Compositions and methods for regulatable modulation of transcription

Abstract

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

Description

[0001] Cross-reference of related applications

[0002] This application claims 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 above applications are incorporated herein by reference.

[0003] Reference for sequence lists

[0004] This application contains a sequence list, which is submitted electronically in ASCII format and is hereby incorporated in its entirety by reference. The ASCII copy was created on January 8, 2021, named 268052_483267_SL.txt, and is 241,815 bytes in size. Technical Field

[0005] This disclosure relates to systems, compositions, and methods for regulating protein expression driven by regulated transcriptional activity. This disclosure provides modular transcription factor systems, polynucleotides, peptides, vectors, cells, compositions, and methods for regulating transcription and regulated protein expression driven by regulated transcriptional activity. Background Technology

[0006] Gene and cell therapies are revolutionizing medicine and offering new hope for treating previously intractable diseases. However, most current technologies do not allow for the tuning of the timing or level of target protein induction. This makes it difficult or impossible to deploy many potential gene and cell therapy applications safely and effectively.

[0007] Inadequate control of exogenous and / or endogenous genes is a key issue in many gene and cell therapy settings. This lack of regulatory control also makes it difficult to safely express proteins with narrow or uncertain therapeutic windows, or proteins requiring more tuned or transient expression.

[0008] One approach to regulating protein expression or function is the use of drug-responsive domains (DRDs). A drug-responsive domain is a small protein domain that can be attached to a target protein of interest. Without a DRD-binding ligand, the attached DRD destabilizes the protein of interest, leading to its rapid degradation by the ubiquitin-proteasome system in the cell. However, when a specific small-molecule DRD-binding ligand binds to the DRD, the attached protein of interest becomes stable and performs its function.

[0009] DRD technology forms the basis for a new class of cell and gene therapies, providing tunable and temporally controlled gene expression and function, thereby expanding the range of protein therapeutics that can be safely and effectively incorporated into cell and gene therapy modalities. However, in fusion proteins produced by current DRD technology, the protein of interest binds to the DRD, which may not be suitable for certain indications. Therefore, there remains a need to develop cell and gene therapies capable of expressing naturally occurring proteins of interest in a regulated manner. Summary of the Invention

[0010] This invention provides modified cells, nucleic acid molecules, vectors, and cell and gene therapies that can modulate the timing or level of natural therapeutic proteins by administering oral small molecule drugs.

[0011] Furthermore, this disclosure provides compositions, systems, and methods for the tunable regulation of transcription. The compositions relate to transcription factor systems and reagents for inducing transcriptional activity of polynucleotides encoding proteins of interest. The compositions provided in this disclosure include nucleic acid molecules, peptides, and cells associated with transcription factor systems. Methods associated with the transcription factor systems provided in this disclosure include methods for generating modified cells and methods for treating or preventing diseases.

[0012] This document provides a transcription factor system. The transcription factor system disclosed herein is a combination of one or more polynucleotides comprising: (1) one or more nucleic acid sequences encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; (2) a nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor or a portion thereof is operatively linked to the DRD; and (3) a nucleic acid sequence encoding a payload and operatively linked to an inducible promoter containing the specific polynucleotide binding site.

[0013] This disclosure provides modified cells associated with transcription factor systems.

[0014] In some aspects, this disclosure provides modified cells capable of regulating the expression or transcription of a payload. The modified cells comprise: a first polynucleotide containing a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD). At least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of both is operatively linked to the DRD. The transcription factor activation domain interacts with the transcription factor DNA-binding domain to form a transcription factor that, upon binding to the specific polynucleotide binding site, is capable of activating transcription of a fourth nucleic acid sequence encoding a protein of interest and operatively linked to the specific polynucleotide binding site, an exogenous inducible promoter containing the specific polynucleotide binding site, or both. In some embodiments, the protein of interest is a heterologous protein. In some embodiments, the fourth nucleic acid sequence is located on the first polynucleotide. In some implementations, the modified cells also contain a second polynucleotide that contains a fourth nucleic acid sequence.

[0015] In some aspects, this disclosure provides a modified cell comprising a polynucleotide containing a first nucleic acid sequence encoding a drug-responsive domain (DRD) and a second nucleic acid sequence encoding a transcription factor. The transcription factor is operatively linked to the DRD and is capable of binding to a specific polynucleotide binding site and activating transcription of a third nucleic acid sequence encoding a protein of interest, the third nucleic acid sequence being operatively linked to the specific polynucleotide binding site, an exogenous inducible promoter containing the specific polynucleotide binding site, or both. In some embodiments, the protein of interest is a heterologous protein. In some embodiments, the third nucleic acid sequence is located on the polynucleotide containing the first and second nucleic acid sequences. In some embodiments, the modified cell further comprises a second polynucleotide containing the third nucleic acid sequence.

[0016] In another aspect, this disclosure provides a modified cell comprising (a) a first polynucleotide comprising: a first nucleic acid sequence encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; and a second nucleic acid sequence encoding a drug-responsive domain (DRD); ​​wherein the transcription factor or a portion thereof is operatively linked to the DRD; and (b) a second polynucleotide comprising a third nucleic acid sequence encoding a protein of interest, the third nucleic acid sequence being operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site.

[0017] In another aspect, this disclosure provides a modified cell comprising (a) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription of a second nucleic acid sequence encoding a protein of interest; wherein the second nucleic acid sequence is operatively 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 operatively linked to the DRD.

[0018] In another aspect, this disclosure provides a modified cell comprising (a) a first polynucleotide comprising: a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD); ​​wherein at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD; and (b) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site; wherein the transcription factor activation domain interacts with the transcription factor DNA-binding domain to form a transcription factor that, upon binding to the specific polynucleotide binding site, is capable of activating transcription.

[0019] In another aspect, this 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, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site located on an exogenous inducible promoter; and (c) a third polynucleotide comprising a nucleic acid sequence encoding a drug-responsive domain (DRD); ​​wherein at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD. In one aspect, the transcription factor activation domain interacts with the transcription factor DNA-binding domain to form a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription of a nucleic acid sequence encoding a protein of interest, said nucleic acid sequence being operatively linked to an exogenous inducible promoter.

[0020] In various embodiments, one or more of the transcription factor DNA-binding domain, transcription factor activation domain, and DRD are derived from a parental protein. In some embodiments, the transcription factor DNA-binding domain is derived from a parental protein selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL. In some embodiments, the transcription factor activation domain is derived from a parental protein, wherein the parental protein is p65. In some embodiments, the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2), human DHFR, E. coli DHFR (ecDHFR), human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5.

[0021] In some embodiments, the DRD is stable in the presence of a ligand selected from the group consisting of acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). In some embodiments, the DRD reacts with or interacts with a ligand selected from the group consisting of acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP).

[0022] In some implementations, the protein of interest is a wild-type protein.

[0023] In some implementations, the protein of interest is a therapeutic protein.

[0024] In some implementations, the protein of interest is selected from the group consisting of: cytokines, antibodies or their antigen-binding fragments, coagulation factors, enzymes, gene-edited proteins, T-cell receptors (TCRs), and chimeric antigen receptors (CARs).

[0025] In some implementations, the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

[0026] In some implementations, the protein of interest is a secretory protein.

[0027] In some implementations, the cells are T cells, natural killer cells (NK cells), or tumor-infiltrating lymphocytes (TILs).

[0028] In some implementations, the cells are stem cells, liver cells, blood cells, pancreatic cells, neuronal cells, eye cells, muscle cells, or bone cells.

[0029] This disclosure also provides nucleic acid molecules related to the transcription factor system.

[0030] In one aspect, this disclosure provides a nucleic acid molecule comprising (a) a first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and (b) a second nucleic acid sequence encoding a drug-responsive domain (DRD). In some embodiments, the nucleic acid molecule further comprises (c) a third nucleic acid sequence encoding a transcription factor activation domain; wherein (i) the transcription factor DNA-binding domain is operatively linked to the DRD; (ii) the transcription factor activation domain is operatively linked to the DRD; or (iii) a combination of the transcription factor DNA-binding domain and the transcription factor activation domain is operatively linked to the DRD. In some embodiments, the transcription factor DNA-binding domain is derived from a parental protein selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL. In some embodiments, the transcription factor activation domain is derived from a parental protein, wherein the parental protein is p65.

[0031] In one aspect, this disclosure provides a nucleic acid molecule comprising (a) a first nucleic acid sequence encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; and (b) a second nucleic acid sequence encoding a drug-responsive domain (DRD); ​​wherein the transcription factor is operatively linked to the DRD. In some embodiments, the nucleic acid molecule further comprises (c) a third nucleic acid sequence encoding a protein of interest, the third nucleic acid sequence being operatively linked to the specific polynucleotide binding site, an exogenous inducible promoter comprising the specific polynucleotide binding site, or both.

[0032] In some implementations, the specific polynucleotide binding site is located on an exogenous inducible promoter.

[0033] In some implementations, the DRD is derived from a parental protein selected from the group consisting of: human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5.

[0034] In some embodiments, the DRD is stable in the presence of a ligand selected from the group consisting of acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). In some embodiments, the DRD reacts with or interacts with a ligand selected from the group consisting of acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP).

[0035] In some implementations, the protein of interest is a wild-type protein.

[0036] In some implementations, the protein of interest is a therapeutic protein.

[0037] In some implementations, the protein of interest is selected from the group consisting of: cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T-cell receptors (TCRs), and chimeric antigen receptors (CARs).

[0038] In some implementations, the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

[0039] In some implementations, the protein of interest is a secretory protein.

[0040] This document also provides vectors containing the nucleic acid molecules described herein. The vectors provided in this disclosure include plasmids or viral vectors. In some aspects, the viral vectors are derived from adenoviruses, adeno-associated viruses (AAVs), alphaviruses, flaviviruses, herpesviruses, measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease virus (NDV), poxviruses, and picornaviruses. In some aspects, the viral vectors are selected from the group consisting of lentiviral vectors, gamma retroviral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

[0041] This disclosure also provides a first polynucleotide and a second polynucleotide, the first polynucleotide and the second polynucleotide comprising nucleic acid sequences encoding one or more components of a transcription factor system.

[0042] In one aspect, this disclosure provides a first polynucleotide and a second polynucleotide, the first polynucleotide comprising: a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD); ​​wherein at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD; and the second polynucleotide comprises: a fourth nucleic acid sequence encoding a protein of interest, the fourth nucleic acid sequence being operatively linked to an inducible promoter containing the specific polynucleotide binding site; wherein the transcription factor activation domain interacts with the transcription factor DNA-binding domain to form a transcription factor that, upon binding to the specific polynucleotide binding site, is capable of activating transcription, 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.

[0043] In one aspect, this disclosure provides a first polynucleotide and a second polynucleotide, the first polynucleotide comprising: a first nucleic acid sequence encoding a transcription factor and a second nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is operatively linked to the DRD, and wherein the transcription factor is capable of activating transcription upon binding to a specific polynucleotide binding site; and the second polynucleotide comprising: a third nucleic acid sequence encoding a protein of interest, the third nucleic acid sequence being operatively linked to an inducible promoter containing a 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.

[0044] In some embodiments, the DRD is derived from a parental protein selected from the group consisting of: human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. In some embodiments, the DRD is stable in the presence of a ligand selected from the group consisting of: acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP).

[0045] 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: cytokines, antibodies, coagulation factors, enzymes, gene-editing proteins, T-cell receptors (TCRs), and chimeric antigen receptors (CARs). 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.

[0046] This disclosure also provides methods related to transcription factor systems.

[0047] In one aspect, this disclosure provides a method for generating modified cells, the method comprising introducing a nucleic acid molecule into the cell, the nucleic acid molecule comprising: (a) a first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and (b) a second nucleic acid sequence encoding a drug-responsive domain (DRD). In one embodiment, the nucleic acid molecule further comprises a third nucleic acid sequence encoding a transcription factor activation domain. In some embodiments, (i) the transcription factor DNA-binding domain is operatively linked to the DRD; (ii) the transcription factor activation domain is operatively linked to the DRD; or (iii) a combination of the transcription factor DNA-binding domain and the transcription factor activation domain is operatively linked to the DRD.

[0048] In some embodiments, the method further includes introducing into the cell a fourth nucleic acid sequence encoding a protein of interest, the fourth nucleic acid sequence being operatively linked to an inducible promoter containing a specific polynucleotide binding site. In some embodiments, the protein of interest is a heterologous protein. In one embodiment, the fourth nucleic acid sequence is 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.

[0049] In some implementations, the protein of interest is selected from the group consisting of: cytokines, antibodies or their antigen-binding fragments, coagulation factors, enzymes, gene-edited proteins, T-cell receptors (TCRs), and chimeric antigen receptors (CARs).

[0050] In some implementations, the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

[0051] In some implementations, the protein of interest is a secretory protein.

[0052] In some implementations, nucleic acid molecules are introduced into cells via plasmids or viral vectors. In one implementation, the viral vector is derived from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus. In one implementation, the viral vector is selected from the group consisting of lentiviral vectors, gamma retroviral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

[0053] In some implementations, nucleic acid molecules are introduced into cells via non-viral delivery methods.

[0054] In some implementations, the cells are T cells, natural killer cells (NK cells), or tumor-infiltrating lymphocytes (TILs). In some implementations, the cells are stem cells, hepatocytes, blood cells, pancreatic cells, neurons, eye cells, muscle cells, or bone cells.

[0055] This disclosure also provides methods related to the treatment or prevention of diseases.

[0056] In one aspect, this disclosure provides a method for treating or preventing a disease in a subject in need, the method comprising: (a) providing a cell population; (b) introducing at least one nucleic acid molecule into at least one cell in the cell population, wherein the at least one nucleic acid molecule comprises: (i) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD; and (ii) a second polynucleotide, the second... The polynucleotide contains a fourth nucleic acid sequence encoding a protein of interest that prevents or treats a disease or its symptoms, said fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing a specific polynucleotide binding site; (c) the cell is delivered to a subject; and (d) a ligand is administered to the subject that makes the DRD sufficiently stable to express at least one of a transcription factor activation domain and a transcription factor DNA binding domain sufficient to form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the protein of interest in the cell; wherein the expression of the protein of interest is regulated by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0057] In one aspect, this disclosure provides a method for introducing modified cells into a subject requiring disease treatment or prevention, the method comprising: (a) providing a cell population; (b) introducing at least one nucleic acid molecule into at least one cell in the cell population, wherein the at least one nucleic acid molecule comprises: (i) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain binding to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD; and (ii) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site; and (c) delivering the cell into a subject.

[0058] In one aspect, this disclosure provides a method for introducing modified cells into a subject in need of disease treatment or prevention, the method comprising: (a) providing a cell population; (b) introducing at least one nucleic acid molecule or a first polynucleotide and a second polynucleotide from any of the foregoing aspects into at least one cell in the cell population; and delivering the cell into the subject.

[0059] In one embodiment, this disclosure provides a method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, the method comprising: (a) introducing at least one nucleic acid molecule into at least one cell of the subject, wherein the at least one nucleic acid molecule comprises: (i) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain binding to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD; and (ii) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site.

[0060] In one aspect, this disclosure provides a method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, the method comprising: (a) introducing at least one nucleic acid molecule into at least one cell of the subject, wherein the at least one nucleic acid molecule comprises: (i) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD upon expression in the cell. D; and (ii) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing a specific polynucleotide binding site; and (b) administration of a ligand to the subject that stabilizes the DRD sufficiently to express at least one of a transcription factor activation domain and a transcription factor DNA binding domain 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 the expression of the protein of interest is regulated by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0061] In one aspect, this disclosure provides a method for treating a disease in a subject in need, the method comprising: (a) providing a cell population; (b) introducing at least one first nucleic acid molecule and at least one second nucleic acid molecule into at least one cell in the cell population, wherein: (i) the first nucleic acid molecule comprises a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD after expression in the cell; and (ii) The second nucleic acid molecule contains a fourth nucleic acid sequence encoding a protein of interest that treats a disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing a specific polynucleotide binding site; (c) the cell is delivered to a subject; and (d) a ligand is administered to the subject that makes the DRD sufficiently stable to express an amount sufficient to form a transcription factor activation domain and a transcription factor DNA binding domain that bind to the specific polynucleotide binding site and enables the expression of the protein of interest in the cell; wherein the expression of the protein of interest is regulated by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0062] In one aspect, this disclosure provides a method for treating a disease in a subject in need, the method comprising: (a) providing a cell population; (b) introducing at least one first nucleic acid molecule and at least one second nucleic acid molecule into at least one cell in the cell population, wherein: (i) the first nucleic acid molecule comprises a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein at least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD after expression in the cell; and (ii) the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein of interest that prevents and / or treats the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing a specific polynucleotide binding site; and (c) delivering the cell to a subject.

[0063] In one related embodiment, this disclosure provides a method for preventing and / or treating a disease in a subject in need. The method includes: (a) providing a cell population; and (b) introducing at least one first nucleic acid molecule and at least one second nucleic acid molecule into at least one cell in the cell population. In this example of the method, the first nucleic acid molecule comprises a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD). At least one of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD after expression in the cell; and the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein of interest that prevents and / or treats the disease in the subject in need. The fourth nucleic acid sequence is operatively linked to an exogenous inducible promoter containing a specific polynucleotide binding site. The method further includes the steps of: (c) delivering cells to a subject; and (d) administering a ligand to the subject that makes the DRD sufficiently stable to express an amount sufficient to form a transcription factor activation domain and a transcription factor DNA-binding domain, which binds to a specific polynucleotide binding site and enables the expression of the protein of interest in the cells. In this example of the method, the expression of the protein of interest is regulated by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0064] In relevant embodiments, the treatment and prevention methods of this disclosure can be implemented by introducing a single vector into a cell, wherein the vector carries a first nucleic acid molecule and a second nucleic acid molecule, wherein: (i) the first nucleic acid molecule comprises a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor activation domain and / or the transcription factor DNA-binding domain are operatively linked to the DRD after expression in the cell; and the second nucleic acid molecule comprises a fourth nucleic acid sequence encoding a protein of interest that treats or prevents the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing a specific polynucleotide binding site.

[0065] In some alternative embodiments, the treatment and prevention methods of this disclosure can be implemented by introducing a first vector and a second vector into cells, wherein the first vector comprises: a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor activation domain and / or the transcription factor DNA-binding domain are operatively linked to the DRD after expression in cells; and the second vector comprises a fourth nucleic acid sequence encoding a protein of interest that prevents and / or treats the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing a specific polynucleotide binding site.

[0066] In some implementations, nucleic acid molecules are introduced into cells via plasmids or viral vectors. In some embodiments, the viral vectors are derived from adenoviruses, adeno-associated virus (AAV), alphaviruses, flaviviruses, herpesviruses, measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease virus (NDV), poxviruses, and picornaviruses. In other embodiments, the viral vectors are selected from the group consisting of lentiviral vectors, gamma retroviral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

[0067] In some implementations, nucleic acid molecules are introduced into cells via non-viral delivery methods.

[0068] This disclosure also provides a system for the regulated expression of a protein of interest in cells, the system comprising: (a) a first polynucleotide encoding a transcription factor linked to a drug response domain (DRD), the transcription factor selectively transcribing a polynucleotide sequence encoding the protein of interest; (b) a second polynucleotide containing an exogenous transcription factor binding site located upstream of and adjacent to the nucleic acid sequence encoding the protein of interest; (c) introducing the first and second polynucleotides into the cell under conditions that stably integrate the first and second polynucleotides into the cellular genome; and (d) regulating the expression of the transcription factor by adding a ligand that stabilizes the DRD; wherein the transcription factor specifically binds to the transcription factor binding site located upstream of and adjacent to the polynucleotide sequence encoding the protein of interest, and wherein the expression of the protein of interest is regulated by the amount of the transcription factor present in the cell.

[0069] This disclosure also provides pharmaceutical compositions comprising the compositions described herein and pharmaceutically acceptable excipients. Attached Figure Description

[0070] Figure 1A-Figure 1B A schematic diagram of the transcription factor system design scheme is shown. Figure 1A This diagram shows a transcription factor construct, referred to as the "DRD-TF construct," which contains nucleic acid sequences encoding a transcription factor DNA-binding domain, a transcription factor activation domain, and a drug-responsive domain (DRD). Figure 1B A schematic diagram of a payload construct is shown, which contains an inducible promoter that includes a binding site for a transcription factor DNA-binding domain.

[0071] Figures 2A-2B The ligand-dependent activity of a transcription factor system containing transcription factors with different DRDs was demonstrated. Figure 2A The images show Western blots of lysate 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 operatively linked to a DRD derived from a CA2, ecDHFR, ER, or hDHFR parental protein. Detailed information on treatment conditions for each construct and ligand is provided in Tables 4 and 6. The upper plot of the Western blots shows banding of endogenous p65, transcription factors and DRD peptides encoded by each DRD-TF construct, and transcription factor peptides encoded by the constitutive construct ZFHD-055. Figure 2B Showing Figure 2A The quantification of protein blots was normalized with the constitutive condition set to 1.0.

[0072] Figures 3A-3E The ligand-dependent activity of a transcription factor system containing transcription factors regulated by ecDHFR DRD was demonstrated. Figure 3A A schematic diagram showing the transcription factor construct ZFHD-005. Figure 3B A schematic diagram showing the payload construct ZFHD-007. Figure 3C A schematic diagram showing the constitutive transcription factor construct ZFHD-004. Figure 3D This image shows a Western blot of the lysate from U2OS cells stably integrated with the specified construct and treated with 10 μM TMP or 0.1% DMSO. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 44.3 kDa represents the transcription factor and DRD peptide encoded by the transcription factor construct ZFHD-005. The band appearing at approximately 26.5 kDa represents the transcription factor peptide encoded by the construct ZFHD-004. Figure 3EThe median fluorescence intensity (MFI) of GFP in U2OS cells treated with 10 μM TMP or 0.1% DMSO is shown for stable integration of the specified construct as assessed by flow cytometry.

[0073] Figures 4A-4C The dose-response relationship of ligands in transcription factor systems containing transcription factors regulated by ecDHFR DRD is shown. Figure 4A This image shows a Western blot of the lysate from U2OS cells stably integrated with constructs ZFHD-005 and ZFHD-007 and treated with DMSO or a specified concentration of TMP. Lanes labeled "U2OS" represent untransduced U2OS cells treated with TMP. Bands appearing at approximately 60 kDa represent endogenous p65. Bands appearing at approximately 44.3 kDa represent transcription factors and DRD peptides encoded by the transcription factor construct ZFHD-005. Figure 4B show Figure 4A The quantification of the "ZFHD-005 peptide" band was specified in the Western blot. Fluorescence was normalized for endogenous p65. Figure 4C The median fluorescence intensity (MFI) of GFP in U2OS cells stably integrated with constructs ZFHD-005 and ZFHD-007 and treated with a specified concentration of TMP is shown by flow cytometry. Figure 4C The highest concentration of TMP used was 33 μM. The data shown are from three replicate experiments. Error bars represent standard deviations.

[0074] Figures 5A-5B This demonstrates the ligand-dependent activity of a transcription factor system containing ecDHFR DRD-regulated transcription factors in T cells. Figure 5A This image shows a Western blot of the lysate from untransduced T cells or T cells transduced with the virus (OTLV-ZFHD-005 or OTLV-ZFHD-007) and treated with TMP or DMSO. Bands appearing at approximately 60 kDa represent endogenous p65. Bands appearing at approximately 44.3 kDa (indicated by arrows) represent transcription factors and DRD peptides encoded by the transcription factor construct ZFHD-005. Figure 5B The data displayed are the median fluorescence intensity (MFI) of GFP in untransduced T cells or T cells transduced with a specified construct and treated with TMP or DMSO, as assessed by flow cytometry. Data shown are from three replicates. Error bars represent the standard deviation from the mean.

[0075] Figures 6A-6D The ligand-dependent activity of a transcription factor system containing CA2 DRD-regulated transcription factors was demonstrated in ARPE-19 cells. Figure 6AA schematic diagram of the transcription factor construct ZFHD-019 is shown. Figure 6B Western blots show protein lysates from untransduced ARPE-19 cells or stable integrated constructs ZFHD-019 and ZFHD-007, treated with 10 μM ACZ or 1% DMSO. Bands appearing at approximately 60 kDa represent endogenous p65. Bands appearing at approximately 55.8 kDa represent transcription factors and DRD peptides encoded by the transcription factor construct ZFHD-019. Figure 6C show Figure 6B The quantification of the "ZFHD-019 peptide" band was specified in the Western blot. Fluorescence was normalized for endogenous p65. Figure 6D The figure shows the mean fluorescence intensity (MFI) of GFP in untransduced ARPE-19 cells or ARPE-19 cells stably integrated with the specified construct and untreated or treated with 10 μM ACZ or 1% DMSO, as assessed by flow cytometry. The data shown are from three replicates. Error bars represent the standard deviation from the mean. The untransduced ARPE-19 cells and ARPE-19 cells stably integrated with construct ZFHD-007 shown in the figure were treated with DMSO.

[0076] Figures 7A-7B The dose-response relationship of ligands in transcription factor systems involving CA2 DRD regulation is shown. Figure 7A This image shows a Western blot of the lysate from ARPE-19 cells stably integrated with constructs ZFHD-007 and ZFHD-019 and treated with a specified concentration of ACZ. The band appearing at approximately 60 kDa represents endogenous p65. The band appearing at approximately 55.8 kDa represents the transcription factor and DRD polypeptide encoded by the transcription factor construct ZFHD-019. Figure 7B show Figure 7A The quantification of the "ZFHD-019 peptide" band was specified in the Western blot. Fluorescence was normalized for the endogenous P65 band.

[0077] Figure 8 This figure shows the dose-response relationship of ligands in a transcription factor system containing CA2 DRD-regulated transcription factors. The plot displays the median fluorescence intensity (MFI) of GFP in U2OS cells treated with specified concentrations of ACZ, using stable integrative constructs ZFHD-007 and ZFHD-019 as assessed by flow cytometry. Data shown are from two replicates. Error bars represent standard deviations.

[0078] Figures 9A-9C The ligand-dependent activity of a transcription factor system containing CA2 DRD-regulated transcription factors was demonstrated in Jurkat cells. Figure 9A A schematic diagram showing the transcription factor construct ZFHD-048. Figure 9B A schematic diagram of the payload construct ZFHD-022 is shown. Figure 9C The median fluorescence intensity (MFI) of GFP in Jurkat cells treated with DMSO (0.1%) or ACZ (10 μM final concentration) using stable integrated constructs ZFHD-048 and ZFHD-022, as assessed by flow cytometry, is shown. The data presented are for cells that are positive for the transduction marker.

[0079] Figures 10A-10F The ligand-dependent activity of a single-vector transcription factor system containing transcription factors regulated by ecDHFR DRD was demonstrated. Figure 10A A schematic diagram of the ZFHD-012 construct is shown. Figure 10B A schematic diagram of the ZFHD-018 construct is shown. Figure 10C and Figure 10D Western blots of lysates from U2OS cells transduced with lentivirus and treated with 10 μM TMP or 0.1% DMSO are shown, the lentivirus being prepared from a specified construct. The band appearing at approximately 44.3 kDa represents the transcription factor and DRD polypeptide encoded by the specified construct. In the single-vector construct, 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, resulting in a band of approximately 44.3 kDa representing the transcription factor and DRD polypeptide. Figure 10E and Figure 10F The median fluorescence intensity (MFI) of GFP in U2OS cells transduced with lentivirus and treated with 10 μM TMP or 0.1% DMSO is shown by flow cytometry. The lentivirus was prepared from a specified construct.

[0080] Figures 11A-11B The ligand-dependent activity of a single-vector transcription factor system containing a transcription factor regulated by CA2 DRD was demonstrated. Figure 11A A schematic diagram of a single-carrier system is shown, depicted as construct ZFHD-036. Figure 11B The figure shows the median fluorescence intensity (MFI) of GFP in Jurkat cells transduced with lentivirus and treated with 10 μM ACZ or 0.1% DMSO, as evaluated by flow cytometry. The lentivirus was prepared from the specified construct. ZFHD-036.1 and ZFHD-036.2 in the figure represent two cell lines, each transduced with lentivirus prepared from construct ZFHD-036.

[0081] Figures 12A-12B The ligand-dependent activity of a transcription factor system containing transcription factor construct variants was demonstrated. Figure 12A A schematic diagram of transcription factor construct variants is shown.

[0082] Figure 12B The median fluorescence intensity (MFI) of GFP in U2OS cells with stable integration of the specified construct, as assessed by flow cytometry, and treated with 0.1% DMSO or 10 μM TMP is shown.

[0083] Figure 13 The ligand reaction time progression analysis of transcription factor systems containing transcription factor construct variants is shown. The figure displays the median fluorescence intensity (MFI) of GFP in U2OS cells that stably integrated the specified construct as assessed by flow cytometry and were treated with 0.1% DMSO or 10 μM TMP for specified time periods.

[0084] Figures 14A-14D The ligand-dependent activity of transcription factor systems containing payload construct variants was demonstrated. Figure 14A A schematic diagram of the payload construct ZFHD-007 is shown. Figure 14B A schematic diagram of the payload construct ZFHD-017 is shown. Figures 14C-14D The median fluorescence intensity (MFI) of GFP in U2OS cells with stable integration of the specified construct, as assessed by flow cytometry, and treated with 0.1% DMSO or 10 μM TMP is shown.

[0085] Figure 15 The figure illustrates the ligand-dependent activity of a transcription factor system containing a payload construct encoding a secreted IL12 payload. The figure shows the concentration of secreted IL12 in the supernatant collected from U2OS cells stably integrated with the specified construct and treated with 0.1% DMSO or 10 μM TMP.

[0086] Figures 16A-16D This study demonstrates the ligand-dependent regulatory role of different transcription factors that can be operatively linked to the DRD derived from the parental CA2 protein. Figure 16A Western blots of lysate from untransfected (“mock”) HEK293T cells and HEK293T cells transfected with the following constructs are shown. (1) cjun-001 (“001”), (2) cjun-002 (“002”), or (3) cjun-003 (“003”). Cell populations transfected with each construct after treatment with DMSO or ACZ are shown (indicated by “+” symbols). The labeled band “c-Jun-001 and -002 peptides” identifies the CA2-linker-C-jun peptide encoded by the cjun-001 and cjun-002 constructs. The labeled band “c-Jun-003 peptide” identifies the c-Jun peptide encoded by the cjun-003 construct. Figure 16B Showing Figure 16A Quantification of protein blots. Figure 16C Western blots of lysate from untransfected (“mock”) HEK293T cells and HEK293T cells transfected with the following constructs are shown. (1) FOXP3-013 (“013”), (2) FOXP3-014 (“014”), or (3) FOXP3-015 (“015”). Cell populations transfected with each construct after treatment with DMSO or ACZ are shown (indicated by “+” symbols). The labeled band “FOXP3-013 and -014 polypeptides” identifies the CA2-FOXP3 polypeptide encoded by the FOXP3-013 and FOXP3-014 constructs. The labeled band “FOXP3-015 polypeptide” identifies the FOXP3 polypeptide encoded by the FOXP3-015 construct. Figure 16D Showing Figure 16C Quantification of protein blots.

[0087] Figures 17A-17B This study demonstrated the ligand-dependent regulatory role of the c-Jun transcription factor construct stably integrated into Jurkat cells. Figure 17A Western blots of lysate from untransduced (“mock”) Jurkat cells and Jurkat cells transduced with lentiviruses prepared from constructs cjun-001 (“001”) and cjun-002 (“002”) are shown. Cell lines transfected with each construct after treatment with DMSO or ACZ are shown (indicated by “+” symbols). Bands of c-Jun peptide and phosphorylated c-Jun peptide are shown. Figure 17B Showing Figure 17A Quantification of proteins in Western blots.

[0088] Figure 18 The nucleotide sequence of the pELDS-puro transfer vector is shown (SEQ ID NO: 68).

[0089] Figure 19 The nucleotide sequence of the pELNS-puro transfer vector is shown (SEQ ID NO: 69). Detailed Implementation

[0090] Transcription factor system

[0091] According to this disclosure, a transcription factor system is a combination of one or more polynucleotides comprising (1) one or more nucleic acid sequences encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; (2) a nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is operatively linked to the DRD; and (3) a nucleic acid sequence encoding a payload operatively linked to an inducible promoter containing the specific polynucleotide binding site.

[0092] In some implementations, a combination of one or more polynucleotides of a transcription factor system can be used to modify cells, such as immune cells for treating diseases, and to generate a system that can regulate the expression of a protein of interest by modulating the presence of a transcription factor that acts on a polynucleotide encoding a payload or a protein of interest.

[0093] In some implementations, the combination of one or more polynucleotides in the transcription factor system includes a polynucleotide containing a first nucleic acid sequence encoding a transcription factor and a second nucleic acid sequence encoding a DRD.

[0094] This disclosure also provides a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises: a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD). In this example, at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD illustrated herein. The second polynucleotide comprises a fourth nucleic acid sequence encoding a protein of interest, which is operatively linked to an inducible promoter containing a specific polynucleotide binding site. In this example, the transcription factor activation domain and the transcription factor DNA-binding domain interact to form a transcription factor that, upon binding to a specific polynucleotide binding site, is capable of activating transcription, and the first and second polynucleotides are each carried in a single vector, or the first and second polynucleotides are carried in separate vectors.

[0095] In one related example, this disclosure provides compositions and nucleic acids operable for regulating transcription. For example, this disclosure provides a first polynucleotide and a second polynucleotide of a tunable transcription factor system. The first polynucleotide comprises a first nucleic acid sequence encoding a transcription factor and a second nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is operablely linked to the DRD, and wherein the transcription factor is capable of activating transcription upon binding to a specific polynucleotide binding site. The second polynucleotide comprises a third nucleic acid sequence encoding a protein of interest, the third nucleic acid sequence being operablely linked to an inducible promoter containing a specific polynucleotide binding site; thereby enabling the first and second polynucleotides to be carried in separate vectors, each in a single vector.

[0096] In some embodiments, a combination of one or more polynucleotides of a transcription factor system comprises: a first nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; a second nucleic acid sequence encoding a transcription factor activation domain; and a third nucleic acid sequence encoding a DRD. In some aspects, the combination of one or more polynucleotides of a transcription factor system comprises a polynucleotide containing a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence. In some aspects, the combination of one or more polynucleotides of a transcription factor system comprises a polynucleotide containing 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 containing a first nucleic acid sequence; a second polynucleotide containing a second nucleic acid sequence; and a third polynucleotide containing a third nucleic acid sequence. On one hand, the transcription factor DNA-binding domain is operatively linked to a DRD. On another aspect, the transcription factor activation domain is operatively linked to a DRD. On yet another aspect, both the transcription factor DNA-binding domain and the transcription factor activation domain are operatively linked to a DRD. In some aspects, the transcription factor DNA-binding domain and the transcription factor activation domain are expressed as a transcription factor fusion protein.

[0097] According to this disclosure, the transcription factor system encodes transcription factors capable of driving payload expression. In some embodiments, the transcription factor is encoded by a first nucleic acid sequence encoding a transcription factor activation domain and a second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site. The transcription factor activation domain and the transcription factor DNA-binding domain interact to form a transcription factor, which, upon binding to the specific polynucleotide binding site, activates the transcription of the nucleic acid sequence encoding the payload.

[0098] In some embodiments, the specific polynucleotide binding site comprises at least one nucleic acid site having a specific sequence recognized and bound by a transcription factor DNA-binding domain. In some embodiments, the specific polynucleotide binding site comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten nucleic acid sites recognized by the DNA-binding domain of this disclosure. In some embodiments, the specific polynucleotide binding site comprises eight nucleic acid sites recognized by the DNA-binding domain. In some embodiments, the specific polynucleotide binding site comprises two or more tandem nucleic acid sites, each having a specific sequence recognized and bound by a transcription factor DNA-binding domain. In some aspects, the tandem nucleic acid sites comprise the same nucleic acid sequence. In some embodiments, the specific polynucleotide binding site comprises a tandem repeat nucleic acid site recognized by the DNA-binding domain of this disclosure.

[0099] As described herein, a transcription factor or a portion thereof is operatively linked to a DRD in the transcription factor system of this disclosure. The presence, absence, or amount of a ligand that binds to or interacts with the DRD can modulate the stability of the transcription factor and thus its function following such binding or interaction. Thus, the transcription factor system can exhibit ligand-dependent activity.

[0100] In some embodiments, the transcription factor system is present in the cell or cell population. In some embodiments, one or more polynucleotides of the transcription factor system are introduced into the cell or cell population.

[0101] Transcription factor system construct

[0102] A combination of one or more polynucleotides of a transcription factor system may also be referred to herein as a combination of one or more nucleic acid constructs. A polynucleotide or nucleic acid construct may comprise nucleic acid sequences arranged in different ways, and / or may be uniquely combined as part of a transcription factor system, provided that the resulting combination of polynucleotides or nucleic acid constructs comprises (1) one or more nucleic acid sequences encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; (2) a nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is operatively linked to the DRD; and (3) a nucleic acid sequence encoding a payload operatively linked to an inducible promoter containing a specific polynucleotide binding site.

[0103] In some embodiments, the transcription factor system comprises multiple constructs. In some embodiments, the transcription factor system comprises a transcription factor construct and a payload construct. On one hand, the transcription factor construct contains a nucleic acid sequence encoding a transcription factor. On the other hand, the transcription factor construct contains a nucleic acid sequence encoding a transcription factor activation domain and a nucleic acid sequence encoding a transcription factor DNA-binding domain.

[0104] In some embodiments, the transcription factor system comprises a single construct. This single construct contains a transcription factor encoding the transcription factor system, a DRD, and a payload nucleic acid sequence. In some embodiments, such a single-construct transcription factor system can be introduced into cells as 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.

[0105] In addition to the nucleic acid sequences for transcription factor systems described herein, the nucleic acid constructs disclosed herein may also contain additional nucleic acid sequences. These additional nucleic acid sequences include, but are not limited to, regulatory elements, polyadenylation sequences, adapters, and cleavage sites.

[0106] In some embodiments, the transcription factor construct may include nucleic acid sequences encoding a promoter, a transcription factor DNA-binding domain, a transcription factor activation domain, and a transcription factor activation domain (DRD). In some embodiments, the nucleic acid sequence encoding the DRD is adjacent to a nucleic acid sequence encoding at least one transcription factor domain. In some embodiments, the nucleic acid sequence encoding the DRD is located between the nucleic acid sequences encoding the transcription factor DNA-binding domain and the transcription factor activation domain.

[0107] In some implementations, the transcription factor construct may contain nucleic acid sequences encoding the following: a promoter, a transcription factor DNA-binding domain, a transcription factor activation domain, a linker, and a DRD. In some aspects, the linker is located between the nucleic acid sequence encoding the transcription factor domain and the nucleic acid sequence encoding the DRD.

[0108] In some embodiments, the promoter in the transcription factor construct is EF1a. In some embodiments, the transcription factor DNA-binding domain encoded in the transcription factor construct is ZFHD1. In some embodiments, the transcription factor activation domain encoded in the transcription factor construct is p65.

[0109] In some implementations, the payload construct may include a nucleic acid sequence encoding: a specific polynucleotide binding site containing at least one nucleic acid site having a specific sequence that is recognized and bound by a transcription factor DNA-binding domain; a promoter; and a payload. An exemplary binding site contains eight (8) nucleic acid sites recognized by the ZFHD1 DNA-binding domain.

[0110] In some embodiments, the constructs of this disclosure, such as transcription factor constructs or payload constructs, are integrated into plasmids or viral vectors. In some embodiments, the plasmid or viral vector includes one or more regulatory elements that become operatively linked to one or more components of the construct integrated into the plasmid or viral vector. In some embodiments, the plasmid or viral vector includes regulatory elements well known in the art, including, for example, promoters, introns, spacers, filler sequences, etc. In some embodiments, the transcription factor construct is integrated into the plasmid or viral vector such that components of the transcription factor construct are operatively linked to regulatory elements of the plasmid or viral vector. In some embodiments, such a transcription factor construct includes 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 the expression of the transcription factor DNA-binding domain, the transcription factor activation domain, and the DRD. Such a promoter may be selected from constitutive promoters, tissue-specific promoters, cell-specific promoters, cell differentiation-specific promoters, and / or disease-specific promoters. Optionally, the promoter can be selected from EF1a, CMV, EFS, RSV, SFFV, PGK, CAG, and SV40.

[0111] Components of the transcription factor system

[0112] As described above, the polynucleotide or nucleic acid constructs of a transcription factor system may comprise nucleic acid sequences arranged in different ways, and / or may be uniquely combined as part of a transcription factor system, provided that the resulting combination of polynucleotide or nucleic acid constructs comprises (1) one or more nucleic acid sequences encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; (2) a nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor is operatively linked to the DRD; and (3) a nucleic acid sequence encoding a payload operatively linked to an inducible promoter containing a specific polynucleotide binding site. Thus, the transcription factor system is a modular system, and each component of the transcription factor system can be selected separately.

[0113] The nucleic acid sequence encoding the drug-reactive domain (DRD) may be selected from the DRD sequences described in more detail in the "Drug-reactive Domain (DRD)" section below.

[0114] The nucleic acid sequences encoding the one or more transcription factors may be selected from one or more sequences encoding existing transcription factors, engineered transcription factors derived from existing transcription factors, or engineered transcription factors containing a DNA-binding domain and an activation domain. As used herein, "engineered transcription factor derived from an existing transcription factor" means an engineered transcription factor that is at least partially derived from a parental (natural) transcription factor molecule or sequence and retains the ability to bind to a specific polynucleotide binding site and activate transcription. For example, an engineered transcription factor may be derived from a parental transcription factor containing one or more zinc finger domains capable of specifically contacting a DNA sequence. Engineered TAL effector transcription factors may be designed to include a TAL effector repeat region that recognizes a specific DNA binding site, a mammalian nuclear localization signal (NLS), and a synthetic transcription activation domain. If the transcription factor is an engineered transcription factor containing a DNA-binding domain and an activation domain, the DNA-binding domain and the activation domain may be selected separately and combined to form the complete transcription factor.

[0115] The transcription factor DNA-binding domain can be derived from existing nucleic acid binding proteins. For example, the DNA-binding sequence or domain of an existing DNA-binding protein can be used as the transcription factor DNA-binding domain of this disclosure or can be modified to produce the transcription factor DNA-binding domain of this disclosure.

[0116] In some respects, the DNA-binding domain of transcription factors is derived from parental proteins selected from the following group: ZFHD1, Cas9, Cas12, and TAL.

[0117] In some embodiments, the transcription factor DNA-binding domain is derived from the parent protein ZFHD1. ZFHD1 is a zinc finger homology domain fusion protein designed by Pomerantz, JL et al. (Pomerantz, JL et al., "Structure-Based Design of Transcription Factors." Science, Vol. 267, No. 5194, 1995). ZFHD1 contains zinc fingers 1 and 2 of Zif268, a gly-gly-arg-arg linker, and an OCT-1 homology domain. ZFHD1 can bind nucleic acid sequences containing the sequence TAATGATGGGCG (SEQ ID NO: 70). In some embodiments, the transcription factor DNA-binding domain consists of or contains the amino acid sequence of ZFHD1.

[0118] In some embodiments, this disclosure provides methods for regulating target genes and their corresponding functional proteins (e.g., payloads or proteins of interest) using a Cas / guide RNA system. It should be understood that those skilled in the art will be able to design suitable guide RNAs to form co-localization complexes with target nucleic acids, including target genes as described herein.

[0119] Various Cas proteins are known to those skilled in the art, including CasI (Cas3), CasIA (Cas8a), CasIB (Cas8b), CasIC (Cas8c), CasID (Cas10d), CasIE (Cse1), CasIF (Csy1), CasIU, CasII (Cas9), CasIIA (Csn2), CasIIB (Cas4), CasIIC, CasIII (Cas10), CasIIIA (Csm2), CasIIIB (Cmr5), CasIIIC, CasIIBD, CasIV (Csf1), CasIVA, CasIVB, CasV (Cpf1), C2c2, and C2c1, etc.

[0120] In some implementations, the transcription factor DNA-binding domain is derived from a Cas protein selected from the group consisting of: C2C1, C2C3, Cpf1 (also known as Cas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, C as10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

[0121] According to one aspect, the Cas9 protein includes the sequence shown for naturally occurring Cas9 from Staphylococcus aureus, Streptococcus thermophilus, Streptococcus pyogenes, or Neisseria meningitidis, and a protein sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology to that sequence and serving as a DNA-binding protein, such as an RNA-guided DNA-binding protein.

[0122] According to one aspect, the Cas12 protein includes the sequence shown for naturally occurring Cas12 from the new culprits Francisella novicida, Acidaminococcus, Lachnospiraceae, or Prevotella, and a protein sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology to that sequence and serving as a DNA-binding protein, such as an RNA-guided DNA-binding protein.

[0123] In some embodiments, the transcription factor DNA-binding domain is derived from the parental Cas protein, such as parental Cas9 or Cas12. In some embodiments, the transcription factor DNA-binding domain is or comprises Cas9 modified to lack nuclease activity. In some embodiments, the transcription factor DNA-binding domain is or comprises Cas12 modified to lack nuclease activity.

[0124] Naturally occurring Cas9 contains two nuclease domains: an HNH-like nuclease domain that cleaves the DNA strand complementary to the guide RNA sequence (the target strand); and a RuvC-like nuclease domain that cleaves the DNA strand opposite to the complementary strand (the non-target strand). By simultaneously mutating both the HNH and RuvC nuclease domains (producing so-called "inactive Cas9" or "dCas9"), the resulting dCas9 retains its RNA-guided DNA targeting ability but loses its endonuclease activity. In some embodiments, the transcription factor DNA-binding domain is a dCas9 containing mutated HNH and RuvC nuclease domains. In some embodiments, the transcription factor DNA-binding domain is a dCas9 containing mutated HNH and RuvC nuclease domains and is derived from parental Staphylococcus aureus, Streptococcus thermophilus, Streptococcus pyogenes, or Neisseria meningitidis Cas9.

[0125] Naturally occurring Cas12 (e.g., Cas12a and Cas12b) contains a RuvC-like domain that cleaves DNA. Catalytically inactive Cas12 (DNase-inactive, also referred to herein as "dCas12") can be derived from the parental Cas12 protein by mutating the RuvC nuclease domain. In some embodiments, the transcription factor DNA-binding domain is or contains catalytically inactive Cas12 (dCas12).

[0126] In some embodiments, the transcription factor DNA-binding domain is derived from a parental 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 lacking nuclease activity or modified to lack nuclease activity. In some embodiments, the transcription factor DNA-binding domain is or comprises a type II Cas homolog containing mutant HNH and RuvC nuclease domains.

[0127] According to one exemplary embodiment, Cas9 is altered or otherwise modified to inactivate nuclease activity. Such alteration or modification includes changing one or more amino acids to inactivate nuclease activity or nuclease domains. Such modifications include removing one or more polypeptide sequences exhibiting nuclease activity, i.e., nuclease domains, thereby eliminating one or more polypeptide sequences exhibiting nuclease activity, i.e., nuclease domains, from the Cas9 DNA-binding protein. Other modifications to inactivate nuclease activity will be apparent to those skilled in the art. Therefore, nuclease-free DNA-binding proteins include polypeptide sequences modified to inactivate nuclease activity or by removing one or more polypeptide sequences to inactivate nuclease activity. Even with inactivated nuclease activity, nuclease-free DNA-binding proteins retain the ability to bind to DNA. Therefore, DNA-binding proteins include one or more polypeptide sequences required for DNA binding but may lack one or more or all nuclease sequences exhibiting nuclease activity. See Jinek et al. (2012) Science 337, 816-821. Cas9 proteins lacking nuclease activity are referred to as nuclease-free Cas9 (“Cas9Nuc”, “inactivated Cas9”, or “dCas9”) and exhibit reduced or eliminated nuclease activity, or have no or substantially no nuclease activity at the detection level. Based on this, the nuclease activity of Cas9Nuc may be undetectable using known assays, i.e., its nuclease activity is below the detection level of known assays.

[0128] In some embodiments, the transcription factor DNA-binding domain is derived from the Cas9 parent protein. In some embodiments, the transcription factor DNA-binding domain comprises Cas9 with a mutated nuclease domain (referred to as "inactivated Cas9" or "dCas9"). The resulting dCas9 retains its RNA-guided DNA targeting capability but loses its endonuclease activity. In some embodiments, the transcription factor DNA-binding domain is dCas9.

[0129] This disclosure provides for the use of guide RNA to target a Cas protein, such as the nuclease-free Cas9 operatively linked to a DRD, to the polynucleotide binding sequence described herein. Such guide RNAs can be readily designed by those skilled in the art when a particular polynucleotide binding sequence is known. Guide RNAs may include one or more of a spacer sequence, a tracr pairing sequence, and a tracr sequence. The term spacer sequence is as understood by those skilled in the art and may include any polynucleotide that is sufficiently complementary to the polynucleotide binding sequence to hybridize with it and guide the CRISPR complex to sequence-specific binding to the polynucleotide binding sequence. Guide RNAs may be formed by covalently linking a spacer sequence with a tracr pairing sequence (which may be referred to as crRNA) and a separate tracr sequence, wherein the tracr pairing sequence hybridizes with a portion of the tracr sequence. According to some aspects, the tracr pairing sequence and the tracr sequence may be covalently linked or joined using an adapter sequence, and the construct may be referred to as a fusion of the tracr pairing sequence and the tracr sequence. The adapter sequence referred to herein is a nucleotide sequence linking the tracr pairing sequence and the tracr sequence, referred to herein as a nucleic acid sequence. Therefore, guide RNA can be a two-component species (i.e., independent crRNA and tracrRNA hybridized together) or a single-molecule species (i.e., crRNA-tracrRNA fusion, commonly referred to as sgRNA).

[0130] In some embodiments, the guide RNA can be delivered directly to cells as a natural species or as a species transcribed from its homologous DNA by methods known to those skilled in the art, including injection or lipid transfection, wherein the homologous DNA is introduced into the cells by electroporation, transient and stable transfection (including lipid transfection), and viral transduction.

[0131] In some embodiments, the transcription factor system comprises one or more polynucleotides encoding a transcription factor regulated by a DRD, wherein the transcription factor contains a DNA-binding domain, which is or contains a nuclease-free Cas9. When a DRD stabilizing ligand is added, the DRD and the transcription factor become stable, and the nuclease-free Cas9 is expressed and can be used to bind guide RNA. Upon binding to the guide RNA, the Cas9-gRNA system binds a polynucleotide binding sequence that is operatively linked to a protein of interest. When the Cas9-gRNA system binds the polynucleotide binding sequence, the gene of the protein of interest is transcribed due to the presence of the transcription factor activation domain. Therefore, when a regulated transcription factor expression construct comprises a Cas9-gRNA system, RNA-guided DNA regulation is achieved in cells, such as human cells, by tethering or linking a DRD to a nuclease-free Cas9 or a transcription factor activation domain. Therefore, aspects of this disclosure include methods and materials for localizing transcription regulatory domains to target loci by fusing, linking, or conjugating a DRD to a Cas9Nuc or a transcription factor activation domain, or both.

[0132] In some embodiments, the transcription factor DNA-binding domain is derived from the parental TAL protein. Transcription activator-like (TAL) effectors (also known as “TALEs”) are proteins secreted by Xanthomonas bacteria to regulate gene expression in host plants and aid bacterial infection. TAL effectors have a repeating region consisting of a tandem repeat sequence primarily having 33 or 34 amino acid residues. The repeat monomers differ primarily at amino acid positions 12 and 13, and there is a strong correlation between the unique amino acid pairs at positions 12 and 13 and the corresponding nucleotides at the TALE binding site. The transcription factor DNA-binding domain of this disclosure may contain all or part of the repeating region of a TAL effector capable of binding to a specific DNA binding site. In some embodiments, the DNA-binding domain contains a synthetic TAL effector capable of recognizing a desired nucleic acid sequence. Methods for assembling custom TAL effectors are readily available to those skilled in the art. “Engineering TAL effector” herein refers to a polypeptide derived from a parental TAL effector protein that contains the repeating region of a TAL effector and / or a synthetic TAL effector or a 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 be a C2H2 zinc finger protein. In some embodiments, the transcription factor DNA-binding domain may include one or more zinc finger domains that make sequence-specific contact with DNA. In some embodiments, the transcription factor DNA-binding domain may include at least two, at least three, at least four, or at least five zinc finger domains forming a zinc finger array capable of specifically recognizing DNA sites. In some embodiments, the transcription factor DNA-binding domain comprises a three-finger array. Engineered DNA-binding domains containing one or more zinc finger domains are referred to herein as "engineered zinc finger binding proteins".

[0133] In some implementations, the transcription factor DNA-binding domain may be selected from engineered zinc finger binding proteins, engineered TAL effectors, or other natural or engineered DNA-binding domains.

[0134] Zinc finger domains and TALE DNA-binding domains can be "engineered" to bind predetermined nucleotide sequences, for example, by engineering the recognition regions of naturally occurring zinc finger or TALE proteins (altering one or more amino acids). Therefore, engineered DNA-binding proteins (zinc fingers or TALEs) are non-naturally occurring proteins. A non-limiting example of a method for engineering DNA-binding proteins is design and selection. The designed DNA-binding protein is a protein that does not exist in nature, and its design / composition is primarily based on reasonable criteria. Reasonable design criteria include applying substitution rules and computerized algorithms to process information in a database storing existing ZFP and / or TALE design and binding data. See, for example, U.S. Patent Nos. 8,586,526, 6,140,081, 6,453,242, 6,534,261, and 8,586,526; see also WO 98 / 53058, WO98 / 53059, WO 98 / 53060, WO 02 / 016536, and WO 03 / 016496, which relate to the design and selection of DNA-binding proteins derived from existing ZFP and / or TALE proteins and the disclosure of their associated binding data, which are incorporated herein by reference in their entirety.

[0135] The activation domain of the engineered transcription factor according to this disclosure can be derived from regions or domains of existing transcription factors. In some embodiments, the activation domain is a transcriptionally activating region of an existing transcription factor. In some embodiments, the activation domain of the transcription factor can 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 C-terminal region of the human transcription factor NF-κβp65 protein (referred to herein as "p65"). In some embodiments, the activation domain comprises the C-terminal region of the human transcription factor NF-κβp65 protein.

[0136] One consideration in the transcription factor system design presented herein is that the encoded transcription factor is capable of binding to a specific polynucleotide binding site, and the nucleic acid sequence encoding the payload is operatively linked to an inducible promoter containing that specific polynucleotide binding site. In various embodiments, the inducible promoter is an exogenous inducible promoter. Pairings of transcription factors (including engineered transcription factors) with their corresponding polynucleotide binding sites are known in the art. DNA-binding domains of DNA-binding proteins with their corresponding polynucleotide binding sites, as well as methods for identifying novel DNA-binding domain sequences and corresponding polynucleotide binding sites that can be used to design synthetic transcription factors and corresponding synthetic promoters, are also known. For example, Khalil AS et al. provided zinc finger arrays that can be used as core building blocks for constructing synthetic transcription factors, and also provided corresponding nucleic acid binding sequences that can be inserted into synthetic promoters and recognized by the zinc finger array (Khalil A.S. et al., Cell 2012, 150, 647-658, incorporated herein by reference in their entirety). Khalil AS et al. also identified synthetic transcription factor-promoter pairs and provided design strategies to improve transcriptional output by altering promoters (e.g., polymerizing zinc finger binding sequences to produce promoters with repetitive operons) and by modifying synthetic transcription factors (e.g., by generating variants). Any transcription factor-promoter pairs or engineered zinc finger arrays and their corresponding nucleic acid binding sites disclosed by Khalil AS et al. can be used in the transcription factor systems disclosed herein. For example, Khalil AS et al.'s... Figure 3AA library of amino acid residues of the recognition helix of a zinc finger array and corresponding DNA-binding sequences is provided, which can be used to design the transcription factor DNA-binding domain and specific polynucleotide binding sites of the present disclosure. Those skilled in the art will be able to modify the sequences of these transcription factors or arrays by cloning the sequences of transcription factors or zinc finger arrays provided by Khalil, AS, et al. into the constructs of the transcription factor systems provided herein. As another example, Zhang, F, et al. describe methods for designing and fabricating engineered TAL effectors with corresponding nucleic acid binding sites. These can be used to prepare engineered transcription factors and their specific polynucleotide binding sites. Any of the TAL effectors provided by Zhang, F, et al. can be used to prepare the transcription factor DNA-binding domain in the transcription factor systems of the present disclosure. For example, Zhang, F, et al. disclosed the construction of 17 artificial TAL effectors targeting specific DNA binding sites and also provided the sequences of the repeat regions of the TAL effectors and the corresponding nucleic acid binding sequences in Figure 2a. The TAL effectors or their DNA-binding portions disclosed by Zhang, F, et al. can be used to construct the DNA-binding domain and corresponding nucleic acid binding sequences of the inducible promoters of the present disclosure. Those skilled in the art will recognize that there are several options for the selection and design of the DNA-binding domains of this disclosure. In addition to selecting well-known DNA-binding proteins and domains, the DNA-binding domains of this disclosure can also be designed based on the frameworks of existing DNA-binding proteins. For example, those skilled in the art can use a method based on the Cys2His2 zinc finger protein framework to select DNA-binding domains (Pabo, CO et al., Annu. Rev. Biochem. 2001. 70: 313-40).

[0137] In some implementations, an inducible promoter operatively linked to a nucleic acid sequence encoding a payload comprises a minimal promoter (also known as a “min promoter” or “core promoter”) and a specific polynucleotide binding site. In this case, both the minimal promoter and the specific polynucleotide binding site are operatively linked to the nucleic acid sequence encoding the payload. The term “minimum promoter” refers to the smallest structure capable of forming an initiation complex. A minimal promoter may contain an RNA polymerase binding site, a TATA box, and a transcription initiation site. A minimal promoter may be coupled to one or more reactive elements, such as enhancers or transcription factor binding sites, to produce an inducible promoter. Further details about minimal promoters and their coupling with reactive elements are provided by Ede et al. (Ede et al., ACS Synth Biol. 20 May 2016; 5(5):395-404). In some embodiments, the inducible promoter of the transcription factor system or its components disclosed herein comprises a minimum promoter selected from the following minimum promoters: minCMV, CMV53 (minCMV with an upstream GC box added), minSV40 (minimum simian virus 40 promoter), miniTK (the -33 to +32 region of the herpes simplex virus thymidine kinase promoter), MLP (the -38 to +6 region of the adenovirus major late promoter), pJB42CAT5 (the minimum promoter derived from the human junB gene), YB_TATA (a synthetic minimum promoter developed by Benenson and colleagues (Hansen, J. et al., Proc Natl Acad SciUSA. 2014; 111:15705-15710)) and a separate TATA box.

[0138] In some embodiments, the specific polynucleotide binding site may comprise at least one nucleic acid site having a specific sequence that is recognized and bound by a transcription factor DNA-binding domain. In some embodiments, the specific polynucleotide binding site comprises two or more nucleic acid sites, each having a specific sequence that is recognized and bound by a transcription factor DNA-binding domain. The pairing of DNA-binding domains with their corresponding polynucleotide binding sites has been discussed above.

[0139] You can choose the nucleic acid sequence that encodes the payload to encode any payload or protein of interest. Further details about the payload are provided in the “Payload” section below.

[0140] Exemplary nucleic acid constructs that can be used alone (as a single construct) or in combination as part of a transcription factor system are described in Table 1. An asterisk ("*") in Table 1 indicates the translation of a stop codon.

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] Table 2 provides other illustrative constructs containing structurally different transcription factor components. An asterisk ("*") in Table 2 indicates the translation of a stop codon. Corresponding control constructs that do not contain the regulated transcription factor, as well as individual construct components, are also provided. As described in the descriptions of constructs cjun-001 and cjun-002, the peptide linker is located between the CA2 and c-Jun components in each construct. Furthermore, all constructs contain the P2A peptide.

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] Characterization of ligand-dependent activity of transcription factor systems

[0183] The ligand-dependent activity of transcription factor systems can be characterized by a variety of methods.

[0184] In some embodiments, the ligand-dependent activity of the transcription factor system is characterized by the ligand-dependent regulation of transcription factor peptides (e.g., transcription factor DNA-binding domains, transcription factor activation domains, or both) encoded by the transcription factor system. In some embodiments, the ligand-dependent activity of the transcription factor system is characterized by the ligand dose-dependent regulation of transcription factor peptides encoded by the transcription factor system. On one hand, the transcription factor peptide is a peptide containing a transcription factor activation domain. On the other hand, the transcription factor peptide is a peptide containing a transcription factor DNA-binding domain. Furthermore, the transcription factor peptide is a peptide containing both a transcription factor activation domain and a transcription factor DNA-binding domain. The ligand-dependent regulation of transcription factor peptides can be characterized by various methods. In some aspects, the ligand-dependent regulation of transcription factor peptides can be assessed by measurement, such as measuring the level of the transcription factor peptide or its domains using an immunoassay.

[0185] In some implementations, the ligand-dependent activity of the transcription factor system is characterized by the ligand-dependent expression of the payload encoded by the transcription factor system. Payload expression can be assessed using various methods. In some aspects, payload expression is assessed by measuring payload mRNA levels. In other aspects, payload expression is assessed by measuring payload peptide levels.

[0186] In some embodiments, the transcription factor system can be compared with a control transcription factor system lacking a DRD. In some embodiments, the ligand-dependent activity of the transcription factor system can be analyzed or characterized relative to the activity of the transcription factor system containing a control transcription factor construct lacking a DRD. An example of a control transcription factor construct is the construct ZFHD-004 described in this disclosure (as shown in Table 1).

[0187] transcription factors

[0188] Transcription factors are proteins that bind to DNA, preferably to sequence-specific sites (transcription factor polynucleotide binding sites) on or near the promoter. These proteins facilitate the binding of the transcription mechanism to the promoter, thereby activating the transcription of the DNA sequence. Such entities are also called transcription regulatory proteins.

[0189] In various embodiments, the transcription factors used in the transcription factor systems, compositions, and methods described herein include a transcription factor DNA-binding domain and a transcription factor activation domain. In some embodiments, the combination of the transcription factor DNA-binding domain and the transcription factor activation domain produces a functional transcription factor. In various embodiments, the transcription factor DNA-binding domain and / or the transcription factor activation domain may interact with other transcriptional regulatory elements.

[0190] In some implementations, transcription factors are illustrated as proteins that recognize and bind to specific short DNA sequences, thereby influencing gene expression. The recognition of DNA sequences by transcription factors occurs through the chemical interaction between the amino acid side chains of the transcription factor protein and the base pairs of DNA residues that act as regulatory sequences. The transcription factor thus "reads" the genome sequence; this mechanism provides sequence recognition functionality, upon which information about regulatory interactions is used to control gene expression.

[0191] Transcription factors typically consist of DNA-binding domains and effector or activation domains, mediating interactions with other proteins required for transcription, including interactions with other transcription factors. Transcription factors perform numerous functions, including gene activation. They are transcribed in the nucleus, translated in the cytoplasm, and re-enter the nucleus via nuclear localization sites mediated by all transcription factor protein sequences, finding their target sites in genomic DNA. Transcription factors comprise basic domains that allow them to non-specifically concentrate near the DNA, thereby facilitating the diffusion-restricted discovery of their target sites.

[0192] In various embodiments of this disclosure, the transcription factor system utilizes a transcription factor comprising and / or containing a transcription factor DNA-binding domain and a transcription factor effector or activation domain or protein (which may be used interchangeably herein). A combination of a transcription factor activation domain, a transcription factor DNA-binding domain, and / or a transcription factor activation domain and a transcription factor DNA-binding domain is operatively linked to a DRD (any of which is a DRD-TF). After stabilizing the linked DRD by binding an exogenous stabilizing ligand, the stabilized DRD-TF is capable of transcribing the protein of interest.

[0193] The DNA sequence to which the transcription factor DNA-binding domain binds is called the transcription factor binding site or response element, or, as used interchangeably herein, a specific polynucleotide binding site; these binding sites are located in or near the promoter of the regulated DNA sequence. The promoter containing the specific polynucleotide binding site can be a foreign promoter. In some embodiments, the promoter can be a foreign 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 a foreign nucleic acid sequence.

[0194] In various embodiments of this disclosure, suitable transcription factors for use in synthesizing transcription factor systems may include any known transcription factor with a known transcription factor binding site. Examples of such transcription factors include, but are not limited to, the STAT family (STAT 1, 2, 3, 4, 5a, 5b, and 6), c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD, fos / jun, NFκB, HIV-TAT, the E2F family, the T-Box gene family, helical-loop-helical 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, homology domain + POU, Myb / SANT, THAP finger, CENPB, E2F, and BED. ZF, GATA, Rel, CxxC, IRF, SAND, SMAD, HSF, MBD, RFX, CUT+ homology domain, DM, STAT, ARID / BRIGHT, Grainyhead, MADS box, AP-2, CSD, and homology domain+PAX. Exemplary transcription factor DNA-binding domains may include one or more DNA-binding domains derived from parental proteins selected from the group consisting of ZFHD1, Cas9, Cas12, and TAL.

[0195] In each embodiment, the transcription factor system provides tunable transcription of the protein of interest or the payload (which may be used interchangeably herein). In each embodiment, the nucleic acid sequence encoding the protein of interest is operatively linked to an exogenous inducible promoter containing a specific polynucleotide binding site, i.e., a defined DNA polynucleotide sequence that specifically binds to a transcription factor DNA-binding domain. The combination of the transcription factor binding domain and the transcription factor DNA activation domain then regulates the transcription of the protein of interest.

[0196] When cells or organisms containing DRD-TF are exposed to an exogenous stable ligand, the DRD-TF becomes stable. The stable DRD-TF then binds to the specific polynucleotide binding site to which it binds, thereby regulating the transcription of the polynucleotide encoding the protein of interest. In some embodiments, binding to a stable DRD-TF activates the transcription of the polynucleotide encoding the protein of interest, thereby inducing protein expression in the cell or organism. In the absence of an exogenous stable ligand, the DRD-TF degrades and cannot activate transcription. Therefore, the amount and timing of protein expression can be controlled by administering an exogenous stable ligand to cells or organisms.

[0197] In various embodiments, the transcription factor DNA-binding domain and the transcription factor activation domain are typically operatively linked or may be separated by one or more insert sequences, such as adapters or cleavage sites. In various embodiments, the first polynucleotide may include a first nucleic acid sequence encoding the transcription factor DNA-binding domain; a second nucleic acid sequence encoding the transcription factor activation domain; and a third nucleic acid sequence encoding a drug-responsive domain (DRD). In such embodiments, the transcription factor activation domain and / or the transcription factor DNA-binding domain are operatively linked to the DRD after expression in the cell. Furthermore, the cell will also include a second polynucleotide containing a fourth nucleic acid sequence that can be specifically bound by the transcription factor DNA-binding domain and a fifth nucleic acid sequence encoding a protein of interest or payload as described herein.

[0198] The transcription factor DNA-binding domain, the transcription factor activation domain, and the protein of interest or payload can be provided on the same vector or on different vectors for use in the methods of this disclosure.

[0199] In some embodiments, the vector comprises the polynucleotides described herein. In some embodiments, the vector comprises at least a first nucleic acid sequence encoding at least one of a transcription factor DNA-binding domain and a transcription factor activation domain, and a second nucleic acid sequence encoding a drug-responsive domain (DRD); ​​wherein the transcription factor DNA-binding domain and / or the transcription factor activation domain are operatively linked to the DRD. Optionally, in some embodiments, the first vector comprises a transcription factor linked to the DRD, and the second vector comprises a protein of interest or payload operatively linked to a transcription factor polynucleotide binding site. In another embodiment, a single vector comprises: a first nucleic acid sequence encoding a transcription factor capable of binding to a specific polynucleotide binding site and activating transcription; a second nucleic acid sequence encoding a drug-responsive domain (DRD); ​​wherein the transcription factor is operatively linked to the DRD; and optionally, a third nucleic acid sequence encoding a protein of interest, the third nucleic acid sequence being operatively linked to an inducible promoter comprising a transcription factor polynucleotide binding site. In some embodiments, the first vector comprises at least a first nucleic acid sequence encoding at least one of a transcription factor DNA-binding domain and a transcription factor activation domain, and a second nucleic acid sequence encoding a drug-responsive domain (DRD); ​​wherein the transcription factor DNA-binding domain and / or the transcription factor activation domain are operatively linked to the DRD, and the second vector comprises a third nucleic acid sequence that can be specifically bound by the transcription factor DNA-binding domain and a fourth nucleic acid sequence encoding a protein of interest or payload as described herein.

[0200] In some implementations, the vector also has a replication origin (ori) that allows the vector to amplify, for example, in bacteria. Additionally or alternatively, the vector includes selective markers such as antibiotic resistance genes, colored marker genes, and suicide genes.

[0201] Drug-reactive domain (DRD)

[0202] Drug-reactive domains (DRDs) are a class of protein domains that are unstable and degrade in the absence of a ligand, but regain their stability by binding to a corresponding DRD-binding ligand. The term drug-reactive domain (DRD) is interchangeable with the term destabilizing domain (DD). DRDs can attach to peptides or proteins and destabilize the linked peptide or protein in the absence of a DRD-binding ligand. DRDs transfer their destabilizing properties to the linked peptide or protein through protein degradation. Without being bound by any theory, in the absence of a DRD-binding ligand, the attached peptide or protein is rapidly degraded by the cell's ubiquitin-proteasome system. Ligands that bind to or interact with a DRD can modulate the stability of the attached peptide or protein after such binding or interaction. When the ligand binds to its intended DRD, the instability is reversed, and the function of the attached peptide or protein can be restored. The conditional stability of DRDs allows for rapid and undisturbed switching from stable proteins to unstable substrates for degradation. Furthermore, the dependence of DRD on its ligand concentration provides further tunable control over the degradation rate.

[0203] In some embodiments, the DRD of this disclosure may be derived from a known polypeptide capable of post-translational regulation of a protein. In some embodiments, the DRD of this disclosure may be developed or derived from a known protein. Regions, portions, or domains of wild-type proteins may be used wholly or partially as DRDs. They may be combined or rearranged to generate new peptides, proteins, regions, or domains, any of which may serve as a starting point for the design of a DRD or other DRDs.

[0204] In some embodiments, the DRD may be derived from a parental protein or from a mutant protein having one, two, three, or more amino acid mutations compared to the parental protein. In some embodiments, the parental protein may be selected from, but is not limited to, FKBP; human protein FKBP; human DHFR (hDHFR); Escherichia coli DHFR (ecDHFR); PDE5 (phosphodiesterase 5); CA2 (carbonic anhydrase II); and ER (estrogen receptor). Examples of proteins that can be used to develop the DRD and its ligands are listed in Table 3.

[0205] Table 3: Proteins and their binding ligands

[0206]

[0207]

[0208]

[0209] In some embodiments, the sequence of the protein used to generate the DRD may comprise all, part, or a region of the protein sequences in Table 3. In some embodiments, the protein that can be used to generate the DRD includes isoforms of the proteins listed in Table 3.

[0210] hPDE5DRD

[0211] In some embodiments, the DRD of this disclosure is derived from hPDE5. In some embodiments, the DRD of this disclosure is derived from hPDE5 isoform 2. In some embodiments, the DRD of this disclosure is derived from hPDE5 isoform 3. In some embodiments, the DRD of this disclosure is derived from hPDE5 isoform X1.

[0212] In some embodiments, the DRD of this disclosure is derived from cGMP-specific 3',5'-cyclic phosphodiesterase (hPDE5), which contains the amino acid sequence of SEQ ID NO.71.

[0213] In some embodiments, the DRD of this disclosure may include the entire hPDE5 (SEQ ID NO. 71). In some embodiments, the DRD derived from hPDE5 may include the catalytic domain of hPDE5 (e.g., amino acids 535-860 of SEQ ID NO. 71). In some embodiments, the hPDE5 DRD of this disclosure may include a methionine at the N-terminus of the catalytic domain of hPDE5 (i.e., amino acids 535-860 of the wild-type (WT) hPDE5).

[0214] In some embodiments, the DRD of this disclosure comprises, in whole or in part, a cGMP-specific 3',5'-cyclic phosphodiesterase (hPDE5; SEQ ID NO. 71) and also includes a mutation at amino acid position 732 (R732) of SEQ ID NO. 71. In some embodiments, the mutation at amino acid position 732 (R732) is selected from the group consisting of: R732L, R732A, R732G, R732V, R732I, R732P, R732F, R732W, R732Y, R732H, R732S, R732T, R732D, R732E, R732Q, R732N, R732M, R732C, and R732K.

[0215] In some embodiments, the hPDE5DRD of this disclosure may further comprise one or more mutations independently selected from the group consisting of: H653A, F736A, D764A, D764N, Y612F, Y612W, Y612A, W853F, I821A, Y829A, F787A, D656L, Y728L, M625I, E535D, E536G, Q541R, K555R, F559L, F561L, F564L, F564S, K591E, N587S, K604E, K608E, N609H, K630R, K633E, N636 S, N661S, Y676D, Y676N, C677R, H678R, D687A, T712S, D724N, D724G, L738H, N742S, A762S, D764G, D764V, S766F, K795E, L797F, I799T, T8 02P, S815C, M816A, I824T, C839S, K852E, S560G, V585A, I599V, I648V, S663P, L675P, T711A, F744L, L746S, F755L, L804P, M816T and F840S.

[0216] In some embodiments, the DRD of this disclosure comprises, in whole or in part, a cGMP-specific 3',5'-cyclic phosphodiesterase (hPDE5; SEQ ID NO. 71) and also includes a mutation at amino acid position 732 (R732) of SEQ ID NO. 71. In some such embodiments, the DRD further comprises (i) a mutation at amino acid position 764 (D764) of SEQ ID NO. 71, wherein the mutation at D764 is selected from D764N and D764A; (ii) a mutation at amino acid 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) a mutation F736A at amino acid position 736 (F736) of SEQ ID NO. 71; or (iv) a mutation H653A at amino acid position 653 (H653) of SEQ ID NO. 71.

[0217] In some embodiments, the DRD of this disclosure comprises, in whole or in part, a cGMP-specific 3',5'-cyclic phosphodiesterase (hPDE5; SEQ ID NO. 71) and further comprises an amino acid mutation at a position relative to SEQ ID NO. 71, the mutation being selected from the group consisting of: W853F, I821A, Y829A, F787A, F736A, D656L, Y728L, M625I, and H653A.

[0218] In some embodiments, the hPDE5DRD of this disclosure may contain one or more mutations independently selected from the group consisting of: T537A, E539G, V548E, D558G, F559S, E565G, C574N, R577Q, R577W, N583S, Q586R, Q589L, K591R, K591R, L595P, C596R, W615R, F619S, Q623R, K6 33I, Q635R, N636S, T639S, D640N, E642G, I643T, L646S, A649V, A650T, S652G, H653A, D654G, V660 A. V660A, L672P, A673T, C677Y, M681T, E682G, H685R, F686S, Q688R, M691T, S695G, G697D, S702I, I 706T, E707K, Y709H, Y709C, I715V, I720V, A722V, D724G, Y728C, K730E, R732L, L738I, I739M, K74 1N, K741R, F744L, D748N, K752E, K752E, K752E, E753K, L756V, M758T, M760T, A762V, C763R, D764N, D764N, I774V, L781F, L781P, E785K, R794G, M805T, R807G, K812R, I813T, I813T, M816R, Q817R, V818A, F820S, I821V, C825R, Y829C, E830K, L832P, S836L, C846Y, C846S, L856P, L856P, A857T, or E858G.

[0219] In some implementations, the hPDE5DRD of this disclosure may contain two independent mutations selected from the following: E536K, I739W; H678F, S702F; E669G, I700T; G632S, I648T; T639S, M816R; Q586R, D724G; E539G, L738I; L672P, S836L; M691T, D764N; I720V, F820S; E682G, D748N; S652G, Q688R; Y728C, Q817R; H653, R732L; L595P, K741R; R732D, F736S; R732E, F736D; R732V, F736G; 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.

[0220] In some implementations, the hPDE5DRD of this disclosure may contain two independent mutations selected from Q623R, D654G, K741N; A673T, L756V, C846Y; E642G, G697D, I813T; C677Y, H685R, A722V; Q635R, E753K, I813T; Y709H, K812R, L832P; N583S, K752E, C846S; K591R, I643T, L856P; F619S, V818A, Y829C; and F559S, Y709C, M760T. In some implementations, the hPDE5DRD of this disclosure may contain two independent mutations selected from the following: S695G, E707K, I739M, C763R; A649V, A650T, K730E, E830K; and R577W, W615R, M805T, I821V.

[0221] In some implementations, the hPDE5DRD of this disclosure may contain a plurality of mutations independently selected from the following: V660A, L781F, R794G, C825R, E858G; T537A, D558G, I706T, F744L, D764N; R577Q, C596R, V660A, I715V, E785K, L856P; and V548E, Q589L, K633I, M681T, S702I, K752E, L781P, A857T.

[0222] hDHFR DRD

[0223] In some embodiments, the DRD of this disclosure is derived from human dihydrofolate reductase (hDHFR) protein, such as, but not limited to, human dihydrofolate reductase 1 (hDHFR1), human dihydrofolate reductase 2 (hDHFR2), or fragments or variants thereof.

[0224] In some embodiments, the DRD may be derived from the hDHFR protein and include at least one mutation. In some embodiments, the DRD may be derived from the hDHFR protein and include more than one mutation. In some embodiments, the DRD may be derived from the hDHFR protein and include two, three, four, or five mutations.

[0225] In some embodiments, the DRD of this disclosure may include the entire hDHFR (SEQ ID NO.2). In some embodiments, the DRD derived from hDHFR may contain amino acids 2-187 of the parental hDHFR sequence (e.g., amino acids 2-187 of SEQ ID NO.2). This is referred to herein as the hDHFR M1del mutation.

[0226] In some embodiments, the DRD of this disclosure comprises one or all of the region of hDHFR (SEQ ID NO.2), and also comprises, relative to SEQ ID NO.2, a mutation selected from the following: I17V, F59S, N65D, K81R, Y122I, N127Y, M140I, K185E, N186D, and M140I.

[0227] In some embodiments, the DRD of this disclosure contains one or all of the hDHFR (SEQ ID NO.2) region and also contains two or more mutations relative to SEQ ID NO.2.

[0228] In some embodiments, the hDHFR DRD of this disclosure contains two or more mutations selected from the following: (A10V, H88Y); (C7R / Y163C); (I17V, Y122I); (Q36H, Y122I); (Q36K, Y122I); (Q36R, Y122I); (Q36S, Y122I); (Q36T, Y122I); (N65H, Y122I); (N65L, Y122I); (N65R, Y122I); (N65W, Y122I); (Q103E, Y122I); (Q103S, Y122I); (Q103S, Y122I); (Q103E ... 122I); (N108D; Y122I); (V121A, Y122I); (Y122I, K174N); (Y122I, E162G); (A125F, Y122I); (N127Y, Y122I); (H131R / E144G); (E162G / I176F); (K55R, N65K, Y122I); (Q36E, Q103H, Y122I); (Q36F, N65F, Y122I); and (V110A / V136M / K177R).

[0229] In some embodiments, the hDHFR DRD of this disclosure contains two or more mutations selected from the following: (I17V, Y122I); (G21T, Y122N); (Q36H, Y122I); (Q36K, Y122I); (Q36R, Y122I); (Q36S, Y122I); (Q36T, Y122I); (N65H, Y122I); (N65L, Y122I); (N65R, Y122I); (N65W, Y122I); (L74N, Y122I); (G21T, Y122I); (G36H, Y122I); (G36K, Y122I); (G36R, Y122I); (G36S, Y122I); (G36 ...K, Y122I); (G36K, Y122I); (G36K, Y122I); (G36K, Y122I); (G36K, Y122I); (G36K, Y122I); (G36K, Y122I); (G36K, Y 2I); (Q103E, Y122I); (Q103S, Y122I); (N108D; Y122I); (V121A, Y122I); (Y122I, K174N); (Y122I, E162G); (A125F, Y122I); (N127Y, Y122I); (K55R, N65K, Y122I); (Q36E, Q103H, Y122I); and (Q36F, N65F, Y122I).

[0230] In some embodiments, the DRD of this disclosure comprises, in whole or in part, human dihydrofolate reductase (hDHFR; SEQ ID NO. 2), and further comprises the Y122I mutation at amino acid position 122 (Y122) of SEQ ID NO. 2. In some such embodiments, the DRD further comprises: (i) the Q36K mutation at amino acid position 36 (Q36) of SEQ ID NO. 2; (ii) the A125F mutation at amino acid position 125 (A125) of SEQ ID NO. 2; (iii) the N65F mutation at amino acid position 65 (N65) of SEQ ID NO. 2; and an F or K substitution at amino acid position 36 (Q36) of SEQ ID NO. 2.

[0231] In some embodiments, the hDHFR DRD of this disclosure may contain one or more mutations independently selected from the group consisting of: M1del, V2A, C7R, I8V, V9A, A10T, A10V, Q13R, N14S, G16S, I17N, I17V, K19E, N20D, G21T, G21E, D22S, L23S, P24S, L28P, N30D, N30H, N30S, E31G, E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E , K56R, T57A, F59S, I61T, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N73G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F 89L, R92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, N108D, K109E, K109R, V110A, D111N, M1 12T, M112V, V113A, W114R, I115V, I115L, V116I, G117D, V121A, Y122C, Y12 2D, Y122I, K123R, K123E, A125F, M126I, N127R, N127S, N127Y, H128R, H128 Y, H131R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, T137R , R138G, R138I, I139T, I139V, M140I, M140V, Q141R, D142G, F143S, F143L, E 144G, D146G, T147A, F148S, F148L, F149L, P150L, ​​E151G, I152V, D153A, D1 53G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y16 3C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, I176 A. I176F, I176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C,Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.

[0232] In some embodiments, the DRD of this disclosure includes hDHFR(C7R, Y163C); hDHFR(E162G, I176F); hDHFR(G21T, Y122I); hDHFR(H131R, E144G); hDHFR(I17V, Y122I); hDHFR(L74N, Y122I; hDHFR(L94A, T147A); hDHFR(M53T, R138I); hDHFR(N127Y, Y122I); hDHFR(Q36K, Y122I); hDHFR(T137R, F143L); hDHFR(T57A, I72A); hDHFR(V121A). hDHFR(Y122I); hDHFR(V75F, Y122I); hDHFR(Y122I, A125F); hDHFR(Y122I, M140I); hDHFR(Y178H, E181G); hDHFR(Y183H, K185E); hDHFR(WT amino acid 2-187)(G21T, Y122I); hDHFR(WT amino acid 2-187)(I17V, Y122I); hDHFR(WT amino acid 2-187)(L74N, Y122I); hDHFR(WT amino acid 2-187)(L94A, T147A); hDHFR(WT amino acid 2-187)( M53T, R138I); hDHFR (amino acids 2-187 of WT) (N127Y, Y122I); hDHFR (amino acids 2-187 of WT) (Q36K, Y122I); hDHFR (amino acids 2-187 of WT) (V121A, Y122I); hDHFR (amino acids 2-187 of WT) (V75F, Y122I); hDHFR (amino acids 2-187 of WT) (Y122I, A125F); hDHFR (amino acids 2-187 of WT) (Y122I, M140I); hDHFR (E31D, F32M, V116I); hDHFR (G21E, I72V, I176) T); hDHFR (I8V, K133E, Y163C); hDHFR (K19E, F89L, E181G); hDHFR (L23S, V121A, Y157C); hDHFR (N49D, F59S, D153G); hDHFR (Q36F, N65F, Y122I); hDHFR FR (Q36F, Y122I, A125F); hDHFR (V110A, V136M, K177R); hDHFR (V9A, S93R, P150L); hDHFR (Y122I, H131R, E144G); hDHFR (G54R, I115L, M140V, S168C);hDHFR (amino acids 2-187 of WT) (E31D, F32M, V116I); hDHFR (amino acids 2-187 of WT) (Q36F, N65F, Y122I); hDHFR (amino acids 2-187 of WT) (Q36F, Y122I, A125F); hDHFR (amino acids 2-187 of WT) (Y122I, H131R, E144G); hDHFR (V2A, R33G, Q36R, L100P, K185R); hDHFR (D22S, F32M, R33S, Q36S, N65S); hDHFR (amino acids 2-187 of WT) (D2 2S, F32M, R33S, Q36S, N65S); hDHFR (I17N, L98S, K99R, M112T, E151G, E162G, E172G); hDHFR (G16S, I17V, F89L, D96G, K123E, M140V, D146G, K15 6R); hDHFR (K81R, K99R, L100P, E102G, N108D, K123R, H128R, D142G, F180L, K185E); hDHFR (R138G, D142G, F143S, K156R, K158E, E162G, V166A, hD HFR (F35L, R37G, N65A, L68S, K69E, R71G, L80P, K99G, G117D, L132P, I139V, M140I, D142G, D146G, E173G, D187G); hDHFR (L28P, N30H, M38V, V44 A, L68S, N73G, R78G, A97T, K99R, A107T, K109R, D111N, L134P, F135V, T147A, I152V, K158R, E172G, V182A, E184R); hDHFR (V2A, I17V, N30D, E31 G, Q36R, F59S, K69E, I72T, H88Y, F89L, N108D, K109E, V110A, I115V, Y1 22D, L132P, F135S, M140V, E144G, T147A, Y157C, V170A, K174R, N186S);hDHFR (L100P, E102G, Q103R, P104S, E105G, N108D, V113A, W114R, Y122C, M126I, N127R, H128Y, L132P, F135P, I139T, F148S, F149L, I152V, D153A, D169G, V170A, I176A, K177R, V182A, K185R, N186S); and hDHFR (A10T, Q13R, N14S) , N20D, P24S, N30S, M38T, T40A, K47R, N49S, K56R, I61T, K64R, K69R, I72A, R78G, E82G, F89L, D96G, N108D, M112V, W114R, Y122D, K123E, I139V, Q141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y183C, E184G, K185del, D187N). ;

[0233] ecDHFR DRD

[0234] In some embodiments, the DRD of this disclosure is derived from *E. coli* dihydrofolate reductase (ecDHFR). In some embodiments, the DRD may be derived from the ecDHFR protein and include at least one mutation. In some embodiments, the DRD may be derived from the ecDHFR protein and include more than one mutation. In some embodiments, the DRD may be derived from the ecDHFR protein and include two, three, four, or five mutations. In some embodiments, the DRD may be derived from the ecDHFR protein and contain at least one mutation selected from Y100I, F103L, and G121V. In some embodiments, the DRD may be derived from the ecDHFR protein and contain at least two mutations selected from: R12Y, Y100I; R12H, E129K; H12Y, Y100I; H12L, Y100I; R98H, F103S; M42T, H114R; N18T, A19V; and I61F, T68S.

[0235] FKBP DRD

[0236] In some embodiments, the DRD of this disclosure is derived from the FK506-binding protein (FKBP) protein or fragments or variants thereof. In some embodiments, the DRD may be derived from the FKBP protein and include at least one mutation. In some embodiments, the DRD may be derived from the FKBP protein and include more than one mutation. In some embodiments, the DRD may be derived from the FKBP protein and include two, three, four, or five mutations.

[0237] In some embodiments, the DRD of this disclosure is wholly or partially derived from human FKBP protein (SEQ ID NO.3) and contains at least one mutation selected from the following: F36V, F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K105I. In some embodiments, the FKBP DRD of this disclosure contains more than one mutation selected from the following: F36P, L106P; and E31G, F36V, R71G, and K105E.

[0238] ER DRD

[0239] In some embodiments, the DRD of this disclosure is derived from the estrogen receptor (ER) protein or fragments or variants thereof. In some embodiments, the DRD may be derived from the ER protein and include at least one mutation. In some embodiments, the DRD may be derived from the ER protein and include more than one mutation. In some embodiments, the DRD may be derived from the ER protein and include two, three, four, or five mutations.

[0240] In some embodiments, the DRD of this disclosure comprises the ligand-binding domain of ER (amino acids 305 to 509 of SEQ ID NO: 6). In some embodiments, the DRD may include at least one mutation relative to the ER ligand-binding domain, wherein the mutation occurs at position 413 (N413) and / or position 502 (Q502). In some embodiments, the mutation is at position N413 and is N413D, N413T, N413H, N413A, N413Q, N413V, N413C, N413K, N413M, N413R, N413S, N413W, N413I, N413E, N413L, N413P, N413F, N413Y, or N413G. In some implementations, the mutation is at position Q502 and is Q502H, Q502D, Q502E, Q502V, Q502A, Q502T, Q502N, Q502K, Q502S, Q502L, Q502Y, Q502W, Q502F, Q502I, Q502G, Q502P, Q502M, or Q502C. In some implementations, the DRD includes mutations at positions N413 and Q502, wherein the mutation at position N413 is selected from N413D, N413T, N413H, N413A, N413Q, N413V, N413C, N413K, N413M, N413R, N413S, N413W, N413I, N413E, N413L, and N413P. The mutation at position Q502 is selected from Q502H, Q502D, Q502E, Q502V, Q502A, Q502T, Q502N, Q502K, Q502S, Q502L, Q502Y, Q502W, Q502F, Q502I, Q502G, Q502P, Q502M, or Q502C.

[0241] 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 ERDRD contains at least two mutations and is N413T, Q502H or N413D, Q502H.

[0242] In some implementations, the ER DRD may also contain one or more mutations independently selected from L384M, M421G, G521R, or Y537S.

[0243] In some embodiments, the DRD of this disclosure comprises the following: ER(amino acids 305-549, L384M, N413F, M421G, G521R, Y537S of WT), ER(amino acids 305-549, L384M, N413L, M421G, G521R, Y537S of WT), ER(amino acids 305-549, L384M, N413Y, M421G, G521R, Y537S of WT), ER(amino acids 305-549, L384M, N413H, M421G, G521R, Y537S of WT), ER(amino acids 305-549, L384M, N413Q, M421G, G521R, Y537S of WT), ER(amino acids 305-549, L384M, N413Q, M421G, G521R, G537S of WT). 521R, Y537S), ER (WT amino acids 305-549, L384M, N413I, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413M, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413K, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413V, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413S, M421G, G521R, Y537S) ), ER (WT amino acids 305-549, L384M, N413C, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413W, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413P, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413R, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413T, M421G, G521R, Y537S), ER (WT amino acids) Amino acids 305-549, L384M, N413A, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413E, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, N413G, M421G, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502F, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502L, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502L, G521R, Y537S), ER (WT amino acids 305-549,L384M, M421G, Q502Y, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502H, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502I, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502M, G521R, Y537S), ER (WT amino acids 305 -549, L384M, M421G, Q502N, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502K, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502V, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502S, G521R, Y537S), ER (WT amino Acids 305-549, L384M, M421G, Q502C, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502W, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502P, G521R, Y537S), ER (WT amino acids 305-549, L384M, M421G, Q502T, G521R, Y537S), ER (W The amino acids in T (305-549, L384M, M421G, Q502A, G521R, Y537S), ER (amino acids in WT (305-549, L384M, M421G, Q502D, G521R, Y537S), ER (amino acids in WT (305-549, L384M, M421G, Q502E, G521R, Y537S), and ER (amino acids in WT (305-549, L384M, M421G, Q502G, G521R, Y537S)).

[0244] CA2 DRD

[0245] In some embodiments, the DRD of this disclosure may be derived from human carbonic anhydrase 2 (hCA2), an enzyme that is a member of the metalloenzyme superfamily of carbonic anhydrases. In some embodiments, the DRD may be derived from the hCA2 protein and include at least one mutation. In some embodiments, the DRD may be derived from the hCA2 protein and include more than one mutation. In some embodiments, the DRD may be derived from the hCA2 protein and include two, three, four, or five mutations.

[0246] In some embodiments, the DRD of this disclosure may be derived from amino acids 1-260 of CA2 (SEQ ID NO. 5). In some embodiments, the DRD is derived from CA2 containing amino acids 2-260 of the parental CA2 sequence (e.g., amino acids 2-260 of SEQ ID NO. 5). This is referred to herein as the CA2M1del mutation. In one embodiment, the DRD derived from CA2 may contain amino acids 2-237 of the parental CA2 sequence (e.g., amino acids 2-237 of SEQ ID NO. 5).

[0247] In some embodiments, the DRD of this disclosure comprises one or all of a region of human carbonic anhydrase 2 (CA2; SEQ ID NO.5), and further comprises, relative to SEQ ID NO.5, a mutation selected from the following: E106D, G63D, H122Y, I59N, L156H, L183S, L197P, S56F, S56N, W208S, Y193I, and Y51T.

[0248] In some embodiments, the DRD of this disclosure comprises one or all of human carbonic anhydrase 2 (CA2; SEQ ID NO.5), and is relative to SEQ ID NO. NO.5 also contains mutations selected from the following: A115L, A116Q, A116V, A133L, A133T, A141P, A152D, A152L, A152R, A173C, A173G, A173L, A173T, A23P, A247L, A247S, A257L, A257S, A38P, A38V, A54Q, A54V, A54X, A65L, A65N, A65V, A77I, A77P, A77Q, C205M, C205R, C205V, C205W, C205Y, D101G, D101M, D110I, D129I, D13 8G, D138M, D138N, D161*, D161M, D161V, D164G, D164I, D174*, D174T, D 179E, D179I, D179R, D189G, D189I, D19T, D19V, D242G, D242T, D32T, D34 T, D41T, D52I, D52L, D71F, D71G, D71K, D71M, D71S, D71Y, D72I, D72S, D7 2T, D72X, D75T, D75V, D85M, E106D, E106G, E106S, E117*, E117N, E14N, E 186*, E186N, E204A, E204D, E204G, E204N, E213*, E213G, E213N, E220K , E220R, E220S, E233D, E233G, E233R, E235*, E235G, E235N, E237K, E237 R, E238*, E238N, E238R, E26S, E69D, E69K, E69S, F130L, F146V, F175I, F 175L, F175S, F178L, F178S, F20L, F20S, F225I, F225L, F225S, F225Y, F2 30I, F230L, F230S, F259L, F259S, F66S, F70I, F70L, F95Y, G102D, G104 R, G104V, G128R, G12D, G12E, G131E, G131R, G131W, G139D, G144D, G144V , G150A, G150S, G150W, G155A, G155C, G155D, G155S, G170A, G170D, G182 A. G182W, G195A, G195R, G232R, G232W, G234L, G234V, G25E, G63D, G63V,G81E、G81V、G82D、G86A、G86D、G98V、H107I、H107Q、H119T、H119Y、H122T、H122Y、H15L、H15T、H15Y、H17D、H17I、H36I、H36Q、H64M、H94T、H96T、I145F、I145M、I166H、I166L、I209D、I209L、I215H、I215S、I22L、I255N、I255S、I33S、I59F、I59N、I59S、I91F、K111E、K111N、K112R、K113I、K113N、K126N、K132E、K132R、K148E、K148R、K153*、K153N、K158E、K158N、K167*、K169N、K169R、K171Q、K171R、K18R、K212N、K212Q、K212R、K212W、K224E、K224N、K227*、K227N、K24R、K251E、K251R、K256Q、K260F、K260L、K260Q、K39S、K45N、K45S、K80M、K80R、L118F、L120W、L140V、L140W、L143*、L147*、L147F、L156F、L156H、L156P、L156Q、L163A、L163W、L183P、L183S、L184F、L184P、L188P、L188W、L197*、L197M、L197P、L197R、L197T、L202F、L202H、L202I、L202P、L202R、L202S、L203P、L203S、L203W、L211*、L211A、L211S、L223*、L223I、L223V、L228F、L228H、L228T、L239*、L239F、L239T、L250*、L250P、L250T、L44*、L44M、L47C、L47V、L57*、L57X、L60S、L79F、L79S、L84W、L90*、L90V、M240D、M240L、M240R、M240W、N11D、N11K、N124T、N177*、N177T、N229*、N229T、N231D、N231F、N231K、N231L、N231M、N231Q、N231T、N243Q、N243T、N252E、N252T、N61R、N61T、N61Y、N62K、N62M、N67D、N67T、P137L、P13A、P13H、P13L、P13S、P154L、P154R、P154T、P180L、P180S、P185L、P185S、P185V、P194Q、P200A、P200L、P200S、P200T、P201A、P201L、P201R、P201S、P214T、P236L、P236T、P246L、P246Q、P249A、P249F、P249H、P249I、P249X、P30L、P30S、P42L、P83A、Q103K、Q135S、Q136N、Q157R、Q157S、Q221A、Q221R、Q248F、Q248L、Q248S、Q254A、Q254K、Q28S、Q53H、Q53K、Q53N、Q74R、Q92H、Q92S、R181H、R181S、R181V、R226H、R226P、R226V、R245A、R253G、R253Q、R27A、R58G、R89D、R89F、R89I、R89X、R89Y、S105L、S105Q、S151A、S151I、S151Q、S165F、S165P、S172E、S172V、S187I、S187P、S196H、S196L、S216A、S216Q、S218A、S218Q、S219A、S219Q、S258F、S258P、S29C、S29P、S43P、S43T、S48L、S50P、S56F、S56N、S56P、S56X、S73L、S73N、S73X、S99H、T108L、T125I、T125P、T168K、T168N、T168Q、T176H、T176L、T192D、T192F、T192I、T192N、T192P、T192X、T198D、T198I、T198P、T199A、T199H、T199P、T207D、T207I、T207P、T207S、T35I、T35L、T37Q、T55L、T87L、V109M、V109W、V121F、V134C、V134F、V142F、V149G、V149L、V159L、V159S、V160C、V160L、V162A、V162C、V206*、V206C、V206M、V210C、V217L、V217R、V217S、V222A、V222C、V222G、V241G、V241W、V241X、V31L、V49F、V68L、V68W、V78C、W123G、W123R、W16G、W191*、W191G、W191L、W208G、W208L、W208S、W244*、W244G、W244L、W97C、W97G、Y114H、Y114M, Y127M, Y190*, Y190L, Y190T, Y193C, Y193F, Y193I, Y193L, Y193T, Y193V, Y193X, Y40M, Y51F, Y51M, Y51T, Y51X, Y88T, K9N, and S29A. As used herein, “*” indicates the translation of a stop codon, and X represents any amino acid.

[0249] In some embodiments, the DRD of this disclosure comprises one or all of a region of human carbonic anhydrase 2 (CA2; SEQ ID NO.5) and further comprises two or more mutations relative to SEQ ID NO.5.

[0250] In some embodiments, the DRD of this disclosure comprises CA2 (amino acids 2-260 of WT, R27L, H122Y), CA2 (amino acids 2-260 of WT, T87I, H122Y), CA2 (amino acids 2-260 of WT, H122Y, N252D), CA2 (amino acids 2-260 of WT, D72F, V241F), CA2 (amino acids 2-260 of WT, V241F, P249L), CA2 (amino acids 2-260 of WT, D72F, P249L), CA2 (amino acids 2-260 of WT, D71L, L250R), CA2 (amino acids 2-260 of WT, D72F, P249F), C A2 (WT amino acid 2-260, T55K, G63N, Q248N), CA2 (WT amino acid 2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT amino acid 2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT amino acid 2-260, W4Y, L156H), CA2 (WT amino acid 2-260, L156H, G234del, E235del, P236del), CA2 (WT amino acid 2-260, L156H, F225L), CA2 (WT amino acid... Acid 2-260, D70N, D74N, D100N, L156H), (CA2(WT amino acid 2-260, I59N, G102R), CA2(WT amino acid 2-260, G63D, E69V, N231I), CA2(WT amino acid 2-260, R27L, T87I, H122Y, N252D), CA2(WT amino acid 2-260, D72F, V241F, P249L), CA2(WT amino acid 2-260, D71L, T87N, L250R), CA2(WT amino acid 2-260, L156H, S172C, F178Y, E186D), CA2(WT The amino acids 2-260, A77I, P249F), CA2 (WT amino acids 2-260, E106D, C205S), CA2 (WT amino acids 2-260, C205S, W208S), CA2 (WT amino acids 2-260, S73N, R89Y), CA2 (WT amino acids 2-260, D71K, T192F), CA2 (WT amino acids 2-260, S73N, R89F), CA2 (WT amino acids 2-260, G63D, M240L), CA2 (WT amino acids 2-260, V134F, L228F) or CA2 (WT amino acids 2-260, S56F, D71S).

[0251] In some embodiments, the DRD of this disclosure comprises CA2 (amino acids 2-260 of WT, R27L, H122Y), CA2 (amino acids 2-260 of WT, T87I, H122Y), CA2 (amino acids 2-260 of WT, H122Y, N252D), CA2 (amino acids 2-260 of WT, D72F, V241F), CA2 (amino acids 2-260 of WT, V241F, P249L), CA2 (amino acids 2-260 of WT, D72F, P249L), CA2 (amino acids 2-260 of WT, D71L, L250R), CA2 (amino acids 2-260 of WT, D72F, P249F), CA2 (W (T amino acids 2-260, T55K, G63N, Q248N), CA2 (WT amino acids 2-260, L156H, A257del, S258del, F259del, K260del), CA2 (WT amino acids 2-260, L156H, S2del, H3del, H4del, W5del), CA2 (WT amino acids 2-260, W4Y, L156H), CA2 (WT amino acids 2-260, L156H, G234del, E235del, P236del), CA2 (WT amino acids 2-260, L156H, F225L), CA2 (WT amino acids 2-260, D) 70N, D74N, D100N, L156H), (CA2 (WT amino acid 2-260, I59N, G102R), CA2 (WT amino acid 2-260, G63D, E69V, N231I), CA2 (WT amino acid 2-260, R27L, T87I, H122Y, N252D), CA2 (WT amino acid 2-260, D72F, V241F, P249L), CA2 (WT amino acid 2-260, D71L, T87N, L250R), CA2 (WT amino acid 2-260, L156H, S172C, F178Y, E186D), CA2 (WT amino acid 2-260, D7 1F, N231F), CA2 (WT amino acid 2-260, A77I, P249F), CA2 (WT amino acid 2-260, D71K, P249H), CA2 (WT amino acid 2-260, D72F, P249H), CA2 (WT amino acid 2-260, Q53N, N61Y), CA2 (WT amino acid 2-260, E106D, C205S), CA2 (WT amino acid 2-260, C205S, W208S), CA2 (WT amino acid 2-260, S73N, R89Y), CA2 (WT amino acid 2-260, D71K, T192F), CA2 (WT amino acid 2-260,Y193L, K260L), CA2 (WT amino acid 2-260, D71F, V241F, P249L), CA2 (WT amino acid 2-260, L147F, Q248F), CA2 (WT amino acid 2-260, D52I, S258P), CA2 (WT amino acid 2-260, D72S, T192N), CA2 (WT amino acid 2-260, D179E, T192I), CA2 (WT amino acid 2-260, S56N, Q103K), CA2 (WT amino acid 2-260, D71Y, Q248L), CA2 (WT amino acid 2-260, S73N) 、R89F), CA2(WT amino acid 2-260, D71K, N231L, E235G, L239F), CA2(WT amino acid 2-260, D72F, P249I), CA2(WT amino acid 2-260, D72X, V241X, P249X), CA2(WT amino acid 2-260, A54X, S56X, L57X, T192X), CA2(WT amino acid 2-260, Y193V, K260F), CA2(WT amino acid 2-260, G63D, M240L), CA2(WT amino acid 2-260, V134F, L228F), CA2( WT amino acid 2-260, D71G, N231K), CA2 (WT amino acid 2-260, S56F, D71S), CA2 (WT amino acid 2-260, D52L, G128R, Q248F), CA2 (WT amino acid 2-260, S73X, R89X), CA2 (WT amino acid 2-260, Y51X, D72X, V241X, P249X), CA2 (WT amino acid 2-260, D72I, W97C), CA2 (WT amino acid 2-260, D71K, T192F, N231F), CA2 (WT amino acid 2-260, H36Q, S43T) Y51F, N67D, G131W, R226H), CA2 (WT amino acid 2-260, F70I, F146V), CA2 (WT amino acid 2-260, K45N, V68L, H119Y, K169R, D179E), CA2 (WT amino acid 2-260, H15L, A54V, K111E, E220K, F225I), CA2 (WT amino acid 2-260, P13S, P83A, D101G, K111N, F230I), CA2 (WT amino acid 2-260, G63D, W123R, E220K), CA2 (WT amino acid 2-260,N11D, E69K, G86D, V109M, K113I, T125I, D138G, G155S), CA2 (WT amino acid 2-260, I59N, G102R, A173T), CA2 (WT amino acid 2-260, L79F, P180S), CA2 (WT amino acid 2-260, A77P, G102R, D138N), CA2 (WT amino acid 2-260, F20L, K45N, G63D, E69V, N2) 31I), CA2 (WT amino acid 2-260, T199N, L202P, L228F), CA2 (WT amino acid 2-260, K9N, H122Y, T168K), CA2 (WT amino acid 2-260, Q53H, L90V, Q92H, G131E), CA2 (WT amino acid 2-260, L44M, L47V, N62K, E69D), CA2 (WT amino acid 2-260, D75V, K169N, F259L) CA2 (WT amino acid 2-260, T207S, V222A, N231D), CA2 (WT amino acid 2-260, I59F, V206M, G232R), CA2 (WT amino acid 2-260, P13A, A133T), CA2 (WT amino acid 2-260, I59N, R89I), CA2 (WT amino acid 2-260, A65N, G86D, G131R, G155D, K158N, V162A, G170D), P236L), CA2 (WT amino acid 2-260, G12R, H15Y, D19V), CA2 (WT amino acid 2-260, A65V, F95Y, E106G, H107Q, I145M, F175I), CA2 (WT amino acid 2-260, G63D, E69V, N231I), CA2 (WT amino acid 2-260, S29A, C205S) and / or CA2 (WT amino acid 2-260, S29C, C205S).

[0252] In some embodiments, the DRD of this disclosure comprises all or part of human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises an H122Y mutation at amino acid position 122 (H122) of SEQ ID NO. 5. In some such embodiments, the DRD further comprises: (i) an R27L mutation at amino acid position 27 (R27) of SEQ ID NO. 5; (ii) a T87I mutation at amino acid position 87 (T87) of SEQ ID NO. 5; (iii) an N252D mutation at amino acid position 252 (N252) of SEQ ID NO. 5; or a combination of (i), (ii), and / or (iii).

[0253] In some embodiments, the DRD of this disclosure comprises all or part of human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and also contains an E106D mutation at amino acid position 106 (E106) of SEQ ID NO. 5. In some such embodiments, the DRD also contains a C205S mutation at amino acid position 205 (C205) of SEQ ID NO. 5.

[0254] In some embodiments, the DRD of this disclosure comprises all or part of human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and also contains a W208S mutation at amino acid position 208 (W208) of SEQ ID NO. 5. In some such embodiments, the DRD also contains a C205S mutation at amino acid position 205 (C205) of SEQ ID NO. 5.

[0255] In some embodiments, the DRD of this disclosure comprises all or part of human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and also contains an I59N mutation at amino acid position 59 (I59) of SEQ ID NO. 5. In some such embodiments, the DRD also contains a G102R mutation at amino acid position 102 (G102) of SEQ ID NO. 5.

[0256] In some embodiments, the DRD of this disclosure comprises all or part of human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises an L156H mutation at amino acid position 156 (L156) of SEQ ID NO. 5. In some such embodiments, the DRD further comprises (i) a W4Y mutation at amino acid position 4 (W4) of SEQ ID NO. 5; (ii) an F225L mutation at amino acid position 225 (F225) of SEQ ID NO. 5; (iii) a deletion of amino acids 257-260 of SEQ ID NO. 5; (iv) a deletion of amino acids 1-5 of SEQ ID NO. 5; or (v) a deletion of amino acids G234, E235, and P236 of SEQ ID NO. 5.

[0257] In some embodiments, the DRD of this disclosure comprises all or part of human carbonic anhydrase 2 (CA2; SEQ ID NO. 5) and also contains four mutations relative to SEQ ID NO. 5, the mutations corresponding to: (i) L156H, S172C, F178Y and E186D; (ii) D70N, D74N, D100N and L156H.

[0258] In some embodiments, the DRD of this disclosure comprises all or part of 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 an S73N mutation at amino acid position 73 (S73) of SEQ ID NO. 5; and (ii) the second mutation is a substitution of F or Y at amino acid position 89 (R89) of SEQ ID NO. 5.

[0259] In some embodiments, the DRD of this disclosure comprises all or part of human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises an N or F substitution at amino acid position 56 (S56) of SEQ ID NO. 5. In some such embodiments, the DRD comprises two substitutions corresponding to S56F and D71S relative to SEQ ID NO. 5.

[0260] In some embodiments, the DRD of this disclosure comprises all or part of 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 D or N substitution at 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 N231I; or (iv) T55K, G63N and Q248N.

[0261] In some embodiments, the DRD of this disclosure comprises all or part of 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 an L or K substitution at 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.

[0262] In some embodiments, the DRD of this disclosure comprises all or part of 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) an F substitution at amino acid position 241 (V241) of SEQ ID NO. 5; (ii) an F or L substitution at 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) A77I and P249F; or (vi) V241F and P249L.

[0263] In some embodiments, the DRD of this disclosure comprises all or part of human carbonic anhydrase 2 (CA2; SEQ ID NO. 5), and further comprises one or more substitutions selected from the group consisting of Y51T, L183S, Y193I, L197P, and combinations of V134F and L228F relative to SEQ ID NO. 5.

[0264] The amino acid sequence of the DRD covered by this disclosure has at least about 70% identity with the amino acid sequence of the parent protein from which the sequence is derived, preferably at least about 75% or 80% identity, more preferably at least about 85%, 86%, 87%, 88%, 89% or 90% identity, and even more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In some embodiments, the amino acid sequence of the DRD covered by this disclosure is at least about 70% identical to the parent protein from which it is derived (e.g., a parent protein having the amino acid sequence of any one of SEQ ID No: 1, 2, 3, 4, 5, 6 and 71), preferably at least about 75% or 80% identical, more preferably at least about 85%, 86%, 87%, 88%, 89% or 90% identical, and even more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

[0265] Examples of DRDs disclosed herein include those derived from: human carbonic anhydrase 2 (CA2), human DHFR, ecDHFR, human estrogen receptor (ER), FKBP, human protein FKBP, and human PDE5. Suitable DRDs, which may be referred to as destabilizing domains or ligand-binding domains, are also known in the art. See, for example, WO2018 / 161000; WO2018 / 231759; WO2019 / 241315; US8,173,792; US8,530,636; WO2018 / 237323; WO2017 / 181119; US2017 / 0114346; US2019 / 0300864; WO2017 / 156238; Miyazaki et al., J Am Chem Soc, 134:3942 (2012); Banazz ynski et al. (2006) Cell 126:995-1004; Stankunas, K. et al. (2003) Mol. Cell 12:1615-1624; Banazzynski 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 daSilva et al. (2009) Proc. Natl. Acad. Sci. USA 106:7583-7588; Pruett-Miller et al. (2009) PLoS Gene t. 5:e1000376; and Feng et al. (2015) Elife 4:e10606.

[0266] As provided above in the “Transcription Factor Systems” section, a transcription factor system comprises one or more polynucleotide combinations containing a nucleic acid sequence encoding a drug-responsive domain (DRD), wherein a transcription factor (e.g., a transcription factor DNA-binding domain, a transcription factor activation domain, or both) is operatively linked to the DRD. The nucleic acid sequence encoding the DRD may be selected from the DRD sequences described herein. Constructs containing DRD sequences are provided in Table 1 above. Additional constructs containing different DRDs are provided in Table 4. An asterisk (“*”) in Table 4 indicates the translation of a stop codon.

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275] Stimulants of the transcription factor system

[0276] The transcription factor system disclosed herein can respond to stimuli.

[0277] In some embodiments, the stimulant is a ligand. The ligand can be nucleic acid-based, protein-based, lipid-based, organic, inorganic, or any combination thereof. In some embodiments, the ligand can be a synthetic molecule. In some embodiments, the ligand can be a small molecule therapeutic compound. In some embodiments, the ligand can be a small molecule drug previously approved by a regulatory agency, such as the U.S. Food and Drug Administration (FDA).

[0278] As described in this disclosure, transcription factor systems can exhibit ligand-dependent activity. Ligands can bind to DRDs and stabilize transcription factors or domains of transcription factors encoded by the transcription factor system. The effect of ligands known to bind to candidate DRDs on the activity of the transcription factor system can be tested.

[0279] In some implementations, the ligand is cell-permeable. In some implementations, the ligand can be engineered to be lipophilic to improve cell permeability.

[0280] In some implementations, the ligand is a small molecule. Clinically approved small molecule ligands can be safe and have appropriate pharmacokinetics and distribution.

[0281] In some embodiments, the ligand may be complexed or bound to one or more other molecules, such as, but not limited to, another ligand, protein, peptide, nucleic acid, lipid, lipid derivative, sterol, steroid, metabolite, metabolite derivative, or small molecule. In some embodiments, the ligand stimulant is complexed or bound to one or more other molecules of different kinds and / or numbers. In some embodiments, the ligand stimulant is a polymer of the same kind of ligand. In some embodiments, the ligand stimulant polymer comprises 2, 3, 4, 5, 6, or more monomers.

[0282] CA2 ligands

[0283] In some embodiments, the ligand of this disclosure binds to carbonic anhydrase. In some embodiments, the ligand binds to and inhibits the function of carbonic anhydrase and is referred to herein as a carbonic anhydrase inhibitor.

[0284] In some embodiments, the ligand is a small molecule that binds to carbonic anhydrase 2. In one embodiment, the small molecule is a CA2 inhibitor. Examples of CA2 inhibitors include, but are not limited to, celecoxib (also known as Celebrex), valdecoxib, rofecoxib, acetazolamide, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, zonisamide, dansylamide, and dichlorphenamide.

[0285] In some implementations, the ligand may comprise a small molecule moiety known to mediate binding to CA2. The ligand may also be modified to reduce off-target binding to carbonic anhydrases other than CA2 and increase specific binding to CA2.

[0286] In some embodiments, the stimulant may be a ligand that binds to more than one carbonic anhydrase. In one embodiment, the stimulant is a pan-carbonic anhydrase inhibitor that can bind to two or more carbonic anhydrases.

[0287] DHFR ligands

[0288] In some embodiments, the ligand of this disclosure binds to dihydrofolate reductase. In some embodiments, the ligand binds to and inhibits dihydrofolate reductase function and is referred to herein as a dihydrofolate inhibitor.

[0289] In some embodiments, the ligand can be a selective inhibitor of human DHFR. The ligands disclosed herein can also be selective inhibitors of dihydrofolate reductase from bacteria and parasitic organisms such as *Pneumocystis* spp., *Toxoplasma* spp., *Trypanosoma* spp., *Mycobacterium* spp., and *Streptococcus* spp. Ligands specific to other DHFRs can be modified to improve binding to human dihydrofolate reductase.

[0290] Examples of dihydrofolate inhibitors include, but are not limited to, trimethoprim (TMP), methotrexate (MTX), pralatrexate, piritrexim, pyrimethamine, talotrexin, chlorguanide, pentamidine, trimetrexate, aminopterin, C1898 trihydrochloride, pemetrexed disodium, raltitrexed, sulfaguanidine, folotyn, ilaprin, and diaveridine.

[0291] In some embodiments, the ligands of this disclosure may include dihydrofolate or any derivative thereof capable of binding human DHFR. In some embodiments, the ligands of this disclosure may be 2,4,diamino heterocyclic compounds. In some embodiments, the 4-oxo group in dihydrofolate may be modified to produce a DHFR inhibitor. In one example, the 4-oxo group may be replaced by a 4-amino group. Various diamino heterocycles, including pteridine, quinazoline, pyridopyrimidine, pyrimidine, and triazine, may also be used as a skeleton for developing DHFR inhibitors and may be used according to this disclosure.

[0292] In some embodiments, the ligand comprises a TMP-derived ligand containing a ligand moiety known to mediate binding to DHFR. The ligand may also be modified to reduce off-target binding to other folate-metabolizing enzymes and increase specific binding to DHFR.

[0293] ER ligands

[0294] In some embodiments, the ligands of this disclosure bind to the ER. The ligands may be agonists or antagonists. In some embodiments, the ligands bind to and inhibit ER function and are referred to herein as ER inhibitors. In some embodiments, the ligands may be selective inhibitors of human ER. The ligands of this disclosure may also be selective inhibitors of ER from other species. Ligands specific to other ERs may be modified to improve binding to human ER.

[0295] The ligand can be an ER agonist, such as, but not limited to, the endogenous estrogen 17β-estradiol (E2) and the synthetic nonsteroidal estrogen diethylstilbestrol (DES). In some embodiments, the ligand can be an ER antagonist, such as ICI-164,384, RU486, tamoxifen, 4-hydroxytamoxifen (4-OHT), fulvestrant, oremifene, lasofoxifene, cromifene, femarelle, and ormeloxifene and raloxifene (RAL).

[0296] In some embodiments, the stimulant disclosed herein may be an ER antagonist, such as, but not limited to, bardoxifene and / or raloxifene.

[0297] In some embodiments, the ligand comprises a bardoxifene-derived ligand containing a ligand moiety known to mediate binding to the ER. The ligand may also be modified to reduce off-target binding to other folate-metabolizing enzymes and increase specific binding to ER-derived DRDs.

[0298] Phosphodiesterase ligand

[0299] In some embodiments, the ligands of this disclosure bind to phosphodiesterases. In some embodiments, the ligands bind to and inhibit phosphodiesterase function and are referred to herein as phosphodiesterase inhibitors.

[0300] In some embodiments, the ligand is a small molecule that binds to phosphodiesterase 5. In one embodiment, the small molecule is an hPDE5 inhibitor. Examples of hPDE5 inhibitors include, but are not limited to, sildenafil, vardenafil, tadalafil, avanafil, lodenafil, mirodenafil, udenafil, benzamidenafil, dasantafil, beminafil, SLx-2101, LAS 34179, UK-343,664, UK-357903, UK-371800, and BMS-341400.

[0301] In some implementations, the ligand comprises a sildenafil-derived ligand containing a ligand moiety known to mediate binding to hPDE5. The ligand may also be modified to reduce off-target binding to phosphodiesterases and increase specific binding to hPDE5.

[0302] In some embodiments, the stimulant may be a ligand that binds to more than one phosphodiesterase. In one embodiment, the stimulant is a panphosphodiesterase inhibitor that can bind to two or more hPDEs, such as aminophyline, paraxanthine, pentoxifylline, theobromine, dipyridamole, theophylline, zaprinast, icariin, CDP-840, etazolate, and glaucine.

[0303] In some embodiments, the ligand is an hPDE1 inhibitor. In some embodiments, the ligand is an hPDE2 inhibitor. In some embodiments, the ligand is an hPDE3 inhibitor.

[0304] In some embodiments, the ligand is an hPDE4 inhibitor. In some embodiments, the ligand is an hPDE6 inhibitor. In some embodiments, the ligand is an hPDE7 inhibitor. In some embodiments, the ligand is an hPDE8 inhibitor. In some embodiments, the ligand is an hPDE9 inhibitor. In some embodiments, the ligand is an hPDE10 inhibitor.

[0305] FKBP ligands

[0306] In some embodiments, the ligands of this disclosure bind to FKBP, including human FKBP. In some embodiments, the ligand is SLF or Shield-1.

[0307] Payload

[0308] The payload may include any polypeptide or protein or a fragment thereof. The payload may be a wild-type sequence, a fragment of a wild-type sequence, and / or contain one or more mutations. The payload may be a natural protein derived from the genome of an organism, or a variant, mutant, or derivative thereof. Natural proteins may be derived from, for example, mammalian organisms, bacteria, and viruses. The payload may be a protein or polypeptide encoded by a recombinant nucleic acid molecule, a fusion or chimeric polypeptide, or a polypeptide that functions as part of a protein complex.

[0309] In one instance, the payload could be a polypeptide encoded by a nucleic acid sequence from the human genome.

[0310] In some embodiments, the payload may be a variant sequence of the parent polypeptide. In some aspects, the variant sequence may have the same or similar activity as the reference sequence. Alternatively, the variant may have altered (e.g., increased or decreased) activity relative to the reference sequence. Generally, as determined using sequence alignment procedures known to those skilled in the art, a variant of a particular polypeptide of this 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 with that particular reference polypeptide.

[0311] Therapeutic agent as effective payload

[0312] In some embodiments, the payload of this disclosure may be a therapeutic agent. For example, the payload may be a cancer therapeutic agent, a therapeutic agent for autoimmune diseases, an immunotherapeutic agent, an anti-inflammatory agent, an antipathogenic agent, or a gene therapy agent. In some aspects, the immunotherapeutic agent may be an antibody and its fragments and variants, a T-cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric switch receptor, an antagonist of a co-inhibitory molecule, an agonist of a 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.

[0313] In some embodiments, the payload of this disclosure may be an immunotherapeutic agent that induces an immune response in an organism. The immunotherapeutic agent may be, but is not limited to, antibodies and their fragments and variants, TCRs, chimeric antigen receptors (CARs), chimeric switch receptors, cytokines, chemokines, cytokine receptors, chemokine receptors, cytokine-cytokine receptor fusion peptides, or any agent that induces an immune response. In one embodiment, the immunotherapeutic agent induces an anticancer immune response in cells or in a subject.

[0314] Cytokines, chemokines, and other soluble factors as the effective payload

[0315] In some embodiments, the payload of this disclosure may be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells, and other cell types, which act as chemical communication agents between cells and tissues in vivo. These proteins mediate a variety of physiological functions, ranging from their effects on cell growth, differentiation, migration, and survival to numerous effector activities. For example, activated T cells produce a variety of cytokines with cytotoxic functions that eliminate tumor cells.

[0316] In some embodiments, the payload of this disclosure may be cytokines and their fragments, variants, analogs, and derivatives, including but not limited to interleukins, tumor necrosis factor (TNF), interferon (IFN), TGFβ, and chemokines. In some embodiments, the payload of this invention may be a cytokine that stimulates an immune response. In other embodiments, the payload of this invention may be an antagonist of cytokines that negatively affect anticancer immune responses.

[0317] In some embodiments, the payload of this disclosure may be a cytokine receptor, a recombinant receptor, a variant thereof, an analogue, or a derivative thereof; or a signaling component of a cytokine. In various embodiments, the payload of this disclosure may include secreted cytokines or membrane-bound forms of cytokines. Illustrative examples of membrane cytokines may include cytokines operatively fused, linked, or coupled to transmembrane domains, such as the CD8α transmembrane domain, B7-1 transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, or the human IgG4 Fc region (e.g., immunostimulatory cytokines, such as IL12, IL2, IL15, and IL18). In various embodiments, cytokines may be fused or coupled to transmembrane domains via intermediate peptides or protein sequences, such as linkers, hinges, transmembrane tails, etc.

[0318] In one embodiment, the payload of this disclosure may be a cytokine fused to the extracellular domain of TNFα. Such payloads are produced in the form of membrane-associated cytokines fused to the extracellular domain of TNF. In one embodiment, the cytokines may be detached from the cell surface by the action of membrane-associated proteases and / or proteases in the extracellular space (e.g., MMP9).

[0319] In some embodiments, the payload of this disclosure may be an interleukin (IL) cytokine. Interleukins (ILs) are a class of glycoproteins produced by leukocytes that regulate immune responses. As used herein, the term "interleukin (IL)" refers to an interleukin polypeptide of any species or origin and includes both full-length proteins and fragments or portions of such proteins.

[0320] In some embodiments, the payload of this disclosure may comprise IL12. IL12 is a heterodimeric protein consisting of two subunits (p35 and p40) secreted by antigen-presenting cells (such as macrophages and dendritic cells). Expression of IL12 requires simultaneous expression of both subunits to produce a biologically active heterodimer. In some embodiments, the payload of this disclosure may be either the p35 subunit or the p40 subunit.

[0321] In some implementations, the payload of this disclosure may include all or part of IL12.

[0322] In some implementations, IL12 may be Flexi IL12, where the p35 and p40 subunits are encoded by a single cDNA that produces the single-chain polypeptide. The single-chain polypeptide can be generated by placing the p35 subunit at the N-terminus or C-terminus of the single-chain polypeptide. Similarly, the p40 subunit may be located at the N-terminus or C-terminus of the single-chain polypeptide.

[0323] The format of the IL12 payload disclosed herein can be optimized. In one embodiment, the payload may be a bicistronic IL12 containing p40 and p35 subunits, these subunits being separated by internal ribosome entry sites or cleavage sites such as P2A or furin protease, to allow independent expression of both subunits from a single vector. In another embodiment, the payload may be either the p40 subunit or the p35 subunit of IL12.

[0324] In some embodiments, the payload may be membrane-bound IL12. IL12 binds to the membrane via 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 via the transmembrane domain. In some aspects, the membrane-bound IL12 can be detached from or cleaved from the cell surface by a protease. In some embodiments, the transmembrane domain of this disclosure may be derived from a natural or synthetic source. The transmembrane domain may be derived from any natural membrane-bound or transmembrane protein. Alternatively, the transmembrane domain of this disclosure may be synthetic. In some aspects, the synthetic sequence may primarily contain hydrophobic residues, such as leucine and valine. In some aspects, the selected transmembrane and / or hinge domains may be resistant to protease activity.

[0325] In some embodiments, the payload of this disclosure may include IL15. Interleukin-15 is a potent immunostimulatory cytokine and an important survival factor for T cells and natural killer cells.

[0326] In some embodiments, the payload of this disclosure may contain all or a portion of IL15. Any portion of IL15 that retains one or more functions of full-length or mature IL15 may be used in this disclosure. These functions include promoting NK cell survival, regulating NK cell and T cell activation and proliferation, and supporting NK cell development from hematopoietic stem cells.

[0327] In some cases, IL15 is linked in whole or in part to one or more transmembrane proteins.

[0328] The IL15 payload can be designed to be secreted (using, for example, an IL2 signal sequence) or membrane-bound (using, for example, an IgE or CD8a signal sequence).

[0329] A unique feature of IL15-mediated activation is the trans-presentation mechanism, in which IL15 is presented as a complex with the α subunit of the IL15 receptor (IL15Ra), which binds to and activates membrane-bound IL15β / γ receptors on the same or different cells. In some embodiments, the payload of this disclosure is membrane-bound IL15. In some embodiments, the payload of this disclosure may include an IL15 / IL15Ra fusion peptide. In some embodiments, the payload may be whole or a portion of IL15 fused to whole or a portion of IL15Ra. Any portion of IL15 and IL15Ra, respectively retaining one or more functions of full-length or mature IL15 or IL15Ra, may be used.

[0330] In some respects, IL15 molecules are extracellular and tethered to the cell via transmembrane domains. In other respects, membrane-bound IL15 can be detached from or cleaved from the cell surface by proteases.

[0331] The membrane-bound IL15 or IL15 / IL15Ra fusion peptide disclosed herein can be detached, either wholly or partially, into the extracellular space. As used herein, detachment refers to the release of membrane-associated biomolecules from the membrane to which they are tethered. In some cases, detachment may be caused by proteolytic cleavage.

[0332] The payload of this disclosure may contain an amino acid sequence similar to that of human IL15, such as UniProtKB-P40933(IL15_HUMAN).

[0333] In some embodiments, the payload of this disclosure can be used to improve the expansion, survival, persistence, and potency of immune cells, such as CD8+ TEM cells, natural killer cells, and tumor-infiltrating lymphocytes (TILs), as well as CAR T cells for immunotherapy. On one hand, this disclosure provides a payload for minimizing toxicity associated with cytokine therapy. In some embodiments, the payload of this disclosure may contain all or a portion of IL2. Any portion of IL2 retaining one or more functions of full-length or mature IL2 may be used in this disclosure.

[0334] It should be understood in this art that the nomenclature of certain genes and / or proteins may or may not include punctuation marks such as dashes "-"; or symbols such as Greek letters. Whether these are included or excluded herein, their meaning is not intended to be altered as would be understood by those skilled in the art. For example, IL2, IL-2, and IL 2 refer to the same interleukin. Similarly, IL15, IL 15, and IL-15 refer to the same interleukin. Likewise, TNFalpha, TNFα, TNF-alpha, TNF-α, TNF alpha, and TNFα all refer to the same protein.

[0335] Antibodies and antibody fragments as payload

[0336] In some implementations, the payload of this disclosure may be an antibody, an antibody fragment, or a variant thereof.

[0337] Antibodies can be complete antibodies, antibody light chains, antibody heavy chains, antibody fragments, antibody variants, or antibody derivatives.

[0338] For the purposes of this article, an "antibody" may contain a heavy variable domain, a light variable domain, and an Fc region.

[0339] In some implementations, the payload may be a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homologous cells (clones), meaning that the individual antibodies constituting the population are identical and / or bind to the same epitopes, except for possible variants that may arise during monoclonal antibody production; such variants are generally present in trace amounts. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody targets a single determinant on the antigen.

[0340] In one embodiment, the payload of this disclosure may be a humanized antibody. As used herein, the term "humanized antibody" refers to a chimeric antibody comprising a minimal portion derived from one or more non-human (e.g., mouse) antibody sources and the remainder derived from one or more human immunoglobulin sources. In most cases, a humanized antibody is a antibody in which residues of a hypervariable region derived from a receptor antibody are replaced by residues of a hypervariable region derived from an antibody (donor antibody) of a non-human species, such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or ability. In one embodiment, the antibody may be a humanized full-length antibody.

[0341] As used herein, the term "antibody variant" refers to a modified antibody (as opposed to a natural or initiating antibody) or a biomolecule (e.g., an antibody mimic) that is structurally and / or functionally similar to a natural or initiating antibody. The amino acid sequence, composition, or structure of an antibody variant may be altered compared to a natural antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.

[0342] In some embodiments, antibody fragments and variants may contain antigen-binding regions derived from intact antibodies. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; bifunctional antibodies; linear antibodies; single-chain antibody molecules, such as single-chain variable fragments (scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, referred to as "Fab" fragments, each having a single antigen-binding site. A residual "Fc" fragment is also produced, its name reflecting its tendency to crystallize. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites and remains capable of cross-linking antigens. The payload of this disclosure may contain one or more of these fragments.

[0343] In some implementations, the payload of this disclosure may be a therapeutic antibody.

[0344] Chimeric antigen receptor payload

[0345] In some embodiments, the payload of this disclosure may be a chimeric antigen receptor (CAR). As used herein, the term "chimeric antigen receptor (CAR)" refers to a synthetic receptor that mimics the T cell receptor (TCR) on the surface of T cells. Generally, a CAR consists of an extracellular targeting domain, a transmembrane domain / region, and an intracellular signaling / activation domain. Cells, such as T cells engineered to express a CAR, can be redirected to attack target cells expressing molecules that can be recognized by the targeting portion of the CAR. In a standard CAR receptor, the following components are linearly constructed as a single fusion protein: an extracellular targeting domain, a transmembrane domain, and an intracellular signaling / activation domain. The extracellular region contains the targeting domain / region (e.g., scFv) that recognizes specific tumor antigens or other tumor cell surface molecules. The intracellular region may contain a signaling domain of the TCR complex (e.g., the CD3ζ signaling region) and / or one or more co-stimulatory signaling domains, such as those from CD28, 4-1BB (CD137), and OX-40 (CD134). For example, first-generation CARs only have a CD3ζ signaling domain, while to enhance T cell persistence and proliferation, a co-stimulatory intracellular domain is added, resulting in second-generation CARs with a CD3ζ signaling domain and one co-stimulatory signaling domain, and third-generation CARs with a CD3ζ signaling domain and two or more co-stimulatory signaling domains. When expressed by T cells, the CAR confers antigen specificity to the T cells determined by the extracellular targeting portion of the CAR. Fourth-generation CARs involve adding one or more components, such as homing and suicide genes, to develop more capable and safer CAR architectures.

[0346] In some implementations, when transduced into immune cells (e.g., T cells and NK cells), the CAR payload can redirect the immune cells toward a target (e.g., tumor cells) that expresses a molecule recognized by the extracellular target portion of the CAR.

[0347] Nucleic acid modifiers as effective load

[0348] In some implementations, the payload of this disclosure may be a nucleic acid modifier.

[0349] In some embodiments, the payload of this disclosure may be a component of a gene editing system. In some embodiments, the payload of this disclosure may be a Cas protein (CRISPR-associated protein), including Cas9 and Cas12. Cas proteins may be altered or otherwise modified. For example, the Cas protein may be deadCas9. In some embodiments, the Cas9 protein may be an enzymatically active Cas9 protein, a wild-type Cas9 protein, a Cas9 nickase, or a nuclease-free or nuclease-deficient Cas9 protein. In some embodiments, the payload of this disclosure may be a zinc finger nuclease, a TALEN (transcription activator-like effector nuclease), or a wide range of nucleases.

[0350] In some embodiments, the payload of this disclosure may be a recombinase, such as Cre recombinase.

[0351] Reagents used to treat autoimmune diseases as the effective load

[0352] In some embodiments, the payload of this disclosure may be an agent for treating, improving, or preventing autoimmune diseases.

[0353] In some embodiments, the payload of this disclosure includes anti-cytokines, such as neutralizing antibodies against tumor necrosis factor (TNF)-α, IL-1, and IL-6. In some embodiments, the payload of this disclosure targets B cell depletion, such as neutralizing antibodies against CD20, CD22, CD28, CTLA-4, and B lymphocyte stimulators (BLyS).

[0354] Pharmaceutical compositions and formulations

[0355] This teaching also includes pharmaceutical compositions comprising one or more of the following: the transcription factor system of this disclosure, nucleic acid, polynucleotide, modified cell or payload; and optionally at least one pharmaceutically acceptable excipient or inert ingredient.

[0356] As used herein, the term "pharmaceutical composition" means a formulation 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, and optionally other chemical components, such as physiologically suitable carriers and excipients.

[0357] The terms “excipient” or “inactive ingredient” refer to inert or inactive substances added to a pharmaceutical composition to further facilitate the application of the compound.

[0358] In some embodiments, the composition is administered to humans, human patients, or subjects. For the purposes of this disclosure, the phrase "active ingredient" generally refers to any one or more transcription factor system components delivered as described herein.

[0359] Although the description of the pharmaceutical compositions provided herein is primarily directed toward pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as non-human animals, including non-human mammals. Subjects considering administration of the pharmaceutical compositions include, but are not limited to, non-human mammals, including agricultural animals such as cattle, horses, chickens, and pigs; domestic animals such as cats and dogs; or research animals such as mice, rats, rabbits, dogs, and non-human primates.

[0360] The pharmaceutical compositions according to this disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or in multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient administered to a subject and / or a convenient fraction of such a dose, such as half or one-third of such a dose.

[0361] The relative amounts of the active ingredient, pharmaceutically acceptable excipients or inert ingredients and / or any additional ingredients in the pharmaceutical compositions according to this disclosure will vary depending on the identity, physical condition and / or status of the treated subject and, additionally, on the route of administration of the composition. For example, the composition may contain at least 80% (w / w) of the active ingredient, for example, between 0.1% and 100%, such as between 0.5% and 50%, between 1% and 30%, between 5% and 80%.

[0362] The efficacy of treatment or improvement in a disease can be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dosage of medication required to maintain treatment effectiveness, disease marker levels, or any other measurable parameter applicable to the given disease being treated or serving as a target for prevention. Healthcare practitioners in the art can monitor the efficacy of treatment or prevention by measuring any one or any combination of these parameters. Regarding the administration of the compositions of this disclosure, "effectively targeted" for example, cancer indicators, when administered in a clinically appropriate manner, results in beneficial effects on at least a substantial proportion of patients, such as symptom improvement, cure, reduction of disease burden, reduction of tumor or cell count, extended life, improved quality of life, or other effects generally considered positive by physicians familiar with treating a particular type of cancer.

[0363] The therapeutic or preventative effect is evident when one or more parameters of a disease state show statistically significant improvement, or when there is no worsening or development of symptoms that were originally expected. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50%, or higher percentage, in a measurable parameter of the disease indicates effective treatment. The efficacy of a given composition or formulation of this disclosure can also be determined using experimental animal models of a given disease known in the art. When using experimental animal models, the efficacy of the treatment is confirmed when a statistically significant change is observed.

[0364] formulation

[0365] The polynucleotide and carrier compositions disclosed herein can be formulated by any suitable delivery method. Formulations can be, but are not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipids, lipid complexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, and combinations thereof.

[0366] In one embodiment, the polynucleotide and carrier formulation are nanoparticles that may contain at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, and polyethylene glycol-modified lipids. On the other hand, 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.

[0367] For the polynucleotides of this disclosure, the formulation may be selected from any formulation taught, for example, in international application PCT / US2012 / 069610.

[0368] Inactive ingredients

[0369] In some embodiments, the drug or other formulation may contain at least one excipient that is an inactive ingredient. As used herein, the term "inactive ingredient" refers to one or more inactive agents included in a formulation. In some embodiments, all, none, or some of the inactive ingredients that may be used in formulations of this disclosure may be approved by the U.S. Food and Drug Administration (FDA).

[0370] Drug administration, delivery and application

[0371] The compositions disclosed herein can be delivered to cells or subjects via one or more routes and methods. Viral vectors containing one or more of the transcription factor systems, nucleic acids, polynucleotides, payloads, and other components described herein can be used to deliver them to cells and / or subjects. Other methods, such as mRNA, plasmids, and recombinant proteins, can also be used.

[0372] deliver

[0373] naked delivery

[0374] The pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads disclosed herein can be delivered to cells, tissues, organs, and / or organisms in a naked form. As used herein, the term "naked" means that the pharmaceutical composition, transcription factor system, nucleic acid, polynucleotide, or payload is delivered without any agents or modifications that promote transfection or penetration. Naked pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads can be delivered to cells, tissues, organs, and / or organisms using administration routes known in the art and described herein. In some embodiments, naked delivery may include formulations in simple buffers such as saline or PBS.

[0375] Preparation delivery

[0376] In some embodiments, the pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads of this disclosure can be formulated using the methods described herein. Formulations may comprise modified and / or unmodified pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads. Formulations may also include, but are not limited to, cell permeabilizers, pharmaceutically acceptable carriers, delivery agents, bioerectible or biocompatible polymers, solvents, and / or sustained-release delivery reservoirs. Formulations of this disclosure can be delivered to cells using administration routes known in the art and described herein.

[0377] Pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, or payloads may also be formulated for direct delivery to organs or tissues in any of the several ways in the art, including but not limited to direct immersion or bathing; delivery via catheter; delivery via gel, powder, ointment, cream, lotion, and / or drops; delivery via substrates such as fabrics coated or impregnated with the composition or biodegradable materials.

[0378] Delivery to cells

[0379] In another aspect of this disclosure, the transcription factor system of this disclosure, or its components and compositions thereof, as well as the vectors containing said polynucleotides, can be introduced into cells, such as immune effector cells.

[0380] In one aspect of this disclosure, the polynucleotides of the transcription factor system or its components and compositions thereof can be packaged in plasmids, viral vectors, or integrated into a viral genome, thereby allowing transient or stable expression of the polynucleotides. Preferred viral vectors are retroviral vectors, including lentiviral vectors and gamma retroviral vectors. To construct a retroviral vector, a polynucleotide molecule of the transcription factor system is inserted into the viral genome in place of certain viral sequences to produce a replication-defective virus. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol, and env genes but lacking the LTR and packaging components. The recombinant retroviral particles are secreted into a culture medium, then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are particularly preferred because they are capable of infecting both dividing and non-dividing cells.

[0381] The vector can also be transferred to cells via non-viral methods, using physical methods such as needle, electroporation, acoustic perforation, and hydroporation; or using chemical carriers, such as inorganic particles (e.g., calcium phosphate, silica, gold) and / or chemical methods. In some embodiments, delivery can be performed using synthetic or naturally biodegradable agents, such as cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based carriers, or polymer-based carriers. In some embodiments, the vector can be transferred to cells through temporary membrane disruption, for example, by high-speed cell deformation.

[0382] In some embodiments, the peptides of this disclosure can be delivered directly to cells. In one embodiment, the peptides of this disclosure can be delivered using a synthetic peptide comprising an endosomal leakage domain (ELD) fused to a cell penetration domain (CLD). The peptides of this disclosure are introduced into cells together with the ELD-CLD-synthetic peptide. The ELD facilitates the escape of proteins 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 protein transmembrane transport and have also been described in the art. The ELD-CLD fusion protein synergistically increases transduction efficiency when compared to co-transduction using either domain alone. In some embodiments, a histidine-rich domain may optionally be added to the shuttle construct as an additional method to allow the payload to escape from the endosome into the cytosol. The shuttle may also include cysteine ​​residues at the N or C terminus to produce a multimer of the fusion peptide. When compared to single fusion peptide constructs, multimers of ELD-CLD fusion peptides generated by adding cysteine ​​residues to the peptide terminus exhibit even higher transduction efficiency. The peptides of this disclosure can also be attached to appropriate localization signals to guide the delivery vehicle to suitable subcellular locations, such as the cell nucleus. In some embodiments, any of the ELD, CLD, or fusion ELD-CLD synthetic peptides taught in International Patent Publications WO2016161516 and WO2017175072 may be used in this disclosure (the contents of which are incorporated herein by reference in their entirety).

[0383] Delivery method and / or carrier

[0384] The transcription factor system or components thereof disclosed herein can be delivered in one or more ways. This disclosure also provides vectors for packaging the polynucleotides of this disclosure, which encode transcription factors and their portions, DRDs, or payload constructs and combinations thereof. The vectors of this disclosure can also be used to deliver the packaged polynucleotides to cells, local tissue sites, or subjects. These vectors can 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 that can be used as vectors include, but are not limited to, adenoviruses, adeno-associated viruses (AAV), alphaviruses, flaviviruses, herpesviruses, measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease virus (NDV), poxviruses, and microRNA viruses. In one embodiment, the viral vector is selected from lentiviral vectors, gamma retroviral vectors, adeno-associated virus (AAV) vectors, adenoviral vectors, and herpesvirus vectors.

[0385] Generally, a vector contains a replication origin that functions in at least one organism, a promoter sequence and a suitable restriction endonuclease site, as well as one or more selective markers, such as drug resistance genes.

[0386] In some implementations, the recombinant expression vector may contain regulatory sequences, such as transcription and translation start and stop codons, which are specific to the host cell type to which the vector is to be introduced.

[0387] In some embodiments, the carriers of this disclosure may comprise one or more effective loads taught herein, wherein two or more effective loads may be included in a ligand reaction. In this case, the two or more effective loads are simultaneously modulated by the same ligand or reactive agent.

[0388] Lentiviral agents / particles

[0389] In some implementations, lentiviral agents / particles can be used as a delivery method. Lentivirals are a subgroup of viruses in the family Retroviridae, so named because their viral RNA genome needs to be reverse transcribed into DNA before integration into the host genome. Therefore, the most important characteristic of lentiviral agents / particles is the integration of their genetic material into the genome of the target / host cell. Some examples of lentiviruses include human immunodeficiency virus (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi, and caprine arthritis encephalitis virus (CAEV).

[0390] Typically, the lentiviral particles that constitute gene delivery agents are themselves defective in replication (also known as "self-inactivating"). Lentivirals are able to infect both dividing and non-dividing cells via entry mechanisms that cross the intact host nuclear envelope. Recombinant lentiviral agents / particles have been produced by multiple attenuation of HIV virulence genes, for example, by deleting genes Env, Vif, Vpr, Vpu, Nef, and Tat, thereby making the vector biologically safe. Accordingly, for example, lentiviral vectors derived from HIV-1 / HIV-2 can mediate the efficient delivery, integration, and long-term expression of transgenes in non-dividing cells.

[0391] Lentiviral particles can be generated by co-expressing viral packaging elements and the vector genome itself in production cells such as human HEK293T cells. These elements are typically provided as three or four separate plasmids. Production cells are co-transfected with plasmids encoding lentiviral components and plasmids containing foreign transgenes to be transferred to target cells, i.e., the medium itself (also called the transfer vector), said lentiviral components including the viral core components (i.e., structural proteins) and enzyme components, as well as envelope proteins (called the packaging system). Generally, the plasmid or vector is included in the production cell line. The plasmid / vector is introduced into the production cell line by transfection, transduction, or infection. Methods of transfection, transduction, or infection are well known to those skilled in the art. As a non-limiting example, packaging and transfer constructs can generally be introduced into the production cell line with a dominant-selective marker such as neo, DHFR, Gln synthase, or ADA by calcium phosphate transfection, lipid transfection, or electroporation, followed by selection and isolation of clones in the presence of appropriate drugs.

[0392] The production cells generate recombinant viral particles containing foreign genes, such as components of the transcription factor system or their polynucleotides disclosed herein. The recombinant viral particles are recovered from the culture medium and titrated using standard methods used by those skilled in the art. The recombinant lentiviral vector can be used to infect target cells.

[0393] Cells that can be used to produce high-titer lentiviral particles include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol. Ther., 2005, 11:452-459), and FreeStyle cells. TM HEK293 expression system (ThermoFisher, Waltham, MA) and other HEK293T-based production cell lines (e.g., Stewart et al., HumGene 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 are incorporated herein by reference in their entirety).

[0394] In some respects, the envelope protein can be a heterologous envelope protein from other viruses, such as the G protein of vesicular stomatitis virus (VSV G) or the envelope protein of baculovirus gp64.VSV-G glycoproteins may be selected, in particular, from species classified in the genus *VesicularVirula*: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV), and Vesicular stomatitis New Jersey virus (VSNJV), and / or virus strains temporarily classified in the genus *VesicularVirula*, such as Grass carp rhabdovirus and BeAn 157575 virus (BeAn 157575), Botekevirus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Gray Lodge virus (GLOV), Jurona virus (JURY), Klamath virus (KLAV), Kwatta virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tupaia virus The viruses include TUPV ​​(ulcerative rhabdovirus), UDRV (ulcerative disease rhabdovirus), and YBV (Yug Bogdanovac virus).gp64 or other baculovirus env proteins may originate from AcMNPV (Actovira californica), Anagrapha falcifera, Bombyx mori, Choristoneura fumiferana, Orgyia pseudotsugata, Epiphyas postvittana, Hyphantria cunea, Galleria mellonella, Dhori virus, Thogoto virus, Antheraeapemyi, or Batken virus. In some respects, the envelope protein can be RD114, RD115, or a glycoprotein derived from gibbon leukemia virus (GaLV) or baboon retrovirus envelope (BaEV).

[0395] Other elements provided in the lentiviral particle may include a retroviral LTR (long terminal repeat sequence) located at the 5' or 3' end, a retroviral output element, optionally a lentiviral reverse response element (RRE), a promoter or its active portion, and a locus control region (LCR) or its active portion.

[0396] Methods for generating recombinant lentiviral particles are discussed in the art, for example in U.S. Patent Nos. 8,846,385, 7,745,179, 7,629,153, 7,575,924, 7,179,903 and 6,808,905.

[0397] The lentiviral vectors used may be selected from, but are not limited to, pLVX, pLenti, pLenti6, pLJM1, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJM1-EGFP, pULTRA, pInducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionII.

[0398] Adeno-associated virus particles

[0399] The delivery of polynucleotides from any of the transcription factor systems, transcription factor constructs, or payload constructs disclosed herein can be achieved using recombinant adeno-associated virus (rAAV) vectors. Such vectors or viral particles can be designed to utilize capsids of any known serotype or combinations of serotype capsids.

[0400] AAV vectors include not only single-stranded vectors but also self-complementary AAV vectors (scAAV). scAAV vectors contain DNA annealed together to form a double-stranded vector genome. By skipping the synthesis of the second strand, scAAV allows for rapid expression in cells.

[0401] The rAAV vector can be manufactured using 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.

[0402] Transcription factor constructs and payload constructs can be encoded in one or more viral genomes to be packaged in the AAV capsid taught in this paper.

[0403] In addition to at least one or two ITRs (inverted terminal repeats), such vectors or viral genomes may also include 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, filler sequences, etc.

[0404] The transcription factor constructs or payload constructs disclosed herein can be administered in the form of one or more or independent AAV particles.

[0405] In some implementations, the transcription factor system construct can be administered in the form of one or more AAV particles. In some implementations, more than one transcription factor system construct can be encoded in the viral genome.

[0406] Retroviral vector / particle (γ-retroviral vector)

[0407] In some embodiments, retroviral vectors / particles can be used to deliver the transcription factor systems, transcription factor constructs, or payload constructs disclosed herein. Retroviral vectors (RVs) allow for the permanent integration of transgenes into target cells. In addition to lentiviral vectors based on compound HIV-1 / 2, simple gamma-retrovirus-based retroviral vectors have been widely used for delivering therapeutic genes and have clinically proven to be among the most efficient and powerful gene delivery systems capable of transducing multiple cell types. Example species of gamma-retroviruses include murine leukemia virus (MLV) and feline leukemia virus (FeLV).

[0408] In some implementations, gamma retrovirus vectors derived from mammalian gamma retroviruses such as murine leukemia virus (MLV) are recombinant. The MLV family of gamma retroviruses includes monotropy, bitropy, heterotropy, and polytropy subfamilies. Monotropy viruses can only infect mouse cells using the mCAT-1 receptor. Examples of monotropy viruses are Moloney MLV and AKV. Bitropy viruses infect mice, humans, and other species via the Pit-2 receptor. An example of a bitropy virus is 4070A virus. Heterotropy and polytropy viruses utilize the same (Xpr1) receptor, but their species affinity differs. Heterotropy viruses such as NZB-9-1 infect humans and other species but not rodents, while polytropy viruses such as focal formation virus (MCF) infect rodents, humans, and other species.

[0409] Gamma-retroviral vectors can be generated in packaging cells by co-transfecting cells with several plasmids, including a plasmid encoding a retroviral structural and enzyme (gag-pol) polyprotein, a plasmid encoding an envelope (env) protein, and a plasmid encoding a vector mRNA containing a polynucleotide encoding the composition disclosed herein, which will be packaged in newly formed viral particles.

[0410] In some respects, recombinant gamma-retroviral vectors are pseudotyped using envelope proteins derived from other viruses. The envelope glycoprotein is incorporated into the outer lipid layer of the viral particle, thereby increasing / altering cell tropism. In some respects, the envelope protein can be RD114, RD115, or a glycoprotein derived from gibbon leukemia virus (GaLV) or baboon retrovirus envelope glycoprotein (BaEV).

[0411] In some embodiments, the recombinant gamma-retroviral vector is a self-inactivating (SIN) gamma-retroviral vector. The vector cannot replicate. The SIN vector may have a deletion within the 3'U3 region, which initially contains enhancer / promoter activity. Furthermore, the 5'U3 region may be replaced with a strong promoter derived from cytomegalovirus or RSV (required in packaging cell lines) or a selected internal promoter and / or enhancer element. The internal promoter can be selected according to the specific requirements of gene expression desired for the particular purpose of this disclosure.

[0412] In some implementations, polynucleotides of transcription factor systems, transcription factor constructs, or payload constructs are inserted into the recombinant viral genome. Other components of the viral mRNA of the recombinant gamma-retroviral vector can be modified by inserting or removing naturally occurring sequences (e.g., inserting IRES, inserting heterologous polynucleotides encoding polypeptides of interest or repressive nucleic acids, shuffling of more effective promoters from different retroviruses or viruses to replace the wild-type promoter, etc.). In some instances, the recombinant gamma-retroviral vector may contain modified packaging signals and / or primer binding sites (PBS), and / or 5'-enhancer / promoter element elements in the U3 region of the 5'-long terminal repeat (LTR), and / or modified 3'-SIN elements in the U3 region of the 3'-LTR. These modifications may increase titer and infectivity.

[0413] Oncolytic virus vector

[0414] In some embodiments, the polynucleotides of this disclosure can 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 a vaccine virus. Oncolytic viruses can be naturally occurring or can be genetically modified viruses, such as oncolytic adenoviruses and oncolytic herpesviruses.

[0415] In some embodiments, the oncolytic vaccine virus may include viral particles of a thymidine kinase (TK) deficient, granulocyte-macrophage (GM) colony-stimulating factor (CSF)-expressing, replicative vaccinia virus vector sufficient to induce oncolysis of cells in a tumor; see, for example, U.S. Patent No. 9,226,977.

[0416] Messenger RNA (mRNA)

[0417] In some embodiments, the transcription factor system, transcription factor construct, or payload construct of this disclosure may be designed in the form of messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and is capable of translation to produce, in vitro, in vivo, in situ, or ex vivo, the polypeptide of interest. Such mRNA molecules may have any structural components or features of any mRNA molecule taught in International Application No. PCT / US2013 / 030062.

[0418] In some implementations, the transcription factor system or its components may be engineered to be in the form of self-amplifying RNA. As used herein, “self-amplifying RNA” refers to an RNA molecule that can replicate in the host, resulting in an increase in the amount of RNA and the protein encoded by that RNA. Such self-amplifying RNA may have any of the structural features or components of RNA taught in International Patent Application Publication No. WO2011005799.

[0419] Dosage

[0420] This disclosure provides methods for administering components or compositions of any one or more transcription factor systems to a subject in need. These can be administered to the subject in any amount and via any route of administration for the effective prevention or treatment of diseases, conditions and / or disorders (e.g., diseases, conditions and / or disorders associated with cancer or autoimmune diseases) or for imaging. The exact amount required will vary depending on the subject's species, age and general condition, the severity of the disease, the specific composition, its administration method, its mode of activity, etc.

[0421] The compositions according to this disclosure are typically formulated in dosage units to facilitate administration and ensure uniformity of dosage. However, it should be understood that the total daily dosage of the compositions of this disclosure will be determined by the attending physician within the bounds of reasonable medical judgment. For any given subject, the specific therapeutic, prophylactic, or appropriate imaging dose level will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field.

[0422] In some embodiments, the compositions of this disclosure can be used in cancer immunotherapy at different doses to avoid T-cell exhaustion, prevent cytokine release syndrome, and minimize immunotherapy-related toxicities. For example, low doses of the compositions of this disclosure can be used as initial treatment for patients with a high tumor burden, while patients with a low tumor burden can be treated with high and repeated doses of the compositions of this disclosure to ensure minimal tumor antigen load is identified. In another case, the compositions of this disclosure can be delivered pulsatilely to reduce tetanic T-cell signaling and enhance persistence in vivo. In some aspects, toxicity can be minimized by initially using a low dose of the compositions of this disclosure, followed by a high dose. The dose can be altered if serum markers such as ferritin, serum C-reactive protein, IL6, IFN-γ, and TNF-α are elevated.

[0423] In some embodiments, neurotoxicity may be associated with CAR or TIL therapy. This neurotoxicity may be related to CD19-CAR. The toxicity may be caused by excessive T cell infiltration into the brain. In some embodiments, neurotoxicity can be mitigated by preventing T cells from crossing the blood-brain barrier. This can be achieved by targeting gene deletion of an endogenous α-4 integrin inhibitor, such as Tysabri / natalizumab, which is also used in this disclosure.

[0424] This document also provides methods for administering the ligands or DRD ligands according to this disclosure to subjects in need. In some embodiments, the ligand is selected from acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP). The ligand can be administered to the subject or cells using any amount and any route of administration that effectively modulates the transcription factor system, DRD, or payload of this disclosure. In some embodiments, ACZ can be used with hCA2 DRD, methotrexate can be used with hDHFRDRD, and trimethoprim can be used with ecDHFR DRD. The exact amount required will vary depending on the subject's species, age and general condition, disease severity, the specific composition, its administration mode, its mode of activity, etc. The subject can be human, mammal, or animal. The compositions according to this disclosure are typically formulated in unit dosage forms to facilitate administration and uniformity of dosage. However, it should be understood that the total daily dose of the compositions of this disclosure will be determined by the attending physician within the bounds of reasonable medical judgment. In some embodiments, the ligand according to this disclosure can be delivered at doses sufficient to accommodate the subject's weight, such as about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, or about 0.1 mg / kg per day. Dosage levels of approximately 40 mg / kg, approximately 0.5 mg / kg, approximately 30 mg / kg, approximately 0.01 mg / kg, approximately 10 mg / kg, approximately 0.1 mg / kg, approximately 10 mg / kg, approximately 25 mg / kg, approximately 10 mg / kg, approximately 100 mg / kg, approximately 50 mg / kg, approximately 500 mg / kg, or approximately 100 mg / kg to approximately 1000 mg / kg may be administered once or more daily to achieve the desired effect. In some implementations, the dosage level, based on the subject's weight, can be 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg, 150 mg / kg, 160 mg / kg, 170 mg / kg, 180 mg / kg, 190 mg / kg, or mg / kg daily or once or multiple times daily to achieve the desired effect.

[0425] This disclosure provides a method for delivering any ligand described herein to cells or tissues, the method comprising contacting the cells or tissues with the ligand and can be performed in vitro, ex vivo, or in vivo. In some embodiments, the ligands according to this disclosure can be administered to cells at dose levels sufficient to deliver about 1 nM to about 10 nM, about 5 nM to about 50 nM, about 10 nM to about 100 nM, about 50 nM to about 500 nM, about 100 nM to about 1000 nM, about 1 μM to about 10 μM, about 5 μM to about 50 μM, about 10 μM to about 100 μM, about 25 μM to about 250 μM, and about 50 μM to about 500 μM. In some embodiments, the ligand may be administered to cells at doses selected from, but not limited to, the following: 0.00064 μM, 0.0032 μM, 0.016 μM, 0.08 μM, 0.4 μM, 1 μM, 2 μM, 10 μM, 50 μM, 75 μM, 100 μM, 150 μM, 175 μM, 200 μM, and 250 μM.

[0426] The desired dose of the ligands disclosed herein can be delivered once, three times a day, twice a day, once a day, every other day, every three days, once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, the desired dose can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When using multiple administrations, a split dosing regimen as described herein can be used. As used herein, “fractionated dosing” means dividing a “single unit dose” or total daily dose into two or more doses, such as administering a “single unit dose” in two or more separate administrations. As used herein, “single unit dose” refers to the dose of any therapeutic agent administered in a single dose / one-time / single route / single point of contact, i.e., a single administration event. The desired dose of the ligands disclosed herein can 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 agent administered over a period of time at a set frequency. As used herein, “continuous flow” refers to a dose of a therapeutic agent administered continuously over a period of time via a single route / single point of contact, i.e., a continuous administration event. The total daily dose, i.e., the amount given or prescribed over 24 hours, can be administered by any of these methods, or by a combination of these methods, or by any other method suitable for drug administration.

[0427] application

[0428] In some embodiments, the composition for cancer immunotherapy or treatment of autoimmune diseases can be administered ex vivo to cells and subsequently given to a subject. In other embodiments, the cells are selected from B cells, T cells, natural killer cells (NK cells), or tumor-infiltrating lymphocytes (TILs). Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods for isolating cytotoxic T cells are described in U.S. Patent Nos. 6,805,861 and 6,531,451. The isolation of NK cells is described in U.S. Patent No. 7,435,596.

[0429] In some embodiments, depending on the nature of the cells, they can be introduced into a host organism, such as a mammal, in a variety of ways, including injection, transfusion, infusion, local infusion, or implantation. In some aspects, the cells of this disclosure can be introduced into tumor sites. The number of cells used depends on a variety of factors, including the purpose of introduction, cell lifespan, the protocol used (e.g., number of administrations), cell doubling capacity, etc. The cells can be in physiologically acceptable culture media.

[0430] In some embodiments, the cells of this disclosure can be administered to a subject suffering from a disease or ailment in multiple doses. Administration generally achieves improvement and / or treatment or prevention of one or more symptoms or clinical conditions of cancer or its clinical condition or symptoms.

[0431] In some embodiments, the composition for immunotherapy or treatment of autoimmune diseases can be administered in vivo. In some embodiments, the polynucleotide of this disclosure, comprising the transcription factor system, payload, and composition, can be delivered in vivo to a subject via gene therapy.

[0432] Delivery route

[0433] The pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, payloads, carriers, and cells disclosed herein can be administered via any route to achieve therapeutic efficacy. These include, but are not limited to, enteric (to the intestines), gastrointestinal tract, epidural (to the dura mater), oral (via the mouth), transdermal, peridural space, intracerebral (to the brain), intravenous (to the ventricles), epidermal (applied to the skin), intradermal (to the skin itself), subcutaneous (below the skin), nasal (administered via the nose), intravenous (to the vein), intravenous bolus, intravenous infusion, intra-arterial (to the artery), intramuscular (to the muscle), intracardiac (to the heart), intraosseous infusion (to the bone marrow), intrathecal (to the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (via the eye), intracavitary injection (to the pathological cavity), and intracavitary (to the eye). Application methods include: penile root, vaginal, intrauterine, extraamniotic, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), vaginal, inhalation (nasal), sublingual, sublipal, enema, eye drops (applied to the conjunctiva), ear drops, auricle (applied to the ear or via the ear), cheek (applied to the cheek), conjunctiva, skin, teeth (applied to one or more teeth), electroosmosis, intracervical, intrasinus, intratrachea, in vitro, hemodialysis, infiltration, interstitium, intraperitoneum, intraamniotic, intraarticular, intrabile duct, intrabronchus, intrabursa, intracartilage, intracochlear, intracaudal, intracerebellomedullary cistern, intracorneal, and intracoronal. Intracoronary artery (within the coronary artery), corpus cavernosum (within the expandable space of the corpus cavernosum), intervertebral disc (within the intervertebral disc), duct (within the glandular duct), duodenum (within the duodenum), dura mater (within or below the dura mater), epidermis (applied to the epidermis), esophagus (applied to the esophagus), stomach (within the stomach), gingiva (within the gingiva), ileum (within the ileum), lesion (within the lesion or directly introduced into the lesion), lumen (within the lumen), lymphatic vessel (within the lymphatic system), medullary cavity (within the medullary cavity), meninges (within the meninges), myocardium (within the myocardium), eye (within the eye), ovary (within the ovary), pericardium (within the pericardium). Intrapleural (within the pleura), intraprostatic (within the prostate), intrapulmonary (within the lung or its bronchi), intrasinus (within the sinuses or periorbital sinuses), intraspinal (within the spine), intrasynovial (within the synovial cavity of a joint), intratendinous (within the tendon), intratesticular (within the testis), intrasheath (within the cerebrospinal fluid at any level of the brain-spinal axis), intrapleural (within the pleural cavity), intraductal (within the tubes of an organ), intratumoral (within the tumor), tympanic cavity (within the middle ear), intravascular (within one or more blood vessels), intraventricular (within the ventricular cavity), iontophoresis (using an electric current that migrates soluble salt ions into body tissues), irrigation (cleansing or flushing open wounds or body cavities), laryngeal (directly applied to the larynx).Nasogastric (through the nose and into the stomach), occlusive dressing technique (surface application followed by covering with a dressing to seal the area), ophthalmic (application to the external eye), oropharyngeal (direct application to the mouth and pharynx), extra-gastric, percutaneous, periarticular, peridural, perineurial, periodontal, rectal, respiratory (application via oral or nasal inhalation to the respiratory tract for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, surface, transplacental (through or across the placenta), transtracheal (through the tracheal wall), transtympanic (across or through the tympanic cavity), ureter (application to the ureter), urethra (application to the urethra), vagina, sacral block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photodissociation, or spinal cord.

[0434] Parenteral and injectable administration

[0435] In some embodiments, the pharmaceutical compositions, transcription factor systems, nucleic acids, polynucleotides, payloads, carriers, and cells of this disclosure can be administered parenterally. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active compound, the liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers. In some embodiments for parenteral administration, the composition is mixed with a solubilizer, such as... Alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof. In other embodiments, surfactants such as hydroxypropyl cellulose are included.

[0436] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, and / or emulsions in non-toxic, parenteral-acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable mediators and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile, non-volatile oils are typically used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid can also be used in the preparation of injectable solutions.

[0437] Injectable formulations may be sterilized, for example, by filtering through a bacterial trap and / or by incorporating a sterilizing agent in the form of a sterile solid composition, which may be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0438] Detectable reagents and labels

[0439] The transcription factor systems, nucleic acids, polynucleotides, payloads, vectors, and cells disclosed herein can be linked to or combined with one or more radioactive or detectable reagents.

[0440] These reagents include various small organic molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and jellyfish luminescent proteins), chemiluminescent materials, and radioactive materials (e.g., 18 F, 67 Ga、 81m Kr、 82 Rb、 111 In、 123 I, 133 Xe, 201 Tl、 125 I, 35 S, 14 C 3 H or 99m Tc (e.g., present as high technetium salts (technetium salt (VII), TcO4) - (Forms), and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), single-crystal iron oxide nanoparticles (MION) and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast agents (iohexol), microbubbles or perfluorocarbons).

[0441] In some embodiments, the detectable reagent may be an undetectable precursor that becomes detectable upon activation (e.g., a fluorescent tetrazine-fluorophore construct (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or an enzyme-activated fluorescent agent (e.g., (VisEn Medical))) In vitro assays that can use enzyme-labeled compositions include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation assay, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.

[0442] Applications and uses

[0443] The transcription factor systems, constructs, ligands, or compositions disclosed herein can be used in a variety of applications, including but not limited to treatment, diagnosis and prognosis, bioengineering, bioprocessing, biomanufacturing, research reagents, metabolomics, gene expression, enzyme substitutes, etc.

[0444] This disclosure provides a method of administering a composition, such as a pharmaceutical composition comprising one or more components of a transcription factor system, to a subject in need.

[0445] While several uses may not involve medical treatment, such as generating cell lines and reagents for scientific research, one use involves administering the compositions of this disclosure to generate in vivo gene therapy or modified cells for adoptive cell therapy, such as for treating cancer, autoimmune diseases, and other diseases. Illustrative methods for the medical treatment or prevention of a disease, ailment, or condition in a subject in need may include the following steps: (a) providing a cell population (human cells, animal cells, primary cells, or cell cultures, including autologous cells, allogeneic cells, or syngeneic cells); (b) introducing at least one nucleic acid molecule into at least one cell in the cell population, wherein the at least one nucleic acid molecule comprises: (i) a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain that binds to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD), wherein the transcription factor activation domain and / or the transcription factor DNA-binding domain bind to the drug-responsive domain. (i) a domain operatively linked to the DRD; and (ii) a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest for treating the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter comprising a specific polynucleotide binding site; (c) delivery of the cell to the subject; and (d) administration of a ligand to the subject to stabilize the DRD sufficiently to express an amount of transcription factor activation domain and transcription factor DNA binding domain that form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the protein of interest in the cell; wherein the expression of the protein of interest is regulated by the presence of the ligand in the subject, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

[0446] In the above methods, the protein of interest can be used to improve, cure, prevent or alleviate one or more symptoms of a disease, ailment or condition.

[0447] The compositions disclosed herein may be administered to a subject in any amount and via any route of administration for the effective prevention or treatment of diseases, conditions and / or ailments (e.g., diseases, conditions and / or ailments associated with cancer or autoimmune diseases and other diseases) or for imaging. The exact amount required will vary depending on the subject’s species, age and general condition, severity of disease, the specific composition, its administration method, mode of activity, etc.

[0448] The compositions according to this disclosure are typically formulated in dosage units to facilitate administration and ensure uniformity of dosage. However, it should be understood that the total daily dosage of the compositions of this disclosure will be determined by the attending physician within the bounds of reasonable medical judgment. For any given subject, the specific therapeutic, prophylactic, or appropriate imaging dose level will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field.

[0449] This document also provides a method for administering one or more stabilizing ligands (as used herein, a ligand stabilizing a DRD may be referred to as a stabilizing ligand or simply a ligand, and it should be understood that the ligand effectively stabilizes the DRD used in the transcription factor system according to this disclosure) to a subject in need. The ligand can be administered to a subject or cell in any amount and via any route of administration that effectively modulates the expression level of the transcription factor of this disclosure in cells containing the transcription factor system. The exact amount of stabilizing ligand required will vary depending on the subject's species, age and general condition, disease severity, specific composition, administration mode, mode of activity, etc. The subject may be a human, mammal, or animal.

[0450] Therapeutic uses

[0451] Cancer immunotherapy

[0452] Cancer immunotherapy aims to induce or restore the immune system's responsiveness to cancer. Significant advances in immunotherapy research have led to the development of various strategies, broadly categorized as active and passive immunotherapy. Generally, these strategies can be used to directly kill cancer cells or counteract the immunosuppressive tumor microenvironment. Active immunotherapy aims to induce an endogenous, durable tumor antigen-specific immune response. This response can be further enhanced through non-specific stimulation by immune response modulators such as cytokines. In contrast, passive immunotherapy involves administering effector immune molecules, such as tumor antigen-specific cytotoxic T cells or antibodies, to the host. This approach is short-lived and requires multiple applications.

[0453] Despite significant progress, the efficacy of current immunotherapy strategies is limited by associated toxicities. These are often related to the narrow therapeutic window associated with immunotherapy, partly because therapeutic doses need to be pushed to the edge of potentially fatal toxicities to achieve clinically meaningful therapeutic effects. Furthermore, the dosage in vivo often increases due to the unpredictable proliferation of adopted immune cells within the patient's body.

[0454] A major risk involved in immunotherapy is the on-target but off-tumor side effect caused by T cell activation in response to normal tissue expression of tumor-associated antigens (TAAs). Clinical trials using T cells expressing T cell receptors targeting specific TAAs have reported rashes, colitis, and hearing loss in response to immunotherapy.

[0455] When tumor cells are killed in response to immunotherapy, the therapy can also produce on-target and tumor-targeting toxicity. Adverse effects include tumor lysis syndrome, cytokine release syndrome, and associated macrophage activation syndrome. Importantly, these adverse effects can occur during tumor destruction, so even successful tumor-targeting immunotherapies can lead to toxicity. Therefore, methods for controlling immunotherapy through immunomodulation are particularly needed, as they have the potential to reduce toxicity and maximize efficacy.

[0456] This disclosure provides systems, compositions, immunotherapeutic agents, and methods for use in immunotherapy. These compositions provide modulotropic regulation of gene expression and function in immunotherapies, for example, for the prevention and treatment of cancer.

[0457] On the one hand, the systems, compositions, immunotherapeutic agents, and other components of this disclosure can be controlled by individually added stable ligands, which provides significant flexibility for modulating cancer immunotherapy. Furthermore, the systems, compositions, and methods of this disclosure can also be combined with therapeutic agents such as chemotherapeutic agents, small molecules, gene therapies, and antibodies to prevent and / or treat diseases, such as cancer.

[0458] The tunable properties of the systems and compositions disclosed herein have the potential to enhance the potency and duration of immunotherapy efficacy. Using the compositions of this disclosure to reversibly silence the bioactivity of adoptive transfer cells allows for maximizing the potential of cell therapy without irreversibly killing and terminating the therapy.

[0459] This disclosure provides a method for finely modulating immunotherapy after administration to patients. This further improves the safety and efficacy of immunotherapy and increases the population of subjects who may benefit from it.

[0460] In some embodiments, the immune cells of this disclosure may be T cells modified to express a payload or protein of interest, such as an antigen-specific T cell receptor (TCR) or an antigen-specific chimeric antigen receptor (CAR) as taught herein (referred to as CAR T cells). Thus, at least one polynucleotide encoding a protein of interest, such as the CAR system (or TCR) described herein, or a vector containing such polynucleotide, is introduced into the T cell. T cells expressing a CAR or TCR bind to a specific antigen via the extracellular targeting portion of the CAR or TCR, thereby transmitting a signal to the T cell through an intracellular signal transduction domain, and thus activating the T cell. Activated CAR T cells alter their behavior, including releasing cytotoxic cytokines (such as tumor necrosis factor and lymphotoxin), increasing cell proliferation, and altering cell surface molecules. These changes disrupt target cells expressing the antigen recognized by the CAR or TCR. Furthermore, the release of cytokines or the changes in cell surface molecules stimulate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0461] The CAR introduced into T cells can be a first-generation CAR comprising only the intracellular signaling domain from the TCR CD3ζ, a second-generation CAR comprising both the intracellular signaling domain and the co-stimulatory signaling domain from the TCR CD3ζ, a third-generation CAR comprising the intracellular signaling domain and two or more co-stimulatory signaling domains from the TCR CD3ζ, a split CAR system, or an on / off CAR system. In one embodiment, the expression of the CAR or TCR is controlled by a transcription factor, wherein the transcription factor or a component thereof is operatively linked to the DRD, which results in little or no accumulation of the transcription factor in the absence of a stable ligand. The payload has a polynucleotide binding sequence specific to the transcription factor or a component thereof, thus producing almost no protein of interest in the absence of a stable ligand. When a stable ligand is applied to a cell containing a transcription factor system, the transcription factor is protected from degradation upon coupling with the DRD, and then the transcription factor binds to its homologous polynucleotide binding sequence adjacent to the protein of interest, followed by transcription. The transcribed mRNA is then translated to produce the polypeptide / protein of interest. In some exemplary embodiments, the presence or absence of a DRD stabilizing ligand is used to modulate the expression of CAR or TCR in transduced T cells or NK cells.

[0462] In some embodiments, the CAR T cells of this disclosure may be further modified to express another, two, three, or more immunotherapeutic agents. The immunotherapeutic agents may be another CAR or TCR specific to different target molecules; cytokines such as IL2, IL12, IL15, and IL18, or cytokine receptors such as IL15Ra; chimeric switch receptors that convert inhibitory signals into stimulatory signals; homing receptors that guide adopted cells to target sites such as tumor tissue; agents that optimize the metabolism of immune cells; or safety switch genes (e.g., suicide genes) that kill activated T cells when a serious event is observed after adoptive cell transfer or when the transferred immune cells are no longer needed. These molecules may be contained in the same construct or in separate constructs.

[0463] In one embodiment, the CAR T cells (including TCR T cells) of this disclosure can be “armed” CAR T cells, which are transfected or transduced with one or more components of a transcription factor system containing a CAR payload and encoding cytokines under the control of the same or different transcription factors operably linked to the same or different DRDs. Inducible or constitutively secreted active cytokines further arm the CAR T cells to improve efficacy and durability. Hereinafter, such CAR T cells are also referred to as “armored CAR T cells.” The “armoring” molecules can be selected based on the tumor microenvironment and other elements of the innate and adaptive immune system. In some embodiments, the molecule can be a stimulating factor, such as IL2, IL12, IL15, IL18, type I IFN, CD40L, and 4-1BBL, which have been shown to further enhance the efficacy and durability of CAR T cells in confronting a hostile tumor microenvironment through various mechanisms.

[0464] Chimeric antigen receptor-engineered T-cell (CAR-T) therapy has not yet been successfully applied to solid tumors. Enhancing CAR-T cell function and selectively delivering payloads to solid tumors are key strategies for achieving effective CAR-T therapy targeting solid tumors. In one embodiment, the payload or protein of interest may include interleukin-12 (IL12), which can be used to enhance the efficacy of CAR-T cells, particularly because of its potential to remodel the tumor microenvironment. IL12 has previously been shown in preclinical and clinical models to effectively enhance the efficacy of CAR or TCR-modified T cells as well as tumor-infiltrating lymphocytes (TILs). However, constitutive production of IL12 may compromise safety and / or efficacy; therefore, local delivery of cytokines may be a preferred approach if needed. In some embodiments, the transcription factor system of this disclosure or components thereof can be used to exogenously control IL12 expression to enable the use of IL12 in adoptive cell therapy.

[0465] In some embodiments, the transcription factor regulatory system of this disclosure can be used to regulate the expression of payloads such as Flexi IL12 (or other IL12 constructs, such as membrane-bound IL12) in transformed immune cells to improve CAR efficacy, particularly in solid tumor settings, by providing controlled local signaling for tumor microenvironment remodeling and epitope diffusion. With the addition of a DRD-specific stabilizing ligand, the transcription factor regulation described herein also provides rapid, dose-dependent, and localized IL12 production.

[0466] In some respects, the armed CAR T cells of this disclosure are modified to express CD19CAR and a payload such as IL12, the expression of which is regulated using the transcription factor system or composition of this disclosure. Upon CAR-mediated activation in a tumor, such T cells release inducible IL12, thereby enhancing T cell activation and attracting and activating innate immune cells to eliminate CD19-positive cancer cells.

[0467] In one embodiment, the T cells of this disclosure may be modified to incorporate into a transcription factor system comprising a CAR payload encoded by the transcription factor system or its components and a nucleic acid sequence encoding a suicide gene.

[0468] In one embodiment, the CAR T cells (including TCR T cells) of this disclosure can be transfected or transduced using one or more components of a transcription factor system containing cytokines and safety switch genes (e.g., suicide genes). The suicide gene can be an inducible caspase, such as caspase 9, which induces apoptosis, when activated by an extracellular stable ligand of a DRD encoded by the transcription factor system. If necessary, this induced apoptosis eliminates metastatic cells to reduce the risk of direct toxicity and uncontrolled cell proliferation.

[0469] In one embodiment, transcription factor systems and components thereof that modulate the expression level and activity of any of the said payloads or proteins of interest (which may be used interchangeably) can be used in immunotherapy. As a non-limiting example, immunotherapeutic agents can be antibodies and fragments thereof and variants, cancer-specific T-cell receptors (TCRs) and variants thereof, antitumor-specific chimeric antigen receptors (CARs), chimeric switch receptors, inhibitors of co-inhibitory receptors or ligands, agonists of co-stimulatory receptors and ligands, cytokines, chemokines, cytokine receptors, chemokine receptors, soluble growth factors, metabolic factors, suicide genes, homing receptors, or any agent that induces an immune response in cells and a subject.

[0470] In some embodiments, the composition for inducing or inhibiting an immune response may comprise one or more components of a transcription factor system, or one or more polypeptides encoded by the transcription factor system. In some embodiments, the transcription factor system may comprise: a first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain; a second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site; and a third nucleic acid sequence encoding a drug-responsive domain (DRD); ​​wherein at least one of the transcription factor activation domain, the transcription factor DNA-binding domain, and a combination of the transcription factor activation domain and the transcription factor DNA-binding domain is operatively linked to the DRD; and a second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest, the fourth nucleic acid sequence being operatively linked to an inducible promoter comprising the specific polynucleotide binding site; wherein the transcription factor activation domain interacts with the transcription factor DNA-binding domain to form a transcription factor; and wherein the binding of the transcription factor to the specific polynucleotide binding site is required for the transcription factor to regulate the transcription of the fourth nucleic acid sequence.

[0471] On the one hand, the effective payload can be an immunotherapeutic agent.

[0472] In some embodiments, the transcription factor systems and compositions of this disclosure relate to the transcriptional regulation of protein (protein of interest or payload) function, including, for example, antitumor immune responses of immunotherapeutic agents. In some embodiments, the immunotherapeutic agent may include cytokines, chemokines, antibodies, integrins, integrins, membrane proteins, extracellular proteins, which may be used to upregulate or improve the function of one or more immune cell types, or downregulate the activity of one or more immune cell types. In various embodiments, immunotherapeutic agents that may be used to treat diseases, disorders, or conditions may include cytokines, such as interleukins. In various embodiments, the transcription factor system provides a protein of interest or payload comprising interleukins, such as IL-2, IL-6, IL-12, IL-15, IL-18, and other immunotherapeutic agents that promote or upregulate the lifespan and activity of one or more immune cell types that may be used to treat diseases, disorders, or conditions or symptoms associated with any of these diseases, disorders, or conditions.

[0473] In some implementations, cells genetically modified to encode and express at least one transcription factor can be used in adoptive cell therapy (ACT, also known as “adoptive cell transfer”), where the at least one transcription factor is operable to allow transcription of a protein of interest (immunotherapy agent) linked to a transcription factor polynucleotide binding site. As used herein, adoptive cell transfer refers to the administration of immune cells (derived from a host, allogeneic, or genetically modified) with direct anticancer activity. ACT has shown promise in clinical applications for malignant and infectious diseases. For example, genetically engineered T cells that 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 ACTs using autologous lymphocytes genetically modified to express anti-tumor T-cell receptors have been used to treat metastatic melanoma (Rosenberg and Dudley, Curr. Opin. Immunol. 2009, 21:233-240).

[0474] According to this disclosure, one or more components of a transcription factor system can be used to develop and administer cell therapies, such as adoptive cell therapy. In some embodiments, one or more components of a transcription factor system can be used in cell therapy to achieve CAR therapy; in the manipulation or regulation of TILs; in allogeneic cell therapy; in combination with other lines of treatment (e.g., radiation, cytokines) to encode engineered or modified TCRs, or to enhance T cells other than TCRs (e.g., by introducing cytokine genes, checkpoint inhibitors PD1, CTLA4 genes).

[0475] This document provides a method for adoptive cell therapy. The method includes pretreatment of a subject in need; modulation of immune cells with one or more components of a transcription factor system and / or a composition of this disclosure; administration to the subject engineered immune cells expressing a composition of this disclosure; and successful transplantation of the engineered cells into the subject.

[0476] In some embodiments, the tunable transcription factor expression constructs and compositions of this disclosure can be used to minimize pretreatment regimens associated with adoptive cell therapy. As used herein, “pretreatment” refers to any treatment regimen administered to a subject to improve the outcome of adoptive cell therapy. Pretreatment strategies include, but are not limited to, whole-body irradiation and / or chemotherapy to clear lymph nodes. Adoptive therapy clinical trials without pretreatment have failed to demonstrate any clinical benefit, highlighting the importance of pretreatment in ACT. However, pretreatment can be associated with significant toxicity and limits suitable subject cohorts for ACT. In some cases, immune cells used for ACT can be engineered using the transcription factors described herein to express cytokines such as IL-2, IL-6, IL12, and IL15 as a payload, thereby allowing selective expression of a protein of interest that can be modulated using stable ligands of this disclosure to reduce the need for pretreatment (Pengram et al. (2012) Blood 119(18):4133-41; the contents of which are incorporated herein by reference in their entirety).

[0477] In some embodiments, the immune cells used 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, the immunostimulatory cells used for ACT may be derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In some embodiments, autologous or allogeneic immune cells are used for ACT.

[0478] In some embodiments, the cells used for ACT may be T cells engineered to express CAR, said CAR containing an antigen-binding domain specific to antigens on tumor cells of interest. In some embodiments, the cells used for ACT may be NK cells engineered to express CAR, said CAR containing an antigen-binding domain specific to antigens on tumor cells of interest. In addition to adoptive transfer of genetically modified T cells (e.g., CAR T cells) for immunotherapy, alternative types of CAR-expressing leukocytes, 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. According to this disclosure, the expression level of CAR in T cells and NK cells is regulated and controlled by a small molecule binding to a DRD operatively linked to a transcription factor or a component thereof, which enables selective transcription of CAR in transfected or transduced T cells and NK cells. In this case, the CAR is encoded by a nucleic acid sequence operatively linked to an inducible promoter containing a specific polynucleotide binding site for the transcription factor.

[0479] In some implementations, NK cells engineered to express one or more components of a transcription factor system can be used for ACT. NK cell activation induces perforin / granzyme-dependent apoptosis of target cells. NK cell activation also induces the secretion of cytokines such as IFNγ, TNF-α, and GM-CSF. These cytokines enhance the phagocytic function and antimicrobial activity of macrophages and enhance adaptive immune responses by upregulating antigen presentation by antigen-presenting cells such as dendritic cells (DCs) (reviewed in Vivier et al., Nat. Immunol., 2008, 9(5):503-510).

[0480] Other examples of gene modification may include the introduction of chimeric antigen receptors (CARs) and the downregulation of inhibitory NK cell receptors, such as NKG2A.

[0481] NK cells can also circumvent inhibitory signals that they interact with tumor cells through gene reprogramming. For example, genetically modifying NK cells using CRISPR, ZFN, or TALEN to silence their inhibitory receptors may enhance their anti-tumor capabilities.

[0482] Immune cells can be isolated and expanded in vitro using a variety of methods known in the art. For example, methods for isolating and expanding cytotoxic T cells are described in U.S. Patent Nos. 6,805,861 and 6,531,451; U.S. Patent Publication No. US20160348072A1; and International Patent Publication No. WO2016168595A1; the contents of each are incorporated herein by reference in their entirety. Isolation and expansion of NK cells are described in U.S. Patent Publication No. US20150152387A1, U.S. Patent No. 7,435,596; and Oyer, JL (2016). Cytotherapy. 18(5):653-63; the contents of each are incorporated herein by reference in their entirety. Specifically, primary human NK cells can be expanded in the presence of feeder cells, such as bone marrow cell lines genetically modified to express membrane-bound IL15, IL21, IL12, and 4-1BBL.

[0483] In some cases, immune cell subsets can be enriched for ACT. Methods for enriching immune cells are taught in International Patent Publication No. WO2015039100A1. In another example, cancer-responsive T cells can be enriched using T cells positive for B and T lymphocyte attenuation markers (BTLA), as described in U.S. Patent No. 9,512,401 (the contents of which are incorporated herein by reference in their entirety).

[0484] In some implementations, the immune cells used for ACT can be depleted to select subsets to enhance T cell proliferation. For example, the method taught in U.S. Patent Publication No. US20160298081A1 can be used to deplete Foxp3+ T lymphocytes to minimize the antitumor immune response; the contents of that patent are incorporated herein by reference in their entirety.

[0485] In some embodiments, activation and expansion of T cells for ACT are achieved by antigen stimulation of chimeric antigen receptors (CARs) transiently expressed on the cell surface. This activation method is taught in International Patent No. WO2017015427, the contents of which are incorporated herein by reference in their entirety.

[0486] In some embodiments, immune cells can be activated by antigens associated with antigen-presenting cells (APCs). In some embodiments, APCs can be antigen-specific or non-specific dendritic cells, macrophages, or B cells. APCs in their organs may be autologous or homologous. In some embodiments, APCs can be artificial antigen-presenting cells (aAPCs), such as cell-based aAPCs or cell-free aAPCs. Cell-based aAPCs can be selected from genetically modified allogeneic cells such as human erythroleukemia cells, or xenogeneic cells such as mouse fibroblasts and Drosophila cells. Alternatively, APCs can be cell-free, wherein the antigen or co-stimulatory domain is presented on a synthetic surface, such as latex beads, polystyrene beads, lipid vesicles, or exogenous bodies.

[0487] In some embodiments, the cells of this disclosure, particularly T cells, can be expanded using an artificial cell platform. In one embodiment, mature T cells can be generated using artificial thymus organoids (ATOs) as described in 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 stromal cell lines expressing delta-like canonical notch ligand (DLL1). In this approach, stromal cells are aggregated with hematopoietic stem cells and progenitor cells by centrifugation and deployed at an air-fluid interface on a cell culture chamber to generate organoid cultures. ATO-derived T cells exhibit an immature phenotype, a diverse T cell receptor (TCR) repertoire, and TCR-dependent function.

[0488] In some implementations, adoptive cell therapy is performed via autologous transfer, where cells are derived from the subject in need of treatment and are administered to the same subject after isolation and processing. In other cases, ACT may involve allogeneic transfer, where cells are isolated and / or prepared from a donor subject other than the recipient subject who ultimately receives the cell therapy. The donor and recipient subjects may be genetically identical or similar, or may express the same HLA class or subtype.

[0489] In some embodiments, multiple immunotherapeutic agents introduced into immune cells (e.g., T cells and NK cells) for ACT can be controlled by the same or different transcription factor systems. In one example, each of two payloads, such as a cytokine like IL12 and a CAR construct like CD19CAR, is transcribed by one or more transcription factors on the same or different transcription factor systems, wherein the one or more transcription factors are linked to the same or different DRDs. The payload is transcribed and translated when the DRD is stabilized with a stable ligand specific to the DRD. The expression of IL12 and CD19CAR is regulated using one or more stable ligands. In other embodiments, multiple immunotherapeutic agents introduced into immune cells (e.g., T cells and NK cells) for ACT can be controlled by different transcription factor systems. In one example, a cytokine like IL12 and a CAR construct like CD19CAR are each transcribed by one of two different transcription factors, each operatively linked to a different DRD, and thus can be independently regulated using different stimuli. In another example, a suicide gene and a CAR construct can be transcribed and activated by two different transcription factors.

[0490] After gene regulation using one or more components of the transcription factor system and composition disclosed herein, cells are administered to a subject in need. Methods of administering cells for adoptive cell therapy are known and can be used in conjunction with the methods and compositions provided. For example, adoptive T-cell therapy methods are described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85. See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338; the contents of which are incorporated herein by reference in their entirety.

[0491] In some implementations, immune cells used for ACT can be modified to express one or more immunotherapeutic agents (proteins of interest) that promote immune cell activation, infiltration, expansion, survival, and anti-tumor function. The immunotherapeutic agent can be a second CAR or TCR specific to different target molecules; cytokines or cytokine receptors; chimeric switch receptors that convert inhibitory signals into stimulatory signals; homing receptors that guide adopted cells to target sites such as tumor tissue; agents that optimize the metabolism of immune cells; or safety switch genes (e.g., suicide genes) that kill activated T cells when serious events are observed after adopted cell transfer or when the transferred immune cells are no longer needed.

[0492] In some embodiments, immune cells used for adoptive cell transfer can be genetically manipulated to improve their persistence, cytotoxicity, tumor targeting ability, and ability to hom to disease sites in vivo, with the overall goal of further improving their ability to kill tumors in cancer patients. One example is the introduction of one or more components of the transcription factor system of this disclosure, encoding cytokines such as gamma-cytokines (e.g., IL2 and IL15), into immune cells to promote immune cell proliferation and survival. Transducing cytokine genes (e.g., gamma-cytokines IL2 and IL15) encoded by the transcription factor system into immune cells will enable immune cells (e.g., NK cells) to proliferate without the addition of exogenous cytokines, thereby giving NK cells expressing cytokines enhanced tumor cell cytotoxicity.

[0493] In some implementations, one or more components of the transcription factor system can be utilized to prevent T cell exhaustion. As used herein, “T cell exhaustion” refers to the gradual 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 exhibit low proliferative and cytokine-producing capacity, along with a high rate of apoptosis and high surface expression of multiple inhibitory receptors. T cell activation leading to exhaustion can occur in the presence or absence of antigens.

[0494] In some embodiments, one or more components of the transcription factor system can be used to prevent T cell exhaustion in the context of chimeric antigen receptor-T cell therapy (CAR-T). In this context, exhaustion may, in some cases, be caused by oligomerization of CAR scFvs on the cell surface leading to the continuous activation of the CAR's intracellular domains. As a non-limiting example, the CAR of this disclosure may include scFvs that cannot oligomerize. As another non-limiting example, a CAR that is rapidly internalized and re-expressed after antigen exposure may also be selected to prevent chronic scFv oligomerization on the cell surface. In one embodiment, the framework region of the scFv may be modified to prevent constitutive CAR signaling (Long et al., 2014. Cancer Research. 74(19)S1; the contents of which are incorporated herein by reference in their entirety). One or more components of the transcription factor system of this disclosure may also be used to regulate the surface expression of CARs on the T cell surface to prevent chronic T cell activation. The CAR of this disclosure may also be engineered to minimize exhaustion. As a non-limiting example, the 41-BB signaling domain may be incorporated into the CAR design to improve T cell exhaustion. In some implementations, any of the strategies disclosed by Long HA et al. can be used to prevent exhaustion (Long AH et al. (2015) Nature Medicine 21, 581-590; the contents of which are incorporated herein by reference in their entirety).

[0495] In some embodiments, the tunable properties of the transcription factor system disclosed herein can be used to reverse human T cell exhaustion observed under tetanic CAR signaling. Reversible silencing of the bioactivity of adoptively transferred cells using the compositions of this disclosure can be used to reverse tetanic signaling, which in turn can revitalize T cells. Reversal of exhaustion can be measured by downregulating a variety of exhaustion-associated inhibitory receptors.

[0496] In some embodiments, T cell metabolic pathways can be modified to reduce T cell sensitivity to exhaustion. Metabolic pathways may include, but are not limited to, glycolysis, the urea cycle, the citric acid cycle, β-oxidation, fatty acid biosynthesis, the pentose phosphate pathway, nucleotide biosynthesis, and glycogen metabolism. As a non-limiting example, a payload that reduces the rate of glycolysis can be used to limit or prevent T cell exhaustion (Long et al., Journal for Immunotherapy of Cancer 2013, 1(Supplement 1): P21; the contents of which are incorporated herein by reference in their entirety). In one embodiment, the T cells of this disclosure can be used in combination with glycolysis inhibitors such as 2-deoxyglucose and rapamycin.

[0497] In some embodiments, the payload or protein of interest disclosed herein may be used in combination with an antibody or fragment targeting 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.

[0498] In some embodiments, the compositions of this disclosure can be used to alter the tumor-infiltrating lymphocyte (TIL) population in a subject. In one embodiment, any of the payloads described herein can be used to alter the ratio of CD4-positive cells to CD8-positive cells. In some embodiments, TILs can be sorted in vitro and engineered to express any of the cytokines described herein. The payloads of this disclosure can be used to amplify CD4 and / or CD8 TIL populations to enhance TIL-mediated immune responses.

[0499] Parameters used to improve CAR-T therapy outcomes are described in Finney et al., JCI. 2019; 129(5):2123-2132 (the contents of which are incorporated herein by reference in their entirety). The levels of the biomarker LAG3 (high) / TNF-α (low) in peripheral blood CD8+ T cells at the time of apheresis can also predict subsequent dysfunctional responses in subjects with high antigen loads who do not achieve a complete response lasting more than several weeks. The intrinsic characteristics of T cells as a result of the initiating T cell pool, and the effects of the manufacturing process combined with CD19 antigen-induced activation following adoptive transfer, can also play a role in CAR-T therapy outcomes. The initiating T cell pool may be partially influenced by the timing of apheresis. In one implementation, apheresis can be performed prior to chemotherapy. The cumulative burden of CD19-expressing leukemia and normal B cells evaluated in the bone marrow prior to lymphocyte-clearing chemotherapy may be important in determining CAR-T therapy outcomes. According to Finney et al., increased antigen burden improves CAR-T therapy outcomes. To increase the CD19 antigen burden in the body, subjects can also be infused with amplified subject-derived T cells (also known as T-APCs) that are genetically modified to express CD19.

[0500] In some embodiments, the regulated transcription factor expression constructs, payloads of interest (e.g., immunotherapeutic agents), vectors, cells, and compositions disclosed herein can be used in combination with cancer vaccines.

[0501] In some implementations, cancer vaccines may contain peptides and / or proteins derived from tumor-associated antigens (TAAs). Such strategies can be used to elicit an immune response in a subject, which in some cases may be a cytotoxic T-lymphocyte (CTL) response. Peptides used for cancer vaccines may also be modified to match the mutation profile of the subject. For example, EGFR-derived peptides with mutations matching those found in subjects in need of therapy have been successfully used in lung cancer patients (Li F et al. (2016) Oncoimmunology. Oct 7; 5(12):e1238539; the contents of which are incorporated herein by reference in their entirety).

[0502] In one embodiment, the cancer vaccine of this disclosure may include a hyperagonist-modified peptide ligand (APL) derived from a tumor-associated antigen (TAA). These are mutant peptide ligands that differ from the native peptide sequence by one or more amino acids and activate a specific CTL clone more effectively than the native epitope. These modifications can enable the peptide to bind better to restrictive class I MHC molecules or to interact more favorably with the TCRs of a given subset of tumor-specific CTLs. The APL can be selected using the methods taught in U.S. Patent No. US20160317633A1, the contents of which are incorporated herein by reference in their entirety.

[0503] In some implementations, the effector immune cells genetically modified to encode components of the disclosed transcription factor system and payload can be combined with the biological adjuvants described herein. The dual regulation of CARs with cytokines and ligands separates the kinetic control of target-mediated activation from intrinsic T cell expansion. This dual regulation also minimizes the patient's need for pretreatment regimens. As a non-limiting example, DRD-regulated transcription factors of transcriptional payloads (e.g., CARs, such as CD19CARs) can be combined with cytokines (e.g., IL12) to enhance the antitumor efficacy of CARs (Pegram H.J. et al., Tumor-targeted T cells modified to secrete IL12 eradicate systemictumors without need for prior conditioning. Blood. 2012; 119:4133-41; the contents of which are incorporated herein by reference in their entirety). As another non-limiting example, Merchant et al. combined dendritic cell-based vaccination with recombinant human IL7 to improve outcomes in high-risk pediatric sarcomas (Merchant, MS et al., Adjuvant immunotherapy to Improve Outcome in High-Risk Pediatric Sarcomas. Clin Cancer Res. 2016. 22(13):3182-91; the contents of which are incorporated herein by reference in their entirety).

[0504] In some embodiments, effector immune cells modified to express one or more antigen-specific TCRs or CARs can be combined with compositions disclosed herein, which comprise immunotherapeutic agents that transform an immunosuppressive tumor microenvironment.

[0505] On the one hand, effector immune cells can be combined to express CARs that are specific to different target molecules on the same cell. On the other hand, different immune cells modified to express the same CAR construct, such as NK cells and T cells, can be combined for tumor therapy. For example, T cells modified to express CD19CAR can be combined with NK cells modified to express the same CD19CAR to treat B-cell malignancies.

[0506] In other implementations, immune cells modified to express CAR can be combined with checkpoint blockers.

[0507] In some embodiments, effector immune cells that are genetically modified to express one or more components of a transcription factor system (such as the payload of this disclosure) may be combined with cancer vaccines and other immunotherapeutic agents and adjuvant therapies of this disclosure.

[0508] In some embodiments, the methods of this disclosure may include combining the compositions of this disclosure with other agents (e.g., anticancer agents) effective in treating cancer, infectious diseases, and other immunodeficiency diseases. As used herein, the term "anticancer agent" means any agent capable of negatively affecting a subject's cancer, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the occurrence or number of cancer metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting cancer progression, or prolonging the life of a subject with cancer.

[0509] In some embodiments, the anticancer agent or therapy may be a chemotherapy agent, or a radiotherapy, immunotherapy agent, surgery, or any other therapeutic agent combined with this disclosure to improve the efficacy of treatment.

[0510] In one embodiment, one or more components of a transcription factor system comprising CD19CAR may be used in combination with an aminopyrimidine derivative, such as a Burkitt's tyrosine receptor kinase (BTK) inhibitor, using the methods taught in International Patent Application No. WO2016164580, the contents of which are incorporated herein by reference in their entirety.

[0511] In some embodiments, the compositions disclosed herein may be used in combination with immunotherapeutic agents other than the therapies of the present invention described herein, such as antibodies that are specific to certain target molecules on the surface of tumor cells.

[0512] Exemplary chemotherapy therapies include, but are not limited to, acivicin; aclarubicin; aodazole hydrochloride; acronine; adozelesin; aldesleukin; atratamine; ambomycin; and ametantrone. Acetate); Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperrin; Sulindac; Curcumin; Alkylating agents, including: Nitrogenmustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; Nitrosoureas, such as carmustine (BCU), lomustine (CCNU), and semustine (methyl-CCNU). U); ethyleneimine / methylmelamine, such as triethylenemelamine (TEM), triethylene, thiotepa, hexamethylmelamine (HMM, atratamine); alkyl sulfonates, such as busulfan; triazines, such as dacarbazine (DTIC); antimetabolites, including folic acid analogs such as methotrexate and trimethotropic acid, pyrrolidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine. abine), cytarabine (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; epipodophyllotoxins such as etoposide and teniposide; and antibiotics such as actinomycin D. D) Daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes such as L-asparaginase; cytokines such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, TNF-β, and GM-CSF; anti-angiogenic factors such as angiostatin and endostatin; FGF or VEGF inhibitors, such as angiostatin receptor blockers. Soluble forms of the body, including soluble VGF / VEGF receptors; platinum coordination complexes, such as cisplatin and carboplatin; anthraquinones, such as mitoxantrone; substituted ureas, such as hydroxyurea; methylhydrazine derivatives, including N-methylhydrazine (MIFf) and procarbazine; adrenocortical inhibitors, such as mitotane (o,p'-DDD) and aminoglutethimide; hormones and antagonists, including adrenocortical hormone antagonists, such as prednisone and its equivalents, dexamethasone and aminoglutethimide; progestins, such as hydroxyprogesterone caproate. Caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens, such as diethylstilbestrol and ethinylestradiol equivalents; anti-estrogens, such as tamoxifen; androgens, including testosterone propionate and fluoxymesterone / equivalents;Antiandrogens, such as flutamide, gonadotropin-releasing hormone analogs, and leuprolide; nonsteroidal antiandrogens, such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, antioxidants, telomerase inhibitors, BH3 mimics, ubiquitin ligase inhibitors, stat inhibitors, and receptor tyrosine kinase inhibitors, such as imatinib mesylate (marketed as Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor, now marketed as Tarveca); antiviral agents, such as oseltamivir phosphate, amphotericin B, and palivizumab; Sdi 1 mimics; simustine; senescence-derived inhibitors; sparfosic acid. (acid); Spiramycin D; Spiromustine; Splenopentin; Spongostrigin 1; Squalamine; Stipiamide; Hemolysin inhibitor; Sulfinosine; Super-active vasoactive intestinal peptide antagonist; Velaresol; Veramine; Verdins; Verteporfin; Vinorelbine; Vinxaltine; Vitaxin; Vorozole; Zanoterone; Zeniplatin; Zilascorb; Zinostatinstimalamer; PI3Kβ small molecule inhibitor, GSK2636771; Pan-PI3K inhibitor (BKM120); BRAF inhibitor. Vemurafenib (Zelboraf) and dabrafenib (Tafinlar); or any of the foregoing analogues, derivatives, and variants.

[0513] Radiation therapy agents and factors include radiation and waves that induce DNA damage, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, and radioactive isotopes. Therapy can be achieved by irradiating the local tumor site with these forms of radiation. It is highly likely that all of these factors will cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from 50 to 200 roentgens per day over a longer period (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. Radioactive isotope doses vary considerably and depend on the isotope's half-life, the intensity and type of emitted radiation, and the absorption by the proliferating cells.

[0514] In some implementations, the chemotherapy agent can be an immunomodulator, such as lenalidomide (LEN). Recent studies have shown that lenalidomide can enhance the antitumor function of CAR-modified T cells (Otahal et al., Oncoimmunology, 2015, 5(4):e1115940). Some examples of antitumor antibodies include tocilizumab (ocilizumab) and cetuximab (siltuximab).

[0515] Other agents that can be used in combination with the compositions disclosed herein may include, but are not limited to, agents that upregulate cell surface receptors and their ligands, such as Fas / Fas ligands, DR4 or DR5 / TRAIL GAP linkages, cell growth inhibitors and differentiation agents, cell adhesion inhibitors such as focal adhesion kinase (FAK) inhibitors and lovastatin, or agents that increase the sensitivity of overproliferating cells to apoptosis inducers such as antibody C225.

[0516] Combinations may include the simultaneous or separate administration of the compositions of this disclosure and other agents. Alternatively, the immunotherapy of this invention may be administered before or after other agents / therapies, with intervals ranging from minutes, days, weeks to months.

[0517] This disclosure provides a method for reducing tumor volume or burden in a subject in need, the method comprising introducing the composition of this disclosure into the subject.

[0518] This disclosure also provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of effector immune cells that are genetically modified to contain the transcription factor system of this disclosure.

[0519] cancer

[0520] Various cancers can be treated with the pharmaceutical compositions, transcription factor system components, and modulated transcription factor expression constructs disclosed herein, including DRDs or payloads. As used herein, the term "cancer" refers to any of a variety of malignant tumors characterized by the proliferation of anaplastic cells that tend to invade surrounding tissues and metastasize to new body sites, and also refers to a pathological condition characterized by the growth of such malignant tumors. Cancer can be a tumor or a hematologic malignancy, and includes, but is not limited to, all types of lymphoma / leukemia, carcinomas, and sarcomas, such as cancers or tumors found in the anus, bladder, bile ducts, bones, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lungs, mediastinum (chest), mouth, ovary, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, coccyx, testis, thyroid, and uterus.

[0521] The types of cancers that can be treated with the compositions disclosed herein include, but are not limited to, papilloma / carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma / adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, hemangioma, osteoma, chondroma, glioma, lymphoma / leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, basal cell carcinoma, and undifferentiated sinus carcinoma.

[0522] The types of sarcomas that can be treated with the compositions disclosed herein include, but are not limited to, soft tissue sarcomas, such as alveolar soft tissue sarcoma, angiosarcoma, dermatofibrosarcoma, destenoid tumor, fibroproliferative small round cell tumor, extraosseous chondrosarcoma, extraosseous osteosarcoma, fibrosarcoma, hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

[0523] Infectious diseases

[0524] In some embodiments, the transcription factor system of this disclosure can be used to treat infectious diseases. The transcription factor system of this disclosure can be introduced into cells suitable for adoptive cell transfer, such as macrophages, dendritic cells, natural killer cells, and / or T cells. Infectious diseases treated with the transcription factor system of this disclosure can include diseases caused by viruses, bacteria, fungi, and / or parasites. The IL15-IL15Ra payload of this disclosure can be used to increase the proliferation and / or persistence of immune cells that can be used to treat infectious diseases.

[0525] "Infectious disease" in this document refers to a disease caused by any pathogen or agent that infects mammalian cells, preferably human cells, and causes a disease condition. Examples include bacteria, yeast, fungi, protozoa, mycoplasma, viruses, prions, and parasites. Examples include those involving: (a) viral diseases, such as those caused by adenoviruses, herpesviruses (e.g., HSV-I, HSV-II, CMV, or VZV), poxviruses (e.g., orthopoxviruses, such as smallpox or cowpox, or molluscum contagiosum), microRNA viruses (e.g., rhinoviruses or enteroviruses), orthomyxoviruses (e.g., influenza viruses), paramyxoviruses (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), coronaviruses (e.g., SARS), papillomaviruses (e.g., papillomaviruses, such as those causing genital warts, common warts, or plantar warts), liver... (a) Illnesses caused by viral infections (e.g., hepatitis B virus), flaviviruses (e.g., hepatitis C virus or dengue virus), or retroviruses (e.g., lentiviruses such as HIV); (b) bacterial diseases, such as those caused by Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, or Helicobacter. Diseases caused by bacterial infections of the genus Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella.(c) Other infectious diseases, such as chlamydia, fungal diseases (including but not limited to candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis), parasitic diseases (including but not limited to malaria, Pneumocystis carinii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosomiasis), and prion diseases causing human illnesses such as Creutzfeldt-Jakob disease (CJD), variant CJD (vCJD), Gerstmann-Straüssler-Scheinker syndrome, fatal familial insomnia, and kuru.

[0526] Immuno-oncology and Cell Therapy

[0527] Recent advances in cancer immunology have allowed for the development of several approaches to help the immune system fight cancer. These immunotherapeutic approaches include targeting cancer antigens via monoclonal antibodies or by adoptive transfer of engineered T cells (e.g., cells containing chimeric antigen receptors or engineered T cell receptors).

[0528] In some embodiments, the pharmaceutical compositions, transcription factor systems, modifiable transcription factor expression constructs, modifiable transcription factor expression construct components, and modifiable transcription factor expression constructs including their payloads can be used to modulate or alter or utilize the immune system to target one or more cancers. This approach can also be considered in conjunction with other such biological methods, such as immunomodulatory therapies, such as administration of interferon, interleukin, colony-stimulating factors, other monoclonal antibodies, vaccines, and gene therapy. Furthermore, it is envisioned that nonspecific immunomodulators can also be used as anticancer therapies in combination with the pharmaceutical compositions, transcription factor systems, modifiable transcription factor expression constructs, modifiable transcription factor expression construct components, and modifiable transcription factor expression constructs including their payloads.

[0529] Cancer immunotherapy refers to a group of different treatment strategies designed to induce a patient's own immune system to fight cancer. In some embodiments, the pharmaceutical compositions, transcription factor systems, modulotropic transcription factor expression constructs, modulotropic transcription factor expression construct components, and modulotropic transcription factor expression constructs including their transcription factors and / or payloads are designed as immuno-oncology therapeutic agents.

[0530] Cell therapy

[0531] There are several types of cell immunotherapy, including tumor-infiltrating lymphocyte (TIL) therapy, genetically engineered T cells carrying chimeric antigen receptors (CARs), and recombinant TCR technology.

[0532] According to this disclosure, transcription factor systems can be used to develop and administer cell therapies, such as adoptive cell therapy. Transcription factor systems, their transcription factors, and payloads can be used in cell therapy to achieve TCR removal—TCR gene disruption, TCR engineering; regulation of epitope-marked receptors; in APC platforms for stimulating T cells; as tools to enhance ex vivo APC stimulation; to improve T cell expansion methods; for ex vivo stimulation with antigens; in TCR / CAR combinations; for manipulation or regulation of TILs; in allogeneic cell therapy; in combinations of T cell therapy with other lines of treatment (e.g., radiation, cytokines); to encode engineered or modified TCRs; or to enhance T cells other than TCRs (e.g., by introducing cytokine genes, checkpoint inhibitors PD1, CTLA4 genes).

[0533] In some implementations, an improved response rate is achieved to support cell therapy.

[0534] Cell population expansion and persistence can be achieved by modulating or finely adjusting payload components, such as receptors or pathway components in T cells, NK cells, or other immune-related cells. In some embodiments, the transcription factor system of this disclosure is designed to spatially and / or temporally control the expression of proteins that enhance T cell or NK cell responses. In some embodiments, the transcription factor system is designed to spatially and / or temporally control the expression of proteins that inhibit T cell or NK cell responses.

[0535] In some embodiments, cells genetically modified to contain transcription factor systems as described herein can be engineered to reduce, mitigate, or eliminate CAR cytokine storms. In some embodiments, such reduction, mitigation, and / or elimination occurs in solid tumors or the tumor microenvironment.

[0536] In some implementations, the transcription factor system may encode one or more cytokines, such as interleukins, such as IL2, IL6, IL12, IL15, and IL21.

[0537] In one embodiment, the payload of this disclosure may include IL2. On the other hand, the transcription factor system of this disclosure may encode transcription factors that selectively transcribe IL2, IL12, IL15, and other interleukin immunotherapeutic agents, said transcription factors being carefully tuned using stable ligands selective to the DRD used in the transcription factor system.

[0538] On one hand, the transcription factor system disclosed herein can encode transcription factors, such as IL12 fusion peptides, for selective transcription payloads. The regulated IL12 fusion peptides can be used directly as immunotherapeutic agents or transduced into effector immune cells (T cells and TIL cells) to generate modified T cells with greater in vivo expansion and viability for adoptive cell transfer. Using regulated IL12 minimizes the need for stringent pretreatment protocols in current adoptive cell therapies. IL12 can be used to alter the tumor microenvironment and increase the persistence of solid tumors that are untreatable by current tumor antigen-targeting therapies. In some embodiments, CAR-expressing T cells can be armed with transcription factor-regulated IL12 to alleviate immunosuppression without systemic toxicity.

[0539] In some implementations, IL12 may be Flexi IL12, where the p35 and p40 subunits are encoded by a single cDNA that produces a single-chain polypeptide.

[0540] In some embodiments, the illustrative transcription factor system may encode, be regulated, or induced to produce one or more cytokines for expanding the cells of this disclosure. In such cases, the actual expansion of cells can be tested. The expansion may be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher percentages. In some embodiments, the cytokine is IL15. The transcription factor system encoding IL15 may be programmed to induce the proliferation of a cytotoxic population and avoid stimulation of T regs. In other embodiments, the transcription factor system inducing the proliferation of a cytotoxic population may also stimulate NK and NKT cells. Interleukin-15 is a potent immunostimulatory cytokine and an important survival factor for T cells and natural killer cells. Preclinical studies comparing IL2 and IL15 have shown that IL15 is associated with lower toxicity compared to IL2. In some embodiments, the transcription factor system of this disclosure may encode an IL15 fusion peptide. The IL15 peptide may also be modified to increase its binding affinity to the IL15 receptor. For example, asparagine at position 72 of IL15 can be replaced by aspartic acid (SEQ ID NO.2 of US Patent Publication US20140134128; the contents of which are incorporated herein by reference in their entirety).

[0541] The immune system can be used to treat diseases other than cancer. Transcription factor systems, their components, or regulated transcription factor expression constructs can be used in immunotherapy to treat diseases, including but not limited to autoimmune diseases, allergies, graft-versus-host disease, and diseases and conditions that may lead to immunodeficiency, such as acquired immunodeficiency syndrome (AIDS).

[0542] In some embodiments, the payload of this disclosure may be a chimeric antigen receptor (CAR), which, when transduced into immune cells (e.g., T cells and NK cells), can redirect immune cells toward a target (e.g., tumor cells) that expresses a molecule recognized by the extracellular target portion of the CAR.

[0543] In some embodiments, pharmaceutical compositions comprising transcription factor systems, including their payloads of interest or proteins, can be used to modulate or alter or exploit the immune system to target one or more autoreactive immune components, such as autoantibodies and autoreactive immune cells, to alleviate autoimmune diseases.

[0544] In some implementations, transcription factor systems can be used in immunotherapy-based treatments to attenuate or mitigate graft-versus-host disease (GVHD). GVHD is a condition that occurs after stem cell or bone marrow transplantation, in which allogeneic donor immune cells respond to host tissue. In some implementations, the transcription factor system may be programmed to encode cytokines or immunomodulators designed to modulate Tregs to treat GVHD.

[0545] In some implementations, the immunogenicity of transcription factor systems can be significantly lower than that of other biological circuits or switches in the art, attributable to the expression of human natural or wild-type proteins of interest.

[0546] Pharmaceutical compositions comprising the transcription factor system disclosed herein can be used to treat a variety of autoimmune diseases and autoimmune-related diseases. As used herein, the term "autoimmune disease" refers to a disease in which the body produces antibodies that attack its own tissues.

[0547] Autoimmune diseases include, but are not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy, Balo's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, and Castleman's disease. Diseases including celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, Coxsackie myocarditis, CREST disease, primary mixed cryoglobulinemia, demyelinating neuropathy, herpetic dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, and Evans syndrome. Syndrome, fibrotic alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatous polyangiitis (GPA) (formerly known as Wegener's granulomatosis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditisThyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosis, immunomodulatory lipoprotein, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytic clotting vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD), lupus erythematosus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease. Diseases including multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devrwick's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatic disease, streptococcal-associated childhood autoimmune neuropsychiatric disorders (PANDAS), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, and Parsonnage-Turner syndrome. Symptoms of rheumatoid arthritis, perinic body plana inflammation (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II and III autoimmune polyglandular syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosa, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome Syndrome, sperm and testicular autoimmunity, stiff person syndrome, Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritisArteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, bullous dermatitis, and vitiligo.

[0548] Various kidney diseases can be treated with pharmaceutical compositions containing one or more components of the transcription factor system disclosed herein.

[0549] Various cardiovascular diseases can be treated with pharmaceutical compositions comprising one or more components of the transcription factor system disclosed herein.

[0550] Various antibody deficiencies can be treated with pharmaceutical compositions containing one or more components of the transcription factor system disclosed herein.

[0551] Various neurological diseases can be treated with pharmaceutical compositions containing one or more components of the transcription factor system disclosed herein.

[0552] Various lung diseases can be treated with pharmaceutical compositions containing one or more components of the transcription factor system disclosed herein.

[0553] Various bone diseases can be treated with pharmaceutical compositions containing one or more components of the transcription factor system disclosed herein.

[0554] Various hematologic disorders can be treated with pharmaceutical compositions comprising one or more components of the transcription factor system disclosed herein.

[0555] In some embodiments, a pharmaceutical composition comprising one or more components of the transcription factor system disclosed herein can be used to modulate or alter or utilize proteins in the central nervous system, including cerebrospinal fluid (CSF) proteins.

[0556] In some instances, pharmaceutical compositions comprising one or more components of the transcription factor system disclosed herein can be used to deliver modulated ERT (enzyme replacement therapy) products to the central nervous system. Many lysosomal storage diseases (LSDs) involve CNS symptoms such as intellectual disability, seizures, severe neurodegeneration, behavioral abnormalities, and psychomotor deficits. ERT for LSD is one of the real-world successes in modern molecular medicine. The successful application of ERT relies on controlled delivery of lysosomal proteins (e.g., enzymes) to CNS cells.

[0557] Gene editing

[0558] In some embodiments, the transcription factor system includes a payload comprising a nuclease containing a DNA-binding domain to selectively target specific DNA sequences for gene editing and gene therapy. In some embodiments, the transcription factor system comprises a polynucleotide encoding a zinc finger nuclease, TALES, or CRISPR nuclease as a payload, said polynucleotide being regulated by a transcription factor in the system.

[0559] The CRISPR-Cas9 system is a novel genome editing system that has been rapidly developed and implemented in various model organisms and cell types, replacing other genome editing technologies such as TALEN and ZFN. CRISPR consists of sequence motifs found in the genomes of bacteria and archaea, composed of short (approximately 24–48 nucleotides) direct repeat sequences separated by unique spacers of similar size (Grissa et al., BMC Bioinformatics 8, 172 (2007)). These are typically flanked by a set of genes encoding CRISPR-associated (Cas) proteins, which are essential for the maintenance and function of CRISPR (Barrangou et al., Science 315, 1709 (2007); Brouns et al., Science 321, 960 (2008); Haft et al., PLoS Comput Biol 1, e60 (2005)). CRISPR-Cas systems provide adaptive immunity against invasive genetic elements such as viruses, bacteriophages, and plasmids (Horvath and Barrangou, Science, 2010, 327:167-170; Bhaya et al., Annu. Rev. Genet., 2011, 45:273-297; and Brangou R, RNA, 2013, 4:267-278). Three different types of CRISPR-Cas systems have been classified in bacteria, with type II CRISPR-Cas systems being the most studied. In bacterial type II CRISPR-Cas systems, small CRISPR RNA (crRNA), processed from pre-crRNA (repetitive spacer transcript) in the presence of trans-activating RNA (tracrRNA) / Cas9, can form a doublet with the tracrRNA / Cas9 complex. The duplex recruits the mature complex to a target double-stranded DNA sequence complementary to the spacer sequence in the tracrRNA:crRNA duplex for cleavage of the target DNA by the Cas9 endonuclease (Garneau et al., Nature, 2010, 468:67-71; Jinek et al., Science, 2012, 337:816-821; Gasinas et al., Proc. Natl Acad. Sci. USA., 109: E2579-2586; and Haurwitz et al., Science, 2010, 329:1355-1358).In type II CRISPR-Cas systems, target recognition and cleavage induced by the crRNA:tracrRNA / Cas9 complex requires not only a sequence complementary to the target sequence in the tracrRNA:crRNA duplex (also known as the "protospacer" sequence), but also a protospacer adjacent motif (PAM) sequence located at the 3' end of the protospacer sequence of the target polynucleotide. The PAM motif can vary between different CRISPR-Cas systems.

[0560] The CRISPR-Cas9 system has been developed and improved for gene editing and has proven to be an effective and unique technique for editing nucleic acid sequences, even in eukaryotic cells.

[0561] However, controlling the function and activity of CRISPR-Cas systems (such as guide RNA and nucleases) has always been a challenge and often leads to problems.

[0562] The transcription factor systems and / or any components thereof disclosed herein can be used to regulate or modulate the CRISPR / Cas9 system to optimize its utility.

[0563] In some embodiments, the payload of the tunable transcription factor expression construct of this disclosure may include alternative isoforms or orthologs of the Cas9 enzyme.

[0564] The most commonly used Cas9 is derived from *Streptococcus pyogenes*, and the RuvC domain can be inactivated by the D10A mutation, while the HNH domain can be inactivated by the H840A mutation. Besides Cas9 derived from *Streptococcus pyogenes*, other RNA-guided endonucleases (RGENs) can also be used for programmable genome editing. Cas9 sequences have been identified in more than 600 bacterial strains. Although the Cas9 family exhibits high diversity in amino acid sequences and protein sizes, all Cas9 proteins share a common structure, possessing a central HNH nuclease domain and a separate RuvC / RHase H domain.

[0565] In some implementations, the payload of this disclosure may be a separate Cas-9 (Zetsche B et al., Asplit-Cas9 architecture for inducible genome editing and transcriptionmodulation. Nat Biotechnol. 2015 Feb; 33(2):139-42; the contents of which are incorporated herein by reference in their entirety).

[0566] In addition to Cas9 orthologs, other Cas9 variants, such as inactive dCas9 and fusion proteins with effector domains having different functions, can also be used as platforms for gene regulation. Any of the aforementioned enzymes can be used in this disclosure.

[0567] CRISPR / Cas9-based modulatory transcription factor expression constructs can be generated by any of the methods taught in International Publication No. WO2016106244 and Gao Y et al. (Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat Methods. Dec 2016; 13(12):1043-1049; the contents of which are incorporated herein by reference in their entirety).

[0568] The CRISPR / Cas9 system can also be used to regulate gene expression, which can be combined with its gene-editing utility. In some embodiments, the payload of the tunable transcription factor system disclosed herein may include a CRISPR-associated transcriptional activator, such as VP64-p65-Rta (VPR), associated with the CRISPR / Cas9 system.

[0569] Other applications and uses

[0570] Stem cell applications

[0571] The transcription factor systems and / or components thereof disclosed herein may be useful for controlled reprogramming of cells, stem cell transplantation, or other applications where controlled or regulated expression of these reprogramming factors would be beneficial.

[0572] The tunable transcription factor expression constructs disclosed herein can be used to reprogram cells, including stem cells or induced pluripotent stem cells. The induction of induced pluripotent stem cells (iPSCs) was first achieved by Takahashi and Yamanaka (Cell, 2006, 126(4):663-76; incorporated herein by reference in its entirety) using viral vectors to express KLF4, c-MYC, OCT4, and SOX2 (collectively referred to as KMOS).

[0573] Excisable lentiviruses and transposon vectors, repeated use of transient plasmids, free-type and adenovirus vectors have also been used to attempt to obtain iPSCs (Chang, C.-W. et al., Stem Cells, 2009.27(5):1042-1049; Kaji, K. et al., Nature, 2009.458(7239):771-5; Okita, K. et al., Science, 2008.322(5903):949-53; Stadtfeld, M. et al., Science, 2008.322(5903):945-9; Woltjen, K. et al., Nature, 2009; Yu, J. et al., Science, 2009:1172482; Fusaki, N. et al., Proc Jpn Acad Ser B Phys Biol Sci, 2009, 85(8):348-62; each is incorporated herein by reference in its entirety.

[0574] DNA-free methods for generating human iPSCs have also been obtained using sequential protein transduction with recombinant proteins incorporating cell-penetrating peptides (Kim, D. et al., Cell Stem Cell, 2009.4(6):472-476; Zhou, H. et al., Cell Stem Cell, 2009.4(5):381-4; each incorporated herein by reference in its entirety), and infectious transgene delivery using Sendaivirus (Fusaki, N. et al., Proc Jpn Acad Ser B Phys Biol Sci, 2009.85(8): pp.348-62; incorporated herein by reference in its entirety).

[0575] The tunable transcription factor expression constructs disclosed herein may include a payload containing any gene supporting reprogramming of cells, including but not limited to OCT, such as OCT4; SOX, such as SOX1, SOX2, SOX3, SOX15, and SOX18; NANOG; KLF, such as KLF1, KLF2, KLF4, and KLF5; MYC, such as c-MYC and n-MYC; REM2; TERT; and LIN28, and variants thereof. Sequences of such reprogramming factors are taught, for example, in international application PCT / US2013 / 074560, the contents of which are incorporated herein by reference in their entirety.

[0576] The tunable transcription factor expression construct disclosed herein may include a payload containing any factors that facilitate stem cell mobilization. In autologous stem cell therapy, sources of stem cells for transplantation may include bone marrow, peripheral blood mononuclear cells, and umbilical cord blood. Stem cells from these sources (e.g., bone marrow) are stimulated to enter the bloodstream. Therefore, sufficient stem cells are available for collection for future re-infusion. A cytokine strategy or combination of cytokine strategies may be used to mobilize stem cells, including but not limited to G-CSF (filgrastim), GM-CSF, and chemotherapy prior to cytokine administration (chemomobilization).

[0577] Metabolic peptides and hormones

[0578] In some embodiments, the transcription factor systems and / or any components thereof disclosed herein can be used to regulate natural or synthetic peptides. Naturally occurring peptides may include, but are not limited to, peptide hormones, natriuretic peptides, food peptides, and their derivatives and precursors.

[0579] The transcription factor systems and / or any components thereof disclosed herein may also be used for the pulsed release of hormones or other peptide drugs.

[0580] Enzyme replacement therapy (ERT)

[0581] Enzyme replacement therapy (ERT) is a medical treatment that replaces enzymes in a patient's body. ERT provides a therapeutic intervention to address underlying metabolic defects in many conditions caused by enzyme deficiency. These conditions include, but are not limited to, lysosomal storage diseases (LSD), congenital glycosylation disorders, and metabolic disorders characterized by the absence or reduction of enzyme activity in the cytoplasm.

[0582] Blood clotting

[0583] Coagulation defects often lead to bleeding and / or thrombosis. The most well-known coagulation factor disorder is hemophilia. Its three main forms are hemophilia A (deficiency of factor VIII), hemophilia B (deficiency of factor IX or "Christmas disease"), and hemophilia C (deficiency of factor XI, moderate bleeding tendency). Other conditions caused by coagulation factor deficiencies include, but are not limited to, Von Willebrand disease (caused by deficiency of Von Willebrand factor (vWF)), Bernard-Soulier syndrome (caused by deficiency or absence of vWF receptor GPIb), thrombophlebitis (caused by factor XII mutation), congenital afibrinogenemia, familial amyloid nephropathy (caused by factor I mutation), congenital prothrombin-accelerating factor precursor / factor VII deficiency, thrombotic tendency (caused by factor II deficiency), congenital factor X deficiency, congenital factor XIIIa / b deficiency, and prokallikrein / Fletcher factor deficiency. Factor deficiency, kininogen deficiency, glomerulonephropathy with fibronectin deposition, heparin cofactor II deficiency, protein C deficiency, protein S deficiency, protein Z deficiency, antithrombin III deficiency, plasminogen deficiency, type I (woody conjunctivitis), antifibrinolytic enzyme deficiency, plasminogen activator inhib...

Claims

1. A modified cell, said cell comprising a first polynucleotide, the first polynucleotide comprising The first nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65; A second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site, wherein the transcription factor DNA-binding domain is derived from ZFHD1; and The third nucleic acid sequence encoding a drug-reactive domain (DRD), wherein the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2) and human DHFR; At least one of the transcription factor activation domain, the transcription factor DNA binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA binding domain is operatively linked to the DRD. The transcription factor activation domain interacts with the transcription factor DNA binding domain to form a transcription factor, which, after binding to the specific polynucleotide binding site, can activate the transcription of a fourth nucleic acid sequence. and The fourth nucleic acid sequence encodes the protein of interest and is operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site or operatively linked to the specific polynucleotide binding site.

2. The modified cell of claim 1, wherein the protein of interest is a heterologous protein.

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

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

5. The modified cell according to any one of claims 1 to 4, wherein the DRD is stable in the presence of a ligand selected from the group consisting of acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP).

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

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

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

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

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

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

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

13. A nucleic acid molecule, said nucleic acid molecule comprising: a. A first nucleic acid sequence encoding a transcription factor DNA-binding domain, wherein the transcription factor DNA-binding domain binds to a specific polynucleotide binding site, wherein the transcription factor DNA-binding domain is derived from ZFHD1; b. A second nucleic acid sequence encoding a drug-reactive domain (DRD), wherein the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2) and human DHFR; c. A third nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65; Wherein (i) the transcription factor DNA-binding domain is operatively linked to the DRD; (ii) the transcription factor activation domain is operatively linked to the DRD; or (iii) a combination of the transcription factor DNA-binding domain and the transcription factor activation domain is operatively linked to the DRD; d. A fourth nucleic acid sequence encoding a protein of interest, said fourth nucleic acid sequence being operatively linked to an inducible promoter containing said specific polynucleotide binding site.

14. The nucleic acid molecule of claim 13, wherein the DRD is stable in the presence of a ligand selected from the group consisting of acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP).

15. The nucleic acid molecule of claim 13 or 14, wherein the protein of interest is a wild-type protein.

16. The nucleic acid molecule of claim 13 or 14, wherein the protein of interest is a therapeutic protein.

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

18. The nucleic acid molecule of claim 13 or 14, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN and Cre.

19. The nucleic acid molecule of claim 13 or 14, wherein the protein of interest is a secretory protein.

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

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

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

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

24. A first polynucleotide and a second polynucleotide, the first polynucleotide comprising: The first nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65; A second nucleic acid sequence encoding a transcription factor DNA-binding domain, said transcription factor DNA-binding domain binding to a specific polynucleotide binding site, wherein said transcription factor DNA-binding domain is derived from ZFHD1; and The third nucleic acid sequence encoding a drug-reactive domain (DRD), wherein the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2) and human DHFR; At least one of the transcription factor activation domain, the transcription factor DNA binding domain, or a combination of the transcription factor activation domain and the transcription factor DNA binding domain is operatively linked to the DRD; and The second polynucleotide contains: A fourth nucleic acid sequence encoding a protein of interest, said fourth nucleic acid sequence being operatively linked to an inducible promoter containing said specific polynucleotide binding site; The transcription factor activation domain and the transcription factor DNA binding domain interact to form a transcription factor, which, upon binding to the specific polynucleotide binding site, is capable of activating transcription, 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.

25. The first and second polynucleotides of claim 24, wherein the DRD is stable in the presence of a ligand selected from the group consisting of acetazolamide (ACZ), methotrexate (MTX), and trimethoprim (TMP).

26. The first and second polynucleotides as described in claim 24 or 25, wherein the protein of interest is a wild-type protein.

27. The first polynucleotide and the second polynucleotide as described in claim 24 or 25, wherein the protein of interest is a therapeutic protein.

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

29. The first and second polynucleotides as claimed in claim 24 or 25, wherein the protein of interest is selected from the group consisting of IL2, IL12, IL15, Cas9, ZFN, and Cre.

30. The first polynucleotide and the second polynucleotide as described in claim 24 or 25, wherein the protein of interest is a secretory protein.

31. A method for producing modified cells, the method comprising introducing a nucleic acid molecule into the cells, the nucleic acid molecule comprising: a. A first nucleic acid sequence encoding a transcription factor DNA-binding domain, wherein the transcription factor DNA-binding domain binds to a specific polynucleotide binding site, wherein the transcription factor DNA-binding domain is derived from ZFHD1; b. A second nucleic acid sequence encoding a drug-reactive domain (DRD), wherein the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2) and human DHFR. The nucleic acid molecule further comprises a third nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65. Wherein (i) the transcription factor DNA-binding domain is operatively linked to the DRD; (ii) the transcription factor activation domain is operatively linked to the DRD; or (iii) a combination of the transcription factor DNA-binding domain and the transcription factor activation domain is operatively linked to the DRD. The method also includes introducing into the cells: A fourth nucleic acid sequence encoding a protein of interest, said fourth nucleic acid sequence being operatively linked to an inducible promoter containing said specific polynucleotide binding site.

32. The method of claim 31, wherein the protein of interest is a heterologous protein.

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

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

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

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

37. The method according to any one of claims 31 to 34, wherein the protein of interest is a secretory protein.

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

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

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

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

42. The method of any one of claims 31 to 41, wherein the cell is a T cell, a natural killer cell (NK cell), or a tumor-infiltrating lymphocyte (TIL).

43. The method of any one of claims 31 to 41, wherein the cell is a stem cell, hepatocyte, blood cell, pancreatic cell, neuron, eye cell, muscle cell, or bone cell.

44. Use of at least one nucleic acid molecule in the preparation of a medicament, said medicament being used in a method for treating or preventing a disease in a subject of need, said method comprising: a. Provide cell population; b. Introducing at least one nucleic acid molecule into at least one cell in the cell population, wherein the at least one nucleic acid molecule comprises: i. A first polynucleotide, the first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65; A second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site, wherein the transcription factor DNA-binding domain is derived from ZFHD1; and The third nucleic acid sequence encoding the drug-reactive domain (DRD), wherein the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2) and human DHFR. At least one of the transcription factor activation domain and the transcription factor DNA binding domain is operatively linked to the DRD; and ii. A second polynucleotide, the second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that prevents or treats the disease or its symptoms, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site; c. Deliver the cells into the subject; and d. Administering a ligand to the subject, the ligand stabilizing the DRD sufficiently to express 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, the transcription factor binding to the specific polynucleotide binding site and enabling the expression of the protein of interest in the cell; The expression of the protein of interest is regulated by the presence of a ligand in the subject's body, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

45. Use of at least one nucleic acid molecule in the preparation of a medicament for use in a method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, the method comprising: a. Introducing at least one nucleic acid molecule into at least one cell of the subject, wherein the at least one nucleic acid molecule comprises: i. A first polynucleotide, the first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65; A second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site, wherein the transcription factor DNA-binding domain is derived from ZFHD1; and The third nucleic acid sequence encoding the drug-reactive domain (DRD), wherein the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2) and human DHFR. The transcription factor activation domain and the transcription factor DNA binding domain, at least one of which is expressed in the cell, are operatively linked to the DRD; and ii. A second polynucleotide, the second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site.

46. ​​Use of at least one nucleic acid molecule in the preparation of a medicament for use in a method for genetically modifying one or more cells in a subject requiring treatment or prevention of a disease, the method comprising: a. Introducing at least one nucleic acid molecule into at least one cell of the subject, wherein the at least one nucleic acid molecule comprises: i. A first polynucleotide, the first polynucleotide comprising a first nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65; A second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site, wherein the transcription factor DNA-binding domain is derived from ZFHD1; and The third nucleic acid sequence encoding the drug-reactive domain (DRD), wherein the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2) and human DHFR. The transcription factor activation domain and the transcription factor DNA binding domain, at least one of which is expressed in the cell, are operatively linked to the DRD; and ii. A second polynucleotide, the second polynucleotide comprising a fourth nucleic acid sequence encoding a protein of interest that treats the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site; and b. Administering a ligand to the subject, the ligand stabilizing the DRD sufficiently to express 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, the transcription factor binding to the specific polynucleotide binding site and enabling the expression of the protein of interest in the cell; The expression of the protein of interest is regulated by the presence of a ligand in the subject's body, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

47. Use of at least one first nucleic acid molecule and at least one second nucleic acid molecule in the preparation of a medicament, said medicament being used in a method for treating a disease in a subject of need, said method comprising: a. Provide cell population; b. Introducing at least one first nucleic acid molecule and at least one second nucleic acid molecule into at least one cell in the said cell population, wherein: i. The first nucleic acid molecule contains a first nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65; A second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site, wherein the transcription factor DNA-binding domain is derived from ZFHD1; and The third nucleic acid sequence encoding the drug-reactive domain (DRD), wherein the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2) and human DHFR. At least one of the transcription factor activation domain and the transcription factor DNA binding domain, after being expressed in the cell, is operatively linked to the DRD; and ii. The second nucleic acid molecule contains a fourth nucleic acid sequence encoding a protein of interest that treats the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site; c. Deliver the cells into the subject; and d. Administering a ligand to the subject, the ligand stabilizing the DRD sufficiently to express an amount of the transcription factor activation domain and the transcription factor DNA binding domain sufficient to form a transcription factor that binds to the specific polynucleotide binding site and enables the expression of the protein of interest in the cell; The expression of the protein of interest is regulated by the presence of a ligand in the subject's body, and the amount and / or duration of ligand administration is sufficient to produce a therapeutically effective amount of the protein of interest.

48. Use of at least one first nucleic acid molecule and at least one second nucleic acid molecule in the preparation of a medicament, said medicament being used in a method for treating a disease in a subject in need, said method comprising: a. Provide cell population; b. Introducing at least one first nucleic acid molecule and at least one second nucleic acid molecule into at least one cell in the said cell population, wherein: i. The first nucleic acid molecule contains a first nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65; A second nucleic acid sequence encoding a transcription factor DNA-binding domain, the transcription factor DNA-binding domain binding to a specific polynucleotide binding site, wherein the transcription factor DNA-binding domain is derived from ZFHD1; and The third nucleic acid sequence encoding the drug-reactive domain (DRD), wherein the DRD is derived from a parental protein selected from the group consisting of human carbonic anhydrase 2 (CA2) and human DHFR. At least one of the transcription factor activation domain and the transcription factor DNA binding domain, after being expressed in the cell, is operatively linked to the DRD; and ii. The second nucleic acid molecule contains a fourth nucleic acid sequence encoding a protein of interest that treats the disease, the fourth nucleic acid sequence being operatively linked to an exogenous inducible promoter containing the specific polynucleotide binding site; and c. Deliver the cells into the subject.

49. The use according to any one of claims 44 to 48, wherein the nucleic acid molecule is introduced into the cell via a plasmid or viral vector.

50. The use according to claim 49, wherein the viral vector is derived from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpesvirus, measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and picornavirus.

51. The use according to claim 49, wherein the viral vector is selected from the group consisting of: lentiviral vectors, gamma retroviral vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, and herpesvirus vectors.

52. The use according to any one of claims 44 to 48, wherein the nucleic acid molecule is introduced into the cell by a non-viral delivery method.

53. A system for the tunable expression of a protein of interest in cells, the system comprising: a. A first polynucleotide encoding a transcription factor linked to a drug response domain (DRD), said transcription factor selectively transcribing a polynucleotide sequence encoding the protein of interest, wherein said DRD is derived from a parental protein selected from the group consisting of: human carbonic anhydrase 2 (CA2) and human DHFR, The first polynucleotide contains The first nucleic acid sequence encoding a transcription factor activation domain, wherein the transcription factor activation domain is derived from a parent protein, wherein the parent protein is p65; as well as A second nucleic acid sequence encoding a transcription factor DNA-binding domain, wherein the transcription factor DNA-binding domain binds to a specific polynucleotide binding site, and wherein the transcription factor DNA-binding domain is derived from ZFHD1; b. A second polynucleotide containing a foreign transcription factor binding site, wherein the foreign transcription factor binding site is located upstream and near the nucleic acid sequence encoding the protein of interest; c. Introducing the first polynucleotide and the second polynucleotide into the cell under conditions that the first polynucleotide and the second polynucleotide are stably integrated into the genome of the cell; d. Regulation of the expression of the transcription factor by adding a ligand that stabilizes the DRD; The transcription factor specifically binds to a transcription factor binding site located upstream and near the polynucleotide sequence encoding the protein of interest, and the expression of the protein of interest is regulated by the amount of transcription factor present in the cell.

54. The modified cell according to any one of claims 1-12, wherein the DRD is derived from CA2.

55. The modified cells according to any one of claims 1-12, wherein the DRD is derived from human DHFR.

56. The nucleic acid molecule according to any one of claims 13-19, wherein the DRD is derived from CA2.

57. The nucleic acid molecule according to any one of claims 13-19, wherein the DRD is derived from human DHFR.

58. The first polynucleotide and the second polynucleotide according to any one of claims 24-30, wherein the DRD is derived from CA2.

59. The first polynucleotide and the second polynucleotide according to any one of claims 24-30, wherein the DRD is derived from human DHFR.

60. The method according to any one of claims 31-43, wherein the DRD is derived from CA2.

61. The method according to any one of claims 31-43, wherein the DRD is derived from human DHFR.

62. The use according to any one of claims 44-52, wherein the DRD is derived from CA2.

63. The use according to any one of claims 44-52, wherein the DRD is derived from human DHFR.

64. The system of claim 53, wherein the DRD is derived from CA2.

65. The system of claim 53, wherein the DRD is derived from human DHFR.

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