments, a DRD of the present disclosure comprises the following: ER (aa 305-549 of WT, L384M, N413F, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413L, M421G, G521R, Y5378), ER (aa 305-549 of WT, L384M, N413Y, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413H, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413Q, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N4131, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413M, M421G, G521R, Y5378S), ER (aa 305-549 of WT, L384M, N413K, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413V, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413S, M421G, G521R, Y5378S), ER (aa 305-549 of WT, L384M, N413C, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413W, M421G, G521R, Y5378), ER (aa 305-549 of WT, L384M, N413P, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413R, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413T, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413A, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413E, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, N413G, M421G, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502F, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502L, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502Y, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502H, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502I, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502M, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502N, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502K, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502V, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502S, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502C, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502W, G521R, Y537S), ER (aa 305- 549 of WT, L384M, M421G, Q502P, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502T, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502A, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502D, G521R, Y537S), ER (aa 305-549 of WT, L384M, M421G, Q502E, G521R, Y537S), and ER (aa 305-549 of WT, L384M, M421G, Q502G, G521R, Y537S). CA2 DRDs

[00186] In some embodiments, a DRD of the present disclosure may be derived from human carbonic anhydrase 2 (hCA2), which is a member of the carbonic anhydrases, a superfamily of metalloenzymes. In some embodiments, the DRD may be derived from a hCA2 protein and include at least one mutation. In some embodiments, the DRD may be derived from a hCA2 protein and include more than one mutation. In some embodiments, the DRD may be derived from an hCA2 protein and include two, three, four or five mutations.

[00187] In some embodiments, a DRD of the present disclosure may be derived from amino acids 1-260 of CA2 (SEQ ID NO. 5). In some embodiments, DRDs are derived from CA2 comprising 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 a CA2 M1del mutation. In one embodiment, DRDs derived from CA2 may comprise amino acids 2-237 of the parent CA2 sequence (e.g., amino acids 2-237 of SEQ ID NO. 5).

[00188] In some embodiments, a DRD of the present disclosure comprises a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises a mutation relative to SEQ ID NO. 5 selected from E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193L and Y5IT.

[00189] In some embodiments, a DRD of the present disclosure comprises a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises a mutation relative to SEQ ID NO. 5 selected from A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, AS54Q, A54V, A54X, A65L, A65N, A65V, A771, A77P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D1101, D129], D138G, D138M, D138N, D161*, D161M, D161V, D164G, D164], D174*, D174T, D179E, D179], D179R, D189G, D189], D19T, D19V, D242G, D242T, D32T, D34T, D41T, D521, D52L, D71F, D71G, D71K, D71M, D718, D71Y, D721, D72S, D72T, D72X, D75T, D75V, D85M, E106D, E106G, E1068, E117*, E117N, E14N, E186*, E186N, E204A, E204D, E204G, E204N, E213*, E213G, E213N, E220K, E220R, E2208, E233D, E233G, E233R, E235%, E235G, E235N, E237K, E237R, E238%, E238N, E238R, E26S, E69D, E69K, E69S, FI130L, F146V, F175, F175L, F175, F178L, F178S, F20L, F208, F225I, F225L, F225S, F225Y, F2301, F230L, F230S, F259L, F259S, F66S, F701, F70L, F95Y, G102D, G104R, G104V, G128R, G12D, G12E, GI31E, G131R, G131W, G139D, G144D, G144V, G1504, G150S, G150W, G155A, G155C, G155D, G155S, G170A, G170D, G182A, G182W, G195A, G195R, G232R, G232W, G234L, G234V, G25E, G63D, G63V, G81E, G81V, G82D, G86A, G86D, G98V, H1071, H107Q, HI19T, H119Y, H122T, H122Y, H15L, HIST, H15Y, H17D, H17L H36I, H36Q, H64M, HO4T, HO6T, I145F, 1145M, 1166H, I166L, 1209D, 1209L, I215H, 1215S, 221, 1255N, 12558, 1338, ISOF, ISON, 1598, I91F, K111E, K111N, K112R, K1131, 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, 1.2021, L202P, L202R, 1.2028, L203P, L203S, L203W, L211* L211A, L211S, L223* 12231, L223V, L228F, L228H, 1.228T, L239*, L239F, L239T, L250*, L250P, L250T, L44*, L44M, L47C, L47V, L57*, L57X, L60S, L79F, L79S, L84W, L90*, LO0V, M240D, M240L, M240R, M240W, N11D, N11K, N124T, N177%, N177T, N229*, N229T, N231D, N23 1F, N23 1K, N231L, N231M, N231Q, N231T, N243Q, N243T, N252E, N252T, N61R, N61T, N61Y, N62K, N62M, N67D, N67T, P137L, P13A, P13H, PI3L, P13S, P154L, P154R, P154T, P180L, P180S, P185L, P185S, P185V, P194Q, P200A, P200L, P200S, P200T, P2014, P201L, P201R, P2018, P214T, P236L, P236T, P246L, P246Q, P249A, P249F, P249H, P2491, P249X, P30L, P30S, P42L, P83A, Q103K, Q135S, Q136N, Q157R, Q157S, Q221A, Q221R, Q248F, Q248L, Q248S, Q254A, Q254K, Q28S, Q53H, Q53K, Q53N, Q74R, QO2H, Q928, R181H, R181S, R181V, R226H, R226P, R226V, R245A, R253G, R253Q, R27A, R58G, R89D, R89F, R89I, R89X, R8OY, S105L, $105Q, S151A, S151I, S151Q, S165F, S165P, S172E, $172V, S1871, S187P, S196H, S196L, S216A, S216Q, S218A, S218Q, S219A, $219Q, S258F, S258P, S29C, S29P, S43P, S43T, S48L, S50P, S56F, S56N, S56P, S56X, S73L, ST3N, $73X, S99H, T108L, T125I T125P, T168K, T168N, T168Q, T176H, T176L, T192D, T192F, T1921, T192N, T192P, T192X, T198D, T1981, T198P, T199A, T199H, T199P, T207D, T207I, T207P, T207S, T35I, T35L, T37Q, T55L, T87L, VI09M, V109W, V121F, V134C, V134F, V142F, V149G, V149L, V159L, V159S, V160C, V160L, V162A, V162C, V206*, V206C, V206M, V210C, V217L, V217R, V217S, V222A, V222C, V222G, V241G, V241W, V241X, V31L, V49F, V6SL, V68W, V78C, W123G, W123R, W16G, W191* W191G, W191L, W208G, W208L, W208S, W244* W244G, W244L, WOTC, W97G, Y114H, Y114M, Y127M, Y190%, Y190L, Y190T, Y193C, Y193F, Y193L, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y5IM, Y5IT, Y51X, Y88T, K9N, and S29A. As used herein “*” indicates the translation of the stop codon and X indicates any amino acid.

[00190] In some embodiments, a DRD of the present disclosure comprises a region of or the whole human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises two or more mutations relative to SEQ ID NO. 5.

[00191] In some embodiments, a DRD of the present disclosure comprises CA2 (aa 2-260 of WT, R27L, H122Y), CA2 (aa 2-260 of WT, T87I, H122Y), CA2 (aa 2-260 of WT, H122Y, N252D), CA2 (aa 2-260 of WT, D72F, V241F), CA2 (aa 2-260 of WT, V241F, P249L), CA2 (aa 2-260 of WT, D72F, P249L), CA2 (aa 2-260 of WT, D71L, L250R), CA2 (aa 2-260 of WT, D72F, P249F), CA2 (aa 2-260 of WT, TS5K, G63N, Q248N), CA2 (aa 2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 of WT, L156H, S2del, H3del, Hadel, W5del), CA2 (aa 2-260 of WT, W4Y, L156H), CA2 (aa 2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 of WT, L156H, F225L), CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R), CA2 (aa 2-260 of WT, G63D, E69V, N231I), CA2 (aa 2-260 of WT, R27L, T871, H122Y, N252D), CA2 (aa 2-260 of WT, D72F, V241F, P249L), CA2 (aa 2-260 of WT, D7IL, T87N, L250R), CA2 (aa 2-260 of WT, L156H, S172C, F178Y, E186D), CA2 (aa 2-260 of WT, A771, P249F), CA2 (aa 2-260 of WT, E106D, C205S), CA2 (aa 2-260 of WT, C205S, W208S), CA2 (aa 2-260 of WT, S73N, R89Y), CA2 (aa 2-260 of WT, D71K, T192F), CA2 (aa 2-260 of WT, S73N, R89F), CA2 (aa 2-260 of WT, G63D, M240L), CA2 (aa 2-260 of WT, V134F, L228F), or CAZ2 (aa 2-260 of WT, S56F, D718).

[00192] In some embodiments, a DRD of the present disclosure comprises CA2 (aa 2-260 of WT, R27L, H122Y), CA2 (aa 2-260 of WT, T87I, H122Y), CA2 (aa 2-260 of WT, H122Y, N252D), CA2 (aa 2-260 of WT, D72F, V241F), CA2 (aa 2-260 of WT, V241F, P249L), CA2 (aa 2-260 of WT, D72F, P249L), CA2 (aa 2-260 of WT, D71L, L250R), CA2 (aa 2-260 of WT, D72F, P249F), CA2 (aa 2-260 of WT, T55K, G63N, Q248N), CA2 (aa 2-260 of WT, L156H, A257del, S258del, F259del, K260del), CA2 (aa 2-260 of WT, L156H, S2del, H3del, H4del, W5del), CA2 (aa 2-260 of WT, W4Y, L156H), CA2 (aa 2-260 of WT, L156H, G234del, E235del, P236del), CA2 (aa 2-260 of WT, L156H, F225L), CA2 (aa 2-260 of WT, D70N, D74N, D100N, L156H), (CA2 (aa 2-260 of WT, I59N, G102R), CA2 (aa 2-260 of WT, G63D, E69V, N231I), CA2 (aa 2-260 of WT, R27L, T871, H122Y, N252D), CA2 (aa 2-260 of WT, D72F, V241F, P249L), CA2 (aa 2-260 of WT, D71L, T87N, L250R), CA2 (aa 2-260 of WT, L156H, S172C, F178Y, E186D), CA2 (aa 2-260 of WT, D71F, N231F), CA2 (aa 2-260 of WT, A771, P249F), CA2 (aa 2-260 of WT, D71K, P249H), CA2 (aa 2-260 of WT, D72F, P249H), CA2 (aa 2-260 of WT, Q53N, N61Y), CA2 (aa 2-260 of WT, E106D, C2058), CA2 (aa 2-260 of WT, C205S, W208S), CA2 (aa 2-260 of WT, S73N, R89Y), CA2 (aa 2-260 of WT, D71K, T192F), CA2 (aa 2-260 of WT, Y193L, K260L), CA2 (aa 2-260 of WT, D7IF, V241F, P249L), CA2 (aa 2-260 of WT, L147F, Q248F), CA2 (aa 2-260 of WT, D521, $258P), CA2 (aa 2-260 of WT, D728, T192N), CA2 (aa 2-260 of WT, D179E, T1921), CA2 (aa 2- 260 of WT, S56N, Q103K), CA2 (aa 2-260 of WT, D71Y, Q248L), CA2 (aa 2-260 of WT, ST3N, R89F), CA2 (aa 2-260 of WT, D71K, N231L, E235G, L239F), CA2 (aa 2-260 of WT, D72F, P2491), CA2 (aa 2-260 of WT, D72X, V241X, P249X), CA2 (aa 2-260 of WT, A54X, S56X, L57X, T192X), CA2 (aa 2-260 of WT, Y193V, K260F), CA2 (aa 2-260 of WT, G63D, M240L), CA2 (aa 2-260 of WT, V134F, L228F), CA2 (aa 2-260 of WT, D71G, N231K), CA2 (aa 2-260 of WT, S56F, D718), CA2 (aa 2-260 of WT, D52L, G128R, Q248F), CA2 (aa 2-260 of WT, S73X, R89X), CA2 (aa 2-260 of WT, Y51X, D72X, V241X, P249X), CA2 (aa 2-260 of WT, D721, W97C), CA2 (aa 2- 260 of WT, D71K, T192F, N231F), CA2 (aa 2-260 of WT, H36Q, S43T, YSIF, N67D, G131W, R226H), CA2 (aa 2-260 of WT, F701, F146V), CA2 (aa 2-260 of WT, K45N, V68L, H119Y, K169R, D179E), CA2 (aa 2-260 of WT, H15L, AS4V, K111E, E220K, F225I), CA2 (aa 2-260 of WT, P13S, P83A, D101G, K111N, F230I), CA2 (aa 2-260 of WT, G63D, W123R, E220K), CA2 (aa 2-260 of WT, N11D, E69K, G86D, V109M, K113L, T125L, D138G, G155S), CA2 (aa 2-260 of WT, IS9N, G102R, A173T), CA2 (aa 2-260 of WT, L79F, P180S), CA2 (aa 2-260 of WT, A77P, G102R, D138N), CA2 (aa 2-260 of WT, F20L, K45N, G63D, E69V, N231I), CA2 (aa 2-260 of WT, T199N, L202P, L228F), CA2 (aa 2-260 of WT, KON, H122Y, T168K), CA2 (aa 2-260 of WT, Q53H, LOV, Q2H, GI31E), CA2 (aa 2-260 of WT, L44M, L47V, N62K, E69D), CA2 (aa 2-260 of WT, D75V, K160N, F259L), CA2 (aa 2-260 of WT, T207S, V222A, N231D), CA2 (aa 2-260 of WT, ISOF, V206M, G232R), CA2 (aa 2-260 of WT, P13A, A133T), CA2 (aa 2-260 of WT, ISON, R89I), CA2 (aa 2-260 of WT, A65N, G86D, G131R, G155D, K158N, V162A, G170D, P236L), CA2 (aa 2-260 of WT, G12R, H15Y, D19V), CA2 (aa 2-260 of WT, A65V, F95Y, E106G, H107Q, 1145M, F175T), CA2 (aa 2-260 of WT, G63D, E69V, N231I), CA2 (aa 2-260 of WT, S29A, C2058) and / or CA2 (aa 2-260 of WT, $29C, C2058).

[00193] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises a H122Y mutation in the amino acid at position 122 (H122) of SEQ ID NO. 5. In some such embodiments, the DRD further comprises: (i) a R27L mutation in the amino acid at position 27 (R27) of SEQ ID NO. 5; (ii) a T871 mutation in the amino acid at position 87 (T87) of SEQ ID NO. 5; (iii) a N252D mutation in the amino acid at position 252 (N252) of SEQ ID NO. 5; or a combination of (i), (ii) and / or (iii).

[00194] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises an E106D mutation in the amino acid at position 106 (E106) of SEQ ID NO. 5. In some such embodiments, the DRD further comprises a C205S mutation in the amino acid at position 205 (C205) of SEQ ID NO. 5.

[00195] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises a W208S mutation in the amino acid at position 208 (W208) of SEQ ID NO. 5. In some such embodiments, the DRD further comprises a C2058 mutation in the amino acid at position 205 (C205) of SEQ ID NO. 5.

[00196] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises a ISON mutation in the amino acid at position 59 (I59) of SEQ ID NO. 5. In some such embodiments, the DRD further comprises a G102R mutation in the amino acid at position 102 (G102) of SEQ ID NO. 5.

[00197] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises a L156H mutation in the amino acid at position 156 (L156) of SEQ ID NO. 5. In some such embodiments, the DRD further comprises (i) a W4Y mutation in the amino acid at position 4 (W4) of SEQ ID NO. 5; (ii) a F225L mutation in the amino acid at position 225 (F225) of SEQ ID NO. 5; (iii) a deletion of amino acids at positions 257-260 of SEQ ID NO. 5; (iv) a deletion of amino acids at positions 1-5 of SEQ ID NO. 5; or (v) a deletion of amino acids G234, E235 and P236 of SEQ ID NO. 5.

[00198] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises four mutations relative to SEQ ID NO. 5, the mutations corresponding to: (i) L156H, S172C, F178Y, and E186D; or (ii) D70N, D74N, D100ON, and L156H.

[00199] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises a first mutation and a second mutation relative to SEQ ID NO. 5, wherein: (i) the first mutation is a S73N mutation in the amino acid at position 73 (873) of SEQ ID NO. 5; and (ii) the second mutation is a substitution of F or Y at the amino acid position 89 (R89) of SEQ ID NO. 5.

[00200] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises a substitution of N or F at the amino acid position 56 (S56) of SEQ ID NO. 5. In some such embodiments, the DRD comprises two substitutions relative to SEQ ID NO. 5 that correspond to S56F and D718.

[00201] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises one or more substitutions relative to SEQ ID NO. 5, wherein at least one substitution is a substitution of D or N at the amino acid position 63 (G63) of SEQ ID NO. 5, and wherein the one or more substitutions correspond to: (i) G63D;, (ii) G63D and M240L; (iii) G63D, E69V and N2311 or (iv) T55K, G63N and Q248N.

[00202] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises two or more substitutions relative to SEQ ID NO. 5, wherein one of the two or more substitutions is a substitution of L or K at the amino acid position 71 (D71) of SEQ ID NO. 5, and wherein 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.

[00203] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises two or more substitutions relative to SEQ ID NO. 5, wherein at least one of the two or more substitutions is: (i) a substitution of F at the amino acid position 241 (V241) of SEQ ID NO. 5; or (ii) a substitution of F or L at the amino acid position 249 (P249) of SEQ ID NO. 5; and wherein 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) A771 and P249F; or (vi) V241F and P249L.

[00204] In some embodiments, a DRD of the present disclosure comprises, in whole or in part, a human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises one or more substitutions relative to SEQ ID NO. 5, selected from Y51T, L183S, Y1931, L197P and the combination of V134F and L228F.

[00205] The amino acid sequences of the DRDs encompassed in the present disclosure have 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 further preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of a parent protein from which it is derived. In some embodiments, the amino acid sequence of the DRDs encompassed in the present disclosure have 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 further preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of a parent protein (for example, a parent protein having an amino acid sequence of any one of SEQ ID Nos: 1,2, 3, 4, 5, 6, and 71) from which it is derived.

[00206] Examples of DRDs of the present disclosure include those derived from: human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDES. Suitable DRDs, which may be referred to as destabilizing domains or ligand binding domains, are also known in the art. See, e.g, W02018 / 161000; W02018 / 231759; W02019 / 241315; US8,173,792; US8,530,636; W02018 / 237323; W02017 / 181119; US2017 / 0114346, US2019 / 0300864; W02017 / 156238; Miyazaki et al., ] 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. (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:¢10606.

[00207] As provided above in the “Transcription Factor System” section, the combination of one or more polynucleotides of a transcription factor system comprises a nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor (for example, a transcription factor DNA binding domain, a transcription factor activation domain, or both) is operably linked to the DRD. The nucleic acid sequence that encodes a DRD may be selected from sequences of DRDs described herein. Constructs comprising DRD sequences are provided in Table 1 above. Additional constructs comprising different DRDs are provided in Table 4. An asterisk (“*”) in Table 4 indicates the translation of a stop codon. g 22 <glc a Srasy BzdzBes3zEYY F2ZEBEZER 22222 gEZ 55582 EERE gLz282523823%¢ Bo EIiEiirEerais gxigEighed EEE ELT Ze EERE EEE gz gigs Sow 9E¥EZS SHEE SEEESEIR321E g% AERC EE EE EEE EERE EEE , foEgioigeias :: 2 Ee¥ 5529A32%58 § EEEgs9z5gEzE%us 5 |¥z=23%2 ggs&igsas & |EE2EERS 352 FERRER f EEERiczifiizica SoEUEZES89z EEE gE Mm - go Zz Z 28 388% Sess EEivesd 798820 zsnsiugrtannairat nisl Ty PELL IRI ETL RE 8% SHB 89838838°% BRIEFS H L208 ei SOEERES2508T 5283 SEES REE R RL. SOSEHEPERa2a RES Sge880sssg8224 38% » SR oEiTan anEiiiiaocd SEETSIESESES 3538 Bongsolpp nie aRodas BEd 2555555588 2 SIRE FER a HHH EE SO HHH] g lEEiarnanrnittiliiEinl ll 8 £3 DE en 5 8 3 BEES SST ROYE SERIREEL £ PHiEiiigEs Tine 3 5 EEEEEES EEE BEisoEsREEEe EEEEEEEgE Za gigs zas gEI8g sy 558 $8389582955 80 8 £8 |3F2528% PRiTaEiEaIsiiiEZos FE252E 8s 3 |8 BEoiiiiconotnaias THE SHEE 515 [8858S P2BS50583 BEC rEfnEocRiiiaizect 2 BRHE 3 ZES50352893 & 8 2 ls Ba gz iri z 8 EER ERIIEIEEI In asta TE 8 o SRBRSSERFILBE558 5 2 8388 3 Ss = & S 43.8 § |g §58az ElEEsst 2 |S4fk8=2 = " oe ° wv - Slge2 GREER 3 a SEe<2e3zPId9oxd 7 >a 25s 289 EaiEpetifeaiEiit. = ade 0E7358 ZogpER zz hon cgzaLE D0x% 28g=9747aL 2>>T SZ EEUSEILEIRLUDOEERY 08USEgbR Ar S ERE 9 gz SoB8553982x% SBE: OBSE5084%2 cigdesiiizsituct EEE ZfIggiiiiiies, TEER naBEIYD Q SEoE%pitEans 522: SEiiilietiniani Zi SZ233Eige 2 £E Bz EO —HEEZZ2A HEE ERE EEE = £12fc ane HEHE HIE HH HHI £3 $F: 5 Hi TH I, 2x cores Hi gees I — §gC: E58 ERLE 5 ¥5¥ §e028% Hi Hi 88838 BiiEs g TH HiT in {Hi LH lin ini i an ois I 83 53 i 8 3 iis EERE iin 8 583 3 o E 2 2 88 838 3 Hii Ih 8B 8 g EXE ES ERY Ho nn 1H nia Li g Sis gos EpEsic di Hi EEE Ha 11H 3 38303 Poesy BE HH = He nin tin gsi 98 g wn , 3 oF jm géag 37g 5308 : a t N Qo ess o SE 2Egces £3858 £9 ne E 2zE szigt aE a ge sgioee Q S <2 9 20 i ea S5il a giz Os fgegs = © < Oo 53 a ® S< 05 =< < 03 +] ho 8 $5 s 288 go eo =x Ee S5BES 53 EZ3 3 g g ie =] Bnet SEs Lo SES iE 83 FEED ELEEZE 25g = E255 3 = RZ Egg Q = <Z = 380 £5 8552 g 29 Oo BEERS = E <2E gc gids Ha HE SEESES ee iii Hie eg SsEris Hil Hit SiiEss a Lili Ii HE IE £558 z £5 HEE g g s< Hin i S50 Ege SEs 5 ipec ete ro Hi £3Ee fay 3g 2583 g855 £859 a 8s 2 a Hi Hi i Hi HE gooEgcs Ain Ha o9 dl Hee Hie A 53% 8855 ne Hi HHH 38% 2 Hiri Bl 25525 288s Es << 2 Oras Xe<2sSB¥50 PEiEcEiiaiest %= >a gecEny £L73828539°222 825 EEE oZEZ38 =52R30&C RE2RE9TETEL8ES EEEE9Z20E EEE: AEY=KD A>SESAQT g82zEe5as g5g2 zo Z g83cg203% TEEREIRSIRAE g FEEEEEHEEIS Eg8EEZs2258287 z = EL86=2 po<a ne Ha o Hu SHEM2ZES%EAs SE, ls — 2 goce’ HH Hi 53% 2 AO HH I Hi HH " EEE I 2 538 fh 28 eg 3 gs 25 Ha Biol ii 88 Ti 1 Hin HH nay fii Li Ha g 252 £83 38 Hi 8 Xs e £58 gEygs Hon $8 5 288% He 5 g pains lean §9 ens so Ef Hi iil Hi Ha li Fina £830 g HH Began Hin 58 La 3 HEE T3% HH 582 ih 1 Hii Saifd iti thi 255% 223% Hi Bogs £88 58 88 TE] F538 = g§8¢8 8 Hii Hi Hi Alia 2 i Ha Hn Ha Hi HHI ged Hi HE Hi gg fiagd Hi £2 2 gs [i Hi Hilti HI HEE 58% = = > & gx if a E23 Noo 8 58929588 PE 2385 g g 25% “= _ fzigises BEBeaTazanEipestynaitonlsgts 28852858 pRIRRRi iE Blan nll SpPoiB8s |FePEe T2223855888 goRBEEC oes 28 §aopiad: |iiF SoefoaroREoaliiicEciaios 352253 Eni Ei §E285522 eooirEEsaiacys Bi5c2 E55%E80053 TI5ETE 38 ESETEEri anni iliia fadRiys 53FFFEP2 5S CIEE EET EE EEL S5528388§ SPT HSS §C5588985885 8 SoFisads s3fzasst |g SEoiiEoigidiiiipiid Braids Bilis HEH HEE EH IZ PBOG 8 Qo DH 0F QO BE £8 BEER g8: giiEEiass ga88388 BEE3859855¢ BIERPRALE 8 325853 BSS 858085502 808 g§ TRESESRED (8 228323s Bo iiiieniieiizios 585823502 §edgtgsesatadasies 52285855 g 5% |2¥¥¥ESS53 Fo85f822i55252 S8g508 588 85359 5555552383889 S93830838cB0 0 5s BEizaics Bil nnittiail ent §55Esias ini ani 22 z 5 BS. 88 DEHN S20E55:55% [3558 58588 TEIIITEIASREEEESESCE £3 S55288 SiiGopecigilEE nina anty 388% Fuizoats lb SH8 2EE5ECRE589 08583 = 588 i a g N 2 a £ N Stimuli of transcription factor systems

[00208] A transcription factor system of the present disclosure can be responsive to a stimulus.

[00209] In some embodiments, a stimulus is a ligand. Ligands may be nucleic acid-based, protein- based, lipid based, organic, inorganic or any combination of the foregoing. In some embodiments, ligands may be synthetic molecules. In some embodiments, ligands may be small molecule therapeutic compounds. In some embodiments, ligands may be small molecule drugs previously approved by a regulatory agency, such as the US Food and Drug Administration (FDA).

[00210] As described in the present disclosure, a transcription factor system can exhibit ligand- dependent activity. A ligand can bind to a DRD and stabilize a transcription factor or a domain of a transcription factor encoded by the transcription factor system. Ligands that are known to bind candidate DRDs can be tested for their effect on the activity of a transcription factor system.

[00211] In some embodiments, a ligand is cell permeable. In some embodiments, a ligand may be designed to be lipophilic to improve cell permeability.

[00212] In some embodiments, a ligand is a small molecule. A small molecule ligand may be clinically approved to be safe and have appropriate pharmaceutical kinetics and distribution.

[00213] In some embodiments, the ligand may be complexed or bound to one or more other molecules such as, but not limited to, another ligand, a protein, peptide, nucleic acid, lipid, lipid derivative, sterol, steroid, metabolite, metabolite derivative or small molecule. In some embodiments, the ligand stimulus is complexed or bound to one or more different kinds and / or numbers of other molecules. In some embodiments, the ligand stimulus is a multimer of the same kind of ligand. In some embodiments, the ligand stimulus multimer comprises 2, 3, 4, 5, 6, or more monomers. CA? ligands

[00214] In some embodiments, a ligand of the present disclosure binds to carbonic anhydrases. In some embodiments, the ligand binds to and inhibits carbonic anhydrase function and is herein referred to as carbonic anhydrase inhibitor.

[00215] 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 referred to as Celebrex), Valdecoxib, Rofecoxib, Acetazolamide, Methazolamide, Dorzolamide, Brinzolamide, Diclofenamide, Ethoxzolamide, Zonisamide, Dansylamide, and Dichlorphenamide.

[00216] In some embodiments, the ligands may comprise portions of small molecules know to mediate binding to CA2. Ligands may also be modified to reduce off-target binding to carbonic anhydrases other than CA2 and increase specific binding to CA2.

[00217] In some embodiments, the stimulus may be a ligand that binds to more than one carbonic anhydrase. In one embodiment, the stimulus is a pan carbonic anhydrase inhibitor that may bind to two or more carbonic anhydrases. DHFR ligands

[00218] In some embodiments, a ligand of the present disclosure binds to dihydrofolate reductase. In some embodiments, the ligand binds to and inhibits dihydrofolate reductase function and is herein referred to as a dihydrofolate inhibitor.

[00219] In some embodiments, the ligand may be a selective inhibitor of human DHFR. Ligands of the disclosure may also be selective inhibitors of dihydrofolate reductases of bacteria and parasitic organisms such as Pneumocystis spp., Toxoplasma spp., Trypanosoma spp., Mycobacterium spp., and Streptococcus spp. Ligands specific to other DHFR may be modified to improve binding to human dihydrofolate reductase.

[00220] Examples of dihydrofolate inhibitors include, but are not limited to, Trimethoprim (TMP), Methotrexate (MTX), Pralatrexate, Piritrexim, Pyrimethamine, Talotrexin, Chloroguanide, Pentamidine, Trimetrexate, aminopterin, C1 898 trihydrochloride, Pemetrexed Disodium, Raltitrexed, Sulfaguanidine, Folotyn, Iclaprim and Diaveridine.

[00221] In some embodiments, ligands of the present disclosure may include dihydrofolic acid or any of its derivatives that may bind to human DHFR. In some embodiments, the ligands of the present disclosure may be 2,4, diaminohetrocyclic compounds. In some embodiments, the 4-oxo group in dihydrofolate may be modified to generate DHFR inhibitors. In one example, the 4 -oxo group may be replaced by 4-amino group. Various diamino heterocycles, including pteridines, quinazolines, pyridopyrimidines, pyrimidines, and triazines, may also be used as scaffolds to develop DHFR inhibitors and may be used according to the present disclosure.

[00222] In some embodiments, ligands include TMP-derived ligands containing portions of the ligand known to mediate binding to DHFR. Ligands may also be modified to reduce off-target binding to other folate metabolism enzymes and increase specific binding to DHFR. ER ligands

[00223] In some embodiments, a ligand of the present disclosure binds to ER. Ligands may be agonists or antagonists. In some embodiments, the ligand binds to and inhibits ER function and is herein referred to as an ER inhibitor. In some embodiments, the ligand may be a selective inhibitor of human ER. Ligands of the disclosure may also be selective inhibitors of ER of other species. Ligands specific to other ER may be modified to improve binding to human ER.

[00224] Ligands may be ER agonists such as but not limited to endogenous estrogen 17b-estradiol (E2) and the synthetic nonsteroidal estrogen diethylstilbestrol (DES). In some embodiments. The ligands may be ER antagonists, such as ICI-164,384, RU486, tamoxifen, 4-hydroxytamoxifen (4- OHT), fulvestrant, oremifene, lasofoxifene, clomifene, femarelle and ormeloxifene and raloxifene (RAL).

[00225] In some embodiments, the stimulus of the current disclosure may be ER antagonists such as, but not limited to, Bazedoxifene and / or Raloxifene.

[00226] In some embodiments, ligands include Bazedoxifene- derived ligands containing portions of the ligand known to mediate binding to ER. Ligands may also be modified to reduce off-target binding to other folate metabolism enzymes and increase specific binding to ER derived DRDs. Phosphodiesterase ligands

[00227] In some embodiments, ligands of the present disclosure bind to phosphodiesterases. In some embodiments, the ligands bind to and inhibit phosphodiesterase function and are herein referred to as phosphodiesterase inhibitors.

[00228] In some embodiments, the ligand is a small molecule that binds to phosphodiesterase 5. In one embodiment, the small molecule is a hPDES5 inhibitor. Examples of hPDES inhibitors include, but are not limited to, Sildenafil, Vardenafil, Tadalafil, Avanafil, Lodenafil, Mirodenafil, Udenafil, Benzamidenafil, Dasantafil, Beminafil, SLx-2101, LAS 34179, UK-343,664, UK-357903, UK- 371800, and BMS-341400.

[00229] In some embodiments, ligands include sildenafil-derived ligands containing portions of the ligand known to mediate binding to hPDES. Ligands may also be modified to reduce off-target binding to phosphodiesterases and increase specific binding to hPDES.

[00230] In some embodiments, the stimulus may be a ligand that binds to more than one phosphodiesterase. In one embodiment, the stimulus is a pan-phosphodiesterase inhibitor that may bind to two or more hPDEs such as Aminophyline, Paraxanthine, Pentoxifylline, Theobromine, Dipyridamole, Theophyline, Zaprinast, Icariin, CDP-840, Etazolate and Glaucine.

[00231] In some embodiments, the ligand is a hPDEI inhibitor. In some embodiments, the ligand is a hPDE2 inhibitor. In some embodiments, the ligand is a hPDE3 inhibitor. In some embodiments, the ligand is a hPDE4 inhibitor. In some embodiments, the ligand is a hPDES6 inhibitor. In some embodiments, the ligand is a hPDE7 inhibitor. In some embodiments, the ligand is a hPDE8 inhibitor. In some embodiments, the ligand is a hPDE9 inhibitor. In some embodiments, the ligand is a hPDE10 inhibitor. FKBP Ligands

[00232] In some embodiments, ligands of the present disclosure bind to FKBP, including human FKBP. In some embodiments, the ligand is SLF or Shield-1. Payloads

[00233] Payloads may include any polypeptide or any protein or fragment thereof. A payload may be a wild-type sequence, a fragment of a wild-type sequence and / or comprise one or more mutations. A payload may be a natural protein from an organism genome, or variants, mutants, and derivatives thereof. The natural protein may be from, for example, a mammalian organism, a bacterium, and a virus. A 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.

[00234] In one example, a payload may be a polypeptide encoded by a nucleic acid sequence from a human genome.

[00235] In some embodiments, a payload may be a variant sequence of a parent polypeptide. In some aspects, the variant sequence may have the same or a similar activity as the reference sequence. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to a reference sequence. Generally, variants of a particular polypeptide of the 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%, 99% but less than 100% sequence identity to that particular reference polypeptide as determined by sequence alignment programs known to those skilled in the art. Therapeutic agents as payloads

[00236] In some embodiments, payloads of the present disclosure may be therapeutic agents. For example, a payload may be a cancer therapeutic agent, a therapeutic agent for an autoimmune disease, an immunotherapeutic agent, an anti-inflammatory agent, an anti-pathogen agent or a gene therapy agent. In some aspects, the immunotherapeutic agent may be an antibody and fragments and variants thereof, a T-cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric switch receptor, an antagonist of a co-inhibitory molecule, an agonist of a co-stimulatory molecule, a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, a metabolic factor, a coagulation factor, an enzyme, a homing receptor and a safety switch.

[00237] In some embodiments, payloads of the present disclosure may be immunotherapeutic agents that induce immune responses in an organism. The immunotherapeutic agent may be, but is not limited to, an antibody and fragments and variants thereof, a TCR, a chimeric antigen receptor (CAR), a chimeric switch receptor, a cytokine, chemokine, a cytokine receptor, a chemokine receptor, a cytokine-cytokine receptor fusion polypeptide, or any agent that induces an immune response. In one embodiment, the immunotherapeutic agent induces an anti-cancer immune response in a cell, or in a subject. Cytokines, chemokines and other soluble factors as payloads

[00238] In some embodiments, payloads of the present disclosure may be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells and other cell types, which act as chemical communicators between cells and tissues within the body. These proteins mediate a wide range of physiological functions, from effects on cell growth, differentiation, migration and survival, to a number of effector activities. For example, activated T cells produce a variety of cytokines for cytotoxic function to eliminate tumor cells.

[00239] In some embodiments, payloads of the present disclosure may be cytokines, and fragments, variants, analogs and derivatives thereof, including but not limited to interleukins, tumor necrosis factors (TNFs), interferons (IFNs), TGF beta and chemokines. In some embodiments, payloads of the present invention may be cytokines that stimulate immune responses. In other embodiments, payloads of the invention may be antagonists of cytokines that negatively impact anti- cancer immune responses.

[00240] In some embodiments, payloads of the present disclosure may be cytokine receptors, recombinant receptors, variants, analogs and derivatives thereof; or signal components of cytokines. In various embodiments, payloads of the present disclosure may include secreted cytokines or membrane bound form of cytokines. An illustrative example of a membrane cytokine, may include a cytokine (for example, an immune stimulatory cytokine, for example, IL12, IL2, IL15 and IL18) that is operably fused, linked or connected to a transmembrane domain, for example, a CD8a transmembrane domain, a B7-1 transmembrane domain, a CD4 transmembrane domain, a CD 28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, or a human IgG4 Fc region. In various embodiments, the cytokine may be fused or connected to a transmembrane domain via an intervening peptide or protein sequence, such as a linker, a hinge, a transmembrane tail etc.

[00241] In one embodiment, payloads of the present disclosure may be cytokines fused to TNF alpha ectodomain. Such payloads are produced as membrane associated cytokines fused to the TNF ectodomain. In one embodiment, the cytokine may be shed from the cell surface by the action of membrane associated proteases, and / or proteases in the extracellular space e.g. MMP9.

[00242] In some embodiments, payloads of the present disclosure may be an interleukin (IL) cytokine. Interleukins (ILs) are a class of glycoproteins produced by leukocytes for regulating immune responses. As used herein, the term “interleukin (IL)” refers to an interleukin polypeptide from any species or source and includes the full-length protein as well as fragments or portions of the protein.

[00243] In some embodiments, a payload of the disclosure may comprise IL12. IL12 is a heterodimeric protein of two subunits (p35, p40) that is secreted by antigen presenting cells, such as macrophages and dendritic cells. Expression of IL12 requires the simultaneous expression of the two subunits to produce a biologically active heterodimer. In some embodiments, payloads of the disclosure may be the p35 subunit or the p40 subunit.

[00244] In some embodiments, a payload of the disclosure may comprise whole or a portion of n.12.

[00245] In some embodiments, the [L12 may be a Flexi IL12, wherein both p35 and p40 subunits are encoded by a single cDNA that produces a single chain polypeptide. The single chain polypeptide may be generated by placing the p35 subunit at the N terminus or the C terminus of the single chain polypeptide. Similarly, the p40 subunit may be at the N terminus or C terminus of the single chain polypeptide.

[00246] The format of an IL12 payload of the present disclosure may be optimized. In one embodiment, the payload may be a bicistronic IL12 containing p40 and p35 subunits separated by an internal ribosome entry site or a cleavage site such as P2A or Furin to allow independent expression of both subunits from a single vector. In another embodiment, the payload may be the p40 subunit of IL12 or the p35 subunit of IL12.

[00247] In some embodiments, the payload may be IL12 that is membrane bound. IL12 may be bound to the membrane by a transmembrane domain. The transmembrane domain may also include an optional hinge domain. In some aspects, the IL12 molecule is extracellular and tethered to the cell by the transmembrane domain. In some aspects, the membrane bound IL12 may be shed or cleaved from the cell surface by the action of proteases. In some embodiments, the transmembrane domain of the present disclosure may be derived either from a natural or from a synthetic source. The transmembrane domain may be derived from any naturally membrane-bound or transmembrane protein. Alternatively, the transmembrane domain of the present disclosure may be synthetic. In some aspects, the synthetic sequence may comprise predominantly hydrophobic residues such as leucine and valine. In some aspects, transmembrane and / or hinge domains that are resistant to the activity of proteases may be selected.

[00248] In some embodiments, a payload of the disclosure may comprise IL15. Interleukin 15 is a potent immune stimulatory cytokine and an essential survival factor for T cells and Natural Killer cells.

[00249] In some embodiments, a payload of the disclosure may comprise whole or a portion of IL15. Any portion of IL15 that retains one or more functions of full-length or mature IL15 may be useful in the present disclosure. Such functions include the promotion of NK cell survival, regulation of NK cell and T cell activation and proliferation as well as the support of NK cell development from hematopoietic stem cells.

[00250] In some instances, whole or a portion of the IL15 is linked to the whole or a portion of one or more transmembrane proteins.

[00251] An IL15 payload may be designed to be secreted (using e.g. IL2 signal sequence) or membrane bound (using e.g. IgE or CD8a signal sequence).

[00252] A unique feature of IL15 mediated activation is the mechanism of trans-presentation in which IL15 is presented as a complex with the alpha subunit of IL15 receptor (IL15Ra) that binds to and activates membrane bound IL15 beta / gamma receptor, either on the same cell or a different cell. In some embodiments, a payload of the present disclosure is a membrane bound IL15. In some embodiments, a payload of the present disclosure may include an IL15 / IL15Ra fusion polypeptide. In some embodiments, the payload may be a whole or a portion of IL15 fused to the whole or a portion of IL15Ra. Any portion of IL15 and IL15Ra that retains one or more functions of full-length or mature IL15 or IL15Ra respectively may be used.

[00253] In some aspects, the IL15 molecule is extracellular and tethered to the cell by the transmembrane domain. In some aspects, the membrane bound IL15 may be shed or cleaved from the cell surface by the action of proteases.

[00254] The whole or a portion of the membrane bound IL15 or IL15 / IL15Ra fusion polypeptides of the disclosure may be shed into the extracellular space. Shedding as used herein refers to the release of membrane associated biomolecules from the membrane to which they are tethered. In some instances, shedding may be induced by proteolytic cleavage.

[00255] Payloads of the present disclosure may comprise amino acid sequences similar to the amino acid sequence of human IL15, for example, UniProtKB - P40933 (IL15_HUMAN).

[00256] In some embodiments, payloads of the present disclosure may be utilized to improve expansion, survival, persistence, and potency of immune cells such as CD8+TEM, natural killer cells and tumor infiltrating lymphocytes (TIL) cells, and CAR T cells used for immunotherapy. In one aspect, the present disclosure provides payloads to minimize toxicity related to cytokine therapy. In some embodiments, a payload of the disclosure may comprise whole or a portion of IL2. Any portion of IL2 that retains one or more functions of full-length or mature IL2 may be useful in the present disclosure.

[00257] It is understood in the art that certain gene and / or protein nomenclature for the same gene or protein may be inclusive or exclusive of punctuation such as a dash “-” or symbolic such as Greek letters. Whether these are included or excluded herein, the meaning is not meant to be changed as would be understood by one of skill in the art. For example, IL2, IL-2 and IL 2 refer to the same interleukin. Likewise, IL15, IL 15 and IL-15 refer to the same interleukin. Likewise, TNFalpha, TNFa, TNF-alpha, TNF-a, TNF alpha and TNF « all refer to the same protein. Antibodies and antibody fragments as payloads

[00258] In some embodiments, payloads of the present disclosure may be antibodies, antibody fragments and variants thereof.

[00259] The antibody may be an intact antibody, an antibody light chain, antibody heavy chain, an antibody fragment, an antibody variant, or an antibody derivative.

[00260] For the purposes herein, an “antibody” may comprise a heavy and light variable domain as well as an Fc region.

[00261] In some embodiments, the payload maybe a monoclonal antibody. As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibodies, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[00262] In one embodiment, the payload of the present disclosure may be a humanized antibody. As used herein, the term “humanized antibody” refers to a chimeric antibody comprising a minimal portion from one or more non-human (e.g., murine) antibody source(s) with the remainder derived from one or more human immunoglobulin sources. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and / or capacity. In one embodiment, the antibody may be a humanized full-length antibody.

[00263] As used herein, the term “antibody variant” refers to a modified antibody (in relation to a native or starting antibody) or a biomolecule resembling a native or starting antibody in structure and / or function (e.g., an antibody mimetic). Antibody variants may be altered in their amino acid sequence, composition or structure as compared to a native antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgG1, 1gG2, IgG3, 1gG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.

[00264] In some embodiments, antibody fragments and variants may comprise antigen binding regions from intact antibodies. Examples of antibody fragments and variants may include, but are not limited to Fab, Fab', F(ab")2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as single chain variable fragment (scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen- binding fragments, called “Fab” fragments, each with a single antigen-binding site. Also produced is a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking with the antigen. Payloads of the present disclosure may comprise one or more of these fragments.

[00265] In some embodiments, antibody payloads of the present disclosure may be therapeutic antibodies. Chimeric antigen receptor payloads

[00266] In some embodiments, payloads of the present disclosure may be chimeric antigen receptors (CARs). As used herein, the term “chimeric antigen receptor (CAR)” refers to a synthetic receptor that mimics a T-cell receptor (TCR) on the surface of T cells. In general, a CAR is composed of an extracellular targeting domain, a transmembrane domain / region and an intracellular signaling / activation domain. Cells such as T cells engineered to express a CAR can be redirected to attack target cells that express a molecule which can be recognized by the targeting moiety of the CAR. In a standard CAR receptor, the components: the extracellular targeting domain, transmembrane domain and intracellular signaling / activation domain, are linearly constructed as a single fusion protein. The extracellular region comprises a targeting domain / moiety (e.g., a scFv) that recognizes a specific tumor antigen or other tumor cell-surface molecules. The intracellular region may contain a signaling domain of a TCR complex (e.g., the signal region of CD3(), and / or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX-40 (CD134). For example, a “first-generation CAR” only has the CD3( signaling domain, whereas in an effort to augment T-cell persistence and proliferation, costimulatory intracellular domains are added, giving rise to second generation CARs having a CD3( signal domain plus one costimulatory signaling domain, and third generation CARs having CD3( signal domain plus two or more costimulatory signaling domains. A CAR, when expressed by a T cell, endows the T cell with antigen specificity determined by the extracellular targeting moiety of the CAR. A fourth-generation CAR includes addition of one or more elements such as homing and suicide genes to develop a more competent and safer architecture of CAR.

[00267] In some embodiments, a CAR payload, when transduced into immune cells (e.g., T cells and NK cells), can re-direct the immune cells against the target (e.g., a tumor cell) which expresses a molecule recognized by the extracellular target moiety of the CAR. Nucleic acid modifying agents as payloads

[00268] In some embodiments, payloads of the present disclosure may be nucleic acid modifying agents.

[00269] In some embodiments, payloads of the present disclosure may be components of gene editing systems. In some embodiments, payloads of the present disclosure may be a Cas protein (CRISPR-associated protein), including Cas9 and Cas12. The Cas protein may be altered or otherwise modified. For example, the Cas protein may be a deadCas9. In some embodiments, the Cas9 protein is an enzymatically active Cas9 protein, a Cas9 protein wild-type protein, a Cas9 protein nickase or a nuclease null or nuclease deficient Cas9 protein. In some embodiments, payloads of the present disclosure may be Zinc finger nucleases, TALEN (Transcription activator- like effector-based nucleases) and meganucleases.

[00270] In some embodiments, payloads of the present disclosure may be a recombinase, such as Cre recombinase. Agents for treating autoimmune disorders as payloads

[00271] In some embodiments, payloads of the present disclosure may be agents for treating, ameliorating or preventing autoimmune disorders.

[00272] In some embodiments, payloads of the present disclosure include anti-cytokines, such as neutralizing antibodies to tumor necrosis factor (TNF)-a, IL-1 and IL-6. In some embodiments, payloads of the present disclosure target B-cell depletion, such as neutralizing antibodies to CD20, CD22, CD28, CTLA-4, and B-lymphocyte stimulator (BLyS). Pharmaceutical compositions and formulations

[00273] The present teachings further comprise pharmaceutical compositions comprising one or more of the transcription factor systems, nucleic acids, polynucleotides, modified cells or payloads of the present disclosure, and optionally at least one pharmaceutically acceptable excipient or inert ingredient.

[00274] As used herein the term “pharmaceutical composition” refers to a preparation of one or more of the transcription factor systems, nucleic acids, polynucleotides, modified cells, payloads or transcription factor system components described herein, or pharmaceutically acceptable salts thereof, optionally with other chemical components such as physiologically suitable carriers and excipients.

[00275] The term “excipient” or “inactive ingredient” refers to an inert or inactive substance added to a pharmaceutical composition to further facilitate administration of a compound.

[00276] In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to any one or more transcription factor system components to be delivered as described herein.

[00277] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, non-human mammals, including agricultural animals such as cattle, horses, chickens and pigs, domestic animals such as cats, dogs, or research animals such as mice, rats, rabbits, dogs and non- human primates.

[00278] A pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[00279] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient or inert ingredient, and / or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w / w) active ingredient.

[00280] Efficacy of treatment or amelioration of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. A healthcare practitioner skilled in the art may monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of compositions of the present disclosure, “effective against” for example a cancer, indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of cancer.

[00281] A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given composition or formulation of the present disclosure can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change is observed. Formulations

[00282] The polynucleotide and vector compositions of the present disclosure may be formulated in any manner suitable for delivery. The formulation may be, but is not limited to, nanoparticles, poly (lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids and combinations thereof.

[00283] In one embodiment, the polynucleotide and vector formulation is a nanoparticle which may comprise 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 aspect, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA and DODMA.

[00284] For polynucleotides of the disclosure, the formulation may be selected from any of those taught, for example, in International Application PCT / US2012 / 069610. Inactive ingredients

[00285] In some embodiments, pharmaceutical or other formulations may comprise at least one excipient which is an inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more inactive agents included in formulations. In some embodiments, all, none or some of the inactive ingredients which may be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA). Dosing, delivery and administration

[00286] The compositions of the disclosure may be delivered to a cell or a subject through one or more routes and modalities. The 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 a cell and / or a subject. Other modalities may also be used such as mRNAs, plasmids, and as recombinant proteins. Delivery Naked delivery

[00287] Pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads of the present disclosure may be delivered to cells, tissues, organs and / or organisms in naked form. As used herein in, the term “naked” refers to pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads delivered free from agents or modifications which promote transfection or permeability. The naked pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads may be delivered to the cells, tissues, organs and / or organisms using routes of administration known in the art and described herein. In some embodiments, naked delivery may include formulation in a simple buffer such as saline or PBS. Formulated delivery

[00288] In some embodiments, pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads of the present disclosure may be formulated, using methods described herein. Formulations may comprise pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads which may be modified and / or unmodified. Formulations may further include, but are not limited to, cell penetration agents, pharmaceutically acceptable carriers, delivery agents, bioerodible or biocompatible polymers, solvents, and / or sustained-release delivery depots. Formulations of the present disclosure may be delivered to cells using routes of administration known in the art and described herein.

[00289] Pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads may also be formulated for direct delivery to organs or tissues in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and / or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with compositions, and the like. Delivery to Cells

[00290] In another aspect of the disclosure, polynucleotides of a transcription factor system or components thereof and compositions of the disclosure and vectors comprising the polynucleotides may be introduced into cells such as immune effector cells.

[00291] In one aspect of the disclosure, polynucleotides of a transcription factor system or components thereof and compositions of the disclosure, may be packaged into plasmids, viral vectors or integrated into viral genomes allowing transient or stable expression of the polynucleotides. Preferable viral vectors are retroviral vectors including lentiviral vectors and gamma retroviral vectors. In order to construct a retroviral vector, a polynucleotide molecule of a transcription factor system is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. 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 the culture media, then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are especially preferred as they are capable of infecting both dividing and non-dividing cells.

[00292] Vectors may also be transferred to cells by non-viral methods by physical methods such as needles, electroporation, sonoporation, hydroporation; chemical carriers such as inorganic particles (e.g. calcium phosphate, silica, gold) and / or chemical methods. In some embodiments, synthetic or natural biodegradable agents may be used for delivery such as cationic lipids, lipid nano emulsions, nanoparticles, peptide based vectors, or polymer based vectors. In some embodiments, vectors may be transferred to cells by temporary membrane disruption, for example, by high speed cell deformation.

[00293] In some embodiments, the polypeptides of the disclosure may be delivered to the cell directly. In one embodiment, the polypeptides of the disclosure may be delivered using synthetic peptides comprising an endosomal leakage domain (ELD) fused to a cell penetration domain (CLD). The polypeptides of the disclosure are co introduced into the cell with the ELD-CLD-synthetic peptide. ELDs facilitate the escape of proteins that are trapped in the endosome, into the cytosol. Such domains are derived proteins of microbial and viral origin and have been described in the art. CPDs allow the transport of proteins across the plasma membrane and have also been described in the art. The ELD-CLD fusion proteins synergistically increase the transduction efficiency when compared to the co-transduction with either domain alone. In some embodiments, a histidine rich domain may optionally be added to the shuttle construct as an additional method of allowing the escape of the cargo from the endosome into the cytosol. The shuttle may also include a cysteine residue at the N or C terminus to generate multimers of the fusion peptide. Multimers of the ELD- CLD fusion peptides generated by the addition of cysteine residue to the terminus of the peptide show even greater transduction efficiency when compared to the single fusion peptide constructs. The polypeptides of the disclosure may also be appended to appropriate localization signals to direct the cargo to the appropriate sub-cellular location e.g. nucleus. In some embodiments any of the ELDs, CLDs or the fusion ELD-CLD synthetic peptides taught in the International Patent Publication, W02016161516 and WO2017175072 may be useful in the present disclosure (the contents of each of which are herein incorporated by reference in their entirety). Delivery Modalities and / or Vectors

[00294] The transcription factor systems or components thereof of the present disclosure may be delivered using one or more modalities. The present disclosure also provides vectors that package polynucleotides of the disclosure encoding transcription factors and parts thereof, DRDs, or payload constructs, and combinations thereof. Vectors of the present disclosure may also be used to deliver the packaged polynucleotides to a cell, a local tissue site or a subject. These vectors may be of any kind, including DNA vectors, RNA vectors, plasmids, viral vectors and particles. Viral vector technology is well known and described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Viruses, which are useful as vectors include, but are not limited to an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes virus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus. In some embodiments, the virus is selected from a lentivirus vector, a gamma retrovirus vector, adeno-associated virus (AAV) vector, adenovirus vector, and a herpes virus vector.

[00295] In general, vectors contain an origin of replication functional in at least one organism, a promoter sequence and convenient restriction endonuclease site, and one or more selectable markers e.g. a drug resistance gene.

[00296] In some embodiments, the recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell into which the vector is to be introduced.

[00297] In some embodiments, the vector of the disclosure may comprise one or more payloads taught herein, wherein the two or more payloads may be included in one ligand response. In this case, the two or more payloads are tuned by the same ligand or responsive agent simultaneously. Lentiviral vehicles / particles

[00298] In some embodiments, lentiviral vehicles / particles may be used as delivery modalities. Lentiviruses are subgroup of the Retroviridae family of viruses, named because reverse transcription of viral RNA genomes to DNA is required before integration into the host genome. As such, the most important features of lentiviral vehicles / particles are the integration of their genetic material into the genome of a target / host cell. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1 and HIV-2, the Simian Immunodeficiency Virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (IDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi and caprine arthritis encephalitis virus (CAEV).

[00299] Typically, lentiviral particles making up the gene delivery vehicle are replication defective on their own (also referred to as “self-inactivating”). Lentiviruses are able to infect both dividing and non-dividing cells by virtue of the entry mechanism through the intact host nuclear envelope. Recombinant lentiviral vehicles / particles have been generated by multiply attenuating the HIV virulence genes, for example, the genes Env, Vif, Vpr, Vpu, Nef and Tat are deleted making the vector biologically safe. Correspondingly, lentiviral vehicles, for example, derived from HIV-1 / HIV- 2 can mediate the efficient delivery, integration and long-term expression of transgenes into non- dividing cells.

[00300] Lentiviral particles may be generated by co-expressing the virus packaging elements and the vector genome itself in a producer cell such as human HEK293T cells. These elements are usually provided in three or four separate plasmids. The producer cells are co-transfected with plasmids that encode lentiviral components including the core (i.e. structural proteins) and enzymatic components of the virus, and the envelope protein(s) (referred to as the packaging systems), and a plasmid comprising a foreign transgene to be transferred to the target cell, the vehicle itself (also referred to as the transfer vector). In general, the plasmids or vectors are included in a producer cell line. The plasmids / vectors are introduced via transfection, transduction or infection into the producer cell line. Methods for transfection, transduction or infection are well known by those of skill in the art. As non-limiting example, the packaging and transfer constructs can be introduced into producer cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gln synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.

[00301] The producer cell produces recombinant viral particles that contain the foreign gene, for example, the transcription factor systemcomponents or polynucleotides thereof of the present disclosure. The recombinant viral particles are recovered from the culture media and titrated by standard methods used by those of skill in the art. The recombinant lentiviral vehicles can be used to infect target cells.

[00302] Cells that can be used to produce high-titer lentiviral particles may 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 producer 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 contents of each of which are incorporated herein by reference in their entirety).

[00303] In some aspects, the envelope proteins may be heterologous envelope proteins from other viruses, such as the G protein of vesicular stomatitis virus (VSV G) or baculoviral gp64 envelope proteins. The VSV-G glycoprotein may especially be chosen among species classified in the vesiculovirus genus: Carajas virus (CISV), 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 (VSNIV) and / or stains provisionally classified in the vesiculovirus genus as Grass carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Gray Lodge virus (GLOV), Jurona virus (JURY), Klamath virus (KLAV), Kwatta virus (KWAV), La Joya virus (LIV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tupaia virus (TUPV), Ulcerative disease rhabdovirus (UDRV) and Yug Bogdanovac virus (YBV). The gp64 or other baculoviral env protein can be derived from Autographa californica nucleopolyhedrovirus (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fumiferana nucleopolyhedrovirus, Orgyia pseudotsugata single capsid nuclear polyhedrosis virus, Epiphyas postvittana nucleopolyhedrovirus, Hyphantria cunea nucleopolyhedrovirus, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nucleopolyhedrovirus or Batken virus. In some aspects, the envelope proteins may be RD114, RD115 or derived from gibbon ape leukemia virus (GaLV) or a baboon retroviral envelope glycoprotein (BaEV).

[00304] Other elements provided in lentiviral particles may comprise retroviral LTR (long- terminal repeat) at either 5° or 3° terminus, a retroviral export element, optionally a lentiviral reverse response element (RRE), a promoter or active portion thereof, and a locus control region (LCR) or active portion thereof.

[00305] Methods for generating recombinant lentiviral particles are discussed in the art, for example, U.S. Pat. NOs.: 8, 846, 385; 7,745, 179; 7,629,153; 7,575,924; 7,179, 903; and 6, 808, 905.

[00306] Lentivirus vectors used may be selected from, but are not limited to pLVX, pLenti, pLenti6, pLIM1, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLIM1-EGFP, pULTRA, pInducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionIl. Adeno-associated viral particles

[00307] Delivery of polynucleotides of any of the transcription factor systems, transcription factor constructs, or payload constructs of the present disclosure may be achieved using recombinant adeno-associated viral (rAAV) vectors. Such vectors or viral particles may be designed to utilize any of the known serotype capsids or combinations of serotype capsids.

[00308] AAV vectors include not only single stranded vectors but self-complementary AAV vectors (scAAVs). scAAV vectors contain DNA which anneals together to form double stranded vector genome. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.

[00309] The rAAV vectors may be manufactured by standard methods in the art such as by triple transfection, in sf9 insect cells or in suspension cell cultures of human cells such as HEK293 cells.

[00310] The transcription factor constructs and payload constructs may be encoded in one or more viral genomes to be packaged in the AAV capsids taught herein.

[00311] Such vector or viral genomes may also include, in addition to at least one or two ITRs (inverted 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.

[00312] The transcription factor constructs or payload constructs of the disclosure may be administered in one or more or separate AAV particles.

[00313] In some embodiments, the transcription factor system constructs may be administered in one or more AAV particles. In some embodiments, more than one transcription factor system construct may be encoded in a viral genome. Retroviral vehicles / particles (y-retroviral vectors)

[00314] In some embodiments, retroviral vehicles / particles may be used to deliver the transcription factor systems, transcription factor constructs or payload constructs of the present disclosure. Retroviral vectors (RVs) allow the permanent integration of a transgene in target cells. In addition to lentiviral vectors based on complex HIV-1 / 2, retroviral vectors based on simple gamma- retroviruses have been widely used to deliver therapeutic genes and demonstrated clinically as one of the most efficient and powerful gene delivery systems capable of transducing a broad range of cell types. Example species of Gamma retroviruses include the murine leukemia viruses (MLVs) and the feline leukemia viruses (FeLV).

[00315] In some embodiments, gamma-retroviral vectors derived from a mammalian gamma- retrovirus such as murine leukemia viruses (MLVs), are recombinant. The MLV families of gamma retroviruses include the ecotropic, amphotropic, xenotropic and polytropic subfamilies. Ecotropic viruses are able to infect only murine cells using mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect murine, human and other species through the Pit-2 receptor. One example of an amphotropic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (Xpr1) receptor, but differ in their species tropism. Xenotropic viruses such as NZB-9-1 infect human and other species but not murine species, whereas polytropic viruses such as focus-forming viruses (MCF) infect murine, human and other species.

[00316] Gamma-retroviral vectors may be produced in packaging cells by co-transfecting the cells with several plasmids including one encoding the retroviral structural and enzymatic (gag-pol) polyprotein, one encoding the envelope (env) protein, and one encoding the vector mRNA comprising polynucleotide encoding the compositions of the present disclosure that is to be packaged in newly formed viral particles.

[00317] In some aspects, the recombinant gamma-retroviral vectors are pseudotyped with envelope proteins from other viruses. Envelope glycoproteins are incorporated in the outer lipid layer of the viral particles which can increase / alter the cell tropism. In some aspects, the envelope proteins may be RD114, RD115 or derived from gibbon ape leukemia virus (GaLV) or a baboon retroviral envelope glycoprotein (BaEV).

[00318] In some embodiments, the recombinant gamma-retroviral vectors are self-inactivating (SIN) gammaretroviral vectors. The vectors are replication incompetent. SIN vectors may harbor a deletion within the 3° U3 region initially comprising enhancer / promoter activity. Furthermore, the 5 U3 region may be replaced with strong promoters (needed in the packaging cell line) derived from Cytomegalovirus or RSV, or an internal promotor of choice, and / or an enhancer element. The choice of the internal promotors may be made according to specific requirements of gene expression needed for a particular purpose of the disclosure.

[00319] In some embodiments, polynucleotides of the transcription factor systems, transcription factor constructs, or payload constructs are inserted within the recombinant viral genome. The other components of the viral mRNA of a recombinant gamma-retroviral vector may be modified by insertion or removal of naturally occurring sequences (e.g., insertion of an IRES, insertion of a heterologous polynucleotide encoding a polypeptide or inhibitory nucleic acid of interest, shuffling of a more effective promoter from a different retrovirus or virus in place of the wild-type promoter and the like). In some examples, the recombinant gamma-retroviral vectors may comprise modified packaging signal, and / or primer binding site (PBS), and / or 5'-enhancer / promoter elements in the U3- region of the 5'- long terminal repeat (LTR), and / or 3'-SIN elements modified in the U3-region of the 3'-LTR. These modifications may increase the titers and the ability of infection. Oncolytic Viral vector

[00320] In some embodiments, polynucleotides of present disclosure may be packaged into oncolytic viruses. As used herein, the term “oncolytic virus” refers to a virus that preferentially infects and kills cancer cells such as vaccine viruses. An oncolytic virus can occur naturally or can be a genetically modified virus such as oncolytic adenovirus, and oncolytic herpes virus.

[00321] In some embodiments, oncolytic vaccine viruses may include viral particles of a thymidine kinase (TK)-deficient, granulocyte macrophage (GM)-colony stimulating factor (CSF)- expressing, replication-competent vaccinia virus vector sufficient to induce oncolysis of cells in the tumor; See e.g., US Pat. NO.: 9,226,977. Messenger RNA (mRNA)

[00322] In some embodiments, the transcription factor systems, transcription factor constructs, or payload constructs of the disclosure may be designed as messenger RNAs (nRNAs). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. Such mRNA molecules may have the structural components or features of any of those taught in International Application number PCT / US2013 / 030062.

[00323] In some embodiments, the transcription factor systems or components thereof may be designed as self-amplifying RNA. “Self-amplifying RNA” as used herein refers to RNA molecules that can replicate in the host resulting in the increase in the amount of the RNA and the protein encoded by the RNA. Such self-amplifying RNA may have structural features or components of any of those taught in International Patent Application Publication No. W02011005799. Dosing

[00324] The present disclosure provides methods comprising administering any one or more or component or composition of a transcription factor system to a subject in need thereof. These may be administered to a subject using any amount and any route of administration effective for preventing or treating or imaging a disease, disorder, and / or condition (e.g, a disease, disorder, and / or condition relating to cancer or an autoimmune disease). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.

[00325] Compositions in accordance with the disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[00326] In some embodiments, compositions of the disclosure may be used for cancer immunotherapy in varying doses to avoid T cell exhaustion, prevent cytokine release syndrome and minimize toxicity associated with immunotherapy. For example, low doses of the compositions of the present disclosure may be used to initially treat patients with high tumor burden, while patients with low tumor burden may be treated with high and repeated doses of the compositions of the disclosure to ensure recognition of a minimal tumor antigen load. In another instance, the compositions of the present disclosure may be delivered in a pulsatile fashion to reduce tonic T cell signaling and enhance persistence in vivo. In some aspects, toxicity may be minimized by initially using low doses of the compositions of the disclosure, prior to administering high doses. Dosing may be modified if serum markers such as ferritin, serum C-reactive protein, IL6, IFN-y, and TNF-a are elevated.

[00327] In some embodiments, the neurotoxicity may be associated with CAR or TIL therapy. Such neurotoxicity may be associated CD19-CARs. Toxicity may be due to excessive T cell infiltration into the brain. In some embodiments, neurotoxicity may be alleviated by preventing the passage of T cells through the blood brain barrier. This can be achieved by the targeted gene deletion of the endogenous alpha-4 integrin inhibitors such as tysabri / natalizumab may also be useful in the present disclosure.

[00328] Also provided herein are methods of administering ligands or DRD ligands in accordance with the disclosure to a subject in need thereof. In some embodiments, the ligand is selected from Acetazolamide (ACZ), Methotrexate (MTX), and Trimethoprim (TMP). The ligand may be administered to a subject or to cells, using any amount and any route of administration effective for tuning the transcription factor system, DRD, or payloads of the disclosure. In some embodiments, ACZ may be used with a hCA2 DRD, methotrexate may be used with an hDHFR DRD, and trimethoprim may be used with an ecDHFR DRD. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The subject may be a human, a mammal, or an animal. Compositions in accordance with the disclosure are typically formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope of sound medical judgment. In certain embodiments, the ligands in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg, from about 10 mg / kg to about 100 mg / kg, from about 50 mg / kg to about 500 mg / kg, from about 100 mg / kg to about 1000 mg / kg, of subject body weight per day, one or more times a day, to obtain the desired effect. In some embodiments, the dosage levels may be Img / kg, 5 mg / kg, 10mg / kg, 20mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg, 150 mg / kg, 160 mg / kg, 170 mg / kg, 180 mg / kg, 190 mg / kg or mg / kg of subject body weight per day, or one or more times a day, to obtain the desired effect.

[00329] The present disclosure provides methods for delivering to a cell or tissue any of the ligands described herein, comprising contacting the cell or tissue with the ligand and can be accomplished in vitro, ex vivo, or in vivo. In certain embodiments, the ligands in accordance with the present disclosure may be administered to cells at dosage levels sufficient to deliver from about 1 nM to about 10 nM, from about 5 nM to about 50 nM, from about 10 nM to about 100 nM, from about 50 nM to about 500 nM, from about 100 nM to about 1000 nM, from about 1 uM to about 10 uM from about 5 pM to about 50 uM from about 10 uM to about 100 pM from about 25 uM to about 250 pM from about 50 pM to about 500 uM. In some embodiments, the ligand may be administered to cells at doses selected from but not limited to 0.00064 uM, 0.0032 pM, 0.016 uM, 0.08 uM, 0.4 uM, 1 uM 2 pM, 10 uM, 50 uM, 75, uM, 100 uM , 150 uM, 175 uM, 200 pM, 250 uM.

[00330] The desired dosage of the ligands of the present disclosure may be delivered only once, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. As used herein, a “split dose” is the division of “single unit dose” or total daily dose into two or more doses, e.g., two or more administrations of the “single unit dose”. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event. The desired dosage of the ligand of the present disclosure may be administered as a “pulse dose” or as a “continuous flow”. As used herein, a “pulse dose” is a series of single unit doses of any therapeutic administered with a set frequency over a period of time. As used herein, a “continuous flow” is a dose of therapeutic administered continuously for a period of time in a single route / single point of contact, i.e., continuous administration event. A total daily dose, an amount given or prescribed in 24-hour period, may be administered by any of these methods, or as a combination of these methods, or by any other methods suitable for a pharmaceutical administration. Administration

[00331] In some embodiments, the compositions for cancer immunotherapy or treatment of autoimmune disease may be administered to cells ex vivo and subsequently administered to the subject. In further embodiments, the cell is selected from a B cell, a T cell, a natural killer cell (NK cell), or a tumor infiltrating lymphocyte (TIL). Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating cytotoxic T cells are described in U.S. Pat. Nos. 6,805,861 and 6,531, 451. Isolation of NK cells is described in U.S. Pat. Nos. 7.435, 596.

[00332] In some embodiments, depending upon the nature of the cells, the cells may be introduced into a host organism e.g. a mammal, in a wide variety of ways including by injection, transfusion, infusion, local instillation or implantation. In some aspects, the cells of the disclosure may be introduced at the site of the tumor. The number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, or the like. The cells may be in a physiologically-acceptable medium.

[00333] In some embodiments, the cells of the disclosure may be administrated in multiple doses to subjects having a disease or condition. The administrations generally effect an improvement in one or more symptoms of cancer or a clinical condition and / or treat or prevent cancer or clinical condition or symptom thereof.

[00334] In some embodiments, the compositions for immunotherapy or treatment of autoimmune disease may be administered in vivo. In some embodiments, polynucleotides of the present disclosure comprising transcription factor systems, payloads and compositions of the disclosure may be delivered in vivo to the subject via gene therapy. Routes of delivery

[00335] The pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, payloads, vectors and cells of the present disclosure may be administered by any route to achieve a therapeutically effective outcome. These include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal. Parenteral and injectable administration

[00336] In some embodiments, pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, payloads, vectors and cells of the present disclosure may be administered parenterally. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof. In other embodiments, surfactants are included such as hydroxypropylcellulose.

[00337] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[00338] Injectable formulations may be sterilized, for example, by filtration through a bacterial- retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Detectable agents and Labels

[00339] The transcription factor systems, nucleic acids, polynucleotides, payloads, vectors and cells of the present disclosure may be associated with or bound to one or more radioactive agents or detectable agents.

[00340] These agents include various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials (e.g., '°F, ¢’Ga, ®1™Kr, 82Rb, In, 12°], 133Xe, 20171, 12°], 3S, 14C, °H, or *™Tc (e.g, as pertechnetate (technetate(VII), TcO4)), and contrast agents (e.g., gold (e.g, gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONS), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexol), microbubbles, or perfluorocarbons).

[00341] In some embodiments, the detectable agent may be a non-detectable precursor that becomes detectable upon activation (e.g., fluorogenic tetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). In vitro assays in which the enzyme labeled compositions can be used include, but are not limited to, enzyme linked immunosorbent assays (ELISAs), immunoprecipitation assays, immunofluorescence, enzyme immunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis. Applications and uses

[00342] The transcription factor systems, constructs, ligands, or compositions of the present disclosure may be utilized in a large variety of applications including, but not limited to, therapeutics, diagnosis and prognosis, bioengineering, bioprocessing, biomanufacturing, research agents, metabolomics, gene expression, enzyme replacement, etc.

[00343] The present disclosure provides methods comprising administering a composition, for example, a pharmaceutical composition comprising one or more components of a transcription factor system to a subject in need thereof.

[00344] While there may be several uses that do not involve a medical treatment, for example, to generate cell lines and reagents for scientific research, one use involves the administration of the compositions of the present disclosure to generate in vivo gene therapy or modified cells for adoptive cell therapy, for example, the treatment of cancer, autoimmune diseases and other diseases. In an illustrative method of medical treatment or prevention of a disease, condition or disorder in a subject in need thereof, can include the following steps: (a) providing a population of cells (either human, animal, primary or cell culture, including autologous, allogenic or syngeneic); (b) introducing at least one nucleic acid molecule into at least one cell in the population of cells, wherein the at least one nucleic acid molecule comprises: (i) a first polynucleotide comprising a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor activation domain and / or the transcription factor DNA binding domain is operably linked to the DRD; and (ii) a second polynucleotide that comprises a fourth nucleic acid sequence that encodes a protein of interest that treats the disease, the fourth nucleic acid sequence being operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; (c) delivering the cell into the subject; and (d) administering a ligand to the subject that stabilizes the DRD sufficiently to enable 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 expression of the protein of interest in the cell; wherein expression of the protein of interest is regulated by the presence of ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[00345] In the above method, the protein of interest can be used to ameliorate, cure, prevent or reduce one or more symptoms of the disease, condition or disorder.

[00346] The compositions of the present disclosure may be administered to a subject using any amount and any route of administration effective for preventing or treating or imaging a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition relating to cancer, autoimmune diseases and other diseases). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.

[00347] Compositions in accordance with the disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[00348] Also provided herein, are methods of administering one or more stabilizing ligands (as used herein, the ligand that stabilizes the DRD, may be called a stabilizing ligand or simply a ligand, with the understanding that the ligand is effective in stabilizing the DRD used in the transcription factor systems in accordance with the disclosure) to a subject in need thereof. The ligand may be administered to a subject or to cells, using any amount and any route of administration effective for tuning the amount of transcription factor expression of the present disclosure in a cell comprising the transcription factor system. The exact amount of stabilizing ligand required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The subject may be a human, a mammal, or an animal. Therapeutic uses Cancer immunotherapy

[00349] Cancer immunotherapy aims at the induction or restoration of the reactivity of the immune system towards cancer. Significant advances in immunotherapy research have led to the development of various strategies which may broadly be classified into active immunotherapy and passive immunotherapy. In general, these strategies may be utilized to directly kill cancer cells or to counter the immunosuppressive tumor microenvironment. Active immunotherapy aims at induction of an endogenous, long-lasting tumor-antigen specific immune response. The response can further be enhanced by non-specific stimulation of immune response modifiers such as cytokines. In contrast, passive immunotherapy includes approaches where effector immune molecules such as tumor- antigen specific cytotoxic T cells or antibodies are administered to the host. This approach is short lived and requires multiple applications.

[00350] Despite significant advances, the efficacy of current immunotherapy strategies is limited by associated toxicities. These are often related to the narrow therapeutic window associated with immunotherapy, which in part, emerges from the need to push therapy dose to the edge of potentially fatal toxicity to get a clinically meaningful treatment effect. Further, dose expands in vivo since adoptively transferred immune cells continue to proliferate within the patient, often unpredictably.

[00351] A major risk involved in immunotherapy is the on-target but off-tumor side effects resulting from T-cell activation in response to normal tissue expression of the tumor associated antigen (TAA). Clinical trials utilizing T cells expressing T-cell receptor against specific TAA reported skin rash, colitis and hearing loss in response to immunotherapy.

[00352] Immunotherapy may also produce on target, on-tumor toxicities that emerge when tumor cells are killed in response to the immunotherapy. The adverse effects include tumor lysis syndrome, cytokine release syndrome and the related macrophage activation syndrome. Importantly, these adverse effects may occur during the destruction of tumors, and thus even a successful on-tumor immunotherapy might result in toxicity. Approaches to control immunotherapy via immunotherapeutic agent regulation are thus highly desirable since they have the potential to reduce toxicity and maximize efficacy.

[00353] The present disclosure provides systems, compositions, immunotherapeutic agents and methods for immunotherapy. These compositions provide tunable regulation of gene expression and function in immunotherapy, for example for the prevention and treatment of cancer.

[00354] In one aspect, the systems, compositions, immunotherapeutic agents and other components of the disclosure can be controlled by a separately added stabilizing ligand, which provides a significant flexibility to regulate cancer immunotherapy. Further, the systems, compositions and the methods of the present disclosure may also be combined with therapeutic agents such as chemotherapeutic agents, small molecules, gene therapy, and antibodies to prevent and / or treat a disease, for example, cancer.

[00355] The tunable nature of the systems and compositions of the disclosure has the potential to improve the potency and duration of the efficacy of immunotherapies. Reversibly silencing the biological activity of adoptively transferred cells using compositions of the present disclosure allows maximizing the potential of cell therapy without irretrievably killing and terminating the therapy.

[00356] The present disclosure provides methods for fine tuning of immunotherapy after administration to patients. This in turn improves the safety and efficacy of immunotherapy and increases the subject population that may benefit from immunotherapy.

[00357] In some embodiments, immune cells of the disclosure may be T cells modified to express a payload or protein of interest, for example, an antigen-specific T cell receptor (TCR), or an antigen specific chimeric antigen receptor (CAR) taught herein (known as CAR T cells). Accordingly, at least one polynucleotide encoding a protein of interest, for example, a CAR system (or a TCR) described herein, or a vector comprising the polynucleotide is introduced into a T cell. The T cell expressing the CAR or TCR binds to a specific antigen via the extracellular targeting moiety of the CAR or TCR, thereby a signal via the intracellular signaling domain (s) is transmitted into the T cell, and as a result, the T cell is activated. The activated CAR T cell changes its behavior including release of a cytotoxic cytokine (e.g., a tumor necrosis factor, and lymphotoxin, etc.), improvement of a cell proliferation rate, change in a cell surface molecule, or the like. Such changes cause destruction of a target cell expressing the antigen recognized by the CAR or TCR. In addition, release of a cytokine or change in a cell surface molecule stimulates other immune cells, for example, a B cell, a dendritic cell, a NK cell, and a macrophage.

[00358] The CAR introduced into a T cell may be a first-generation CAR including only the intracellular signaling domain from TCR CD?3zeta, or a second-generation CAR including the intracellular signaling domain from TCR CD3zeta and a costimulatory signaling domain, or a third- generation CAR including the intracellular signaling domain from TCR CD3zeta and two or more costimulatory signaling domains, or a split CAR system, or an on / off switch CAR system. In one example, the expression of the CAR or TCR is controlled by a transcription factor, wherein the transcription factor or component thereof is operably linked to a DRD, which in the absence of a stabilizing ligand will result in the little to no accumulation of transcription factor. The payloadhas a polynucleotide binding sequence specific to the transcription factor or component thereof, therefore, without the stabilizing ligand, little to no protein of interest is produced. When stabilizing ligand is administered to the cell comprising the transcription factor system, the transcription factor is rescued from degradation when coupled to the DRD, and the transcription factor then binds to its cognate polynucleotide binding sequence immediately adjacent the protein of interest, which then is transcribed. The transcribed mRNA is then translated to produce the polypeptide / protein of interest. In some exemplary embodiments, the presence or absence of the DRD stabilizing ligand is used to tune the CAR or TCR expression in transduced T cells or NK cells.

[00359] In some embodiments, CAR T cells of the disclosure may be further modified to express another one, two, three or more immunotherapeutic agents. The immunotherapeutic agents may be another CAR or TCR specific to a different target molecule; a cytokine such as IL2, IL12, IL15 and IL18, or a cytokine receptor such as IL15Ra; a chimeric switch receptor that converts an inhibitory signal to a stimulatory signal; a homing receptor that guides adoptively transferred cells to a target site such as the tumor tissue; an agent that optimizes the metabolism of the immune cell; or a safety switch gene (e.g., a suicide gene) that kills activated T cells when a severe event is observed after adoptive cell transfer or when the transferred immune cells are no-longer needed. These molecules may be included in the same constructs or in separate constructs.

[00360] In one embodiment, the CAR T cell (including TCR T cell) of the disclosure may be an “armed” CAR T cell which is transfected or transduced with one or more components of the transcription factor system comprising a CAR payload and either the same or a different transcription factor system encoding a cytokine under control of the same or different transcription factor operably linked to the same or different DRD. The inducible or constitutively secreted active cytokines further arm CAR T cells to improve efficacy and persistence. In this context, such CAR T cell is also referred to as “armored CAR T cell”. The “armor” molecule may be selected based on the tumor microenvironment and other elements of the innate and adaptive immune systems. In some embodiments, the molecule may be a stimulatory factor such as IL2, IL12, IL15, IL18, type I IFN, CDA40L and 4-1BBL which have been shown to further enhance CAR T cell efficacy and persistence in the face of a hostile tumor microenvironment via different mechanisms.

[00361] Chimeric Antigen Receptor engineered T cells (CAR-T) therapies have yet to be successfully applied to solid tumors. Enhancing CAR-T cell functionality and selectively delivering cargo to the site of solid tumors represent key tactics to achieve effective CAR-T therapy for solid tumors. In one embodiment, a payload or protein of interest may include Interleukin 12 (IL12) may be utilized to enhance the effectiveness of CAR-T cells, especially since it has the potential to remodel the tumor microenvironment. IL12 has been previously shown to be effective in enhancing efficacy of CAR or TCR modified T-cells as well as tumor infiltrating lymphocytes (TILs) in preclinical and clinical models. However, constitutive production of IL12 can compromise safety and / or efficacy; therefore, on demand, local delivery of the cytokine may be a preferred approach. In some embodiments, transcription factor system of the present disclosure, or components thereof, may be utilized to exogenously control IL12 expression to enable the use of IL12 in adoptive cell therapy.

[00362] In some embodiments, transcription factor regulated systems of the present disclosure may be used to regulate a payload such as Flexi IL12 (or other IL12 constructs such as membrane bound IL12) expression in transformed immune cells to improve the efficacy of the CARs, especially in solid tumor settings, by providing a controlled local signal for tumor microenvironment remodeling and epitope spreading. Transcription factor regulation as described herein also provides rapid, dose dependent, and local production of IL12, upon addition of DRD specific stabilizing ligands.

[00363] In some aspects, the armed CAR T cell of the disclosure is modified to express a CD19 CAR and a payload such as IL12, which is regulated using a transcription factor system or composition of the present disclosure. Such T cells, after CAR mediated activation in the tumor, release inducible IL12 which augments T-cell activation and attracts and activates innate immune cells to eliminate CD19-positive cancer cells.

[00364] In one embodiment, T cells of the disclosure may be modified to incorporate a transcription factor system comprising a CAR payload encoded by the transcription factor system or component thereof and a nucleic acid sequence encoding a suicide gene.

[00365] In one embodiment, the CAR T cell (including TCR T cell) of the disclosure may be transfected or transduced with one or more components of a transcription factor system comprising a cytokine and a safety switch gene (e.g, suicide gene). The suicide gene may be an inducible caspase such as caspase 9 which induces apoptosis, when activated by an extracellular stabilizing ligand of a DRD encoded by the transcription factor system. Such induced apoptosis eliminates transferred cell as required to decrease the risk of direct toxicity and uncontrolled cell proliferation.

[00366] In one embodiment, the transcription factor system, and components thereof that tune expression levels and activities of any described payloads or proteins of interest (used interchangeably) may be used for immunotherapy. As non-limiting examples, an immunotherapeutic agent may be an antibody and fragments and variants thereof, a cancer specific T cell receptor (TCR) and variants thereof, an anti-tumor specific chimeric antigen receptor (CAR), a chimeric switch receptor, an inhibitor of a co-inhibitory receptor or ligand, an agonist of a co-stimulatory receptor and ligand, a cytokine, chemokine, a cytokine receptor, a chemokine receptor, a soluble growth factor, a metabolic factor, a suicide gene, a homing receptor, or any agent that induces an immune response in a cell and a subject.

[00367] In some embodiments, the 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 a first polynucleotide comprising a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD); wherein at least one of the transcription factor activation domain, the transcription factor DNA binding domain, and the combination of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; and a second polynucleotide comprising a fourth nucleic acid sequence that encodes a protein of interest, the fourth nucleic acid sequence being operably linked to an inducible promoter comprising the specific polynucleotide binding site; wherein the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor; and wherein binding of the transcription factor to the specific polynucleotide binding site is required for regulating transcription of the fourth nucleic acid sequence by the transcription factor.

[00368] In one aspect, the payload may be an immunotherapeutic agent.

[00369] In some embodiments, a transcription factor system, and compositions of the present disclosure relate to transcriptional regulation of protein (protein of interest or payload) function, including for example, anti-tumor immune responses of immunotherapeutic agents. In some embodiments, the immunotherapeutic agents may include cytokines, chemokines, antibodies, integrins, integral proteins, membrane proteins, extracellular proteins that may be used to upregulate, or improve the function of one or more immune cell types, or down regulate the activity of one or more immune cell types. In various embodiments, the immunotherapeutic agents useful in the treatment of a disease, condition or disorder can include cytokines, for example interleukins. In various embodiments, the transcription factor system provides a protein of interest or payload that includes an interleukin, for example, IL-2, IL-6, IL12, IL15, IL18 and other immunotherapeutic agents that promotes or upregulates the longevity and activity of one or more immune cell types useful to treat a disease, condition or disorder or a symptom associate with any of these.

[00370] In some embodiments, cells which are genetically modified to encode and express at least one transcription factor operable to permit transcription of a protein of interest linked to a transcription factor polynucleotide binding site (immunotherapeutic agent) may be used for adoptive cell therapy (ACT, also referred to as “adoptive cell transfer”). As used herein, adoptive cell transfer refers to the administration of immune cells (from autologous, allogenic or genetically modified hosts) with direct anticancer activity. ACT has shown promise in clinical application against malignant and infectious disease. For example, T cells genetically engineered to recognize CD19 have been used to treat follicular B cell lymphoma (Kochenderfer et al., Blood, 2010, 116:4099- 4102; and Kochenderfer and Rosenberg, Nat Rev Clin Oncol., 2013, 10(5): 267-276) and ACT using autologous lymphocytes genetically-modified to express anti-tumor T cell receptors has been used to treat metastatic melanoma (Rosenberg and Dudley, Curr. Opin. Immunol. 2009, 21: 233-240).

[00371] According to the present disclosure, the 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 therapies to effect CAR therapies, in the manipulation or regulation of TILs, in allogeneic cell therapy, in combination T cell therapy with other treatment lines (e.g. radiation, cytokines), to encode engineered TCRs, or modified TCRs, or to enhance T cells other than TCRs (e.g. by introducing cytokine genes, genes for the checkpoint inhibitors PD1, CTLA4).

[00372] Provided herein are methods for use in adoptive cell therapy. The methods involve preconditioning a subject in need thereof, modulating immune cells with one or more components of a transcription factor system, and / or compositions of the present disclosure; administering to a subject engineered immune cells expressing compositions of the disclosure and the successful engraftment of engineered cells within the subject.

[00373] In some embodiments, regulatable transcription factor expression constructs and compositions of the present disclosure, may be used to minimize preconditioning regimens associated with adoptive cell therapy. As used herein “preconditioning” refers to any therapeutic regimen administered to a subject to improve the outcome of adoptive cell therapy. Preconditioning strategies include but are not limited to total body irradiation and / or lymphodepleting chemotherapy. Adoptive therapy clinical trials without preconditioning have failed to demonstrate any clinical benefit, indicating its importance in ACT. Yet, preconditioning is associated with significant toxicity and limits the subject cohort that is suitable for ACT. In some instances, immune cells for ACT may be engineered to express cytokines such as IL-2, IL-6, IL12 and IL15 as payload using transcription factors described herein to permit selective expression of the protein of interest which may be tuned using a stabilizing ligand of the present disclosure to reduce the need for preconditioning (Pengram etal. (2012) Blood 119 (18): 4133-41, the contents of which are incorporated by reference in their entirety).

[00374] In some embodiments, immune cells for 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, immune stimulatory cells for ACT may be generated from embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC). In some embodiments, autologous or allogeneic immune cells are used for ACT.

[00375] In some embodiments, cells used for ACT may be T cells engineered to express CARs comprising an antigen-binding domain specific to an antigen on tumor cells of interest. In other embodiments, cells used for ACT may be NK cells engineered to express CARs comprising an antigen-binding domain specific to an antigen on tumor cells of interest. In addition to adoptive transfer of genetically modified T cells (e.g., CAR T cells) for immunotherapy, alternate types of CAR-expressing leukocytes, either alone, or in combination with CAR T cells may be used for adoptive immunotherapy. In one example, a mixture of T cells and NK cells may be used for ACT. The expression level of CARs in T cells and NK cells, according to the present disclosure, is tuned and controlled by a small molecule that binds to the DRD(s) operably linked to a transcription factor or components thereof, which enables selective transcription of the CAR in the transfected or transduced T cells and NK cells. In this scenario, the CAR is encoded by a nucleic acid sequence operably inked to an inducible promoter comprising the specific polynucleotide binding site of the transcription factor.

[00376] In some embodiments, NK cells engineered to express one or more components of a transcription factor system may be used for ACT. NK cell activation induces perforin / granzyme- dependent apoptosis in target cells. NK cell activation also induces cytokine secretion such as IFN vy, TNF-a and GM-CSF. These cytokines enhance the phagocytic function of macrophages and their antimicrobial activity and augment the adaptive immune response via up-regulation of antigen presentation by antigen presenting cells such as dendritic cells (DCs) (Reviewed by Vivier et al., Nat. Immunol., 2008, 9(5): 503-510).

[00377] Other examples of genetic modification may include the introduction of chimeric antigen receptors (CARs) and the down-regulation of inhibitory NK cell receptors such as NKG2A.

[00378] NK cells may also be genetically reprogrammed to circumvent NK cell inhibitory signals upon interaction with tumor cells. For example, using CRISPR, ZFN, or TALEN to genetically modify NK cells to silence their inhibitory receptors may enhance the anti-tumor capacity of NK cells.

[00379] Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating and expanding cytotoxic T cells are described in U.S. Pat. Nos. 6,805,861 and 6,531,451; US Patent Publication NO. US20160348072A1 and International Patent Publication NO. WO2016168595A1; the contents of each of which are incorporated herein by reference in their entirety. Isolation and expansion of NK cells is described in US Patent Publication NO. US20150152387A1, U.S. Patent NO. 7,435,596; and Oyer, J.L. (2016). Cytotherapy.18(5):653- 63; the contents of each of which are incorporated by reference herein in its entirety. Specifically, human primary NK cells may be expanded in the presence of feeder cells e.g. a myeloid cell line that has been genetically modified to express membrane bound IL15, IL21, IL12 and 4-1BBL.

[00380] In some instances, sub populations of immune cells may be enriched for ACT. Methods for immune cell enrichment are taught in International Patent Publication No. WO02015039100A1. In another example, T cells positive for B and T lymphocyte attenuator marker BTLA) may be used to enrich for T cells that are anti-cancer reactive as described in U.S. Pat. NO. 9,512,401 (the content of each of which are incorporated herein by reference in their entirety).

[00381] In some embodiments, immune cells for ACT may be depleted of select sub populations to enhance T cell expansion. For example, immune cells may be depleted of Foxp3+ T lymphocytes to minimize the anti-tumor immune response using methods taught in US Patent Publication NO. US 20160298081A1; the contents of which are incorporated by reference herein in their entirety.

[00382] In some embodiments, activation and expansion of T cells for ACT is achieved antigenic stimulation of a transiently expressed Chimeric Antigen Receptor (CAR) on the cell surface. Such activation methods are taught in International Patent NO. W02017015427, the content of which are incorporated herein by reference in their entirety.

[00383] In some embodiments, immune cells may be activated by antigens associated with antigen presenting cells (APCs). In some embodiments, the APCs may be dendritic cells, macrophages or B cells that are antigen specific or nonspecific. The APCs may autologous or homologous in their organ. In some embodiments, the APCs may be artificial antigen presenting cells (aAPCs) such as cell based aAPCs or acellular aAPCs. Cell based aAPCs may be selected from either genetically modified allogeneic cells such as human erythroleukemia cells or xenogeneic cells such as murine fibroblasts and Drosophila cells. Alternatively, the APCs maybe be acellular wherein the antigens or costimulatory domains are presented on synthetic surfaces such as latex beads, polystyrene beads, lipid vesicles or exosomes.

[00384] In some embodiments, cells of the disclosure, specifically T cells may be expanded using artificial cell platforms. In one embodiment, the mature T cells may be generated using artificial thymic organoids (ATOs) described by Seet CS et al. 2017. Nat Methods. 14, 521-530 (the contents of which are incorporated herein by reference in their entirety). ATOs are based on a stromal cell line expressing delta like canonical notch ligand (DLL1). In this method, stromal cells are aggregated with hematopoietic stem and progenitor cells by centrifugation and deployed on a cell culture insert at the air—fluid interface to generate organoid cultures. ATO-derived T cells exhibit naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function.

[00385] In some embodiments, adoptive cell therapy is carried out by autologous transfer, wherein the cells are derived from a subject in need of a treatment and the cells, following isolation and processing are administered to the same subject. In other instances, ACT may involve allogenic transfer wherein the cells are isolated and / or prepared from a donor subject other than the recipient subject who ultimately receives cell therapy. The donor and recipient subject may be genetically identical, or similar or may express the same HLA class or subtype.

[00386] In some embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by the same or different transcription factor systems. In one example, each of the two payloads, for example, a cytokine such as IL12 and a CAR construct such as CD19 CAR that are transcribed by one or more transcription factor(s) on the same or different transcription factor systems, wherein the transcription factors(s) are linked to the same or different DRDs. The payloads are transcribed and translated when the DRD(s) is / are stabilized with a stabilizing ligand specific for the DRD(s). The expression of IL12 and CD19 CAR is tuned using one or more stabilizing ligands. In other embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by different transcription factor systems. In one example, a cytokine such as IL12 and a CAR construct such as CD19 CAR are each transcribed by one of two different transcription factors, each transcription factor is operably linked to a different DRDs, and thereby can be tuned separately using different stimuli. In another example, a suicide gene and a CAR construct may be transcriptionally activated by two different transcription factors.

[00387] Following genetic modulation using one or more components of a transcription factor system and compositions of the disclosure, cells are administered to the subject in need thereof. Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338; the contents of each of which are incorporated herein by reference in their entirety.

[00388] In some embodiments, immune cells for ACT may be modified to express one or more immunotherapeutic agents (proteins of interest) which facilitate immune cells activation, infiltration, expansion, survival and anti-tumor functions. The immunotherapeutic agents may be a second CAR or TCR specific to a different target molecule; a cytokine or a cytokine receptor; a chimeric switch receptor that converts an inhibitory signal to a stimulatory signal; a homing receptor that guides adoptively transferred cells to a target site such as the tumor tissue; an agent that optimizes the metabolism of the immune cell; or a safety switch gene (e.g., a suicide gene) that kills activated T cells when a severe event is observed after adoptive cell transfer or when the transferred immune cells are no-longer needed.

[00389] In some embodiments, immune cells used for adoptive cell transfer can be genetically manipulated to improve their persistence, cytotoxicity, tumor targeting capacity, and ability to home to disease sites in vivo, with the overall aim of further improving upon their capacity to kill tumors in cancer patients. One example is to introduce one or more components of a transcription factor system of the disclosure encoding a cytokine, such as a gamma-cytokine (e.g. IL2 and IL15) into immune cells to promote immune cell proliferation and survival. Transduction of cytokine genes (e.g., gamma-cytokines IL2 and IL15) encoded by a transcription factor system into immune cells will enable the immune cells, e.g. NK cells to propagate without addition of exogenous cytokines such that the cytokine expressing NK cells have enhanced tumor cytotoxicity.

[00390] In some embodiments, one or more components of a transcription factor system may be utilized to prevent T cell exhaustion. As used herein, “T cell exhaustion” refers to the stepwise and progressive loss of T cell function caused by chronic T cell activation. T cell exhaustion is a major factor limiting the efficacy of antiviral and antitumor immunotherapies. Exhausted T cells have low proliferative and cytokine producing capabilities concurrent with high rates of apoptosis and high surface expression of multiple inhibitory receptors. T cell activation leading to exhaustion may occur either in the presence or absence of the antigen.

[00391] In some embodiments, one or more components of a transcription factor system may be utilized to prevent T cell exhaustion in the context of Chimeric Antigen Receptor -T cell therapy (CAR-T). In this context, exhaustion in some instances, may be caused by the oligomerization of the scFvs of the CAR on the cell surface which leads to continuous activation of the intracellular domains of the CAR. As a non-limiting example, CARs of the present disclosure may include scFvs that are unable to oligomerize. As another non-limiting example, CARs that are rapidly internalized and re-expressed following antigen exposure may also be selected to prevent chronic scFv oligomerization on cell surface. In one embodiment, the framework region of the scFvs may be modified to prevent constitutive CAR signaling (Long et al. 2014. Cancer Research. 74(19) S1; the contents of which are incorporated by reference in their entirety). One or more components of a transcription factor system of the present disclosure may also be used to regulate the surface expression of the CAR on the T cell surface to prevent chronic T cell activation. The CARs of the disclosure may also be engineered to minimize exhaustion. As a non-limiting example, the 41-BB signaling domain may be incorporated into CAR design to ameliorate T cell exhaustion. In some embodiments, any of the strategies disclosed by Long H A et al. may be utilized to prevent exhaustion (Long A H et al. (2015) Nature Medicine 21, 581-590; the contents of which are incorporated herein by reference in their entirety).

[00392] In some embodiments, the tunable nature of the transcription factor system of the present disclosure may be utilized to reverse human T cell exhaustion observed with tonic CAR signaling. Reversibly silencing the biological activity of adoptively transferred cells using compositions of the present disclosure may be used to reverse tonic signaling which, in turn, may reinvigorate the T cells. Reversal of exhaustion may be measured by the downregulation of multiple inhibitory receptors associated with exhaustion.

[00393] In some embodiments, T cell metabolic pathways may be modified to diminish the susceptibility of T cells to exhaustion. Metabolic pathways may include, but are not limited to glycolysis, urea cycle, citric acid cycle, beta oxidation, fatty acid biosynthesis, pentose phosphate pathway, nucleotide biosynthesis, and glycogen metabolic pathways. As a non-limiting example, payloads that reduce the rate of glycolysis may be utilized to restrict or prevent T cell exhaustion (Long et al. Journal for Inmunotherapy of Cancer 2013, 1(Suppl 1): P21; the contents of which are incorporated by reference in their entirety). In one embodiment, T cells of the present disclosure may be used in combination with inhibitors of glycolysis such as 2-deoxyglucose, and rapamycin.

[00394] In some embodiments, payloads or proteins of interest of the disclosure may be used in conjunction with antibodies or fragments that target T cell surface markers associated with T cell exhaustion. T-cell surface markers associated with T cell exhaustion that may be used include, but are not limited to, CTLA-1, PD-1, TGIT, LAG-3, 2B4, BTLA, TIM3, VISTA, and CD96. In some embodiments, one or more components of a transcription factor system may be utilized to prevent T cell exhaustion.

[00395] In some embodiments, the compositions of the present disclosure may be utilized to alter TIL (tumor infiltrating lymphocyte) populations in a subject. In one embodiment, any of the payloads described herein may be utilized to change the ratio of CD4 positive cells to CD8 positive populations. In some embodiments, TILs may be sorted ex vivo and engineered to express any of the cytokines described herein. Payloads of the disclosure may be used to expand CD4 and / or CD8 populations of TILs to enhance TIL mediated immune response. Parameters for improving CAR-T therapy outcome are described in Finney et al. JCI. 2019;129(5):2123-2132 (the contents of which are herein incorporated by reference in their entirety). The levels of biomarker LAG3 (high) / TNF-a (low) in peripheral blood CD8+ T cells at the time of apheresis may also predict a subsequent dysfunctional response in subjects with high antigen load who do not achieve complete response that is durable for more than a few weeks. T cell-intrinsic features that are a consequence of the starting T cell repertoire and the effects of the manufacturing process converge with CD19 antigen-induced activation following adoptive transfer may also play a role in the outcome of CAR-T therapy. The starting T cell repertoire may in part be affected by the timing of the apheresis. In one embodiment, the apheresis may be performed prior to chemotherapy. Cumulative burden of CD19 expressing leukemic and normal B cells, as evaluated in the bone marrow prior to lymphodepleting chemotherapy may be important for determining CAR-T therapy outcome. According to Finney et al., increase antigen burden improves CAR-T therapy outcome. To increase CD19 antigen burden in vivo, subjects may also be infused with expanded subject derived T cells genetically modified to express CD19 (also referred to as T-APCs).

[00396] In some embodiments, regulatable transcription factor expression constructs, payloads of interest (e.g., immunotherapeutic agents), vectors, cells and compositions of the present disclosure may be used in conjunction with cancer vaccines.

[00397] In some embodiments, cancer vaccine may comprise peptides and / or proteins derived from tumor associated antigen (TAA). Such strategies may be utilized to evoke an immune response in a subject, which in some instances may be a cytotoxic T lymphocyte (CTL) response. Peptides used for cancer vaccines may also modified to match the mutation profile of a subject. For example, EGFR derived peptides with mutations matched to the mutations found in the subject in need of therapy have been successfully used in patients with lung cancer (Li F et al. (2016) Oncoimmunology. Oct 7;5(12): €1238539; the contents of which are incorporated herein by reference in their entirety).

[00398] In one embodiment, cancer vaccines of the present disclosure may include superagonist altered peptide ligands (APL) derived from tumor associated antigens (TAAs). These are mutant peptide ligands deviate from the native peptide sequence by one or more amino acids, which activate specific CTL clones more effectively than native epitopes. These alterations may allow the peptide to bind better to the restricting Class I MHC molecule or interact more favorably with the TCR of a given tumor-specific CTL subset. APLs may be selected using methods taught in US Patent Publication NO. US20160317633A1, the contents of which are incorporated herein by reference in their entirety.

[00399] In some embodiments, effector immune cells genetically modified to encode the components of the transcription factor system, and payloads of the disclosure may be combined with the biological adjuvants described herein. Dual regulation of CAR and cytokines and ligands to segregate the kinetic control of target-mediated activation from intrinsic cell T cell expansion. Such dual regulation also minimizes the need for pre-conditioning regimens in patients. As a non-limiting example, DRD regulated transcription factors which transcribe a payload, for example, a CAR e.g. CD19 CAR may be combined with cytokines e.g. IL12 to enhance the anti-tumor efficacy of the CAR (Pegram H.J., et al. Tumor-targeted T cells modified to secrete IL12 eradicate systemic tumors without need for prior conditioning. Blood.2012;119:4133—41; the contents of each of which are incorporated herein by reference in their entirety). As another non-limiting example, Merchant et al. combined dendritic cell-based vaccinations with recombinant human IL7 to improve outcome in high-risk pediatric sarcomas patients (Merchant, M.S. et. al. Adjuvant immunotherapy to Improve Outcome in High-Risk Pediatric Sarcomas. Clin Cancer Res. 2016. 22(13):3182-91; the contents of each of which are incorporated herein by reference in their entirety).

[00400] In some embodiments, effector immune cells modified to express one or more antigen-specific TCRs or CARs may be combined with compositions of the disclosure comprising immunotherapeutic agents that convert the immunosuppressive tumor microenvironment.

[00401] In one aspect, effector immune cells modified to express CARs specific to different target molecules on the same cell may be combined. In another aspect, different immune cells modified to express the same CAR construct such as NK cells and T cells may be used in combination for a tumor treatment, for instance, a T cell modified to express a CD19 CAR may be combined with a NK cell modified to express the same CD19 CAR to treat B cell malignancy.

[00402] In other embodiments, immune cells modified to express CARs may be combined with checkpoint blockade agents.

[00403] In some embodiments, effector immune cells genetically modified to express one or more components of the transcription factor system, for example a payload of the disclosure, may be combined with cancer vaccines and other immunotherapeutics and adjuvant treatments of the disclosure.

[00404] In some embodiments, methods of the disclosure may include combination of the compositions of the disclosure with other agents effective in the treatment of cancers, infection diseases and other immunodeficient disorders, such as anti-cancer agents. As used herein, the term “anti-cancer agent” refers to any agent which is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[00405] In some embodiments, anti-cancer agent or therapy may be a chemotherapeutic agent, or radiotherapy, immunotherapeutic agent, surgery, or any other therapeutic agent which, in combination with the present disclosure, improves the therapeutic efficacy of treatment.

[00406] In one embodiment, one or more components of a transcription factor system comprising a CD19 CAR may be used in combination with amino pyrimidine derivatives such as the Burkit's tyrosine receptor kinase (BTK) inhibitor using methods taught in International Patent Application NO. W02016164580, the contents of which are incorporated herein by reference in their entirety.

[00407] In some embodiments, compositions of the present disclosure may be used in combination with immunotherapeutics other than the inventive therapy described herein, such as antibodies specific to some target molecules on the surface of a tumor cell.

[00408] Exemplary chemotherapies include, without limitation, Acivicin; Aclarubicin; Acodazole hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone acetate; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperrin, Sulindac, Curcumin, alkylating agents including: Nitrogen mustards such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas such as carmustine (BC U), lomustine (CCNU), and semustine (methyl-CC U); thylenimines / methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrrolidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6- thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins such as etoposide and teniposide; antibiotics, such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase, cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta and GM-CSF, anti-angiogenic factors, such as angiostatin and endostatin, inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors, including soluble VGF / VEGEF receptors, platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIFf) and procarbazine, adrenocortical suppressants such as mitotane (0,p'-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; non-steroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosin kinase inhibitors such as imatinib mesylate (marketed as Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor) now marketed as Tarveca; anti-virals such as oseltamivir phosphate, Amphotericin B, and palivizumab; Sdi 1 mimetics; Semustine; Senescence derived inhibitor 1; Sparfosic acid; Spicamycin D; Spiromustine; Splenopentin; Spongistatin 1; Squalamine; Stipiamide; Stromelysin inhibitors; Sulfinosine; Superactive vasoactive intestinal peptide antagonist; Velaresol; Veramine; Verdins; Verteporfin; Vinorelbine; Vinxaltine; Vitaxin; Vorozole; Zanoterone; Zeniplatin; Zilascorb; and Zinostatin stimalamer; PI3Kp small-molecule inhibitor, GSK2636771; pan-PI3K inhibitor (BKM120); BRAF inhibitors. Vemurafenib (Zelboraf) and dabrafenib (Tafinlar); or any analog or derivative and variant of the foregoing.

[00409] Radiotherapeutic agents and factors include radiation and waves that induce DNA damage for example, y-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, radioisotopes, and the like. Therapy may be achieved by irradiating the localized tumor site with the above described forms of radiations. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[00410] In some embodiments, the chemotherapeutic agent may be an immunomodulatory agent such as lenalidomide (LEN). Recent studies have demonstrated that lenalidomide can enhance antitumor functions of CAR modified T cells (Otahal et al., Oncoimmunology, 2015, 5(4): €1115940). Some examples of anti-tumor antibodies include tocilizumab, siltuximab.

[00411] Other agents may be used in combination with compositions of the disclosure may also include, but not limited to, agents that affect the upregulation of cell surface receptors and their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion such as focal adhesion kinase (FAKs) inhibitors and Lovastatin, or agents that increase the sensitivity of the hyper proliferative cells to apoptotic inducers such as the antibody C225.

[00412] The combinations may include administering the compositions of the disclosure and other agents at the same time or separately. Alternatively, the present immunotherapy may precede or follow the other agent / therapy by intervals ranging from minutes, days, weeks to months.

[00413] Provided in the present disclosure is a method of reducing a tumor volume or burden in a subject in need, the method comprising introducing into the subject a composition of the disclosure.

[00414] The present disclosure also provides methods for treating a cancer in a subject, comprising administering to the subject an effective amount of effector immune cells genetically modified to comprise a transcription factor system of the present disclosure. Cancer

[00415] Various cancers may be treated with pharmaceutical compositions, transcription factor system components, regulatable transcription factor expression constructs including their DRDs or payloads of the present disclosure. As used herein, the term “cancer” refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the p...

Claims

CLAIMS What is claimed is® 1. A modified cell comprising a first polynucleotide, said first polynucleotide comprising a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD); wherein at least one of the transcription factor activation domain, the transcription factor DNA binding domain, or the combination of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; wherein the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that is able to activate transcription of a fourth nucleic acid sequence upon binding to the specific polynucleotide binding site; and wherein the fourth nucleic acid sequence encodes a protein of interest and is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site. 2 A modified cell comprising a first polynucleotide, said first polynucleotide comprising a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD); wherein at least one of the transcription factor activation domain, the transcription factor DNA binding domain, or the combination of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; wherein the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that is able to activate transcription of a fourth nucleic acid sequence upon binding to the specific polynucleotide binding site; and wherein the fourth nucleic acid sequence encodes a protein of interest and is operably linked to the specific polynucleotide binding site.

3. The modified cell of claim 2, wherein the fourth nucleic acid sequence is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site.

4. The modified cell of any one of claims 1-3, wherein the protein of interest is a heterologous protein.

5. The modified cell of any one of claims 1-4, wherein the fourth nucleic acid sequence is located on the first polynucleotide.

6. The modified cell of any one of claims 1-4, further comprising a second polynucleotide, said second polynucleotide comprising the fourth nucleic acid sequence. % The modified cell of any one of claims 1-6, wherein the transcription factor DNA binding domain is derived from a parent protein selected from the group consisting of: ZFHD1, Cas9, Casl2, and TAL.

8. The modified cell of any one of claims 1-7, wherein the transcription factor activation domain is derived from a parent protein, wherein said parent protein is p65.

9. A modified cell 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, wherein the transcription factor is operably linked to the DRD; and wherein the transcription factor is able to bind to a specific polynucleotide binding site and activate transcription of 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.

10. A modified cell 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, wherein the transcription factor is operably linked to the DRD; and wherein the transcription factor is able to bind to a specific polynucleotide binding site and activate transcription of a third nucleic acid sequence encoding a protein of interest, wherein the third nucleic acid sequence is operably linked to the specific polynucleotide binding site.

11. The modified cell of claim 10, wherein the third nucleic acid sequence is operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site.

12. The modified cell of any one of claims 9-11, wherein the protein of interest is a heterologous protein.

13. The modified cell of any one of claims 9-12, wherein the third nucleic acid sequence is located on the polynucleotide that comprises the first nucleic acid sequence and the second nucleic acid sequence.

14. The modified cell of any one of claims 9-12, further comprising a second polynucleotide that comprises the third nucleic acid sequence.

15. The modified cell of any one of claims 1-14, wherein the DRD is derived from a parent protein selected from the group comprising: human carbonic anhydrase 2 (CA2), human DHFR, E. coli DHFR (ecDHFR), human estrogen receptor (ER), FKBP, human protein FKBP, and human PDES.

16. The modified cell of any one of claims 1-15, wherein the DRD is stabilized in the presence of a ligand selected from the group comprising: Acetazolamide (ACZ), Methotrexate (MTX), and Trimethoprim (TMP).

17. The modified cell of any one of claims 1-16, wherein the protein of interest is a wild-type protein.

18. The modified cell of any one of claims 1-17, wherein the protein of interest is a therapeutic protein.

19. The modified cell of claim 18, wherein the protein of interest is selected from the group consisting of a cytokine, an antibody, a coagulation factor, an enzyme, a gene editing protein, a T cell receptor (TCR) and a chimeric antigen receptor (CAR).

20. The modified cell of any one of claims 1-17, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

21. The modified cell of any one of claims 1-17, wherein the protein of interest is a secreted protein.

22. The modified cell of any one of claims 1-21, wherein the cell is a T cell, a natural killer cell (NK cell), or a tumor infiltrating lymphocyte (TIL). 23 The modified cell of any one of claims 1-21, wherein the cell is a stem cell, a liver cell, a blood cell, a pancreatic cell, a neuronal cell, an ocular cell, a muscle cell, or a bone cell.

24. A nucleic acid molecule comprising: a. a first nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and b. a second nucleic acid sequence that encodes a drug responsive domain (DRD).

25. The nucleic acid molecule of claim 24, further comprising: @; a third nucleic acid sequence that encodes a transcription factor activation domain; wherein either (i) the transcription factor DNA binding domain is operably linked to the DRD; (ii) the transcription factor activation domain is operably linked to the DRD; or (iii) the combination of the transcription factor DNA binding domain and the transcription factor activation domain is operably linked to the DRD.

26. The nucleic acid molecule of claim 24 or 25, further comprising: d. a fourth nucleic acid sequence encoding a protein of interest, said fourth nucleic acid sequence being operably linked to an inducible promoter comprising the specific polynucleotide binding site.

27. The nucleic acid molecule of any one of claims 24-26, wherein the transcription factor DNA binding domain is derived from a parent protein selected from the group consisting of: ZFHD1, Cas9, Casl2, and TAL.

28. The nucleic acid molecule of any one of claims 24-27, wherein the transcription factor activation domain is derived from a parent protein, wherein said parent protein is p65.

29. A nucleic acid molecule comprising: a. a first nucleic acid sequence encoding a transcription factor able to bind to a specific polynucleotide binding site and activate transcription; and b. a second nucleic acid sequence encoding a drug responsive domain (DRD); wherein the transcription factor is operably linked to the DRD.

30. The nucleic acid molecule of claim 29, further comprising: ¢. a third nucleic acid sequence encoding a protein of interest, said third nucleic acid sequence being operably linked to an inducible promoter comprising the specific polynucleotide binding site.

31. The nucleic acid molecule of any one of claims 24-30, wherein the DRD is derived from a parent protein selected from the group comprising: human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDES.

32. The nucleic acid molecule of any one of claims 24-31, wherein the DRD is stabilized in the presence of a ligand selected from the group comprising: Acetazolamide (ACZ), Methotrexate (MTX), and Trimethoprim (TMP).

33. The nucleic acid molecule of any one of claims 26-28 or 30-32, wherein the protein of interest is a wild-type protein. 34, The nucleic acid molecule of any one of claims 26-28 or 30-32, wherein the protein of interest is a therapeutic protein.

35. The nucleic acid molecule of claim 34, wherein the protein of interest is selected from the group consisting of a cytokine, an antibody, a coagulation factor, an enzyme, a gene editing protein, a T cell receptor (TCR) and a chimeric antigen receptor (CAR).

36. The nucleic acid molecule of any one of claims 26-28 or 30-32, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

37. The nucleic acid molecule of claim 26-28 or 30-32, wherein the protein of interest is a secreted protein.

38. A vector comprising the nucleic acid molecule according to any of claims 24-37.

39. The vector according to claim 38, wherein the vector is a plasmid or a viral vector. 40, The vector according to claim 39, wherein the viral vector is derived from an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes virus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus.

41. The vector according to claim 39, wherein the viral vector is selected from the group consisting of a lentivirus vector, a gamma retrovirus vector, adeno-associated virus (AAV) vector, adenovirus vector, and a herpes virus vector.

42. A first polynucleotide and second polynucleotide, the first polynucleotide comprising: a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD); wherein at least one of the transcription factor activation domain, the transcription factor DNA binding domain, or the combination of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; and a second polynucleotide comprising: a fourth nucleic acid sequence that encodes a protein of interest, the fourth nucleic acid sequence being operably linked to an inducible promoter comprising the specific polynucleotide binding site; wherein the transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor that is able to activate transcription upon binding to the specific polynucleotide binding site, and wherein the first polynucleotide and the second polynucleotide are each carried in a single vector, or the first polynucleotide and the second polynucleotide are carried in separate vectors.

43. A first polynucleotide and second polynucleotide, the first polynucleotide comprising: a first nucleic acid sequence that encodes a transcription factor and a second nucleic acid sequence that encodes a drug responsive domain (DRD), wherein the transcription factor is operably linked to the DRD and wherein the transcription factor is able to activate transcription upon binding to a specific polynucleotide binding site; and a second polynucleotide comprising: a third nucleic acid sequence that encodes a protein of interest, the third nucleic acid sequence being operably linked to an inducible promoter comprising the specific polynucleotide binding site; wherein the first polynucleotide and the second polynucleotide are each carried in a single vector, or the first polynucleotide and the second polynucleotide are carried in separate vectors.

44. The first polynucleotide and second polynucleotide of claim 42 or 43, wherein the DRD is derived from a parent protein selected from the group comprising: human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDES.

45. The first polynucleotide and second polynucleotide of any one of claims 42-44, wherein the DROD is stabilized in the presence of a ligand selected from the group comprising: Acetazolamide (ACZ), Methotrexate (MTX), and Trimethoprim (TMP).

46. The first polynucleotide and second polynucleotide of any one of claims 42-45, wherein the protein of interest is a wild-type protein.

47. The first polynucleotide and second polynucleotide of any one of claims 42-45, wherein the protein of interest is a therapeutic protein.

48. The first polynucleotide and second polynucleotide of claim 47, wherein the protein of interest is selected from the group consisting of a cytokine, an antibody, a coagulation factor, an enzyme, a gene editing protein, a T cell receptor (TCR) and a chimeric antigen receptor (CAR).

49. The first polynucleotide and second polynucleotide of any one of claims 42-45, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

50. The first polynucleotide and second polynucleotide of any one of claims 42-45, wherein the protein of interest is a secreted protein.

51. A method of producing a modified cell, said method comprising introducing into a cell a nucleic acid molecule comprising: a. a first nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and b. a second nucleic acid sequence that encodes a drug responsive domain (DRD).

52. The method of claim 51, wherein the nucleic acid molecule further comprises a third nucleic acid sequence that encodes a transcription factor activation domain.

53. The method of claim 52, wherein either (i) the transcription factor DNA binding domain is operably linked to the DRD; (ii) the transcription factor activation domain is operably linked to the DRD,; or (iii) the combination of the transcription factor DNA binding domain and the transcription factor activation domain is operably linked to the DRD.

54. The method according to claim 53, further comprising introducing into the cell: a fourth nucleic acid sequence encoding a protein of interest, said fourth nucleic acid sequence being operably linked to an inducible promoter comprising the specific polynucleotide binding site.

55. The method according to claim54, wherein the protein of interest is a heterologous protein.

56. The method according to claim 54 or 55, wherein the fourth nucleic acid sequence is on the same nucleic acid molecule as the first, second and third nucleic acid sequences.

57. The method according to claim 54 or 55, wherein the fourth nucleic acid sequence is on a different nucleic acid molecule than the first, second and third nucleic acid sequences.

58. The method according to any one of claims 54-57, wherein the protein of interest is selected from the group consisting of a cytokine, an antibody, a coagulation factor, an enzyme, a gene editing protein, a T cell receptor (TCR) and a chimeric antigen receptor (CAR).

59. The method according to any one of claims 54-57, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

60. The method according to any one of claims 54-57, wherein the protein of interest is a secreted protein.

61. The method according to any one of claims 51-60, wherein the nucleic acid molecule is introduced into the cell by a plasmid or a viral vector.

62. The method according to claim 61, wherein the viral vector is derived from an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes virus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus.

63. The method according to claim 61, wherein the viral vector is selected from the group consisting of a lentivirus vector, a gamma retrovirus vector, adeno-associated virus (AAV) vector, adenovirus vector, and a herpes virus vector.

64. The method according to any one of claims 51-60, wherein the nucleic acid molecule is introduced into the cell by a non-viral delivery method.

65. The method of any one of claims 51-64, wherein the cell is a T cell, a natural killer cell (NK cell), or a tumor infiltrating lymphocyte (TIL).

66. The method of any one of claims 51-64, wherein the cell is a stem cell, a liver cell, a blood cell, a pancreatic cell, a neuronal cell, an ocular cell, a muscle cell, or a bone cell.

67. A method for treating or preventing a disease in a subject in need thereof, the method comprising: a. providing a population of cells; b. introducing at least one nucleic acid molecule into at least one cell in the population of cells, wherein the at least one nucleic acid molecule comprises:

1. a first polynucleotide comprising a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; and ii. a second polynucleotide that comprises a fourth nucleic acid sequence that encodes a protein of interest that prevents or treats the disease, or a symptom thereof, said fourth nucleic acid sequence being operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; 2. delivering the cell into the subject; and d. administering a ligand to the subject that stabilizes the DRD sufficiently to enable expression of the 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 expression of the protein of interest in the cell; wherein expression of the protein of interest is regulated by the presence of ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

68. A method for introducing a modified cell into a subject in need of disease treatment or prevention, the method comprising: a. providing a population of cells; b. introducing at least one nucleic acid molecule into at least one cell in the population of cells, wherein the at least one nucleic acid molecule comprises:

1. a first polynucleotide comprising a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain is operably linked to the DRD; and ii. a second polynucleotide that comprises a fourth nucleic acid sequence that encodes a protein of interest that treats the disease, said fourth nucleic acid sequence being operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; and 8: delivering the cell into the subject.

69. A method for introducing a modified cell into a subject in need of disease treatment or prevention, the method comprising: a. providing a population of cells; b. introducing at least one nucleic acid molecule of any one of claims 24-37 or the first polynucleotide and second polynucleotide of any one of claims 42-50 into at least one cell in the population of cells; and 2. delivering the cell into the subject.

70. A method for genetically modifying one or more cells in a subject in need of disease treatment or prevention, the method comprising: a. introducing at least one nucleic acid molecule into at least one cell of the subject, wherein the at least one nucleic acid molecule comprises:

1. a first polynucleotide comprising a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain, upon expression in the cell, is operably linked to the DRD; and ii. a second polynucleotide that comprises a fourth nucleic acid sequence that encodes a protein of interest that treats the disease, said fourth nucleic acid sequence being operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site.

71. A method for genetically modifying one or more cells in a subject in need of disease treatment or prevention, the method comprising: a. introducing at least one nucleic acid molecule into at least one cell of the subject, wherein the at least one nucleic acid molecule comprises:

1. a first polynucleotide comprising a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain, upon expression in the cell, is operably linked to the DRD; and ii. a second polynucleotide that comprises a fourth nucleic acid sequence that encodes a protein of interest that treats the disease, said fourth nucleic acid sequence being operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; and b. administering a ligand to the subject that stabilizes the DRD sufficiently to enable 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 expression of the protein of interest in the cell; wherein expression of the protein of interest is regulated by the presence of ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest. 72 A method for treating a disease in a subject in need thereof, the method comprising: a. providing a population of cells; 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 population of cells, wherein:

1. the first nucleic acid molecule comprises a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain, upon expression in the cell, is operably linked to the DRD; and ii. the second nucleic acid molecule comprises a fourth nucleic acid sequence that encodes a protein of interest that treats the disease, said fourth nucleic acid sequence being operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; e delivering the cell into the subject; and d. administering a ligand to the subject that stabilizes the DRD sufficiently to enable 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 expression of the protein of interest in the cell; wherein expression of the protein of interest is regulated by the presence of ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

73. A method for treating a disease in a subject in need thereof, the method comprising: a. providing a population of cells; 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 population of cells, wherein:

1. the first nucleic acid molecule comprises a first nucleic acid sequence that encodes a transcription factor activation domain; a second nucleic acid sequence that encodes a transcription factor DNA binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence that encodes a drug responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA binding domain, upon expression in the cell, is operably linked to the DRD; and ii. the second nucleic acid molecule comprises a fourth nucleic acid sequence that encodes a protein of interest that treats the disease, said fourth nucleic acid sequence being operably linked to an exogenous inducible promoter comprising the specific polynucleotide binding site; and 8. delivering the cell into the subject.

74. The method according to any one of claims 67-73, wherein the nucleic acid molecule is introduced into the cell by a plasmid or a viral vector.

75. The method according to claim 74, wherein the viral vector is derived from an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes virus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus.

76. The method according to claim 74, wherein the viral vector is selected from the group consisting of a lentivirus vector, a gamma retrovirus vector, adeno-associated virus (AAV) vector, adenovirus vector, and a herpes virus vector.

77. The method according to any one of claims 67-73, wherein the nucleic acid molecule is introduced into the cell by a non-viral delivery method.

78. A system for the tunable expression of a protein of interest in a cell, the system comprising: a. a first polynucleotide encoding a transcription factor linked to a drug response domain (DRD), the transcription factor selectively transcribes a polynucleotide sequence encoding the protein of interest; b. a second polynucleotide comprising an exogenous transcription factor binding site positioned upstream from and adjacent to a nucleic acid sequence encoding the protein of interest; c. introducing the first polynucleotide and the second polynucleotide to the cell under conditions to stably integrate the first polynucleotide and the second polynucleotide into the genome of the cell; d. tuning the expression of the transcription factor by adding a ligand which stabilizes the DRD; wherein the transcription factor specifically binds to a transcription factor binding site positioned upstream from and adjacent to the polynucleotide sequence which encodes the protein of interest, and wherein the expression of the protein of interest is regulated by the quantity of transcription factor present in the cell.

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