Gene regulation via conditional nuclear localization of a polypeptide

Abstract

The present disclosure provides systems for modulating expression of a target polynucleotide in a cell. The system can comprise a chimeric polypeptide comprising a gene modulating polypeptide in-frame fused to a heterologous nuclear localization domain. The heterologous nuclear localization domain can be operable to translocate the chimeric polypeptide to the nucleus of a cell upon activation by an active cellular signaling pathway. The cellular signaling pathway can be inducible in response to an extracellular signal. In response to the extracellular signal, the chimeric polypeptide can be localized to the nucleus of the cell, and the gene modulating polypeptide can modulate expression of a target polynucleotide in the nucleus of the cell.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 647,543, filed March 23, 2018, and U.S. Provisional Application No. 62 / 675,134, filed May 22, 2018, each of which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] [0001.1] This application contains a sequence list that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on March 22, 2019, is named 50489-711_601_SL.txt and is 137,422 bytes in size. Background Technology

[0005] Regulation of cellular activity can involve the binding of ligands to membrane-bound receptors that contain both an extracellular ligand-binding domain and an intracellular (e.g., cytoplasmic) signaling domain. The formation of the complex between the ligand and its binding domain can lead to conformational changes and / or chemical modifications of the receptor, which can result in intracellular signal transduction. In some cases, intracellular signal transduction leads to phosphorylation of downstream targets, thereby altering their activity. These downstream targets can then be activated and perform various functions within the cell.

[0006] In some cases, the extracellular domain of a protein (e.g., a ligand-binding domain) can be attached to the intracellular domain (e.g., a signal transduction domain) of another protein involved in signal transduction to create a chimeric molecule (e.g., a chimeric receptor) that combines the ligand recognition of the former with the signal transduction of the latter.

[0007] Regulation of cellular activity can involve light-responsive peptides (e.g., transmembrane or intracellular proteins). Activation of some light-responsive proteins by light can lead to conformational changes in the peptide, which can result in signal transduction within the cell. In some cases, peptides can interact with one or more additional substances to transduce signals within the cell.

[0008] These methods of modulating cell activity (e.g., via ligand and / or photoactivation) can be used for a variety of purposes, such as modulating immune cells in immunotherapy. Immunotherapy may involve modifying a patient's own immune cells to express chimeric receptors, where arbitrary ligands are specifically transplanted onto immune cell signaling domains. Immune cell signaling domains can participate in activating and / or inactivating immune cells in response to diseases such as cancer.

[0009] Conventional immunotherapy methods suffer from several limitations. These limitations include insufficient signaling from costimulatory receptors to generate a sustained and / or adequate immune response for therapeutic effect, insufficient specificity of modified immune cells for diseased cells such as cancer cells (e.g., on-target, off-tumor effects, and toxicity), and side effects such as cytokine release syndrome (CRS). Signal transduction in immune cells can involve various receptors, including costimulatory receptors. Insufficient signaling from costimulatory receptors can lead to reduced immune cell responses and decreased efficacy of immunotherapy. Off-target effects and side effects, such as cytokine release syndrome, can lead to further medical complications, including inflammatory responses, organ failure, and even death in extreme cases. Summary of the Invention

[0010] This article discloses a system for regulating the expression of a target polynucleotide in cells. The system comprises a chimeric polypeptide containing a gene-regulating polypeptide fused to a heterologous nuclear localization domain (NMR) frame, wherein the NMR is operable to translocate the chimeric polypeptide to the cell nucleus upon activation by an active cellular signaling pathway that is responsive to an extracellular signal, wherein, in response to the extracellular signal, the chimeric polypeptide localizes to the cell nucleus, and the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0011] This document discloses a system for regulating the expression of a target polynucleotide in cells, the system comprising: a) a chimeric receptor polypeptide that activates a cell signaling pathway upon binding to a ligand; and b) a chimeric polypeptide comprising a gene regulatory polypeptide fused within a heterologous nuclear localization domain (HNU), the HNU being operable to translocate the chimeric polypeptide to the cell nucleus upon activation of the cell signaling pathway, wherein, upon binding of the ligand to the chimeric receptor polypeptide, the chimeric polypeptide is localized to the cell nucleus via the induced HNU, and the gene regulatory polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0012] This document discloses a system for regulating the expression of target polynucleotides in cells, the system comprising: a) a cell signaling pathway activator comprising a chemical compound; and b) a chimeric polypeptide comprising a gene regulatory polypeptide fused within a heterologous nuclear localization domain frame, the heterologous nuclear localization domain being operable to translocate the chimeric polypeptide to the cell nucleus upon induction by a cell signaling pathway, wherein, upon application of the activator to the cell, the chimeric polypeptide is localized to the cell nucleus via the activated heterologous nuclear localization domain, and the gene regulatory polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0013] In some embodiments, the nuclear localization domain comprises at least one nuclear localization sequence. In some embodiments of any of the systems of the present invention, activation of the nuclear localization domain includes chemical modification of the nuclear localization sequence. In some embodiments of any of the systems of the present invention, the chemical modification is a chemical modification of at least one amino acid of the nuclear localization sequence. In some embodiments, the chemical modification results in a conformational change and exposure of the nuclear localization sequence. In some embodiments, the chemical modification includes dephosphorylation. In some embodiments, the chemical modification includes phosphorylation. In some embodiments, the chemical modification includes acetylation. In some embodiments, the chemical modification includes methylation. In some embodiments, the chemical modification includes ubiquitination. In some embodiments, the chemical modification includes proteolytic processing. In some embodiments of any of the systems of the present invention, activation of the nuclear localization domain includes binding to a second messenger or signal transduction pathway protein. In some embodiments, the activated signal transduction pathway activates a calcium-dependent phosphatase. In some embodiments, the nuclear localization domain comprises a member or fragment of the activating T cell nuclear factor (NFAT) transcription factor family. In some embodiments, the gene regulatory polypeptide comprises an actuating moiety. In some embodiments of any of the systems of the present invention, the actuation moiety comprises a Cas protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a wide range of nucleases, a recombinase, a flipper enzyme, a transposase, or an Argonaute (Ago) protein (e.g., prokaryotic Argonaute (pAgo), archaea Argonaute (aAgo), and eukaryotic Argonaute (eAgo)). In some embodiments, the actuation moiety comprises a Cas protein. In some embodiments, the Cas protein is complexed with a guide RNA. In some embodiments, the Cas protein is Cas9, Cpf1, C2c1, or C2c3. In some embodiments, the Cas protein is C2c2, Cas13b, Cas13c, or Cas13d. In some embodiments, the Cas protein is substantially lacking in DNA cleavage activity. In some embodiments, the gene regulatory polypeptide further comprises a heterologous functional domain. In some embodiments of any of the systems of the present invention, the heterologous functional domain comprises a transcription activator. In some embodiments, the transcription activator includes VP16, VP32, VP64, VPR, p65, or P65HSF1. In some embodiments of any system of the present invention, the functional domain comprises a transcription repressor. In some embodiments of any system of the present invention, the transcription repressor comprises a KRAB domain. In some embodiments of any system of the present invention, the functional domain comprises a chromosome-modifying enzyme.In some embodiments of any of the systems of the present invention, the chromosome-modifying enzyme comprises a ubiquitinase, protease, methyltransferase, demethylase, acetylase, deacetylase, deaminase, phosphorylase, or dephosphorylase. In some embodiments of any of the systems of the present invention, the chromosome-modifying enzyme modifies one or more nucleotides. In some embodiments of any of the systems of the present invention, the chromosome-modifying enzyme modifies one or more histones. In some embodiments of any of the systems of the present invention, the target polynucleotide is genomic DNA. In some embodiments of any of the systems of the present invention, the target polynucleotide is RNA. In some embodiments, the extracellular signal comprises a ligand, and the binding of the ligand to a transmembrane receptor activates the cellular signaling pathway. In some embodiments, the chimeric receptor polypeptide comprises a Notch receptor, a G protein-coupled receptor (GPCR), an integrin receptor, a cadherin receptor, a tyrosine kinase receptor, a death receptor, an immune receptor, or a chimeric antigen receptor. In some embodiments, the chemical compound increases intracellular calcium concentration relative to baseline levels.

[0014] This article discloses a method for regulating the expression of target polynucleotides in cells, comprising: translocating a gene-regulating polypeptide from the cytoplasm to the nucleus in response to activation of a cell signaling pathway, wherein activation of the cell signaling pathway activates a nuclear localization domain coupled to the gene-regulating polypeptide.

[0015] This article discloses a method for regulating the expression of a target polynucleotide in cells, comprising: a) activating a cell signaling pathway, wherein activating the cell signaling pathway activates a nuclear localization domain linked to a gene-regulating polypeptide; b) localizing the gene-regulating polypeptide to the cell nucleus via the activated nuclear localization domain, wherein after localizing the gene-regulating polypeptide to the cell nucleus, the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell.

[0016] This article discloses a method for regulating the expression of a target polynucleotide in cells, comprising: a) contacting a ligand with a transmembrane receptor, wherein a cell signaling pathway is activated upon contact, and wherein the activated cell signaling pathway activates a nuclear localization domain coupled to a gene-regulating polypeptide; b) translocating the gene-regulating polypeptide from the cytoplasm to the nucleus through the activated nuclear localization domain, wherein the gene-regulating polypeptide regulates the expression of the target polynucleotide after translocation to the nucleus.

[0017] In some embodiments of any method of the present invention, the nuclear localization domain comprises at least one nuclear localization sequence. In some embodiments of any method of the present invention, activation of the nuclear localization domain includes chemical modification of the nuclear localization sequence. In some embodiments of any method of the present invention, the chemical modification is a chemical modification of at least one amino acid of the nuclear localization sequence. In some embodiments of any method of the present invention, the chemical modification results in a conformational change and exposure of the nuclear localization sequence. In some embodiments of any method of the present invention, the chemical modification includes dephosphorylation. In some embodiments of any method of the present invention, the chemical modification includes phosphorylation. In some embodiments of any method of the present invention, the chemical modification includes acetylation. In some embodiments of any method of the present invention, the chemical modification includes methylation. In some embodiments of any method of the present invention, the chemical modification includes ubiquitination. In some embodiments of any method of the present invention, the chemical modification includes proteolytic processing. In some embodiments of any method of the present invention, activation of the nuclear localization domain includes binding to a second messenger or signal transduction pathway protein. In some embodiments of any method of the present invention, the activated signal transduction pathway activates a calcium-dependent phosphatase. In some embodiments of any method of the present invention, the nuclear localization domain comprises a member or fragment of an activated T-cell nuclear factor (NFAT). In some embodiments of the method of the present invention, the gene-regulating polypeptide comprises an actuating moiety. In some embodiments of the method of the present invention, the actuating moiety comprises a Cas protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a wide range of nucleases, a recombinase, a flipper enzyme, a transposase, or an Argonaute (Ago) protein (e.g., prokaryotic Argonaute (pAgo), archaea Argonaute (aAgo), and eukaryotic Argonaute (eAgo)). In some embodiments of the method of the present invention, the gene-regulating polypeptide comprises a Cas protein. In some embodiments of the method of the present invention, the Cas protein is complexed with a guide RNA. In some embodiments of the method of the present invention, the Cas protein is Cas9, Cpf1, C2c1, or C2c3. In some embodiments of the method of the present invention, the Cas protein is C2c2, Cas13a, Cas13b, Cas13c, or Cas13d. In some embodiments of the method of the present invention, the Cas protein is substantially lacking in DNA cleavage activity. In some embodiments of the method of the present invention, the gene-regulating polypeptide comprises a heterologous functional domain. In some embodiments of any of the methods of the present invention, the heterologous functional domain comprises a transcriptional activator. In some embodiments of any of the methods of the present invention, the transcriptional activator comprises VP16, VP32, VP64, VPR, or P65HSF1.In some embodiments of any method of the present invention, the functional domain comprises a transcriptional repressor. In some embodiments of any method of the present invention, the transcriptional repressor comprises a KRAB domain. In some embodiments of any method of the present invention, the functional domain comprises a chromosome-modifying enzyme. In some embodiments of any method of the present invention, the chromosome-modifying enzyme comprises a methyltransferase, demethylase, acetyltransferase, deacetylase, deaminase, phosphorylase, or dephosphorylase. In some embodiments of any method of the present invention, the chromosome-modifying enzyme modifies one or more nucleotides. In some embodiments of any method of the present invention, the chromosome-modifying enzyme modifies one or more histones. In some embodiments of any method of the present invention, the target polynucleotide is genomic DNA. In some embodiments of any method of the present invention, the target polynucleotide is RNA. In some embodiments of any method of the present invention, activating the cell signaling pathway comprises administering a cell signaling pathway activator to the cell, wherein the activator comprises a chemical compound. In some embodiments of any method of the present invention, the chemical compound increases intracellular calcium concentration relative to baseline levels. In some embodiments of any of the methods of the present invention, the transmembrane receptor includes Notch receptor, G protein-coupled receptor (GPCR), integrin receptor, cadherin receptor, tyrosine kinase receptor, death receptor, immune receptor, or chimeric antigen receptor.

[0018] This article discloses a system for regulating the expression of a target polynucleotide in cells. The system comprises a chimeric polypeptide containing a gene-regulating polypeptide fused to a heterologous nuclear localization domain (NMR) frame, wherein the NMR is operable to translocate the chimeric polypeptide to the cell nucleus upon activation of a cell signaling pathway induced by an extracellular signal, wherein the extracellular signal is electromagnetic radiation, and wherein, in response to the extracellular signal, the chimeric polypeptide localizes to the cell nucleus, and the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0019] In some embodiments, the electromagnetic radiation includes X-rays, ultraviolet (UV) rays, visible light, infrared rays, microwaves, or any combination thereof. In some embodiments, the system of the present invention includes a signal transduction unit that activates the cell signal transduction pathway upon application of the extracellular signal.

[0020] In some embodiments, the signal transduction unit comprises a transmembrane protein, wherein the transmembrane protein induces the cell signal transduction pathway upon application of the extracellular signal. In some embodiments, the signal transduction unit comprises an intracellular protein, wherein the intracellular protein induces the cell signal transduction pathway upon application of the extracellular signal.

[0021] In some embodiments, the signal transduction unit comprises a transmembrane protein and an intracellular protein. In some embodiments, the application of the extracellular signal activates the transmembrane protein, which in turn activates the intracellular protein to induce the cell signaling pathway. In some embodiments, the application of the extracellular signal activates the intracellular protein, which in turn activates the transmembrane protein to induce the cell signaling pathway. In some embodiments, the intracellular protein comprises a first portion and a second portion, and wherein the application of the extracellular signal induces a conformational change in the intracellular protein, thereby exposing an active site of at least one of the first portion and the second portion. In some embodiments, the exposed active site activates the transmembrane protein to induce the cell signaling pathway. In some embodiments, the exposed active site binds to the transmembrane protein to activate the transmembrane protein.

[0022] In some embodiments, the cell signaling pathway includes calcium. In some embodiments, at least one of the first and second portions of the intracellular protein includes an LOV domain. In some embodiments, the signaling unit further includes an α-helical peptide domain located between the first and second portions of the intracellular protein, wherein application of an extracellular signal induces a conformational change in at least a portion of the α-helical domain. In some embodiments, at least one of the first and second portions of the intracellular protein includes a SOAR domain. In some embodiments, the transmembrane protein includes a calcium channel. In some embodiments, the transmembrane protein includes an ORAI1 domain.

[0023] In some embodiments, the cells are not kidney cells or kidney cell lines. In some embodiments, the cells are not cervical cancer cells or cervical cancer cell lines.

[0024] In some embodiments, the extracellular signal increases intracellular calcium concentration relative to baseline levels. In some embodiments, the nuclear localization domain comprises at least one nuclear localization sequence. In some embodiments, activation of the nuclear localization domain includes chemical modification of the nuclear localization sequence. In some embodiments, the chemical modification is a chemical modification of at least one amino acid of the nuclear localization sequence. In some embodiments, the chemical modification results in a conformational change and exposure of the nuclear localization sequence. In some embodiments, the chemical modification includes dephosphorylation. In some embodiments, the chemical modification includes phosphorylation. In some embodiments, the chemical modification includes acetylation. In some embodiments, the chemical modification includes methylation. In some embodiments, the chemical modification includes ubiquitination. In some embodiments, the chemical modification includes proteolytic processing. In some embodiments, activation of the nuclear localization domain includes binding to a second messenger or signal transduction pathway protein. In some embodiments, the activated signal transduction pathway activates a calcium-dependent phosphatase. In some embodiments, the nuclear localization domain comprises a member or fragment of the activating T cell nuclear factor (NFAT) transcription factor family. In some embodiments, the gene regulatory polypeptide comprises an actuating moiety. In some embodiments, the actuation moiety comprises a Cas protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a wide range of nucleases, a recombinase, a flipper enzyme, a transposase, or an Argonaute (Ago) protein (e.g., prokaryotic Argonaute (pAgo), archaea Argonaute (aAgo), and eukaryotic Argonaute (eAgo)). In some embodiments, the actuation moiety comprises a Cas protein. In some embodiments, the Cas protein is complexed with a guide RNA. In some embodiments, the Cas protein is Cas9, Cpf1, C2c1, or C2c3. In some embodiments, the Cas protein is C2c2, Cas13b, Cas13c, or Cas13d. In some embodiments, the Cas protein is substantially lacking in DNA cleavage activity. In some embodiments, the gene regulatory polypeptide further comprises a heterologous functional domain. In some embodiments, the heterologous functional domain comprises a transcription activator. In some embodiments, the transcription activator includes VP16, VP32, VP64, VPR, p65, or P65HSF1. In some embodiments, the functional domain comprises a transcription repressor. In some embodiments, the transcription repressor comprises a KRAB domain. In some embodiments, the functional domain comprises a chromosome-modifying enzyme. In some embodiments, the chromosome-modifying enzyme includes a methyltransferase, demethylase, acetyltransferase, deacetylase, deaminase, phosphorylase, or dephosphorylase. In some embodiments, the chromosome-modifying enzyme modifies one or more nucleotides.In some embodiments, the chromosome-modifying enzyme modifies one or more histones. In some embodiments, the target polynucleotide is genomic DNA. In some embodiments, the target polynucleotide is RNA.

[0025] This article discloses a method for regulating the expression of a target polynucleotide in a cell, comprising: (a) applying electromagnetic radiation to the cell, wherein a cell signaling pathway is activated by the electromagnetic radiation, and wherein the activated cell signaling pathway activates a nuclear localization domain coupled to a gene-regulating polypeptide; and (b) translocating the gene-regulating polypeptide from the cytoplasm to the nucleus through the activated nuclear localization domain, wherein the gene-regulating polypeptide regulates the expression of the target polynucleotide after translocation to the nucleus.

[0026] In some embodiments, the electromagnetic radiation includes X-rays, ultraviolet (UV) radiation, visible light, infrared radiation, microwaves, or any combination thereof. In some embodiments, the method of the present invention further includes applying the electromagnetic radiation to activate signal transduction units, wherein activating the signal transduction units activates the cell signal transduction pathway. In some embodiments, the electromagnetic radiation increases intracellular calcium concentration relative to baseline levels.

[0027] In some embodiments, the signal transduction unit comprises a transmembrane protein, wherein the transmembrane protein induces the cell signal transduction pathway upon application of the electromagnetic radiation. In some embodiments, the signal transduction unit comprises an intracellular protein, wherein the intracellular protein induces the cell signal transduction pathway upon application of the electromagnetic radiation.

[0028] In some embodiments, the signal transduction unit comprises a transmembrane protein and an intracellular protein. In some embodiments, the application of electromagnetic radiation activates the transmembrane protein, which in turn activates the intracellular protein to induce the cell signaling pathway. In some embodiments, the application of electromagnetic radiation activates the intracellular protein, which in turn activates the transmembrane protein to induce the cell signaling pathway. In some embodiments, the intracellular protein comprises a first portion and a second portion, and wherein the application of electromagnetic radiation induces a conformational change in the intracellular protein, thereby exposing an active site of at least one of the first portion and the second portion. In some embodiments, the exposed active site activates the transmembrane protein to induce the cell signaling pathway. In some embodiments, the exposed active site binds to the transmembrane protein to activate the transmembrane protein.

[0029] In some embodiments, the cell signaling pathway includes calcium. In some embodiments, at least one of the first and second portions of the intracellular protein includes an LOV domain. In some embodiments, the intracellular protein further includes an α-helical peptide domain located between the first and second portions of the intracellular protein, wherein the application of electromagnetic radiation induces a conformational change in at least a portion of the α-helical domain. In some embodiments, at least one of the first and second portions of the intracellular protein includes a SOAR domain. In some embodiments, the transmembrane protein includes a calcium channel. In some embodiments, the transmembrane protein includes an ORAI1 domain.

[0030] In some embodiments, the method of the present invention further includes applying the electromagnetic radiation to the cells for a period of time, thereby providing temporal and / or spatial control over the activation of the cell signaling pathway. In some embodiments, the method further includes: (a) infusing the cells into an individual; and (b) directing an electromagnetic radiation source to apply the electromagnetic radiation to at least a portion of the individual, thereby activating the cell signaling pathway in a spatially controlled manner. In some embodiments, the electromagnetic radiation source is implanted in a therapeutically significant site within the individual. In some embodiments, the method further includes: (a) culturing the cells in the absence of the electromagnetic radiation; (b) applying the electromagnetic radiation to the cells for a period of time to activate the regulation of target polynucleotide expression; and (c) infusing the activated cells into the individual.

[0031] In some embodiments, the cells are not kidney cells. In some embodiments, the cells are not cervical cancer cells.

[0032] In some embodiments, the electromagnetic radiation increases intracellular calcium concentration relative to baseline levels. In some embodiments, the nuclear localization domain comprises at least one nuclear localization sequence. In some embodiments, activation of the nuclear localization domain includes chemical modification of the nuclear localization sequence. In some embodiments, the chemical modification is a chemical modification of at least one amino acid of the nuclear localization sequence. In some embodiments, the chemical modification results in a conformational change and exposure of the nuclear localization sequence. In some embodiments, the chemical modification includes dephosphorylation. In some embodiments, the chemical modification includes phosphorylation. In some embodiments, the chemical modification includes acetylation. In some embodiments, the chemical modification includes methylation. In some embodiments, the chemical modification includes ubiquitination. In some embodiments, the chemical modification includes proteolytic processing. In some embodiments, activation of the nuclear localization domain includes binding to a second messenger or signal transduction pathway protein. In some embodiments, the activated signal transduction pathway activates a calcium-dependent phosphatase. In some embodiments, the nuclear localization domain comprises a member or fragment of an activated T-cell nuclear factor (NFAT). In some embodiments, the gene regulatory polypeptide comprises an actuating moiety. In some embodiments, the actuating portion comprises a Cas protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a wide range of nucleases, a recombinase, a flipper enzyme, a transposase, or an Argonaute (Ago) protein (e.g., prokaryotic Argonaute (pAgo), archaea Argonaute (aAgo), and eukaryotic Argonaute (eAgo)). In some embodiments, the gene regulatory polypeptide comprises a Cas protein. In some embodiments, the Cas protein is complexed with a guide RNA. In some embodiments, the Cas protein is Cas9, Cpf1, C2c1, or C2c3. In some embodiments, the Cas protein is C2c2, Cas13a, Cas13b, Cas13c, or Cas13d. In some embodiments, the Cas protein is substantially lacking in DNA cleavage activity. In some embodiments, the gene regulatory polypeptide comprises a heterologous functional domain. In some embodiments, the heterologous functional domain comprises a transcription activator. In some embodiments, the transcription activator includes VP16, VP32, VP64, VPR, or P65HSF1. In some embodiments, the functional domain contains a transcription repressor. In some embodiments, the transcription repressor contains a KRAB domain. In some embodiments, the functional domain contains a chromosome-modifying enzyme. In some embodiments, the chromosome-modifying enzyme includes a methyltransferase, demethylase, acetyltransferase, deacetylase, deaminase, phosphorylase, or dephosphorylase. In some embodiments, the chromosome-modifying enzyme modifies one or more nucleotides.In some embodiments, the chromosome-modifying enzyme modifies one or more histones. In some embodiments, the target polynucleotide is genomic DNA. In some embodiments, the target polynucleotide is RNA.

[0033] Incorporation

[0034] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually cited and incorporated herein by reference. Attached Figure Description

[0035] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be gained by referring to the following detailed description of illustrative embodiments utilizing the principles of the invention, along with the accompanying drawings, in which:

[0036] Figure 1 An example of inducible gene regulation controlled by receptor activation is depicted. In the depicted example, the interaction between a ligand and its corresponding receptor activates an intrinsic signal transduction pathway, the receptor comprising an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain (ICD). The signaling cascade leads to biochemical or structural changes in a fusion protein, which consists of a gene regulatory polypeptide (GMP) and a heterologous nuclear localization domain, to translocate the fusion protein into the nucleus to regulate the expression of a target gene. In this example, the ability of the heterologous nuclear localization domain to translocate into the nucleus can be controlled by the presence or absence of ligand-receptor interactions.

[0037] Figure 2 An example of inducible gene regulation controlled by receptor activation is depicted. In the depicted example, the interaction between an antigen and its corresponding CAR activates the intrinsic TCR signaling pathway, the CAR consisting of an extracellular single-chain antibody variable fragment (scFv), a spacer region, a transmembrane (TM) domain, and intracellular signaling domains 1 and 2. The signaling cascade leads to dephosphorylation of the NFAT component of the NFAT-dCas9-VP64 fusion protein, thereby inducing a conformational change in the inhibitory binding partner or its dissociation from the NFAT moiety. Consequently, the nuclear localization signal (NLS) peptide becomes exposed, causing the fusion protein to translocate into the nucleus. Subsequently, the dCas9-VP64 moiety of the fusion protein combines with a target-specific single guide RNA (sgRNA) to regulate the expression of the target gene. In this example, the ability of the NFAT protein domain to translocate into the nucleus is controlled by CAR activation signaling.

[0038] Figure 3A and Figure 3B Depicting the use Figure 2The example data generated by the system is depicted in the figure.

[0039] Figure 4A A diagram illustrating the functional domains of the NFATc2 protein is provided. The NFATc2 protein comprises four functional domains: an N-terminal transactivation domain (TAD-N), an NFAT homology region (NHR), a DNA-binding domain (DBD), and a C-terminal transactivation domain (TAD-C). The N-terminal portion of NFATc2 (nNFATc2) is used as a component in some embodiments disclosed herein.

[0040] Figure 4B The amino acid sequence of an exemplary nNFATc2-dCas9-VP64 construct (SEQ ID NO:1) is depicted. In this example, the N-terminal portion of NFATc2 fused with the dCas9 protein is underlined.

[0041] Figure 5 Depicting the use Figure 2 The example data is generated by an example system for gene downregulation as depicted in the example, where KRAB is used as the effector domain instead of VP64.

[0042] Figure 6A and Figure 6B Example data is depicted using dCas9 generated by fusing with smaller NFATc2 variants or other NFAT family proteins.

[0043] Figure 7 Example data generated using dCas9 fused with the RelA protein instead of NFATc2 is depicted.

[0044] Figure 8 An example of inducible gene regulation controlled by electromagnetic radiation is depicted. In the depicted example, electromagnetic radiation activates a signaling unit composed of a transmembrane protein (ORAI1) and an intracellular chimeric protein (LOV2-Jα-SOAR / CAD). The application of electromagnetic radiation induces a conformational change in the intracellular chimeric protein to expose an active site. The active site activates the transmembrane protein, which in turn induces cellular signaling pathways (e.g., calcium-dependent activation of calcium-dependent phosphatases). The induced cellular signaling pathway leads to the dephosphorylation of the NFAT component of the NFAT-dCas9-repressor / activator fusion protein. The nuclear localization signaling (NLS) domain of the NFAT component becomes exposed, allowing the fusion protein to translocate into the nucleus. Subsequently, the dCas9-repressor / activator portion of the fusion protein combines with a target-specific single guide RNA (sgRNA) to regulate the expression of a target gene. Detailed Implementation

[0045] Unless otherwise stated, the practice of some of the methods disclosed herein employs conventional techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the scope of the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols in Molecular Biology (F.M.A. Susubel et al.); Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.G. Taylor (1995)); Harlow and Lane (1988); Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th edition (R.R. Freshney (2010)).

[0046] definition

[0047] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used in the specification and claims include plural references. For example, the term “a chimeric transmembrane receptor polypeptide” includes multiple chimeric transmembrane receptor polypeptides.

[0048] The terms "about" or "approximately" refer to a range of acceptable error for a particular value as determined by a person skilled in the art, the range of error depending in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may refer to one or more standard deviations. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly for biological systems or processes, the term may refer to a range of an order of magnitude, preferably within 5 times the value, more preferably within 2 times. Where a particular value is described in this application and claims, unless otherwise stated, the term "about" should be assumed to refer to a range of acceptable error for the particular value.

[0049] As used in this article, "cell" generally refers to a biological cell. A cell can be the basic structural, functional, and / or biological unit of a living organism. Cells can originate from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaea cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycophytes, hornworts, bryophytes, mosses), and algal cells (e.g., *Botryococcus braunii*, *Chlamydomonas reinhardtii*, *Nannochloropsis gaditana*, *Chlorella pyrenoidosa*, and *Sargassum patens*). Cells derived from various organisms include: seaweed (e.g., kelp), fungal cells (e.g., yeast cells, mushroom cells), animal cells, cells derived from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells derived from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and cells derived from mammals (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Sometimes, cells do not originate from natural organisms (e.g., cells can be synthetically produced, sometimes called artificial cells).

[0050] As used herein, the term "antigen" refers to a molecule or fragment thereof that can be bound by a selective binder. As an example, an antigen can be a ligand that can be bound by a selective binder such as a receptor. As another example, an antigen can be an antigen molecule that can be bound by a selective binder such as an immunoprotein (e.g., an antibody). An antigen can also refer to a molecule or fragment thereof that can be used in an animal to produce antibodies that can bind to that antigen.

[0051] As used herein, the term "antibody" refers to a protein-binding molecule that has immunoglobulin-like function. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies) and their derivatives, variants, and fragments. Antibodies include, but are not limited to, different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.) of immunoglobulins (Ig). Derivatives, variants, or fragments may refer to functional derivatives or fragments that retain the binding specificity (e.g., intact and / or partially) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), microantibodies, biantibodies, and single-domain antibodies ("sdAb" or "nanobody" or "cameloid antibody"). The term antibody includes antibodies that have been optimized, engineered, or chemically conjugated, and antigen-binding fragments of antibodies. Examples of optimized antibodies include affinity-matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (e.g., antibodies optimized for crystallizable regions of fragments) and multispecific antibodies (e.g., bispecific antibodies).

[0052] As used herein, the term "Fc receptor" or "FcR" generally refers to a receptor or any derivative, variant, or fragment thereof that can bind to the Fc region of an antibody. In some embodiments, an FcR is a receptor (γ receptor, FcγR) that binds to IgG antibodies and includes receptors of the FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The term "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus.

[0053] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates—adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-denitro-dGTP, and 7-denitro-dATP, as well as nucleotide derivatives that confer nuclease resistance on nucleic acid molecules containing them. As used herein, the term nucleotide may refer to dideoxynucleotide triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxynucleotide triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled using known techniques. They can also be labeled with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides can include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides may include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, all available from Perkin Elmer, Foster City, Calif.; and FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, and FluoroLink, all available from Amersham, Arlington Heights, Ill. Cy3-dUTP and FluoroLinkCy5-dUTP; luciferin-15-dATP, luciferin-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, luciferin-12-ddUTP, luciferin-12-UTP and luciferin-15-2'-dATP, available from Boehringer Mannheim, Indianapolis, Ind.; and luciferin-15-2'-dATP, available from Molecular The nucleotides for chromosome markers obtained by Probes, Eugene, and Oreg include: BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Waterfall Blue-7-UTP, Waterfall Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides can also be labeled or marked by chemical modifications. A single chemically modified nucleotide can be a biotin-dNTP.Some non-limiting examples of biotinylated dNTPs may include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0054] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs, and whether single-stranded, double-stranded, or multi-stranded. Polynucleotides can be exogenous or endogenous to the cell. Polynucleotides can exist in cell-free environments. Polynucleotides can be genes or segments thereof. Polynucleotides can be DNA. Polynucleotides can be RNA. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Polynucleotides may include one or more analogs (e.g., modified backbones, sugars, or nucleobases). Where modifications are present, the nucleotide structure can be modified before or after polymer assembly. Some non-limiting examples of analogues include: 5-bromouracil, peptide nucleic acids, heteronucleic acids, morpholino nucleic acids, locked nucleic acids, diol nucleic acids, threonine nucleic acids, dideoxynucleotides, cordycepin, 7-denitro-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogues, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, brassinoside, and woyoside. Non-restricted examples of polynucleotides include coding or non-coding regions of genes or gene segments, one or more loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides (including cell-free DNA (cfDNA) and cell-free RNA (cfRNA)), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0055] As used herein, the term "gene" refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) involved in encoding RNA transcripts and its corresponding nucleotide sequence. As used herein with respect to genomic DNA, the term includes intercalated non-coding regions as well as regulatory regions, and may include 5' and 3' ends. In some uses, the term includes the transcribed sequence, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region will contain an "open reading frame" encoding a polypeptide. In some uses of the term, "gene" contains only the coding sequence necessary to encode a polypeptide (e.g., "open reading frame" or "coding region"). In some cases, a gene does not encode a polypeptide, such as ribosomal RNA (rRNA) genes and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or a natural gene located at its native position in an organism's genome. A gene may refer to an "exogenous gene" or a non-natural gene. Non-natural genes can refer to genes that are not normally found in the host organism but are introduced into the host organism through gene transfer. Non-natural genes can also refer to genes that are not located at their natural position in the organism's genome. Furthermore, non-natural genes can refer to naturally occurring nucleic acid or polypeptide sequences that contain mutations, insertions, and / or deletions (e.g., non-natural sequences).

[0056] As used herein, the terms “target polynucleotide” and “target nucleic acid” refer to a nucleic acid or polynucleotide targeted by the actuating portion of this disclosure. A target polynucleotide may be DNA (e.g., endogenous or exogenous). DNA may refer to a template for generating mRNA transcripts and / or various regulatory regions that regulate the transcription of mRNA from a DNA template. A target polynucleotide may be a larger polynucleotide, such as a chromosome or a region of a chromosome. A target polynucleotide may refer to an extrachromosomal sequence (e.g., an appendage sequence, microloop sequence, mitochondrial sequence, chloroplast sequence, etc.) or a region of an extrachromosomal sequence. A target polynucleotide may be RNA. RNA may be, for example, mRNA that can serve as a template for encoding a protein. Target polynucleotides containing RNA may contain various regulatory regions that regulate the translation of proteins from an mRNA template. Target polynucleotides may encode gene products (e.g., DNA encoding RNA transcripts or RNA encoding protein products) or contain regulatory sequences that regulate the expression of gene products. Generally, the term “target sequence” refers to a nucleic acid sequence on a single strand of the target nucleic acid. The target sequence can be a portion of a gene, a regulatory sequence, genomic DNA, cell-free nucleic acids (including cfDNA and / or cfRNA), cDNA, fusion genes, and RNA (including mRNA, miRNA, rRNA), etc. When targeted by the actuated portion, the target polynucleotide can lead to altered gene expression and / or activity. When targeted by the actuated portion, the target polynucleotide can lead to edited nucleic acid sequences. The target nucleic acid may contain nucleic acid sequences that may not be associated with any other sequences in the nucleic acid sample due to single nucleotide substitutions. The target nucleic acid may contain nucleic acid sequences that may not be associated with any other sequences in the nucleic acid sample due to 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions. In some embodiments, the substitution may not occur within 5, 10, 15, 20, 25, 30, or 35 nucleotides at the 5' end of the target nucleic acid. In some embodiments, the substitution may not occur within 5, 10, 15, 20, 25, 30, or 35 nucleotides at the 3' end of the target nucleic acid.

[0057] The term "expression" refers to one or more processes by which polynucleotides (e.g., transcribed into mRNA or other RNA transcripts) are transcribed from a DNA template and / or by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoding polypeptides are collectively referred to as "gene products." If the polynucleotides are derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells. Regarding expression, "upregulation" generally refers to an increase in the expression level of polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequences relative to their expression levels in the wild-type state, while "downregulation" generally refers to a decrease in the expression level of polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequences relative to their expression levels in the wild-type state.

[0058] As used herein, the terms “complementary,” “complementary,” “complementary,” and “complementarity” generally refer to a sequence that is completely complementary to and hybridizes with a given sequence. In some cases, a sequence that hybridizes with a given nucleic acid is referred to as a “complementary” or “reverse complementary” of a given molecule, provided that its base sequence in a given region can bind complementary to the base sequence of its binding partner, such that, for example, AT, AU, GC, and GU base pairs are formed. Typically, a first sequence that hybridizes with a second sequence can hybridize specifically or selectively with the second sequence, such that, during hybridization, hybridization with the second sequence or a set of second sequences (e.g., more thermodynamically stable under a given set of conditions, such as stringent conditions commonly used in the art) is preferred over hybridization with a non-target sequence. Generally, hybridizable sequences share a certain degree of sequence complementarity, such as 25%-100% complementarity, across their entire or partial lengths, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity. For example, for the purpose of assessing the percentage of complementarity, sequence identity can be measured using any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally using the default settings), the BLAST algorithm (see, for example, the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally using the default settings), or the Smith-Waterman algorithm (see, for example, the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, optionally using the default settings). Optimal alignment can be evaluated using any suitable parameters of the chosen algorithm, including the default parameters.

[0059] Complementarity can be complete complementarity or near-complementarity / sufficient complementarity. Complete complementarity between two nucleic acids means that they can form a double helix, where each base in the double helix is ​​bonded to a complementary base via Watson-Crick pairing. Near-complementarity or sufficient complementarity means that the sequence in one strand is not perfectly and / or imperfectly complementary to the sequence in the opposing strand, but under a set of hybridization conditions (e.g., salt concentration and temperature), sufficient bonding occurs between the bases on the two strands to form a stable hybrid complex. Such conditions can be predicted either by using the sequence and standard mathematical calculations to predict the Tm of the hybridized strand, or by empirically determining Tm using conventional methods.

[0060] As used herein, the term “regulation” in relation to expression or activity refers to alterations in the level of expression or activity. Regulation can occur at the transcriptional, post-transcriptional, translational, and / or post-translational levels.

[0061] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of at least two amino acid residues linked by peptide bonds. This term does not indicate a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring. This term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be non-amino acid-interval. This term includes amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structures (e.g., domains). This term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, oxidation, and any other operations such as conjugation with a labeled component. As used herein, the term “amino acid” generally refers to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids may include natural and non-natural amino acids that have been chemically modified to include groups or chemical moieties not naturally present on the amino acid. Amino acid analogs may refer to amino acid derivatives. The term “amino acid” includes D-amino acids and L-amino acids.

[0062] When used in this document with respect to polypeptides, the term "variant" refers to a polypeptide that is related to but not identical to a wild-type polypeptide, for example, in terms of amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Variants include polypeptides that contain one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild-type polypeptide. Variants also include derivatives of wild-type polypeptides and fragments of wild-type polypeptides.

[0063] As used herein, the term "percentage of identity (%)" refers to the percentage of amino acid (or nucleic acid) residues in the candidate sequence that are identical to those in the reference sequence after alignment and, where necessary, the introduction of vacancies to achieve the maximum percentage of identity (i.e., vacancies may be introduced in one or both of the candidate and reference sequences to achieve optimal alignment, and non-homologous sequences may be ignored for comparison purposes). For the purpose of determining the percentage of identity, alignment can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. The percentage of identity between the two sequences can be calculated by aligning the test sequence with the comparison sequence using BLAST, determining the number of identical amino acids or nucleotides at the same position in the test sequence as in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0064] As used herein, the term "gene regulatory peptide" or "GMP" refers to a peptide that contains at least an actuating portion capable of regulating gene expression or activity and / or editing nucleic acid sequences. GMPs may contain additional peptide sequences that are not directly involved in regulating gene expression, such as linker sequences, target sequences, etc.

[0065] As used herein, the term "actuated portion" refers to a portion of a gene that can regulate its expression or activity and / or edit a nucleic acid sequence, whether exogenous or endogenous. Actuated portions can regulate gene expression at the transcriptional, post-transcriptional, translational, and / or post-translational levels. Actuated portions can regulate gene expression at the transcriptional level, for example by regulating the production of mRNA from DNA such as chromosomal DNA or cDNA. In some embodiments, the actuated portion recruits at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. The actuated portion itself can bind to DNA and regulate transcription by physically hindering it, for example by preventing proteins such as RNA polymerases and other related proteins from assembling on a DNA template. Actuated portions can regulate gene expression at the translational level, for example by regulating the production of proteins from an mRNA template. In some embodiments, the actuated portion regulates gene expression at the post-transcriptional level by affecting the stability of the mRNA transcript. In some embodiments, the actuated portion regulates gene expression at the post-translational level by altering peptide modifications such as glycosylation of newly synthesized proteins. In some embodiments, the actuated portion regulates gene expression by editing a nucleic acid sequence (e.g., a region of the genome). In some implementations, the actuation portion regulates gene expression by editing the mRNA template. In other cases, editing the nucleic acid sequence can alter the underlying template for gene expression.

[0066] The Cas protein mentioned in this article can be a type of protein or polypeptide. Cas protein can refer to a nuclease. Cas protein can refer to an endonuclease. Cas protein can refer to any modified (e.g., shortened, mutated, extended) polypeptide sequence or homolog of a Cas protein. Cas protein can be codon-optimized. Cas protein can be a codon-optimized homolog of a Cas protein. Cas protein can be enzymatically inactive, partially active, constitutively active, fully active, inducible active, and / or more active (e.g., more active than the wild-type homolog of the protein or polypeptide). Cas protein can be a type II Cas protein. Cas protein can be Cas9. Cas protein can be a type V Cas protein. Cas protein can be Cpf1 or Cas12a. Cas protein can be C2c1. Cas protein can be C2c3. Cas protein can be a type VI Cas protein. Cas protein can be C2c2 or Cas13a. Cas protein can be Cas13b. Cas protein can be Cas13c. Cas protein can be Cas13d. Cas proteins (e.g., variants, mutants, enzymatically inactivated, and / or conditionally enzymatically inactivated site-directed peptides) can bind to target nucleic acids. Cas proteins (e.g., variants, mutants, enzymatically inactivated, and / or conditionally enzymatically inactivated endonucleases) can bind to target RNA or DNA.

[0067] As used herein, the term "crRNA" generally refers to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary crRNA (e.g., crRNA from *Streptococcus pyogenes*). crRNA generally refers to a nucleic acid having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary crRNA (e.g., crRNA from *Streptococcus pyogenes*, *Staphylococcus aureus*, etc.). crRNA can refer to modified forms of crRNA, which may include nucleotide changes such as deletions, insertions or substitutions, variants, mutations, or chimeras. crRNA can be a nucleic acid that has at least about 60% sequence identity with a wild-type exemplary crRNA sequence (e.g., crRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over at least 6 consecutive nucleotides. For example, the crRNA sequence over at least 6 consecutive nucleotides can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the wild-type exemplary crRNA sequence (e.g., crRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.).

[0068] As used herein, the term "tracrRNA" generally refers to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from *Streptococcus pyogenes*). tracrRNA can also refer to a nucleic acid having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from *Streptococcus pyogenes*, *Staphylococcus aureus*, etc.). tracrRNA can also refer to a modified form of tracrRNA, which may include nucleotide changes such as deletions, insertions or substitutions, variants, mutations, or chimeras. A tracrRNA can refer to a nucleic acid that is at least about 60% identical to the sequence of a wild-type exemplary tracrRNA (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) for at least six consecutive nucleotides. For example, the tracrRNA sequence may be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the sequence of a wild-type exemplary tracrRNA (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.).

[0069] As used herein, “guide nucleic acid” can refer to a nucleic acid that can hybridize with another nucleic acid. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. A guide nucleic acid can be programmed to bind to a nucleic acid sequence specifically at a site. The target nucleic acid or the nucleic acid to be targeted may contain nucleotides. A guide nucleic acid may contain nucleotides. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid may be called the complementary strand. The strand of a double-stranded target polynucleotide that is complementary to the complementary strand and therefore may not be complementary to the guide nucleic acid may be called the non-complementary strand. A guide nucleic acid may contain one polynucleotide chain and may be called a “single guide nucleic acid.” A single guide nucleic acid may contain crRNA. A single guide nucleic acid may contain both crRNA and tracrRNA. A guide nucleic acid may contain two polynucleotide chains and may be called a “double guide nucleic acid.” A double guide nucleic acid may contain both crRNA and tracrRNA. Unless otherwise stated, the term “guide nucleic acid” may be inclusive, referring to both single and double guide nucleic acids.

[0070] The guiding nucleic acid may contain segments that can be referred to as "nucleic acid targeting regions" or "nucleic acid targeting sequences." Nucleic acid targeting regions may contain sub-segments that can be referred to as "protein-binding regions," "protein-binding sequences," or "Cas protein-binding regions."

[0071] As used herein, the term "targeting sequence" refers to the nucleotide and corresponding amino acid sequence encoding a targeting polypeptide that mediates the localization (or retention) of a protein to a subcellular location, such as the plasma membrane or the membrane of a given organelle, the nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi apparatus, chloroplasts, apoplasts, peroxisomes, or other organelles. For example, targeting sequences can guide proteins (e.g., receptor or adaptor polypeptides) to the nucleus using nuclear localization signals (NLS); guide proteins out of the nucleus, for example, to the cytoplasm using nuclear export signals (NES); guide proteins to mitochondria using mitochondrial targeting signals; guide proteins to the endoplasmic reticulum (ER) using ER retention signals; guide proteins to peroxisomes using peroxisome targeting signals; guide proteins to the plasma membrane using membrane localization signals; or combinations thereof.

[0072] As used herein, a “nuclear localization domain” can refer to a nuclear localization signal or other sequence or domain capable of crossing the nuclear membrane to enter the nucleus. A nuclear localization domain can fuse with a polypeptide frame; in this case, the nuclear localization domain may be referred to as a “heterologous nuclear localization domain.” A nuclear localization domain can be in an inactive state, in which it cannot cross the nuclear membrane and therefore cannot enter the nucleus. A nuclear localization domain can be in an active state, in which it can cross the nuclear membrane and therefore enter the nucleus. When a heterologous nuclear domain is active and enters the nucleus, the polypeptide fused with the heterologous nuclear domain also enters the nucleus. In response to extracellular or intracellular signals, a nuclear localization domain can switch between an inactive and an active state.

[0073] As used herein, “fusion” can refer to a protein and / or nucleic acid containing one or more non-natural sequences (e.g., portions). Fusions can contain one or more identical non-natural sequences. Fusions can contain one or more different non-natural sequences. Fusions can be chimeras. Fusions can contain nucleic acid affinity tags. Fusions can contain barcodes. Fusions can contain peptide affinity tags. Fusions can provide site-directed subcellular localization of peptides (e.g., nuclear localization signals (NLS) for targeting to the nucleus, mitochondrial localization signals for targeting to mitochondria, chloroplast localization signals for targeting to chloroplasts, endoplasmic reticulum (ER) retention signals, etc.). Fusions can provide non-natural sequences (e.g., affinity tags) that can be used for tracking or purification. Fusions can be small molecules, such as biotin or dyes, such as Alexa fluor dye, Cyanine3 dye, and Cyanine5 dye.

[0074] Fusion can refer to any protein with a functional role. For example, fusion proteins may contain methyltransferase activity, demethylase activity, superoxide dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylation activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthase activity, or demyristylation activity. Effector proteins can modify genomic loci. Fusion proteins can be fusions of Cas proteins and heterologous functional domains. Fusion proteins can be non-natural sequences fused to Cas proteins.

[0075] As used herein, "heterologous functional domain" can refer to a domain within a fusion protein that contains functional activity. A heterologous functional domain can be a transcriptional activator. A heterologous functional domain can be a transcriptional repressor. A heterologous functional domain can include methyltransferase activity, demethylase activity, superoxide dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylation activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitin activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthase activity, or demyristylation activity. The heterologous functional domain can be a chromosome-modifying enzyme, such as a methyltransferase, demethylase, acetyltransferase, deacetylase, deaminase, phosphorylase, dephosphorylase, histone-modifying enzyme, or nucleotide-modifying enzyme. The heterologous functional domain can be a histone-modifying enzyme. The heterologous functional domain can be a nucleotide-modifying enzyme.

[0076] As used herein, “non-natural” can refer to a nucleic acid or polypeptide sequence not found in native nucleic acids or proteins. “Non-natural” can refer to an affinity tag. “Non-natural” can refer to a fusion. “Non-natural” can refer to a naturally occurring nucleic acid or polypeptide sequence containing mutations, insertions, and / or deletions. Non-natural sequences can exhibit and / or encode activities (e.g., enzyme activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.), which can also be exhibited by nucleic acid and / or polypeptide sequences fused to a non-natural sequence. Non-natural nucleic acid or polypeptide sequences can be linked to naturally occurring nucleic acid or polypeptide sequences (or variants thereof) through genetic engineering to generate chimeric nucleic acid and / or polypeptide sequences encoding chimeric nucleic acids and / or polypeptides.

[0077] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to vertebrates, preferably mammals such as humans. Mammals include, but are not limited to, rats, apes, humans, farm animals, racing animals, and pets. This also includes tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro.

[0078] As used herein, the terms “treatment” and “treatment” refer to methods for obtaining a beneficial or desired outcome, including but not limited to therapeutic and / or preventive benefits. For example, a treatment may include the administration of a system or cell population disclosed herein. A therapeutic benefit is any treatment-related improvement or effect on a disease, condition, or symptom in response to one or more treatments. For preventive benefits, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject who has reported one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet been manifested.

[0079] The term "effective amount" or "therapeutic effective amount" refers to an amount of composition, such as an amount of a composition containing immune cells, such as lymphocytes (e.g., T lymphocytes and / or NK cells) constituting the system of this disclosure, sufficient to produce the desired activity when administered to a subject in need. In the context of this disclosure, the term "therapeutic effective" means an amount of composition sufficient to delay the onset of at least one symptom of a condition treated by the methods of this disclosure, to halt its progression, or to cause relief or mitigation.

[0080] As used herein, the term "electromagnetic radiation" refers to one or more wavelengths from the electromagnetic spectrum, including but not limited to X-rays (about 0.1 nanometers (nm) to about 10.0 nm; or about 10 nm). 18 Hertz (Hz) to approximately 10 16 Hz), ultraviolet (UV) light (approximately 10.0 nm to approximately 380 nm; or approximately 8 × 10⁻⁶ Hz). 16 Hz to approximately 10 15Hz), visible light (approximately 380 nm to approximately 750 nm; or approximately 8 × 10⁻⁶ Hz), and visible light (approximately 380 nm to approximately 750 nm; or approximately 8 × 10⁻⁶ Hz). 14 Hz to approximately 4×10 14 Hz), infrared (approximately 750 nm to approximately 0.1 cm; or approximately 4 × 10⁻⁶ Hz). 14 Hz to approximately 5×10 11 Hz) and microwaves (approximately 0.1 cm to approximately 100 cm; or approximately 10 Hz). 8 Hz to approximately 5×10 11 (Hz). In some cases, within the ultraviolet wavelength range, wavelengths from approximately 300 nm to approximately 380 nm can be referred to as "near" ultraviolet, from approximately 200 nm to approximately 300 nm as "far" ultraviolet, and from approximately 10 nm to approximately 200 nm as "extreme" ultraviolet. In some cases, within the visible light wavelength range, wavelengths from approximately 380 nm to approximately 490 nm can be referred to as "blue" light.

[0081] As used in this article, "electromagnetic radiation source" refers to a source that emits electromagnetic radiation. An electromagnetic radiation source can emit one or more wavelengths from the electromagnetic spectrum.

[0082] Extracellular signal-mediated gene regulation

[0083] In one aspect, this disclosure provides a system for regulating the expression of a target polynucleotide in a cell, the system comprising: a chimeric polypeptide comprising a gene-regulating polypeptide fused to a heterologous nuclear localization domain frame, wherein the nuclear localization domain is operable to translocate the chimeric polypeptide to the cell nucleus upon activation by an active cellular signaling pathway responsive to an extracellular signal, wherein the chimeric polypeptide is localized to the cell nucleus in response to the extracellular signal, and the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0084] In one aspect, this disclosure provides a system for regulating the expression of a target polynucleotide in a cell, the system comprising: a) a chimeric receptor polypeptide that activates a cell signaling pathway upon binding to a ligand; and b) a chimeric polypeptide comprising a gene regulatory polypeptide fused within a heterologous nuclear localization domain frame, the heterologous nuclear localization domain being operable to translocate the chimeric polypeptide to the cell nucleus upon activation of the cell signaling pathway, wherein, upon binding of the ligand to the chimeric receptor polypeptide, the chimeric polypeptide is localized to the cell nucleus via the activated heterologous nuclear localization domain, and the gene regulatory polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0085] In one aspect, this disclosure provides a system for regulating the expression of a target polynucleotide in a cell, the system comprising: a) a cell signaling pathway activator comprising a chemical compound; and b) a chimeric polypeptide comprising a gene regulatory polypeptide fused to a heterologous nuclear localization domain frame, the heterologous nuclear localization domain being operable to translocate the chimeric polypeptide to the cell nucleus upon activation of an activated cell signaling pathway, wherein, upon application of the activator to the cell, the chimeric polypeptide is localized to the cell nucleus via the activated heterologous nuclear localization domain, and the gene regulatory polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0086] In one aspect, this disclosure provides a system for regulating the expression of a target polynucleotide in a cell, the system comprising: a chimeric polypeptide comprising a gene-regulating polypeptide fused to a heterologous nuclear localization domain frame, wherein the nuclear localization domain is operable to translocate the chimeric polypeptide to the cell nucleus upon activation by an active cellular signaling pathway, the cellular signaling pathway being responsive to an extracellular signal inducible, wherein, in response to the extracellular signal, the chimeric polypeptide localizes to the cell nucleus, and the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0087] In one aspect, this disclosure provides a system for regulating the expression of a target polynucleotide in a cell, the system comprising: a) a chimeric receptor polypeptide that activates a cell signaling pathway upon binding to a ligand; and b) a chimeric polypeptide comprising a gene regulatory polypeptide fused within a heterologous nuclear localization domain frame, the heterologous nuclear localization domain being operable to translocate the chimeric polypeptide to the cell nucleus upon induction by a cell signaling pathway, wherein, upon binding of the ligand to the chimeric receptor polypeptide, the chimeric polypeptide is localized to the cell nucleus via the induced heterologous nuclear localization domain, and the gene regulatory polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0088] In one aspect, this disclosure provides a system for regulating the expression of a target polynucleotide in cells, the system comprising: a) a cell signaling pathway activator comprising a chemical compound; and b) a chimeric polypeptide comprising a gene regulatory polypeptide fused within a heterologous nuclear localization domain frame, the heterologous nuclear localization domain being operable to translocate the chimeric polypeptide to the cell nucleus upon induction by a cell signaling pathway, wherein, upon application of the activator to the cell, the chimeric polypeptide is localized to the cell nucleus via the activated heterologous nuclear localization domain, and the gene regulatory polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0089] In some embodiments, the gene-regulating peptide is fused within a frame of an adjustable localization domain (e.g., a nuclear localization domain). In some embodiments, the gene-regulating peptide includes an actuating portion, and this actuating portion is fused within a frame of an adjustable localization domain. The adjustable localization domain may include a localization domain that can be induced or activated, whereby the induction or activation allows the localization domain to be localized to a desired location.

[0090] The adjustable positioning domain may include a heterogeneous core positioning domain. The heterogeneous core positioning domain may include core positioning signals.

[0091] Heterologous nuclear localization domains (HNUs) can exist in active and inactive states. In the inactive state, the HNU may be unable to enter the cell nucleus. In the active state, the HNU may be able to enter the cell nucleus.

[0092] The heterologous nuclear localization domain can be derived from a transcription factor. This transcription factor can be a regulated transcription factor that is active solely in response to a signal or signal transduction pathway and is capable of translocating into the nucleus. This transcription factor can be a regulated transcription factor that is primarily active and capable of translocating into the nucleus in response to a signal or signal transduction pathway. This transcription factor can be a regulated transcription factor that is generally active and capable of translocating into the nucleus in response to a signal or signal transduction pathway.

[0093] In some embodiments described herein, the heterologous nuclear localization domain may be derived from members of the activated T cell nuclear factor (NFAT) family. For example, the heterologous nuclear localization domain may be derived from NFATP, NFAT1, NFATC1, NFATC2, NFATC3, NFAT4, NFATCx, NFATC4, NFAT3, or NFAT5.

[0094] In some embodiments described herein, the heterologous nuclear localization domain may be derived from nuclear factor κB ((NF-κB), NFKB1p50, activator protein 1 (AP-1), signal transducer and activator of transcription 1 (STAT1), STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B) and STAT6, sterol response element binding protein (SREBP; e.g., SREBP-1 or SREBF1) or other transcription factors or signal transducers.

[0095] In some embodiments described herein, the heterologous nuclear localization domain may be derived from an intracellular receptor. The intracellular receptor may be a hormone-activated receptor. Hormone-activated receptors may be estrogen receptors, thyroid hormone receptors, steroid hormone receptors, second messenger receptors, inositol triphosphate (IP3) receptors, endocrine peptide hormone receptors, or neurosteroid receptors.

[0096] In some embodiments described herein, the heterologous nuclear localization domain may be derived from light or circadian rhythm or electromagnetic sensing proteins, such as cryptochromes (e.g., CRY1, CRY2), Timeless (TIM), and the PAS domain of PER proteins (e.g., PER1, PER2, and PER3).

[0097] Adjustable localization domains or heteronuclear localization domains can switch between inactive and active states in response to a signal. This switching can be the result of chemical modification. Chemical modification can include dephosphorylation, phosphorylation, demethylation, methylation, acetylation, deacetylation, deamination, ubiquitination, deubiquitination, proteolytic processing, or other suitable chemical modifications. In some cases, chemical modification leads to a switch from an inactive to an active state. In others, chemical modification results in structural or conformational changes, thus leading to a switch from an inactive to an active state. In still others, this structural or conformational change exposes a portion of a localization domain that would not be exposed in the inactive state, thereby creating an active state.

[0098] A tunable localization domain or heteronuclear localization domain can switch between an inactive and an active state in response to a signal. This signal can be an inducing or activating signal. The signal can be a result of a signaling pathway or signal transduction pathway. In some instances, the signaling pathway is activated or induced in the cell. The signal or signal transduction pathway can cause chemical modification of the tunable localization domain or heteronuclear localization domain. Chemical modification can be dephosphorylation, phosphorylation, demethylation, methylation, acetylation, deacetylation, deamination, ubiquitination, deubiquitination, proteolytic processing, or other suitable chemical modifications. In some cases, chemical modification leads to a switch from an inactive state to an active state. In some cases, chemical modification leads to a structural or conformational change, thereby resulting in a switch from an inactive state to an active state. In some cases, the structural or conformational change exposes a portion of the localization domain that would not be exposed in the inactive state, thus producing an active state.

[0099] In some implementations, extracellular signals (e.g., ligands, chemical compounds, etc.) can increase the concentration of intracellular ions (e.g., sodium, potassium, chloride, bicarbonate, calcium, phosphate, etc.) in the cell relative to the baseline level of intracellular ions in the absence of extracellular signals. In some cases, the extracellular ions may include calcium. In some cases, the extracellular ions may be calcium. For certain localization domains or portions (e.g., nuclear localization domains, such as NFAT), regulation of intracellular ion signaling may be crucial for activation of the localization domain. For example, in the case of NFAT proteins, calmodulin (CaM) (which may be a calcium sensor protein) can be activated by an increase in intracellular calcium levels in the cell. After one or more calcium ions bind to CaM, CaM subsequently activates serine / threonine phosphatase calcium-dependent phosphatase (CN). Subsequently, the activated CN rapidly modifies one or more regions of the NFAT protein (e.g., phosphorylating the serine-rich region (SRR) and SP repeat sequence at the N-terminus of the NFAT protein), resulting in a conformational change that exposes the nuclear localization signal, thereby leading to NFAT nuclear input.

[0100] In some embodiments, the signal transduction pathway is activated or induced by a signal or the signal transduction pathway itself. In some embodiments, the signal transduction pathway is activated or induced by a chimeric receptor or a transmembrane receptor of the subject. In some embodiments, the signal transduction pathway is activated or induced by a signal transduction cascade, which in turn is activated by a chimeric receptor or a transmembrane receptor of the subject.

[0101] In some embodiments, the cellularly activated or induced signaling pathway that can switch between inactive and active states of a heterologous nuclear localization domain is the PI3K / AKT pathway. In some embodiments, the transmembrane receptors in this pathway include tyrosine kinase receptors, integrins, B cell receptors, T cell receptors, cytokine receptors, or G protein-coupled receptors, and the signaling pathway further involves PRKCE, ITGAM, ITGA5, IRAK1, PRKAA2, EIF2AK2, PTEN, EIF4E, PRKCZ, GRK6, MAPK1, TSC1, PLK1, AKT2, IKBKB, PIK3CA, CDK8, CDKN1B, NFKB2, BCL2, PIK3CB, PPP2R1A, MAPK8, BCL2L1, MAPK3, TSC2, ITGA1, KRAS, EIF4EBP1, RELA, PR KCD, NOS3, PRKAA1, MAPK9, CDK2, PPP2CA, PIM1, ITGB7, YWHAZ, ILK, TP53, RAF1, IKBKG, RELB, DYRK1A, CDKN1A, ITGB1, MAP2K2, JAK1, AKT1, JAK2, PIK3R1, CHUK , PDPK1, PPP2R5C, CTNNB1, MAP2K1, NFKB1, PAK3, ITGB3, CCND1, GSK3A, FRAP1, SFN, ITGA2, TTK, CSNK1A1, BRAF, GSK3B, AKT3, FOXO1, SGK, HSP90AA1, or RPS6KB1.

[0102] In some embodiments, the cellularly activated or induced signaling pathway that can switch between inactive and active states of a heterologous nuclear localization domain is the ERK / MAPK pathway. In some embodiments, the transmembrane receptors in this pathway include EGFR, Trk A / B, fibroblast growth factor receptor (FGFR), or platelet-derived growth factor receptor (PDGFR), and the signaling pathway further involves PRKCE, ITGAM, ITGA5, HSPB1, IRAK1, PRKAA2, EIF2AK2, RAC1, RAP1A, TLN1, EIF4E, ELK1, GRK6, MAPK1, RAC2, PLK1, AKT2, PIK3CA, CDK8, CREB1, PRKCI, PTK2, FOS, RPS6KA4, PIK3CB, PPP2R1A, PIK3C3, MAPK8, MAPK3, ITGA1, and ETS. 1. KRAS, MYCN, EIF4EBP1, PPARG, PRKCD, PRKAA1, MAPK9, SRC, CDK2, PPP2CA, PIM1, PIK3C2A, ITGB7, YWHAZ, PPP1CC, KSR1, PXN, RAF1, FYN, DYRK1A, ITGB1, MAP2K2, PAK4, PIK3R1, STAT3, PPP2R5C, MAP2K1, PAK3, ITGB3, ESR1, ITGA2, MYC, TTK, CSNK1A1, CRKL, BRAF, ATF4, PRKCA, SRF, STAT1, or SGK.

[0103] In some embodiments, the signaling pathway that can switch between inactive and active states by switching heterologous nuclear localization domains activated or induced in cells is the glucocorticoid receptor signaling pathway. In some embodiments, the transmembrane receptor in this pathway includes glucocorticoid receptors, and the signaling pathway further involves RAC1, TAF4B, EP300, SMAD2, TRAF6, PCAF, ELK1, MAPK1, SMAD3, AKT2, IKBKB, NCOR2, UBE2I, PIK3CA, CREB1, FOS, HSPA5, NFKB2, BCL2, MAP3K14, STAT5B, PIK3CB, PIK3C3, MAPK8, BCL2L1, MAPK3, TSC22D3, MAPK10, NRIP1, KRAS, MAPK13, RELA, STAT5A, MAPK9, NOS2A, PBX1, NR3C1, PIK3C2A, CDKN1C, TRAF2, SERPINE1, NCOA3, MAPK14, TNF, RAF1, IKBKG, MAP3K7, CREBBP, CDKN1A, MAP2K2, JA K1, IL8, NCOA2, AKT1, JAK2, PIK3R1, CHUK, STAT3, MAP2K1, NFKB1, TGFBR1, ESR1, SMAD4, CEBPB, JUN, AR, AKT3, CCL2, MMP1, STAT1, IL6, or HSP90AA1.

[0104] In some embodiments, the signaling pathway that can switch between inactive and active states and is activated or induced in the cell by a heterologous nuclear localization domain is the B cell receptor signaling pathway. In some embodiments, the transmembrane receptors in this pathway include B cell receptors, and the signaling pathway further involves RAC1, PTEN, LYN, ELK1, MAPK1, RAC2, PTPN11, AKT2, IKBKB, PIK3CA, CREB1, SYK, NFKB2, CAMK2A, MAP3K14, PIK3CB, PIK3C3, MAPK8, BCL2L1, ABL1, MAPK3, ETS1, and KRA. S, MAPK13, RELA, PTPN6, MAPK9, EGR1, PIK3C2A, BTK, MAPK14, RAF1, IKBKG, RELB, MAP3K7, MAP2K2, AKT1, P IK3R1, CHUK, MAP2K1, NFKB1, CDC42, GSK3A, FRAP1, BCL6, BCL10, JUN, GSK3B, ATF4, AKT3, VAV3, or RPS6KB1.

[0105] In some implementations, the signaling pathway that can switch between inactive and active states and is activated or induced in the cell by a heterologous nuclear localization domain is the integrin signaling pathway. In some embodiments, the transmembrane receptors in this pathway include integrins or integrin subunits, and the signaling pathway further involves ACTN4, ITGAM, ROCK1, ITGA5, RAC1, PTEN, RAP1A, TLN1, ARHGEF7, MAPK1, RAC2, CAPNS1, AKT2, CAPN2, PIK3CA, PTK2, PIK3CB, PIK3C3, MAPK8, CAV1, CAPN1, ABL1, MAPK3, ITGA1, KRAS, RHOA, SRC, PIK3C2A, ITGB7, PPP1CC, ILK, PXN, VASP, RAF1, FYN, ITGB1, MAP2K2, PAK4, AKT1, PIK3R1, TNK2, MAP2K1, PAK3, ITGB3, CDC42, RND3, ITGA2, CRKL, BRAF, GSK3B, or AKT3.

[0106] In some embodiments, the cellularly activated or induced signaling pathway that can switch between inactive and active states of a heterologous nuclear localization domain is the insulin receptor signaling pathway. In some embodiments, the transmembrane receptor in this pathway includes the insulin receptor, and the signaling pathway further involves PTEN, INS, EIF4E, PTPN1, PRKCZ, MAPK1, TSC1, PTPN11, AKT2, CBL, PIK3CA, PRKCI, PIK3CB, PIK3C3, MAPK8, IRS1, MAPK3, TSC2, KRAS, EIF4, EBP1, SLC2A4, PIK3C2A, PPP1CC, INSR, RAF1, FYN, MAP2K2, JAK1, AKT1, JAK2, PIK3R1, PDPK1, MAP2K1, GSK3A, FRAP1, CRKL, GSK3B, AKT3, FOXO1, SGK, or RPS6KB1.

[0107] In some embodiments, the signaling pathway that can switch between inactive and active states and is activated or induced in the cell by a heterologous nuclear localization domain is the T-cell receptor signaling pathway. In some embodiments, the transmembrane receptor in this pathway includes the T-cell receptor, and the signaling pathway further involves RAC1, ELK1, MAPK1, IKBKB, CBL, PIK3CA, FOS, NFKB2, PIK3CB, PIK3C3, MAPK8, MAPK3, KRAS, RELA, PIK3C2A, BTK, LCK, RAF1, IKBKG, RELB, FYN, MAP2K2, PIK3R1, CHUK, MAP2K1, NFKB1, ITK, BCL10, JUN, or VAV3.

[0108] In some embodiments, the cellularly activated or induced signaling pathway that can switch heterologous nuclear localization domains between inactive and active states is the G protein-coupled receptor (GPCR) signaling pathway. In some embodiments, the transmembrane receptor in this pathway includes GPCRs, and the signaling pathway further involves PRKCE, RAP1A, RGS16, MAPK1, GNAS, AKT2, IKBKB, PIK3CA, CREB1, GNAQ, NFKB2, CAMK2A, PIK3CB, PIK3C3, MAPK3, KRAS, RELA, SRC, PIK3C2A, RAF1, IKBKG, RELB, FYN, MAP2K2, AKT1, PIK3R1, CHUK, PDPK1, STAT3, MAP2K1, NFKB1, BRAF, ATF4, AKT3, or PRKCA.

[0109] Chimeric transmembrane receptors, resulting from the conjugation of various regions or domains from different molecules, may differ from the molecules from which the domains originate, for example, structurally and functionally. However, in some cases, the individual domains may retain their native structure and / or activity. For example, the individual domains may retain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of their native structure and / or activity. For example, the extracellular region containing the ligand-binding domain may retain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the binding affinity of the molecule from which the extracellular region originates. For example, compared to molecules from which the intracellular region originates, the intracellular region containing the signal transduction domain may retain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the ability to activate cellular signal transduction pathways.

[0110] An exemplary configuration of a chimeric transmembrane receptor polypeptide may include (a) an extracellular region, (b) a transmembrane region, and (c) an intracellular region. In some embodiments, the extracellular region may include a ligand interaction domain for binding ligands such as antigens. In some embodiments, the intracellular region includes a cell signaling domain. In some embodiments, the intracellular region includes an immune cell signaling domain.

[0111] The ligand-interaction domain of a chimeric transmembrane receptor peptide may comprise any protein or molecule capable of binding to a ligand, such as an antigen. The ligand-interaction domain of the chimeric transmembrane receptor peptides disclosed herein may be a monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, or functional derivative, variant, or fragment thereof, including but not limited to Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), microantibodies, biantibodies, and single-domain antibodies such as the heavy chain variable domain (VH), light chain variable domain (VL), and the variable domain (VHH) of camel-derived nanobodies. In some embodiments, the ligand-interaction domain comprises at least one of Fab, Fab', F(ab')2, Fv, and scFv. In some embodiments, the ligand F(ab')2 comprises an antibody mimic. Antibody mimics are molecules capable of binding to target molecules with an affinity comparable to that of antibodies, and include single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectin), lipid transport protein scaffolds, calixarene scaffolds, A domains, and other scaffolds. In some embodiments, the ligand interaction domain comprises a transmembrane receptor or any derivative, variant, or fragment thereof. For example, the interaction domain may include at least the ligand-binding domain of a transmembrane receptor.

[0112] In some embodiments, the ligand-interacting domain comprises a humanized antibody. Humanized antibodies can be generated using a variety of techniques, including but not limited to CDR transplantation, veneering or resurfacing, chain shuffling, and other techniques. Selective inclusion of human variable domains, including both light and heavy chains, can reduce the immunogenicity of the humanized antibody. In some embodiments, the ligand-interacting domain of a chimeric transmembrane receptor polypeptide comprises a humanized antibody fragment that binds to the antigen with high affinity and possesses other advantageous biological properties, such as reduced and / or minimal immunogenicity. The humanized antibody or antibody fragment may retain antigen specificity similar to that of the corresponding non-humanized antibody.

[0113] In some implementations, the ligand-interacting domain comprises a single-chain variable fragment (scFv). The scFv molecule can be linked to the heavy chain (V) of an immunoglobulin using flexible linkers such as peptide linkers. H ) and light chains (V L The scFv is generated by connecting the regions together. It can be prepared using various methods.

[0114] In some implementations, the ligand-interacting domain is engineered to bind to a specific target antigen. For example, the ligand-interacting domain may be an engineered scFv. The ligand-interacting domain containing the scFv can be engineered using a variety of methods, including but not limited to display libraries such as phage display libraries, yeast display libraries, cell-based display libraries (e.g., mammalian cells), protein-nucleic acid fusions, ribosome display libraries, and / or E. coli periplasmic display libraries. In some implementations, the engineered ligand-interacting domain can bind to the antigen with a higher affinity than similar antibodies or unengineered antibodies.

[0115] In some embodiments, the ligand-interacting domain binds to multiple antigens, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigens. The ligand-interacting domain may bind to two related antigens, such as two subtypes of botulinum toxin (e.g., botulinum neurotoxin subtype A1 and subtype A2). The ligand-interacting domain may bind to two unrelated proteins, such as receptor tyrosine kinase erbB-2 (also known as Neu, ERBB2, and HER2) and vascular endothelial growth factor (VEGF). An antigen-interacting domain capable of binding to two antigens may include an antibody engineered to bind to two unrelated protein targets at different but overlapping sites. In some embodiments, the antigen-interacting domain binding to multiple antigens includes a bispecific antibody molecule. The bispecific antibody molecule may have a first immunoglobulin variable domain sequence with binding specificity to a first epitope and a second immunoglobulin variable domain sequence with binding specificity to a second epitope. In some embodiments, the first and second epitopes are on the same antigen, such as the same protein (or a subunit of a polyprotein). The first and second epitopes may overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are located on different antigens, such as different proteins (or different subunits of a polyprotein). In some embodiments, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the second epitope. In some embodiments, the bispecific antibody molecule comprises a hapten having binding specificity to the first epitope and a hapten having binding specificity to the second epitope. In some embodiments, the bispecific antibody molecule comprises a hapten or a fragment thereof having binding specificity to the first epitope and a hapten or a fragment thereof having binding specificity to the second epitope.

[0116] In some embodiments, the extracellular region of the chimeric transmembrane receptor polypeptide contains multiple ligand interaction domains, such as at least two ligand interaction domains (e.g., at least three, four, five, six, seven, eight, nine, or ten ligand interaction domains). These multiple ligand interaction domains may exhibit binding to the same or different ligands. In some embodiments, the extracellular region contains at least two ligand interaction domains, such as at least two tandemly linked scFv fragments. In some embodiments, the two scFv fragments are linked via a peptide linker.

[0117] The ligand-interaction domain of the extracellular region of a chimeric transmembrane receptor polypeptide can bind to membrane-bound antigens, such as antigens on the extracellular surface of a cell (e.g., a target cell). In some embodiments, the ligand-interaction domain binds to non-membrane-bound antigens, such as extracellular antigens secreted by the cell (e.g., a target cell) or antigens located in the cytoplasm of the cell (e.g., a target cell). The antigens (e.g., membrane-bound and non-membrane-bound antigens) may be associated with diseases such as viral, bacterial, and / or parasitic infections; inflammatory diseases and / or autoimmune diseases; or tumors such as cancer and / or tumors. Non-limiting examples of antigens that can be bound by the ligand-interaction domain of the chimeric transmembrane receptor polypeptide of the system of the present invention include, but are not limited to, 1-40-β-amyloid protein, 4-1BB, 5AC, 5T4, 707-AP, kinase-anchored protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenergic receptor β3 (ADRB3), AGS-22M6, α-folate receptor, alpha-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, and angiopoietin-binding cell surface receptor 2 (Tie 2) Anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis, B cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), imprinted site regulator brother factor (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCCTC-binding factor (CTCF), CCL11 (eosinophil chemokine-1), CCR4, C CR5, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (α chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3ε, CD30, CD300 molecular-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44 v6, CD5, CD51CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-associated antigen, CFD, ch4D5, X chromosome open reading frame 61 (CXORF61), tight junction protein 18.2 (CLDN18.2), tight junction protein 6 (CLDN6), Clostridium difficile (difficile), aggregation factor A, CLCA2, colony-stimulating factor 1 receptor (CSF1R), CSF2, CTLA-4, C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CXC chemokine receptor type 4, cyclin B1, cytochrome P450 1B1 (CYP1B1), cyp-B, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin EGF type 2, extracellular ADP-ribosyltransferase 4 (ART4), EGF-like motif mucin receptor-like 2 (EMR2), EGF-like domain protein 7 (EGFL7), mutant elongation factor 2 (ELF2M), endotoxin, hepatin A2, hepatin B2, hepatin A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial salivary proteins, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2 ERBB3, ERBB4, ERG (transmembrane serine protease 2 (TMPRSS2) ETS fusion gene), E. coli, ETS translocation variant gene 6 (ETV6-AML) located on chromosome 12p, respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II β chain, fibroblast activating protein α (FAP), fibronectin extracellular domain B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein Folate (FBP), folate hydrolase, folate receptor 1, folate receptor α, folate receptor β, Fos-associated antigen 1, coiled receptor, fucose GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C5 member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), phosphatidylinositol proteoglycan 3 (GPC3), GMCSF receptor α chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, mutant heat shock protein 70-2 (mut hsp70-2), hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1).Hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, hexose moiety of globoH glycoceramide (GloboH), HGF, HHGFR, high molecular weight melanoma-associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPVE7), human dispersive factor receptor kinase, human telomerase reverse transcriptase (hTERT), human TNF, ICAM-1 (CD54), iCE, IFN-α, IFN-β, IFN-γ, IgE, IgE Fc region, IGF-1, IGF-1 receptor, IGH, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1β, IL-20, IL-22, IL-23, IL-31, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, immunoglobulin lamina-like peptide-1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin Integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor, interferon γ-inducible protein, interleukin-11 receptor α (IL-11Rα), interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxyesterase, kinase domain (KDR), KIR2D, KIT (CD117), L1 cell adhesion molecule (L1-CAM), pod Proteins, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, phosphatidylcholine, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex, locus K9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or tumor necrosis factor-α β (TNF-β), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, breast differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitor (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, mucin 1, cell surface-associated protein (MUC1), MUC-2, mucin CanAg, myelin-associated glycoprotein, myostatin,N-acetylglucosamine transferase V (NA17), NCA-90 (granulocyte antigen), nerve growth factor (NGF), neurotrophic proteolytic enzyme 1, neuronal cell adhesion molecule (NCAM), neurogenesis inhibitors (e.g., NOGO-A, NOGO-B, NOGO-C), neurofellin-1 (NRP1), N-hydroxyacetylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), carcinoembryonic antigen (h5T4), oncogene fusion protein (bcr-abl) composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl), and rabbit (Oryctolagus) cuniculus), OX-40, oxLDL, p53 mutant, Pax-3 (PAX3), PAX-5 (PAX5), pannexin 3 (PANX3), phosphate-sodium cotransporter, phosphatidylserine, placenta-specific 1 (Pal1), platelet-derived growth factor receptor α (PDGF-Rα), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, proapocrine-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), proteoproteotransferase subtilisin / kexin Prostate cancer antigen-1 (PCSK9), prostase, prostate cancer tumor antigen-1 (PCTA-1 or galactagogue 8), melanoma antigen-1 recognized by T cells (MelanA or MART1), p15, p53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate cancer cells, prostate-specific protein (prostein), protease serine 21 (Testisin or PRSS21), proteasome (precursor, macroprotein factor) β subunit type 9 (LMP2), Pseudomonas aeruginosa Rabies virus glycoprotein (RAGE), Ras homology family member C (RhoC), receptor activator of nuclear factor κ-B ligand (RANKL), receptor for advanced glycation end products (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitin 1 (RU1), renal ubiquitin 2 (RU2), respiratory syncytial virus (RSV), Rh blood group D antigen, rhesus monkey factor, sarcoma translocation breakpoint, osteosclerosing protein (SOST), select protein P, sialic acid Lewis adhesion molecule (sLe), spermin 17 (SPA17), sphingosine-1-phosphate, squamous cell carcinoma antigens recognized by T cells 1, 2, and 3 (SART1, SART2, and SART3), and stage-specific embryonic antigen-4 (SSEA-4).Staphylococcus aureus, STEAP1, surviving protein, cohesinic polysaccharide 1 (SDC1) + A314, SOX10, surviving protein, surviving protein 2B, synovial sarcoma, SSX2 breakpoint, T cell receptor, TCR1 alternating reading frame protein (TARP), telomerase, TEM1, tendinin C, TGF-β (e.g., TGF-β1, TGF-β2, TGF-β3), thyroid-stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen (Tn) (Ag) or (GalNAcα-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), TNF-α, TRAIL-R1, TRAIL-R2, TRG, transglutaminase 5 (TGS5), tumor antigen CTAA16.88, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tumor protein p53 (p53), tumor-specific glycosylation of MUC1, tumor-associated calcium signaling protein 2, tumor-associated glycoprotein 72 (TAG72), tumor-associated glycoprotein 72 (TAG72) The following proteins are listed: G-72)+A327, TWEAK receptor, tyrosinase, tyrosinase-associated protein 1 (TYRP1 or glycoprotein 75), tyrosinase-associated protein 2 (TYRP2), urothrombolysin 2 (UPK2), vascular endothelial growth factors (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), vimentin, v-myc avian myeloma virus oncogene neurocytoma-derived homolog (MYCN), von Willebrand factor (VWF), Wilms tumor protein (WT1), X antigen family member 1A (XAGE1), β-amyloid protein, and κ-light chain.

[0118] In some implementations, the ligand-interacting domain binds to an antigen selected from: 707-AP, biotinylated molecules, α-actin-4, abl-bcr alb-b3 (b2a2), abl-bcr alb-b4 (b3a2), adipogenesis-associated protein, AFP, AIM-2, annexin II, ART-4, BAGE, β-annexin, bcr-abl, bcr-abl p190 (e1a2), bcr-abl p210 (b2a2), bcr-abl p210(b3a2), BING-4, CAG-3, CAIX, CAMEL, Caspase-8, CD171, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44 v7 / 8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, Ep hA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6 / AML, FBP, fetal acetylcholine receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2(FGF6), HST-2 / neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR / FUT, LewisY, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, malic acid oxidase, mammary globin-A, MART-1 / Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, myosin, NA88-A, Neo-PAP, NKG2D, NPM / ALK, N-RAS, NY-ESO-1, OA1 OGT, carcinoembryonic antigen (h5T4), OS-9, P peptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, survival protein, survival protein-2B, SYT / SSX, TAG-72, TEL / AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2 / INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ-light chain. In some embodiments, the ligand-interacting domain binds to the tumor-associated antigen.

[0119] In some embodiments, the transmembrane receptor includes an endogenous receptor. Any suitable endogenous receptor may be used in the system of the present invention. Transmembrane receptors may include Notch receptors; G protein-coupled receptors (GPCRs); integrin receptors; cadherin receptors; catalytic receptors, including receptors with enzymatic activity and receptors that do not have intrinsic enzymatic activity but act by stimulating non-covalently bound enzymes (e.g., kinases); death receptors, such as members of the tumor necrosis factor receptor (TNFR) superfamily; immune receptors; or any variant thereof. In some embodiments, the transmembrane receptor of the system includes a GPCR. In some embodiments, the transmembrane receptor of the system includes an integrin subunit.

[0120] In some embodiments, the transmembrane receptor of the system of the present invention includes an exogenous receptor. In some embodiments, the exogenous receptor is a synthetic receptor. In some embodiments, the synthetic receptor is a chimeric receptor. The transmembrane receptor may include a chimeric antigen receptor (CAR), a synthetic integrin receptor, a synthetic Notch receptor, or a synthetic GPCR receptor.

[0121] In some embodiments, the transmembrane receptor includes a chimeric antigen receptor (CAR). The ligand-binding domain (e.g., extracellular region) of the CAR may include Fab, a single-stranded variable fragment (scFv), the extracellular region of an endogenous receptor (e.g., GPCR, integrin receptor, T-cell receptor, B-cell receptor, etc.), or an Fc-binding domain. The CAR may include a transmembrane domain that positions the receptor within a cell membrane (e.g., plasma membrane, organelle membrane, etc.). In some embodiments, the signaling domain (e.g., intracellular region) of the CAR includes at least one activating motif (ITAM) based on an immunoreceptor tyrosine residue. In some embodiments, the signaling domain (e.g., intracellular region) of the CAR includes at least one inhibitory motif (ITIM) based on an immunoreceptor tyrosine residue. In some embodiments, the CAR includes both an ITAM motif and an ITIM motif. In some embodiments, the CAR includes at least one co-stimulatory domain.

[0122] Upon binding of a ligand to the ligand-binding domain of a transmembrane receptor, whether it is an endogenous or exogenous transmembrane receptor (e.g., a synthetic receptor, such as a chimeric receptor), the receptor's signaling domain can activate at least one signaling pathway in the cell. Through translational regulation; transcriptional regulation; and epigenetic modifications, including regulation of methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, riboylation, and citrullination, signaling pathways and their associated proteins can participate in regulating (e.g., activating and / or deactivating) cellular responses, such as programmed alterations in gene expression.

[0123] In some cases, cellular responses induced by activation of signaling pathways include activation of the nuclear localization domain (NMR). A cellular response induced by activation of a signaling pathway can remove or release an inhibitor that would otherwise render the NMR inactive. Alternatively, a cellular response induced by activation of a signaling pathway can inactivate the NMR. A cellular response induced by activation of a signaling pathway can also activate or recruit an inhibitor that switches the NMR to an inactive state or renders it inactive.

[0124] In some cases, transcriptional regulation in response to activation of signaling pathways can be used in the systems provided herein to express gene regulatory peptides (GMPs). The nucleic acid sequence encoding a GMP, or GMP-coding sequence, can be placed under the control of a promoter that responds to activation of signaling pathways in response to ligand-receptor binding in the cell.

[0125] In various embodiments of the present invention, transmembrane receptors include endogenous receptors. Non-limiting examples of endogenous receptors include Notch receptors; G protein-coupled receptors (GPCRs); integrin receptors; cadherin receptors; catalytic receptors, including receptors with enzymatic activity and receptors that do not have intrinsic enzymatic activity but act by stimulating non-covalently bound enzymes (e.g., kinases); death receptors, such as members of the tumor necrosis factor receptor (TNFR) superfamily; and immune receptors.

[0126] In various embodiments of the present invention, transmembrane receptors include exogenous receptors. In some cases, exogenous receptors are receptors from different organisms or species. In some cases, exogenous receptors may include synthetic receptors not naturally found in cells. In some embodiments, synthetic transmembrane receptors are chimeric receptors constructed by linking multiple domains (e.g., extracellular domain, transmembrane domain, intracellular domain, etc.) from different molecules (e.g., different proteins, homologous proteins, orthologous proteins, etc.).

[0127] The chimeric transmembrane receptor of the present invention may include an endogenous receptor or any variant thereof. The chimeric transmembrane receptor may, for example, bind specifically to at least one ligand via a ligand-binding domain. The ligand-binding domain typically forms part of the extracellular region of the transmembrane receptor and can sense extracellular ligands. In response to ligand binding, the intracellular region of the chimeric transmembrane receptor may activate cellular signaling pathways. In some cases, the receptor's signaling domain activates cellular signaling pathways.

[0128] In some embodiments, the transmembrane receptor includes the Notch receptor or any variant thereof (e.g., synthetic or chimeric receptors). The Notch receptor is a transmembrane protein that mediates cell-cell contact signaling and plays an important role in the development of cell-cell communication (e.g., communication between two contacting cells (receiving and sending cells)) and other aspects. The Notch receptor expressed on the receiving cell recognizes its ligand (a member of the δ family of proteins) expressed on the sending cell. The binding of Notch and δ on these contacting cells leads to a two-step proteolytic process of the Notch receptor, ultimately resulting in the release of the intracellular portion of the receptor from the membrane into the cytoplasm.

[0129] In some embodiments, the transmembrane receptor includes a Notch receptor selected from Notch1, Notch2, Notch3, and Notch4, any homologs thereof, and any variants thereof. In some embodiments, the chimeric receptor includes at least an extracellular region (e.g., a ligand-binding domain) of the Notch receptor or any variant thereof. In some embodiments, the chimeric receptor includes at least a transmembrane region of Notch or any variant thereof. In some embodiments, the chimeric receptor includes at least an intracellular region (e.g., a cytoplasmic domain) of Notch or any variant thereof. Chimeric receptor peptides containing Notch or any variant thereof can bind to Notch ligands. In some embodiments, the binding of the ligand to a chimeric receptor containing Notch or any variant thereof leads to activation of the Notch signaling pathway.

[0130] In some implementations, transmembrane receptors include G protein-coupled receptors (GPCRs) or any variant thereof (e.g., synthetic or chimeric receptors). GPCRs are typically characterized by a seven-span α-helix and may be arranged in a barrel-like tertiary structure, with the seven-span helix forming a lumen within the plasma membrane that serves as a ligand-binding domain. Ligands may also bind to GPCRs elsewhere, such as to extracellular loops and / or N-terminal tails. Ligand binding can activate the associated G protein, which subsequently functions in various signaling pathways. To deactivate this signaling pathway, the GPCR can first be chemically modified by phosphorylation. Phosphorylation can then recruit co-adaptor proteins (e.g., repressor proteins) for additional signaling.

[0131] In some implementations, transmembrane receptors include GPCRs selected from orphan receptors of class A; orphan receptors of class B; orphan receptors of class C; type I taste receptors; type II taste receptors; serotonin receptors; acetylcholine receptors (muscarinic); adenosine receptors; adhesion-type GPCRs; adrenaline receptors; angiotensin receptors; apelin receptors; bile acid receptors; bufotenoid receptors; bradykinin receptors; calcitonin receptors; calcium-sensing receptors; cannabinoid receptors; chemerin receptors; chemokine receptors; cholecystokinin receptors; frizzled GPCRs (e.g., Wnt receptors); complement peptide receptors; corticotropin-releasing factor receptors; dopamine receptors; endothelin receptors; G protein-coupled estrogen receptors; formyl peptide receptors; free fatty acid receptors; GABAB receptors; glycoprotein receptors; auxin-releasing peptide receptors; glucagon receptor family; glycoprotein hormone receptors; gonadotropin-releasing hormone receptors; GPR18, GPR55, and GPR11. 9; Histamine receptor; Hydroxycarboxylic acid receptor; Kisspeptin receptor; Leukotriene receptor; Lysophospholipid (LPA) receptor; Lysophospholipid (S1P) receptor; Melanin-accumulating hormone receptor; Melanocortin receptor; Melatonin receptor; Metabolic glutamate receptor; Motilin receptor; Neuroregulatory peptide U receptor; Neuropeptide FF / Neuropeptide AF receptor; Neuropeptide S receptor; Neuropeptide W / Neuropeptide B receptor; Neuropeptide Y receptor; Neurotensin receptor; Opioid receptor; Orexin receptor; Oxyglutarate receptor; P2Y receptor; Parathyroid hormone receptor; Platelet-activating factor receptor; Prokineticin receptor; Prolactin-releasing peptide receptor; Prostaglandin receptor; Proteolytic enzyme-activating receptor; QRFP receptor; Relaxin family peptide receptor; Somatostatin receptor; Succinate receptor; Tachykinin receptor; Thyrotropin-releasing hormone receptor; Trace amine receptor; Fish styramine receptor; Vasopressin and oxytocin receptors; VIP and PACAP receptors.

[0132] In some embodiments, the transmembrane receptor includes a GPCR selected from: serotonin receptor 1A (HTR1A), serotonin receptor 1B (HTR1B), serotonin receptor 1D (HTR1D), serotonin receptor 1E (HTR1E), serotonin receptor 1F (HTR1F), serotonin receptor 2A (HTR2A), serotonin receptor 2B (HTR2B), serotonin receptor 2C (HTR2C), serotonin receptor 4 (HTR4), serotonin receptor 5A (HTR5A), serotonin receptor 5B (HTR5BP), serotonin receptor 6 (HTR6), and adenylate cyclase-coupled serotonin receptors. The following receptors are associated with serotonin receptor 7 (HTR7), muscarinic cholinergic receptor 1 (CHRM1), muscarinic cholinergic receptor 2 (CHRM2), muscarinic cholinergic receptor 3 (CHRM3), muscarinic cholinergic receptor 4 (CHRM4), muscarinic cholinergic receptor 5 (CHRM5), adenosine A1 receptor (ADORA1), adenosine A2a receptor (ADORA2A), adenosine A2b receptor (ADORA2B), adenosine A3 receptor (ADORA3), adhesion G protein-coupled receptor A1 (ADGRA1), adhesion G protein-coupled receptor A2 (ADGRA2), adhesion G protein-coupled receptor A3 (ADGRA3), adhesion G protein-coupled receptor B1 (ADGRB1), adhesion G protein-coupled receptor B2 (ADGRB2), adhesion G protein-coupled receptor B3 (ADGRB3), and cadherin EGF. LAG G-receptor 1 (CELSR1), cadherin EGF, LAG G-receptor 2 (CELSR2), cadherin EGF The LAG receptor consists of G protein-coupled receptors: G protein-coupled receptor 3 (CELSR3), G protein-coupled receptor D1 (ADGRD1), G protein-coupled receptor D2 (ADGRD2), G protein-coupled receptor E1 (ADGRE1), G protein-coupled receptor E2 (ADGRE2), G protein-coupled receptor E3 (ADGRE3), G protein-coupled receptor E4 (ADGRE4P), G protein-coupled receptor E5 (ADGRE5), G protein-coupled receptor F1 (ADGRF1), G protein-coupled receptor F2 (ADGRF2), G protein-coupled receptor F3 (ADGRF3), G protein-coupled receptor F4 (ADGRF4), G protein-coupled receptor F5 (ADGRF5), G protein-coupled receptor G1 (ADGRG1), G protein-coupled receptor G2 (ADGRG2), G protein-coupled receptor G3 (ADGRG3), and G protein-coupled receptor G4 (ADGRG4).Adhesion G protein-coupled receptor G5 (ADGRG5), Adhesion G protein-coupled receptor G6 (ADGRG6), Adhesion G protein-coupled receptor G7 (ADGRG7), Adhesion G protein-coupled receptor L1 (ADGRL1), Adhesion G protein-coupled receptor L2 (ADGRL2), Adhesion G protein-coupled receptor L3 (ADGRL3), Adhesion G protein-coupled receptor L4 (ADGRL4), Adhesion G protein-coupled receptor V1 (ADGRV1), Adrenergic receptor α1A (ADRA1A), Adrenergic receptor α1B (ADRA1B), Adrenergic receptor α1D (ADRA1D), Adrenergic receptor α2A (ADRA2A), Adrenergic receptor α2B (ADRA2B), Adrenergic receptor α2C (ADRA2C), Adrenergic receptor β1 (ADRB1), Adrenergic receptor β2 (ADRB2), Adrenergic receptor β3 (ADRB3), Angiotensin II type 1 receptor (AGTR1), Angiotensin II type 2 receptor (AGTR2), Apelin receptor (APLNR), G protein-coupled bile acid receptor 1 (GPBAR1), Neuroregulatory peptide B receptor (NMBR), Gastrin-releasing peptide receptor (GRPR), Bufota peptide-like receptor 3 (BRS3), Bradykinin receptor B1 (BDKRB1), Bradykinin receptor B2 (BDKRB2), Calcitonin receptor (CALCR), Calcitonin receptor Chemokine receptor-like receptor (CALCRL), calcium-sensing receptor (CASR), G protein-coupled receptor class C (GPRC6A), cannabinoid receptor 1 (brain) (CNR1), cannabinoid receptor 2 (CNR2), chemerin chemokine-like receptor 1 (CMKLR1), chemokine (CC motif) receptor 1 (CCR1), chemokine (CC motif) receptor 2 (CCR2), chemokine (CC motif) receptor 3 (CCR3), chemokine (CC motif) receptor 4 (CCR4), chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5), chemokine (CC motif) receptor 6 (CCR6), chemokine (CC motif) receptor 7 (CCR5). 7) Chemokine (CC motif) receptor 8 (CCR8), chemokine (CC motif) receptor 9 (CCR9), chemokine (CC motif) receptor 10 (CCR10), chemokine (CXC motif) receptor 1 (CXCR1), chemokine (CXC motif) receptor 2 (CXCR2), chemokine (CXC motif) receptor 3 (CXCR3), chemokine (CXC motif) receptor 4 (CXCR4), chemokine (CXC motif) receptor 5 (CXCR5), chemokine (CXC motif) receptor 6 (CXCR6), chemokine (C-X3-C motif) receptor 1 (CX3CR1), chemokine (C motif) receptor 1 (XCR1).Atypical chemokine receptor 1 (Duffy blood type) (ACKR1), Atypical chemokine receptor 2 (ACKR2), Atypical chemokine receptor 3 (ACKR3), Atypical chemokine receptor 4 (ACKR4), Chemokine (CC motif) receptor-like 2 (CCRL2), Cholecystokinin A receptor (CCKAR), Cholecystokinin B receptor (CCKBR), G protein-coupled receptor 1 (GPR1), Bufotin-like receptor 3 (BRS3), G protein-coupled receptor 3 (GPR3), G protein-coupled receptor 4 (GPR4), G protein-coupled receptor 6 (GPR6), G protein-coupled receptor 12 (GPR12), G protein-coupled receptor 15 (GPR15), G protein-coupled receptor... G protein-coupled receptor 17 (GPR17), G protein-coupled receptor 18 (GPR18), G protein-coupled receptor 19 (GPR19), G protein-coupled receptor 20 (GPR20), G protein-coupled receptor 21 (GPR21), G protein-coupled receptor 22 (GPR22), G protein-coupled receptor 25 (GPR25), G protein-coupled receptor 26 (GPR26), G protein-coupled receptor 27 (GPR27), G protein-coupled receptor 31 (GPR31), G protein-coupled receptor 32 (GPR32), G protein-coupled receptor 33 (gene / pseudogene) (GPR33), G protein-coupled receptor 34 (GPR34), G protein-coupled receptor 35 (GPR35), G protein-coupled receptor 37 ( Endothelin receptor type B-like (GPR37), G protein-coupled receptor 37-like 1 (GPR37L1), G protein-coupled receptor 39 (GPR39), G protein-coupled receptor 42 (gene / pseudogene) (GPR42), G protein-coupled receptor 45 (GPR45), G protein-coupled receptor 50 (GPR50), G protein-coupled receptor 52 (GPR52), G protein-coupled receptor 55 (GPR55), G protein-coupled receptor 61 (GPR61), G protein-coupled receptor 62 (GPR62), G protein-coupled receptor 63 (GPR63), G protein-coupled receptor 65 (GPR65), G protein-coupled receptor 68 (GPR68), G protein-coupled receptor 75 (GPR75). G protein-coupled receptor 78 (GPR78), G protein-coupled receptor 79 (GPR79), G protein-coupled receptor 82 (GPR82), G protein-coupled receptor 83 (GPR83), G protein-coupled receptor 84 (GPR84), G protein-coupled receptor 85 (GPR85), G protein-coupled receptor 87 (GPR87), G protein-coupled receptor 88 (GPR88), G protein-coupled receptor 101 (GPR101), G protein-coupled receptor 119 (GPR119), G protein-coupled receptor 132 (GPR132), G protein-coupled receptor 135 (GPR135), G protein-coupled receptor 139 (GPR139), G protein-coupled receptor 141 (GPR141).G protein-coupled receptor 142 (GPR142), G protein-coupled receptor 146 (GPR146), G protein-coupled receptor 148 (GPR148), G protein-coupled receptor 149 (GPR149), G protein-coupled receptor 150 (GPR150), G protein-coupled receptor 151 (GPR151), G protein-coupled receptor 152 (GPR152), G protein-coupled receptor 153 (GPR153), G protein-coupled receptor 160 (GPR160), G protein-coupled receptor 161 (GPR161), G protein-coupled receptor 162 (GPR162), G protein-coupled receptor 171 (GPR171), G protein-coupled receptor 173 (GPR173), G protein G protein-coupled receptor 174 (GPR174), G protein-coupled receptor 176 (GPR176), G protein-coupled receptor 182 (GPR182), G protein-coupled receptor 183 (GPR183), leucine-rich G protein-coupled receptor 4 (LGR4), leucine-rich G protein-coupled receptor 5 (LGR5), leucine-rich G protein-coupled receptor 6 (LGR6), MAS1 proto-oncogene (MAS1), MAS1 proto-oncogene-like gene (MAS1L), MAS-associated GPR family member D (MRGPRD), MAS-associated GPR family member E (MRGPRE), MAS-associated GPR family member F (MRGPRF), MAS-associated GPR Family members G (MRGPRG), MAS-associated GPR family member X1 (MRGPRX1), MAS-associated GPR family member X2 (MRGPRX2), MAS-associated GPR family member X3 (MRGPRX3), MAS-associated GPR family member X4 (MRGPRX4), opsin 3 (OPN3), opsin 4 (OPN4), opsin 5 (OPN5), purinergic receptor P2Y (P2RY8), purinergic receptor P2Y (P2RY10), trace amine-associated receptor 2 (TAAR2), trace amine-associated receptor 3 (gene / pseudogene) (TAAR3), trace amine-associated receptor 4 (TAAR4P), and trace amine-associated receptor 5 (TAAR5). Trace amine-associated receptor 6 (TAAR6), trace amine-associated receptor 8 (TAAR8), trace amine-associated receptor 9 (gene / pseudogene) (TAAR9), G protein-coupled receptor 156 (GPR156), G protein-coupled receptor 158 (GPR158), G protein-coupled receptor 179 (GPR179), G protein-coupled receptor class C (GPRC5A), G protein-coupled receptor class C (GPRC5B), G protein-coupled receptor class C (GPRC5C), G protein-coupled receptor class C (GPRC5D), coiled receptor 1 (FZD1), coiled receptor 2 (FZD2), coiled receptor 3 (FZD3), coiled receptor 4 (FZD4), coiled receptor 5 (FZD5)Coiloid receptor 6 (FZD6), Coiloid receptor 7 (FZD7), Coiloid receptor 8 (FZD8), Coiloid receptor 9 (FZD9), Coiloid receptor 10 (FZD10), Smooth Coiloid receptor (SMO), Complement component 3a receptor 1 (C3AR1), Complement component 5a receptor 1 (C5AR1), Complement component 5a receptor 2 (C5AR2), Corticotropin-releasing hormone receptor 1 (CRHR1), Corticotropin-releasing hormone receptor 2 (CRHR2), Dopamine receptor D1 (DRD1), Dopamine receptor D2 (DRD2), Dopamine receptor D3 (DRD3), Dopamine receptor D4 (DRD4), Dopamine receptor D5 (DRD5), Endothelin Receptor type A (EDNRA), endothelin receptor type B (EDNRB), formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (FPR2), formyl peptide receptor 3 (FPR3), free fatty acid receptor 1 (FFAR1), free fatty acid receptor 2 (FFAR2), free fatty acid receptor 3 (FFAR3), free fatty acid receptor 4 (FFAR4), G protein-coupled receptor 42 (gene / pseudogene) (GPR42), γ-aminobutyric acid (GABA) B receptor 1 (GABBR1), γ-aminobutyric acid (GABA) B receptor 2 (GABBR2), glycopropyl peptide receptor 1 (GALR1), glycopropyl peptide receptor 2 (GALR2), glycopropyl peptide receptor 3 (GALR3), growth hormone Glucagon receptor (GHSR), growth hormone-releasing hormone receptor (GHRHR), enteroprotective peptide receptor (GIPR), glucagon-like peptide-1 receptor (GLP1R), glucagon-like peptide-2 receptor (GLP2R), glucagon receptor (GCGR), secretin receptor (SCTR), follicle-stimulating hormone receptor (FSHR), luteinizing hormone / human chorionic gonadotropin receptor (LHCGR), thyroid-stimulating hormone receptor (TSHR), gonadotropin-releasing hormone receptor (GNRHR), gonadotropin-releasing hormone receptor 2 (pseudogene) (GNRHR2), G protein-coupled receptor 18 (GPR18), G protein-coupled receptor 55 (GPR55), G protein-coupled receptor 11 9 (GPR119), G protein-coupled estrogen receptor 1 (GPER1), histamine receptor H1 (HRH1), histamine receptor H2 (HRH2), histamine receptor H3 (HRH3), histamine receptor H4 (HRH4), hydroxycarboxylic acid receptor 1 (HCAR1), hydroxycarboxylic acid receptor 2 (HCAR2), hydroxycarboxylic acid receptor 3 (HCAR3), KISS1 receptor (KISS1R), leukotriene B4 receptor (LTB4R), leukotriene B4 receptor 2 (LTB4R2), cysteyl leukotriene receptor 1 (CYSLTR1), cysteyl leukotriene receptor 2 (CYSLTR2), oxoeicosanoid (OXE) receptor 1 (OXER1)Formoyl phosphatidyl receptor 2 (FPR2), lysophosphatidyl receptor 1 (LPAR1), lysophosphatidyl receptor 2 (LPAR2), lysophosphatidyl receptor 3 (LPAR3), lysophosphatidyl receptor 4 (LPAR4), lysophosphatidyl receptor 5 (LPAR5), lysophosphatidyl receptor 6 (LPAR6), sphingosine-1-phosphate receptor 1 (S1PR1), sphingosine-1-phosphate receptor 2 (S1PR2), sphingosine-1-phosphate receptor 3 (S1PR3), sphingosine-1-phosphate receptor 4 (S1PR4), sphingosine-1-phosphate receptor 5 (S1PR5), melanin condensing hormone receptor 1 (MCHR1), melanin condensing hormone receptor 2 (MCHR2), melanocortin 1 receptor (α) Melatonin receptor (MC1R), melanocortin 2 receptor (MC2R), melanocortin 3 receptor (MC3R), melanocortin 4 receptor (MC4R), melanocortin 5 receptor (MC5R), melatonin receptor 1A (MTNR1A), melatonin receptor 1B (MTNR1B), metabotropic glutamate receptor 1 (GRM1), metabotropic glutamate receptor 2 (GRM2), metabotropic glutamate receptor 3 (GRM3), metabotropic glutamate receptor 4 (GRM4), metabotropic glutamate receptor 5 (GRM5), metabotropic glutamate receptor 6 (GRM6), metabotropic glutamate receptor 7 (GRM7), metabotropic glutamate receptor 8 (GRM8), motilin receptor ( MLNR), neuroregulatory peptide U receptor 1 (NMUR1), neuroregulatory peptide U receptor 2 (NMUR2), neuropeptide FF receptor 1 (NPFFR1), neuropeptide FF receptor 2 (NPFFR2), neuropeptide S receptor 1 (NPSR1), neuropeptide B / W receptor 1 (NPBWR1), neuropeptide B / W receptor 2 (NPBWR2), neuropeptide Y receptor Y1 (NPY1R), neuropeptide Y receptor Y2 (NPY2R), neuropeptide Y receptor Y4 (NPY4R), neuropeptide Y receptor Y5 (NPY5R), neuropeptide Y receptor Y6 (pseudogene) (NPY6R), neurotensin receptor 1 (high affinity) (NTSR1), neurotensin receptor 2 (NTSR2), opioids Opioid receptor δ1 (OPRD1), opioid receptor κ1 (OPRK1), opioid receptor μ1 (OPRM1), opioid receptor-like 1 (OPRL1), hypothalamic orexin receptor 1 (HCRTR1), hypothalamic orexin receptor 2 (HCRTR2), G protein-coupled receptor 107 (GPR107), G protein-coupled receptor 137 (GPR137), olfactory receptor family 51 subfamily E member 1 (OR51E1), adipocyte-associated transmembrane protein 1 (TPRA1), G protein-coupled receptor 143 (GPR143), G protein-coupled receptor 157 (GPR157), α-ketoglutarate receptor 1 (OXGR1).Purinergic receptor P2Y (P2RY1), purinergic receptor P2Y (P2RY2), pyrimidine receptor P2Y (P2RY4), pyrimidine receptor P2Y (P2RY6), purinergic receptor P2Y (P2RY11), purinergic receptor P2Y (P2RY12), purinergic receptor P2Y (P2RY13), purinergic receptor P2Y (P2RY14), parathyroid hormone 1 receptor (PTH1R), parathyroid hormone 2 receptor (PTH2R), platelet-activating factor receptor (PTAFR), prodylinin receptor 1 (PROKR1), prodylinin receptor 2 (PROKR2), prolactin-releasing hormone receptor (PRLHR), prostaglandin D2 receptor (DP)(PT GDR, prostaglandin D2 receptor 2 (PTGDR2), prostaglandin E receptor 1 (PTGER1), prostaglandin E receptor 2 (PTGER2), prostaglandin E receptor 3 (PTGER3), prostaglandin E receptor 4 (PTGER4), prostaglandin F receptor (PTGFR), prostaglandin I2 (prostacyclin) receptor (IP) (PTGIR), thromboxane A2 receptor (TBXA2R), factor II thrombin receptor (F2R), F2R-like trypsin receptor 1 (F2RL1), factor II thrombin receptor-like 2 (F2RL2), F2R-like thrombin / trypsin receptor 3 (F2RL3), pyroglutamyl RF amide peptide receptor (QRFPR), and pine Relaxin / insulin-like peptide receptor 1 (RXFP1), relaxin / insulin-like peptide receptor 2 (RXFP2), relaxin / insulin-like peptide receptor 3 (RXFP3), relaxin / insulin-like peptide receptor 4 (RXFP4), somatostatin receptor 1 (SSTR1), somatostatin receptor 2 (SSTR2), somatostatin receptor 3 (SSTR3), somatostatin receptor 4 (SSTR4), somatostatin receptor 5 (SSTR5), succinate receptor 1 (SUCNR1), tachykinin receptor 1 (TACR1), tachykinin receptor 2 (TACR2), tachykinin receptor 3 (TACR3), taste receptor 1 member 1 (TAS1R1) Taste receptor 1 member 2 (TAS1R2), taste receptor 1 member 3 (TAS1R3), taste receptor 2 member 1 (TAS2R1), taste receptor 2 member 3 (TAS2R3), taste receptor 2 member 4 (TAS2R4), taste receptor 2 member 5 (TAS2R5), taste receptor 2 member 7 (TAS2R7), taste receptor 2 member 8 (TAS2R8), taste receptor 2 member 9 (TAS2R9), taste receptor 2 member 10 (TAS2R10), taste receptor 2 member 13 (TAS2R13), taste receptor 2 member 14 (TAS2R14), taste receptor 2 member 16 (TAS2R16), taste receptor 2 member 19 (TAS2R19).Taste receptor 2 member 20 (TAS2R20), taste receptor 2 member 30 (TAS2R30), taste receptor 2 member 31 (TAS2R31), taste receptor 2 member 38 (TAS2R38), taste receptor 2 member 39 (TAS2R39), taste receptor 2 member 40 (TAS2R40), taste receptor 2 member 41 (TAS2R41), taste receptor 2 member 42 (TAS2R42), taste receptor 2 member 43 (TAS2R43), taste receptor 2 member 45 (TAS2R45), taste receptor 2 member 46 (TAS2R46), taste receptor 2 member 5 Taste receptor 2 receptor 60 (TAS2R50), thyrotropin-releasing hormone receptor (TRHR), trace amine-associated receptor 1 (TAAR1), ostrich stomatine receptor 2 (UTS2R), arginine vasopressin receptor 1A (AVPR1A), arginine vasopressin receptor 1B (AVPR1B), arginine vasopressin receptor 2 (AVPR2), oxytocin receptor (OXTR), adenylate cyclase-activated peptide 1 (pituitary) receptor type I (ADCYAP1R1), vasoactive intestinal peptide receptor 1 (VIPR1), vasoactive intestinal peptide receptor 2 (VIPR2), and any variants thereof.

[0133] In some embodiments, the chimeric receptor comprises a G protein-coupled receptor (GPCR) or any variant thereof. In some embodiments, the chimeric receptor includes at least an extracellular region (e.g., a ligand-binding domain) of the GPCR or any variant thereof. In some embodiments, the chimeric receptor includes at least a transmembrane region of the GPCR or any variant thereof. In some embodiments, the chimeric receptor includes at least an intracellular region (e.g., a cytoplasmic domain) of the GPCR or any variant thereof. A chimeric receptor containing a GPCR or any variant thereof can bind to a GPCR ligand. In some embodiments, the binding of a ligand to a chimeric receptor containing a GPCR or any variant thereof leads to activation of the GPCR signaling pathway.

[0134] In some embodiments, transmembrane receptors include integrin receptors, integrin receptor subunits, or any variant thereof (e.g., synthetic or chimeric receptors). Integrin receptors are transmembrane receptors that can act as bridges for cell-cell and cell-extracellular matrix (ECM) interactions. Integrin receptors are typically formed as heterodimers composed of non-covalently bound α and β subunits. At least 18 α subunits and at least 8 β subunits are present. Each subunit typically comprises an extracellular region (e.g., a ligand-binding domain), a transmembrane region, and an intracellular region (e.g., a cytoplasmic domain).

[0135] In some embodiments, the transmembrane receptor comprises an integrin receptor α subunit or any variant thereof, selected from: α1, α2, α3, α4, α5, α6, α7, α8, α9, α10, α11, αV, αL, αM, αX, αD, αE, and αIIb. In some embodiments, the transmembrane receptor comprises an integrin receptor β subunit or any variant thereof, selected from: β1, β2, β3, β4, β5, β6, β7, and β8. The transmembrane receptor of the inventive system comprising an α subunit, a β subunit, or any variant thereof can heterodimerize (e.g., an α subunit dimerizes with a β subunit) to form an integrin receptor or any variant thereof. Non-limiting examples of integrin receptors include α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α8β1, α9β1, α10β1, αVβ1, αLβ1, αMβ1, αXβ1, αDβ1, αIIbβ1, αEβ1, α1β2, α2β2, α3β2, α4β2, α5β2, α6β2, α7β2, α8β2, α9β2, α10β2, αVβ2, αLβ2, αMβ2, αXβ2, αDβ2, αIIbβ2, αEβ2, α1β3, α2β3, α3β3, α4β3, α5β3, α6β3, α7β3, α8β3, α9β3, α10β3, αVβ3, αLβ3, αMβ3, αXβ3, αDβ3, αIIbβ3, αEβ3, α1β4, α2β4, α3β4, α4β4, α5β4, α6β4, α7β4, α8β4, α9β4, α10β4, αVβ4, αLβ4, αMβ4, αXβ4, αDβ4, αIIbβ4, αEβ4, α1β5, α2β5, α3β5, α4β5, α5β5, α6β5, α7β5, α8β5, α9β5, α10β5, αVβ5, αLβ5, αMβ5, αXβ5, αDβ5, αIIbβ5, αEβ5, α1β6, α2β6, α3β6, α4β6, α5β6, α6β6, α7β6, α8β6, α9β6, α10β6, αVβ6, αLβ6, αMβ6, αXβ6, αDβ6, αIIbβ6, αEβ6, α1β7, α2β7, α3β7, α4β7, α5β7, α6β7, α7β7, α8β7, α9β7, α10β7, αVβ7, αLβ7, αMβ7, αXβ7, αDβ7, αIIbβ7, αEβ7, α1β8, α2β8, α3β8, α4β8, α5β8, α6β8, α7β8, α8β8, α9β8, α10β8, αVβ8, αLβ8, αMβ8, αXβ8, αDβ8, αIIbβ8, and αEβ8 receptors.

[0136] In some embodiments, the chimeric receptor includes at least an extracellular region (e.g., a ligand-binding domain) of an integrin subunit (e.g., an α-subunit or a β-subunit) or any variant thereof. In some embodiments, the chimeric receptor includes at least a transmembrane region of an integrin subunit (e.g., an α-subunit or a β-subunit) or any variant thereof. In some embodiments, the chimeric receptor includes at least an intracellular region (e.g., a cytoplasmic domain) of an integrin subunit (e.g., an α-subunit or a β-subunit) or any variant thereof. Chimeric receptors containing integrin subunits or any variant thereof can bind to integrin ligands. In some embodiments, the binding of a ligand to a chimeric receptor containing integrin subunits or any variant thereof leads to activation of integrin signaling pathways.

[0137] In some implementations, transmembrane receptors include cadherin molecules or any variants thereof (e.g., synthetic or chimeric receptors). Cadherin molecules, which can act as both ligands and receptors, refer to certain proteins involved in mediating cell adhesion. Cadherin molecules typically consist of an extracellular domain with five tandem repeats, a single transmembrane segment, and a cytoplasmic domain. For example, E-cadherin, or CDH1, consists of five repeats in the extracellular domain, a transmembrane domain, and an intracellular domain. When E-cadherin is phosphorylated in the intracellular domain, adaptor proteins such as β-catenin and p120-catenin can bind to the receptor.

[0138] In some embodiments, the transmembrane receptor comprises a cadherin or any variant thereof, selected from classical cadherins, desmosome cadherins, protocadherins, and unconventional cadherins. In some embodiments, the transmembrane receptor comprises a classical cadherin or any variant thereof, selected from CDH1 (E-cadherin, epithelial), CDH2 (N-cadherin, neural), CDH12 (cadherin 12, type 2, N-cadherin 2), and CDH3 (P-cadherin, placenta). In some embodiments, the transmembrane receptor comprises desmosome cadherins or any variant thereof, selected from desmosome core glycoproteins (DSG1, DSG2, DSG3, DSG4) and desmosome glial proteins (DSC1, DSC2, DSC3). In some embodiments, the transmembrane receptor includes procadherin or any variant thereof, selected from PCDH1, PCDH10, PCDH11X, PCDH11Y, PCDH12, PCDH15, PCDH17, PCDH18, PCDH19, PCDH20, PCDH7, PCDH8, PCDH9, PCDHA1, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHA2, PCDHA3, PCDHA4, PCDHA5, PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHAC1, PCDHAC2, PCDHB1, PCDHB10, PCDHB11, PCDHB12, PCDHB13, PCDHB14, P CDHB15, PCDHB16, PCDHB17, PCDHB18, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHB7, PCDHB8, PCDHB9, PCDHGA1, PCDHGA10, PCDHGA11, PCDHGA12, PCDHGA2, PCDHGA3, P CDHGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGB1, PCDHGB2, PCDHGB3, PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, FAT, FAT2 and FAT.In some embodiments, the transmembrane receptor includes unconventional cadherins selected from CDH4 (R-cadherin, retina), CDH5 (VE-cadherin, vascular endothelium), CDH6 (K-cadherin, kidney), CDH7 (cadherin 7, type 2), CDH8 (cadherin 8, type 2), CDH9 (cadherin 9, type 2, T1-cadherin), CDH10 (cadherin 10, type 2, T2-cadherin), CDH11 (OB-cadherin, osteoblasts), CDH13 (T-cadherin, H-cadherin, heart), CDH15 (M... - cadherin, myotube), CDH16 (KSP cadherin), CDH17 (LI cadherin, hepato-gut), CDH18 (cadherin 18, type 2), CDH19 (cadherin 19, type 2), CDH20 (cadherin 20, type 2), CDH23 (cadherin 23, neurosensory epithelium), CDH24, CDH26, CDH28, CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, LOC389118, PCLKC, RESDA1, and RET.

[0139] In some embodiments, the chimeric receptor comprises a cadherin molecule or any variant thereof. In some embodiments, the chimeric receptor comprises at least an extracellular region of a cadherin or any variant thereof. In some embodiments, the chimeric receptor comprises at least a transmembrane region of a cadherin or any variant thereof. In some embodiments, the chimeric receptor comprises at least an intracellular region (e.g., a cytoplasmic domain) of a cadherin or any variant thereof. The chimeric receptor polypeptide comprising a cadherin or any variant thereof may bind to a cadherin ligand. In some embodiments, the binding of the ligand to the chimeric receptor comprising a cadherin or any variant thereof leads to activation of the cadherin signaling pathway.

[0140] In some embodiments, transmembrane receptors include catalytic receptors or any variant thereof (e.g., synthetic or chimeric receptors). Examples of catalytic receptors include, but are not limited to, receptor tyrosine kinases (RTKs) and receptor threonine / serine kinases (RTSKs). Catalytic receptors such as RTKs and RTSKs possess certain enzymatic activities. For example, an RTK can phosphorylate a substrate protein on a tyrosine residue, which can then serve as a binding site for an adaptor protein. RTKs typically contain an N-terminal extracellular ligand-binding domain, a single transmembrane α-helix, and a cytosol C-terminal domain with protein tyrosine kinase activity. Some RTKs consist of a single polypeptide, while others are dimers composed of two pairs of polypeptide chains, such as the insulin receptor and some associated receptors. Binding of the ligand to the extracellular domain of these receptors can activate the cytoplasmic kinase domain, leading to phosphorylation of the receptor itself and intracellular target proteins, thereby propagating the signal induced by ligand binding. In some RTKs, ligand binding induces receptor dimerization. Some ligands (e.g., growth factors such as PDGF and NGF) are themselves dimers composed of two identical polypeptide chains. These growth factors can be directly induced to dimerize by simultaneously binding to two different receptor molecules. Other growth factors (e.g., EGF) are monomers but have two different receptor-binding sites that can cross-link the receptor. Ligand-induced dimerization can lead to receptor autophosphorylation, in which the dimerized polypeptide chains cross-phosphorylate each other. Some receptors can multiply.

[0141] In some implementations, transmembrane receptors include class I RTKs (e.g., the epidermal growth factor (EGF) receptor family, including EGFR; the ErbB family, including ErbB-2, ErbB-3, and ErbB-4), class II RTKs (e.g., the insulin receptor family, including INSR, IGF-1R, and IRR), class III RTKs (e.g., the platelet-derived growth factor (PDGF) receptor family, including PDGFR-α, PDGFR-β, CSF-1R, KIT / SCFR, and FLK2 / FLT3), and class IV RTKs. K-type RTKs (e.g., fibroblast growth factor (FGF) receptor families, including FGFR-1, FGFR-2, FGFR-3, and FGFR-4), V-type RTKs (e.g., vascular endothelial growth factor (VEGF) receptor families, including VEGFR1, VEGFR2, and VEGFR3), VI-type RTKs (e.g., hepatocyte growth factor (HGF) receptor families, including hepatocyte growth factor receptor (HGFR / MET) and RON), and VII-type RTKs (e.g., tropomyosin receptor kinase (Trk) receptor families, including TRKA). Class VIII RTKs (e.g., the Eph receptor family, including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6), Class IX RTKs (e.g., the AXL receptor family, such as AXL, MER, and TRYO3), Class X RTKs (e.g., the LTK receptor family, such as LTK and ALK), and Class XI RTKs (e.g., T... IE receptor families, such as TIE and TEK), XII class RTKs (e.g., ROR receptor families ROR1 and ROR2), XIII class RTKs (e.g., DDR receptor families, such as DDR1 and DDR2), XIV class RTKs (e.g., RET receptor families, such as RET), XV class RTKs (e.g., KLG receptor families, including PTK7), XVI class RTKs (e.g., RYK receptor families, including Ryk), XVII class RTKs (e.g., MuSK receptor families, such as MuSK), or any variant thereof.

[0142] In some embodiments, the chimeric receptor includes at least an extracellular region (e.g., a ligand-binding domain) of a catalytic receptor such as RTK or any variant thereof. In some embodiments, the chimeric receptor includes at least a transmembrane region of a catalytic receptor such as RTK or any variant thereof. In some embodiments, the chimeric receptor includes at least an intracellular region (e.g., a cytoplasmic domain) of a catalytic receptor such as RTK or any variant thereof. Chimeric receptors containing RTK or any variant thereof can bind to RTK ligands. In some embodiments, the binding of a ligand to a chimeric receptor containing RTK or any variant thereof leads to activation of the RTK signaling pathway.

[0143] In some embodiments, the chimeric receptor includes at least an extracellular region (e.g., a ligand-binding domain) of a catalytic receptor such as RTSK or any variant thereof. In some embodiments, the chimeric receptor includes at least a transmembrane region of a catalytic receptor such as RTSK or any variant thereof. In some embodiments, the chimeric receptor includes at least an intracellular region (e.g., a cytoplasmic domain) of a catalytic receptor such as RTSK or any variant thereof. Chimeric receptors containing RTSK or any variant thereof can bind to RTSK ligands. In some embodiments, the binding of a ligand to a chimeric receptor containing RTSK or any variant thereof leads to activation of the RTSK signaling pathway.

[0144] In some embodiments, transmembrane receptors including RTSK or any variant thereof can phosphorylate substrates at serine and / or threonine residues, and specific residues can be selected based on a shared sequence. Transmembrane receptors may include type I RTSK, type II RTSK, or any variant thereof. In some embodiments, transmembrane receptors including type I receptor serine / threonine kinases are inactive unless complexed with a type II receptor. In some embodiments, transmembrane receptors including type II receptor serine / threonine kinases contain a constitutively active kinase domain that can phosphorylate and activate the type I receptor when complexed with it. Type II receptor serine / threonine kinases can phosphorylate the kinase domain of the type I partner, leading to protein partner substitution.

[0145] Replacement of the protein partner can allow other proteins (e.g., certain members of the SMAD family) to bind and phosphorylate. Transmembrane receptors may include type I receptors or any variant thereof, selected from: ALK1 (ACVRL1), ALK2 (ACVR1A), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFβR1), ALK6 (BMPR1B), and ALK7 (ACVR1C). Transmembrane receptors may include type II receptors or any variant thereof, selected from: TGFβR2, BMPR2, ACVR2A, ACVR2B, and AMHR2 (AMHR).

[0146] In some embodiments, transmembrane receptors include receptors or any variants thereof that stimulate non-covalently associated intracellular kinases such as Src kinases (e.g., c-Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn, and Frk) or JAK kinases (e.g., JAK1, JAK2, JAK3, and TYK2) without intrinsic enzymatic activity. These include the cytokine receptor superfamily, such as cytokine receptors and peptide hormone receptors. Cytokine receptors typically contain an N-terminal extracellular ligand-binding domain, a transmembrane α-helix, and a C-terminal cytoplasmic domain. The cytoplasmic domain of cytokine receptors typically lacks any known catalytic activity. Instead, cytokine receptors may co-act with non-receptor kinases (e.g., tyrosine kinases or threonine / serine kinases) that can be activated by ligand binding to the receptor.

[0147] In some embodiments, the chimeric receptor includes at least an extracellular region (e.g., a ligand-binding domain) of a catalytic receptor or any variant thereof that is non-covalently associated with an intracellular kinase (e.g., a cytokine receptor). In some embodiments, the chimeric receptor includes at least a transmembrane region of a catalytic receptor or any variant thereof that is non-covalently associated with an intracellular kinase (e.g., a cytoplasmic domain). A chimeric receptor containing a catalytic receptor or any variant thereof that is non-covalently associated with an intracellular kinase can bind to a ligand. In some embodiments, the binding of a ligand to a chimeric receptor containing a catalytic receptor or any variant thereof that is non-covalently associated with an intracellular kinase leads to activation of a signaling pathway.

[0148] Cytokine receptors typically contain an N-terminal extracellular ligand-binding domain, a transmembrane α-helix, and a C-terminal cytoplasmic domain. The cytoplasmic domain of cytokine receptors generally lacks any known catalytic activity. Instead, cytokine receptors can interact in combination with non-receptor kinases (e.g., tyrosine kinases or threonine / serine kinases), which can be activated by ligand binding to the receptor.

[0149] In some embodiments, the transmembrane receptor includes a cytokine receptor, such as a type I cytokine receptor or a type II cytokine receptor or any variant thereof. In some embodiments, the transmembrane receptor includes interleukin receptors (e.g., IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-11R, IL-12R, IL-13R, IL-15R, IL-21R, IL-23R, IL-27R, and IL-31R), colony-stimulating factor receptors (e.g., erythropoietin receptor, CSF-1R, CSF-2R, GM-CSFR, and G-CSFR), hormone receptors / neuropeptide receptors (e.g., growth hormone receptor, prolactin receptor, and leptin receptor) or any variant thereof. In some embodiments, the transmembrane receptor includes a type II cytokine receptor or any variant thereof. In some implementations, transmembrane receptors include interferon receptors (e.g., IFNAR1, IFNAR2, and IFNGR), interleukin receptors (e.g., IL-10R, IL-20R, IL-22R, and IL-28R), tissue factor receptors (also known as platelet tissue factor), or any variant thereof.

[0150] In some implementations, transmembrane receptors include death receptors, receptors containing a death domain, or any variant thereof. Death receptors are often involved in regulating apoptosis and inflammation. Death receptors include members of the TNF receptor family such as TNFR1, Fas receptor, DR4 (also known as TRAIL receptor 1 or TRAILR1), and DR5 (also known as TRAIL receptor 2 or TRAILR2).

[0151] In some embodiments, the chimeric receptor includes at least an extracellular region (e.g., a ligand-binding domain) of the death receptor or any variant thereof. In some embodiments, the chimeric receptor includes at least a transmembrane region (e.g., a cytoplasmic domain) of the death receptor or any variant thereof. Chimeric receptors containing the death receptor or any variant thereof may undergo receptor oligomerization in response to ligand binding, which may subsequently lead to the recruitment of specialized adaptor proteins and activation of signaling cascades such as the caspase cascade.

[0152] In some implementations, transmembrane receptors include immune receptors or any variants thereof. Immune receptors include members of the immunoglobulin superfamily (IgSF) that share structural features with immunoglobulins, such as domains referred to as immunoglobulin domains or folded domains. IgSF members include, but are not limited to, cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, and molecules involved in presenting antigens to lymphocytes.

[0153] In some embodiments, the ligand-interacting domain binds to an antigen containing an antibody, such as an antibody that binds to a cell surface protein or peptide. The cell surface protein or peptide bound by the antibody may include disease-associated antigens, such as viral, bacterial, and / or parasitic infections; inflammatory diseases and / or autoimmune diseases; or vegetations such as cancer and / or tumors. In some embodiments, the antibody binds to a tumor-associated antigen (e.g., a protein or peptide). In some embodiments, the ligand-interacting domain of the chimeric transmembrane receptor peptides disclosed herein may bind to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or functional derivatives, variants, or fragments thereof, including but not limited to Fab, Fab', F(ab')2, Fc, Fv, scFv, microantibodies, biantibodies, and single-domain antibodies such as the heavy chain variable domain (VH), light chain variable domain (VL), and the variable domain (VHH) of camel-derived nanobodies. In some embodiments, the ligand-interacting domain may bind to at least one of Fab, Fab', F(ab')2, Fc, Fv, and scFv. In some implementations, the ligand-interacting domain binds to the Fc domain of the antibody.

[0154] In some implementations, the ligand-interacting domain binds to an antibody selected from: 20-(74)-(74) (mirazolizumab; vetuzumab), 20-2b-2b, 3F8, 74-(20)-(20) (mirazolizumab; vetuzumab), 8H9, A33, AB-16B5, abavoxel, abciximab, abituzumab, ABP 494 (cetuximab biosimilar), abrilumab, ABT-700, ABT-806, actitumab-A (actinic acid Ac-225 lintuzumab), actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adenomyumab, aducanumab, afimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, pegciarizumab, ALD518, alenumab, alicurumab, atamuximab-pentetic acid, amatuximab, AMG 228, AMG 820, anetumab ravtansine, anifrolumab, anifrolumab, APN301, APN311, apizhuzumab, APX003 / SIM-BD0801 (sevacizumab), APX005M, acipimozide, ARX788, ascrinvacumab, acecilizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also known as tocilizumab), atorumab, avelumab B-701, Bavixizumab, Baliximab, Bavexizumab, BAY1129980, BAY1187982, Betumomab, Begelomab, Belixizumab, Benralizumab, Betumomab, Betalutin (177Lu-tetraxetan-tetulomab), Bevacizumab, BEVZ92 (bevacizumab biosimilar), Bezlotoxumab, BGB-A317, BHQ880, BI836880, BI-505, Bimagrumab, Bimekizumab, Bivatuzumab Mertansine, BIW-8962, Blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, Bococizumab, Bentuximab, BrevaRex, Brodalumab, Brolucizumab, Brontictuzumab, C2-2b-2b, Cantuzumab, Mecanizumab, Cantuzumab ravtansine, caplacizumab, carlumab, captuzumab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab), CC-90002, CDX-014, CDX-1401, sildenafil, pecimenopazine, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, pocituzumab, cetuximab, clazakizumab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, Cotara (I-131 derlotuximab biotin), cR6261, crenezumab, DA-3111 (trastuzumab biosimilar), dasizumab, dalizumab, dalotuzumab, dapirolizumab, daratumumab Enhanze, daratumumab, ddectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab, depatuxizumabMafodotin, derlotuximab, dermo-B4, dartuximab, diridavumab, DKN-01, DMOT4039A, atordotin, drozitumab, DS-1123, DS-8895, duligotumab, dupilumab, dusigitumab, emexizumab, eculizumab, edazolizumab, efalizumab, edazolizumab, eldelumab, elgemtumab, erlotuximab, esimozumab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab, vedotin, enoblituzumab, enocituzumab, enoticumab, ensituximab, epitumomab cituxetan, epazolizumab, erlizumab, ertoxumab, ertoxumab, etrolizumab, evinacumab, evolocumab, evinocumab, fanolesomab, falamomab, falizumab, fasinumab, FBTA05, fazolizumab, fezanumab, FF-21101, FGFR2 antibody-drug conjugate, Fibromun, ficlatuzumab, fentuzumab, firivuma b. flanvotumab, fletikumab, fentozumab, foralumab, fresolimumab, FPA144, fresolimumab, FS102, fulranumab, futuximab, galliximab, gantenerumab, gavimumab, gerilimzumab-ozomicin, gerilimzumab, geovokizumab, glembatumumab vedotin, GNR-006, GNR-011, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, gusekumab, Hu14.18K322AMAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, Iparizumab, Irimomab, icrucumab, idarucizumab, IGN002, IGN523, Igovovomab, IMAB362, IMAB362 (claudiximab), Imarumab, IMC-CS4, IMC-D11, Ingazumab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epazumab-tetraxetan), IMMU-114, ImmuTune MP701 antagonist antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab Vedotin, Infliximab, Innomomab, Oxytocinumab, Intolimab, Ipafricept, IPH4102, Ipilimumab, Itolimab, Ixartuzumab, Istiratumab, Itolizumab, Ixekizumab, JNJ-56022473, JNJ-61610588, Keliximab, KTN3379, L19IL2 / L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, Lambrolizumab, Lampalizumab, L-DOS47, Lebrikizumab, Lemasomumab, Lenzilumab, Leukotuximab, Lesamumab, Riviremab, Verivatutumab, Lintoxumab, Lifastuzumabvedotin, Lintoxumab, Lilotomab satetraxetan, LKZ145, losecuzumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumretuzumab, LY3164530, mapamumab, margetuximab, masmosumab, mastozumab, mastozumab, mavliglimab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (inebilizumab), MEDI-565, MEDI6469, mepolizumab, metemumab, MGB453, MGD006 / S80880, MGD007, MGD009, MGD011, milatuzumab, milatuzumab-SN-38, mirvetuximabsoravtansine, mitomumab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, moromulumab, moxicillin, moxetumomab pasudotox, morotumab-CD3, tanacozymeb, namilumab, inatumab, narnatumab, natazumab, nebakumab, nemolizumab, neremozumab, nesvacumab, nimozumab, nivolumab, nivolumab, nivolumab, NOV-10, obiltoxaximab, ocaratuzumab, ocrelizumab, odumozumab, ofamumab, olamatuzumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, anatozumab, ontuxizumab, opicinumab, oportuzumabmonatox, ocovuduzumab, orticumab, otlertuzumab ab, 0X002 / MEN1309, ozanezumab, ozoralizumab, pagibaximab, pallizumab, pankomab, PankoMab-GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pemtumomab, perakizumab, pertuzumab, pemetizumab, PF-05082566 (utomilumab), PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab Vedotin, placulumab, polatuzumab, vedotin, ponezumab, pritoxaximab, pro 140, Proxinium, PSMAADC, quilizumab, radretumab, ralpancizumab, ramucirumab, ranibizumab, refanezumab, regavir, REGN1400, REGN2810 / SAR439684, rallizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, RO7009789, rostrumab, roledumab, romosozumab, rontalizumab, roveizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, Satumomab, SCT200, SCT400, SEA-CD40, Secukinumab, Seribantumab, Setoxaximab, Sevimetaab, SGN-CD19A, SGN-CD19B, SGN-CD33A, SGN-CD70A, SGN-LIV1A, Sirozumab, Sifamumab, Secuximab, Simtuzumab, Cialis, Cilukumab, Sofituzumab, Vedotin, Solanezumab, Solitomab, Sonepizumab, Sotuzumab, Stamolu, Thioxamab, Suvizumab, SYD985, SYM004 (futuximab and modotuximab), Sym015, TAB08, Tabalumab, Tacatuzumab Tetraxetan, Taduzumab, Talizumab, Tanibirumab, Pertapromumab, Taretozumab, TB-403, Tefibazumab, Teleukin, Atemumab, Tenatumomab, Tenesimumab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, TG-1303, TGN1412, Thorium-227-Epazalumab, Tiglimumab, Tigazumab, TisotumabVedotin, TNX-650, Tocilizumab, Tosatoxumab, Tosatoxumab, Tovetumab, Tralokinumab, Trastuzumab-Metansin, TRBS07, TRC105, Tregalizumab, Trevogrumab, TRPH 011, TRX518, TSR-042, TTI-200.7, Tucotuzumab celmoleukin, Tovirazumab, U3-1565, U3-1784, Ublituximab, Ulocuplumab, Urelumab, Ustenumab, Vadastuximab Talirine, Vandortuzumab The following antibodies are included: vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vaporizumab, VB6-845, vedotin, vetozumab, vepamozumab, vesenkumab, vexizumab, voroxizumab, mafodotin, vorsetuzumab, YYB-101, zarumumab, zanolimumab, zaloxizumab, ziralimumab, and azomozumab. In some embodiments, the ligand-interacting domain binds to the Fc domain of the aforementioned antibodies.

[0155] In some implementations, the ligand-interacting domain binds to the antibody, which in turn binds to an antigen selected from the group consisting of: 1-40-β-amyloid protein, 4-1BB, 5AC, 5T4, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1), B7-H3, Bacillus anthracis, BAFF, β-amyloid protein, B-lymphoma cells, C242 antigen, C5, CA-125, canine IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (a cytotoxic agent). Eosinophil chemokine-1), CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3ε, CD30, CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-associated antigen, CFD, ch4D5, CLDN18.2, Clostridium difficile, aggregation factor A, CSF1R, CSF2, CTLA-4, CXC chemokine receptor type 4, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2, EGFL7, EGFR, endotoxin, EpCAM, epithelial salivary protein, ERBB3, Escherichia coli, respiratory syncytial virus F protein, FAP, fibrin I Iβ chain, fibronectin extracellular domain B, folate hydrolase, folate receptor 1, folate receptor α, coiled receptor, ganglioside GD2, GD2, GD3 ganglioside, phosphatidylinositol proteoglycan 3, GMCSF receptor α chain, GPNMB, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, Hsp90, human dispersive factor receptor kinase, human TNF, human β-amyloid protein, ICAM-1 (CD54), IFN-α, IFN-γ, IgE, IgE Fc region, IGF-1 receptor, IGF-1, IGHE, IL-17A, IL-17F, IL20. IL-12, IL-13, IL-17, IL-1β, IL-22, IL-23, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, ILGF2, influenza A hemagglutinin, influenza A virus hemagglutinin, insulin-like growth factor I receptor, integrin α4β7, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor, interferon γ-inducible protein ITGA2, ITGB2 (CD18), KIR2D, Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, phosphatidylcholine, LOXL2, L-selectin (CD62L), LTA, MCP-1, mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen), neurotrophic regulatory protease 1, NGF, N-hydroxyacetylneuraminic acid, NOGO-A, Notch receptor, NRP1, rabbit, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-Rα, phosphate-sodium cotransporter, phosphatidylserine, platelet-derived growth factor receptor β, prostate cancer cells, Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, rhesus monkey factor, RON, RTN4, osteosclerosing protein, SDC1, selector protein P, SLAMF7, SOST, sphingosine-1 Phosphate, Staphylococcus aureus, STEAP1, TAG-72, T cell receptor, TEM1, tendinin C, TFPI, TGF-β1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, tumor-specific glycosylation of MUC1, tumor-associated calcium signaling protein 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA, VEGFR1, VEGFR2, vimentin, and VWF.

[0156] In some embodiments, the ligand-interacting domain may bind to an antibody mimic. As described elsewhere herein, the antibody mimic may bind to the target molecule with an affinity comparable to that of an antibody. In some embodiments, the ligand-interacting domain may bind to a humanized antibody as described elsewhere herein. In some embodiments, the ligand-interacting domain of a chimeric transmembrane receptor polypeptide may bind to a fragment of a humanized antibody. In some embodiments, the ligand-interacting domain may bind to a single-chain variable fragment (scFv).

[0157] In some embodiments, the ligand-interacting domain binds to the Fc moiety of a suitable mammalian immunoglobulin (e.g., IgG, IgA, IgM, or IgE). The suitable Fc-binding domain can be derived from naturally occurring proteins, such as mammalian Fc receptors or certain bacterial proteins (e.g., A and G proteins). Alternatively, the Fc-binding domain can be a synthetic peptide specifically engineered to bind to the Fc moiety of any Ig molecule described herein with the desired affinity and specificity. For example, such an Fc-binding domain can be an antibody or its antigen-binding fragment that specifically binds to the Fc moiety of an immunoglobulin. Examples include, but are not limited to, single-chain variable fragments (scFvs), domain antibodies, and nanobodies. Alternatively, the Fc-binding domain can be a synthetic peptide that specifically binds to the Fc moiety, such as a Kunitz domain, a small modular immunopharmaceutical (SMIP), adnectin, avimer, affibody, DARPin, or anticalin, which can be identified by screening for Fc-binding activity in a peptide library.

[0158] In some embodiments, the ligand interaction domain includes an Fc binding domain comprising the extracellular ligand binding domain of a mammalian Fc receptor. Fc receptors are typically cell surface receptors expressed on the surface of many immune cells, including B cells, dendritic cells, natural killer (NK) cells, macrophages, neutrophils, mast cells, and eosinophils, and exhibit binding specificity to the Fc domain of an antibody. In some cases, binding of the Fc receptor to the Fc moiety of an antibody can trigger antibody-dependent cell-mediated cytotoxicity (ADCC) effects. The Fc receptor used to construct the chimeric transmembrane receptor peptide described herein can be a naturally occurring polymorphic variant, such as a variant that may have altered (e.g., increased or decreased) affinity for the Fc domain compared to its wild-type counterpart. Alternatively, the Fc receptor can be a functional variant of the wild-type counterpart carrying one or more mutations (e.g., substitutions of up to 10 amino acid residues) that alter the binding affinity to the Fc moiety of an Ig molecule. In some implementations, mutations can alter the glycosylation pattern of the Fc receptor, thereby changing the binding affinity to the Fc domain.

[0159] Table 1 lists some exemplary polymorphisms in the extracellular domain of the Fc receptor (see, for example, Kim et al., J.Mol.Evol.53:1-9, 2001).

[0160] Table 1. Exemplary polymorphisms in Fc receptors

[0161] amino acid sequence number 19 48 65 89 105 130 134 141 142 158 FCR10 R S D I D G F Y T V P08637 R S D I D G F Y I F S76824 R S D I D G F Y I V J04162 R N D V D D F H I V M31936 S S N I D D F H I V M24854 S S N I E D S H I V X07934 R S N I D D F H I V X14356(FcγRII) N N N S E S S S I I M31932(FcγRI) S T N R E A F T I G X06948(FcαεI) R S E S Q S E S I V

[0162] Fc receptors can typically be classified based on the isotypes of antibodies they can bind. For example, Fc-γ receptors (FcγR) typically bind to IgG antibodies (e.g., IgG1, IgG2, IgG3, and IgG4); Fc-α receptors (FcαR) typically bind to IgA antibodies; and Fc-ε receptors (FcεR) typically bind to IgE antibodies. In some embodiments, the ligand interaction domain comprises the Fcγ receptor or any derivative, variant, or fragment thereof. In some embodiments, the ligand interaction domain includes an Fc-binding domain comprising an FcR selected from FcγRI (CD64), FcγRIa, FcγRIb, FcγRIc, FcγRIIA (CD32) including allotypes H131 and R131, FcγRIIB (CD32) including FcγRIIB-1 and FcγRIIB-2, FcγRIIIA (CD16a) including allotypes V158 and F158, FcγRIIIB (CD16b) including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2, any derivatives thereof, any variants thereof, and any fragments thereof. The FcγR can be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2). In some embodiments, the ligand interaction domain comprises an Fcε receptor or any derivative, variant, or fragment thereof. In some embodiments, the ligand interaction domain comprises an FcR selected from FcεRI, FcεRII (CD23), any derivative thereof, any variant thereof, and any fragment thereof. In some embodiments, the ligand interaction domain comprises an Fcα receptor or any derivative, variant, or fragment thereof. In some embodiments, the ligand interaction domain comprises an FcR selected from FcαRI (CD89), Fcα / μR, any derivative thereof, any variant thereof, and any fragment thereof. In some embodiments, the ligand interaction domain comprises an FcR selected from FcRn, any derivative thereof, any variant thereof, and any fragment thereof. The choice of ligand-binding domain for Fc receptors of chimeric transmembrane receptor peptides may depend on various factors, such as the isotype of the antibody to which the Fc binding domain is expected to bind and the desired affinity of the binding interaction.

[0163] In some embodiments, the ligand interaction domain includes an extracellular ligand-binding domain of CD16, which may incorporate a naturally occurring polymorphism that modulates the affinity for the Fc domain. In some embodiments, the ligand interaction domain includes an extracellular ligand-binding domain of CD16, which incorporates a polymorphism (e.g., valine or phenylalanine) at position 158. In some embodiments, the ligand interaction domain is generated under conditions that alter its glycosylation state and its affinity for the Fc domain. In some embodiments, the ligand interaction domain comprises an extracellular ligand-binding domain of modified CD16, which makes the chimeric transmembrane receptor polypeptide incorporated therein specific for a subset of IgG antibodies.

[0164] For example, mutations that increase or decrease affinity for IgG subtypes (e.g., IgG1) may be incorporated. In some embodiments, the ligand interaction domain includes an extracellular ligand-binding domain of CD32, which may incorporate naturally occurring polymorphisms that modulate affinity for the Fc domain. In some embodiments, the ligand interaction domain includes an extracellular ligand-binding domain of modified CD32, which makes the chimeric transmembrane receptor polypeptide incorporated therein specific for a subset of IgG antibodies. For example, mutations that increase or decrease affinity for IgG subtypes (e.g., IgG1) may be incorporated.

[0165] In some embodiments, the ligand interaction domain includes an extracellular ligand-binding domain of CD64, which may incorporate naturally occurring polymorphisms that modulate the affinity for the Fc domain. In some embodiments, the ligand interaction domain is generated under conditions that alter its glycosylation state and its affinity for the Fc domain. In some embodiments, the ligand interaction domain includes an extracellular ligand-binding domain of modified CD64, which makes the chimeric transmembrane receptor polypeptide incorporated therein specific for a subset of IgG antibodies. For example, mutations that increase or decrease affinity for IgG isotypes (e.g., IgG1) may be incorporated.

[0166] In other embodiments, the ligand interaction domain comprises a naturally occurring bacterial protein (e.g., protein A, protein G) capable of binding to the Fc portion of an IgG molecule or any derivative, variant, or fragment thereof. In some embodiments, the ligand interaction domain comprises protein A or any derivative, variant, or fragment thereof. Protein A refers to a 42 kDa surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It consists of five domains, each folded into a triple helix bundle, and is capable of binding to IgG by interacting with the Fc region of most antibodies and the Fab region of human VH3 family antibodies. In some embodiments, the ligand interaction domain comprises protein G or any derivative, variant, or fragment thereof. Protein G refers to an approximately 60-kDa protein expressed in group C and group G streptococci that binds to the Fab and Fc regions of mammalian IgG. Although native protein G also binds to albumin, recombinant variants have been engineered to eliminate albumin binding.

[0167] Ligand-interacting domains can also be created de novo using combinatorial biology or directed evolution methods. Starting with a protein scaffold (e.g., scFv derived from IgG, Kunitz domain derived from a Kunitz-type protease inhibitor, ankyrin repeat sequences, Z domain from protein A, lipid transport proteins, fibronectin type III domains, SH3 domain from Fyn, or other domains), a set of amino acid side chains on the surface can be randomly substituted to create a large variant scaffold library. From the large library, variants with affinity for targets such as Fc domains can be isolated by first selecting for binding, followed by amplification via phage, ribosome, or cell display. Repeated rounds of selection and amplification can be used to isolate proteins with the highest affinity for the target. Exemplary Fc-binding peptides may contain the amino acid sequences ETQRCTWHMGELVWCEREHN (SEQ ID NO:19), KEASCSYWLGELVWCVAGVE (SEQ ID NO:20), or DCAWHLGELVWCT (SEQ ID NO:21).

[0168] Any Fc conjugate described herein may possess a suitable binding affinity to the Fc domain of an antibody. Binding affinity refers to the apparent association constant, or KA. KA is the reciprocal of the dissociation constant KD. The extracellular ligand-binding domain of the Fc receptor domain of the chimeric transmembrane receptor polypeptide described herein may possess at least a 10-1 affinity for the Fc moiety of the antibody. -5 10 -6 10 -7 10 -8 10 -9 10 -10M or lower binding affinity KD. In some embodiments, the ligand-interacting domain bound to the Fc portion of the antibody has a higher binding affinity to the antibody, the antibody isotype, or the antibody isotype compared to the binding affinity of the ligand-interacting domain to another antibody, an antibody isotype, or a subtype thereof.

[0169] In some implementations, the extracellular ligand-binding domain of the Fc receptor is specific to an antibody, an antibody isotype, or an antibody isotype compared to the binding of the extracellular ligand-binding domain of the Fc receptor to another antibody, an antibody isotype, or a isotype. Fcγ receptors with relatively high binding affinity include CD64A, CD64B, and CD64C. Fcγ receptors with relatively low binding affinity include CD32A, CD32B, CD16A, and CD16B. Fcε receptors with relatively high binding affinity include FcεRI, and Fcε receptors with relatively low binding affinity include FcεRII / CD23.

[0170] The binding affinity or binding specificity of an Fc receptor or any of its derivatives, variants or fragments or chimeric transmembrane receptor peptides containing an Fc binding domain can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, and spectroscopy.

[0171] In some embodiments, the ligand interaction domain including the extracellular ligand-binding domain of the Fc receptor comprises an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identical to the amino acid sequence of the extracellular ligand-binding region of a naturally occurring Fcγ receptor, Fcα receptor, Fcε receptor, or FcRn. The “percentage of identity” or “% identity” of the two amino acid sequences can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, and corrected according to Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J.Mol.Biol.215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of this disclosure. In cases where gaps exist between two sequences, nick BLAST can be used, as described by Altsurl et al., Nucleic AcidsRes.25(17):3389-3402, 1997. When using the BLAST and nick BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0172] In some embodiments, the ligand interaction domain includes an Fc binding domain containing a variant of the extracellular ligand binding domain of the Fc receptor. In some embodiments, the variant extracellular ligand binding domain of the Fc receptor may contain a variation of up to 10 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) in the amino acid sequence relative to a reference extracellular ligand binding domain. In some embodiments, the variant may be a naturally occurring variant due to genetic polymorphism. In other embodiments, the variant may be a non-naturally occurring modifying molecule. For example, a mutation may be introduced into the extracellular ligand binding domain of the Fc receptor to alter its glycosylation pattern, thereby changing its binding affinity to the corresponding Fc domain.

[0173] In some instances, the ligand interaction domain includes Fc binding, which comprises an Fc receptor selected from CD16A, CD16B, CD32A, CD32B, CD32C, CD64A, CD64B, CD64C, or variants, fragments, or derivatives thereof as described herein. The extracellular ligand binding domain of the Fc receptor may contain up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) relative to the amino acid sequence of the extracellular ligand binding domain of the CD16A, CD16B, CD32A, CD32B, CD32C, CD64A, CD64B, and CD64C described herein. Mutations in the amino acid residues of the extracellular ligand binding domain of the Fc receptor, relative to the non-mutated Fc receptor domain, can lead to an increased binding affinity of the Fc receptor domain to antibodies, antibody isotypes, or subtypes thereof. For example, a mutation at residue 158 of the Fcγ receptor CD16A can lead to an increased binding affinity of the Fc receptor to the Fc moiety of an antibody. In some embodiments, this mutation involves replacing phenylalanine with valine at residue 158 of the Fcγ receptor CD16A. Various suitable substitutions or additional mutations can be generated in the extracellular ligand-binding domain of the Fc receptor, which can enhance or decrease the binding affinity to the Fc moiety of molecules such as antibodies.

[0174] The extracellular region, including the ligand-interacting domain, can be linked to the intracellular region, for example, via a transmembrane segment. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide linking the extracellular and intracellular regions of a chimeric transmembrane receptor can have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises a polypeptide sequence of the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitution, deletion, and insertion, compared to the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as a polypeptide linker sequence. The polypeptide linker can be flexible or rigid. The polypeptide linker can be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from the extracellular region of the receptor to the intracellular region, such as a signal indicating ligand binding.

[0175] The intracellular region of the chimeric transmembrane receptor polypeptide in the system of the present invention may include a primary signaling domain. The primary signaling domain can be any signaling domain or its derivatives, variants, or fragments involved in immune cell signaling. For example, the signaling domain may participate in regulating the primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary signaling domains may include signaling domains for Fcγ receptor (FcγR), Fcε receptor (FcεR), Fcα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the primary signaling domain includes an activation motif based on an immune receptor tyrosine, or ITAM. The primary signaling domain containing ITAM may contain two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids, where each x is any amino acid independently, generating the conserved motif YxxL / Ix. (6-8) YxxL / I. When the ligand-interacting domain binds to an antigen, the primary signaling domain containing the ITAM can be modified, for example, by phosphorylation. Phosphorylated ITAM can serve as a docking site for other proteins, such as those involved in various signaling pathways. In some embodiments, the primary signaling domain includes a modified ITAM domain, such as a mutant, truncated, and / or optimized ITAM domain, which has altered (e.g., increased or decreased) activity compared to the native ITAM domain.

[0176] In some embodiments, the primary signal transduction domain includes an FcγR signal transduction domain (e.g., ITAM). The FcγR signal transduction domain may be selected from FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). In some embodiments, the primary signal transduction domain includes an FcεR signal transduction domain (e.g., ITAM). The FcεR signal transduction domain may be selected from FcεRI and FcεRII (CD23). In some embodiments, the primary signal transduction domain includes an FcαγR signal transduction domain (e.g., ITAM). The FcαR signal transduction domain may be selected from FcαRI (CD89) and Fcα / μR. In some embodiments, the primary signal transduction domain includes a CD3ζ signal transduction domain. In some embodiments, the primary signal transduction domain includes an ITAM with CD3ζ.

[0177] In some implementations, the primary signaling domain comprises an immunoreceptor tyrosine-based inhibitory motif, or ITIM. The ITIM-containing primary signaling domain may contain a conserved amino acid sequence (S / I / V / LxYxxI / V / L) found in the cytoplasmic tails of some inhibitory receptors of the immune system. The ITIM-containing primary signaling domain can be modified, for example, by phosphorylation, with enzymes such as members of the Src kinase family (e.g., Lck). Following phosphorylation, other proteins, including enzymes, can be recruited to the ITIM. These other proteins include, but are not limited to, enzymes such as phosphotyrosine phosphatases SHP-1 and SHP-2, inositol phosphatases known as SHIPs, and proteins with one or more SH2 domains (e.g., ZAP70).Primary signal transduction domains may include the following signal transduction domains (e.g., ITIM): BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, FcγRIIB (CD32), Fc receptor-like protein 2 (FCRL2), Fc receptor-like protein 3 (FCRL3), Fc receptor-like protein 4 (FCRL4), Fc receptor-like protein 5 (FCRL5), Fc receptor-like protein 6 (FCRL6), protein G6b (G6B), interleukin-4 receptor (IL4R), translocation-associated immunoglobulin superfamily receptor 1 (IRTA1), translocation-associated immunoglobulin superfamily receptor 2 (IRTA1), and translocation-associated immunoglobulin superfamily receptor 2 (IRTA1). TA2), killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1), killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2), killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), killer cell immunoglobulin-like receptor 2DL5 (KIR2DL5), killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1), killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2), leukocyte immunoglobulin-like receptor subfamily B member 1 (LIR1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LI) R2), Leukocyte Immunoglobulin-like Receptor Subfamily B Member 3 (LIR3), Leukocyte Immunoglobulin-like Receptor Subfamily B Member 5 (LIR5), Leukocyte Immunoglobulin-like Receptor Subfamily B Member 8 (LIR8), Leukocyte-associated Immunoglobulin-like Receptor 1 (LAIR-1), Mast Cell Function-associated Antigen (MAFA), NKG2A, Innate Cytotoxicity Triggering Receptor 2 (NKp44), NTB-A, Programmed Cell Death Protein 1 (PD-1), PILR, SIGLEC1, Sialic Acid-Binding Ig-like Agglutinin 2 (SIGLEC2 or CD22), Sialic Acid-Binding Ig-like Agglutinin 3 (SIGLEC3 or C D33), sialic acid-binding Ig-like lectin 5 (SIGLEC5 or CD170), sialic acid-binding Ig-like lectin 6 (SIGLEC6), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 10 (SIGLEC10), sialic acid-binding Ig-like lectin 11 (SIGLEC11), sialic acid-binding Ig-like lectin 4 (SIGLEC4), sialic acid-binding Ig-like lectin 8 (SIGLEC8), sialic acid-binding Ig-like lectin 9 (SIGLEC9), platelet and endothelial cell adhesion molecule 1 (PECAM-1), signal regulatory protein (SIRP 2), and signal threshold regulating transmembrane adaptor 1 (SIT).In some implementations, the primary signaling domain includes a modified ITIM domain, such as a mutated, truncated, and / or optimized ITIM domain, which has altered (e.g., increased or decreased) activity compared to the native ITIM domain.

[0178] In some embodiments, the immune cell signaling domain comprises multiple primary signaling domains. For example, the immune cell signaling domain may comprise at least two primary signaling domains, such as at least 2, 3, 4, 5, 7, 8, 9, or 10 primary signaling domains. In some embodiments, the immune cell signaling domain comprises at least two ITAM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains). In some embodiments, the immune cell signaling domain comprises at least two ITIM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITIM domains) (e.g., at least two primary signaling domains). In some embodiments, the immune cell signaling domain comprises both ITAM and ITIM domains.

[0179] The intracellular region of a chimeric transmembrane receptor polypeptide may include an immune cell signaling domain that can provide a co-stimulatory domain. In some embodiments, the co-stimulatory domain, for example, from a co-stimulatory molecule, can provide a co-stimulatory signal for immune cell signaling, such as signaling from the ITAM and / or ITIM domains, for example, for the activation and / or inactivation of immune cells. An exemplary configuration of the chimeric transmembrane receptor may include an immune cell signaling domain comprising a primary signaling domain (signal domain 1) and at least one co-stimulatory domain (signal domain 2, etc.). In some embodiments, the co-stimulatory domain is operable to modulate proliferation and / or survival signals in immune cells. In some embodiments, the co-stimulatory signaling domain comprises a signaling domain of an MHC class I protein, an MHC class II protein, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activating molecule (SLAM protein), an activating NK cell receptor, a BTLA, or a Toll ligand receptor. In some implementations, the co-stimulatory domain comprises the signal transduction domain of a molecule selected from the group consisting of: 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, and BAFF. R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30 ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 ligand / TNFSF5, CD40 / TNFRSF 5. CD48 / SLAMF2, CD49a, CD49D, CD49f, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6, GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLAI class, HLA-DR,HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9 (Cd229), Lymphocyte function-associated antigen-1 (LFA-1), Lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp 30. NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1, RELT / TNFRSF19L, SELPLG(CD162) , SLAM(SLAMF1), SLAM / CD150, SLAMF4(CD244), SLAMF6(NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLPR, VLA1 and VLA-6. In some implementations, the signal transduction domain includes multiple co-stimulatory domains, such as at least two, or even at least three, four, or five co-stimulatory domains.

[0180] Transmembrane receptors, including GPCRs or any variants thereof (e.g., synthetic or chimeric receptors comprising at least one of the extracellular, transmembrane, and intracellular domains of GPCRs), can bind to ligands comprising any suitable GPCR ligand or any variant thereof. Non-limiting examples of ligands that can be bound by GPCRs include (-)-adrenaline, (-)-norepinephrine, (hemolysin) phospholipid mediators, [des-Arg10]kinin, [des-Arg9]bradykinin, [des-Gln14]glucagon-releasing peptide, [Hyp3]bradykinin, [Leu]enkephalin, [Met]enkephalin, 12-hydroxyheptadecanoic acid, 12R-HETE, 12S-HETE, 12S-HPETE, 15S-HETE, 17β-estradiol, 20-hydroxy-LTB4, 2-arachidonicylglycerol, 2-oleoyl-LPA, 3-hydroxyoctanoic acid, 5-hydroxytryptamine, 5- Oxy-15-HETE, 5-oxo-ETE, 5-oxo-ETrE, 5-oxo-ODE, 5S-HETE, 5S-HPETE, 7α,25-dihydroxycholesterol, acetylcholine, ACTH, adenosine diphosphate, adenosine, adrenomedullin 2 / pituitaroid hormone, adrenomedullin, amylin, arachidonic acid ethanolamide, angiotensin II, angiotensin III, annexin I, apelin receptor early endogenous ligand, apelin-13, apelin-17, apelin-36, aspirin-triggered lipoxygenin A4, aspirin Triggered resolvin D1, ATP, β-defensin 4A, dapoxetine, bovine adrenal medullary peptide 8-22, bradykinin, C3a, C5a, Ca2+, calcitonin gene-related peptide, calcitonin, cathepsin G, CCK-33, CCK-4, CCK-8, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL7 CCL8, chemerin, chenodeoxycholic acid, bile acid, corticotropin-releasing hormone, CST-17, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12α, CXCL12β, CXCL13, CXCL16, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, cysteine-leukotriene (CysLT), uracil nucleotides, deoxycholic acid, dihydrosphingosine-1-phosphate, dioleoylphosphatidic acid, dopamine, dynorphin A, dynorphin A-(1-13), dynorphin A-(1-8), dynorphin BEndorphin-1, endothelin-1, endothelin-2, endothelin-3, F2L, free fatty acids, FSH, GABA, glycopyridine, glycopyridine-like peptide, gastric inhibitory peptide, gastrin-17, gastrin-releasing peptide, auxin-releasing peptide, GHRH, glucagon, glucagon-like peptide-1-(7-36)amide, glucagon-like peptide-1-(7-37), glucagon-like peptide-2, glucagon-like peptide-2-(3-33), GnRH I, GnRHII, GRP-(18-27), hCG, histamine, humanin, INSL3, INSL5, kallikrein, kisseptin-10, kisseptin-13, kisseptin-14, kisseptin-54, kynurenic acid, large neuromodulatory peptide N, large neurohypotensive peptide, L-glutamate, LH, lithocholic acid, L-lactic acid, long-chain carboxylic acid, LPA, LTB4, LTC4, LTD4, LTE4, LXA4, Lys-[Hyp3]-bradykinin, lysophosphatidylinositol, lysophosphatidylserine, medium-chain fatty acids, melanin-concentrating hormone, melanokinin Melanin, methylcarbamoyl PAF, Mg2+, motilin, N-arachidonicylglycine, neurokinin A, neurokinin B, neuroregulatory peptide B, neuroregulatory peptide N, neuroregulatory peptide S-33, neuroregulatory peptide U-25, neuronostatin, neuropeptide AF, neuropeptide B-23, neuropeptide B-29, neuropeptide FF, neuropeptide S, neuropeptide SF, neuropeptide W-23, neuropeptide W-30, neuropeptide Y, neuropeptide Y-(3-36), neurohypertensive peptide, pain-sensitive peptide / orphan peptide FQ, N-oleoylethanolamide, obesity-inhibiting factor (obesitin) Octopamine, Appetite Peptide A, Appetite Peptide B, Hydroxysterol, Oxytocin, PACAP-27, PACAP-38, PAF, Pancreatic Polypeptide, YY Peptide, PGD2, PGE2, PGF2α, PGI2, PGJ2, PHM, Phosphatidylserine, PHV, Prodyne-1, Prodyne-2, Prodyne-2β, Prostate Serum Acid Phosphatase (prosaposin), PrRP-20, PrRP-31, PTH, PTHrP, PTHrP-(1-36), QRFP43, Relaxin, Relaxin-1, Relaxin-3, Regression D1 Regression E1, RFRP-1, RFRP-3, R-spondins, secretin, serine protease, sphingosine 1-phosphate, sphingosine phosphocholine, SRIF-14, SRIF-28, substance P, succinic acid, thrombin, thromboxane A2, TIP39, T-kinin, TRH, TSH, tyramine, UDP-glucose, uridine diphosphate, uridine corticosteroid 1, uridine corticosteroid 2, uridine corticosteroid 3, ostrich kinin II-related peptide, ostrich kinin II, vasopressin, VIP, Wnt, Wnt-1, Wnt-10a, Wnt-10b, Wnt-11.Wnt-16, Wnt-2, Wnt-2b, Wnt-3, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, XCL1, XCL2, Zn2+, α-CGRP, α-ketoglutarate, α-MSH, α-neoendorphin, β-alanine, β-CGRP, β-D-hydroxybutyric acid, β-endorphin, β-MSH, β-neoendorphin, β-phenylethylamine, and γ-MSH.

[0181] Transmembrane receptors comprising integrin subunits or any variant thereof (e.g., synthetic or chimeric receptors comprising at least one of the extracellular, transmembrane, and intracellular domains of integrin) can bind to ligands comprising any suitable integrin ligand or any variant thereof. Non-limiting examples of ligands that can be bound by integrin receptors include adenovirus pentagon-based proteins, β-glucan, bone sialic acid protein (BSP), Borrelia burgdorferi, Candida albicans, collagen (CN, e.g., CNI-IV), tendin / tenosin-C, decersin, denatured collagen, unlinking protein, E-cadherin, echovirus 1 receptor, epidermal integrin ligand protein, factor X, FcεRII (CD23), fibrin (Fb), fibroinogen (Fg), fibronectin (Fn), heparin, and HIV. Tat protein, iC3b, intercellular adhesion molecules (e.g., ICAM-1, 2, 3, 4, 5), invaginin, L1 cell adhesion molecule (L1-CAM), laminin, lipopolysaccharide (LPS), MAdCAM-1, matrix metalloproteinase-2 (MMPe), neutrophil inhibitory factor (NIF), osteopontin (OP or OPN), plasminogen activator, prothrombin, spermatogenic protein, platelet-reactive protein (TSP), vascular cell adhesion molecule 1 (VCAM-1), vitrinin (VN or VTN), and von Willebrand factor (vWF).

[0182] Transmembrane receptors, including cadherins or any variant thereof (e.g., synthetic or chimeric receptors including at least one of the extracellular, transmembrane, and intracellular domains of cadherins), can bind to ligands including any suitable cadherin ligand or any variant thereof. For example, a cadherin ligand may include another cadherin receptor (e.g., a cellular cadherin receptor).

[0183] Transmembrane receptors, including RTKs or any variants thereof (e.g., synthetic or chimeric receptors including at least one of the extracellular, transmembrane, and intracellular domains of RTKs), can bind to ligands including any suitable RTK ligand or any variant thereof. Non-limiting examples of RTK ligands include growth factors, cytokines, and hormones. Growth factors include, for example, members of the epidermal growth factor family (e.g., epidermal growth factor or EGF, heparin-binding EGF-like growth factor or HB-EGF, transforming growth factor-α or TGF-α, bimodalin or AR, epithelial regulatory protein or EPR, epigen, betacellulin or BTC, neuroregulatory protein-1 or NRG1, neuroregulatory protein-2 or NRG2, neuroregulatory protein-3 or NRG3, and neuroregulatory protein-4 or NRG4), and the fibroblast growth factor family (e.g., FGF1). The vascular endothelial growth factor family (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF) and platelet-derived growth factor family (e.g., PDGFA, PDGFB, PDGFC, and PDGFD) are included. Hormones include, for example, members of the insulin / IGF / relaxin family (e.g., insulin, insulin-like growth factor, relaxin family peptides (including relaxin 1, relaxin 2, relaxin 3), Ledich cell-specific insulin-like peptide (gene INSL3), early placental insulin-like peptide (ELIP) (gene INSL4), insulin-like peptide 5 (gene INSL5), and insulin-like peptide 6).

[0184] Transmembrane receptors, including cytokine receptors or any variant thereof (e.g., synthetic or chimeric receptors including at least one of the extracellular, transmembrane, and intracellular domains of cytokine receptors), can bind to ligands including any suitable cytokine receptor ligand or any variant thereof. Non-limiting examples of cytokine receptor ligands include interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-20, IL-21, IL-22, IL-23, IL-27, IL-28, and IL-31), interferons (e.g., IFN-α, IFN-β, IFN-γ), colony-stimulating factors (e.g., erythropoietin, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor or GM-CSF, and granulocyte colony-stimulating factor or G-CSF), and hormones (e.g., prolactin and leptin).

[0185] Transmembrane receptors, including death receptors or any variant thereof (e.g., synthetic or chimeric receptors including at least one of the extracellular, transmembrane, and intracellular domains of death receptors), can bind to ligands including any suitable death receptor ligand or any variant thereof. Non-limiting examples of ligands that bind via death receptors include TNFα, Fas ligands, and TNF-associated apoptosis-inducing ligands (TRAIL).

[0186] Transmembrane receptors, including chimeric antigen receptors, can bind to ligands that include membrane-bound ligands (e.g., antigens), such as ligands that bind to the extracellular surface of a cell (e.g., a target cell). In some embodiments, the ligand is non-membrane-bound, such as an extracellular ligand secreted by the cell (e.g., the target cell). The ligand (e.g., membrane-bound and non-membrane-bound) can be antigenic (e.g., evoking an immune response) and associated with diseases such as viral, bacterial, and / or parasitic infections; inflammatory diseases and / or autoimmune diseases; or tumors (e.g., cancer and / or tumors). For example, cancer antigens are proteins produced by tumor cells that can evoke an immune response, particularly a T-cell-mediated immune response. The choice of the antigen-binding portion of the chimeric receptor polypeptide can depend on the specific type of cancer antigen to be targeted. In some embodiments, the tumor antigen comprises one or more antigenic oncogenic epitopes associated with malignancies. Malignancies can express a variety of proteins that can be used as target antigens for immune attack. The antigen-interacting domain can bind to cell surface signals, extracellular matrix (ECM), paracrine signals, procrine signals, endocrine signals, autocrine signals, signals that trigger or control genetic programs in the cell, or any combination thereof. In some embodiments, the interaction between cellular signals bound to the recombinant chimeric receptor peptide involves cell-cell interactions, cell-soluble chemical interactions, and cell-matrix or microenvironment interactions.

[0187] GMPs may include an actuation moiety fused within a heterologous nuclear localization domain frame. This actuation moiety may comprise a nuclease (e.g., a DNA nuclease and / or an RNA nuclease), a modified nuclease (e.g., a DNA nuclease and / or an RNA nuclease) that is nuclease-deficient or has reduced nuclease activity compared to a wild-type nuclease, its derivatives, variants, or fragments thereof. The actuation moiety may regulate gene expression and / or activity, or edit the sequence of nucleic acids (e.g., genes and / or gene products). In some embodiments, the actuation moiety comprises a DNA nuclease, such as an engineered (e.g., programmable or targetable) DNA nuclease, to induce genome editing of a target DNA sequence. In some embodiments, the actuation moiety comprises an RNA nuclease, such as an engineered (e.g., programmable or targetable) RNA nuclease, to induce editing of a target RNA sequence. In some embodiments, the actuation moiety has reduced or minimal nuclease activity. An actuation moiety with reduced or minimal nuclease activity can regulate gene expression and / or activity by physically blocking the target polynucleotide or recruiting other factors that effectively inhibit or enhance the expression of the target polynucleotide. In some embodiments, the actuation moiety comprises a nuclease-free DNA-binding protein derived from a DNA nuclease, which can induce transcriptional activation or repression of the target DNA sequence. In some embodiments, the actuation moiety comprises a nuclease-free RNA-binding protein derived from an RNA nuclease, which can induce transcriptional activation or repression of the target RNA sequence. In some embodiments, the actuation moiety is a nucleic acid-directed actuation moiety. The actuation moiety can regulate gene expression or activity and / or edit nucleic acid sequences, whether exogenous or endogenous.

[0188] Any suitable nuclease can be used in the actuation part. Suitable nucleases include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases, including type I, type II, type III, type IV, type V (e.g., Cpf1 / Cas12a, C2c1, or c2c3) and type VI (e.g., C2c2 / Cas13a, Ca s13b, Cas13c, Cas13d); zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); a wide range of nucleases; RNA-binding proteins (RBP); CRISPR-associated RNA-binding proteins; recombinases; flip enzymes; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaea Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); any derivatives thereof; any variants thereof; and any fragments thereof.

[0189] In some implementations, the actuating portion contains a CRISPR-associated (Cas) protein or Cas nuclease that functions in a non-naturally occurring CRISPR (clustered regularly spaced short palindromic repeats) / Cas (CRISPR-associated) system. In bacteria, this system can provide adaptive immunity against foreign DNA (Barrangou, R. et al., “CRISPR provides acquired resistance against viruses in prokaryotes,” Science (2007) 315:1709-1712; Makarova, KS et al., “Evolution and classification of the CRISPR-Cassystems,” Nat Rev Microbiol (2011) 9:467-477; Garneau, JE et al., “The CRISPR / Casbacterial immune system cleaves bacteriophage and plasmid DNA,” Nature (2010) 468:67-71; Sapranauskas, R. et al., “The Streptococcus thermophilus CRISPR / Cassystem provides immunity in Escherichia coli,” Nucleic Acids Res (2011) 39:9275-9282).

[0190] In a wide range of organisms, including various mammals, animals, plants, and yeast, the CRISPR / Cas system (e.g., modified and / or unmodified) can be used as a tool for genome engineering. A CRISPR / Cas system may contain a guide nucleic acid, such as guide RNA (gRNA), complexed with a Cas protein for targeted regulation of gene expression and / or activity or nucleic acid editing. RNA-guided Cas proteins (e.g., Cas nucleases, such as Cas9 nuclease) can bind specifically to target polynucleotides (e.g., DNA) in a sequence-dependent manner.If the Cas protein has nuclease activity, it can cleave DNA (Gasiunas, G. et al., "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria," Proc Natl Acad Sci USA (2012) 109: E2579-E286; Jinek, M. et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Science (2012) 337: 816-821; Sternberg, SH et al., "DNA interrogation by the CRISPR RNA-guided endonuclease Cas9," Nature (2014) 507: 62; Deltcheva, E. et al., "CRISPR RNA maturation by trans-encoded small RNA and hostfactor RNase III," Nature (2014) 507: 62; 1) 471:602-607), and has been widely used in programmable genome editing in various organisms and model systems (Cong, L. et al., “Multiplex genome engineering using CRISPR-Cas systems,” Science (2013) 339:819-823; Jiang, W. et al., “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nat. Biotechnol. (2013) 31:233-239; Sander, JD and Joung, JK, “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nature Biotechnol. (2014) 32:347-355).

[0191] In some cases, Cas proteins are mutated and / or modified to produce nuclease-deficient proteins or proteins with reduced nuclease activity relative to wild-type Cas proteins. Nuclease-deficient proteins may retain their ability to bind to DNA but may lack or have reduced nucleic acid cleavage activity. The actuating portion containing a Cas nuclease (e.g., retaining wild-type nuclease activity, having reduced nuclease activity, and / or lacking nuclease activity) can function in a CRISPR / Cas system to regulate the level and / or activity (e.g., decrease, increase, or eliminate) of a target gene or protein. Cas proteins can bind to target polynucleotides and physically block transcription or edit nucleic acid sequences to produce non-functional gene products.

[0192] In some embodiments, the actuation portion comprises a Cas protein that forms a complex with a guide nucleic acid such as guide RNA (gRNA). In some embodiments, the actuation portion comprises a Cas protein that forms a complex with a single guide nucleic acid such as a single guide RNA (sgRNA). In some embodiments, the actuation portion comprises an RNA-binding protein (RBP) that optionally complexes with a guide nucleic acid such as guide RNA (e.g., sgRNA), which is capable of forming a complex with the Cas protein.

[0193] Any suitable CRISPR / Cas system can be used. CRISPR / Cas systems can be referenced using various nomenclature systems. Exemplary nomenclature systems are provided in Makarova, KS et al., “An updated evolutionary classification of CRISPR-Cas systems,” Nat Rev Microbiol (2015) 13:722-736 and Shmakov, S. et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems,” Mol Cell (2015) 60:1-13. The CRISPR / Cas system can be type I, type II, type III, type IV, type V, type VI, or any other suitable CRISPR / Cas system. The CRISPR / Cas system used herein can be class 1, class 2, or any other appropriately classified CRISPR / Cas system. Class 1 or class 2 can be determined based on the gene encoding the effector module. Class 1 systems typically have multi-subunit crRNA effector complexes, while class 2 systems typically have a single protein, such as Cas9, Cpf1, C2c1, C2c2, C2c3, or a crRNA effector complex. Class 1 CRISPR / Cas systems can be regulated using complexes of multiple Cas proteins. Class 1 CRISPR / Cas systems may include, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., III, IIIA, IIIB, IIIC, IIID), and type IV (e.g., IV, IVA, IVB) CRISPR / Cas types. Class 2 CRISPR / Cas systems can be regulated using a single large Cas protein. Class 2 CRISPR / Cas systems may include, for example, type II (e.g., II, IIA, IIB) and type V CRISPR / Cas types. CRISPR systems can be complementary to each other and / or can lend trans functional units for CRISPR site targeting.

[0194] The actuating portion of a Cas protein can be a class 1 or class 2 Cas protein. Cas proteins can be type I, II, III, IV, V, or VI Cas proteins. Cas proteins may contain one or more domains. Non-limiting examples of domains include: a directing nucleic acid recognition and / or binding domain, a nuclease domain (e.g., a DNase or RNase domain, RuvC, HNH), a DNA-binding domain, an RNA-binding domain, a helicase domain, a protein-protein interaction domain, and a dimerization domain. The directing nucleic acid recognition and / or binding domain may interact with the directing nucleic acid. The nuclease domain may contain catalytic activity for nucleic acid cleavage. The nuclease domain may lack catalytic activity to prevent nucleic acid cleavage. Cas proteins can be chimeric Cas proteins fused with other proteins or peptides. Cas proteins can be chimeras of various Cas proteins, for example, containing domains from different Cas proteins.

[0195] Non-restricted examples of Cas proteins include c2c1, Cas13a (formerly known as C2c2), Cas13b, Cas13c, Cas13d, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), Cas10, Cas10d, Cas1O, Cas1Od, CasF, CasG, CasH, and Cas12a (formerly known as Cp). f1), Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 and Cul966 and their homologs or modified forms.

[0196] Cas proteins can originate from any suitable organism. Non-limiting examples include *Streptococcus pyogenes*, *Streptococcus thermophilus*, *Streptococcus sp.*, *Staphylococcus aureus*, *Nocardiopsis dassonvillei*, *Streptomyces pristinae spiralis*, *Streptomyces viridochromogenes*, *Streptomyces viridochromogenes*, *Streptosporangium roseum*, *Streptosporangium roseum*, *AlicyclobacHlusacidocaldarius*, *Bacillus pseudomycoides*, *Bacillus selenitireducens*, and *Exiguobacterium*. *Lactobacillus sibiricum*, *Lactobacillus delbrueckii*, *Lactobacillus salivarius*, *Microscilla marina*, *Burkholderiales bacterium*, *Polaromonas naphthalenivorans*, *Polaromonas sp.*, *Crocosphaera watsonii*, *Cyanothece sp.*, *Microcystis aeruginosa*, *Pseudomonas aeruginosa*, and *Synechococcus sp.*), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus halophilus), Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, species of Nostoc, Arthrospira maxima, Arthrospira platensis, species of Arthrospira sp., species of Lyngbya sp., Microcoleuschthonoplastes, species of Oscillatoria sp.), Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, Leptotrichia wadeii, Leptotrichia F0279, Rhodobacter capsulatus SB1003, Rhodobacter capsulatus R121, Rhodobacter capsulatus DE442, Lachnospiraceae bacterium NK4A179, Lachnospiraceae bacterium MA2020, Clostridium aminophilum DSM 10710, Paludibacter propionicigenes WB, Carnobacterium gallinarum DMS4847, Carnobacterium gallinarum DSM4847, and the new culprit Francisella novicida. In some aspects, the organism is *Streptococcus pyogenes*. In some aspects, the organism is *Staphylococcus aureus*. In some aspects, the organism is *Streptococcus thermophilus*.

[0197] Cas proteins can originate from a variety of bacterial species, including but not limited to Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Listeria seeligeri, Listeria weihenstephanensis FSL R90317, Listeria weihenstephanensis FSL M60635, Staphylococcus pseudintermedius, Acidaminococcus intestine, and Olsenella gingivalis. * *Lactobacillus uli*, *Oenococcus kitaharae*, *Bifidobacterium bifidum*, *Lactobacillus rhamnosus*, *Lactobacillus gasseri*, *Finegoldia magna*, *Mycoplasma mobile*, *Mycoplasma gallisepticum*, *Mycoplasma ovipneumoniae*, *Mycoplasma canis*, *Mycoplasma synoviae*, *Eubacterium rectale*, *Streptococcus thermophilus*, *Eubacterium dolichum*, and *Lactobacillus coryniformis subsp.*Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractorsalsuginis, Sphaerochaeta globus, Fibrobacter uccinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella The following bacteria are listed: *Prevotella ruminicola*, *Flavobacterium columnare*, *Aminomonaspaucivorans*, *Rhodospirillum rubrum*, *Candidatus Puniceispirillum marinum*, *Verminephrobacter eiseniae*, *Ralstonia syzygii*, *Dinoroseobacter shibae*, *Azospirillum*, *Nitrobacter hamburgensis*, *Bradyrhizobium*, *Wolinella succinogenes*, and *Campylobacter jejuni subsp.*The following bacteria are listed: *Jejuni*, *Helicobacter mustelae*, *Bacillus cereus*, *Acidovorax ebreus*, *Clostridium perfringens*, *Parvibaculum lavamentivorans*, *Roseburia intestinalis*, *Neisseria meningitidis*, *Pasteurella multocida subsp. Multocida*, *Sutterella wadsworthensis*, *Proteobacterium*, *Legionella pneumophila*, *Parassutterella exrementihominis*, *Wolinella succinogenes*, and *Francisella novicida*.

[0198] The Cas protein used herein may be a wild-type or modified form of the Cas protein. The Cas protein may be an active variant, inactive variant, or fragment of the wild-type or modified Cas protein. Relative to the wild-type form of the Cas protein, the Cas protein may contain amino acid variations, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof. The Cas protein may be a polypeptide having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to the wild-type exemplary Cas protein. The Cas protein may be a polypeptide having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity or sequence similarity to the wild-type exemplary Cas protein. Variant or fragment may contain at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to wild-type or modified Cas proteins or portions thereof. Variant or fragment may target nucleic acid sites with a guide nucleic acid complex, but lacks nucleic acid cleavage activity.

[0199] Cas proteins may contain one or more nuclease domains, such as a DNase domain. For example, the Cas9 protein may contain a RuvC-like nuclease domain and / or an HNH-like nuclease domain. The RuvC and HNH domains can each cleave different strands of double-stranded DNA, thereby creating double-strand breaks in the DNA. Cas proteins may also contain only one nuclease domain (e.g., Cpf1 contains a RuvC domain but lacks an HNH domain).

[0200] The Cas protein may contain an amino acid sequence having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to the nuclease domain (e.g., RuvC domain, HNH domain) of the wild-type Cas protein.

[0201] Cas proteins can be modified to optimize the regulation of gene expression. Cas proteins can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and / or enzymatic activity. Cas proteins can also be modified to alter any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or the Cas protein can be truncated to remove domains not essential for protein function, or to optimize (e.g., enhance or reduce) the Cas protein's activity in regulating gene expression.

[0202] In some embodiments, the actuation moiety comprises a nuclease-free DNA-binding protein derived from a DNA nuclease, which can induce transcriptional activation or repression of the target DNA sequence. In some embodiments, the actuation moiety comprises a nuclease-free RNA-binding protein derived from an RNA nuclease, which can induce transcriptional activation or repression of the target RNA sequence. For example, the actuation moiety may comprise a Cas protein lacking cleavage activity.

[0203] Cas proteins can be fusion proteins. For example, Cas proteins can fuse with heterologous functional domains. Heterologous functional domains may include fusion with cleavage domains, epigenetic modification domains, transcriptional activation domains, or transcriptional repression domains. Cas proteins can also fuse with heterologous peptides that provide increased or decreased stability. The fusion domain or heterologous peptide may be located at the N-terminus, C-terminus, or interior of the Cas protein.

[0204] Gene regulation can be the regulation of any gene of interest. Consider genetic homologs that encompass the genes described herein. For example, a gene may exhibit a certain degree of identity and / or homology with the genes disclosed herein. Therefore, consider genes that can be modified to express or exhibit approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology (at the nucleic acid or protein level). It also takes into account genes that can be modified to express or exhibit approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity (at the nucleic acid or protein level).

[0205] Cas proteins can be provided in any form. For example, Cas proteins can be provided as proteins, either alone or in combination with a guide nucleic acid. Cas proteins can also be provided as nucleic acids encoding Cas proteins, such as RNA (e.g., messenger RNA (mRNA)) or DNA.

[0206] Codon optimization can be performed on nucleic acids encoding Cas proteins to enable efficient translation into proteins in specific cells or organisms.

[0207] The nucleic acid encoding the Cas protein can be stably integrated into the cell's genome. The nucleic acid encoding the Cas protein can be operatively linked to an active promoter in the cell. The nucleic acid encoding the Cas protein can also be operatively linked to a promoter in an expression construct. The expression construct can include any nucleic acid construct capable of directing the expression of a gene or other target nucleic acid sequence (e.g., the Cas gene) and capable of transferring such a target nucleic acid sequence to target cells.

[0208] In some implementations, the Cas protein is a dead Cas protein. A dead Cas protein can be a protein lacking nucleic acid cleavage activity.

[0209] Cas proteins can include modified forms of wild-type Cas proteins. Modified forms of wild-type Cas proteins can include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the Cas protein. For example, modified forms of Cas proteins can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of wild-type Cas proteins (e.g., Cas9 from Streptococcus pyogenes). Modified forms of Cas proteins can lack substantial nucleic acid cleavage activity. When a Cas protein is a modified form lacking substantial nucleic acid cleavage activity, it can be referred to as enzymatically inactive and / or "dead" (abbreviated as "d"). Dead Cas proteins (e.g., dCas, dCas9) can bind to target polynucleotides but may not cleave them. In some respects, a dead Cas protein is a dead Cas9 protein.

[0210] The dCas9 polypeptide can associate with a single guide RNA (sgRNA) to activate or inhibit the transcription of target DNA. sgRNA can be introduced into cells expressing engineered chimeric receptor polypeptides. In some cases, such cells contain one or more different sgRNAs targeting the same nucleic acid. In other cases, the sgRNA targets different nucleic acids within the cell. The nucleic acid targeted by the guide RNA can be any nucleic acid expressed in cells such as immune cells. The targeted nucleic acid can be a gene involved in immune cell regulation. In some implementations, the nucleic acid is cancer-related. Cancer-related nucleic acids can be cell cycle genes, cell response genes, apoptosis genes, or phagocytic genes. Recombinant guide RNA can be recognized by CRISPR proteins, nuclease-free CRISPR proteins, their variants, their derivatives, or fragments thereof.

[0211] Enzymatic inactivity refers to a polypeptide that can bind to the nucleic acid sequence in a sequence-specific manner but does not cleave the target polynucleotide. Site-directed polypeptides with enzymatic inactivity may contain a domain with no enzymatic activity (e.g., a nuclease domain). Enzymatic inactivity can mean no activity. Enzymatic inactivity can mean substantially no activity. Enzymatic inactivity can mean substantially no activity. Enzymatic inactivity can mean activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% of the exemplary wild-type activity (e.g., nucleic acid cleavage activity, wild-type Cas9 activity).

[0212] One or more nuclease domains of a Cas protein (e.g., RuvC, HNH) can be missing or mutated, rendering them inactive or containing reduced nuclease activity (e.g., inactivated or dead Cas, or "dCas"). For example, in a Cas protein containing at least two nuclease domains (e.g., Cas9), if one of the nuclease domains is missing or mutated, the resulting Cas protein (called a nickase) can generate single-strand breaks instead of double-strand breaks at CRISPR RNA (crRNA) recognition sequences in double-stranded DNA. Such a nickase can cleave either the complementary or non-complementary strands, but may not cleave both simultaneously. If all nuclease domains of a Cas protein (e.g., the RuvC and HNH nuclease domains in Cas9; the RuvC nuclease domain in Cpf1) are missing or mutated, the resulting Cas protein may have reduced cleavage capacity or no capacity to cleave either strand of double-stranded DNA. Examples of mutations that can convert Cas9 protein into a nicking enzyme include the D10A mutation in the RuvC domain of Cas9 from *Streptococcus pyogenes* (changing aspartic acid to alanine at position 10). The H939A mutation (changing histidine to alanine at position 839) or the H840A mutation (changing histidine to alanine at position 840) in the HNH domain of Cas9 from *Streptococcus pyogenes* can also convert Cas9 into a nicking enzyme. Examples of mutations that can convert Cas9 protein into dead Cas9 include the D10A mutation in the RuvC domain (changing aspartic acid to alanine at position 10) and the H939A mutation (changing histidine to alanine at position 839) or the H840A mutation (changing histidine to alanine at position 840) in the HNH domain of Cas9 from *Streptococcus pyogenes*.

[0213] The dead Cas protein may contain one or more mutations relative to its wild-type form. Mutations may result in cleavage activity of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% in one or more of the multiple cleavage domains of the wild-type Cas protein. Mutations may result in one or more of the multiple cleavage domains retaining the ability to cleave the complementary strand of the target nucleic acid, but reducing its ability to cleave the non-complementary strand of the target nucleic acid. Mutations may result in one or more of the multiple cleavage domains retaining the ability to cleave the non-complementary strand of the target nucleic acid, but reducing its ability to cleave the complementary strand of the target nucleic acid. Mutations may result in one or more of the multiple cleavage domains lacking the ability to cleave both the complementary and non-complementary strands of the target nucleic acid. The residue to be mutated in the nuclease domain may correspond to one or more catalytic residues of a nuclease. For example, residues such as Asp10, His840, Asn854, and Asn856 in the wild-type exemplary *Streptococcus pyogenes* Cas9 polypeptide can be mutated to inactivate one or more of a plurality of nucleic acid cleavage domains (e.g., nuclease domains). The residues to be mutated in the nuclease domain of the Cas protein may correspond to the residues Asp10, His840, Asn854, and Asn856 in the wild-type *Streptococcus pyogenes* Cas9 polypeptide, as determined, for example, by sequence and / or structural alignment.

[0214] As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 (or any corresponding mutation in the Cas protein) can be mutated. For example, D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A. Mutations other than alanine substitutions may be suitable.

[0215] The D10A mutation can bind to one or more of the H840A, N854A, or N856A mutations to produce a Cas9 protein with essentially no DNA cleavage activity (e.g., the dead Cas9 protein). The H840A mutation can bind to one or more of the D10A, N854A, or N856A mutations to produce a site-directed peptide with essentially no DNA cleavage activity. The N854A mutation can bind to one or more of the H840A, D10A, or N856A mutations to produce a site-directed peptide with essentially no DNA cleavage activity. The N856A mutation can bind to one or more of the H840A, N854A, or D10A mutations to produce a site-directed peptide with essentially no DNA cleavage activity.

[0216] In some embodiments, the Cas protein is a type 2 Cas protein. In some embodiments, the Cas protein is a type II Cas protein. In some embodiments, the Cas protein is a Cas9 protein, a modified form of a Cas9 protein, or a Cas9 protein derived from a Cas9 protein. For example, a Cas9 protein lacking cleavage activity. In some embodiments, the Cas9 protein is a Cas9 protein from *Streptococcus pyogenes* (e.g., SwissProt accession number Q99ZW2). In some embodiments, the Cas9 protein is a Cas9 protein from *Staphylococcus aureus* (e.g., SwissProt accession number J7RUA5). In some embodiments, the Cas9 protein is a modified form of a Cas9 protein from *Streptococcus pyogenes* or *Staphylococcus aureus*. In some embodiments, the Cas9 protein is derived from a Cas9 protein from *Streptococcus pyogenes* or *Staphylococcus aureus*. For example, a *Streptococcus pyogenes* or *Staphylococcus aureus* Cas9 protein lacking cleavage activity.

[0217] Cas9 can generally refer to a polypeptide having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can also refer to a polypeptide having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., from Streptococcus pyogenes). Cas9 can refer to the wild-type or modified form of the Cas9 protein, which may include amino acid changes such as deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.

[0218] In some implementations, the actuating portion comprises a “zinc finger nuclease” or “ZFN”. A ZFN is a fusion of a cleavage domain (such as the cleavage domain of FokI) with at least one zinc finger motif (e.g., at least 2, 3, 4, or 5 zinc finger motifs), which can bind to polynucleotides such as DNA and RNA. Heterodimerization at certain positions in the polynucleotides of two separate ZFNs, with a specific orientation and spacing, can lead to cleavage of the polynucleotide. For example, ZFN binding to DNA can induce double-strand breaks in DNA. To allow the two cleavage domains to dimerize and cleave DNA, two separate ZFNs can bind to the opposite strand of DNA, with their C-termini spaced apart. In some cases, the linker sequence between the zinc finger domain and the cleavage domain may require a 5' edge spacing of approximately 5-7 base pairs at each binding site. In some cases, the cleavage domain is fused to the C-terminus of each zinc finger domain. Exemplary ZFNs include, but are not limited to, Urnov et al., Nature Reviews Genetics, 2010, 11:636-646; Gaj et al., Nat Methods, 2012, 9(8):805-7; U.S. Patents 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; 6,479,626; and those described in U.S. Application Publications 2003 / 0232410 and 2009 / 0203140.

[0219] In some embodiments, the actuating portion containing a ZFN can generate double-strand breaks in target polynucleotides such as DNA. Double-strand breaks in DNA can lead to DNA break repair, which allows for the introduction of gene modifications (e.g., nucleic acid editing). DNA break repair can be performed via non-homologous end joining (NHEJ) or homology-mediated repair (HDR). In HDR, a donor DNA repair template containing a homologous arm flanking the target DNA site can be provided. In some embodiments, the ZFN is a zinc finger nickase that induces site-specific single-strand DNA breaks or nicks, thereby leading to HDR. Descriptions of zinc finger nickases can be found, for example, Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7):1327-33. In some embodiments, the ZFN binds to polynucleotides (e.g., DNA and / or RNA) but cannot cleave them.

[0220] In some embodiments, the cleavage domain comprising the actuating portion of ZFN includes a modified form of the wild-type cleavage domain. The modified form of the cleavage domain may include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the cleavage domain. For example, the modified form of the cleavage domain may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type cleavage domain. The modified form of the cleavage domain may have no substantial nucleic acid cleavage activity. In some embodiments, the cleavage domain is enzymatically inactive.

[0221] In some implementations, the actuating portion comprises a “TALEN” or “TAL effector nuclease.” TALEN refers to an engineered transcription activator-like effector nuclease, which typically contains a central domain of a DNA-binding tandem repeat sequence and a cleavage domain. TALENs can be generated by fusing the DNA-binding domain of a TAL effector with the DNA-cleavage domain. In some cases, the DNA-binding tandem repeat sequence contains 33-35 amino acids in length and two hypervariable amino acid residues at positions 12 and 13, which can recognize at least one specific DNA base pair. Transcription activator-like effector (TALE) proteins can be fused with nucleases such as wild-type or mutant FokI endonucleases or the catalytic domain of FokI. Several mutations of FokI have been used in TALENs, for example, to enhance cleavage specificity or activity. Such TALENs can be engineered to bind to any desired DNA sequence. By generating double-strand breaks in the target DNA sequence, which then undergo NHEJ or HDR, TALENs can be used to generate gene modifications (e.g., nucleic acid sequence editing). In some cases, a single-stranded donor DNA repair template is provided to facilitate HDR. Detailed descriptions of TALEN and its use in gene editing can be found, for example, in U.S. Patents 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Beurdeley et al., Nat Commun, 2013, 4:1762; and Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49-55.

[0222] In some embodiments, the TALEN is engineered to reduce nuclease activity. In some embodiments, the nuclease domain of the TALEN includes a modified form of the wild-type nuclease domain. The modified form of the nuclease domain may include amino acid changes (e.g., deletion, insertion, or substitution) that reduce the nucleic acid cleavage activity of the nuclease domain. For example, the modified form of the nuclease domain may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type nuclease domain. The modified form of the nuclease domain may have no substantial nucleic acid cleavage activity. In some embodiments, the nuclease domain is non-enzymatically active.

[0223] In some embodiments, the transcription activator-like effector (TALE) protein is fused to a domain that regulates transcription and does not contain a nuclease. In some embodiments, the TALE protein is designed to function as a transcription activator. In some embodiments, the TALE protein is designed to function as a transcription repressor. For example, the DNA-binding domain of the TALE protein can be fused (e.g., linked) to one or more transcription activation domains or one or more transcription repression domains. Non-limiting examples of transcription activation domains include the herpes simplex VP16 activation domain and tetrameric repeats of the VP16 activation domain, such as the VP64 activation domain. Other examples include VP16, VP32, VP64, VPR, p65, or P65HSF1. Non-limiting examples of transcription repression domains include Krüppel-associated box domains.

[0224] In some embodiments, the actuating portion includes a broad-spectrum nuclease. A broad-spectrum nuclease generally refers to a highly specific cleavage site-rare endonuclease or a homing endonuclease. A broad-spectrum nuclease can recognize DNA target sites of at least 12 base pairs in length, such as 12 to 40, 12 to 50, or 12 to 60 base pairs. A broad-spectrum nuclease can be a modular DNA-binding nuclease, such as any fusion protein comprising at least one catalytic domain of an endonuclease and at least one DNA-binding domain or protein specifying a nucleic acid target sequence. The DNA-binding domain may contain at least one motif capable of recognizing single-stranded or double-stranded DNA. A broad-spectrum nuclease can be a monomer or a dimer. In some embodiments, the broad-spectrum nuclease is naturally occurring (found in nature) or wild-type, while in others it is non-natural, artificial, engineered, synthetic, rationally designed, or man-made. In some embodiments, the broad range of nucleases disclosed herein includes I-CreI broad range nucleases, I-CeuI broad range nucleases, I-MsoI broad range nucleases, I-SceI broad range nucleases, their variants, their derivatives, and fragments thereof. A detailed description of useful, wide-ranging nucleases and their applications in gene editing can be found, for example, Silva et al., Curr Gene Ther, 2011, 11(1):11-27; Zaslavoskiy et al., BMC Bioinformatics, 2014, 15:191; Takeuchi et al., Proc Natl AcadSci USA, 2014, 111(11):4061-4066, and U.S. Patents 7,842,489; 7,897,372; 8,021,867; 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,36; and 8,129,134.

[0225] In some embodiments, the nuclease domain of the macronext nuclease includes a modified form of the wild-type nuclease domain. This modification may include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the nuclease domain. For example, the modified nuclease domain may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type nuclease domain. The modified nuclease domain may have no substantial nucleic acid cleavage activity. In some embodiments, the nuclease domain is non-enzymatically active. In some embodiments, the macronext nuclease can bind to DNA but cannot cleave it.

[0226] In some embodiments, the actuation moiety is fused with a heterologous functional domain. The heterologous functional domain may include one or more transcriptional repression domains, activation domains, epigenetic domains, recombinase domains, transposase domains, flipase domains, nickase domains, or any combination thereof. The activation domain may include one or more tandem activation domains located at the carboxyl terminus of the enzyme. In other cases, the actuation moiety includes one or more tandem repression domains located at the carboxyl terminus of the protein. Non-limiting exemplary activation domains include GAL4, herpes simplex activation domain VP16, VP64 (a tetramer of herpes simplex activation domain VP16), VP32, VPR, p65, P65HSF1, NF-κBp65 subunit, EBV R transactivator (Rta), and are described in Chavez et al., Nat Methods, 2015, 12(4):326-328 and U.S. Patent Application Publication No. 20140068797. Non-limiting exemplary inhibition domains include the KRAB (Krüppel related box) domain of Kox1, the Mad mSIN3 interaction domain (SID), and the ERF inhibition domain (ERD), and are described in Chavez et al., Nat Methods, 2015, 12(4):326-328 and U.S. Patent Application Publication No. 20140068797. In some embodiments, the heterologous functional domain includes one or more tandem inhibition domains located at the amino terminus of the actuation portion.

[0227] The actuating region can also be fused with a heteropeptide that provides increased or decreased stability. The fusion domain or heteropeptide can be located at the N-terminus, C-terminus, or interior of the actuating region.

[0228] The actuating portion may contain a heterologous polypeptide for easy tracking or purification, such as a fluorescent protein, purification tag, or epitope tag. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), blue-green fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), and red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent protein (mOrange, mKO, Kusabira-Orange, monomer Kusabira-Orange, mTangerine, tdTomato) and any other suitable fluorescent protein. Examples of tags include glutathione S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, SI, T7, V5, VSV-G, histidine (His), biotinylate carboxyl carrier protein (BCCP), and calmodulin.

[0229] In some embodiments, the transmembrane chimeric receptor polypeptide comprises an extracellular domain having a ligand interaction domain, a transmembrane domain, and an intracellular domain containing a cell signaling domain. Figure 1In response to ligand binding, the extracellular domain can signal through the intracellular domain to activate cell signaling pathways. In one instance, ligand binding can lead to modification of the intracellular domain (e.g., phosphorylation), thereby activating cell signaling pathways. Activated cell signaling pathways ultimately lead to activation of the heterologous nuclear localization domain. After activation of the fused heterologous nuclear localization domain, the actuating portion can enter the nucleus to regulate the expression and / or activity of target genes or edit nucleic acid sequences. In some embodiments, the actuating portion is also fused to a heterologous functional domain (e.g., a transcriptional activation or repression domain). In some embodiments, the transmembrane chimeric receptor polypeptide comprises an extracellular region having a ligand interaction domain and an intracellular region containing an immune cell signaling domain. GMP may comprise an actuating portion fused to an inactive heterologous nuclear localization domain. In response to antigen binding, the receptor can be modified in the intracellular region of the receptor ( Figure 2 Following receptor modification (e.g., phosphorylation), an intrinsic signaling cascade is triggered, ultimately leading to the activation of the heterologous nuclear localization domain. After activation of the fused heterologous nuclear localization domain, the actuating portion can enter the nucleus to regulate the expression and / or activity of target genes or edit nucleic acid sequences. In some embodiments, the actuating portion also fuses with a heterologous functional domain (e.g., a transcriptional activation or repression domain).

[0230] In some embodiments, the gene-regulating polypeptide includes at least one targeting sequence that directs the transport of the receptor to a specific region of the cell. The targeting sequence can be used to guide the transport of the polypeptide to which the targeting sequence is attached to to a specific region of the cell. For example, the targeting sequence can use a nuclear localization signal (NLS) to guide the receptor to the nucleus, or a nuclear export signal (NES) to guide the actuating portion out of the nucleus (e.g., the cytoplasm), to mitochondria, endoplasmic reticulum (ER), Golgi apparatus, chloroplasts, apoplasts, peroxisomes, the plasma membrane, or the membrane of various organelles of the cell. In some embodiments, the targeting sequence includes a nuclear export signal (NES) and guides the polypeptide out of the nucleus, for example, to the cytoplasm. The targeting sequence can utilize various nuclear export signals to guide the polypeptide to the cytoplasm. Nuclear export signals are typically short amino acid sequences of hydrophobic residues (e.g., at least about 2, 3, 4, or 5 hydrophobic residues) that target proteins to be exported from the nucleus to the cytoplasm via nuclear transport through nuclear pore complexes. Not all NES substrates can be constitutively exported from the nucleus. In some implementations, the targeting sequence includes a nuclear localization signal (NLS, e.g., SV40 NLS) and guides the peptide to the cell nucleus. The targeting sequence can utilize various nuclear localization signals (NLS) to guide the peptide to the cell nucleus. The NLS can be a single-part sequence or a two-part sequence.

[0231] Non-limiting examples of NLS include NLS sequences derived from: NLS of the SV40 viral large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO:2); NLS from nucleoplasmic proteins (e.g., nucleoplasmic protein bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO:3)); c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:4) or RQRRNELKRSP (SEQ ID NO:5); hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:6); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain of input protein α (SEQ ID NO:7); and the sequences VSRKRPRP (SEQ ID NO:8) and PPKKARED (SEQ ID NO:8) from the fibroid T protein. NO:9); human p53 sequence PQPKKKPL (SEQ ID NO:10); mouse c-abl IV sequence SALIKKKKKMAP (SEQ ID NO:11); influenza virus NS1 sequence DRLRR (SEQ ID NO:12) and PKQKKRK (SEQ ID NO:13); hepatitis virus delta antigen sequence RKLKKKIKKL (SEQ ID NO:14); mouse Mx1 protein sequence REKKKFLKRR (SEQ ID NO:15); human poly(ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:16); and steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK (SEQ ID NO:17).

[0232] In some implementations, the targeting sequence comprises a membrane-targeting peptide and guides the polypeptide to the plasma membrane or organelle membrane. Membrane-targeting sequences can facilitate the transport of chimeric transmembrane receptor polypeptides to cell surface membranes or other cell membranes. Cell membrane-associated molecules contain regions that promote membrane association, and such regions can be incorporated into membrane-targeting sequences. For example, some proteins contain acylated sequences at their N- or C-terminus, and these acyl motifs promote membrane association. Such sequences can be recognized by acyltransferases and typically conform to specific sequence motifs. Some acylation motifs can be modified by a single acyl motif (usually followed by several positively charged residues (e.g., human c-Src) to improve association with anionic lipid head groups), while others can be modified by multiple acyl motifs. For example, the N-terminal sequence of the protein tyrosine kinase Src may contain a single cardiomyol motif. Diacylated regions are located in subsets of certain protein kinases such as members of the Src family (e.g., Yes, Fyn, Lck) and the N-terminal regions of G protein α subunits. Such diacylated regions are typically located within the first 18 amino acids of such proteins and conform to the sequence motif Met-Gly-Cys-Xaa-Cys (SEQ ID NO: 18), where Met is cleaved, Gly is N-acylated, and one of the Cys residues is S-acylated. Gly is frequently myristylated, while Cys can be palmitoylated. Acylated regions conforming to the sequence motif Cys-Ala-Ala-Xaa (the so-called "CAAX box"), which can be modified by the C15 or C10 isoprene moiety from the C-terminus of the γ subunit of G proteins and other proteins, can also be utilized. These and other acylation motifs include, for example, those discussed in Gauthier-Campbell et al., Molecular Biology of the Cell 15:2205-2217 (2004); Glabati et al., Biochem. J. 303:697-700 (1994); and Zlakine et al., J. Cell Science 110:673-679 (1997), and can be incorporated into targeting sequences to induce membrane localization.

[0233] In some embodiments, a native sequence from a protein containing an acylation motif is incorporated into the targeting sequence. For example, in some embodiments, the N-terminal portion of a Lck, Fyn, or Yes protein α subunit, such as the first 25 N-terminal amino acids or fewer from such proteins (e.g., about 5 to 20 amino acids, about 10 to 19 amino acids, or about 15 to 19 amino acids from a native sequence with an optional mutation), may be incorporated into the N-terminus of the chimeric polypeptide. In some embodiments, about 25 amino acids or fewer C-terminal sequences from a G protein γ subunit containing a CAAX box motif sequence (e.g., about 5 to 20 amino acids, about 10 to 18 amino acids, or about 15 to 18 amino acids from a native sequence with an optional mutation) may be linked to the C-terminus of the chimeric polypeptide.

[0234] Any membrane-targeting sequence can be used. In some embodiments, such sequences include, but are not limited to, cardiomylation-targeting sequences, palmitoylation-targeting sequences, isoprenelation sequences (i.e., farnesylation, geranylation, CAAX box), protein-protein interaction motifs, or transmembrane sequences derived from receptors (using signal peptides). Examples include, for instance, those discussed in, for example, ten Klooster, JP et al., Biology of the Cell (2007) 99, 1-12; Vincent, S. et al., Nature Biotechnology 21: 936-40, 1098 (2003).

[0235] Additional protein domains exist that can increase protein retention across various membranes. For example, the Plek substrate protein homology (PH) domain, consisting of approximately 120 amino acids, has been found in over 200 human proteins typically involved in intracellular signaling. The PH domain can bind to various phosphatidylinositol (PI) lipids within the membrane (e.g., PI(3,4,5)-P3, PI(3,4)-P2, PI(4,5)-P2), and thus can play a crucial role in recruiting proteins to different membranes or cellular compartments. Typically, the phosphorylation state of PI lipids is regulated by kinases such as PI-3 kinase or PTEN; therefore, membrane-PH domain interactions may be less stable than those with acyl lipids.

[0236] In some implementations, the targeting sequence for guiding the peptide to the cell membrane can utilize a membrane anchoring signal sequence. Various membrane anchoring sequences are available. For example, membrane anchoring signal sequences of various membrane-binding proteins can be used. Sequences may include those from: 1) class I integrated membrane proteins, such as the IL-2 receptor β chain and the insulin receptor β chain; 2) class II integrated membrane proteins, such as neutral endopeptidase; 3) type III proteins, such as human cytochrome P450 NF25; and 4) type IV proteins, such as human P-glycoprotein.

[0237] In some embodiments, the chimeric receptor peptide is linked to a peptide folding domain that facilitates protein folding. In some embodiments, the actuation moiety is linked to a cell-penetrating domain. For example, the cell-penetrating domain may be derived from the HIV-1 TAT protein, the TLM cell-penetrating motif from human hepatitis B virus, MPG, Pep-1, VP22, a cell-penetrating peptide from herpes simplex virus, or a polyarginine peptide sequence. The cell-penetrating domain may be located at the N-terminus, C-terminus, or any position within the actuation moiety.

[0238] In some implementations, at least two targeting sequences are attached to the actuation moiety. When the actuation moiety is fused with multiple targeting sequences, such as those targeting different locations on the cell, the final localization of the actuation moiety can be determined by the relative strength of the targeting sequences. For example, if the NES is stronger than the NLS, a receptor having both a targeting sequence containing the NES and a targeting sequence containing the NLS can be localized to the cytoplasm. Alternatively, if the NLS is stronger than the NES, the receptor can be localized to the nucleus even if both nuclear localization and nuclear export signals are present on the receptor. The targeting sequences may include, for example, multiple copies of each NLS and NES to fine-tune the degree of cellular localization.

[0239] GMPs, as described elsewhere herein, may include an actuating portion. The actuating portion may include nucleases (e.g., DNA nucleases and / or RNA nucleases) as described elsewhere herein, modified nucleases (e.g., DNA nucleases and / or RNA nucleases) that are nuclease-deficient or have reduced nuclease activity compared to wild-type nucleases, their variants, derivatives, or fragments thereof. The actuating portion may regulate gene expression and / or activity or edit the sequence of nucleic acids (e.g., genes and / or gene products). The actuating portion may regulate gene expression or activity and / or edit nucleic acid sequences, whether exogenous or endogenous. In some embodiments, the actuating portion includes a DNA nuclease, such as an engineered (e.g., programmable or targetable) DNA nuclease, to induce genome editing of a target DNA sequence. In some embodiments, the actuating portion includes an RNA nuclease, such as an engineered (e.g., programmable or targetable) RNA nuclease, to induce editing of a target RNA sequence. In some embodiments, the actuating portion has reduced or minimal nuclease activity. An actuation moiety with reduced or minimal nuclease activity can regulate gene expression and / or activity by physically blocking the expression of the target polynucleotide or by recruiting additional factors that effectively inhibit or enhance the expression of the target polynucleotide. In some embodiments, the actuation moiety comprises a nuclease-free DNA-binding protein derived from a DNA nuclease, which can induce transcriptional activation or repression of the target DNA sequence. In some embodiments, the actuation moiety comprises a nuclease-free RNA-binding protein derived from an RNA nuclease, which can induce transcriptional activation or repression of the target RNA sequence. In some embodiments, the actuation moiety comprises a Cas protein lacking cleavage activity.

[0240] Any suitable nuclease may be used in the actuation part. Suitable nucleases include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases, including type I, type II, type III, type IV, type V, and type VI CRISPR-associated (Cas) peptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); a wide range of nucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flipases; transposases; Argonaute proteins; any derivatives thereof; any variants thereof; and any fragments thereof.

[0241] The actuation portion of the system of this invention, upon entering the cell nucleus, can bind to the target polynucleotide and regulate its expression and / or activity by physically inhibiting the target polynucleotide or recruiting other factors that effectively inhibit or enhance its expression. In some embodiments, the actuation portion comprises a transcriptional activator that effectively increases the expression of the target polynucleotide. The actuation portion may also comprise a transcriptional repressor that effectively decreases the expression of the target polynucleotide. In some embodiments, the actuation portion is operable for editing nucleic acid sequences.

[0242] In some embodiments, the target polynucleotide comprises genomic DNA. In some embodiments, the target polynucleotide comprises a region of a plasmid, such as a plasmid carrying a foreign gene. In some embodiments, the target polynucleotide comprises RNA, such as mRNA. In some embodiments, the target polynucleotide comprises an endogenous gene or gene product. The actuation portion may include one or more copies of a nuclear localization signal that allows the actuator to translocate into the cell nucleus upon activation of the nuclear localization signal or upon release of an inhibitor of the nuclear localization signal.

[0243] In some aspects, methods for regulating the expression of target polynucleotides in cells are disclosed, comprising: translocating a gene-regulating polypeptide from the cytoplasm to the nucleus in response to activation of a cell signaling pathway, wherein activation of the cell signaling pathway activates a nuclear localization domain coupled to the gene-regulating polypeptide.

[0244] In some aspects, methods for regulating the expression of target polynucleotides in cells are disclosed, comprising: a) activating cellular signaling pathways, wherein activating cellular signaling pathways activates a nuclear localization domain linked to a gene-regulating polypeptide; b) localizing the gene-regulating polypeptide to the cell nucleus via the activated nuclear localization domain, wherein, after localizing the gene-regulating polypeptide to the cell nucleus, the gene-regulating polypeptide regulates the expression of target polynucleotides in the cell.

[0245] In some aspects, methods for regulating the expression of target polynucleotides in cells are disclosed, comprising: a) contacting a ligand with a transmembrane receptor, wherein a cell signaling pathway is activated upon contact, and wherein the activated cell signaling pathway activates a nuclear localization domain coupled to a gene-regulating polypeptide; b) translocating the gene-regulating polypeptide from the cytoplasm to the nucleus via the activated nuclear localization domain, wherein the gene-regulating polypeptide regulates the expression of the target polynucleotide after translocation to the nucleus.

[0246] In some aspects, methods for regulating the expression of target polynucleotides in cells are disclosed, comprising: translocating a gene-regulating polypeptide from the cytoplasm to the nucleus in response to induction of a cell signaling pathway, wherein the induction of the cell signaling pathway induces a nuclear localization domain coupled to the gene-regulating polypeptide.

[0247] In some aspects, a method for regulating the expression of a target polynucleotide in a cell is disclosed, comprising: a) inducing a cell signaling pathway in the cell, wherein inducing the cell signaling pathway induces a nuclear localization domain linked to a gene-regulating polypeptide; b) localizing the gene-regulating polypeptide to the cell nucleus via the induced nuclear localization domain, wherein after localizing the gene-regulating polypeptide to the cell nucleus, the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell.

[0248] In some aspects, a method for regulating the expression of a target polynucleotide in a cell is disclosed, comprising: a) contacting a ligand with a transmembrane receptor, wherein a cell signaling pathway is induced after the contact, and wherein the induced cell signaling pathway induces a nuclear localization domain coupled to a gene-regulating polypeptide; b) translocating the gene-regulating polypeptide from the cytoplasm to the nucleus through the induced nuclear localization domain, wherein the gene-regulating polypeptide regulates the expression of the target polynucleotide after translocation to the nucleus.

[0249] The systems and compositions disclosed herein can be used in a variety of applications. For example, the systems and methods disclosed herein can be used in methods for modulating gene expression and / or cell activity. In one aspect, the systems and compositions disclosed herein are used in methods for modulating gene expression and / or cell activity in immune cells. Immune cells modulated using the systems of the present invention can be used in a variety of applications, including but not limited to immunotherapy for treating diseases and conditions. Diseases and conditions that can be treated with the modified immune cells of the present disclosure include inflammatory conditions, cancer, and infectious diseases. In some embodiments, immunotherapy is used to treat cancer.

[0250] The system of the present invention can be introduced into a variety of immune cells, including any cells involved in an immune response. In some embodiments, immune cells include granulocytes, such as basophils, eosinophils, and neutrophils; mast cells; monocytes that can develop into macrophages; antigen-presenting cells, such as dendritic cells; and lymphocytes, such as natural killer cells (NK cells), B cells, and T cells. In some embodiments, the immune cells are immune effector cells. Immune effector cells are immune cells that can perform specific functions in response to stimuli. In some embodiments, the immune cells are immune effector cells that can induce cell death. In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocyte is an NK cell. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is an activated T cell. T cells include naive and memory cells (e.g., central memory or T cells). CM Effect memory or T EM And effect memory RA or T EMRAT cells comprise effector cells (e.g., cytotoxic T cells, CTLs, or Tc cells), helper cells (e.g., Th1, Th2, Th3, Th9, Th7, TFH), regulatory cells (e.g., Treg and Trl cells), natural killer T cells (NKT cells), tumor-infiltrating lymphocytes (TILs), lymphocyte-activated killer cells (LAK), αβT cells, γδT cells, and similar distinct categories of T cell lineages. T cells can be divided into two main categories: CD8+ T cells and CD4+ T cells, based on which protein is present on the cell surface. T cells expressing the system of the present invention can perform multiple functions, including killing infected cells and activating or recruiting other immune cells. CD8+ T cells are referred to as cytotoxic T cells or cytotoxic T lymphocytes (CTLs). CTLs expressing the system of the present invention can participate in the recognition and removal of virus-infected cells and cancer cells. CTLs have specialized compartments or granules containing cytotoxins that induce apoptosis (e.g., programmed cell death). CD4+ T cells can be subdivided into four subsets—Th1, Th2, Th17, and Treg, where "Th" stands for "helper T cells," but other subsets may also exist. Th1 cells coordinate immune responses against intracellular microorganisms, especially bacteria. They produce and secrete molecules that warn and activate other immune cells, such as macrophages that engulf bacteria. Th2 cells participate in coordinating immune responses against extracellular pathogens, such as worms (parasitic worms), by warning B cells, granulocytes, and mast cells. Th17 cells produce interleukin-17 (IL-17), a signaling molecule that activates both immune and non-immune cells. Th17 cells are crucial for recruiting neutrophils.

[0251] In some embodiments, this disclosure provides immune cells expressing the system of the present invention (e.g., at least one of a receptor peptide and a gene regulatory peptide GMP as described herein). In some embodiments, the immune cell is a lymphocyte. When expressed in immune cells, the system of the present invention can be used to conditionally modulate certain activities of the immune cells. Immune cells such as lymphocytes expressing the system of the present invention can participate in cell-mediated immunity to eliminate diseased cells and / or pathogens.

[0252] In some embodiments, the lymphocytes of this disclosure are characterized in that, when the fused heterologous nuclear localization domain is active, the actuating moiety enters the cell nucleus, which occurs after the receptor polypeptide binds to the antigen and thereby triggers an intracellular signaling cascade. When the heterologous nuclear localization domain is active, the actuating moiety translocates into the cell nucleus and then operatively complexes with a target polynucleotide in the lymphocyte. Complexation of the actuating moiety with the target polynucleotide in the lymphocyte can lead to upregulation or increase in the expression of the target polynucleotide (e.g., a gene) in the lymphocyte. In some embodiments, the actuating moiety or the heterologous functional domain fused to the actuating moiety regulates the expression and / or activity of the target polynucleotide comprising an endogenous gene or gene product. The endogenous gene or gene product may participate in an immune response. For example, the actuating moiety or the heterologous functional domain fused to the actuating moiety can lead to increased expression of endogenous genes such as cytokines. Increased cytokine expression can contribute to an effective immune response and / or reduce negative therapeutic effects associated with the immune response.

[0253] In some embodiments, the actuating portion regulates the expression and / or activity of cytokines. Methods of altering cytokine expression can be used to modulate immune cells and / or immune responses, such as altering T cell activation, changing the level of NK cell activation, and various other immune cell activities in immunotherapy. Regulation of cytokine expression can be accomplished through a variety of mechanisms. In some embodiments, the actuating portion regulates the expression of cytokines from target polynucleotides and / or the activity of those cytokines, or edits nucleic acid sequences, such as nucleic acid sequences of genomic DNA encoding cytokines. In some embodiments, the actuating portion or a heterologous functional domain fused to the actuating portion regulates the expression of cytokine receptors from target polynucleotides and / or the activity of those cytokine receptors, or edits nucleic acid sequences, such as nucleic acid sequences of genomic DNA encoding cytokine receptors. The target polynucleotide regulated and / or edited by the actuating portion may comprise an endogenous gene or gene product, such as an endogenous cytokine or cytokine receptor gene (e.g., DNA) or gene product (e.g., RNA). In some embodiments, the actuating portion or a heterologous functional domain fused to the actuating portion alters the expression of cytokines or cytokine receptors (e.g., upregulation and / or downregulation). In some implementations, the actuation portion edits the nucleic acid sequence encoding a cytokine or cytokine receptor. Editing the nucleic acid sequence can produce non-functional gene products, such as truncated and / or non-conforming protein products.

[0254] Cytokines are proteins released by cells that can influence cellular behavior (e.g., chemokines, interferons, lymphokines, interleukins, and tumor necrosis factor). Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by more than one type of cell. Cytokines can participate in the production of systemic or local immune regulation.

[0255] Some cytokines can act as pro-inflammatory cytokines. Pro-inflammatory cytokines are those that participate in inducing or amplifying inflammatory responses. They can work in conjunction with various cells of the immune system, such as neutrophils and leukocytes, to generate an immune response. Some cytokines can also act as anti-inflammatory cytokines. Anti-inflammatory cytokines are those that participate in reducing inflammatory responses. In some cases, anti-inflammatory cytokines can modulate pro-inflammatory cytokine responses. Some cytokines can function as both pro-inflammatory and anti-inflammatory cytokines.

[0256] In some implementations, the expression of pro-inflammatory cytokines can be upregulated in immune cells. Upregulation of pro-inflammatory cytokines can be used, for example, to stimulate an immune response against target cells in immunotherapy. However, in some cases, excessive pro-inflammatory cytokines can lead to harmful effects, such as chronic systemic elevations in the body. In some implementations, the expression of pro-inflammatory cytokines is downregulated. Such downregulation can reduce and / or minimize harmful effects.

[0257] In some embodiments, the expression of cytokines with anti-inflammatory functions can be upregulated. If the inflammatory response is causing harmful effects, upregulating the expression of cytokines with anti-inflammatory functions can be used to, for example, reduce and / or minimize the inflammatory response. In some embodiments, the expression of cytokines with anti-inflammatory functions can be downregulated. If desired, such downregulation can increase and / or enhance the inflammatory response.

[0258] Examples of cytokines that can be regulated by the systems and compositions disclosed herein include, but are not limited to, lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental prolactin; tumor necrosis factor-α; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; and vascular endothelial cells. Growth factors; integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-α; platelet-derived growth factor; transforming growth factor (TGF), such as TGF-α, TGF-β, TGF-β1, TGF-β2, and TGF-β3; insulin-like growth factor-I and-II; erythropoietin (EPO); Flt-3L; stem cell factor (SCF); bone-inducing factor; interferon (IFN), such as IFN-α, IFN-β, and IFN-γ; colony stimulation. Factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); granulocyte-CSF (G-CSF); macrophage-stimulating factor (MSP); interleukins (ILs), such as IL-1, IL-1a, IL-1b, IL-1RA, IL-18, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15. IL-13, IL-14, IL-15, IL-16, IL-17, IL-20; tumor necrosis factors, such as CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE; and other polypeptide factors, including LIF, oncokinetic protein M (OSM), and kit ligand (KL). Cytokine receptors are receptor proteins that bind cytokines. Cytokine receptors can be membrane-bound or soluble.

[0259] In some embodiments, the actuating portion or a heterologous functional domain fused to the actuating portion regulates the expression and / or activity of interleukin (IL-1) family members (e.g., ligands), IL-1 receptor family members, interleukin-6 (IL-6) family members (e.g., ligands), IL-6 receptors, interleukin-10 (IL-10) family members (e.g., ligands), IL-10 receptors, interleukin-12 (IL-12) family members (e.g., ligands), IL-12 receptors, interleukin-17 (IL-17) family members (e.g., ligands), or IL-17 receptors.

[0260] In some embodiments, the actuating portion or a heterologous functional domain fused to the actuating portion regulates the expression and / or activity of cytokines, including but not limited to members of the interleukin-1 (IL-1) family or related proteins; members of the tumor necrosis factor (TNF) family or related proteins; members of the interferon (IFN) family or related proteins; members of the interleukin-6 (IL-6) family or related proteins; and chemokines or related proteins.In some implementations, the actuating component regulates the expression and / or activity of cytokines selected from the following: IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33, BAFF / BLyS / TNFSF138, 4-1BBL, CD153 / CD30L / TNFSF8, CD40LG, CD70, Fas ligand / FASLG / CD95L / CD178, EDA-A1, TNFSF14 / LIGHT / CD258, TNFA, LTA / TNFB / TNFSF1, LTB / TNFC, CD70 / CD27L / TNFSF7, TNFSF10 / TRAIL / APO-2L(CD253), RANKL / OPGL / TNFSF11(CD254), TNFSF12, TNF-alpha / TNFA ,TNFSF13, TL1A / TNFSF15, OX-40L / TNFSF4 / CD252, CD40L / CD154 / TNFSF5, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5 / IFNaG, IFNω / IFNW1, CLCF1, CNTF, IL11, IL31, IL6, Leptin, LIF, OSM, CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP -4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3 , CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXC L17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4 and FAM19A5.

[0261] In some embodiments, the actuating portion or a heterologous functional domain fused to the actuating portion regulates the expression and / or activity of cytokine receptors, including but not limited to members of the interleukin-1 (IL-1) receptor family or related proteins; members of the tumor necrosis factor (TNF) receptor family or related proteins; members of the interferon (IFN) receptor family or related proteins; members of the interleukin-6 (IL-6) receptor family or related proteins; and chemokine receptors or related proteins. In some embodiments, the actuating portion regulates the expression and / or activity of cytokine receptors selected from IL18R1, IL18RAP, IL1R1, IL1R2, IL1R3, IL1R8, IL1R9, IL1RL1, SIGIRR, 4-1BB, BAFFR, TNFRSF7, CD40, CD95, DcR3, TNFRSF21, EDA2R, EDAR, PGLYRP1, TNFRSF19L, TNFR1, TNFR2, TN FRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF25, LTBR, ​​TNFR SF4, TNFRSF8, TRAILR1, TRAILR2, TRAILR3, TRAILR4, IFNAR1, IFNAR2, IFNGR1, IFNGR2, CNTFR, IL11RA, IL6R, LEP R, LIFR, OSMR, IL31RA, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCRL1, CXCR3, CXCR4, CXCR5, CXCR6, CXC R7, CXCR1, CXCR2, ARMCX, BCA-1 / CXCL1, CCL1, CCL12 / MCP-, CCL13 / MCP-, CCL15 / MIP-5 / MIP-1delt, CCL16 / HCC-4 / NCC, CCL17 / TAR, CCL18 / PARC / MIP-, CCL19 / MIP-3, CCL2 / MCP-, CCL20 / MIP-3alpha / MIP3, CCL21 / 6Ckin, CCL22 / MD, CCL23 / MIP, CCL24 / Eotaxin-2 / MPIF-, CCL25, CCL26 / Eotaxin-, CCL27, CCL3, CCL4, CCL4L1 / LAG, CCL5, CCL6,CCL8 / MCP-, CXCL10 / Crg, CXCL12 / SDF-1, CXCL14, CXCL15, CXCL16 / SR-, CXCL2 / MIP-, CXCL3 / GRO, CXCL4, CXCL6 / GCP-, CXCL9, FAM19A4, Fractalkine, I-309 / CCL1 / TC A-, IL-8, MCP-3, NAP-2 / PPBP, XCL2, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCRL1, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 / RDC-1, IL8Ra / CXCR1 and IL8Rb / CXCR2. ,

[0262] In some embodiments, the actuating portion or a heterologous functional domain fused to the actuating portion regulates the expression and / or activity of the following factors: activins (e.g., activin βA, activin βB, activin βC, and activin βE); inhibins (e.g., inhibin-A and inhibin-B); activin receptors (e.g., activin type 1 receptor, activin type 2 receptor); bone morphogenetic proteins (e.g., BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, and BMP15); BM P receptors; growth differentiation factors (e.g., GDF1, GDF2, GDF3, GDF4, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, and GDF15); glial cell-derived neurotrophic factor family ligands (e.g., glial cell line-derived neurotrophic factor (GDNF), neurorankin (NRTN), artemin (ARTN), and persephin (PSPN)); GDNF family receptors; and c-MPL / CD110 / TPOR.

[0263] Cytokine production can be evaluated using a variety of methods. Cytokine production can be evaluated by analyzing the presence of one or more cytokines in a cell culture medium in which modified immune cells are grown (e.g., in vitro production) or in serum obtained from a subject with modified immune cells (e.g., in vivo production). Cytokine levels can be quantified using any suitable assay in a variety of suitable units, including concentration. In some embodiments, cytokine proteins are detected. In some embodiments, mRNA transcripts of cytokines are detected. Examples of cytokine assays include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence assay, radioimmunoassay, antibody arrays that allow parallel detection of various cytokines in a sample, bead-based arrays, quantitative PCR, microarrays, etc. Other suitable methods may include proteomics methods (2-D gel, MS analysis, etc.).

[0264] In some embodiments, the endogenous gene or gene product encodes an immunomodulatory protein. Immunomodulatory proteins include proteins such as immune checkpoint receptors, which, when bound to their homologous ligands, can enhance and / or inhibit immune cell signaling, including but not limited to activation and inhibition signals of immune cells. In some cases, animals can be induced to alter the expression of the regulatory protein (e.g., upregulation and / or downregulation). In some embodiments, animals are induced to edit the nucleic acid sequence encoding the regulatory protein. In some implementations, the endogenous gene or gene product encodes molecules such as: A2AR, B7.1, B7-H3 / CD276, B7-H4 / B7S1 / B7x / Vtcn1, B7-H6, BTLA / CD272, CCR4, CD122, 4-1BB / CD137, CD27, CD28, CD40, CD47, CD70, CISH, CTLA-4 / CD152, DR3, GITR, ICOS / CD278, IDO, KIR, LAG-3, OX40 / CD134, PD-1 / CD279, PD2, PD-L1, PD-L2, TIM-3, and VISTA / Dies1 / Gi24 / PD-1H (C10orf54).

[0265] In some embodiments, the target polynucleotide comprises a heterologous gene or gene product. The heterologous gene or gene product may encode a protein, such as an additional chimeric transmembrane receptor polypeptide. In some embodiments, the additional chimeric transmembrane receptor polypeptide comprises (a) an extracellular region containing an additional ligand-interaction domain that specifically binds to an additional antigen; and (b) a co-stimulatory domain. The additional ligand-interaction domain may bind any suitable antigen. The additional ligand-interaction domain may bind to an antigen previously described. The additional ligand-interaction domain may bind to the same antigen or a different antigen as the chimeric receptor polypeptide. The additional ligand-interaction domain may comprise any suitable ligand-interaction domain. The additional ligand-interaction domain may be any ligand-interaction domain described elsewhere herein. For example, the additional ligand interaction domain may comprise a monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, Fab, Fab', F(ab')2, Fv, single-chain antibody (e.g., scFv), microantibody, biantibody, single-domain antibody (“sdAb” or “nanobody” or “cameloid antibody”), or Fc binding domain. In some embodiments, the additional ligand interaction domain comprises an antibody mimic.

[0266] The additional chimeric transmembrane receptor polypeptide may include a co-stimulatory domain. The co-stimulatory domain can be any of the previously described co-stimulatory domains. The co-stimulatory domain can provide a co-stimulatory signal. In some cases, such co-stimulatory signals provide proliferation and / or survival signals in immune cells expressing the system of the present invention. In some embodiments, the immune cell signaling domain of the chimeric transmembrane receptor polypeptide and the additional chimeric transmembrane receptor polypeptide include at least one co-stimulatory domain. Expression of the additional chimeric transmembrane receptor containing the co-stimulatory domain can provide sufficient cell signal transduction to generate a durable and / or adequate immune response.

[0267] Electromagnetic radiation-mediated gene regulation

[0268] This document provides embodiments and / or modifications of the above-described systems and methods for extracellular signal-mediated gene regulation. Such embodiments and / or modifications, or any further modifications thereof, can utilize one or more components of the above-described systems and methods for extracellular signal-mediated gene regulation.

[0269] In one aspect, this disclosure provides a system for regulating the expression of a target polynucleotide in a cell, the system comprising: a chimeric polypeptide comprising a gene-regulating polypeptide fused to a heterologous nuclear localization domain frame, wherein the nuclear localization domain is operable to translocate the chimeric polypeptide to the cell nucleus upon activation of a cell signaling pathway induced by an extracellular signal, wherein the extracellular signal is electromagnetic radiation, and wherein, in response to the extracellular signal, the chimeric polypeptide is localized to the cell nucleus, and the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0270] In one aspect, this disclosure provides a system for regulating the expression of a target polynucleotide in a cell, the system comprising: a) a chimeric receptor polypeptide that activates a cell signaling pathway upon binding to a ligand; and b) a chimeric polypeptide comprising a gene-regulating polypeptide fused within a heterologous nuclear localization domain frame, the heterologous nuclear localization domain being operable to translocate the chimeric polypeptide to the cell nucleus upon activation by electromagnetic radiation when needed, wherein binding of the ligand to the chimeric receptor polypeptide can further translocate the chimeric polypeptide to the cell nucleus via the activated heterologous nuclear localization domain, and the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0271] In one aspect, this disclosure provides a system for regulating the expression of a target polynucleotide in a cell, the system comprising: a) a cell signaling pathway activator containing an electromagnetic radiation source; and b) a chimeric polypeptide comprising a gene-regulating polypeptide fused to a heterologous nuclear localization domain frame, the heterologous nuclear localization domain being operable to translocate the chimeric polypeptide to the cell nucleus after activation of the cell signaling pathway, wherein, after electromagnetic radiation is applied to the cell, the chimeric polypeptide is localized to the cell nucleus via the activated heterologous nuclear localization domain, and the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

[0272] In some implementations, the electromagnetic radiation used in the system may include one or more wavelengths from the electromagnetic spectrum, including X-rays, ultraviolet (UV) rays, visible light, infrared rays, microwaves, or any combination thereof.

[0273] In some embodiments, the electromagnetic radiation source disclosed herein is used in vitro or in combination with the system. In other embodiments, the electromagnetic radiation source is implanted, attached to, or applied to a subject, including but not limited to mammals and plants. In use, the electromagnetic radiation source can emit at least a portion of the electromagnetic spectrum to one or more specific regions within the subject to provide spatial and / or temporal control in activating cell signaling pathways, thereby providing spatial and / or temporal control in regulating the expression of target polynucleotides within the subject.

[0274] In some implementations, the electromagnetic radiation source can emit at least a portion of an electromagnetic spectrum toward cells or a subject containing cells for a period of time. In some cases, emitting at least a portion of the electromagnetic spectrum over multiple time periods can provide time control in activating cellular signaling pathways, thereby providing time control in regulating the expression of target polynucleotides in cells. In some cases, the electromagnetic radiation source can emit at least a portion of the electromagnetic spectrum for a duration of about 0.1 milliseconds (ms) to about 120 minutes (min). The electromagnetic radiation source can emit at least a portion of the electromagnetic spectrum for a duration of at least 0.1 ms, 0.2 ms, 0.3 ms, 0.4 ms, 0.5 ms, 0.6 ms, 0.7 ms, 0.8 ms, 0.9 ms, 1 ms, 2 ms, 3 ms, 4 ms, 5 ms, 6 ms, 7 ms, 8 ms, 9 ms, 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 200 ms, 300 ms, 40 ... 0ms, 600ms, 700ms, 800ms, 900ms, 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, 20s, 30s, 40s, 50s, 60s, 70s, 80s, 90s, 10 0s, 200s, 300s, 400s, 500s, 600s, 700s, 800s, 900s, 1000s, 2000s, 3000s, 4000s, 5000s, 6000s, 7000s, 7200s or longer. Electromagnetic radiation sources can emit at least a portion of the electromagnetic spectrum for a maximum duration of 7200s, 7000s, 6000s, 5000s, 4000s, 3000s, 2000s, 1000s, 900s, 800s, 700s, 600s, 500s, 400s, 300s, 200s, 100s, 90s, 80s, 70s, 60s, 50s, 40s, 30s, 20s, 10s, 9s, 8s, 7s, 6s, 5s, 4s, 3s, 2s, 1s, or 900ms. , 800ms, 700ms, 600ms, 500ms, 400ms, 300ms, 200ms, 100ms, 90ms, 80ms, 70ms, 60ms, 50ms, 40ms, 30ms, 20ms, 10ms , 9ms, 8ms, 7ms, 6ms, 5ms, 4ms, 3ms, 2ms, 1ms, 0.9ms, 0.8ms, 0.7ms, 0.6ms, 0.5ms, 0.4ms, 0.3ms, 0.2ms, 0.1ms or shorter.

[0275] In some implementations, the electromagnetic radiation source may emit at least a portion of blue light within the visible spectrum to induce cell signaling pathways. The blue light may include wavelengths from about 380 nm to about 490 nm. Alternatively, the blue light may include a wavelength of at least about 380 nm. Or, the blue light may include a wavelength of up to about 490 nm.Blue light can include the following wavelengths: approximately 380nm to approximately 390nm, approximately 380nm to approximately 400nm, approximately 380nm to approximately 410nm, approximately 380nm to approximately 420nm, approximately 380nm to approximately 430nm, approximately 380nm to approximately 440nm, approximately 380nm to approximately 450nm, approximately 380nm to approximately 460nm, approximately 380nm to approximately 470nm, approximately 380nm to approximately 480nm, approximately 380nm to approximately 490nm, approximately 390nm to approximately 400nm, approximately 390nm to approximately 410nm, approximately 390nm to approximately 420nm, approximately 390nm to approximately 430nm, and approximately 390nm to approximately 440nm. m, approximately 390nm to approximately 450nm, approximately 390nm to approximately 460nm, approximately 390nm to approximately 470nm, approximately 390nm to approximately 480nm, approximately 390nm to approximately 490nm, approximately 400nm to approximately 410nm, approximately 400nm to approximately 420nm, approximately 400nm to approximately 430nm, approximately 400nm to approximately 440nm, approximately 400nm to approximately 450nm, approximately 400nm to approximately 460nm, approximately 400nm to approximately 470nm, approximately 400nm to approximately 480nm, approximately 400nm to approximately 490nm, approximately 410nm to approximately 420nm, approximately 410nm to approximately 430nm, approximately 410nm to approximately 440nm, approximately 410nm to approximately 450nm, approximately 410nm to approximately 460nm, approximately 410nm to approximately 470nm, approximately 410nm to approximately 480nm, approximately 410nm to approximately 490nm, approximately 420nm to approximately 430nm, approximately 420nm to approximately 440nm, approximately 420nm to approximately 450nm, approximately 420nm to approximately 460nm, approximately 420nm to approximately 470nm, approximately 420nm to approximately 480nm, approximately 420nm to approximately 490nm, approximately 430nm to approximately 440nm, approximately 430nm to approximately 450nm, approximately 430nm to approximately 460nm, approximately 430nm to approximately 47 ...80nm, approximately 420nm to approximately 480nm, approximately 420nm to approximately 490nm, approximately 430nm to approximately 440nm, approximately 430nm to approximately 450nm, approximately 430nm to approximately 460nm, approximately 430nm to approximately 470nm, approximately 430nm to approximately 480nm, approximately 430nm to approximately 480nm, approximately 420nm to approximately 480nm, approximately 420nm to approximately 490nm, approximately 430nm to approximately 440nm, approximately 430nm to approximately 450nm, approximately 430nm to approximately 460nm, approximately nm to about 480nm, about 430nm to about 490nm, about 440nm to about 450nm, about 440nm to about 460nm, about 440nm to about 470nm, about 440nm to about 480nm, about 440nm to about 490nm, about 450nm to about 460nm, about 450nm to about 470nm, about 450nm to about 480nm, about 450nm to about 490nm, about 460nm to about 470nm, about 460nm to about 480nm, about 460nm to about 490nm, about 470nm to about 480nm, about 470nm to about 490nm, or about 480nm to about 490nm.Blue light can include wavelengths of approximately 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, or 490nm.

[0276] In some embodiments, the electromagnetic radiation source may emit at least a portion of ultraviolet light. The ultraviolet light may include wavelengths from about 10 nm to about 380 nm. The ultraviolet light may include wavelengths of at least about 10 nm. The ultraviolet light may include wavelengths of up to about 380 nm. The ultraviolet light may include the following wavelengths: about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10 nm to about 150 nm, about 10 nm to about 200 nm, about 10 nm to about 250 nm, about 10 nm to about 300 nm, about 10 nm to about 350 nm, about 10 nm to about 380 nm, about 50 nm to about 100 nm, about 50 nm to about 150 nm, about 50 nm to about 200 nm, about 50 nm to about 250 nm, about 50 nm to about 300 nm, about 50 nm to about 350 nm, about 50 nm to about 380 nm, about 100 nm to about 150 nm, about 100 nm to about 200 nm, about 100 nm to about 250 nm, about 1... 00nm to about 300nm, about 100nm to about 350nm, about 100nm to about 380nm, about 150nm to about 200nm, about 150nm to about 250nm, about 150nm to about 300nm, about 150nm to about 350nm, about 150nm to about 380nm, about 200nm to about 250nm, about 200nm to about 300nm, about 200nm to about 350nm, about 200nm to about 380nm, about 250nm to about 300nm, about 250nm to about 350nm, about 250nm to about 380nm, about 300nm to about 350nm, about 300nm to about 380nm or about 350nm to about 380nm. Ultraviolet light can include wavelengths of about 10 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, or about 380 nm.

[0277] In some implementations, the system for regulating the expression of target polynucleotides in cells may further include a signal transduction unit that activates cell signal transduction pathways upon application of an extracellular signal, such that the extracellular signal is electromagnetic radiation.

[0278] In some cases, the signal transduction unit may comprise a transmembrane protein. Upon application of electromagnetic radiation, transmembrane proteins can induce cellular signal transduction pathways. Transmembrane proteins can be retinylidene proteins. Examples of retinylidene proteins include light-gated ion channels (e.g., channelrhodopsin-1 (ChR1), channelrhodopsin-2 (ChR2), bacteriocyanin, halobacterial rhodopsin, proteorhodopsin, etc.), some G protein-coupled receptors (GPCRs) (e.g., opsin, melanin, peropsin, encephalopsin, etc.) and their modifications. In one example, the transmembrane protein may be channelrhodopsin-2 (ChR2). The ChR2 protein may have an L132C mutation to increase its permeability to calcium. When irradiated with blue light (e.g., at a wavelength of 470 nm), the ChR2 protein can be activated and allow calcium ions to flow into the cytoplasm. The increased intracellular calcium ion concentration can then activate calcium and calmodulin-dependent serine / threonine protein phosphatases (e.g., calcium-dependent phosphatases) as cellular signaling pathways. Alternatively, in some cases, the signaling unit may comprise intracellular proteins. Intracellular proteins can induce cellular signaling pathways upon application of electromagnetic radiation.

[0279] In some cases, the signal transduction unit may comprise transmembrane proteins and intracellular proteins. In some instances, the application of an extracellular signal (e.g., electromagnetic radiation) can activate the transmembrane protein of the signal transduction unit, which in turn activates the intracellular protein of the signal transduction unit to induce a cell signaling pathway (e.g., calcium-dependent phosphatase). In some instances, the application of an extracellular signal can activate the intracellular protein of the signal transduction unit, which in turn activates the transmembrane protein of the signal transduction unit to induce a cell signaling pathway. In one instance, the transmembrane protein may be an ion channel protein (e.g., calcium release-activated calcium channel protein 1 (ORAI1)). The intracellular protein may comprise a first part and a second part. The application of electromagnetic radiation can induce a conformational change in the intracellular protein, thereby exposing an active site of at least one of the first and second parts. This active site may be on the first part of the intracellular protein. This active site may be on the second part of the intracellular protein. This active site may be on both the first and second parts of the intracellular protein. Once exposed, the active site of the intracellular protein can activate the transmembrane protein to induce a cell signaling pathway. In some cases, the active sites of intracellu...

Claims

1. A system for regulating the expression of a target polynucleotide in cells, the system comprising: A chimeric polypeptide comprising a gene-regulating polypeptide fused to a heterologous nuclear localization domain (HNU), wherein the HNU is operable to translocate the chimeric polypeptide to the nucleus upon activation of the HNU via a cell signaling pathway induced by an extracellular signal, wherein the HNU consists of the sequence of amino acid residues 1-391 of SEQ ID NO:1, and wherein the chimeric polypeptide does not include the DNA-binding domain (DBD) or C-terminal transactivation domain (TAD-C) of the NFATc2 protein. In response to the extracellular signal, the chimeric polypeptide is localized to the cell nucleus, and the gene-regulating polypeptide regulates the expression of the target polynucleotide in the cell nucleus.

2. The system of claim 1, wherein the system further comprises a signal transduction unit that activates the cell signal transduction pathway after the extracellular signal is applied.

3. The system of claim 1, wherein the system further comprises a chimeric receptor polypeptide.

4. The system of claim 3, wherein the chimeric receptor polypeptide is selected from the group consisting of: Notch receptor, G protein-coupled receptor (GPCR), integrin receptor, cadherin receptor, tyrosine kinase receptor, death receptor, immune receptor, and chimeric antigen receptor.

5. The system of claim 2, wherein the signal transduction unit comprises intracellular proteins.

6. The system of claim 5, wherein in response to the extracellular signal, the intracellular protein activates an intracellular transmembrane protein, wherein the activated transmembrane protein induces the cell signaling pathway.

7. The system of claim 6, wherein the intracellular protein binds to the transmembrane protein in response to the extracellular signal.

8. The system of claim 6, wherein the transmembrane protein comprises a calcium channel.

9. The system of claim 5, wherein the active sites of the intracellular proteins are exposed in response to the extracellular signal.

10. The system of claim 5, wherein the intracellular protein comprises a light-oxygen-voltage (LOV) domain and a STIM-ORAI activation region (SOAR) domain, wherein the LOV domain and the SOAR domain are linked by an α-helical peptide domain.

11. The system of claim 1, wherein the extracellular signal causes an increase in intracellular calcium concentration relative to baseline levels within the cell.

12. The system of claim 1, wherein the cell signaling pathway includes the calcium signaling pathway.

13. The system of claim 1, wherein the extracellular signal comprises a ligand.

14. The system of claim 1, wherein the extracellular signal comprises a chemical compound.

15. The system of claim 1, wherein the extracellular signal comprises electromagnetic radiation.

16. The system of claim 15, wherein the electromagnetic radiation is selected from the group consisting of X-rays, ultraviolet (UV) rays, visible light, infrared rays, and microwaves.

17. The system of claim 1, wherein activation of the heterologous nuclear localization domain includes chemical modification of the heterologous nuclear localization domain.

18. The system of claim 17, wherein the chemical modification is selected from the group consisting of dephosphorylation, phosphorylation, acetylation, methylation, ubiquitination, and protein hydrolysis.

19. The system of claim 1, wherein activation of the heterogeneous nuclear localization domain includes a conformational change of the heterogeneous nuclear localization domain.

20. The system of claim 1, wherein activation of the heterologous nuclear localization domain includes binding of a second signaling pathway protein to the heterologous nuclear localization domain.

21. The system of claim 20, wherein the second signaling pathway protein comprises a calcium-dependent phosphatase.

22. The system according to claim 1, wherein the gene regulatory polypeptide is an endonuclease.

23. The system of claim 1, wherein the gene regulatory polypeptide comprises a Cas protein.

24. The system of claim 23, wherein the Cas protein is substantially lacking in DNA cleavage activity.

25. The system of claim 1, wherein the gene-regulating polypeptide is selected from the group consisting of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), macronucleases, recombinases, flipases, transposases, and Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaea Argonaute (aAgo), and eukaryotic Argonaute (eAgo)).

26. The system of claim 1, wherein the gene regulatory polypeptide comprises a transcription activator.

27. The system of claim 26, wherein the transcriptional activator is selected from the group consisting of VP16, VP32, VP64, VPR, p65 and P65HSF1.

28. The system of claim 1, wherein the gene-regulating polypeptide comprises a transcriptional repressor.

29. The system of claim 28, wherein the transcriptional repressor comprises a KRAB domain.

30. The system of claim 1, wherein the gene-regulating polypeptide comprises a chromosome-modifying enzyme.

31. The system according to claim 30, wherein the chromosome modifying enzyme is selected from the group consisting of methyltransferases, demethylases, acetyltransferases, deacetylases, deaminases, phosphorylases, and dephosphorylases.

32. The system of claim 30, wherein the chromosome-modifying enzyme modifies one or more histones.

33. The system according to claim 1, wherein the target polynucleotide is an endogenous gene of the cell.

34. The system of claim 1, wherein the target polynucleotide is genomic DNA of a cell.

35. The system of claim 1, wherein the target polynucleotide is RNA.

36. The system of claim 1, wherein the cell is a lymphocyte.

37. The system of claim 1, wherein the regulated expression of the target polynucleotide regulates immune cell activity.

38. The system of claim 1, wherein the cell is not a kidney cell.

39. The system of claim 1, wherein the cells are not cervical cancer cells.

40. Lymphocytes expressing any one of claims 1-39.

41. Use of the system according to any one of claims 1-39 or the lymphocyte according to claim 40 in the preparation of a medicament for inducing target cell death.

42. The use according to claim 41, wherein the target cell is a cancer cell.

43. The use according to claim 41, wherein the target cells are disease cells derived from blood disorders, cancer, metabolic disorders, eye diseases, organ diseases, musculoskeletal disorders, or heart diseases.

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