Drug-regulatable transcriptional repressors

Abstract

Provided herein is a chimeric polypeptide for drug-regulated transcriptional repression.Also provided is a method for inhibiting the repression of a gene of interest.Provided herein is a chimeric polypeptide comprising (i) an inducible transcriptional regulator (ITM) comprising a transcriptional repressor domain and a DNA binding domain, and (ii) a degron operably linked to the ITM.The chimeric polypeptide described herein provides a reversible gene on-switch by inhibiting transcriptional repression in response to small molecule drugs.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 171,551, filed April 6, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created in XX month, 20XX, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. [Background technology]

[0003] background Currently available cell and gene therapy products can lack expression control, which can lead to safety concerns, such as toxicity, in subjects receiving such therapies. Thus, additional methods of expression control and regulation of these therapies are needed. Summary of the Invention

[0004] overview Provided herein is a chimeric polypeptide comprising: (i) an inducible transcriptional regulator (ITM) comprising a transcriptional repressor domain and a DNA binding domain; and (ii) a degron operably linked to the ITM, wherein the transcriptional repressor domain is selected from the group consisting of KRAB repression domain, HDAC4 domain, SCX HLH domain, ID1 HLH domain, HERC2 Cyt-b5 domain, TWST1 HLH domain, NKX22 homeodomain, ID3 HLH domain, and TWST2 HLH domain.

[0005] The chimeric polypeptides described herein provide a reversible gene on-switch by inhibiting transcriptional repression in response to small molecule drugs.

[0006] In some embodiments, the transcriptional repressor domain of the chimeric polypeptide comprises a KRAB repression domain. In some embodiments, the KRAB repression domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the KRAB repression domain comprises minKRAB. In some embodiments, the KRAB repression domain is a KRAB repressor domain variant of SEQ ID NO: 2 and comprises one or more amino acid substitutions selected from the group consisting of W27L, K28L, D31A, T32A, Q34A, Q35A, R39E, L43S, T57C, K58C, P59C, V61Y, I62Y, I62A, L63W, L63Y, L63E, R64F, R64W, R64E, L65F, L65E, L65W, E66V, K67F, G68F, and E69Fv. In some embodiments, the KRAB repression domain is a KRAB repressor domain variant of SEQ ID NO:2 and includes one or more amino acid substitutions selected from Q34A / Q35A, I62A, T57C / K58C / P59C, D31A / T32A, L63W / R64W / L65W, E66V, L63E / R64E / L65E, R64F / L65F, W27L / K28L KRAB, R39E, K67F / G68F / E69F, and V61Y / I62Y / L63Y.

[0007] In some aspects, the KRAB repression domain comprises the amino acid sequence of SEQ ID NO: 3. In some aspects, the transcriptional repressor domain comprises the HDAC4 repression domain. In some aspects, the HDAC4 repression domain comprises the amino acid sequence of SEQ ID NO: 4.

[0008] In some embodiments, the DNA binding domain of the chimeric polypeptide comprises a zinc finger (ZF) protein domain. In some embodiments, the ZF protein domain is modular in design and is composed of a zinc finger array (ZFA). In some embodiments, the ZF protein domain comprises 1 to 10 ZFAs. In some embodiments, the ZF protein domain comprises 10 ZFAs.

[0009] In some embodiments, the transcriptional repressor domain is N-terminal to the DNA binding domain. In other embodiments, the transcriptional repressor domain is C-terminal to the DNA binding domain.

[0010] In some embodiments, the transcriptional repressor domain and the DNA binding domain are separated by a first peptide linker. In some embodiments, the first peptide linker comprises the amino acid sequence of GGGGSGGT (SEQ ID NO: 60).

[0011] In some embodiments, the degron of the chimeric polypeptide is selected from the group consisting of HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα), GRR (residues 352-408 of human p105), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeats of SP2 and NB (influenza A or influenza B SP2-NB-SP2), RPB (four copies of residues 1688-1702 of yeast RPB), SPmix (tandem repeats of SP1 and SP2 (A These include SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein), NS2 (three copies of residues 79-93 of influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations), the APC / C degron, and COP1. E3 ligase-binding degron motif, CRL4-Cdt2-binding PIP degron, actinfilin-binding degron, KEAP1-binding degron, KLHL2- and KLHL3-binding degron, MDM2-binding motif, N-degron, hydroxyproline modification in hypoxia signaling, plant hormone-dependent SCF-LRR-binding degron, SCF ubiquitin ligase-binding phosphodegron, plant hormone-dependent SCF-LRR-binding degron, DSGxxS phospho-dependent degron, Siah-binding motif, SPOP In some embodiments, the degron comprises a cereblon (CRBN) polypeptide substrate domain that has the ability to bind to CRBN in response to an immunomodulatory drug (IMiD), thereby promoting the degradation of the chimeric polypeptide via the ubiquitin pathway. In some embodiments, the CRBN polypeptide substrate domain is selected from the group consisting of IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787 and ZN827, or fragments thereof, that have the ability of drug-induced binding of CRBN.In some embodiments, the CRBN polypeptide substrate domain is a chimeric fusion product of a native CRBN polypeptide sequence. In some embodiments, the CRBN polypeptide substrate domain is an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having an amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 6). In some embodiments, the IMiD is an FDA approved drug. In some embodiments, the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide.

[0012] In some embodiments, the inducible transcriptional regulator (ITM) is N-terminal to the degron. In other embodiments, the ITM is C-terminal to the degron.

[0013] In some aspects, the ITM is separated from the degron by a second peptide linker. In some aspects, the second peptide linker comprises an amino acid sequence selected from the group consisting of GSGSGSGS (SEQ ID NO: 7), KEGS (SEQ ID NO: 8), EGK, EAAAK (SEQ ID NO: 9), and AAPAKQE (SEQ ID NO: 10). In some embodiments, the second peptide linker comprises an amino acid sequence selected from the group consisting of AAPAKQEAAAPAKQEAAAPAKQEAAAPAPAAKAEAPAAAPAAKA (SEQ ID NO: 12) and AEAAAKEAAAKEAAAKA (SEQ ID NO: 13).

[0014] Also provided is an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding a chimeric polypeptide as described herein. In some aspects, the promoter operably linked to the polynucleotide sequence encoding the chimeric polypeptide comprises a constitutive promoter. In some aspects, the constitutive promoter is selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1a, hCAGG, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0015] In some embodiments, the promoter operably linked to the polynucleotide sequence encoding the chimeric polypeptide comprises an inducible promoter. In some embodiments, the inducible promoter comprises a minimal promoter and a response element selected from the group consisting of an NFkB response element, a CREB response element, an NFAT response element, an SRF response element 1, an SRF response element 2, an AP1 response element, a TCF-LEF response element promoter fusion, a hypoxia response element, a SMAD binding element, a STAT3 binding site, an inducer molecule responsive promoter, and tandem repeats thereof, an NFkB response element, a CREB response element, an NFAT response element, an SRF response element 1, an SRF response element 2, an AP1 response element, a TCF-LEF response element promoter fusion, a hypoxia responsive element, a SMAD binding element, a STAT3 binding site, an inducer molecule responsive promoter, and tandem repeats thereof.

[0016] In some aspects, the promoter operably linked to the polynucleotide sequence encoding the chimeric polypeptide is a synthetic promoter.

[0017] In some embodiments, the polynucleotide sequence encoding the chimeric polypeptide further encodes a 3' untranslated region (UTR) that comprises an mRNA-destabilizing element. In some embodiments, the mRNA destabilizing element is selected from the group consisting of an AU-rich element and a stem-loop destabilizing element.

[0018] Also provided is an expression system comprising (i) an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding a chimeric polypeptide as described herein, and (ii) a target expression cassette comprising an ITM-responsive promoter operably linked to a gene of interest. In some embodiments, the ITM-responsive promoter comprises a promoter sequence and a sequence that binds to the DNA-binding domain of the ITM. In some embodiments, the sequence that binds to the DNA-binding domain comprises one or more zinc finger binding sites. In some embodiments, the sequence that binds to the DNA-binding domain comprises four or more zinc finger binding sites. In some embodiments, the promoter sequence of the ITM-responsive promoter comprises a constitutive promoter sequence. In some embodiments, the constitutive promoter sequence is selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, EF1a, hCAGG, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb. In some aspects, the promoter sequence of the ITM-responsive promoter comprises a minimal promoter. In some embodiments, the ITM-responsive promoter comprises a synthetic promoter.

[0019] In some embodiments, the gene of interest encodes a therapeutic polypeptide. In some embodiments, the gene of interest encodes a polypeptide selected from the group consisting of cytokines, chemokines, homing molecules, growth factors, cell death regulators, coactivation molecules, tumor microenvironment modifiers a, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0020] In some embodiments, the expression system comprises a heterologous construct comprising both (i) an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding a chimeric polypeptide as described herein, and (ii) a target expression cassette.

[0021] In some embodiments, the expression system comprises (i) a first heterologous construct comprising an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding a chimeric polypeptide as described herein, and (ii) a second heterologous construct comprising a target expression cassette.

[0022] Also provided is an isolated cell comprising (i) an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding a chimeric polypeptide as described herein, or (ii) an expression system as described herein. In some embodiments, the isolated cell is a human cell. In some aspects, the isolated cell is a stem cell. In some aspects, the isolated cell is an immune cell. In some embodiments, the cell is selected from the group consisting of a T cell, a CD8+ T cell, a CD4+ T cell, a gamma delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a virus-specific T cell, a natural killer T (NKT) cell, a natural killer (NK) cell, a B cell, a tumor infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a bone marrow cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.

[0023] Also provided is a gene switch for inhibiting repression of a gene of interest, comprising a chimeric polypeptide as described herein and a ligand, wherein binding of the ligand to the degron induces degradation of the chimeric polypeptide, thereby inhibiting repression of the gene of interest. In some embodiments, the ligand of the gene switch comprises an immunomodulatory drug (IMiD) that promotes degradation of the chimeric polypeptide via the ubiquitin pathway. In some embodiments, the IMiD is an FDA approved drug. In some embodiments, the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide.

[0024] Also provided is a method for inhibiting repression of a gene of interest, comprising: (a) transforming a cell with an expression system comprising (i) an expression cassette encoding a chimeric polypeptide as described herein; and (ii) a target expression cassette comprising an ITM-responsive promoter operably linked to a gene of interest; (b) culturing the transformed cell under conditions suitable for expression of the chimeric polypeptide; and (c) inducing degradation of the chimeric polypeptide by contacting the transformed cell with a ligand that promotes degradation of the chimeric polypeptide. [Brief description of the drawings]

[0025] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings.

[0026] [Figure 1] Figure 2 shows the degradation efficiency of various degron-linked DNA binders following treatment with pomalidomide. The left column represents "No Drug" and the right column represents "+1uM Pom." [Diagram 2] Induction of reporter expression following degradation of various degron-linked transcriptional repressors is shown. The left column represents "no drug" and the right column represents "+1 uM Pom." [Diagram 3] 1 shows the difference in reporter expression in the absence of pomalidomide compared to after treatment with pomalidomide in cells expressing various degron-linked transcriptional repressors. [Figure 4A] Shown is a histogram plot of reporter expression for cells transduced with two degron-linked transcriptional repressor constructs (SB03758 and SB03759, respectively) in the presence or absence of pomalidomide, compared to untransduced cells (lacking reporter) and reporter cells not transduced with the repressor. [Figure 4B]Shown is a histogram plot of reporter expression for cells transduced with two degron-linked transcriptional repressor constructs (SB03758 and SB03759, respectively) in the presence or absence of pomalidomide, compared to untransduced cells (lacking reporter) and reporter cells not transduced with the repressor. [Diagram 5] Figure 2 shows the degradation efficiency of two degron-linked DNA binders, each containing an mRNA destabilization tag, after treatment with pomalidomide. The left column represents "no drug" and the right column represents "1 uM pomalidomide." [Figure 6A] Figure 1 shows reporter expression in cells expressing various degron-linked transcriptional repressors with a degron at the N-terminus in the presence or absence of pomalidomide. Shown is the fold change in geometric mean fluorescence intensity (gMFI) for reporter expression in cells expressing the reporter and the degron-linked transcriptional repressor compared to cells expressing only the reporter but not the degron-linked transcriptional repressor. The left column represents "No Drug" and the right column represents "1uM Pom". [Figure 6B] 6A shows reporter expression in cells expressing various degron-linked transcriptional repressors with a degron at their N-terminus in the presence or absence of pomalidomide. For cells expressing each of the degron-linked transcriptional repressors in FIG. 6A, the fold change in reporter expression is shown comparing expression in the presence of pomalidomide to expression in the absence of pomalidomide. [Figure 7A] Figure 1 shows reporter expression in cells expressing various degron-linked transcriptional repressors with a degron at the C-terminus in the presence or absence of pomalidomide. Shown is the fold change in geometric mean fluorescence intensity (gMFI) for reporter expression in cells expressing the reporter and the degron-linked transcriptional repressor compared to cells expressing only the reporter but not the degron-linked transcriptional repressor. The left column represents "No Drug" and the right column represents "1 uM Pom." [Figure 7B]7A-7D show reporter expression in cells expressing various degron-linked transcriptional repressors with a degron at their C-terminus in the presence or absence of pomalidomide. For cells expressing each of the degron-linked transcriptional repressors in FIG. 7A, the fold change in reporter expression is shown comparing expression in the presence of pomalidomide to expression in the absence of pomalidomide. [Figure 8] Figure 1 shows an IMiD-regulated on-switch controlling expression of IL-12 in a system containing a degron-linked repressor. The top panel shows IL-12 expression levels regulated by the three indicated degron-linked repressors in drug-free conditions or with a titration of pomalidomide (1 nM, 10 nM, 100 nM, and 1 uM). The bottom panel shows IL-12 levels expressed as a fraction of IL12 expression levels quantified in cells transduced with a constitutive IL-12 reporter. [Figure 9] Shown are on-to-off kinetics of the IMiD on-switch with 1 uM pomalidomide and 1 uM iberdomide. Shown are IL-12 expression levels in supernatants collected from cells at the indicated time points from 1 uM pomalidomide (top panel) or 1 uM iberdomide (bottom panel) treatment. SB04640 alone (right column) serves as a reporter-only control where IL-12 expression is constitutive and not regulated by a degron-linked repressor (left column). [Figure 10] Shows the on-to-off kinetics of the IMiD on-switch at 1uM pomalidomide, with additional time points at 12 and 16 hours. IL-12 expression levels in the supernatant are collected from cells at the indicated time points from 1uM pomalidomide treatment. Shows IL-12 expression levels normalized to IL-12 expression levels in cells transduced with the reporter control SB04640 only, where IL-12 expression is constitutive and not regulated by the degron-linked repressor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0028] The term "in vivo" refers to a process that takes place within a living organism.

[0029] As used herein, the term "mammal" includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0030] The term percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have a certain percentage of nucleotides or amino acid residues that are the same, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the entire length of the two sequences being compared.

[0031] For sequence comparison, typically, one sequence acts as a reference sequence to which test sequences are compared. When using sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0032] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., described below).

[0033] One example of an algorithm that is suitable for determining percent sequence identity and percent sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0034] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example an amount sufficient to inhibit transcriptional repression in a cell.

[0035] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0036] Degradable transcriptional repressor chimeric polypeptides The present disclosure provides a chimeric polypeptide comprising an inducible transcriptional regulator (ITM) and a degron. Generally, the degron is operably linked to the ITM to allow degron-based degradation of the chimeric polypeptide to regulate the activity of the ITM.

[0037] Inducible transcription factors The chimeric polypeptides described herein comprise an inducible transcriptional regulator (ITM). The ITM comprises a DNA binding domain and a transcriptional repressor domain.

[0038] In some embodiments, the transcriptional repressor domain can include, but is not limited to, a Krüppel-associated box (KRAB) repression domain, a histone deacetylase 4 (HDAC4) domain, a scleraxis (SCX) HLH domain, an inhibitor of DNA binding 1 (ID1) HLH domain, a HECT domain and RCC1-1-like domain-containing protein 2 (HERC2) Cyt-b5 domain, a twist-related protein 1 (TWST1) HLH domain, a homeobox protein Nkx-2.2 (NKX22) homeodomain, an inhibitor of DNA binding 1 (ID3) HLH domain, or a twist-related protein 2 (TWST2) HLH domain.

[0039] In some embodiments, the transcriptional repressor domain comprises a Kruppel-associated box (KRAB) repression domain. As used herein, "KRAB repression domain" refers to a wild type, variant, or segment of a KRAB domain that has the ability to repress transcription when operably linked to a DNA binding domain. C2H2 / Kruppel-type zinc finger (ZNF) proteins are transcription factors that comprise a KRAB domain and a C-terminal array of zinc fingers that bind to DNA. The KRAB domain is made up of a canonical subdomain A (KRAB-A) with or without auxiliary subdomains such as KRAB-B, KRAB-BL, KRAB-b, or KRAB-C. In some embodiments, the KRAB repressor domain comprises the KRAB domain of the zinc finger protein ZNF10. An exemplary ZNF10 sequence is provided as SEQ ID NO:1. The KRAB domain of ZNF10 comprises residues 2-81 of ZNF10 and is provided as SEQ ID NO:2. In some embodiments, the KRAB repressor domain comprises the amino acid sequence of SEQ ID NO:2.

[0040] In some embodiments, the KRAB repressor domain comprises minKRAB. "MinKRAB" refers to a 45 amino acid segment of the KRAB repression domain that is identified as the minimal repression domain present within the KRAB repression domain. An exemplary minKRAB repressor domain is provided as SEQ ID NO:3.

[0041] In some embodiments, the KRAB repressor domain comprises a KRAB repressor domain variant. As used herein, "KRAB repressor domain variant" refers to a KRAB domain (e.g., SEQ ID NO:2 or SEQ ID NO:3) that retains repressor activity but has one or more amino acid substitutions. In some embodiments, the KRAB repressor domain comprises the amino acid sequence of SEQ ID NO:2 and is a KRAB repressor domain variant and includes one or more amino acid substitutions selected from W27L, K28L, D31A, T32A, Q34A, Q35A, R39E, L43S, T57C, K58C, P59C, V61Y, I62Y, I62A,L63W, L63Y, L63E, R64F, R64W, R64E, L65F, L65E, L65W, E66V, K67F, G68F, and E69F.

[0042] In some embodiments, the KRAB repression domain includes a KRAB repressor domain variant of SEQ ID NO:2, which contains one or more amino acid substitutions selected from Q34A / Q35A, I62A, T57C / K58C / P59C, D31A / T32A, L63W / R64W / L65W, E66V, L63E / R64E / L65E, R64F / L65F, W27L / K28L KRAB, R39E, K67F / G68F / E69F, and V61Y / I62Y / L63Y.

[0043] In some embodiments, the transcriptional repressor domain comprises a histone deacetylase 4 (HDAC4) domain. An exemplary HDAC4 domain is provided as SEQ ID NO:4.

[0044] In some embodiments, the DNA binding domain comprises a zinc finger (ZF) protein domain. The inclusion of a ZF protein domain enables target nucleic acid binding by an inducible transcriptional regulator (ITM).

[0045] In some embodiments, the ZF protein domains are modular in design and are composed of zinc finger arrays (ZFAs).

[0046] A zinc finger array (ZFA) comprises multiple zinc finger protein motifs linked together, optionally separated by a flexible linker sequence. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA with specificity for any desired nucleic acid sequence. The ZF motifs can be directly adjacent to each other or separated by a flexible linker sequence. In some embodiments, a ZFA comprises an array, string, or chain of ZF motifs arranged in tandem. A ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 zinc finger motifs. A ZFA can have 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 zinc finger motifs.

[0047] A ZF protein domain can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more ZFAs. A ZF domain can have 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 10-15, 10-20, or 10-25 ZFAs. In some embodiments, the ZF protein domain comprises 1 to 10 ZFAs. In some embodiments, the ZF protein domain comprises at least 1 ZFA. In some embodiments, the ZF protein domain comprises at least 2 ZFAs. In some embodiments, the ZF protein domain comprises at least 3 ZFAs. In some embodiments, the ZF protein domain comprises at least 4 ZFAs. In some embodiments, the ZF protein domain comprises at least 5 ZFAs. In some embodiments, the ZF protein domain comprises at least 10 ZFAs.

[0048] In some embodiments, the ZFA comprises six zinc finger motifs. An exemplary ZF protein domain comprised of a ZFA containing six zinc finger motifs has the sequence: SRPGERPFQCRICMRNFSRRHGLDRHTRTHTGEKPFQCRICMRNFSDHSSLKRHLRTHTGSQKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDHSNLSRHLKTHTGSQKPFQCRICMRNFSQRSSLVRHLRTHTGEKPFQCRICMRNFSESGHLKRHLRTHLRGS (SEQ ID NO: 5) As shown in the figure.

[0049] Degron In some embodiments, the chimeric polypeptide described herein comprises a degron. In some embodiments, the degron has the ability to induce degradation of ITMs. In some embodiments, the degron includes HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα), GRR (residues 352-408 of human p105), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeats of SP2 and NB (influenza A or influenza B SP2-NB-SP2), RPB (four copies of residues 1688-1702 of yeast RPB), SPmix (tandem repeats of SP1 and SP2 (A These include SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein), NS2 (three copies of residues 79-93 of influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations), the APC / C degron, and COP1. These may include, but are not limited to, an E3 ligase-binding degron motif, a CRL4-Cdt2-binding PIP degron, an actinfilin-binding degron, a KEAP1-binding degron, a KLHL2- and KLHL3-binding degron, an MDM2-binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a plant hormone-dependent SCF-LRR-binding degron, an SCF ubiquitin ligase-binding phosphodegron, a plant hormone-dependent SCF-LRR-binding degron, a DSGxxS phospho-dependent degron, a Siah-binding motif, an SPOP SBC docking motif, or a PCNA-binding PIP box.

[0050] In some embodiments, the degron comprises a cereblon (CRBN) polypeptide substrate domain capable of binding to CRBN in response to an immunomodulatory drug (IMiD), thereby promoting degradation of ITM via the ubiquitin pathway. In some embodiments, the CRBN polypeptide substrate domain may include, but is not limited to, IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787 or ZN827, or fragments thereof, capable of drug-induced binding of CRBN. In some embodiments, the CRBN polypeptide substrate domain comprises a chimeric fusion product of a native CRBN polypeptide sequence. In some embodiments, the CRBN polypeptide substrate domain comprises the amino acid sequence: FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 6) Degrons include IKZF3 / ZFP91 / IKZF3 chimeric fusion products having the following structure: Degrons are described in more detail in International Application Publication No. WO 2019 / 089592 A1, which is incorporated by reference herein for all purposes.

[0051] Peptide Linker In some embodiments, the chimeric polypeptide comprises at least one peptide linker.

[0052] A peptide linker can be any polypeptide sequence that separates two polypeptide domains (e.g., an inducible transcriptional regulator and a degron) without interfering with the function of the polypeptide domains. Examples of peptide linkers include GSGSGSGS (SEQ ID NO:7), KEGS (SEQ ID NO:8), EGK, EAAAK (SEQ ID NO:9), AAPAKQE (SEQ ID NO:10), GSGSGSGSGGAEAAAKEAAAKEAAAKA (SEQ ID NO:11, referred to herein as "concatenated Max Jen linker" or "ConMJ"), AAPAKQEAAAPAKQEAAAPAKQEAAAPAPAAKAEAPAAAPAAKA (SEQ ID NO:12, referred to herein as "ecpd"), AEAAAKEAAAKEAAAKA (SEQ ID NO:13), and GGGGSGGT SEQ ID NO:60).

[0053] In some embodiments, the transcriptional repressor domain and the DNA binding domain are separated by a peptide linker.

[0054] In some embodiments, the inducible transcriptional regulator (ITM) and the degron are separated by a peptide linker. In some embodiments, the peptide linker between the ITM and the degron comprises an amino acid sequence selected from GSGSGSGS (SEQ ID NO: 7), KEGS (SEQ ID NO: 8), EGK, EAAAK (SEQ ID NO: 9), AAPAKQE (SEQ ID NO: 10), and GGGGSGGT (SEQ ID NO: 60). In some embodiments, the peptide linker between the ITM and the degron comprises the amino acid sequence GGGGSGGT (SEQ ID NO: 60).

[0055] In some embodiments, the peptide linker between the ITM and the degron includes an amino acid sequence selected from GSGSGSGSGGAEAAAKEAAAKEAAAKA (SEQ ID NO:11, referred to herein as "concatenated Max Jen linker" or "ConMJ"), AAPAKQEAAAPAKQEAAAPAKQEAAAPAPAAKAEAPAAAPAAKA (SEQ ID NO:12, referred to herein as "ecpd"), AEAAAKEAAAKEAAAKA (SEQ ID NO:13), GSGSGSGS (SEQ ID NO:7), KEGS (SEQ ID NO:8), and EGK.

[0056] In some embodiments, the peptide linker between the ITM and the degron includes an amino acid sequence selected from the group consisting of AAPAKQEAAAPAKQEAAAPAKQEAAAPAPAAKAEAPAAAPAAKA (SEQ ID NO: 12, referred to herein as "ecpd") and AEAAAKEAAAKEAAAKA (SEQ ID NO: 13).

[0057] In some embodiments, the transcriptional repressor domain, the DNA binding domain, and the degrons are each separated by a peptide linker. In some embodiments, the transcriptional repressor domain, the DNA binding domain, and the degrons are each separated by a separate peptide linker.

[0058] Genetic Switch Also provided herein is a gene switch for inhibiting the repression of a gene of interest.The gene switch may comprise (a) a chimeric polypeptide comprising a degron and an inducible transcription regulator capable of repressing the transcription of a gene of interest, and (b) a ligand that binds to the degron of the chimeric polypeptide and induces the degradation of the chimeric polypeptide.The degradation of the chimeric polypeptide releases the repressor activity of the inducible transcription regulator.

[0059] In some embodiments, the ligand comprises an immunomodulatory drug (IMiD) that promotes degradation of the chimeric polypeptide via the ubiquitin pathway.

[0060] In some embodiments, the IMiD is an FDA approved drug.

[0061] In some embodiments, IMiDs can include, but are not limited to, thalidomide, lenalidomide, or pomalidomide.

[0062] Isolated polynucleotide molecules and expression cassettes Also provided herein are polynucleotide molecules (e.g., isolated polynucleotide molecules) and expression cassettes encoding the chimeric polypeptides described herein. In some embodiments, the present disclosure provides an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding a chimeric polypeptide.

[0063] "Isolated" nucleic acid molecule or polynucleotide refers to a polynucleotide molecule, such as DNA or RNA, that is removed from its natural environment. For example, a polynucleotide encoding a chimeric polypeptide contained in a heterologous construct is considered to be isolated. Further examples of isolated polynucleotides include recombinant polynucleotides that are maintained in heterologous host cells, or polynucleotides that are purified (partially or substantially) in solution. Isolated polynucleotides also include polynucleotide molecules that are normally contained in cells that contain the polynucleotide molecule, but where the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0064] Isolated polynucleotide molecules include, but are not limited to, cDNA polynucleotides, RNA polynucleotides, RNAi oligonucleotides (e.g., siRNA, miRNA, antisense oligonucleotides, shRNA, etc.), mRNA polynucleotides, circular plasmids, linear DNA fragments, vectors, minicircles, ssDNA, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), and oligonucleotides.

[0065] In some embodiments, an isolated polynucleotide molecule can include, but is not limited to, DNA, cDNA, RNA, mRNA, and naked plasmids (linear or circular).

[0066] By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to the reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, either individually between the residues in the reference sequence, or interspersed in one or more consecutive groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the present invention can be determined in a conventional manner using known computer programs.

[0067] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide having a set of nucleic acid elements that allow for transcription of a particular polynucleotide in a target cell. Expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the expression cassette portion of an expression vector contains, among other sequences, the nucleic acid sequence to be transcribed and a promoter.

[0068] In some embodiments, the expression cassette comprising a polynucleotide sequence encoding a chimeric polypeptide further encodes a 3' untranslated region (UTR) comprising an mRNA destabilizing element operably linked to the polynucleotide sequence encoding the chimeric polypeptide. In some embodiments, the mRNA destabilizing element comprises an AU-rich element and / or a stem-loop destabilizing element (SLDE).

[0069] In some embodiments, the mRNA destabilizing element comprises an AU-rich element. In some embodiments, the AU-rich element comprises at least two overlapping motifs of the sequence ATTTA (SEQ ID NO: 14). In some embodiments, the AU-rich element comprises ATTTATTTATTTATTTATTTA (SEQ ID NO: 15).

[0070] In some embodiments, the mRNA destabilizing element comprises a stem-loop destabilizing element (SLDE). In some embodiments, the SLDE comprises CTGTTTAATATTTAAACAG (SEQ ID NO: 16).

[0071] In some embodiments, the mRNA destabilizing element comprises at least one AU-rich element and at least one SLDE. As used herein, "AuSLIDE" refers to an AU-rich element operably linked to a stem-loop destabilizing element (SLDE). An exemplary AuSLIDE sequence is provided as SEQ ID NO: 17. In some embodiments, the mRNA destabilizing element comprises 2X AuSLIDE. An exemplary AuSLDE sequence is provided as SEQ ID NO: 18.

[0072] In some embodiments, the present disclosure provides an expression cassette comprising a polynucleotide encoding a chimeric polypeptide as described herein. In some embodiments, the present disclosure provides an expression system comprising a first expression cassette encoding a chimeric polypeptide as described above, and a target expression cassette comprising an ITM-responsive promoter operably linked to a gene of interest.

[0073] "Target expression cassette" refers to an expression cassette that includes a gene having inducible transcriptional regulator (ITM)-controllable expression. Expression is controlled by the ITM based on the presence of a ligand (e.g., pomalidomide).

[0074] The isolated polynucleotide molecules and heterologous constructs as described herein are engineered polynucleotide molecules. An "engineered polynucleotide" is a polynucleotide that does not occur in nature. However, it should be understood that while an engineered polynucleotide does not occur in nature as a whole, it may contain naturally occurring nucleotide sequences. In some embodiments, an engineered polynucleotide contains nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered polynucleotide contains murine nucleotide sequences, bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences. The term "engineered polynucleotide" includes recombinant and synthetic nucleic acids. "Recombinant polynucleotide" refers to a molecule that is constructed by joining nucleotide molecules and, in some embodiments, is capable of replicating in a living cell. "Synthetic polynucleotide" refers to a molecule that is amplified or synthesized chemically or by other means. Synthetic polynucleotides include those that are chemically modified or otherwise modified but are capable of base pairing with naturally occurring nucleotide molecules. Modifications include, but are not limited to, one or more modified internucleotide linkages and non-naturally occurring nucleic acids. Modifications are described in more detail in U.S. Patent No. 6,673,611 and U.S. Patent Application Publication No. 2004 / 0019001, each of which is incorporated by reference in its entirety. The modified internucleotide linkages can be phosphorodithioate or phosphorothioate linkages. The non-natural nucleic acids can be locked nucleic acids (LNAs), peptide nucleic acids (PNAs), glycol nucleic acids (GNAs), phosphorodiamidate morpholino oligomers (PMOs or "morpholinos"), and threose nucleic acids (TNAs). The non-natural nucleic acids are described in more detail in International Application Publication No. WO 1998 / 039352, U.S. Application Publication No. 2013 / 0156849, and U.S. Patent Nos. 6,670,461; 5,539,082; and 5,185,444, each of which is incorporated by reference in its entirety.Recombinant and synthetic polynucleotides also include molecules resulting from the replication of any of the foregoing. The engineered polynucleotides of the present disclosure may be encoded by a single molecule (e.g., contained in the same plasmid or other vector) or by multiple distinct molecules (e.g., multiple distinct independently replicating molecules).

[0075] The engineered polynucleotides of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the engineered nucleic acid constructs are produced using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, DGet al. Nature Methods, 343-345, 2009; and Gibson, DGet al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically uses three enzyme activities in a single tube reaction: a 5' exonuclease, a Y-extension activity of DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity bites back the 5' end sequence, exposing the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed region. DNA ligase then seals the nicks and covalently links the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate assembly, thus resulting in a higher rate of correct assembly. In some embodiments, engineered nucleic acid constructs are produced using IN-FUSION® cloning (Clontech).

[0076] promoter As used herein, a "promoter" refers to a control region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. A promoter may also contain subregions to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. A promoter may be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or drives the transcription of the nucleic acid sequence that it regulates. As used herein, a promoter is considered to be "operably linked" when it is in the correct functional location and orientation relative to the nucleic acid sequence that it regulates, to control (drive) the transcription initiation and / or expression of that sequence.

[0077] A promoter may be a promoter naturally associated with a gene or sequence, such as may be obtained by isolating the 5' non-coding sequence located upstream of the coding segment of a given gene or sequence. Such promoters may be referred to as "endogenous". In some embodiments, a coding nucleic acid sequence may be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the coded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring", such as those that contain different elements and / or mutations of different transcriptional regulatory regions that modify expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including polymerase chain reaction (PCR) (see, for example, U.S. Pat. Nos. 4,683,202 and 5,928,906).

[0078] As used herein, an "inducible promoter" refers to a promoter that is characterized by modulating (e.g., initiating or activating) transcriptional activity when in the presence of, affected by, or contacted by a signal. The signal may be an endogenous or usually exogenous condition (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a protein (e.g., a cytokine) that contacts the inducible promoter in such a way that it is active in modulating transcriptional activity from the inducible promoter. Activation of transcription may include acting directly on the promoter to drive transcription, or indirectly on the promoter by inactivating a repressor that is preventing the promoter from driving transcription. Conversely, inactivation of transcription may include acting directly on the promoter to prevent transcription, or indirectly on the promoter by activating a repressor that then acts on the promoter.

[0079] As used herein, a promoter is "responsive to" or "regulated by" a local tumor condition (e.g., inflammation or hypoxia) or signal when in the presence of that condition or signal, transcription from the promoter is activated or inactivated, increased or decreased. In some embodiments, a promoter contains a response element. A "response element" is a short sequence of DNA within the promoter region that binds to a specific molecule (e.g., a transcription factor) that modulates (regulates) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, but are not limited to, phloretin tunable regulatory element (PEACE), zinc finger DNA binding domain (DBD), interferon gamma activating sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar; 17(3): 121-34, incorporated herein by reference), interferon stimulated response element (ISRE) (Han, KJ et al. J Biol Chem. 2004 Apr 9; 279(15): 15652-61, incorporated herein by reference), NF-kappa B response element (Wang, V. et al. Cell Reports. 2012; 2(4): 824-839, incorporated herein by reference), and STAT3 response element (Zhang, D. et al. J of Biol Chem. 1996; 271: 9503-9509, incorporated herein by reference). Other response elements are encompassed herein. Response elements can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase the sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2x, 3x, 4x, 5x, etc. to indicate the number of repeats present.

[0080] Non-limiting examples of responsive promoters (also called "inducible promoters") (e.g., TGF-beta responsive promoters) are listed in Table 1, which not only shows the design of the promoter and transcription factor, but also the effect of the transcription factor (TF) and the inducer molecule towards transgene transcription (T) (B, binding; D, dissociation; nd, not determined) (A, activation; DA, deactivation; DR, derepression) (see Horner, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Non-limiting examples of components of inducible promoters include those shown in Table 2.

[0081] [Table 1] TIFF2024513232000003.tif252169TIFF2024513232000004.tif100169

[0082] [Table 2]

[0083] As used herein, a "constitutive promoter" refers to a promoter that allows for continuous or unregulated transcriptional activity.

[0084] Other non-limiting examples of constitutive promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EF1a) promoter and EF1a variants hEF1aV1 and hEF1aV2, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the spleen focus forming virus (SFFV) promoter, the simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter. Examples of constitutive promoter amino acid sequences are shown in Table 3.

[0085] [Table 3] TIFF2024513232000007.tif242159TIFF2024513232000008.tif242162TIFF2024513232 000009.tif242158TIFF2024513232000010.tif242158TIFF2024513232000011.tif55170

[0086] A promoter operably linked to a polynucleotide encoding a chimeric polypeptide. In some embodiments, the present disclosure provides a heterologous construct comprising a promoter operably linked to a polynucleotide sequence encoding a chimeric polypeptide as described herein.

[0087] In some embodiments, the promoter operably linked to the polynucleotide sequence encoding the chimeric polypeptide comprises a constitutive promoter, an inducible promoter, and / or a synthetic promoter.

[0088] In some embodiments, the promoter operably linked to the polynucleotide encoding the chimeric polypeptide comprises a constitutive promoter. Examples of constitutive promoters are shown in Table 3. In some embodiments, constitutive promoters can include, but are not limited to, CMV, EFS, SFFV, SV40, MCD, PGK, UbC, EF1a (e.g., wild type or variant such as hEF1aV1 or hEF1aV2), hCAGG, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0089] Heterologous constructs In some embodiments, the polynucleotide molecule as described herein is included in a heterologous construct. The term "vector" or "expression vector" is synonymous with "heterologous construct" and refers to a polynucleotide molecule used to introduce and induce expression of one or more genes operatively associated with the construct in a target cell. This term includes the construct as a self-replicating nucleic acid structure as well as the vector integrated into the genome of the host cell into which it is introduced. A heterologous construct as described herein includes an expression cassette. In some embodiments, provided herein is a heterologous construct comprising an expression cassette comprising a polynucleotide molecule encoding a chimeric polypeptide as described herein. In some embodiments, the heterologous construct further comprises a target expression cassette comprising an inducible transcriptional regulator responsive (ITM responsive) promoter. In some embodiments, provided herein is a first heterologous construct comprising an expression cassette comprising a polynucleotide molecule encoding a chimeric polypeptide, and a second heterologous construct comprising a target expression cassette comprising an inducible transcriptional regulator responsive (ITM responsive) promoter.

[0090] Expression systems containing inducible transcription factor-responsive promoters In some embodiments, provided herein is an expression system comprising a first expression cassette encoding a chimeric polypeptide as described herein, and a target expression cassette comprising an inducible transcription factor-responsive (ITM-responsive) promoter operably linked to a gene of interest.

[0091] Gene of interest In some embodiments, the gene of interest comprises a therapeutic protein. A therapeutic protein is any polypeptide that provides a clinical benefit when provided to a subject. A therapeutic protein may be provided by administering a polypeptide, administering a cell capable of expressing a polypeptide, or administering a polynucleotide encoding the polypeptide.

[0092] In some embodiments, the gene of interest encodes a polypeptide selected from a cytokine, a chemokine, a homing molecule, a growth factor, a cell death regulator, a co-activator molecule, a tumor microenvironment modifier a, a receptor, a ligand, an antibody, a peptide, and an enzyme.

[0093] In some embodiments, the gene of interest comprises a cytokine, which may include, but is not limited to, IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-gamma, and TNF-alpha.

[0094] In some embodiments, the gene of interest comprises a cell death regulator, including cell death-inducing polypeptides and cell survival polypeptides.

[0095] In some embodiments, the gene of interest comprises a cell death-inducing polypeptide. Examples of cell death-inducing polypeptides include caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2-interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1 associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondria-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53- Examples of the cell death-inducing polypeptide include upregulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Art, TNF-related cell death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), vesicular zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, and purine nucleoside phosphorylase. In some embodiments, the cell death-inducing polypeptide is caspase 9, or a functional cleavage thereof. In some embodiments, the cell death-inducing polypeptide comprises the caspase 9 amino acid sequence of SEQ ID NO: 49. In some embodiments, the cell death-inducing polypeptide is diphtheria toxin fragment A (DTA). In some embodiments, the cell death-inducing polypeptide comprises the DTA amino acid sequence of SEQ ID NO: 50. In some embodiments, the cell death-inducing polypeptide is Granzyme B. In some embodiments, the cell death-inducing polypeptide comprises the Granzyme B amino acid sequence of SEQ ID NO: 51. In some embodiments, the cell death-inducing polypeptide is Bax. In some embodiments, the cell death-inducing polypeptide comprises the Bax amino acid sequence of SEQ ID NO: 52.

[0096] In some embodiments, the gene of interest comprises cell survival polypeptide.Examples of cell survival polypeptide include XIAP, Bcl-2, Bcl-xL, Bcl-w, Bcl-2-related protein A1 (BCL2A1), Mcl-1, FLICE-like inhibitory protein (c-FLIP) and adenovirus E1B-19K protein.In some embodiments, the cell survival polypeptide is XIAP.In some embodiments, the cell survival polypeptide comprises the XIAP amino acid sequence of SEQ ID NO:53.

[0097] Inducible transcription factor-responsive promoters In some embodiments, the present disclosure provides a polynucleotide molecule encoding a gene of interest operably linked to an inducible transcriptional regulator responsive promoter (ITM responsive promoter). In some embodiments, the ITM responsive promoter is a synthetic promoter that responds to a chimeric polypeptide comprising an ITM, and the repression of the gene of interest by the ITM can be controlled by the presence of a ligand such as pomalidomide.

[0098] In some embodiments, an ITM-responsive promoter comprises a promoter sequence and an ITM-binding domain that is specifically recognized by an inducible transcription regulator (ITM) as described herein. As used herein, a "core promoter sequence" refers to a portion of a promoter that comprises a core (i.e., "minimal") promoter sequence that is sufficient to interact with RNA polymerase II and initiate transcription. In some embodiments, an ITM-responsive promoter comprises a synthetic promoter that comprises a core promoter sequence (as provided by the promoter sequence) and a transcription regulator response sequence (as provided by the binding domain) that does not naturally co-occur within the promoter region.

[0099] A binding domain may comprise one or more zinc finger binding sites. A zinc finger binding site is a polynucleotide sequence having the ability to bind to a zinc finger protein domain (e.g., the zinc finger protein domain of SEQ ID NO: 5). A binding domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more zinc finger binding sites. An exemplary zinc finger binding site comprises GGCGTAGCCGATGTCGCG (SEQ ID NO: 54). In some embodiments, a binding domain comprises one zinc finger binding site. In some embodiments, a binding domain comprises two or more zinc finger binding sites. The zinc finger binding sites may be separated by a DNA linker. The DNA linker may be 5-40, 5-30, 10-40, 10-30 base pairs in length in some embodiments. In some embodiments, a binding domain comprises two zinc finger binding sites. In some embodiments, a binding domain comprises three zinc finger binding sites. In some embodiments, the binding domain comprises four zinc finger binding sites. An exemplary binding domain that comprises zinc finger binding sites has the sequence: cgggtttcgtaacaatcgcatgaggattcgcaacgccttcGGCGTAGCCGATGTCGCGctcccgtctcagtaaaggtcGGCGTAGCCGATGTCGCGcaatcggactgccttcgtacGGCGTAGCCGATGTCGCGcgtatcagtcgcctcggaacGGCGTAGCCGATGTCGCG (SEQ ID NO: 55) The binding domain of SEQ ID NO:55 contains four binding sites, each of which binds to the zinc finger protein domain of SEQ ID NO:5, and each of the binding sites is separated by a DNA linker.

[0100] In some embodiments, the core promoter sequence comprises a minimal promoter. In some embodiments, the core promoter sequence is derived from a promoter selected from minP, minCMV, YB_TATA, and minTK. An exemplary core promoter sequence comprises TCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO: 56).

[0101] In some embodiments, the core promoter sequence comprises a constitutive promoter sequence. Examples of constitutive promoter sequences are shown in Table 3. In some embodiments, the constitutive promoter sequence can include, but is not limited to, CMV, EFS, SFFV, SV40, MCD, PGK, UbC, EF1a, hCAGG, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0102] An exemplary ITM responsive promoter has the sequence: cgggtttcgtaacaatcgcatgaggattcgcaacgccttcGGCGTAGCCGATGTCGCGctcccgtctcagtaaaggtcGGCGTAGCCGATGTCGCGcaatcggactgccttcgtacGGCGTAGCC GATGTCGCGcgtatcagtcgcctcggaacGGCGTAGCCGATGTCGCGcattcgtaagaggctcactctcccttacacggagtggataACTAGTTCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO: 57) Includes.

[0103] Another exemplary ITM responsive promoter has the sequence: cggggtttcgtaacaatcgcatgaggattcgcaacgccttcGGCGTAGCCGATGTCGCGctcccgtctcagtaaaggtcGGCGTAGCCGATGTCGCGcaatcggactgccttcgtacGGCGTAGCCGATGTCGCGcgtatcagtcgcctcggaacGGCGTAGCCGATGTCGCGcattcgtaagaggctcactctcccttacacggagtggataACTAGTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCG CCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGT GCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC (Sequence number 67) Includes.

[0104] Multicistronic and multiple promoter systems In some embodiments, the engineered polynucleotide or construct of the present disclosure is configured to produce multiple polypeptides. For example, the polynucleotide may be configured to produce two different polypeptides. The polynucleotide molecule may be configured to produce a polypeptide comprising a chimeric protein as described herein, and a polypeptide of interest expressed under the control of a promoter responsive to the chimeric protein.

[0105] In some embodiments, a chimeric polypeptide as described herein and a gene of interest that can be transcriptionally repressed by the chimeric polypeptide may be encoded by the same polynucleotide molecule or a heterologous construct.

[0106] In some embodiments, the engineered nucleic acid can be multicistronic, i.e., two or more separate polypeptides (e.g., multiple exogenous polynucleotides or effector molecules) can be produced from a single transcription product. The engineered nucleic acid can be multicistronic through the use of various linkers, for example, a polynucleotide sequence encoding a first exogenous polynucleotide can be linked to a nucleotide sequence encoding a second exogenous polynucleotide in a 5' to 3' orientation, such as first gene:linker:second gene. The linker polynucleotide sequence can encode one or more 2A ribosomal skipping elements, such as T2A. Other 2A ribosomal skipping elements include, but are not limited to, E2A, P2A, and F2A. The 2A ribosomal skipping elements allow the production of separate polypeptides encoded by the first and second genes to be produced during translation. The linker can encode a cleavable linker polypeptide sequence, such as a furin or TEV cleavage site, and following expression, the cleavable linker polypeptide is cleaved, thereby producing the separate polypeptides encoded by the first and second genes. The cleavable linker can include a polypeptide sequence, such as a flexible linker (e.g., a Gly-Ser-Gly sequence), which further facilitates cleavage.

[0107] The linker can encode an internal ribosome entry site (IRES) such that separate polypeptides encoded by the first and second genes are produced during translation. The linker can encode a splice acceptor, such as a viral splice acceptor.

[0108] The linker can be a combination of linkers, such as a furin-2A linker, which can produce a separate polypeptide through 2A ribosomal skipping, followed by further cleavage of the furin site, which allows complete removal of the 2A residue. In some embodiments, the combination of linkers can include a furin sequence, a flexible linker, and a 2A linker. Thus, in some embodiments, the linker comprises a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker comprises a furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0109] In general, a multicistronic system can express any number of genes or portions thereof using any number or combination of linkers (e.g., an engineered nucleic acid can encode a first, second, and third effector molecule, each separated by a linker, thereby producing separate polypeptides encoded by the first, second, and third effector molecules).

[0110] As used herein, "linker" can refer to a peptide linker that connects a first polypeptide sequence and a second polypeptide sequence, or it can refer to a multicistronic linker as described above.

[0111] Post-transcriptional regulatory elements In some embodiments, the engineered polynucleotide molecules of the present disclosure include a post-transcriptional regulatory element (PRE). A PRE can regulate RNA stability, for example, by destabilizing the RNA (e.g., AU-slide as described above) or by stabilizing the RNA. In some embodiments, a PRE can enhance gene expression through enabling tertiary RNA structure stability and 3' end formation. Non-limiting examples of PREs include Hepatitis B virus PRE (HPRE) and Woodchuck Hepatitis virus PRE (WPRE). In some embodiments, a post-transcriptional regulatory element includes a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, a WPRE includes alpha, beta, and gamma components of a WPRE element. In some embodiments, a WPRE includes an alpha component of a WPRE element. Examples of WPRE sequences include SEQ ID NO:58 and SEQ ID NO:59.

[0112] Engineered cells Also provided herein are cells and methods of producing cells comprising one or more polynucleotide molecules, expression cassettes, or constructs of the present disclosure. These cells are referred to herein as "engineered cells." Typically, these cells, comprising one or more engineered nucleic acids, do not occur in nature. In some embodiments, the cells are isolated cells that recombinantly express one or more engineered polynucleotides. In some embodiments, the engineered polynucleotides are expressed from one or more vectors or selected loci from the genome of the cell. In some embodiments, the cells are engineered to contain a polynucleotide comprising a promoter operably linked to a nucleotide sequence.

[0113] The engineered cells of the present disclosure can include one or more engineered polynucleotides integrated into the genome of the cell (e.g., an expression system including an inducible transcriptional regulator (ITM) responsive promoter). The engineered cells can include one or more engineered polynucleotides that are capable of expression without being integrated into the genome of the cell, e.g., engineered in a transient expression system, such as a plasmid or mRNA.

[0114] Engineered cell types The engineered cells of the present disclosure can be human cells. The engineered cells can be human primary cells. The engineered primary cells can be any somatic cell. The engineered primary cells can be any stem cell. In some embodiments, the engineered cells are derived from a subject. In some embodiments, the engineered cells are allogeneic with respect to the subject.

[0115] The engineered cells of the present disclosure can be isolated from a subject, such as a subject known or suspected to have cancer. Cell isolation methods are known to those skilled in the art and include, but are not limited to, cell surface marker expression-based sorting techniques, such as FACS sorting, positive isolation techniques, and negative isolation, magnetic isolation, and combinations thereof. The engineered cells can be allogeneic with respect to the subject to whom the treatment is being administered. The allogeneic modified cells can be HLA-matched to the subject to whom the treatment is being administered. The engineered cells can be cultured cells, such as ex vivo cultured cells. The engineered cells can be ex vivo cultured cells, such as primary cells isolated from a subject. The cultured cells can be cultured with one or more cytokines.

[0116] In some embodiments, engineered cells of the present disclosure may include, but are not limited to, T cells (e.g., CD8+ T cells, CD4+ T cells, or gamma delta T cells), cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages (e.g., M1 or M2 macrophages), monocytes, dendritic cells, erythrocytes, platelet cells, neurons, oligodendrocytes, astrocytes, placode-derived cells, Schwann cells, cardiomyocytes, endothelial cells, nodal myocytes, microglial cells, hepatocytes, bile duct cells, beta cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0117] In some embodiments, the engineered cells of the present disclosure are T cells (e.g., CD8+ T cells, CD4+ T cells, or gamma-delta T cells). In some embodiments, the engineered cells of the present disclosure are cytotoxic T lymphocytes (CTLs). In some embodiments, the engineered cells of the present disclosure are regulatory T cells. In some embodiments, the engineered cells of the present disclosure are natural killer T (NKT) cells. In some embodiments, the engineered cells of the present disclosure are natural killer cells (NKs). In some embodiments, the engineered cells of the present disclosure are B cells. In some embodiments, the engineered cells of the present disclosure are tumor infiltrating lymphocytes (TILs). In some embodiments, the engineered cells of the present disclosure are innate lymphoid cells. In some embodiments, the engineered cells of the present disclosure are mast cells. In some embodiments, the engineered cells of the present disclosure are eosinophils. In some embodiments, the engineered cells of the present disclosure are basophils. In some embodiments, the engineered cells of the present disclosure are neutrophils. In some embodiments, the engineered cells of the present disclosure are bone marrow cells. In some embodiments, the engineered cell of the present disclosure is a macrophage, e.g., an M1 macrophage or an M2 macrophage). In some embodiments, the engineered cell of the present disclosure is a monocyte. In some embodiments, the engineered or isolated cell of the present disclosure is a dendritic cell. In some embodiments, the engineered cell of the present disclosure is a red blood cell. In some embodiments, the engineered cell of the present disclosure is a platelet cell. In some embodiments, the cell of the present disclosure is a neuron. In some embodiments, the cell of the present disclosure is a microglial cell. In some embodiments, the cell of the present disclosure is an oligodendrocyte. In some embodiments, the cell of the present disclosure is an astrocyte. In some embodiments, the cell of the present disclosure is a placode-derived cell. In some embodiments, the engineered cell of the present disclosure is a Schwann cell. In some embodiments, the engineered cell of the present disclosure is a cardiomyocyte. In some embodiments, the engineered cell of the present disclosure is an endothelial cell. In some embodiments, the engineered cell of the present disclosure is a nodal myocyte. In some embodiments, the engineered cell of the present disclosure is a microglial cell.In some embodiments, the engineered cells of the present disclosure are hepatocytes. In some embodiments, the engineered cells of the present disclosure are cholangiocytes. In some embodiments, the engineered cells of the present disclosure are beta cells. In some embodiments, the engineered cells of the present disclosure are human embryonic stem cells (ESCs). In some embodiments, the engineered cells of the present disclosure are ESC-derived cells. In some embodiments, the engineered cells of the present disclosure are pluripotent stem cells. In some embodiments, the engineered cells of the present disclosure are mesenchymal stromal cells (MSCs). In some embodiments, the engineered cells of the present disclosure are induced pluripotent stem cells (iPSCs). In some embodiments, the engineered cells of the present disclosure are iPSC-derived cells. In some embodiments, the engineered cells are autologous. In some embodiments, the engineered cells are allogeneic. In some embodiments, the engineered cells of the present disclosure are CD34+ cells, CD3+ cells, CD8+ cells, CD16+ cells, and / or CD4+ cells.

[0118] In some embodiments, the cell of the present disclosure is a tumor cell selected from an adenocarcinoma cell, a bladder tumor cell, a brain tumor cell (e.g., a glioma cell or a glioblastoma cell), a breast tumor cell, a cervical tumor cell, a colorectal tumor cell, an esophageal tumor cell, a glioma cell, a kidney tumor cell, a liver tumor cell, a lung tumor cell, a melanoma cell, a mesothelioma cell, an ovarian tumor cell, a pancreatic tumor cell, a prostate tumor cell, a skin tumor cell, a thyroid tumor cell, and a uterine tumor cell.

[0119] Also provided herein are methods that include culturing the engineered cells of the present disclosure. Methods for culturing the engineered cells described herein are known. Those skilled in the art will recognize that the culture conditions will depend on the particular engineered cells of interest. Those skilled in the art will recognize that the culture conditions will depend on the particular downstream use of the engineered cells, for example, the particular culture conditions for the subsequent administration of the engineered cells to a subject.

[0120] How to Manipulate Cells Also provided herein are compositions and methods for engineering cells with any of the polynucleotide molecules or constructs as described herein.

[0121] Generally, cells are engineered by introducing (i.e., delivering) one or more polynucleotides of the present disclosure.Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated delivery, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means.Those skilled in the art will understand that the choice of delivery method may depend on the specific cell type to be engineered.

[0122] Viral-mediated delivery Viral vector-based delivery platforms can be used to engineer cells. In general, viral vector-based delivery platforms engineer cells through introduction (i.e., delivery) into host cells. For example, viral vector-based delivery platforms can engineer cells through introduction of any of the nucleic acids described herein. Viral vector-based delivery platforms can be nucleic acids, and thus engineered nucleic acids can also include engineered virus-derived nucleic acids. Such engineered virus-derived nucleic acids can also be referred to as recombinant viruses or engineered viruses.

[0123] A viral vector-based delivery platform can encode two or more engineered nucleic acids, genes, or transgenes within the same nucleic acid. For example, a nucleic acid from an engineered virus, such as a recombinant virus or engineered virus, can encode one or more transgenes, including but not limited to any of the engineered nucleic acids described herein, that encode one or more effector molecules. The one or more transgenes that encode one or more effector molecules can be configured to express one or more effector molecules. A viral vector-based delivery platform can encode one or more genes, referred to as cis-acting elements or cis-acting genes, in addition to one or more transgenes (e.g., transgenes encoding one or more effector molecules), such as viral genes necessary for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerase, viral, etc.).

[0124] A viral vector-based delivery platform can include two or more viral vectors, such as separate viral vectors encoding engineered nucleic acids, genes, or transgenes, as described herein and referred to as trans-acting elements or genes. For example, a helper-dependent viral vector-based delivery platform can provide additional genes required for viral infectivity and / or virus production on one or more additional separate vectors in addition to a vector encoding one or more effector molecules. One viral vector can deliver two or more engineered nucleic acids, such as one vector delivering engineered nucleic acids configured to produce two or more effector molecules. Two or more viral vectors can deliver two or more engineered nucleic acids, such as two or more vectors delivering one or more engineered nucleic acids configured to produce one or more effector molecules. The number of viral vectors used can depend on the packaging capacity of the viral vector-based vaccine platform described above, and one skilled in the art can select the appropriate number of viral vectors.

[0125] In general, any of the viral vector-based systems can be used for in vitro production of molecules such as effector molecules, or can be used in in vivo and ex vivo gene therapy procedures, for example, to deliver engineered nucleic acids encoding one or more effector molecules in vivo. Selection of an appropriate viral vector-based system depends on a variety of factors, such as the size of the cargo / payload, the immunogenicity of the viral system, the target cells of interest, the strength and timing of gene expression, and other factors that will be appreciated by those skilled in the art.

[0126] Viral vector-based delivery platform can be RNA-based virus or DNA-based virus.Exemplary viral vector-based delivery platform includes but is not limited to herpes simplex virus, adenovirus, measles virus, influenza virus, Indiana vesiculovirus, Newcastle disease virus, vaccinia virus, poliovirus, myxoma virus, reovirus, mumps virus, Maraba virus, rabies virus, rotavirus, hepatitis virus, rubella virus, dengue virus, chikungunya virus, respiratory syncytial virus, lymphocytic choriomeningitis virus, morbillivirus, lentivirus, replicating retrovirus, rhabdovirus, Seneca Valley virus, Sindbis virus, and any variant or derivative thereof.Such vectors may include, but are not limited to, vaccinia, fowlpox, self-replicating alphaviruses, Maraba viruses, adenoviruses (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including but not limited to second, third or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuman et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C Other exemplary viral vector-based delivery platforms, such as the IL-11 promoter, Nucl. Acids Res. (2015) 43(1):682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72(12):9873-9880, are described in the art.

[0127] This sequence may be preceded by one or more sequences that target intracellular compartments. Upon introduction (i.e., delivery) into a host cell, the infected cell (i.e., engineered cell) can express, and in some cases, secrete, one or more effector molecules. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for introduction (i.e., delivery) of engineered nucleic acids, such as Salmonella typhi vectors, and the like, will be apparent to those skilled in the art from the description herein.

[0128] The viral vector-based delivery platform can be a virus that targets tumor cells, referred to herein as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indiana vesiculovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic Maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic morbillivirus, oncolytic lentivirus, oncolytic replicating retrovirus, oncolytic rhabdovirus, oncolytic Seneca Valley virus, oncolytic Sindbis virus, and any variant or derivative thereof. Any of the oncolytic viruses described herein can be a recombinant oncolytic virus that includes another transgene (e.g., an engineered nucleic acid) that encodes one or more effector molecules. The transgene encoding the one or more effector molecules can be configured to express the one or more effector molecules.

[0129] In some embodiments, viruses can include, but are not limited to, lentiviruses, retroviruses, oncolytic viruses, adenoviruses, adeno-associated viruses (AAV), and virus-like particles (VLPs).

[0130] Viral vector-based delivery platforms can be retroviral-based. Generally, retroviral vectors consist of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate one or more engineered nucleic acids (e.g., transgenes encoding one or more effector molecules) into target cells to provide permanent transgene expression. Retrovirus-based delivery systems include, but are not limited to, delivery systems based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retroviral system.

[0131] Viral vector-based delivery platform can be lentivirus-based. In general, lentivirus vector is a retroviral vector that can transduce or infect non-dividing cells and typically produces high viral titers. Lentivirus-based delivery platform can be HIV-based, such as ViraPower system (ThermoFisher) or pLenti system (Cell Biolabs). Lentivirus-based delivery platform can be SIV or FIV-based. Other exemplary lentiviral-based delivery platforms are described in more detail in U.S. Pat. Nos. 7,311,907, 7,262,049, 7,250,299, 7,226,780, 7,220,578, 7,211,247, 7,160,721, 7,078,031, 7,070,993, 7,056,699, and 6,955,919, each of which is incorporated by reference herein for all purposes.

[0132] Viral vector-based delivery platform can be adenovirus-based.Generally, adenovirus-based vector has the ability of very high transduction efficiency in many cell types, does not require cell division, achieves high titer and expression level, and can be produced in large quantities in a relatively simple system.Generally, adenovirus can be used for the transient expression of transgene in infected cells.Because adenovirus is typically not integrated into the genome of the host. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655, each of which is incorporated herein by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 5,585,362, 6,083,716, 7,371,570, 7,348,178, 7,323,177, 7,319,033, 7,318,919, and 7,306,793, and International Patent Application WO 96 / 13597, each of which is incorporated herein by reference for all purposes.

[0133] The viral vector-based delivery platform can be based on adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors can be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). The AAV system can be used for in vitro production of effector molecules or can be used in in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of engineered nucleic acids encoding one or more effector molecules (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. Nos. 4,797,368, 5,436,146, 6,632,670, 6,642,051, 7,078,387, 7,314,912, 6,498,244, 7,906,111; U.S. Patent Publication Nos. 2003-0138772, 2007 / 0036760, and 2009 / 0197338; Gao, et al. al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al, Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003), as well as International Patent Applications WO 2010 / 138263 and WO 93 / 24641, Kotin, Human Gene Therapy 5:793-801 (1994), Muzyczka, J. Clin. Invest. 94:1351 (1994), each of which is incorporated herein by reference for all purposes. Exemplary methods for constructing recombinant AAV vectors are described in U.S. Pat. No. 5,173,414, Tratschin et al, Mol. Cell. Biol. 5:3251-3260 (1985), Tratschin, et al. ah, Mol. Cell. Biol. 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS 81:64666470 (1984), and Samuiski et al., J. Virol. 63:03822-3828 (1989), each of which is incorporated herein by reference for all purposes.Generally, an AAV-based vector comprises a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11 and variants thereof.

[0134] The viral vector-based delivery platform can be a virus-like particle (VLP) platform. Generally, VLPs are constructed by producing viral structural proteins and purifying the resulting viral particles. Then, after purification, cargo / payload (e.g., any of the engineered nucleic acids described herein) is encapsulated ex vivo within the purified particles. Thus, the production of VLPs maintains the separation of nucleic acids encoding viral structural proteins and nucleic acids encoding cargo / payload. The viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo to produce VLPs using methods known to those skilled in the art. The production of VLPs is described in more detail in Seow et al. (Mol Ther. 2009 May;17(5):767-777), which is incorporated herein by reference for all purposes.

[0135] Viral vector-based delivery platforms can be engineered to target (i.e., infect) a range of cells, target a narrow subset of cells, or target specific cells. In general, the envelope protein chosen for a viral vector-based delivery platform determines the viral tropism. The virus used in the viral vector-based delivery platform can be pseudotyped to target specific cells of interest. The viral vector-based delivery platform can be pantropic and can infect a range of cells. For example, a pantropic viral vector-based delivery platform can include a VSV-G envelope. The viral vector-based delivery platform can be amphotropic and can infect mammalian cells. Thus, one skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein to target the desired cell type.

[0136] Lipid Structure Delivery System The engineered nucleic acid of the present disclosure (e.g., a polynucleotide molecule encoding a chimeric polypeptide) can be introduced into cells using lipid-mediated delivery systems. In general, lipid-mediated delivery systems use structures that are composed of an external lipid membrane that encases an internal compartment. Examples of lipid-based structures include, but are not limited to, lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. The lipid structure delivery system can deliver cargo / payload (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0137] Lipid-based nanoparticles can include, but are not limited to, unilamellar liposomes, multilamellar liposomes, and lipid preparations. As used herein, "liposome" is a generic term that encompasses in vitro preparations of lipid vesicles formed by encapsulating a desired cargo, e.g., an engineered nucleic acid, such as any of the engineered nucleic acids described herein, within a lipid shell or lipid aggregate. Liposomes may be characterized as having a vesicular structure with a bilayer membrane that generally comprises phospholipids, and an internal medium that generally comprises an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. The liposomes can be unilamellar liposomes. The liposomes can be multilamellar liposomes. The liposomes can be multivesicular liposomes. The liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are neutrally charged. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols such as cholesterol.The selection of lipids is generally guided by consideration of the desired purpose, such as criteria for in vivo delivery, such as liposome size, acid instability, and liposome stability in bloodstream.A variety of methods are available for preparing liposomes, such as those described in, for example, Szokan et al., Ann.Rev.Biophys.Bioeng.9;467(1980), U.S. Patent Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369 (each of which is incorporated herein by reference for all purposes).

[0138] Multilamellar liposomes generally form spontaneously when lipids, including phospholipids, are suspended in an excess of aqueous solution, resulting in multiple lipid layers separated by aqueous media. Water and dissolved solutes are encapsulated in a closed structure between the lipid bilayers after self-rearrangement of the lipid components. The desired cargo (e.g., polypeptides, nucleic acids, small molecule drugs, engineered nucleic acids such as any of the engineered nucleic acids described herein, viral vectors, viral-based delivery systems, etc.) can be encapsulated in the aqueous solution inside the liposome, attached to the liposome via a linking molecule associated with both the liposome and the polypeptide / nucleic acid, interspersed within the lipid bilayer of the liposome, incorporated within the liposome, complexed with the liposome, or otherwise associated with the liposome, thereby allowing it to be delivered to the target entity. Lipophilic molecules or molecules with lipophilic regions can also be dissolved in or associated with the lipid bilayer.

[0139] The liposomes used according to the present embodiment can be produced by different methods, as will be known to those skilled in the art.The preparation of liposomes is described in more detail in International Publication No. 2016 / 201323, International Application No. PCT / US85 / 01161 and PCT / US89 / 05040, and US Pat. Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706, each of which is incorporated herein by reference for all purposes.

[0140] The liposome can be a cationic liposome.Examples of cationic liposomes are described in more detail in U.S. Patent Nos. 5,962,016, 5,030,453, 6,680,068, U.S. Application No. 2004 / 0208921, and International Patent Application Publication Nos. WO 03 / 015757A1, WO 04029213A2, and WO 02 / 100435A1, each of which is incorporated herein by reference in its entirety.

[0141] Lipid-mediated gene delivery methods are described, for example, in WO 96 / 18372, WO 93 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7):682-691 (1988), U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833, WO 91 / 06309, and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987), each of which is incorporated herein by reference for all purposes.

[0142] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after fusion of the multivesicular body with the plasma membrane. The size of exosomes ranges from 30 to 100 nm in diameter. Their surface consists of a lipid bilayer from the plasma membrane of the donor cell, they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parent cell on their surface. Exosomes useful for delivery of nucleic acids are known to those skilled in the art, for example, exosomes described in more detail in U.S. Pat. No. 9,889,210, incorporated herein by reference for all purposes.

[0143] As used herein, the term "extracellular vesicles" or "EVs" refers to cell-derived vesicles that contain a membrane that encloses an internal space. In general, extracellular vesicles include all membrane-bound vesicles that have a diameter smaller than the cell from which they originate. In general, extracellular vesicles range from 20 nm to 1000 nm in diameter and can contain a variety of macromolecular cargo, either displayed on the external surface of the extracellular vesicle and / or within the internal space across the membrane. The cargo can include nucleic acids (e.g., any of the engineered nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example, and not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by successive extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0144] As used herein, the term "exosome" refers to a small (20-300 nm diameter, more preferably 40-200 nm diameter) cell-derived vesicle that includes a membrane surrounding an internal space and is generated from a cell by direct plasma membrane budding or fusion of a late endosome with the plasma membrane. Exosomes contain lipids or fatty acids and polypeptides, and optionally include a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecules. Exosomes can be derived from or isolated from producing cells based on their size, density, biochemical parameters, or a combination thereof. Exosomes are a type of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of the producing cells. Exosomes and preparation of exosomes are described in further detail in WO 2016 / 201323, which is incorporated herein by reference in its entirety.

[0145] As used herein, the term "nanovesicles" (also referred to as "microvesicles") refers to small (20-250 nm diameter, more preferably 30-150 nm diameter) vesicles of cell origin that contain a membrane surrounding an internal space and are generated from cells by direct or indirect manipulation, such that the nanovesicles are not produced by the producing cells without such manipulation. Generally, nanovesicles are a subspecies of extracellular vesicles. Suitable manipulations of producing cells include, but are not limited to, continuous extrusion, treatment with alkaline solutions, sonication, or combinations thereof. The production of nanovesicles may, in some cases, result in the destruction of the producing cells. Preferably, the population of nanovesicles is substantially free of vesicles derived from producing cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. Nanovesicles comprise lipids or fatty acids and polypeptides, and optionally comprise a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecule. Once derived from the production cells according to the engineering, the nanovesicles may be isolated from the production cells based on their size, density, biochemical parameters, or a combination thereof.

[0146] Lipid nanoparticles (LNPs) are generally synthetic lipid structures that rely on the amphiphilic nature of the lipids to form membrane and vesicle-like structures (Riley 2017). Generally, these vesicles deliver cargo / payload, such as any of the engineered nucleic acids or viral systems described herein, by absorbing into the membrane of the target cell and releasing the cargo into the cytosol. The lipids used in LNP formation can be cationic, anionic, or neutral. The lipids can be synthetic or naturally derived, and in some cases can be biodegradable. Lipids can include lipid conjugates, including but not limited to fats, cholesterol, phospholipids, polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins. The lipid composition generally includes a defined mixture of materials such as cationic, neutral, anionic, and amphiphilic lipids. In some cases, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate attachment of additional moieties. Lipid composition can affect overall LNP size and stability. In one example, lipid composition includes dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or MC3-like molecules. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as PEG or PEG-conjugated lipids, sterols, or neutral lipids. In addition, LNPs can be further engineered or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0147] Micelles are generally spherical synthetic lipid structures formed using single-chain lipids, where the hydrophilic heads of the single-chain lipids form the outer layer or membrane, and the hydrophobic tails of the single-chain lipids form the micelle core. Micelles typically refer to lipid structures that contain only a lipid monolayer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5;25(7):1501-1513), which is incorporated herein by reference for all purposes.

[0148] Nucleic acid vectors, such as expression vectors, directly exposed to serum may have some undesirable consequences, including degradation of nucleic acid by serum nucleases or off-target stimulation of the immune system by free nucleic acid. Similarly, viral delivery systems directly exposed to serum may trigger undesirable immune responses and / or neutralization of the viral delivery system. Therefore, encapsulation of engineered nucleic acid and / or viral delivery systems can be used to avoid degradation while also avoiding potential off-target effects. In certain instances, engineered nucleic acid and / or viral delivery systems are fully encapsulated within a delivery vehicle, such as within the aqueous interior of LNPs. Encapsulation of engineered nucleic acid and / or viral delivery systems within LNPs can be performed by techniques well known to those skilled in the art, such as microfluidic mixing and droplet generation performed on a microfluidic droplet generation device. Such devices include, but are not limited to, standard T-junction devices or flow focusing devices. In one example, a desired lipid formulation, such as an MC3 or MC3-like-containing composition, is provided to a droplet generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired agent, such that the delivery vector and desired agent are fully encapsulated within the interior of the MC3 or MC3-like-based LNPs. In one example, the droplet generating device can control the size range and size distribution of the LNPs produced. For example, the LNPs can have sizes ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Following droplet generation, the delivery vehicles (e.g., engineered nucleic acid and / or viral delivery systems) encapsulating the cargo / payload can be further processed or manipulated to prepare them for administration.

[0149] Nanoparticle Delivery Nanomaterials can be used to deliver engineered nucleic acids (e.g., polynucleotide molecules encoding chimeric polypeptides). Importantly, nanomaterial vehicles can be made of non-immunogenic materials and generally avoid eliciting immunity to the delivery vector itself. These materials include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in more detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery--A Review. Nanomaterials 2017,7(5),94), incorporated herein by reference for all purposes.

[0150] Genome editing system Genome editing system can be used to engineer host genome to encode engineered nucleic acid such as polynucleotide molecule encoding chimeric polypeptide of the present disclosure.Generally, "genome editing system" refers to any system for integrating exogenous gene into genome of host cell.Genome editing system includes, but is not limited to, transposon system, nuclease genome editing system, and viral vector-based delivery platform.

[0151] The transposon system can be used to integrate engineered nucleic acids, such as the engineered nucleic acids of the present disclosure, into the host genome. Transposons generally comprise terminal inverted repeats (TIRs) flanking cargo / payload nucleic acid and transposase. The transposon system can provide transposons in cis or in trans with TIR flanking cargo. The transposon system can be a retrotransposon system or a DNA transposon system. Generally, the transposon system randomly integrates cargo / payload (e.g., engineered nucleic acid) into the host genome. Examples of transposon systems include systems using transposons of the Tc1 / Mariner transposon superfamily, such as the Sleeping Beauty transposon system, which are described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug; 52(4): 355-380), and U.S. Patent Nos. 6,489,458, 6,613,752, and 7,985,739, each of which is incorporated herein by reference for all purposes. Another example of a transposon system includes the PiggyBac transposon system, which is described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is incorporated herein by reference for all purposes.

[0152] Nuclease genome editing systems can be used to engineer host genomes to encode engineered nucleic acids, such as the isolated polynucleotides or heterologous constructs of the present disclosure. Without wishing to be bound by theory, generally, nuclease-mediated gene editing systems used to introduce exogenous genes utilize the cells' natural DNA repair mechanisms, particularly the homologous recombination (HR) repair pathway. Briefly, after damage to genomic DNA (typically a double-strand break), cells can eliminate the damage by using another source of DNA with identical or substantially identical sequences at both its 5' and 3' ends as a template during DNA synthesis to repair the damage. In a natural situation, HDR can use other chromosomes present in the cell as templates. In gene editing systems, exogenous polynucleotides are introduced into cells and used as homologous recombination templates (HRT or HR templates). In general, any additional exogenous sequence not naturally found in a chromosome, with the lesion contained between the 5' and 3' complementary ends in the HRT (e.g., a gene or part of a gene), can be incorporated (i.e., integrated) into a given genomic locus during templated HDR. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to a first region of the endogenous genomic target locus, a nucleotide sequence identical to a second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid (e.g., any of the engineered nucleic acids described herein, such as any of the engineered nucleic acids encoding one or more effector molecules).

[0153] In some examples, the HR template can be linear. Examples of linear HR templates include, but are not limited to, linearized plasmid vectors, ssDNA, synthetic DNA, and PCR amplified DNA. In certain examples, the HR template can be circular, such as a plasmid. The circular template can include a supercoiled template.

[0154] The identical or substantially identical sequences found at the 5' and 3' ends of the HR template are generally referred to as arms (HR arms) with respect to the exogenous sequence to be introduced. The HR arms can be identical (i.e., 100% identical) to the region of the endogenous genome target locus. The HR arms can be substantially identical to the region of the endogenous genome target locus in some cases. While substantially identical HR arms can be used, it may be advantageous for the HR arms to be identical, since the efficiency of HDR pathway may be affected by HR arms with less than 100% identity.

[0155] Each HR arm, i.e., the 5' and 3' HR arms, can be the same size or different sizes. The length of each HR arm can be 50, 100, 200, 300, 400, or 500 bases or more, respectively. The HR arms can generally be any length, but practical considerations such as the effect of the HR arm length and the overall template size on the overall editing efficiency can also be taken into account. The HR arms can be identical or substantially identical to the region of the endogenous genome target locus that is immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to the region of the endogenous genome target locus that is immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to the region of the endogenous genome target locus that is within a certain distance from the cleavage site, such as 1 base pair, 10 base pairs or less, 50 base pairs or less, or 100 base pairs or less, from each other.

[0156] Nuclease genome editing systems can cleave target genomic loci using a variety of nucleases, including, but not limited to, clustered regularly interspaced short palindromic repeats (CRISPR) family nucleases or derivatives thereof, transcription activator-like effector nucleases (TALENs) or derivatives thereof, zinc finger nucleases (ZFNs) or derivatives thereof, and homing endonucleases (HEs) or derivatives thereof.

[0157] A CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid encoding one or more of the effector molecules described herein. The CRISPR system is described in more detail in M. Adli ("The CRISPR tool kit for genome editing and beyond" Nature Communications; volume 9 (2018), Article number: 1911), which is incorporated herein by reference for all that it teaches. In general, a CRISPR-mediated gene editing system includes a CRISPR-associated (Cas) nuclease and an RNA that directs cleavage to a specific target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 system, which consists of a Cas9 nuclease and an RNA with a CRISPR RNA (crRNA) domain and a transactivating CRISPR (tracrRNA) domain. A crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity to a target sequence ("defined nucleotide sequence"), e.g., a genomic sequence, through base pair hybridization, and an RNA domain that hybridizes to the tracrRNA. The tracrRNA can interact with a nuclease (e.g., Cas9) and thereby facilitate the recruitment of the nuclease (e.g., Cas9) to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can be a single polynucleotide, also referred to as a single guide RNA (sgRNA). Although the Cas9 system is exemplified herein, other CRISPR systems, such as the Cpf1 system, can be used. The nuclease can include its derivatives, such as Cas9 functional mutants, e.g., Cas9 "nickase" mutants, which generally mediate only a single strand cleavage of a defined nucleotide sequence, as opposed to the complete double strand cleavage typically produced by the Cas9 enzyme.

[0158] In general, the components of the CRISPR system interact with each other to form a ribonucleoprotein (RNP) complex to mediate sequence-specific cleavage. In some CRISPR systems, each component can be produced separately and used to form an RNP complex. In some CRISPR systems, each component can be produced separately in vitro and contacted with each other in vitro to form an RNP complex (i.e., "complexed"). The RNP produced in vitro can then be introduced (i.e., "delivered") into the cytosol and / or nucleus of a cell, for example, the cytosol and / or nucleus of a T cell. The RNP complex produced in vitro can be delivered to a cell by various means, including, but not limited to, electroporation, lipid-mediated introduction, cell membrane deformation by physical means, lipid nanoparticles (LNPs), virus-like particles (VLPs), and sonication. In a particular example, the RNP complex produced in vitro can be delivered to cells using Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, MaxCyte electroporation system, Miltenyi CliniMACS electroporation system, Neon electroporation system, and BTX electroporation. CRISPR nucleases, such as Cas9, can be produced in vitro (i.e., synthesized and purified) using various protein production techniques known to those skilled in the art. CRISPR system RNAs, such as sgRNAs, can be produced in vitro (i.e., synthesized and purified) using various RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

[0159] The RNP complex produced in vitro can be complexed with different ratios of nuclease and gRNA.The RNP complex produced in vitro can also be used in different amounts in CRISPR-mediated editing system.For example, depending on the number of cells that are desired to be edited, the total amount of RNP that is added can be adjusted, such as reducing the amount of RNP complex that is added when editing a large number of cells in reaction.

[0160] In some CRISPR systems, each component (e.g., Cas9 and sgRNA) can be encoded by a polynucleotide separately, and each polynucleotide can be introduced into the cell together or separately. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multi-cistronic vector, see the description of an exemplary multi-cistronic system below) and can be introduced into the cell. After the expression of the CRISPR components encoded by each polynucleotide in the cell (e.g., translation of nuclease and transcription of CRISPR RNA), the RNP complex can be formed in the cell and then directed to site-specific cleavage.

[0161] Some RNPs can be engineered to have moieties that facilitate delivery of the RNP into the nucleus. For example, Cas9 nuclease can have a nuclear localization signal (NLS) domain, whereby when the Cas9 RNP complex is delivered into the cytosol of a cell, or after translation of Cas9 and subsequent RNP formation, the NLS can facilitate further transport of the Cas9 RNP into the nucleus.

[0162] The cells described herein can be engineered using non-viral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using non-viral methods. The cells described herein can be engineered using viral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using viral methods, such as adenovirus, retrovirus, lentivirus, or any of the other viral-based delivery methods described herein.

[0163] In some CRISPR systems, two or more CRISPR compositions can be provided, each of which targets the same gene or common genomic locus at more than one target nucleotide sequence.For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance from each other.In some CRISPR systems, two or more CRISPR compositions can be provided, each of which targets the opposite strand of the same gene or common genomic locus.For example, two separate CRISPR "nickase" compositions can be provided to direct cleavage at the same gene or common genomic locus at opposite strand.

[0164] Generally, the features of the CRISPR-mediated editing system described herein can be applied to other nuclease-based genome editing systems.TALEN is an engineered site-specific nuclease, which is composed of the DNA binding domain of TALE (transcription activator-like effector) and the catalytic domain of restriction endonuclease Fokl.By changing the amino acids present in the highly variable residue region of the monomer of DNA binding domain, different artificial TALENs can be made to target different nucleotide sequences.The DNA binding domain then directs nuclease to the target sequence and creates double-strand breaks. TALEN-based systems are described in more detail in U.S. Patent Application Serial No. 12 / 965,590, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, U.S. Patent No. 10,172,880, and U.S. Patent Application Serial No. 13 / 738,381, all of which are incorporated by reference herein in their entirety. ZFN-based editing systems are described in more detail in U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061, all of which are incorporated by reference herein in their entirety for all purposes.

[0165] Other Operation Delivery Systems A variety of additional means for introducing an engineered nucleic acid (e.g., a polynucleotide molecule encoding a chimeric polypeptide as described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein.

[0166] Electroporation can be used to deliver polynucleotides to recipient entities. Electroporation is a method of internalizing cargo / payload into the internal compartment of a target cell or entity through the application of an electric field to temporarily permeabilize the outer membrane or shell of the target cell or entity. In general, this method involves placing a cell or target entity between two electrodes in a solution containing the cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cell is then disrupted, i.e., permeabilized, by applying a transient set voltage, which allows the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the example of a cell, at least a portion, if not the majority, of the cell remains viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., number of cells, concentration of cargo, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, composition of electroporation solution, etc.) vary depending on several factors, including but not limited to, the type of cells or other recipient entity, the cargo delivered, the desired efficiency of internalization, and the desired survival rate. Optimization of such criteria is within the scope of those skilled in the art. A variety of devices and protocols can be used for electroporation. Examples include, but are not limited to, the Neon® Transfection System, MaxCyte® Flow Electroporation™, Lonza® Nucleofector™ System, and Bio-Rad® Electroporation System.

[0167] Other means for introducing an engineered nucleic acid (e.g., a polynucleotide molecule encoding a chimeric polypeptide as described herein) into a cell or other target recipient entity include, but are not limited to, sonication, gene guns, hydrodynamic injection, and cell membrane deformation by physical means.

[0168] Compositions and methods for in vivo delivery of engineered mRNA, such as naked plasmids or mRNA, are described in detail in Kowalski et al. (Mol Ther. 2019 Apr 10;27(4):710-728) and Kaczmarek et al. (Genome Med. 2017;9:60), each of which is incorporated by reference herein for all purposes.

[0169] How to use Methods of using the chimeric polypeptides, polynucleotide molecules, or cells as described herein are also encompassed by the disclosure.

[0170] In some embodiments, the method includes inhibiting the repression of a gene of interest. The method of inhibiting the repression may include providing a transformed cell comprising an expression system comprising (i) an expression cassette encoding a chimeric polypeptide as described herein, and (ii) a target expression cassette comprising an ITM-responsive promoter operably linked to a gene of interest, culturing the transformed cell under conditions suitable for expression of the chimeric polypeptide, and inducing degradation of the chimeric polypeptide by contacting the transformed cell with a ligand that promotes degradation of the chimeric polypeptide.

[0171] In some embodiments, inhibiting repression is measurable as at least a 1.5-fold increase, at least a 2-fold increase, at least a 3-fold increase, at least a 4-fold increase, or at least a 5-fold increase in expression of a gene of interest operably linked to an ITM-responsive promoter in a transformed cell after contacting the transformed cell with a ligand, compared to expression of the gene of interest in a comparable transformed cell not contacted with the ligand.

[0172] In some embodiments, in the absence of a ligand, the expression level of the gene of interest is suppressed by the ITM by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, or at least 10-fold compared to the expression level in the absence of the ITM.

[0173] In vivo methods The methods provided herein also include inhibiting transcriptional repression in vivo, for example, by delivering a ligand that induces degradation of the chimeric polypeptide in vivo.

[0174] In some embodiments, the transformed cell is in a human or animal, and contacting the transformed cell with the ligand comprises administering a pharmacological dose of a ligand to the human or animal. In some embodiments, the ligand administered to the subject is an immunomodulatory drug (IMiD) that promotes degradation of the chimeric polypeptide via the ubiquitin pathway. In some embodiments, the IMiD can include, but is not limited to, thalidomide, lenalidomide, and pomalidomide. In some embodiments, the IMiD is pomalidomide and is administered to the subject at a concentration of about 1 mg per day to about 50 mg per day. In certain embodiments, the non-endogenous ligand is administered to the subject at a concentration of about 4 mg per day.

[0175] Pharmaceutical Compositions The chimeric polypeptides, isolated polynucleotides, and cells of the present disclosure can be formulated into pharmaceutical compositions. These compositions can contain, in addition to one or more of the engineered nucleic acids or engineered cells, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials must be non-toxic and must not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal.

[0176] Regardless of whether what is administered to an individual is a cell, a polypeptide, a nucleic acid, a small molecule, or other pharma- ceutically useful compound according to the present disclosure, the administration is preferably a "therapeutically effective amount" or a "prophylactically effective amount" (in some cases, prophylaxis can also be considered treatment), which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the protein aggregation disorder being treated. Prescription of treatment, e.g., determining dosage, etc., is within the responsibility of general practitioners and other physicians, and typically takes into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0177] The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated.

[0178] Additional Embodiments The following paragraphs provide additional enumerated embodiments.

[0179] Embodiment 1: below: (a) an inducible transcriptional regulator (ITM) that contains a transcriptional repressor domain and a DNA binding domain; (b) Degron and A chimeric polypeptide comprising: the degron is operably linked to the ITM; and The transcriptional repressor domain is selected from the group consisting of KRAB repression domain, HDAC4 domain, SCX HLH domain, ID1 HLH domain, HERC2 Cyt-b5 domain, TWST1 HLH domain, NKX22 homeodomain, ID3 HLH domain, and TWST2 HLH domain; Chimeric polypeptides.

[0180] Embodiment 2: 2. The chimeric polypeptide of embodiment 1, wherein the transcriptional repressor domain comprises a KRAB repression domain.

[0181] Embodiment 3: The chimeric polypeptide of embodiment 2, wherein the KRAB repression domain comprises minKRAB.

[0182] Embodiment 4: 4. The chimeric polypeptide according to any one of embodiments 1 to 3, wherein the KRAB repression domain comprises the amino acid sequence of SEQ ID NO:2.

[0183] Embodiment 5: 5. The chimeric polypeptide of embodiment 4, wherein the KRAB repression domain comprises a KRAB repressor domain variant of SEQ ID NO:2 and comprises one or more amino acid substitutions selected from the group consisting of W27L, K28L, D31A, T32A, Q34A, Q35A, R39E, L43S, T57C, K58C, P59C, V61Y, I62Y, I62A, L63W, L63Y, L63E, R64F, R64W, R64E, L65F, L65E, L65W, E66V, K67F, G68F, and E69F.

[0184] Embodiment 6: 5. The chimeric polypeptide of embodiment 4, wherein the KRAB repression domain comprises a KRAB repressor domain variant of SEQ ID NO:2 and includes one or more amino acid substitutions selected from Q34A / Q35A, I62A, T57C / K58C / P59C, D31A / T32A, L63W / R64W / L65W, E66V, L63E / R64E / L65E, R64F / L65F, W27L / K28L KRAB, R39E, K67F / G68F / E69F, and V61Y / I62Y / L63Y.

[0185] Embodiment 7: 4. The chimeric polypeptide according to any one of embodiments 1 to 3, wherein the KRAB repression domain comprises the amino acid sequence of SEQ ID NO:3.

[0186] Embodiment 8: 2. The chimeric polypeptide of embodiment 1, wherein the transcriptional repressor domain comprises an HDAC4 repression domain.

[0187] Embodiment 9: The chimeric polypeptide of embodiment 8, wherein the HDAC4 repression domain comprises the amino acid sequence of SEQ ID NO:4.

[0188] Embodiment 10: 10. The chimeric polypeptide of any one of embodiments 1 to 9, wherein the DNA-binding domain comprises a zinc finger (ZF) protein domain.

[0189] Embodiment 11: The chimeric polypeptide of embodiment 10, wherein the ZF protein domain is of modular design and composed of zinc finger arrays (ZFAs) of zinc finger motifs.

[0190] Embodiment 12: The chimeric polypeptide of embodiment 11, wherein the ZF protein domain comprises 1 to 10 zinc finger motifs.

[0191] Embodiment 13: The chimeric polypeptide of embodiment 11, wherein the ZF protein domain comprises one to six zinc finger motifs.

[0192] Embodiment 14: The chimeric polypeptide of embodiment 13, wherein the ZF protein domain comprises SEQ ID NO:5.

[0193] Embodiment 15: 15. The chimeric polypeptide of any one of embodiments 1 to 14, wherein the transcriptional repressor domain is N-terminal to the DNA binding domain.

[0194] Embodiment 16: 15. The chimeric polypeptide of any one of embodiments 1 to 14, wherein the transcriptional repressor domain is C-terminal to the DNA binding domain.

[0195] Embodiment 17: 17. The chimeric polypeptide according to any one of embodiments 1 to 16, wherein the transcriptional repressor domain and the DNA binding domain are separated by a first peptide linker.

[0196] Embodiment 18: 18. The chimeric polypeptide of embodiment 17, wherein the first peptide linker comprises the amino acid sequence of GGGGSGGT (SEQ ID NO: 60).

[0197] Embodiment 19: 19. The chimeric polypeptide of any one of embodiments 1 to 18, wherein the ITM is a synthetic transcription regulator.

[0198] Embodiment 20: The degrons are HCV NS4 degron, PEST (two copies of human IκBα residues 277–307), GRR (residues 352–408 of human p105), DRR (residues 210–295 of yeast Cdc34), SNS (tandem repeats of SP2 and NB (influenza A or B SP2-NB-SP2), RPB (four copies of yeast RPB residues 1688–1702), SPmix (tandem repeats of SP1 and SP2 (A These include SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein), NS2 (three copies of residues 79-93 of influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations), the APC / C degron, and COP1. 20. The chimeric polypeptide of any one of embodiments 1 to 19, wherein the chimeric polypeptide is selected from the group consisting of an E3 ligase-binding degron motif, a CRL4-Cdt2-binding PIP degron, an actinfilin-binding degron, a KEAP1-binding degron, a KLHL2- and KLHL3-binding degron, an MDM2-binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a plant hormone-dependent SCF-LRR-binding degron, an SCF ubiquitin ligase-binding phosphodegron, a plant hormone-dependent SCF-LRR-binding degron, a DSGxxS phospho-dependent degron, a Siah-binding motif, an SPOP SBC docking motif, and a PCNA-binding PIP box.

[0199] Embodiment 21: 21. The chimeric polypeptide of any one of embodiments 1 to 20, wherein the degron comprises a cereblon (CRBN) polypeptide substrate domain having the ability to bind CRBN in response to an immunomodulatory drug (IMiD).

[0200] Embodiment 22: 22. The chimeric polypeptide of embodiment 21, wherein the CRBN polypeptide substrate domain is selected from the group consisting of IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787 and ZN827, or fragments thereof, capable of drug-induced binding of CRBN.

[0201] Embodiment 23: 23. The chimeric polypeptide of embodiment 21 or embodiment 22, wherein the CRBN polypeptide substrate domain comprises a chimeric fusion product of a native CRBN polypeptide sequence.

[0202] Embodiment 24: 22. The chimeric polypeptide of embodiment 21, wherein the CRBN polypeptide substrate domain comprises an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 6).

[0203] Embodiment 25: The chimeric polypeptide of any one of embodiments 21 to 24, wherein the IMiD is an FDA approved drug.

[0204] Embodiment 26: 26. The chimeric polypeptide of any one of embodiments 21 to 25, wherein the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide.

[0205] Embodiment 27: 27. The chimeric polypeptide of any one of embodiments 1 to 26, wherein the ITM is N-terminal to the degron.

[0206] Embodiment 28: 27. The chimeric polypeptide of any one of embodiments 1 to 26, wherein the ITM is C-terminal to the degron.

[0207] Embodiment 29: 29. The chimeric polypeptide of any one of embodiments 1 to 28, wherein the ITM is separated from the degron by a second peptide linker.

[0208] Embodiment 30: 30. The chimeric polypeptide of embodiment 29, wherein the second peptide linker comprises an amino acid sequence selected from the group consisting of GSGSGSGS (SEQ ID NO: 7), KEGS (SEQ ID NO: 8), EGK, EAAAK (SEQ ID NO: 9), and AAPAKQE (SEQ ID NO: 10).

[0209] Embodiment 31: 30. The chimeric polypeptide of embodiment 29, wherein the second peptide linker comprises an amino acid sequence selected from the group consisting of AAPAKQEAAAPAKQEAAAPAKQEAAAPAPAAKAEAPAAAPAAKA (SEQ ID NO: 12) and AEAAAKEAAAKEAAAKA (SEQ ID NO: 13).

[0210] Embodiment 32: An expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding the chimeric polypeptide according to any one of embodiments 1 to 31.

[0211] Embodiment 33: 33. The expression cassette of embodiment 32, wherein the promoter comprises a constitutive promoter.

[0212] Embodiment 34: 34. The expression cassette of embodiment 33, wherein the constitutive promoter is selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1a, hCAGG, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0213] Embodiment 35: 33. The expression cassette of embodiment 32, wherein the promoter comprises an inducible promoter.

[0214] Embodiment 36: 36. The expression cassette of embodiment 35, wherein the inducible promoter comprises a minimal promoter and a response element selected from the group consisting of an NFkB response element, a CREB response element, an NFAT response element, an SRF response element 1, an SRF response element 2, an AP1 response element, a TCF-LEF response element promoter fusion, a hypoxia response element, a SMAD binding element, a STAT3 binding site, an inducer molecule responsive promoter, and tandem repeats thereof.

[0215] Embodiment 37: 37. The expression cassette of embodiment 36, wherein the promoter comprises a synthetic promoter.

[0216] Embodiment 38: 38. The expression cassette according to any one of embodiments 32 to 37, wherein the polynucleotide sequence encoding the chimeric polypeptide further encodes a 3' untranslated region (UTR) comprising an mRNA destabilizing element.

[0217] Embodiment 39: 39. The expression cassette of embodiment 38, wherein the mRNA destabilizing element is selected from the group consisting of an AU-rich element and a stem-loop destabilizing element.

[0218] Embodiment 40: An expression system comprising the expression cassette according to any one of embodiments 32 to 39 and a target expression cassette comprising an ITM-responsive promoter operably linked to a gene of interest.

[0219] Embodiment 41: The expression system of embodiment 40, wherein the ITM responsive promoter comprises a promoter sequence and a sequence that binds to the DNA binding domain of the ITM.

[0220] Embodiment 42: 42. The expression system of embodiment 41, wherein the sequence that binds to the DNA-binding domain comprises one or more zinc finger binding sites.

[0221] Embodiment 43: 43. The expression system of embodiment 42, wherein the sequence that binds to the DNA-binding domain comprises one or more zinc finger binding sites having the amino acid sequence of SEQ ID NO:54.

[0222] Embodiment 44: 44. The expression system of embodiment 42 or embodiment 43, wherein the sequence that binds to the DNA-binding domain comprises more than four zinc finger binding sites.

[0223] Embodiment 45: 45. The expression system of embodiment 44, wherein the sequence that binds to the DNA-binding domain comprises the amino acid sequence of SEQ ID NO:55.

[0224] Embodiment 46: 46. ​​An expression system according to any one of embodiments 40 to 45, wherein the promoter sequence of the ITM responsive promoter comprises a constitutive promoter sequence.

[0225] Embodiment 47: 47. The expression system of embodiment 46, wherein the constitutive promoter sequence is selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1a, hCAGG, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0226] Embodiment 48: 48. An expression system according to any one of embodiments 40 to 47, wherein the promoter sequence of the ITM responsive promoter comprises a minimal promoter.

[0227] Embodiment 49: An expression system according to any one of embodiments 40 to 48, wherein the ITM responsive promoter comprises a synthetic promoter.

[0228] Embodiment 50: 50. The expression system according to any one of embodiments 40 to 49, wherein the gene of interest encodes a therapeutic polypeptide.

[0229] Embodiment 51: An expression system according to any one of embodiments 40 to 50, wherein the gene of interest encodes a polypeptide selected from the group consisting of cytokines, chemokines, homing molecules, growth factors, cell death regulators, co-activation molecules, tumor microenvironment modifiers a, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0230] Embodiment 52: An expression system according to any one of embodiments 40 to 51, comprising a heterologous construct comprising both (i) an expression cassette of any one of embodiments 28 to 35 and (ii) a target expression cassette.

[0231] Embodiment 53: An expression system according to any one of embodiments 40 to 51, comprising a first heterologous construct comprising an expression cassette according to any one of embodiments 32 to 39, and a second heterologous construct comprising a target expression cassette.

[0232] Embodiment 54: An isolated cell comprising an expression cassette according to any one of embodiments 32 to 39.

[0233] Embodiment 55: An isolated cell comprising an expression system according to any one of embodiments 40 to 53.

[0234] Embodiment 56: 56. The isolated cell of embodiment 54 or 55, wherein the cell is a human cell.

[0235] Embodiment 57: 57. The isolated cell of any one of embodiments 54 to 56, wherein the cell is a stem cell.

[0236] Embodiment 58: 57. The isolated cell of any one of embodiments 54 to 56, wherein the cell is an immune cell.

[0237] Embodiment 59: 57. The isolated cell of any one of embodiments 54-56, wherein the cell is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0238] Embodiment 60: A gene switch for inhibiting repression of a gene of interest, comprising the chimeric polypeptide of any one of embodiments 1 to 31 and a ligand, wherein binding of the ligand to a degron induces degradation of the chimeric polypeptide, thereby inhibiting repression of the gene of interest, and the gene of interest is operably linked to an ITM-responsive promoter.

[0239] Embodiment 61: The gene switch of embodiment 60, wherein the ligand comprises an immunomodulatory drug (IMiD) that promotes degradation of the chimeric polypeptide via the ubiquitin pathway.

[0240] Embodiment 62: The gene switch of embodiment 61, wherein the IMiD is an FDA approved drug.

[0241] Embodiment 63: The gene switch of embodiment 61 or 62, wherein the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide.

[0242] Embodiment 64: 1. A method for inhibiting repression of a gene of interest, comprising: (a) providing a cell comprising an expression system comprising: (i) an expression cassette encoding the chimeric polypeptide according to any one of embodiments 1 to 31; and (ii) a target expression cassette comprising an ITM-responsive promoter operably linked to a gene of interest; (b) inducing degradation of the chimeric polypeptide by contacting the transformed cell with a ligand that promotes degradation of the chimeric polypeptide; A method comprising:

[0243] Embodiment 65: 65. The method of embodiment 64, further comprising culturing the cells under conditions suitable for expression of the chimeric polypeptide.

[0244] Embodiment 66: The method according to embodiment 64 or embodiment 65 or embodiment 65, wherein the expression cassette encoding the chimeric polypeptide comprises an expression cassette according to any one of embodiments 32 to 39.

[0245] Embodiment 67: The method according to any one of embodiments 64 to 66, wherein the expression system comprises an expression system according to any one of embodiments 40 to 53.

[0246] 68. Embodiment 68 A method for producing a cell having the ability of drug-regulated transcriptional repression, the method comprising a step of transforming the cell with an expression cassette according to any one of embodiments 32 to 39 or an expression system according to any one of embodiments 40 to 56. EXAMPLES

[0247] Below are examples of specific embodiments for carrying out the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but it is understood that some experimental error and deviation should be allowed for.

[0248] The practice of this disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature, see, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (East, Pennsylvania: Mack Publishing Company, 1992); rd Ed. (Plenum Press) Vols A and B (1992).

[0249] Example 1 Efficient degradation of degron-linked DNA binders The degradation efficiency of the degron-linked DNA binders after drug treatment was evaluated.

[0250] material and method U87-MG cells were transduced (50,000 cells / transduced) with lentiviruses encoding each construct (50,000 pg each, titered by p24 assay) as shown in Table 4. Each construct format is described 5' to 3' (ORF from N-terminus to C-terminus) with the sequences described in Table 4.

[0251] [Table 4]

[0252] Two days after transduction, each cell group was cultured in the presence of 1 μM pomalidomide or in the absence of drug for 7 days, and blue fluorescent protein (BFP) expression was assessed by fluorescence-activated cell sorting (FACS). BFP expression in each cell group (with or without drug) is shown in Figure 1.

[0253] result Degradation via the ubiquitin pathway in response to the immunomodulatory drug (IMiD) pomalidomide was evaluated for constructs containing the cereblon (CRBN) polypeptide substrate domain (referred to as "degron" in Table 4) in various orientations and using various linkers. As shown in FIG. 1, for each construct tested, at least 40% of the cells were BFP positive in the absence of drug, whereas in the presence of pomalidomide, 10% or less of the cells were BFP positive. For each construct, the percentage of BFP positive cells in the presence of drug was compared to the percentage of BFP positive cells in the absence of drug to determine percent degradation. Table 5 shows the percent degradation for each of the constructs tested. The results demonstrate IMiD-dependent degradation of the constructs.

[0254] [Table 5]

[0255] Example 2 Degradation of a degron-linked repressor to induce transcription A reporter cell line was generated by transducing U87-MG cells with a lentivirus encoding mCherry linked to the ITM-responsive promoter of SEQ ID NO:56, which contains the EF1 core promoter sequence with a zinc finger binding domain composed of four ZF10-1 binding sites upstream of the core promoter sequence.

[0256] The ability of the degron-linked repressors to regulate reporter protein expression was assessed by transduction assays.

[0257] material and method Reporter cell lines were generated by transducing U87-MG cells with lentivirus encoding mCherry linked to the ITM-responsive promoter of SEQ ID NO:56, which contains an EF1a core promoter sequence with a zinc finger binding domain composed of four ZF10-1 binding sites upstream of the core promoter sequence. Reporter cells were transduced (50,000 cells / transduced) with lentivirus encoding each construct (50,000 pg each, titered by p24 assay) as shown in Table 6. Each construct format is described from 5' to 3' (ORF from N-terminus to C-terminus). Constructs SB03757 and SB03760 contain a minKrab repression domain that is codon optimized for E. coli, and constructs SB03758 and SB03761. For constructs SB03758 and SB03761, the entire construct was codon optimized for E. coli.

[0258] [Table 6]

[0259] Two days after transduction, cells were incubated either in the presence of 1 μM pomalidomide or in the absence of drug. On the fifth day of drug treatment, cells were assayed for mCherry expression by fluorescence-activated cell sorting (FACS).

[0260] result Degron-linked promoter regulation was evaluated for constructs containing the cereblon (CRBN) polypeptide substrate domain (d913) in various orientations and with various Krab-derived repression domains, linkers, and mRNA destabilization elements. The geometric mean fluorescence intensity (MFI) of mCherry for each condition is shown in Figure 2. The fold difference between the geometric MFI of mCherry for drug-free versus pomalidomide-treated conditions was calculated as shown in Figure 3. As shown in Figure 3, drug-induced release of repression was possible with constructs SB03758 and SB03759 in response to pomalidomide treatment. Histogram plots of mCherry expression in the presence or absence of pomalidomide for cells transduced with SB03758, as well as compared to untransduced cells (lacking reporter) and reporter cells not transduced with the repressor, are shown in Figure 4A. Histogram plots of mCherry expression in the presence or absence of pomalidomide for cells transduced with SB03759, as compared to untransduced cells (lacking reporter) and reporter cells not transduced with the repressor, are shown in Figure 4B. The histogram plots shown in Figures 4A and 4B demonstrate that repression by SB03758 and SB03759 is tunable in a drug-dependent manner.

[0261] Example 3 Optimization of Degradation of Degron-Linked Repressors Constructs engineered to optimize drug-induced release of repression were assayed to directly test degradation efficiency.

[0262] material and method Reporter cells as described in Example 2 were transduced (50,000 cells / transduction) with lentiviruses encoding each construct (50,000 pg each, titrated by p24 assay) as shown in Table 7, or not transduced as a negative control. Each construct format is described from 5' to 3' (ORF from N-terminus to C-terminus). Both SB03747 and SB03750 contain the mRNA destabilization tag AuSLDE at the 3' end. Seven days after transduction and 5 days of treatment in the presence of 1 μM pomalidomide or in the absence of drug, BFP was measured by fluorescence-activated cell sorting (FACS) analysis.

[0263] [Table 7]

[0264] result As shown in Figure 5, constructs SB03747 and SB03750 showed percent degradation of 81.3% and 72.5%, respectively, following treatment with 1 μM pomalidomide. Hence, both constructs are efficiently degraded following pomalidomide treatment.

[0265] Example 4 Degradation of degron-linked repressors with various construct orientations to induce transcription The ability of degron-linked repressors with different orientations to regulate reporter expression was assessed by transduction assays.

[0266] material and method Reporter cell lines were generated as described in Example 2. Reporter cells were transduced (50,000 cells / transduced) with lentiviruses (50,000 pg each, titered by p24 assay) encoding either constructs with a degron N-terminal to an inducible transcriptional regulator (ITM) (Table 8) or constructs with a degron C-terminal to an ITM (Table 9). Constructs SB03758 and SB03759 as described in Example 2 were also included in this experiment. Each construct format is described 5' to 3' (ORF from N-terminus to C-terminus).

[0267] [Table 8]

[0268] [Table 9]

[0269] Two days after transduction, cells transfected with each construct were incubated either in the presence of 1 μM pomalidomide or in the absence of drug. On the fifth day of drug treatment, cells were assayed by fluorescence-activated cell sorting (FACS) for mCherry expression. The geometric mean fluorescence intensity (MFI) of mCherry for each condition is shown in FIG. 6A for each construct with a degron at the N-terminus. The fold difference between the geometric MFI of mCherry for the drug-free condition versus the pomalidomide-treated condition was calculated for each construct with a degron at the N-terminus, as shown in FIG. 6B. The geometric mean fluorescence intensity (MFI) of mCherry for each condition is shown in FIG. 7A for each construct with a degron at the C-terminus. The fold difference between the geometric MFI of mCherry for the drug-free condition versus the pomalidomide-treated condition was calculated for each construct with a degron at the C-terminus, as shown in FIG. 7B.

[0270] result Degron-linked promoter regulation was evaluated for constructs containing the cereblon (CRBN) polypeptide substrate domain (d913) in various orientations and with various repression domains, linkers, and mRNA destabilization elements. As shown in Figure 6A, the degron-linked repressor with HDAC4 repressor domain and N-terminal degron has the ability to repress reporter expression in the absence of drug, and repression is relieved as shown by induced reporter expression in the presence of drug. In addition, Figure 6A confirms the reversibility of repression by constructs SB03758 and SB03759, as demonstrated in Example 2. As shown in Figure 6B, the degron-linked repressor with HDAC4 repressor domain and N-terminal degron, and constructs SB03758 and SB03759 each demonstrate at least a 4-fold increase in reporter expression in the presence of drug compared to the absence of drug. As shown in Figure 7A, the degron-linked repressor with HDAC4 repressor domain and C-terminal degron has the ability to suppress reporter expression in the absence of drug, and the suppression was reversible as shown by the reporter expression induced in the presence of drug. In addition, Figure 7A provides another confirmation of constructs SB03758 and SB03759 as shown in Figure 6A and also demonstrated in Example 2. As shown in Figure 7B, the degron-linked repressor with HDAC4 repressor domain and C-terminal degron, and constructs SB03758 and SB03759 each demonstrate at least a 4-fold increase in reporter expression in the presence of drug compared to the absence of drug. Thus, the data demonstrates the constructs with the ability to suppress transcription in a drug-dependent, regulatable manner.

[0271] [Table 10] TIFF2024513232000019.tif242156TIFF2024513232000020.tif242153TIFF20245132320 00021.tif242153TIFF2024513232000022.tif242152TIFF2024513232000023.tif196169 TIFF2024513232000024.tif242154TIFF2024513232000025.tif242154TIFF20245132320 00026.tif242154TIFF2024513232000027.tif242153TIFF2024513232000028.tif234165

[0272] Example 5 IMiD-regulated expression of IL-12 payload in the IMiD-On system The efficiency of IMiD-regulated expression of IL-12 payload was assessed in a system in which addition of an IMiD increases expression of IL-12 through degradation of a degron-linked repressor.

[0273] material and method 50,000 U87MG cells were seeded in 24w dishes 18-14 hours prior to transduction and then transduced with 25 kpg of degron-linked repressor (SB03759, SB03936, or SB04397) and 25 kpg of IL12 reporter construct (SB04640) (virus was quantified by p24 ELISA). A construct encoding IL-12 linked to an ITM-responsive promoter containing an EFS promoter sequence with a zinc finger binding domain consisting of four ZF10-1 binding sites upstream of the EFS promoter sequence (SEQ ID NO:67) was used as the reporter.

[0274] For pomalidomide titration experiments, cells were split into drug-free media condition or 1uM pomalidomide condition 2 days after transduction. Then 3 days later, 100k cells of each condition were seeded into 24w plates, and after 24 hours, supernatants were collected and assayed using an IL12 p70 ELISA kit (#D1200 R&D Systems) to quantify IL-12.

[0275] For kinetics studies, transduced cells were seeded at 100k cells per well in 24w plates. Then, 24 hours later, the supernatant was replaced with no drug or 1uM pomalidomide or iverdimide. Supernatants were harvested at the indicated times (3h, 6h, 12h, 16h, and 24h) after drug treatment.

[0276] The constructs evaluated are shown in Table 11. Each construct format is described 5' to 3' (ORF from N-terminus to C-terminus) with the sequences described in Table 11. The sequences of the constructs are shown in Table 12.

[0277] [Table 11]

[0278] result Regulated IL-12 expression in response to the immunomodulatory drug (IMiD) pomalidomide was assessed for constructs containing the d913 degron in various orientations and using various linkers for a drug-regulatable system in which addition of the IMiD leads to degradation of the degron-linked repressor resulting in increased expression of the payload ("IMiD ON").

[0279] A degron-linked repressor was constructed to repress expression of the payload IL-12 under drug-free conditions through an inducible transcriptional regulator (ITM) that features a transcriptional repressor and a DNA-binding domain that binds to a promoter operably linked to IL-12 in the absence of drug. The ITM is linked to a degron such that transcriptional repression is released upon degradation of the degron-linked ITM upon addition of the IMiD pomalidomide.

[0280] Three different degron-linked repressors were evaluated for their ability to regulate IL-12 expression under drug-free conditions or with increasing concentrations of pomalidomide (1 nM, 10 nM, 100 nM, and 1 uM). As shown in FIG. 8, IL-12 expression increased with the addition of IMID to constructs SB03936 and SB04397. IL-12 levels are shown in the top panel. The bottom panel shows quantification of IL-12 restoration expression to unrepressed levels when the degron-linked repressor is degraded with the addition of increasing concentrations of pomalidomide. IL-12 levels are displayed as a fraction of the quantified IL12 expression levels in cells transduced with a constitutive IL-12 reporter. IL-12 expression is regulated by the SB04397 degron-linked repressor and restored to reporter-only levels in cells treated with pomalidomide at concentrations of 10 nM or higher. Results demonstrated IMiD-dependent regulation of IL-12 production, with addition of IMiDs increasing IL-12 expression in an "IMiD-ON" system featuring a degron-linked repressor.

[0281] The kinetics was then evaluated for the degron-linked repressor SB04397. The IMiDs pomalidomide and iberdomide were also compared. As shown in Figure 9, IL-12 expression levels increased over time in the supernatants collected from cells for both 1uM pomalidomide treatment (upper panel) and 1uM iberdomide treatment (lower panel) for cells transduced with both the IL-12 reporter SB04640 and the degron-linked repressor SB04397 (left column) compared to the reporter-only control (right column). The kinetics was further evaluated at additional time points for the degron-linked repressor SB04397 and the IMiD pomalidomide. As shown in Figure 10, IL-12 expression levels (as assessed normalized to IL-12 expression levels in cells transduced with the SB04640 reporter alone) demonstrated increased IL-12 expression at 12 and 16 hours following the addition of 1 uM pomalidomide. These results indicate rapid activation of the IMiD ON switch, with expression of IL-12 detected within 24 hours of adding the IMiD and initiating degradation of the degron-linked repressor.

[0282] [Table 12] TIFF2024513232000031.tif254163TIFF2024513232000032.tif254160TIFF2024513232000033.tif254163TIFF2024513232000034.tif98162

[0283] While the present disclosure has been specifically shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the disclosure and the appended claims.

[0284] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. below: (a) an inducible transcriptional regulator (ITM) comprising a transcriptional repressor domain and a DNA binding domain; (b) degron and A chimeric polypeptide comprising: the degron is operably linked to the ITM; and The transcriptional repressor domain is selected from the group consisting of KRAB repression domain, HDAC4 domain, SCX HLH domain, ID1 HLH domain, HERC2 Cyt-b5 domain, TWST1 HLH domain, NKX22 homeodomain, ID3 HLH domain, and TWST2 HLH domain; Chimeric polypeptides.

2. the transcriptional repressor domain comprises the KRAB repression domain; Optionally, the KRAB repression domain comprises minKRAB; Optionally, the KRAB repression domain comprises the amino acid sequence of SEQ ID NO:2; Optionally, the KRAB repression domain comprises a KRAB repressor domain variant of SEQ ID NO:2, and includes one or more amino acid substitutions selected from the group consisting of W27L, K28L, D31A, T32A, Q34A, Q35A, R39E, L43S, T57C, K58C, P59C, V61Y, I62Y, I62A, L63W, L63Y, L63E, R64F, R64W, R64E, L65F, L65E, L65W, E66V, K67F, G68F, and E69F; Optionally, the KRAB repression domain comprises a KRAB repressor domain variant of SEQ ID NO:2, and includes one or more amino acid substitutions selected from Q34A / Q35A, I62A, T57C / K58C / P59C, D31A / T32A, L63W / R64W / L65W, E66V, L63E / R64E / L65E, R64F / L65F, W27L / K28L KRAB, R39E, K67F / G68F / E69F, and V61Y / I62Y / L63Y; Optionally, the KRAB repression domain comprises the amino acid sequence of SEQ ID NO:

3. The chimeric polypeptide of claim 1.

3. the transcriptional repressor domain comprises the HDAC4 repression domain, Optionally, the HDAC4 repression domain comprises the amino acid sequence of SEQ ID NO:

4. The chimeric polypeptide of claim 1.

4. the DNA binding domain comprises a zinc finger (ZF) protein domain; Optionally, the ZF protein domain is of modular design and composed of zinc finger arrays (ZFAs) of zinc finger motifs; Optionally, the ZF protein domain comprises 1-10 zinc finger motifs; Optionally, the ZF protein domain comprises six zinc finger motifs; Optionally, the ZF protein domain comprises SEQ ID NO:

5. The chimeric polypeptide of claim 1.

5. a. the transcriptional repressor domain is N-terminal to the DNA binding domain or C-terminal to the DNA binding domain; and / or b. the transcriptional repressor domain and the DNA binding domain are separated by a first peptide linker; Optionally, the first peptide linker comprises an amino acid sequence selected from the group consisting of GGGGSGGT (SEQ ID NO: 60), and / or c. The ITM is a synthetic transcription factor; The chimeric polypeptide of claim 1.

6. (a) The degron is selected from the group consisting of HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα), GRR (residues 352-408 of human p105), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeats of SP2 and NB (influenza A or influenza B SP2-NB-SP2), RPB (four copies of residues 1688-1702 of yeast RPB), SPmix (tandem repeats of SP1 and SP2 (influenza A or influenza B SP2-NB-SP2), and SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein), NS2 (three copies of residues 79-93 of influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations), APC / C degron, COP1 and / or is selected from the group consisting of an E3 ligase-binding degron motif, a CRL4-Cdt2-binding PIP degron, an actinfilin-binding degron, a KEAP1-binding degron, a KLHL2- and KLHL3-binding degron, an MDM2-binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a plant hormone-dependent SCF-LRR-binding degron, an SCF ubiquitin ligase-binding phosphodegron, a plant hormone-dependent SCF-LRR-binding degron, a DSGxxS phospho-dependent degron ("DSGxxS" is set forth as SEQ ID NO:68), a Siah-binding motif, a SPOP SBC docking motif, and a PCNA-binding PIP box; (b) the degron comprises a cereblon (CRBN) polypeptide substrate domain having the ability to bind to CRBN in response to an immunomodulatory drug (IMiD); Optionally, said CRBN polypeptide substrate domain is selected from the group consisting of IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or a fragment thereof, that is capable of drug-inducible binding of CRBN; Optionally, the CRBN polypeptide substrate domain comprises a chimeric fusion product of a native CRBN polypeptide sequence; Optionally, the CRBN polypeptide substrate domain comprises an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO:6), and / or (c) the IMiD is an FDA approved drug; Optionally, the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide; Optionally, the ITM is N-terminal to the degron or C-terminal to the degron; Optionally, the ITM is separated from the degron by a second peptide linker; Optionally, the second peptide linker comprises an amino acid sequence selected from the group consisting of GSGGSGSGS (SEQ ID NO:7), KEGS (SEQ ID NO:8), EGK, EAAAK (SEQ ID NO:9), and AAPAKQE (SEQ ID NO:10); or the second peptide linker comprises an amino acid sequence selected from the group consisting of AAPAKQEAAAPAKQEAAAPAKQEAAAPAAAAKAEAPAAAAPAAKA (SEQ ID NO:12) and AEAAAKEAAAKEAAAKA (SEQ ID NO:13). The chimeric polypeptide of claim 1.

7. An expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding the chimeric polypeptide of any one of claims 1 to 6, Optionally, the promoter comprises a constitutive promoter selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1a, hCAGG, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb, or the promoter comprises an inducible promoter, optionally, the inducible promoter comprises a minimal promoter and a response element selected from the group consisting of an NFkB response element, a CREB response element, an NFAT response element, an SRF response element 1, an SRF response element 2, an AP1 response element, a TCF-LEF response element promoter fusion, a hypoxia response element, a SMAD binding element, a STAT3 binding site, an inducer molecule responsive promoter, and tandem repeats thereof; Optionally, the promoter comprises a synthetic promoter; Optionally, the polynucleotide sequence encoding the chimeric polypeptide further encodes a 3' untranslated region (UTR) comprising an mRNA destabilization element; Optionally, the mRNA destabilizing element is selected from the group consisting of an AU-rich element and a stem-loop destabilizing element. Expression cassette.

8. 8. An expression system comprising the expression cassette of claim 7 and a target expression cassette comprising an ITM-responsive promoter operably linked to a gene of interest, Optionally, the ITM-responsive promoter comprises a promoter sequence and a sequence that binds to the DNA-binding domain of the ITM; Optionally, the sequence that binds to the DNA binding domain comprises one or more zinc finger binding sites; Optionally, the sequence that binds to the DNA binding domain comprises one or more zinc finger binding sites having an amino acid sequence of SEQ ID NO:54; Optionally, the sequence that binds to the DNA binding domain comprises more than four zinc finger binding sites; Optionally, the sequence that binds to the DNA binding domain comprises the amino acid sequence of SEQ ID NO:55, Optionally, the promoter sequence of the ITM-responsive promoter comprises a constitutive promoter sequence selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, EF1a, hCAGG, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb; Optionally, the promoter sequence of the ITM responsive promoter comprises a minimal promoter; Optionally, the ITM responsive promoter comprises a synthetic promoter; Optionally, the gene of interest encodes a therapeutic polypeptide selected from the group consisting of a cytokine, a chemokine, a homing molecule, a growth factor, a cell death regulator, a coactivator molecule, a tumor microenvironment modifier a, a receptor, a ligand, an antibody, a polynucleotide, a peptide, and an enzyme; Optionally, the expression system comprises a heterologous construct comprising both (i) the expression cassette of claim 7 and (ii) a target expression cassette; Optionally, the expression system comprises a first heterologous construct comprising the expression cassette of claim 7, and a second heterologous construct comprising a target expression cassette. Expression system.

9. An isolated cell comprising the expression cassette of claim 7.

10. a. the cell is a human cell, and / or b. the cell is a stem cell, and / or c. the cell is an immune cell, and / or d. the cell is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, red blood cells, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells; The isolated cell of claim 9.

11. A gene switch for inhibiting the repression of a gene of interest, comprising: Comprising a chimeric polypeptide according to any one of claims 1 to 6 and a ligand, binding of the ligand to the degron induces degradation of the chimeric polypeptide, thereby preventing repression of the gene of interest; The gene of interest is operably linked to an ITM-responsive promoter; Optionally, the ligand comprises an immunomodulatory drug (IMiD) that promotes degradation of the chimeric polypeptide via the ubiquitin pathway; Optionally, the IMiD is an FDA approved drug; Optionally, the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide. Gene switch.

12. 1. A method for inhibiting repression of a gene of interest, comprising: a. Providing a cell comprising an expression system comprising: (i) an expression cassette encoding the chimeric polypeptide of any one of claims 1 to 6; and (ii) a target expression cassette comprising an ITM-responsive promoter operably linked to a gene of interest; b. inducing degradation of the chimeric polypeptide by contacting the transformed cell with a ligand that promotes degradation of the chimeric polypeptide; A method comprising:

13. a. the method further comprises culturing the cell under conditions suitable for expression of the chimeric polypeptide; and / or b. The expression cassette encoding the chimeric polypeptide comprises the expression cassette of claim 7. The method of claim 12.

14. 1. A method for producing a cell capable of drug-regulated transcriptional repression, comprising: Transforming said cell with the expression cassette of claim 7. A method comprising:

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