Regulating gene expression in gamma delta t cell receptors expressing cells

Abstract

The present disclosure provides a method for regulating an activity of a gamma delta T cell. The method may comprise administering to said gamma delta T cell a gene modulating polypeptide (GMP) comprising an actuator moiety configured to regulate expression of a target polynucleotide in said gamma delta T cell. The target polynucleotide may encode a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolase.

Description

REGULATING GENE EXPRESSION IN GAMMA DELTA T CELL RECEPTORS EXPRESSING CELLS

[0001] Cells that express gamma (γ) and / or delta (δ) T cell receptors (e.g., gamma-delta (γδ) T cells) can participate in one or more immune responses during progression of a disease, such as cancer. In some cases, the γδ T cells can play direct and / or indirect anti-tumor roles (e.g., anti-tumor cytotoxicity, cytokine production, activation of one or more immune cell types, etc.). Thus, the γδ T cells can be used in cancer immunotherapy. In some cases, the γδ T cells may be activated (e.g., by a stimulus) to promote and / or prolong their anti-tumor activity. Alternatively or in addition to, the γδ T cells may be isolated, expanded, and administered for cancer immunotherapy.

[0002] The γδ T cells can be activated via one or more pathways (e.g., one or more cellular receptors). In some cases, interaction of a cellular receptor with a ligand can play a role in sensing a stimulus (e.g., one or more environmental cues) and translating such extracellular stimulation into intracellular signaling. Intracellular signaling can result in the regulation of biochemical processes including transcriptional activation of gene expression and new protein synthesis to control cell activities, such as the anti-tumor activity. However, effectiveness of the γδ T cells in immunotherapy can be reduced by, for example, insufficient activation from an inadequate receptor-ligand interaction and / or homologous immune checkpoint pathways (e.g., apoptotic pathways).

[0003] In view of the foregoing, there exists a considerable need for alternative methods and systems to control an activity (e.g., the anti-tumor activity) of the γδ T cells.

[0004] An aspect of the present disclosure provides a method for regulating an activity of a gamma delta T cell, comprising: administering to the gamma delta T cell a gene modulating polypeptide (GMP) comprising an actuator moiety configured to regulate expression of a target polynucleotide in the gamma delta T cell, wherein the target polynucleotide encodes a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolase.

[0005] In some embodiments, regulating the activity of the gamma delta T cell comprises decreasing fratricide of gamma delta T cells.

[0006] In some embodiments, regulating the activity of the gamma delta T cell comprises prolonging activation of the gamma delta T cell.

[0007] In some embodiments, the actuator moiety is an RNA-guided actuator moiety or a variant thereof, which RNA-guided actuator moiety forms a complex with the target polynucleotide. In some embodiments, the actuator moiety is a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity. In some embodiments, the actuator moiety is Cas9 and / or Cpf1.

[0008] In some embodiments, the actuator moiety comprises an activator effective to increase expression of the target polynucleotide.

[0009] In some embodiments, the actuator moiety comprises a repressor effective to decrease expression of the target polynucleotide.

[0010] In some embodiments, the polypeptide is the death-inducing protein. In some embodiments, the death-inducing protein comprises Fas, Fas ligand, granzyme B, granzyme M, and / or perforin. In some embodiments, the regulation of expression of the target polynucleotide comprises decreasing the expression of the target polynucleotide.

[0011] In some embodiments, the polypeptide is the inhibitor of the death-inducing protein. In some embodiments, the inhibitor comprises serpin B9, serpin B4, and / or c-FLIP. In some embodiments, the regulation of expression of the target polynucleotide comprises increasing the expression of the target polynucleotide.

[0012] In some embodiments, the polypeptide is the hydrolase. In some embodiments, the polypeptide comprises CD39. In some embodiments, the regulation of expression of the target polynucleotide comprises decreasing the expression of the target polynucleotide.

[0013] In some embodiments, the method further comprises treating the gamma delta T cell with a virus to administer the GMP to the gamma delta T cell. In some embodiments, the method further comprises integrating into a genome of the gamma delta T cell a nucleic acid sequence encoding the GMP by using the virus. In some embodiments, the virus is a retrovirus. In some embodiments, the retrovirus is a gamma-retrovirus selected from the group consisting of: Moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), and spleen focus-forming virus (SFFV), and / or variants thereof.

[0014] In some embodiments, the method further comprises treating the gamma delta T cell with a nuclease to administer the GMP to the gamma delta T cell. In some embodiments, the method further comprises inserting into a genome of the gamma delta T cell a nucleic acid sequence encoding the GMP by using the nuclease. In some embodiments, the nuclease comprises a CRISPR- associated polypeptide (Cas), zinc finger nuclease (ZFN), zinc finger associate gene regulation polypeptides, transcription activator-like effector nuclease (TALEN), transcription activator-like effector associated gene regulation polypeptides, meganuclease, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0015] In some embodiments, the method further comprises administering to the gamma delta T cell a chimeric polypeptide comprising the GMP, wherein the chimeric polypeptide is operable to release the GMP from the chimeric polypeptide in response to a stimulant, and wherein the released GMP is operable to regulate expression of the target polynucleotide in the gamma delta T cell.

[0016] In some embodiments, the method further comprises administering to the gamma delta T cell a chimeric polypeptide comprising the GMP and a nuclear localization domain, wherein the nuclear localization domain is operable to translocate the chimeric polypeptide to a nucleus of the gamma delta T cell in response to a stimulant, and wherein the translocated GMP is operable to regulate expression of the target polynucleotide in the gamma delta T cell.

[0017] Another aspect of the present disclosure provides a system for regulating an activity of a gamma delta T cell, comprising: a gene modulating polypeptide (GMP) comprising an actuator moiety configured to regulate expression of a target polynucleotide in the gamma delta T cell, wherein the target polynucleotide encodes a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolase.

[0018] In some embodiments, the GMP is operable to decrease fratricide of gamma delta T cells.

[0019] In some embodiments, the GMP is operable to prolong activation of the gamma delta T cell.

[0020] In some embodiments, the actuator moiety is an RNA-guided actuator moiety or a variant thereof, which RNA-guided actuator moiety forms a complex with the target polynucleotide. In some embodiments, the actuator moiety is a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity. In some embodiments, the actuator moiety is Cas9 and / or Cpf1.

[0021] In some embodiments, the actuator moiety comprises an activator effective to increase expression of the target polynucleotide.

[0022] In some embodiments, the actuator moiety comprises a repressor effective to decrease expression of the target polynucleotide.

[0023] In some embodiments, the polypeptide is the death-inducing protein. In some embodiments, the death-inducing protein comprises Fas, Fas ligand, granzyme B, granzyme M, and / or perforin. In some embodiments, the regulation of expression of the target polynucleotide comprises decreasing the expression of the target polynucleotide.

[0024] In some embodiments, the polypeptide is the inhibitor of the death-inducing protein. In some embodiments, the inhibitor comprises serpin B9, serpin B4, and / or c-FLIP. In some embodiments, the regulation of expression of the target polynucleotide comprises increasing the expression of the target polynucleotide.

[0025] In some embodiments, the polypeptide is the hydrolase. In some embodiments, the polypeptide comprises CD39. In some embodiments, the regulation of expression of the target polynucleotide comprises decreasing the expression of the target polynucleotide.

[0026] In some embodiments, a nucleic acid sequence encoding the GMP is integrated into a genome of the gamma delta T cell by a virus. In some embodiments, the virus is a retrovirus. In some embodiments, the retrovirus is a gamma-retrovirus selected from the group consisting of: Moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), and spleen focus-forming virus (SFFV), and / or variants thereof.

[0027] In some embodiments, a nucleic acid sequence encoding the GMP is integrated into a genome of the gamma delta T cell by a nuclease. In some embodiments, the nuclease comprises a CRISPR- associated polypeptide (Cas), zinc finger nuclease (ZFN), zinc finger associate gene regulation polypeptides, transcription activator-like effector nuclease (TALEN), transcription activator-like effector associated gene regulation polypeptides, meganuclease, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0028] In some embodiments, a nucleic acid sequence encoding the GMP is integrated into a genome of the gamma delta T cell by an endonuclease. In some embodiments, the endonuclease comprises a CRISPR- associated polypeptide (Cas), zinc finger nuclease (ZFN), zinc finger associate gene regulation polypeptides, transcription activator-like effector nuclease (TALEN), transcription activator-like effector associated gene regulation polypeptides, meganuclease, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0029] In some embodiments, the system further comprises a chimeric polypeptide comprising the GMP, wherein the chimeric polypeptide is operable to release the GMP from the chimeric polypeptide in response to a stimulant, and wherein the released GMP is operable to regulate expression of the target polynucleotide in the gamma delta T cell.

[0030] In some embodiments, the system further comprises a chimeric polypeptide comprising the GMP and a nuclear localization domain, wherein the nuclear localization domain is operable to translocate the chimeric polypeptide to a nucleus of the gamma delta T cell in response to a stimulant, and wherein the translocated GMP is operable to regulate expression of the target polynucleotide in the gamma delta T cell.

[0031] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.Incorporation by Reference

[0032] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0033] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:Fig.1

[0034] schematically illustrates examples of vectors and their use for expression of a system comprising a chimeric receptor polypeptide and a chimeric adaptor polypeptide in a γδ T cell. The chimeric receptor polypeptide comprises a CAR, and the chimeric adaptor polypeptide comprises a GMP including a LAT-dCas9-effector. Upon exogenous expression the system in the γδ T cell and tumor antigen recognition by the CAR of the chimeric receptor polypeptide: (1) serpinB9, serpinB4, and / or c-Flip can be upregulated, thereby enhancing survival of the γδ T cell; and / or (2) CD39 can be downregulated, thereby maintaining proliferation and / or activation responsiveness of the γδ T cell to pyrophosphates, pyrophosphate byproducts, and / or other γδ TCR ligand(s). The pyrophosphates, pyrophosphate byproducts, and / or other γδ TCR ligand(s) can be administered after tumor-chimeric receptor polypeptide interaction;Fig.2

[0035] schematically illustrates examples of vectors and their use for pyrophosphate-induced expression of a system comprising a chimeric adaptor polypeptide in a γδ T cell. The chimeric adaptor polypeptide (1) comprises a GMP including a LAT-dCas9-effector, (2) is under the control of exogenous promoters such as LAG3 and / or Fas promoter, and (3) is compatible with a chimeric receptor polypeptide comprising a CAR. The exogenous LAG3 and / or Fas promoters may be identical to or a functional variant of endogenous LAG3 and / or Fas promoters of the γδ T cell. In these examples, the chimeric receptor polypeptide comprising the CAR is constitutively expressed in the γδ T cell. During one or more treatments with pyrophosphates, pyrophosphate byproducts, and / or other γδ TCR ligand(s), the LAG3 and / or Fas promoter (e.g., endogenous and / or exogenous) activities are induced and / or upregulated in the γδ T cell, thereby inducing expression of the chimeric adaptor polypeptide in the γδ T cell. The expressed chimeric adaptor polypeptide and chimeric receptor polypeptide work in conjunction to activate the dCas9-effector that upregulates expression of serpinB9, serpinB4, and / or c-Flip, thereby enhancing survival and activation state of the γδ T cell. Alternatively or in addition to, the expressed chimeric adaptor polypeptide and chimeric receptor polypeptide work in conjunction to activate the dCas9-effector that downregulates CD39 expression. In these examples, the γδ T cell can function with or without the chimeric antigen polypeptide; andFig.3

[0036] [Fig.3] schematically illustrates gene knock-in of a system comprising a GMP under the control of an endogenous promoter of γδ T cell. One or more endogenous genes under the control fo the endogenous promoter of the γδ T cell may be knocked-out. One or more gene editing endonucleases can be used for the gene knock-in and / or knock-out.

[0037] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0038] The practice of some methods disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0039] As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a transmembrane receptor” can include a plurality of transmembrane receptors.

[0040] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0041] As used herein, a “cell” can refer to a biological cell. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g. cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g. kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and etcetera. Sometimes a cell is not orginating from a natural organism (e.g. a cell can be a synthetically made, sometimes termed an artificial cell).

[0042] The terms “gamma delta cell,” “γδ cell,” and “GD cell,” as used interchangeably herein, can refer to a cell that has a distinctive T-cell receptor (TCR) comprised of one gamma (γ) TCR chain and one delta (δ) TCR chain. In some cases, the γδ cell can comprise a lymphocyte that has the γδ TCR, such as, for example, a γδ T cell or T lymphocyte. The terms “gamma delta T cell,” “γδ T cell,” and “GD T cell,” as used interchangeably herein, can refer to a T cell (T lymphocyte) that comprises the distinctive TCR comprised of the γ TCR chain and the δ TCR chain. The γδ T cell may comprise at least a portion of the γδ TCR expressed on the cell membrane. The γ TCR chain can be any one of Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, Vγ10, a functional variant thereof, or a combination thereof. The δ TCR chain can be any one of δ1, δ2, δ3, a functional variant thereof, or a combination thereof. In some examples, the γδ TCR may be Vγ9 / Vδ2 TCR, Vγ10 / Vδ2 TCR, and / or Vγ2 / Vδ2 TCR.

[0043] The γδ T cell can be activated by a γδ T cell activator. The term “γδ T cell activator,” as used herein, can refer to a molecule (natural or synthetic) which can activate or induce activation of a biological activity (e.g., migration, proliferation, synthesis of one or more polypeptides, etc.) of a γδ T cell. In some cases, the γδ T cell activator may be a ligand of the γδ TCRs and / or a different receptor. Alternatively or in addition to, the γδ T cell activator may be an endogenous ligand. The γδ T cell activator may be of various natures, such as a peptide, lipid, small molecule, etc. The γδ T cell activator may be a purified or otherwise artificially produced (e.g., by chemical synthesis, or by microbiological process). In some cases, the γδ T cell activator can comprise an antibody having a functional antigenic specificity as that of another γδ T cell activator.

[0044] The γδ T cell activator may be administered to a subject (e.g., a patient having a cancer or a tumor) in an amount and / or under conditions sufficient to increase the activity of the γδ T cells in the subject. Increasing the activity of the γδ T cells in the subject may increase cytokine secretion by the γδ T cells and / or increase the cytolytic activity of γδ T cells (e.g., the cytolytic activity against one or more tumor or cancer cells).

[0045] The γδ T cell activator can comprise a phosphoantigen. The phosphoantigen may comprise one or more phosphates. The phosphoantigen may comprise an organic pyrophosphate and / or a functional modification thereof. The phosphoantigen may be non-peptidic antigen. Alternatively or in addition to, the phosphoantigen may be linked (e.g., covalently or non-covalently) to one or more amino acids and / or one or more polynucleotides. Examples of such phosphate containing compounds acting as a phosphoantigen can include nucleotides (or polynucleotides), nucleotide analogs (e.g., 7-deaza-dGTP, 7-deaza-dATP, etc.), or pyrophosphonates (e.g., bisphosphonates, such as zolendronate or zolendronic acid).

[0046] The term "bisphosphonate," as used herein, can refer to any compound which is an analog of pyrophosphate (e.g., endogenous pyrophosphate). Examples of pyrophosphates (PP) and / or its functional derivatives may include, for example, isopentenyl pyrophosphate (IPP), dimethyl allyl pyrophosphate (DMAPP), farnesyl pyrophosphate (FPP), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), hexaprenyl pyrophosphate (HPP), neryl pyrophosphate (NPP), octaprenyl pyrophosphate (OPP), solanesyl pyrophosphate (SPP), a functional derivative thereof, or a combination thereof. Additional examples of the phosphoantigen may include BrHPP, BrIAPP, CBrHPP, EpoxPP, HDMAPP, CHDMAPP, NHDMAPP, H-angelylPP, H-tiglylPP, a functional derivative thereof, or a combination thereof. Examples of bisphosphonates may include aminobisphosphonates. Examples of bisphosphonates can include, but are not limited to zoledronic acid, risedronate, alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, piridronate, or pamidronate and functional analogues thereof.

[0047] The phosphoantigen may activate a γδ T cell, thereby, for example, to increase a biological activity (e.g., migration, proliferation, synthesis of one or more polypeptides, etc.) of the γδ T cell. The phosphoantigen may cause proliferation of the γδ T cell. The phosphoantigen may increase cytokine secretion from the γδ T cell. The phosphoantigen may increase the cytolytic activity of the γδ T cell. The phosphoantigen may activate a γδ T cell directly and / or indirectly. In some cases, the phosphoantigen (e.g., IPP) may directly interact with a receptor (e.g., Vγ9Vδ2 TCR) of the γδ T cell to activate the γδ T cell. In some cases, the phosphoantigen may interact with a target cell (e.g., a cancer / tumor cell) of the γδ T cell, and the target cell may metabolize the phosphoantigen into a γδ T cell activator. Subsequently, the target cell may secret and / or present the γδ T cell activator to activate a neighboring γδ T cell. Alternatively, the phosphoantigen may be uptaken by the target cell, and cause the target cell to release a γδ T cell activator. In an example, zolendronate may be an indirect γδ T cell activator. When zolendronate is uptaken by a cancer / tumor cell, zoledronate may inhibit FPP synthase and cause an accumulation of IPP in the cancer / tumor cell. The cancer / tumor cell may secrete or present the IPP on its surface. The IPP may be recognized by γδ T cells (e.g., Vγ9Vδ2 T cells) and activate the γδ T cells to respond to the cancer / tumor cell.

[0048] Further details of design and application of the phosphoantigen and modifications thereof, in the context of treating cancer or tumor, are disclosed in U.S. Patent Application No. 12 / 601,628, which is incorporated in its entirety herein by reference.

[0049] A biological function of the γδ T cell activator can be hindered and / or inhibited by an inhibitor of the γδ T cell activator. The inhibitor of the γδ T cell activator may bind a target (e.g., a target protein) of the γδ T cell activator to block the γδ T cell activator-target binding. Alternatively or in addition to, the inhibitor of the γδ T cell activator may bind a molecule (e.g., a polypeptide or a polynucleotide) involved in a signaling cascade of the γδ T cell activator-target binding, thereby to block the signaling activity of the γδ T cell activator. In another alternative, or additionally, the inhibitor of the γδ T cell activator may degrade or metabolize the γδ T cell activator and / or the target of the γδ T cell activator. The inhibitor of the γδ T cell activator may or may not be a transmembrane protein. The inhibitor of the γδ T cell activator may be an extracellular molecule and / or an intracellular molecule.

[0050] In some cases, the γδ T cell activator may be a phosphoantigen, and the inhibitor of the γδ T cell activator may be a phosphoantigen inhibitor. The phosphoantigen inhibitor may comprise a hydrolase. The term “hydrolase,” as used herein, can refer to a catalyst (e.g., an enzyme) that is configured to break a chemical bond. The hydrolase may divide a molecule into two or more smaller molecules. Examples of the hydrolase may comprise a lipase, phosphatase, glycosidase, peptidase, nucleosidase, nucleotidase, a functional modification thereof, or a combination thereof.

[0051] The phosphoantigen inhibitor (e.g., the hydrolase) hydrolase may comprise a nucleotidase. The nucleotidase may be configured to catalyze nucleotide hydrolysis, in the presence and / or absence of another enzyme. The nucleotidase may comprise a 3’-nucleotidase and a 5’-nucleotidase. The 5’nucleotidase may be configured to cleave off a phosphate from a 5' end of a phosphate-containing molecule (e.g., a nucleotide), while the 3’-nucleotidass may be configured to cleave off a phosphate from a 3' end of a phosphate-containing molecule (e.g., a nucleotide).

[0052] The phosphoantigen inhibitor (e.g., the hydrolase) may comprise an ectonucleotidase. In some cases, the ectonucleotidase may be expressed on a plasma membrane of a cell. At least a portion of an active site (e.g., a catalytic site) of the ectonucleotidase may be on the extracellular domain of such transmembrane ectonucleotidase. The phosphoantigen inhibitor may comprise a hydrolase (e.g., an ectonucleoside triphosphate diphosphohydrolase (NTPDase), such as NTPDase1 / ENTPD1 / CD39), a phosphodiesterase (e.g., a nucleotide pyrophosphatase / phosphodiesterase (NPP)), an alkaline phosphatase, a functional modification thereof, or a combination thereof. Examples of the NTPDase can include NTPDase1 / ENTPD1 / CD39, NTPDase2 / ENTPD2 / CD39L1, NTPDase3 / ENTPD3 / CD39L3, NTPDase4 / ENTPD4 / LALP70, NTPDase5 / ENTPD5 / CD39L4, NTPDase6 / ENTPD6 / CD39L2, NTPDase7 / ENTPD7 / LALP1, NTPDase8 / ENTPD8, a functional modification thereof, or a combination thereof.

[0053] The terms “cell death” or “death of a cell,” as used interchangeably herein, can refer to a process or event that causes a cell to cease and / or diminish normal metabolism in vivo or in vitro. Cell death can be induced by the cell itself (self-induced) or by another cell (e.g., another cell of the same type or a different type). In some cases, cell death can include, but are not limited to, programmed cell death (i.e., apoptosis), gradual death of the cells as occurs in diseased states (i.e., necrosis), and more immediate cell death such as toxicity (e.g., cytotoxicity, such as acute cytotoxicity). In some cases, cell apoptosis can be extrinsic (e.g., via signaling through a cell surface receptor, such as a death receptor) or intrinsic (e.g., via mitochondrial pathway).

[0054] In some cases, cell death can be caused, initiated, and / or prolonged by a compound. In some cases, such compound can be referred to as a death-inducing compound (e.g., a death-inducing polypeptide or protein). In some cases, the death-inducing compound can include an apoptosis-inducing compound and / or a cytotoxic compound. The death-inducing compound can comprise a small molecule (e.g., lipids, sugars, etc.), one or more nucleic acids (e.g., a single nucleic acid, polynucleotide, etc.), one or more amino acids (e.g., a single amino acid, peptides, proteins, polypeptides, etc.), and / or a combination thereof. The death-inducing compound can be endogenous and / or exogenous to the cell that is undergoing cell death. The death-inducing compound can be synthetic or natural. The death-inducing compound can be an intracellular protein, transmembrane protein (e.g., cell surface receptor), and / or extracellular protein. The death-inducing compound can be at least one receptor (e.g., an individual receptor or a complex of multiple receptors), at least one ligand for the at least one receptor, and / or a combination of the at least one receptor and the at least one ligand. In some cases, the death-inducing compound may activate a cell to express the same and / or a different compound that can cause or prolong death of the cell or another cell. Alternatively or in addition to, the death-inducing compound may activate promote death-related (e.g., apoptotic) intracellular signaling, such as, for example, cell apoptosis pathway. The death-inducing compound can comprise an apoptosis regulator, e.g., a pro-apoptotic regulator.

[0055] In some cases, cell death can be slowed down, stopped, and / or prevented by an inhibitor. Such inhibitor can be referred to as a cell death inhibitor. The cell death inhibitor can comprise a small molecule (e.g., lipids, sugars, etc.), one or more nucleic acids (e.g., a single nucleic acid, polynucleotide, etc.), one or more amino acids (e.g., a single amino acid, peptides, proteins, polypeptides, etc.), and / or a combination thereof. In some cases, the cell death inhibitor can be an inhibitor of a death-inducing compound (e.g., a death-inducing protein). The cell death inhibitor may prevent production (e.g., synthesis, metabolism, etc.) of the death-inducing compound. The cell death inhibitor may bind to, deform, and / or degrade the death-inducing compound, thereby preventing function of the death-inducing compound. In some cases, the cell death inhibitor may bind to or degrade an activator (e.g., a protease) of cell death, thereby slowing down, stopping, and / or preventing cell death.

[0056] The cell death inhibitor can comprise an apoptosis regulator, e.g., an anti-apoptotic regulator. In some cases, the cell death inhibitor can reduce or prevent formation of a death-inducing signaling complex (DISC) and subsequent activation of the procaspase and / or caspase cascade. In some cases, the cell death inhibitor can suppress tumor necrosis factor-α (TNF-α), Fas / Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, and / or apoptosis triggered by death-inducing agents (e.g., chemotherapy agents) in tumor / cancer cells. In some cases, the cell death inhibitor can include a cellular FLICE (FADD-like interleukin (IL)-1β-converting enzyme)-inhibitory protein (c-FLIP / CFLAR / CASH), or a functional variant thereof. The c-FLIP may be expressed as long (c-FLIP(L)), short (c-FLIP(S)), and / or c-FLIP(R) splice variants in cells (e.g., human cells).

[0057] In an example, the cell death inhibitor can be a Serpin superfamily polypeptide that (i) binds a protease (e.g., a cell death-inducing serine protease) and (ii) induces a large conformational change in the target protease to disrupt its active site. Examples of the Serpin superfamily may include, for example, Serpin B1, Serpin B2, Serpin B3, Serpin B4, Serpin B6, Serpin B8, Serpin B9alpha 1-antitrypsin, angiotensinogen, ovalbumin, antiplasmin, alpha 1-antichymotrypsin, thyroxine-binding protein, complement 1 inactivators, antithrombin III, heparin cofactor II, plasminogen inactivators, gene Y protein, placental plasminogen activator inhibitor, barley Z protein, a functional variation thereof, or a combination thereof. In some cases, one or more members of the Serpin family may be substrates rather than inhibitors of serine endopeptidases.

[0058] In some cases, the cell death inhibitor can upregulate one or more cytoprotective and / or pro-survival signaling proteins, e.g., Akt, ERK, NF-kB, etc. In an example, the cell death inhibitor amy include c-Flip or its functional variant thereof.

[0059] The death-inducing compound (e.g., the death-inducing protein) can include a tumor necrosis factor receptor superfamily (TNFRSF) and / or one or more respective ligands of the TNFRSF, such as, for example, one or more tumor necrosis factors (TNFs). The TNFRSF can be a transmembrane protein or a soluble protein (e.g., cleaved from transmembrane protein, e.g., TNFR1, or naturally lacking a transmembrane domain, e.g., DcR3). In some cases, the TNFRSF can include one or more TNFRSF adaptor proteins, including, for example, TNF receptor type 1-associated DEATH domain protein (TRADD), TNF receptor associated factors (TRAF), receptor-interacting protein kinases (RIP) (e.g., RIPK1, RIPK2, RIPK3, RIPK4, RIPK5, etc.) and Fas-associated protein with death domain (FADD / MORT1).

[0060] In some cases, the TNFRSF can comprise a death domain (DD). The death domain can be an interaction module (e.g., a protein interaction module) composed of one or more secondary structures (e.g., a bundle of six alpha-helices). The TNFRSF comprising the death domain can be referred to as a death receptor. Examples of the death receptor can comprise TNFR1, Fas receptor, DR4, and / or DR5.

[0061] The TNFRSF can include Tumor necrosis factor receptor 1 (i.e., CD120a), and its ligand can include TNF-alpha (cachectin). The TNFRSF can include Tumor necrosis factor receptor 2 (i.e., CD120b), and its ligand can include TNF-alpha (cachectin). The TNFRSF can include Lymphotoxin beta receptor (i.e., CD18), and its ligand can include Lymphotoxin beta (TNF-C). The TNFRSF can include OX40 (i.e., CD134), and its ligand can include OX40L. The TNFRSF can include CD40 (i.e., Bp50), and its ligand can include CD154. The TNFRSF can include Fas receptor (i.e., Apo-1, CD95), and its ligand can include Fas ligand (FasL). The TNFRSF can include Decoy receptor 3 (i.e., TR6, M68), and its ligand can include FasL, LIGHT, and / or TL1A. The TNFRSF can include CD27 (i.e., S152, Tp55), and its ligand can include CD70 and / or Siva. The TNFRSF can include CD30 (i.e., Ki-1), and its ligand can include CD153. The TNFRSF can include 4-1BB (i.e., CD137), and its ligand can include 4-1BB ligand. The TNFRSF can include Death receptor 4 (i.e., TRAILR1, Apo-2, CD261), and its ligand can include TRAIL. The TNFRSF can include Death receptor 5 (i.e., TRAILR2, CD262), and its ligand can include TRAIL. The TNFRSF can include Decoy receptor 1 (i.e., TRAILR3, LIT, TRID, CD263), and its ligand can include TRAIL. The TNFRSF can include Decoy receptor 2 (i.e., TRAILR4, TRUNDD, CD264), and its ligand can include TRAIL. The TNFRSF can include RANK (i.e., CD265), and its ligand can include RANKL. The TNFRSF can include Osteoprotegerin (i.e., OCIF, TR1), and its ligand can include RANKL. The TNFRSF can include TWEAK receptor (i.e., Fn14, CD266), and its ligand can include TWEAK. The TNFRSF can include TACI (i.e., IGAD2, CD267), and its ligand can include APRIL, BAFF, and / or CAMLG. The TNFRSF can include BAFF receptor (i.e., CD268), and its ligand can include BAFF. The TNFRSF can include Herpesvirus entry mediator (i.e., ATAR, TR2, CD270), and its ligand can include LIGHT. The TNFRSF can include Nerve growth factor receptor (i.e., p75NTR, CD271), and its ligand can include NGF, BDNF, NT-3, and / or NT-4. The TNFRSF can include B-cell maturation antigen (i.e., TNFRSF13A, CD269), and its ligand can include BAFF. The TNFRSF can include Glucocorticoid-induced TNFR-related (i.e., AITR, CD357), and its ligand can include GITR ligand. The TNFRSF can include TROY (i.e., TAJ, TRADE). The TNFRSF can include Death receptor 6 (i.e., CD358The TNFRSF can include Death receptor 3 (i.e., Apo-3, TRAMP, LARD, WS-1), and its ligand can include TL1A. The TNFRSF can include Ectodysplasin A2 receptor (i.e., XEDAR), and its ligand can include EDA-A2.

[0062] The death-inducing compound (e.g., the apoptosis-inducing compound) can comprise a granzyme, a perforin, a defensin, one or more components of a membrane attack complex (MAC) or a terminal complement complex (TCC), a Bcl-2 family member, cytochrome C, a caspase, a human leukocyte antigen (HLA) complex, or a combination thereof. The granzyme may be a protease (e.g., a serine protease) that induces programmed cell death (e.g., apoptosis) in a target cell. In some cases, the granzyme may activate one or more proteins (e.g., caspases, Bid, etc.) that are configured to execute cell death. Examples of the granzyme can include granzyme A, granzyme B, granzyme C, granzyme D, granzyme E, granzyme F, granzyme G, granzyme H, granzyme I, granzyme J, granzyme K, granzyme L, granzyme M, granzyme N, or a combination thereof. The perforin may be a pore-forming polypeptide that binds to a target cell’s plasma membrane and oligomerizes (e.g., in a calcium-dependent manner) to form pores on the target cell’s plasma membrane. Examples of the perforin can include perforin-1, perforin-2, or a combination thereof. The defensin may be a cationic polypeptides (e.g., a cysteine-rich cationic polypeptides) that bind and disrupt a target cell’s plasma membrane. Examples of the defensin can comprise alpha-defensins, beta-defensins, theta-defensins, or a combination thereof. The Bcl-2 family may comprise proteins that share Bcl-2 homology (BH) domains. Examples of the Bcl-2 family member can comprise Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Hrk, Bok, or a combination thereof. Examples of the caspase can include caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase- 11, caspase-12, caspase-13, caspase-14, a respective procaspase, or a combination thereof. The HLA complex may encode one or more major histocompatibility complex (MHC) proteins. The HLA complex can comprise HLA class I, HLA class II, HLA class III, a functional variant thereof, or a combination thereof. The HLA class I may comprise HLA-A, HLA-B, HLA-C, a functional variant thereof, or a combination thereof. The HLA class II may comprise HLA-DP, HLA-DM, HLA-DO, HLA-DQ, HLA-DR, a functional variant thereof, or a combination thereof. The HLA class III may comprise other immune components, such as complement components (e.g., C2, C4, factor B, etc.), cytokines (e.g., TNF- α), heat shot protein (hsp), a functional variant thereof, or a combination thereof.

[0063] In some cases, a family of compounds (e.g., a family of proteins) can comprise both pro-cell death (e.g., pro-apoptosis) and anti-cell death (e.g., anti-apoptosis) proteins. In an example, the Bcl-2 family can include both pro-apoptosis proteins (e.g., Bik, Bid, Bim, Bad, Bak, Bax, Bcl-Xs, Diva, Egl-1, Noxa, etc. ) and anti-apoptosis proteins (e.g., Bcl-2, BC1-XL, Mcl-1, CED-9, A1, Bfl-1, etc.).

[0064] The term “fratricide,” as used herein, can refer to an observation wherein an antigen and / or a receptor associated with a disease (e.g., a cancer or tumor) may be, in addition to diseased cells (e.g., cancer or tumor cells), present on one or effector cells that are configured to target the diseased cells (e.g., lymphocytes, such as T lymphocytes). In such a case, the effector cells may target each other in addition to, or alternative to, targeting the diseased cells. In some cases, an effector cell may target an additional effectir cell of a same type or different types, and induce a biological response in the additional effector cell, e.g., cell death. Such side-effect of inducing the biological response in the additional effector cell can also be referred to as fratricide. In some cases, the terms “fratricide,” “self-inflected injury,” and “a death of a cell by another cell of the same type” may be used interchangeably herein. In an example, the effector cell may be a γδ T cell, and the diseased cell may be a cancer / tumor cell.

[0065] In an example, the effector cell may be a γδ T cell, and the diseased cell may be a cancer / tumor cell. The cancer / tumor cell may express a cell surface receptor (e.g., Fas / CD95 / APO-1 / APT1), and the γδ T cell may express a respective cell surface ligand (e.g., FasL / CD95L / APTL) that is configured to bind and target the cell surface receptor of the cancer / tumor cell, leading to cell death (e.g., apoptosis) of the cancer / tumor cell. However, the γδ T cell may also express both the receptor and the ligand (e.g., Fas and FasL), which may result in interaction of one γδ T cell to another γδ T cell via the receptor-ligand binding, leading to cell death (e.g., apoptosis) of either or both of the γδ T cells.

[0066] The term “antigen,” as used herein, refers to a molecule or a fragment thereof (e.g., ligand) capable of being bound by a selective binding agent. As an example, an antigen can be a ligand that can be bound by a selective binding agent such as a receptor. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent such as an immunological protein (e.g., an antibody). An antigen can also refer to a molecule or fragment thereof capable of being used in an animal to produce antibodies capable of binding to that antigen.

[0067] The term “antibody,” as used herein, refers to a proteinaceous binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies), as well as variants thereof. Antibodies include, but are not limited to, immunoglobulins (Ig’s) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, etc.). A variant can refer to a functional derivative or fragment which retains the binding specificity (e.g., complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), minibodies, diabodies, and single-domain antibodies (“sdAb” or “nanobodies” or “camelids”). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity-matured antibodies. Examples of antibodies that have been engineered include Fc optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies).

[0068] The terms “Fc receptor” or “FcR,” as used herein, generally refers to a receptor, or any variant thereof, that can bind to the Fc region of an antibody. In certain embodiments, the FcR is one which binds an IgG antibody (a gamma receptor, Fcgamma R) and includes receptors of the Fcgamma RI (CD64), Fcgamma RII (CD32), and Fcgamma RIII (CD16) subclasses, including allelic variants and alternatively spliced forms of these receptors. Fcgamma RII receptors include Fcgamma RIIA (an “activating receptor”) and Fcgamma RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. The term “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus.

[0069] The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide can be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides can include but are not limited 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 can 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 available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade 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 available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can 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).

[0070] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three dimensional structure, and can perform any function, known or unknown. A polynucleotide can comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g. rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (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 can be interrupted by non-nucleotide components.

[0071] The term “gene,” as used herein, refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5’ and 3’ ends. In some uses, the term encompasses the transcribed sequences, including 5’ and 3’ untranslated regions (5’-UTR and 3’-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can refer to an “exogenous gene” or a non-native gene. A non-native gene can refer to a gene not normally found in the host organism but which is introduced into the host organism by gene transfer (e.g., transgene). A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and / or deletions (e.g., non-native sequence).

[0072] The terms “target polynucleotide” and “target nucleic acid,” as used herein, refer to a nucleic acid or polynucleotide which is targeted by an actuator moiety of the present disclosure. A target polynucleotide can be DNA (e.g., endogenous or exogenous). DNA can refer to template to generate mRNA transcripts and / or the various regulatory regions which regulate transcription of mRNA from a DNA template. A target polynucleotide can be a portion of a larger polynucleotide, for example a chromosome or a region of a chromosome. A target polynucleotide can refer to an extrachromosomal sequence (e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) or a region of an extrachromosomal sequence. A target polynucleotide can be RNA. RNA can be, for example, mRNA which can serve as template encoding for proteins. A target polynucleotide comprising RNA can include the various regulatory regions which regulate translation of protein from an mRNA template. A target polynucleotide can encode for a gene product (e.g., DNA encoding for an RNA transcript or RNA encoding for a protein product) or comprise a regulatory sequence which regulates expression of a gene product. In general, the term “target sequence” refers to a nucleic acid sequence on a single strand of a target nucleic acid. The target sequence can be a portion of a gene, a regulatory sequence, genomic DNA, cell free nucleic acid including cfDNA and / or cfRNA, cDNA, a fusion gene, and RNA including mRNA, miRNA, rRNA, and others. A target polynucleotide, when targeted by an actuator moiety, can result in altered gene expression and / or activity. A target polynucleotide, when targeted by an actuator moiety, can result in an edited nucleic acid sequence. A target nucleic acid can comprise a nucleic acid sequence that may not be related to any other sequence in a nucleic acid sample by a single nucleotide substitution. A target nucleic acid can comprise a nucleic acid sequence that may not be related to any other sequence in a nucleic acid sample by a 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 of the 5’ end of a target nucleic acid. In some embodiments, the substitution may not occur within 5, 10, 15, 20, 25, 30, 35 nucleotides of the 3’ end of a target nucleic acid.

[0073] The terms “transfection” or “transfected” refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0074] The term “expression” refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. “Up-regulated,” with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression in a wild-type state.

[0075] The term “vector,” as used herein, can refer to a nucleic acid molecule capable transferring or transporting a payload nucleic acid molecule. The payload nucleic acid molecule can be generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell gene (e.g., host cell DNA). Examples of a vector may include, but are not limited to, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0076] A “plasmid,” as used herein, generally refers to a non-viral expression vector, e.g., a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. A “viral vector,” as used herein, generally refers to a viral-derived nucleic acid that is capable of transporting another nucleic acid into a cell. A viral vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples for viral vectors include, but are not limited to Gamma-retroviral, Alpha-retroviral, Foamy viral, lentiviral, adenoviral, or adeno-aasociated viral vectors.

[0077] A vector of any of the embodiments of the present disclosure can comprise exogenous, endogenous, or heterologous control sequences such as promoters and / or enhancers. An “endogenous” control sequence is one which is naturally linked to a given gene in the genome. An “exogenous” control sequence is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A “heterologous” control sequence is an exogenous sequence that is from a different species than the cell being genetically manipulated. A “synthetic” control sequence may comprise elements of one more endogenous and / or exogenous sequences, and / or sequences determined in vitro or in silico that provide optimal promoter and / or enhancer activity for the particular gene therapy.

[0078] The terms “complement,” “complements,” “complementary,” and “complementarity,” as used herein, generally refer to a sequence that is fully complementary to and hybridizable to the given sequence. In some cases, a sequence hybridized with a given nucleic acid is referred to as the “complement” or “reverse-complement” of the given molecule if its sequence of bases over a given region is capable of complementarily binding those of its binding partner, such that, for example, A-T, A-U, G-C, and G-U base pairs are formed. In general, a first sequence that is hybridizable to a second sequence is specifically or selectively hybridizable to the second sequence, such that hybridization to the second sequence or set of second sequences is preferred (e.g. thermodynamically more stable under a given set of conditions, such as stringent conditions commonly used in the art) to hybridization with non-target sequences during a hybridization reaction. Typically, hybridizable sequences share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-100% complementarity, 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. Sequence identity, such as for the purpose of assessing percent complementarity, can be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g. the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally with default settings), the BLAST algorithm (see e.g. the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see e.g. the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, optionally with default settings). Optimal alignment can be assessed using any suitable parameters of a chosen algorithm, including default parameters.

[0079] Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids can mean that the two nucleic acids can form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing. Substantial or sufficient complementary can mean that a sequence in one strand is not completely and / or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations to predict the Tm of hybridized strands, or by empirical determination of Tm by using routine methods.

[0080] The term “regulating” with reference to expression or activity, as used herein, refers to altering the level of expression or activity. Regulation can occur at the transcriptional level, post-transcriptional level, translational level, and / or post-translational level.

[0081] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and / or tertiary structure (e.g., domains). The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids,” as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues. Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. Amino acid analogues can refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids.

[0082] The term “variant,” when used herein with reference to a polypeptide, refers to a polypeptide related, but not identical, to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Variants include polypeptides comprising 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 the wild type polypeptide and fragments of the wild type polypeptide.

[0083] The term “percent (%) identity,” as used herein, refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment, for purposes of determining percent identity, can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Percent identity of two sequences can be calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides in the same position of 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.

[0084] The term “gene modulating polypeptide” or “GMP,” as used herein, refers to a polypeptide comprising at least an actuator moiety capable of regulating expression or activity of a gene and / or editing a nucleic acid sequence. A GMP can comprise additional peptide sequences which are not directly involved in modulating gene expression, for example targeting sequences, polypeptide folding domains, etc.

[0085] The term “actuator moiety,” as used herein, refers to a moiety which can regulate expression or activity of a gene and / or edit a nucleic acid sequence, whether exogenous or endogenous. An actuator moiety can regulate expression of a gene at the transcriptional level, post-transcriptional level, translational level, and / or post-translation level. An actuator moiety can regulate gene expression at the transcription level, for example, by regulating the production of mRNA from DNA, such as chromosomal DNA or cDNA. In some embodiments, an actuator moiety 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. An actuator moiety can itself bind to DNA and regulate transcription by physical obstruction, for example preventing proteins such as RNA polymerase and other associated proteins from assembling on a DNA template. An actuator moiety can regulate expression of a gene at the translation level, for example, by regulating the production of protein from mRNA template. In some embodiments, an actuator moiety regulates gene expression at a post-transcriptional level by affecting the stability of an mRNA transcript. In some embodiments, an actuator moiety regulates gene expression at a post-translational level by altering the polypeptide modification, such as glycosylation of newly synthesized protein. In some embodiments, an actuator moiety regulates expression of a gene by editing a nucleic acid sequence (e.g., a region of a genome). In some embodiments, an actuator moiety regulates expression of a gene by editing an mRNA template. Editing a nucleic acid sequence can, in some cases, alter the underlying template for gene expression.

[0086] A Cas protein referred to herein can be a type of protein or polypeptide. A Cas protein can refer to a nuclease. A Cas protein can refer to an endoribonuclease. A Cas protein can refer to any modified (e.g., shortened, mutated, lengthened) polypeptide sequence or homologue of the Cas protein. A Cas protein can be codon optimized. A Cas protein can be a codon-optimized homologue of a Cas protein. A Cas protein can be enzymatically inactive, partially active, constitutively active, fully active, inducible active and / or more active, (e.g. more than the wild type homologue of the protein or polypeptide.). A Cas protein can be Cas9. A Cas protein can be Cpf1. A Cas protein can be C2c2. A Cas protein can be Cas13a. A Cas protein (e.g., variant, mutated, enzymatically inactive and / or conditionally enzymatically inactive site-directed polypeptide) can bind to a target nucleic acid. A Cas protein (e.g., variant, mutated, enzymatically inactive and / or conditionally enzymatically inactive endoribonuclease) can bind to a target RNA or DNA.

[0087] The terms “deactivated nuclease” or “dead nuclease,” as used interchangeably herein, can refer to a nuclease, wherein the function of the nuclease is entirely or partially deactivated. In a case where the nuclease is a Cas protein, a deactivated / dead Cas nuclease may be referred to as “dCas” (e.g., dCas9).

[0088] The term “crRNA,” as used herein, can generally refer to a nucleic acid with 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., a crRNA from S. pyogenes, S. aureus, etc.). crRNA can generally refer to a nucleic acid with 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., a crRNA from S. pyogenes, S. aureus, etc.). crRNA can refer to a modified form of a crRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera. A crRNA can be a nucleic acid having at least about 60% sequence identity to a wild type exemplary crRNA (e.g., a crRNA from S. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. For example, a crRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100 % identical to a wild type exemplary crRNA sequence (e.g., a crRNA from S. pyogenes S. aureus, etc) over a stretch of at least 6 contiguous nucleotides.

[0089] The term “tracrRNA,” as used herein, can generally refer to a nucleic acid with 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., a tracrRNA from S. pyogenes S. aureus, etc). tracrRNA can refer to a nucleic acid with 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., a tracrRNA from S. pyogenes S. aureus, etc). tracrRNA can refer to a modified form of a tracrRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera. A tracrRNA can refer to a nucleic acid that can be at least about 60% identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. For example, a tracrRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100 % identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides.

[0090] As used herein, a “guide nucleic acid” can refer to a nucleic acid that can hybridize to another nucleic acid. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. The guide nucleic acid can be programmed to bind to a sequence of nucleic acid site-specifically. The nucleic acid to be targeted, or the target nucleic acid, can comprise nucleotides. The guide nucleic acid can comprise nucleotides. A portion of the target nucleic acid can 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 can be called the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore may not be complementary to the guide nucleic acid can be called noncomplementary strand. A guide nucleic acid can comprise a polynucleotide chain and can be called a “single guide nucleic acid.” A guide nucleic acid can comprise two polynucleotide chains and can be called a “double guide nucleic acid.” If not otherwise specified, the term “guide nucleic acid” can be inclusive, referring to both single guide nucleic acids and double guide nucleic acids.

[0091] A guide nucleic acid can comprise a segment that can be referred to as a “nucleic acid-targeting segment” or a “nucleic acid-targeting sequence.” A nucleic acid-targeting segment can comprise a sub-segment that can be referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment”.

[0092] The terms “cleavage recognition sequence” or “cleavage recognition site.” as used herein, with reference to peptides, refers to a site of a peptide at which a chemical bond, such as a peptide bond or disulfide bond, can be cleaved. Cleavage can be achieved by various methods. Cleavage of peptide bonds can be facilitated, for example, by an enzyme such as a protease.

[0093] The term “targeting sequence,” as used herein, refers to a nucleotide sequence and the corresponding amino acid sequence which encodes a targeting polypeptide which mediates the localization (or retention) of a protein to a sub-cellular location, e.g., plasma membrane or membrane of a given organelle, nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi, chloroplast, apoplast, peroxisome or other organelle. For example, a targeting sequence can direct a protein (e.g., a GMP) to a nucleus utilizing a nuclear localization signal (NLS); outside of a nucleus of a cell, for example to the cytoplasm, utilizing a nuclear export signal (NES); mitochondria utilizing a mitochondrial targeting signal; the endoplasmic reticulum (ER) utilizing an ER-retention signal; a peroxisome utilizing a peroxisomal targeting signal; plasma membrane utilizing a membrane localization signal; or combinations thereof.

[0094] As used herein, “fusion” can refer to a protein and / or nucleic acid comprising one or more non-native sequences (e.g., moieties). A fusion can comprise one or more of the same non-native sequences. A fusion can comprise one or more of different non-native sequences. A fusion can be a chimera. A fusion can comprise a nucleic acid affinity tag. A fusion can comprise a barcode. A fusion can comprise a peptide affinity tag. A fusion can provide for subcellular localization of the site-directed polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an endoplasmic reticulum (ER) retention signal, and the like). A fusion can provide a non-native sequence (e.g., affinity tag) that can be used to track or purify. A fusion can be a small molecule such as biotin or a dye such as Alexa fluor dyes, Cyanine3 dye, Cyanine5 dye.

[0095] A fusion can refer to any protein with a functional effect. For example, a fusion protein can comprise methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodelling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, or demyristoylation activity. An effector protein can modify a genomic locus. A fusion protein can be a fusion in a Cas protein. A fusion protein can be a non-native sequence in a Cas protein.

[0096] Thus, in some embodiments, an actuator moiety may comprise a fusion polypeptide. The fusion polypeptide may comprise two or more fragments that each confer at least one activity selected from the group consisting of: nuclease activity, methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodelling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, and demyristoylation activity.

[0097] In some cases, the actuator moiety may comprise a fusion polypeptide, and the fusion polypeptide may comprise two fragments that each confer (i) a nuclease activity (or modifications thereof, e.g., Cas activity or reduced Cas activity) and (ii) a hydrolase activity (e.g., cytidine deaminase activity). In some examples, the actuator moiety comprising the fusion polypeptide may be a nucleobase editor. The term “nucleobase editor” or “base editor,” as used interchangeably herein, can refer to an agent comprising a polypeptide that is capable of making a modification to a nucleobase (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some cases, the base editor (e.g., deaminase) may be capable of deaminating a base within a nucleic acid. In some cases, the base editor may be capable of deaminating a base within a DNA molecule. In some cases, the base editor may be capable of deaminating a cytosine (C) in DNA. In some cases, the base editor may be capable of excising a base within a DNA molecule. In some cases, the base editor may be capable of excising an adenine, guanine, cytosine, thymine or uracil within a nucleic acid (e.g., DNA or RNA) molecule. In some cases, the base editor may be a fusion protein comprising a programmable nucleic acid binding protein (e.g., a nuclease as provided in the present disclosure, such as Cas or dCas) fused to a cytidine deaminase. In some cases, the base editor may be fused to a uracil binding protein (UBP), such as a uracil DNA glycosylase (UDG). In some cases, the base editor may be fused to a nucleic acid polymerase (NAP) domain. In some cases, the NAP domain may be a translesion DNA polymerase. In some cases, the base editor may comprise a programmable nucleic acid binding protein, a cytidine deaminase, and a UBP (e.g., UDG). In some cases, the base editor may comprise a programmable nucleic acid binding protein, a cytidine deaminase, and a nucleic acid polymerase (e.g., a translesion DNA polymerase). In some cases, the base editor comprises a programmable nucleic acid binding protein, a cytidine deaminase, a UBP (e.g., UDG), and a nucleic acid polymerase (e.g., a translesion DNA polymerase).

[0098] In some examples, the base editor may introduce one or more transition mutations (e.g., C to T, G to A, A to G, or T to C) without requiring double stranded breaks in many cell types and organisms, including mammals.

[0099] In some cases, the actuator moiety may comprise a fusion polypeptide, and the fusion polypeptide may comprise two fragments that each confer (i) a nuclease activity (or modifications thereof, e.g., Cas activity or reduced Cas activity) and (ii) a polymerase activity (e.g., DNA or RNA polymerase activity). As used here, the term “polymerase” can refer to a polypeptide that is able to catalyze addition of one or more nucleotides or analogs thereof (e.g., natural or synthetic nucleotides) to a nucleic acid molecule in a template dependent manner. In an example, an DNA insertion sequence encoded by a template RNA molecule may be added to a 3’-end of a target DNA molecule by action of a polymerase (e.g., reverse transcriptase). Examples of a polymerase may include, but are not limited to, (i) polymerases isolated from Thermus aquaticus, Thermus thermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritima, (ii) E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, (iii) T7, T3, SP6 RNA polymerases, and (iv) AMV, M-MLV and HIV reverse transcriptase.

[0100] In some examples, the actuator moiety may comprise a fusion polypeptide, and the fusion polypeptide may comprise (i) a Cas protein or modifications thereof (e.g., deactivated Cas or Cas nickase) that is coupled (e.g., covalently coupled) to (ii) a reverse transcriptase. The Cas protein may be configured to only nick one strand of a target nucleic acid (e.g., one strand of a double stranded DNA molecule). The reverse transcriptase may be configured to generate a new nucleic acid sequence (e.g., a new DNA polynucleotide stand) by coping from a nucleic acid template (e.g., a RNA template). Such actuator moiety may function in conjunction with an engineered gRNA (i.e. prime editing gRNA, or pegRNA). The pegRNA may comprise a plurality of segments. The plurality of segments may comprise (i) a nucleic acid-targeting segment (e.g., spacer region of a gRNA), (ii) a Cas protein-binding segment (e.g., as two separate crRNA and tracrRNA molecules, or as a single scaffold molecule), (iii) a reverse transcriptase template segment encoding a desired nucleic acid edit, and (iv) a binding segment that binds to the nicked strand of the target nucleic acid. In an example, the reverse transcriptase template segment of the pegRNA may encode a desired DNA sequence. Alternatively, the reverse transcriptase template segment of the pegRNA may encode a complimentary DNA sequence having complementarity to a desired DNA sequence, such that when the complimentary DNA sequence is introduced to a first strand of the target gene, the desired DNA sequence may be subsequently added to a second and opposite strand of the target gene (e.g., via one or more DNA repair mechanisms).

[0101] In an example, a fusion complex of (i) an actuator moiety comprising the Cas protein and the reverse transcriptase and (ii) a pegRNA may introduce one or more transition mutations (e.g., C to T, G to A, A to G, or T to C) without requiring double stranded breaks in many cell types and organisms, including mammals. Alternatively or in addition to, such fusion complex may perform one or more transversion mutations (e.g., C to A, C to G, G to C, G to T, A to C, A to T, T to A, and T to G), e.g., for T-A to A-T mutation needed to correct sickle cell disease, without requiring double stranded breaks in many cell types and organisms, including mammals. Alternatively or in addition to, such fusion complex may introduce an indel (e.g., an insertion and / or deletion) to the target nucleic acid or target gene. The fusion complex may introduce an addition of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or more nucleotides to the target gene. The fusion complex may introduce an addition of at most 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide to the target gene. The fusion complex may introduce a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or more nucleotides to the target gene. The fusion complex may introduce a deletion of at most 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide to the target gene. The fusion complex may or may not introduce a frameshift in the gene.

[0102] In some cases, an engineered gRNA (e.g., a pegRNA) may be coupled (e.g., covalently or non-covalently coupled) to a moiety (e.g., a polypeptide molecule) that confers at least one activity selected from the group consisting of: nuclease activity, methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodelling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, and demyristoylation activity. In an example, a pegRNA may be operatively coupled to a nucleic acid polymerase (e.g., a reverse transcriptase) by action of the nucleic acid polymerase recognizing and non-covalently binding to a fragment (e.g., a loop structure) of the pegRNA. In such a case, the nucleic acid polymerase may or may not be covalently coupled to a nuclease (e.g., a Cas protein or a dCas protein).

[0103] As used herein, the “non-native” can refer to a nucleic acid or polypeptide sequence that is not found in a native nucleic acid or protein. Non-native can refer to affinity tags. Non-native can refer to fusions. Non-native can refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and / or deletions. A non-native sequence may exhibit and / or encode for an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.) that can also be exhibited by the nucleic acid and / or polypeptide sequence to which the non-native sequence is fused. A non-native nucleic acid or polypeptide sequence may be linked to a naturally-occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid and / or polypeptide sequence encoding a chimeric nucleic acid and / or polypeptide.

[0104] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0105] The terms “treatment” and “treating,” as used herein, refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. For example, a treatment can comprise administering a system or cell population disclosed herein. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

[0106] The term “effective amount” or “therapeutically effective amount” refers to the quantity of a composition, for example a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells) comprising a system of the present disclosure, that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

[0107] The term “chimeric antigen receptor” or alternatively a “CAR” may be used herein to refer to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as “an intracellular or intrinsic signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule. In some cases, the stimulatory molecule may be the zeta chain associated with the T cell receptor complex. In some cases, the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule. In some cases, the costimulatory molecule may comprise 4-1BB (i.e., CD137), CD27, and / or CD28. In one aspect the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane. In some cases, the CAR may further comprise a GMP, as described in the present disclosure.

[0108] The CAR, as used herein, may be a first-, second-, third-, or fourth-generation CAR system, a functional variant thereof, or any combination thereof. First- generation CARs (e.g., CD19R or CD19CAR) include an antigen binding domain with specificity for a particular antigen (e.g., an antibody or antigen-binding fragment thereof such as an scFv, a Fab fragment, a VHH domain, or a VH domain of a heavy-chain only antibody), a transmembrane domain derived from an adaptive immune receptor (e.g., the transmembrane domain from the CD28 receptor), and a signaling domain derived from an adaptive immune receptor (e.g., one or more (e.g., three) ITAM domains derived from the intracellular region of the CD3 ζ receptor or FcεRIγ). Second-generation CARs modify the first-generation CAR by addition of a co-stimulatory domain to the intracellular signaling domain portion of the CAR (e.g., derived from co-stimulatory receptors that act alongside T-cell receptors such as CD28, CD137 / 4-1BB, and CD134 / OX40), which abrogates the need for administration of a co-factor (e.g., IL-2) alongside a first-generation CAR. Third-generation CARs add multiple co-stimulatory domains to the intracellular signaling domain portion of the CAR (e.g., CD3ζ-CD28-OX40, or CD3ζ-CD28-41BB). Fourth-generation CARs modify second- or third-generation CARs by the addition of an activating cytokine (e.g., IL-12, IL-23, or IL-27) to the intracellular signaling portion of the CAR (e.g., between one or more of the costimulatory domains and the CD3ζ ITAM domain) or under the control of a CAR-induced promoter (e.g., the NFAT / IL-2 minimal promoter).

[0109] The term “conditionally enhancing expression” refers to expression of a polypeptide sequence (e.g., an endogenous polypeptide sequence, a chimeric polypeptide sequence, etc.) that occurs subject to one or more requirements rather than continually. Upon increasing, maintaining, and / or decreasing of the expression of the polypeptide sequence in a cell (e.g., an immune cell, a stem cell, etc.), the cell may be contacted with a stimulant (e.g., a ligand or an antigen) to initiate the conditional enhancement of expressing the polypeptide sequence in the cell. In some cases, the cell may not have begun expression the polypeptide sequence prior to at least a first contact with the stimulant. In some cases, the cell may have begun expression of the polypeptide sequence, and after the expression of the polypeptides sequence is plateaued out or decreased, the cell may be contacted with the stimulant to initiate the conditional enhancement of expressing the polypeptide sequence in the cell. The cell may be ex vivo (e.g., in vitro) or in vivo (e.g., administered to a subject). In some cases, the conditional enhancement of expressing the polypeptide sequence in the cell may be temporary or permanent. In some cases, the cell may be contacted with the stimulant at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. In some cases, the cell may be contacted with the stimulant at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time.

[0110] In some cases, a continual expression of a polypeptide sequence (e.g., Cas or dCas) may have an off-target effect on a host cell, e.g., cell cytotoxicity. In such a case, conditionally promoting and / or enhancing expression of the polypeptide sequence (e.g., via contacting the cell with a stimulant) may be beneficial, at least for a reason that cell cytotoxicity may be controlled (e.g., diminished or prevented). Alternatively or in addition to, conditionally promoting and / or enhancing expression of the polypeptide sequence may be beneficial in that a continual metabolic burden of the host cell to synthesize the polypeptide sequence can be controlled (e.g., diminished or prevented). Without wishing to be bound by theory, controlling the metabolic burden of the host cell can improve viability, proliferation, and / or function of the host cell.

[0111] The terms “operatively linked” and “under the operative control” may be used herein interchangeably to refer to two sequences (e.g., two nucleotide sequences, two polypeptide sequences, a nucleotide sequence and a polypeptide sequence) that are either physically linked or are functionally linked so that at least one of the sequences can act on the other sequence. In some cases, a gene regulatory sequence (e.g., a promoter) and an additional nucleotide sequence (e.g., a gene of interest, a transgene, etc.), are operatively linked if the expression (e.g., transcription and translation) of the additional nucleotide sequence can be governed by the gene regulatory sequence. Accordingly, the gene regulatory sequence and the additional nucleotide sequence to be expressed may be physically linked to each other, e.g., by inserting the gene regulatory sequence at or adjacent to a 5′ end of the additional nucleotide sequence to be expressed. Alternatively, the gene regulatory sequence and the additional nucleotide sequence to be expressed may be merely in physical proximity so that the gene regulatory sequence is functionally linked to the additional nucleotide sequence to be expressed. In some cases, the two sequences that are operatively linked may be separated by at least 5, 10, 20, 40, 60, 80, 100, 300, 500, 1500 bp, or more. In some cases, the two sequences that are operatively linked may be separated by at most 1500, 500, 300, 100, 80, 60, 40, 20, 10, 5 bp, or less.

[0112] The term “promoter” may be used herein to refer to the regulatory DNA region which controls transcription or expression of a gene and which can be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated. A promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene transcription. A ‘basal promoter’, also referred to as a ‘core promoter’, may generally refer to a promoter that contains all the basic necessary elements to promote transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters typically, though not necessarily, contain a TATA-box and / or a CAAT box.

[0113] In one aspect, the present disclosure provides a method for regulating an activity of a gamma-delta (γδ) cell, such as, for example, a γδ T cell. In some cases, the method for regulating an activity of the γδ T cell can comprise administering to the γδ T cell a gene modulating polypeptide (GMP) comprising an actuator moiety configured to regulate expression of a target polynucleotide in the γδ T cell, wherein the target polynucleotide can encode a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolase.

[0114] In some cases, the administration of the GMP to the γδ T cell can increase or decrease the activity of the γδ T cell by at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 5-fold, 10-fold, 100-fold, 1000-fold, or more in comparison to an absence of the administration of the GMP to the γδ T cell. In some cases, the administration of the GMP to the γδ T cell can increase or decrease the activity of the γδ T cell by at most 1000-fold, 100-fold, 10-fold, 5-fold, 4.0-fold, 3.5-fold, 3.0-fold, 2.5-fold, 2.0-fold, 1.9-fold, 1.8-fold, 1.7-fold, 1.6-fold, 1.5-fold, 1.4-fold, 1.3-fold, 1.2-fold, 1.1-fold, or less in comparison to an absence of the administration of the GMP to the γδ T cell.

[0115] In some cases, the administration of the GMP to the γδ T cell can increase or decrease the activity of the γδ T cell for at least 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, 1 year, or more in comparison to an absence of the administration of the GMP to the γδ T cell. In some cases, the administration of the GMP to the γδ T cell can increase or decrease the activity of the γδ T cell for at most 1 year, 6 months, 4 months, 2 months, 4 weeks, 3 weeks, 2 weeks, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 10 minutes, 5 minutes, 1 minute, or less in comparison to an absence of the administration of the GMP to the γδ T cell.

[0116] The regulating the expression of the target polypeptide in the γδ T cell can comprise decreasing, increasing, inhibiting, and / or prolonging the expression of the target polypeptide in the γδ T cell. The regulating the expression of the target polypeptide in the γδ T cell can be decreasing the expression of the target polypeptide in the γδ T cell. The regulating the expression of the target polypeptide in the γδ T cell can be increasing the expression of the target polypeptide in the γδ T cell.

[0117] The regulating the expression of the target polypeptide in the γδ T cell may directly and / or indirectly allow the regulating the activity of the γδ T cell. In some cases, the regulating the activity of the γδ T cell can comprise decreasing and / or inhibiting fratricide of one or more γδ T cells, self-inflicted injury of the γδ T cell, death of the γδ T cell by another γδ T cell, and / or death of another γδ T cell by the γδ T cell, thereby improving (directly and / or indirectly) viability, proliferation, and / or function of the cell.

[0118] In some case, the target polynucleotide can encode a polypeptide, and the polypeptide can comprise the death-inducing protein. The polypeptide may be the death-inducing protein. The death-inducing protein may comprise one or more of the death-inducing compounds provided herein in the present disclosure. Examples of the death-inducing protein can comprise Fas, Fas ligand, granzyme B, granzyme M, and / or perforin.

[0119] In some cases, a γδ T cell comprising the GMP may be operable to target a diseased cell (e.g., a cancer / tumor cell) and induce cell death (e.g., apoptosis) of the targeted diseased cell. The diseased cell may express a cell surface receptor (e.g., Fas / CD95 / APO-1 / APT1), and the γδ T cell may express a respective cell surface ligand (e.g., FasL / CD95L / APTL) that is operable to bind and target the cell surface receptor of the diseased cell.

[0120] In some cases, upon the γδ T cell-diseased cell binding (e.g., via FasL-Fas binding), the γδ T cell may induce one or more intracellular signaling cascades (e.g., one or more apoptosis pathways) to induce cell death of the diseased cell. In one example, the intracellular signaling cascade(s) may be induced via a cellular receptor (e.g., TNFR, Fas, TRAILR, etc.) of the diseased cell. Alternatively or in addition to, the γδ T cell may release one or more first compounds (e.g., proteases, e.g., a granzyme), which first compound(s) are operable to enter into the cytosol of the diseased cell (e.g., via an endosomal uptake and escape) and activate one or more death-inducing compounds (e.g., caspases) inside the diseased cell, thereby inducing diseased cell death. In another alternative, or addition, the γδ T cell may release one or more second compounds, which second compound(s) are operable to bind to the plasma membrane of the diseased cell and form one or more pores on the plasma membrane, thereby lysing and killing the diseased cell. In some cases, the γδ T cell may express both the first compound(s) and the second compound(s), and the second compound(s) (e.g., perforin) may form the pore(s) on the diseased cell membrane to allow passage (e.g., diffusion) of the first compound(s) (e.g., granzyme) into the diseased cell. The first compound(s) and the second compound(s) may be the same or different. The first compound(s) and / or the second compound(s) may be expressed and / or released by the γδ T cell prior to, during, and subsequent to the binding of the γδ T cell and the diseased cell (e.g., via FasL-Fas binding).

[0121] In some cases, the γδ T cells can express both the diseased cell-targeting cell surface ligand (e.g., FasL / CD95L / APTL), as well as its respective cell surface receptor (e.g., Fas / CD95 / APO-1 / APT1). Thus, the γδ T cells may be operable to target and induce cell death of the diseased cell and / or cell death (e.g., fratricide) of other γδ T cells. In some cases, the cell surface ligand of a γδ T cell may bind the respective cell surface receptor of the same γδ T cell and promote self-induced death of the γδ T cell. Thus, the GMP comprising the actuator moiety may be configured to regulate (e.g., decrease or inhibit) expression of the death-inducing protein (e.g., Fas / CD95 / APO-1 / APT1) in γδ T cells, thereby decreasing or inhibiting fratricide of the γδ T cells, while maintaining or increasing their cytotoxic (and / or cytolytic) activity against the diseased cell.

[0122] In some cases, the γδ T cells can express and release death-inducing compounds operable to induce death of the diseased cells. However, the death-inducing compounds may also induce death of the γδ T cells that are releasing such compounds. In an example, a concentration of the death-inducing compounds in the extracellular environment of the γδ T cells and the diseased cells may be sufficient to diffuse into both the diseased cells and the γδ T cells. Thus, the GMP comprising the actuator moiety may be configured to regulate (e.g., decrease or inhibit) expression of the death-inducing protein (e.g., granzyme, perforin, etc.) in γδ T cells, thereby decreasing or inhibiting fratricide of the γδ T cells.

[0123] In some case, the target polynucleotide can encode a polypeptide, and the polypeptide can comprise the inhibitor of the death-inducing protein. The polypeptide may be the inhibitor of the death-inducing protein. The inhibitor of the death-inducing protein may comprise one or more of the cell death inhibitors provided herein in the present disclosure (e.g., the Serpin superfamily, including Serpin B1 and / or Serpin B4). In some cases, the inhibitor of the death-inducing protein may prevent production of, deform, and / or degrade one or more death-inducing compounds. In such a case, an expression of the inhibitor of the death-inducing protein by a γδ T cell may protect the γδ T cell from cell death (e.g., apoptosis) by one or more death-inducing proteins expressed by the same or different γδ T cell. Thus, the GMP comprising the actuator moiety may be configured to regulate (e.g., induce or increase) expression of the inhibitor of the death-inducing protein in γδ T cells, thereby decreasing or inhibiting fratricide of the γδ T cells.

[0124] In some cases, the regulating the activity of the γδ T cell can comprise inducing and / or prolonging activation of the γδ T cell. The activation of the γδ T cell can comprise activation of one or more biological activities (e.g., migration, proliferation, synthesis of one or more polypeptides, etc.) as provided herein in the present disclosure. The inducing / prolonging activation of the γδ T cell may comprise inducing and / or prolonging expression of (1) one or more γδ T cell activators and / or (2) one or more targets of the γδ T cell activator(s). In an example, the γδ T cell activator(s) may be a ligand, and the target(s) of the γδ T cell activator(s) may be a different polypeptide (e.g., a receptor, an intracellular protein, etc.). Alternatively or in addition to, the inducing / prolonging activation of the γδ T cell may comprise (1) decreasing and / or inhibiting expression of one or more inhibitors of the γδ T cell activator and / or (2) degrading and / or metabolizing the inhibitor(s) of the γδ T cell activator.

[0125] In some cases, the γδ T cell activator may be provided (e.g., synthesized) by the γδ T cell or a different type of cell (e.g., other immune cells or non-immune cells). In some cases, the γδ T cell may be exogenous to the subject (e.g., a patient) comprising the γδ T cell.

[0126] In some case, the target polynucleotide can encode a polypeptide, and the polypeptide can comprise the hydrolase. The polypeptide may be the hydrolase. The hydrolase may comprise any of the hydrolase provided herein in the present disclosure. In some cases, the γδ T cell activator may be a phosphoantigen, and the hydrolase may comprise any of the phosphoantigen inhibitor provided herein in the present disclosure. In an example, the hydrolase may be an ectonucleoside triphosphate diphosphohydrolase (e.g., NTPDase1 / ENTPD1 / CD39), which is operable to cleave one or more phosphates in the phosphoantigen, thereby decreasing or inhibiting the phosphoantigen-induced activation of the γδ T cell. As a result, the hydrolase, when it is not disrupted by the GMP of the present disclosure, may indirectly decrease activity (e.g., cytotoxic activity) of the γδ T cell against a diseased cell, such as a cancer / tumor cell.

[0127] Thus, in some cases, the GMP comprising the actuator moiety may be configured to induce and / or prolong expression of (1) one or more γδ T cell activators, (2) one or more substrates or pro-forms (e.g., prodrugs) of the γδ T cell activator(s), and / or (3) one or more regulators (e.g., catalysts, such as enzymes) of the synthesis of the γδ T cell activator(s).

[0128] In some cases, the GMP comprising the actuator moiety may be configured to induce and / or prolong expression of (1) one or more targets of the γδ T cell activator(s), (2) one or more substrates or pro-forms of the γδ T cell activator(s), and / or (3) one or more regulators (e.g., catalysts, such as enzymes) of the synthesis of the target(s) of the γδ T cell activator(s).

[0129] In some cases, the GMP comprising the actuator moiety may be configured to decrease and / or inhibit expression of one or more inhibitors (e.g., enzymes) of the γδ T cell activator(s). In some cases, the GMP comprising the actuator moiety may be configured to degrade and / or metabolize the inhibitor(s) of the γδ T cell activator(s).

[0130] In some cases, the regulating the activity of the γδ T cell can comprise reducing and / or preventing activation of the γδ T cell.

[0131] The GMP may comprise an actuator moiety that regulates expression of a target polynucleotide in the γδ T cell. The target polynucleotide in the γδ T cell may encode a target polypeptide. In some cases, the target polypeptide may induce or inhibit proliferation, differentiation, and / or survival of the immune cell. In some cases, the target polypeptide may comprise (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolase. The actuator moiety can bind to a target polynucleotide to regulate expression and / or activity of a target gene. In some embodiments, the target polynucleotide comprises genomic DNA. In some embodiments, the target polynucleotide comprises a region of a plasmid, for example a plasmid carrying an exogenous gene. In some embodiments, the target polynucleotide comprises RNA, for example mRNA. In some embodiments, the target polynucleotide comprises an endogenous gene or gene product. The actuator moiety can comprise a nuclease (e.g., DNA nuclease and / or RNA nuclease), modified nuclease (e.g., DNA nuclease and / or RNA nuclease) that is nuclease-deficient or has reduced nuclease activity compared to a wild-type nuclease or a variant thereof. The actuator moiety can regulate expression or activity of a gene and / or edit the sequence of a nucleic acid (e.g., a gene and / or gene product). In some embodiments, the actuator 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 actuator moiety comprises a 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 actuator moiety has reduced or minimal nuclease activity. An actuator moiety having reduced or minimal nuclease activity can regulate expression and / or activity of a gene by physical obstruction of a target polynucleotide or recruitment of additional factors effective to suppress or enhance expression of the target polynucleotide. The actuator moiety can physically obstruct the target polynucleotide or recruit additional factors effective to suppress or enhance expression of the target polynucleotide. In some cases, the actuator moiety comprises an activator effective to increase expression of the target polynucleotide. In some embodiments, the actuator moiety comprises a transcriptional activator effective to increase expression of the target polynucleotide. In other cases, the actuator moiety comprises a repressor effective to decrease expression of the target polynucleotide. Non-limiting examples of transcription activators include GAL4, VP16, VP64, p65 subdomain (NFkappaB), and VP64-p65-Rta (VPR). In some embodiments, the actuator moiety comprises a transcriptional repressor effective to decrease expression of the target polynucleotide. Non-limiting examples of transcription repressors include Kruippel associated box (KRAB or SKD), the Mad mSIN3 interaction domain (SID), and the ERF repressor domain (ERD). In some embodiments, the actuator moiety comprises a nuclease-null DNA binding protein derived from a DNA nuclease that can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, the actuator moiety comprises a nuclease-null RNA binding protein derived from a RNA nuclease that can induce transcriptional activation or repression of a target RNA sequence. In some embodiments, the actuator moiety is a nucleic acid-guided actuator moiety. In some embodiments, the actuator moiety is a DNA-guided actuator moiety. In some embodiments, the actuator moiety is an RNA-guided actuator moiety or a variant thereof, which RNA-guided actuator moiety forms a complex with the target polynucleotide. An actuator moiety can regulate expression or activity of a gene and / or edit a nucleic acid sequence, whether exogenous or endogenous.

[0132] Any suitable nuclease can be used in a two receptor system. Suitable nucleases include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); meganucleases; RNA-binding proteins (RBP); CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); and any variant thereof. In some cases, the actuator moiety is a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity (dCas). In some cases, the actuator moiety can be Cas9 and / or Cpf1.

[0133] Any target gene can be regulated by the comprising the actuator moiety. It is contemplated that genetic homologues of a gene described herein are covered. For example, a gene can exhibit a certain identity and / or homology to genes disclosed herein. Therefore, it is contemplated that the expression of a gene that exhibits or exhibits at least about 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) can be regulated. It is also contemplated that the expression of a gene that exhibits or exhibits at least about 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) can be regulated.

[0134] In some cases, the administration of the GMP to the γδ T cell can comprise treating the γδ T cell with a delivery vehicle, which delivery vehicle comprising at least a portion of the GMP and / or a polynucleotide that encodes at least a portion of the GMP. The delivery vehicle may be viral or non-viral. The at least the portion of the GMP and / or the polynucleotide that encodes the at least the portion of the GMP may be attached covalently and / or non-covalently (e.g., ionically, via hydrogen bonds, etc.) to the delivery vehicle. Alternatively or in addition to, the at least the portion of the GMP and / or the polynucleotide that encodes the at least the portion of the GMP may be encapsulated by the delivery vehicle without any physical attachment to the delivery vehicle.

[0135] In some cases, the delivery vehicle may comprise a targeting moiety with an affinity to one or more ligands (e.g., a portion of a cell surface receptor, a polysaccharide chain, one or more extracellular proteins) present on or adjacent to the surface of the γδ T cell. The targeting moiety may enhance targeting and binding of the delivery vehicle to the γδ T cell. The targeting moiety may enhance intracellular entrance, uptake, and / or penetration of the delivery vehicle into the γδ T cell. The targeting moiety may be linked (e.g., via covalent and / or a non-covalent bond) to an external surface of the delivery vehicle. The targeting moiety may be a non-natural molecule, at least a portion of a natural molecule, a functional derivative thereof, or a combination thereof. The targeting moiety may be a small molecule, a polynucleotide (e.g., an aptamer), a polypeptide (e.g., an oligopeptide or a protein), an antibody or a functional fragment thereof, a functional derivative thereof, or a combination thereof.

[0136] In some cases, the delivery vehicle may not comprise such targeting moiety against the γδ T cell.

[0137] Examples of the viral delivery vehicle may comprise an adenovirus, a retrovirus, a lentivirus (e.g., a human immunodeficiency virus (HIV)), an adeno-associated virus (AAV), and / or a Herpes simplex virus (HSV). In an example, the viral delivery vehicle may be a retrovirus. The retrovirus may be a gamma-retrovirus selected from the group consisting of: Feline Leukemia Virus (FLV), Feline Sarcoma Virus (Strain Hardy-Zuckerman 4),  Finkel-Biskis-Jinkins Murine Sarcoma Virus (FBJMSV), Murine leukemia virus (MLV) (e.g. Friend Murine Leukemia Virus (FMLV), Moloney Murine Leukemia Virus (MMLV), Murine Type C Retrovirus (MTCR)), Gibbon Ape Leukemia Virus (GALV), Koala Retrovirus (KR), Moloney Murine Sarcoma Virus (MMSV), Porcine Endogenous Retrovirus E (PERE), Reticuloendotheliosis Virus (RV), Woolly Monkey Sarcoma Virus (WMSV), Baboon Endogenous Virus Strain M7 (BEVSM7), Murine Osteosarcoma Virus (MOV), Mus Musculus Mobilized Endogenous Polytropic Provirus (MMMEPP), PreXMRV-1, RD114 Retrovirus, Spleen Focus-Forming Virus (SFFV), Abelson murine leukemia virus (AMLV), Murine Stem Cell Virus (MSCV), and variants thereof.

[0138] The delivery vehicle may comprise of a nucleotide (e.g., a polynucleotide), an amino acid (e.g., a peptide or polypeptide), a polymer, a metal, a ceramic, a derivative thereof, or a combination thereof. In an example, the delivery vehicle may comprise of a diamond nanoparticle (“nanodiamonds”), a gold nanoparticle, a silver nanoparticle, a calcium phosphate nanoparticle, etc. The delivery vehicle may or may not comprise a fluid (e.g., a liquid or gas). The delivery vehicle may have various shapes and sizes. For example, the delivery vehicle may be in the shape of a sphere, cuboid, or disc, or any partial shape or combination of shapes thereof. The delivery vehicle may have a cross-section that is circular, triangular, square, rectangular, pentagonal, hexagonal, or any partial shape or combination of shapes thereof.

[0139] Examples of the non-viral delivery vehicle may comprise nanoparticles, nanospheres, nanocapsules, microparticies, microspheres, microcapsules, liposomes, nanoemulsions, solid lipid nanoparticles, modifications thereof, or combinations thereof. The non-viral delivery vehicle of the present invention may be prepared by methods, such as, but not limited to, nanoprecipitation, emulsion solvent evaporation method, emuision-crosslinking method, emulsion solvent diffusion method, microemulsion method, gas antisolvent precipitation method, ionic gelation methods milling or size reduction method, PEGylation method, salting-out method, dialysis method, single or double emulsification method, nanospray drying method, layer by layer method, desolvation method, supercritical fluid technology, supramolecular assembly, or combinations thereof.

[0140] In some cases, the method can further comprise integrating into the genome of the γδ T cell a nucleic acid sequence (e.g., a polynucleotide) encoding the GMP. In some cases, a nucleic acid sequence (e.g., a polynucleotide) encoding the GMP may be integrated into the genome of the γδ T cell. Upon administration of the polynucleotide encoding for at least a portion of the GMP (e.g., with or without the delivery vehicle), at least a portion of the polynucleotide may be integrated into the genome of the γδ T cell. The at least the portion of the integrated polynucleotide may be placed under the control of an autologous promoter of the γδ T cell. Alternatively or in addition to, the at least a portion of the integrated polynucleotide may further comprise a promoter that is heterologous (a heterologous promoter) to the γδ T cell. The heterologous promoter may be configured to bind one or more molecules (e.g., an RNA polymerase, a transcription factor, etc.) that are homologous or heterologous to the γδ T cell.

[0141] The γδ T cell may be in vivo and / or ex vivo (e.g., in vitro) during the treatment with the delivery vehicle comprising a payload (e.g., the at least the portion of the GMP and / or the polynucleotide that encodes the at least the portion of the GMP).

[0142] In some cases, the delivery vehicle comprising the payload may be injected into a bodily part of a subject (e.g., a vein, a marrow, etc. of a patient), and the delivery vehicle may interact with (e.g., enter into) the γδ T cell in vivo. Other examples of the injection method may include intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavernous, and / or intravitreal.

[0143] In some cases, the subject may be injected with a dose of the delivery vehicle comprising the payload for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some cases, the subject may be injected with a dose of the delivery vehicle comprising the payload for at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some cases, the subject may be injected with a dose of the delivery vehicle comprising the payload at a frequency of at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, 90, 180, 360, or more days. In some cases, the subject may be injected with a dose of the delivery vehicle comprising the payload at a frequency of at most once every 360, 180, 90, 60, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day.

[0144] In some cases, the γδ T cell may be isolated from the subject, and the isolated γδ T cell may be treated (e.g., cultured in a culture media) with the delivery vehicle comprising the payload. The isolated γδ T cell may be allowed or stimulated to proliferate prior to, during, and / or subsequent to the treatment with the delivery vehicle comprising the payload. Alternatively or in addition to, a cell that is not a γδ T cell (e.g., a stem cell, a skin cell, a blood cell, etc.) may be isolated from the subject, and the isolated cell may be induced to differentiate into a γδ T cell, trans-differentiate into a γδ T cell, and / or express the γδ TCR complex prior to the treatment with the delivery vehicle comprising a payload. In some cases, the cell that is not a γδ T cell may first be de-differentiated into an induced pluripotent stem cell (iPSC) prior to differentiation into a γδ T cell and / or inducing expression of the γδ TCR complex. Following, the isolated and treated γδ T cell may be injected (transplanted) into the subject.

[0145] Any of the cells provided herein that are treated (ex vivo and / or in vivo) with at least the payload to administer the GMR comprising the actuator moiety may be referred to as an engineered γδ T cells.

[0146] In some cases, such engineered γδ T cells may be injected into a bodily part of a subject (e.g., a vein, a marrow, etc. of a patient), and the delivery vehicle may interact with (e.g., enter into) the γδ T cell in vivo. Other examples of the injection method may include intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal.

[0147] In some cases, the subject may be injected with a dose of the treated γδ T cells for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some cases, the subject may be injected with a dose of the treated γδ T celsl for at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some cases, the subject may be injected with a dose of the treated γδ T cells at a frequency of at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, 90, 180, 360, or more days. In some cases, the subject may be injected with a dose of the treated γδ T cells at a frequency of at most once every 360, 180, 90, 60, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day.

[0148] In some cases, the subject may be injected with at least about 0.5, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 (x 109) treated γδ T cells, or more. In other cases, the subject may be injected with at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5 (x 109) treated γδ T cells, or less.

[0149] In some cases, the GMP may be a portion of a chimeric polypeptide. The chimeric polypeptide may or may not be a transmembrane protein. In an example, the chimeric polypeptide may be a CAR, and the GMP may be at least a portion of an intracellular domain of the CAR. In another example, the chimeric polypeptide may be a chimeric transmembrane protein, and the GMP may be at least a portion of an intracellular domain of the chimeric transmembrane protein. In a different example, the chimeric polypeptide comprising the GMP may be an intracellular protein.

[0150] In some cases, the administration of the GMP to the γδ T cell can comprise treating the γδ T cell with at least a portion of the chimeric polypeptide comprising the GMP and / or a polynucleotide that encodes the at least a portion of the chimeric polypeptide comprising the GMP. Such treatment may occur in the presence or absence of one or more delivery vehicles provided herein in the present disclosure. In some cases, the method can further comprise administering to the γδ T cell a chimeric polypeptide comprising the GMP, wherein the chimeric polypeptide is operable to release the GMP from the chimeric polypeptide in response to a stimulant, and wherein the released GMP is operable to regulate expression of the target polynucleotide in the γδ T cell. In some cases, the method can further comprise administering to the γδ T cell a chimeric polypeptide comprising the GMP and a nuclear localization domain, wherein the nuclear localization domain is operable to translocate the chimeric polypeptide to a nucleus of the γδ T cell in response to a stimulant, and wherein the translocated GMP is operable to regulate expression of the target polynucleotide in the γδ T cell.

[0151] In some cases, the nuclear localization domain can be derived from a transcription factor, as abovementioned. The transcription factor can be a regulatable transcription factor that is only active and able to translocate into a nucleus in response to a signal or signaling pathway. The transcription factor can be a regulatable transcription factor that is primarily active and able to translocate into a nucleus in response to a signal or signaling pathway. The transcription factor can be a regulatable transcription factor that is generally active and able to translocate into a nucleus in response to a signal or signaling pathway.

[0152] In some examples, the nuclear localization domain can be derived from the NFAT family members (e.g., NFATp, NFAT1, NFATc1, NFATc2, NFATc3, NFAT4, NFATx, NFATc4, NFAT3, and NFAT5), nuclear factor kappa B (NF-κB), NFKB1 p50, activator protein 1 (AP-1), signal transducer and activator of transcription family members (e.g., STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6), sterol response element-binding proteins (e.g., SREBP-1 and SREBF1), a light or circadian or electromagnetic sensing protein such as cryptochromes (e.g., CRY1, CRY2), Timeless (TIM), PAS domain of PER proteins (e.g., PER1, PER2, and PER3), or other transcription factors or signal transducers.

[0153] In one aspect, the present disclosure provides a system for regulating an activity of a gamma-delta (γδ) cell, such as, for example, a γδ T cell. In some cases, the system for regulating an activity of the γδ T cell can comprise a gene modulating polypeptide (GMP) comprising an actuator moiety configured to regulate expression of a target polynucleotide in the γδ T cell, wherein the target polynucleotide can encode a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolase. The systems disclosed herein may utilize all components and configurations described in methods for regulating an activity of a γδ cell of the present disclosure.

[0154] In some cases, the system (e.g., the GMP) may be expressed by the γδ T cell. In some cases, the system (e.g., the GMP) may be administered to the γδ T cell.

[0155] In some cases, the GMP can increase or decrease the activity of the γδ T cell by at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 5-fold, 10-fold, 100-fold, 1000-fold, or more in comparison to the γδ T cell in the absence of the GMP. In some cases, the GMP can increase or decrease the activity of the γδ T cell by at most 1000-fold, 100-fold, 10-fold, 5-fold, 4.0-fold, 3.5-fold, 3.0-fold, 2.5-fold, 2.0-fold, 1.9-fold, 1.8-fold, 1.7-fold, 1.6-fold, 1.5-fold, 1.4-fold, 1.3-fold, 1.2-fold, 1.1-fold, or less in comparison to the γδ T cell in the absence of the GMP.

[0156] In some cases, the GMP can increase or decrease the activity of the γδ T cell for at least 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, 1 year, or more in comparison to the γδ T cell in the absence of the GMP. In some cases, the GMP can increase or decrease the activity of the γδ T cell for at most 1 year, 6 months, 4 months, 2 months, 4 weeks, 3 weeks, 2 weeks, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 10 minutes, 5 minutes, 1 minute, or less in comparison to the γδ T cell in the absence of the GMP.

[0157] The regulating the expression of the target polypeptide in the γδ T cell can comprise decreasing, increasing, inhibiting, and / or prolonging the expression of the target polypeptide in the γδ T cell. The regulating the expression of the target polypeptide in the γδ T cell can be decreasing the expression of the target polypeptide in the γδ T cell. The regulating the expression of the target polypeptide in the γδ T cell can be increasing the expression of the target polypeptide in the γδ T cell.

[0158] The regulating the expression of the target polypeptide in the γδ T cell may directly and / or indirectly allow the regulating the activity of the γδ T cell. In some cases, the regulating the activity of the γδ T cell can comprise decreasing and / or inhibiting fratricide of one or more γδ T cells, self-inflicted injury of the γδ T cell, death of the γδ T cell by another γδ T cell, and / or death of another γδ T cell by the γδ T cell, thereby improving (directly and / or indirectly) viability, proliferation, and / or function of the cell. In some cases, the GMP may be operable to decrease fratricide of the γδ T cell.

[0159] In some case, the target polynucleotide can encode a polypeptide, and the polypeptide can comprise the death-inducing protein. The polypeptide may be the death-inducing protein. The death-inducing protein may comprise one or more of the death-inducing compounds provided herein in the present disclosure. Examples of the death-inducing protein can comprise Fas, Fas ligand, granzyme B, granzyme M, and / or perforin.

[0160] In some cases, a γδ T cell comprising the GMP may be operable to target a diseased cell (e.g., a cancer / tumor cell) and induce cell death (e.g., apoptosis) of the targeted diseased cell. The diseased cell may express a cell surface receptor (e.g., Fas / CD95 / APO-1 / APT1), and the γδ T cell may express a respective cell surface ligand (e.g., FasL / CD95L / APTL) that is operable to bind and target the cell surface receptor of the diseased cell.

[0161] In some case, the target polynucleotide can encode a polypeptide, and the polypeptide can comprise the inhibitor of the death-inducing protein. The polypeptide may be the inhibitor of the death-inducing protein. The inhibitor of the death-inducing protein may comprise one or more of the cell death inhibitors provided herein in the present disclosure (e.g., the Serpin superfamily, including Serpin B1 and / or Serpin B4). In some cases, the inhibitor of the death-inducing protein may prevent production of, deform, and / or degrade one or more death-inducing compounds. In such a case, an expression of the inhibitor of the death-inducing protein by a γδ T cell may protect the γδ T cell from cell death (e.g., apoptosis) by one or more death-inducing proteins expressed by the same or different γδ T cell. Thus, the GMP comprising the actuator moiety may be configured to regulate (e.g., induce or increase) expression of the inhibitor of the death-inducing protein in γδ T cells, thereby decreasing or inhibiting fratricide of the γδ T cells.

[0162] In some cases, the regulating the activity of the γδ T cell can comprise inducing and / or prolonging activation of the γδ T cell. The activation of the γδ T cell can comprise activation of one or more biological activities (e.g., migration, proliferation, synthesis of one or more polypeptides, etc.) as provided herein in the present disclosure. The inducing / prolonging activation of the γδ T cell may comprise inducing and / or prolonging expression of (1) one or more γδ T cell activators and / or (2) one or more targets of the γδ T cell activator(s). In an example, the γδ T cell activator(s) may be a ligand, and the target(s) of the γδ T cell activator(s) may be a different polypeptide (e.g., a receptor, an intracellular protein, etc.). Alternatively or in addition to, the inducing / prolonging activation of the γδ T cell may comprise (1) decreasing and / or inhibiting expression of one or more inhibitors of the γδ T cell activator and / or (2) degrading and / or metabolizing the inhibitor(s) of the γδ T cell activator.

[0163] In some case, the target polynucleotide can encode a polypeptide, and the polypeptide can comprise the hydrolase. The polypeptide may be the hydrolase. The hydrolase may comprise any of the hydrolase provided herein in the present disclosure. In some cases, the γδ T cell activator may be a phosphoantigen, and the hydrolase may comprise any of the phosphoantigen inhibitor provided herein in the present disclosure. In an example, the hydrolase may be an ectonucleoside triphosphate diphosphohydrolase (e.g., NTPDase1 / ENTPD1 / CD39), which is operable to cleave one or more phosphates in the phosphoantigen, thereby decreasing or inhibiting the phosphoantigen-induced activation of the γδ T cell. As a result, the hydrolase, when it is not disrupted by the GMP of the present disclosure, may indirectly decrease activity (e.g., cytotoxic activity) of the γδ T cell against a diseased cell, such as a cancer / tumor cell.

[0164] Thus, in some cases, the GMP comprising the actuator moiety may be configured to induce and / or prolong expression of (1) one or more γδ T cell activators, (2) one or more substrates or pro-forms (e.g., prodrugs) of the γδ T cell activator(s), and / or (3) one or more regulators (e.g., catalysts, such as enzymes) of the synthesis of the γδ T cell activator(s).

[0165] In some cases, the GMP comprising the actuator moiety may be configured to induce and / or prolong expression of (1) one or more targets of the γδ T cell activator(s), (2) one or more substrates or pro-forms of the γδ T cell activator(s), and / or (3) one or more regulators (e.g., catalysts, such as enzymes) of the synthesis of the target(s) of the γδ T cell activator(s).

[0166] In some cases, the GMP comprising the actuator moiety may be configured to decrease and / or inhibit expression of one or more inhibitors (e.g., enzymes) of the γδ T cell activator(s). In some cases, the GMP comprising the actuator moiety may be configured to degrade and / or metabolize the inhibitor(s) of the γδ T cell activator(s).

[0167] In some cases, the GMP may be configured to reduce and / or prevent activation of the γδ T cell.

[0168] In some cases, the GMP comprising the actuator moiety may be configured to increase or decrease expression of one or more angiogenic factors in the γδ T cell. In some cases, the GMP comprising the actuator moiety may be configured to decrease expression of one or more angiogenic factors in the γδ T cell. In some cases, the GMP comprising the actuator moiety may be configured to decrease expression of one or more angiogenic factors in the γδ T cell. The GMP comprising the actuator moiety may be expressed along with a guide RNA (e.g., sgRNA) against one or more polynucleotide sequences encoding for the one or more angiogenic factors in the γδ T cell. The actuator moiety of the GMP, in conjunction with the guide RNA, may be configured to increase or decrease expression of one or more angiogenic factors in the γδ T cell.

[0169] The one or more angiogenic factors can include pro-angiogenic factors and / or anti-angiogenic factors. Examples of the pro-angiogenic factors can include, but are not limited to, FGF, VEGF, VEGFR, NRP-1, Ang1, Ang2, PDGF (BB-homodimer), PDGFR, TGF-β, endoglin, TGF-βreceptors, MCP-1, Integrins αVβ3, αVβ3, α5β1, VE-Cadherin, CD31, ephrin, plasminogen activators, plasminogen activator inhibitor-1, eNOS, COX-2, AC133, Id1 / Id3, Angiogenin, HGF, Vegf, IL-17, IL-1 alpha, IL-8, IL-6, Cxcl5, Fgfα, Fgfβ, Tgfα, Tgfβ, MMPs (including mmp9), Plasminogen activator inhibitor-1, Thrombospondin, Angiopoietin 1, Angiopoietin 2, Amphiregulin, Leptin, Endothelin-1, AAMP, AGGF1, AMOT, ANGLPTL3, ANGPTL4, BTG1, IL-1β, NOS3, TNFSF12, and / or VASH2.

[0170] In some cases, a nucleic acid sequence encoding the GMP may be integrated into a genome of the γδ T cell.

[0171] In some cases, the GMP may be a portion of a chimeric polypeptide. The chimeric polypeptide may or may not be a transmembrane protein. In an example, the chimeric polypeptide may be a CAR, and the GMP may be at least a portion of an intracellular domain of the CAR. In another example, the chimeric polypeptide may be a chimeric transmembrane protein, and the GMP may be at least a portion of an intracellular domain of the chimeric transmembrane protein. In a different example, the chimeric polypeptide comprising the GMP may be an intracellular protein.

[0172] In some cases, the system may comprise: (a) a chimeric receptor polypeptide that is modified upon binding an antigen, wherein receptor modification comprises a conformational change or chemical modification; (b) a chimeric adaptor polypeptide that binds the receptor in response to the receptor modification; (c) a gene modulating polypeptide (GMP) comprising an actuator moiety linked to a cleavage recognition site, wherein upon cleavage of the cleavage recognition site, the actuator moiety is activated to complex with a target polynucleotide; and (d) a cleavage moiety that cleaves the cleavage recognition site when in proximity to the cleavage recognition site. In some cases, the GMP may form a portion of an intracellular region of the chimeric receptor polypeptide, and the cleavage moiety may form a portion of the chimeric adaptor polypeptide. In another example, the GMP may form a portion of the chimeric adaptor polypeptide, and the cleavage moiety may form a portion of an intracellular region of the chimeric receptor polypeptide. In a different example, the cleavage moiety may be complexed with a second adaptor polypeptide that binds the chimeric receptor polypeptide in response to the receptor modification, and the GMP may form a portion of the chimeric adaptor polypeptide.

[0173] In some cases, the cleavage recognition site may comprise a polypeptide sequence, and the cleavage moiety may comprise protease activity. In some cases, the cleavage recognition site may comprise a disulfide bond, and the cleavage moiety may comprise oxidoreductase activity. In some cases, the cleavage recognition site may comprise a first portion of an intein sequence that reacts with a second portion of the intein sequence to release the actuator moiety.

[0174] In some cases, the cleavage moiety can only cleave the recognition site when in proximity to the cleavage recognition site. The cleavage recognition site can comprise a polypeptide sequence that is a recognition sequence of a protease. The cleavage moiety can comprise protease activity which recognizes the polypeptide sequence. A cleavage moiety comprising protease activity can be a protease, or any derivative, variant or fragment thereof. A protease can refer to any enzyme that performs proteolysis, in which polypeptides are cleaved into smaller polypeptides or amino acids. Various proteases can be suitable for use as a cleavage moiety. Some proteases can be highly promiscuous such that a wide range of protein substrates are hydrolysed. Some proteases can be highly specific and only cleave substrates with a certain sequence, e.g., a cleavage recognition sequence or peptide cleavage domain. In some cases, the cleavage recognitions site can comprise multiple cleavage recognition sequences, and each cleavage recognition sequence can be recognized by the same or different cleavage moiety comprising protease activity (e.g., protease). Sequence-specific proteases that can be used as cleavage moieties include, but are not limited to, superfamily CA proteases, e.g., families C1, C2, C6, C10, C12, C16, C19, C28, C31, C32, C33, C39, C47, C51, C54, C58, C64, C65, C66, C67, C70, C71, C76, C78, C83, C85, C86, C87, C93, C96, C98, and C101, including papain (Carica papaya), bromelain (Ananas comosus), cathepsin K (liverwort) and calpain (Homo sapiens); superfamily CD proteases, e.g., family C11, C13, C14, C25, C50, C80, and C84: such as caspase-1 (Rattus norvegicus) and separase (Saccharomyces cerevisiae); superfamily CE protease, e.g., family C5, C48, C55, C57, C63, and C79 including adenain (human adenovirus type 2); superfamily CF proteases, e.g., family C15 including pyroglutamyl-peptidase I (Bacillus amyloliquefaciens); superfamily CL proteases, e.g., family C60 and C82 including sortase A (Staphylococcus aureus); superfamily CM proteases, e.g. family C18 including hepatitis C virus peptidase 2 (hepatitis C virus); superfamily CN proteases, e.g., family C9 including sindbis virus-type nsP2 peptidase (sindbis virus); superfamily CO proteases, e.g., family C40 including dipeptidyl-peptidase VI (Lysinibacillus sphaericus); superfamily CP proteases, e.g., family C97 including DeSI-1 peptidase (Mus musculus); superfamily PA proteases, e.g., family C3, C4, C24, C30, C37, C62, C74, and C99 including TEV protease (Tobacco etch virus); superfamily PB proteases, e.g., family C44, C45, C59, C69, C89, and C95 including amidophosphoribosyltransferase precursor (homo sapiens); superfamily PC proteases, families C26, and C56 including Ȗ-glutamyl hydrolase (Rattus norvegicus); superfamily PD proteases, e.g., family C46 including Hedgehog protein (Drosophila melanogaster); superfamily PE proteases, e.g., family P1 including DmpA aminopeptidase (Ochrobactrum anthropi); others proteases, e.g., family C7, C8, C21, C23, C27, C36, C42, C53 and C75. Additional proteases include serine proteases, e.g., those of superfamily SB, e.g., families S8 and S53 including subtilisin (Bacillus licheniformis); those of superfamily SC, e.g., families S9, S10, S15, S28, S33, and S37 including prolyl oligopeptidase (Sus scrofa); those of superfamily SE, e.g., families S11, S12, and S13 including D-Ala-D-Ala peptidase C (Escherichia coli); those of superfamily SF, e.g., families S24 and S26 including signal peptidase I (Escherichia coli); those of Superfamily SJ, e.g., families S16, S50, and S69 including lon-A peptidase (Escherichia coli); those of Superfamily SK, e.g., families S14, S41, and S49 including Clp protease (Escherichia coli); those of Superfamily SO, e.g., families S74 including Phage K1F endosialidase CIMCD self-cleaving protein (Enterobacteria phage K1F); those of superfamily SP, e.g., family S59 including nucleoporin 145 (Homo sapiens); those of superfamily SR, e.g., family S60 including Lactoferrin (Homo sapiens); those of superfamily SS, families S66 including murein tetrapeptidase LD-carboxypeptidase (Pseudomonas aeruginosa); those of superfamily ST, e.g., families S54 including rhomboid-1 (Drosophila melanogaster); those of superfamily PA, e.g., families S1, S3, S6, S7, S29, S30, S31, S32, S39, S46, S55, S64, S65, and S75 including Chymotrypsin A (Bos taurus); those of superfamily PB, e.g., families S45 and S63 including penicillin G acylase precursor (Escherichia coli); those of superfamily PC, e.g., families S51 including dipeptidase E (Escherichia coli); those of superfamily PE, e.g., families P1 including DmpA aminopeptidase (Ochrobactrum anthropi); those unassigned, e.g., families S48, S62, S68, S71, S72, S79, and S81 threonine proteases, e.g., those of superfamily PB clan, e.g., families T1, T2, T3, and T6 including archaean proteasome, ȕ component (Thermoplasma acidophilum); and those of superfamily PE clan, e.g., family T5 including ornithine acetyltransferase (Saccharomyces cerevisiae); aspartic proteases, e.g., BACE1, BACE2; cathepsin D; cathepsin E; chymosin; napsin-A; nepenthesin; pepsin; plasmepsin; presenilin; renin; and HIV-1 protease, and metalloproteinases, e.g., exopeptidases, metalloexopeptidases; endopeptidases, and metalloendopeptidases. A cleavage recognition sequence (e.g., polypeptide sequence) can be recognized by any of the proteases disclosed herein.

[0175] In some cases, the cleavage recognition site can comprise a cleavage recognition sequence (e.g., polypeptide sequence or peptide cleavage domain) that is recognized by a protease selected from the group consisting of: achromopeptidase, aminopeptidase, ancrod, angiotensin converting enzyme, bromelain, calpain, calpain I, calpain II, carboxypeptidase A, carboxypeptidase B, carboxypeptidase G, carboxypeptidase P, carboxypeptidase W, carboxypeptidase Y, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin H, cathepsin L, chymopapain, chymase, chymotrypsin, clostripain, collagenase, complement C1r, complement C1s, complement Factor D, complement factor I, cucumisin, dipeptidyl peptidase IV, elastase (leukocyte), elastase (pancreatic), endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, enterokinase, factor Xa, ficin, furin, granzyme A, granzyme B, HIV Protease, IGase, kallikrein tissue, leucine aminopeptidase (general), leucine aminopeptidase (cytosol), leucine aminopeptidase (microsomal), matrix metalloprotease, methionine, aminopeptidase, neutrase, papain, pepsin, plasmin, prolidase, pronase E, prostate specific antigen, protease alkalophilic from Streptomyces griseus, protease from Aspergillus, protease from Aspergillus saitoi, protease from Aspergillus sojae, protease (B. licheniformis) (alkaline or alcalase), protease from Bacillus polymyxa, protease from Bacillus sp, protease from Rhizopus sp., protease S, proteasomes, proteinase from Aspergillus oryzae, proteinase 3, proteinase A, proteinase K, protein C, pyroglutamate aminopeptidase, rennin, rennin, streptokinase, subtilisin, thermolysin, thrombin, tissue plasminogen activator, trypsin, tryptase and urokinase.

[0176] Further details of proteases and associated recognition sequences that can be used in systems and methods of the present disclosure are disclosed in Patent Cooperation Treaty (PCT) Patent Application No. PCT / US17 / 012885 and PCT Patent Application No. PCT / US17 / 012881, each of which is incorporated in its entirety herein by reference.

[0177] In some cases, a system comprising that is administered to the γδ cell (e.g., the γδ T cell) can comprise the chimeric polypeptide comprising the GMP and / or a polynucleotide that encodes the at least a portion of the chimeric polypeptide comprising the GMP. The system comprising the GMP may be administered to the γδ T cell in the presence or absence of one or more delivery vehicles provided herein in the present disclosure. In some cases, the system can further comprise a chimeric polypeptide comprising the GMP, wherein the chimeric polypeptide can be operable to release the GMP from the chimeric polypeptide in response to a stimulant, and wherein the released GMP can be operable to regulate expression of the target polynucleotide in the gamma delta T cell. In some cases, the system can further comprise a chimeric polypeptide comprising the GMP and a nuclear localization domain, wherein the nuclear localization domain can be operable to translocate the chimeric polypeptide to a nucleus of the gamma delta T cell in response to a stimulant, and wherein the translocated GMP can be operable to regulate expression of the target polynucleotide in the gamma delta T cell. The chimeric polypeptide may or may not be a transmembrane receptor. The chimeric polypeptide may or may not be an intracellular protein. The chimeric polypeptide may or may not bind a ligand (e.g., an antigen of a target cell).

[0178] In some cases, the actuator moiety can be an RNA-guided actuator moiety or a variant thereof, which RNA-guided actuator moiety forms a complex with the target polynucleotide. In some cases, the actuator moiety can be a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity. In some cases, the actuator moiety can be Cas9 and / or Cpf1. In some cases, the actuator moiety can comprise an activator effective to increase expression of the target polynucleotide. In some cases, the actuator moiety can comprise a repressor effective to decrease expression of the target polynucleotide.

[0179] The GMP of the system may comprise any of the GMP provided herein in the present disclosure. The actuator moiety of the system may comprise any of the actuator moiety provided herein in the present disclosure.

[0180] In some embodiments, the system can comprise (a) a chimeric transmembrane receptor polypeptide comprising (i) an extracellular region comprising a ligand (e.g., an antigen) interacting domain that binds a ligand (e.g., an antigen) and (ii) an intracellular region comprising a signaling domain (e.g., an immune cell signaling domain); (b) a chimeric adaptor polypeptide comprising a receptor associating moiety (e.g., a receptor binding moiety) that (i) binds the receptor polypeptide when the receptor polypeptide has undergone a receptor modification upon binding to a ligand and / or (ii) moves in proximity to the receptor polypeptide when the receptor polypeptide has undergone a receptor modification upon binding to a ligand; (c) a GMP comprising an actuator moiety linked to a cleavage recognition site; and (d) a cleavage moiety that cleaves the cleavage recognition site when in proximity to the cleavage recognition site to release the actuator moiety from the GMP. In some cases, the cleavage moiety forms a portion of the intracellular region of the receptor polypeptide, and the GMP forms a portion of the chimeric adaptor polypeptide. In some cases, the cleavage moiety is complexed with a second adaptor polypeptide that binds and / or moves in proximity to the receptor polypeptide that has undergone the receptor modification upon binding to an antigen, and the GMP forms a portion of the chimeric adaptor polypeptide. In some cases, the cleavage moiety forms a portion of the chimeric adaptor polypeptide, and the GMP forms a portion of the intracellular region of the receptor polypeptide.

[0181] In some cases, the proximity between the chimeric adaptor polypeptide and the chimeric receptor polypeptide may be sufficient to allow the cleavage moiety to cleave the cleavage recognition site to release the actuator moiety from the GMP.

[0182] In some cases, the chimeric transmembrane receptor polypeptide may comprise at least a portion of a TCR. In such a case, the chimeric adaptor polypeptide may comprise at least a portion of a signaling adaptor (e.g., a transmembrane signaling adaptor) of the TCR. When the chimeric receptor polypeptide comprising the at least the portion of the TCR has undergone a receptor modification upon binding to a ligand, the at least the portion of the signaling adaptor of the TCR may be activated to bind and / or move in proximity to the chimeric receptor polypeptide, thereby to allow the cleavage moiety to cleave the cleavage recognition site to release the actuator moiety from the GMP. In an example, the signaling adaptor of the TCR may be a linker for activation of T cells (LAT).

[0183] In some cases, the chimeric transmembrane receptor polypeptide may comprise at least a portion of a C-type lectin-like receptor, such as, for example, a CD94 family receptor. Examples of the CD95 family receptors can include NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2G. In such a case, the chimeric adaptor polypeptide may comprise at least a portion of a signaling adaptor (e.g., a transmembrane signaling adaptor) of the C-type lectin-like receptor. When the chimeric receptor polypeptide comprising the at least the portion of the C-type lectin-like receptor has undergone a receptor modification upon binding to a ligand, the at least the portion of the signaling adaptor of the C-type lectin-like receptor may be activated to bind and / or move in proximity to the chimeric receptor polypeptide, thereby to allow the cleavage moiety to cleave the cleavage recognition site to release the actuator moiety from the GMP. In an example, the C-type lectin-like receptor may be NKG2D, and the signaling adaptor of NKG2D may be DAP10. In some cases, the chimeric receptor polypeptide comprising the at least the portion of the NKG2D receptor may be activated to induce the release of the actuator moiety from the GMP of the system upon binding of a NKG2D ligand (NKG2DL) to the NKG2DL binding domain of the at least the portion of the NKG2D receptor of the chimeric receptor polypeptide. Examples of the NKG2DL may include, but are not limited to, MICA, MICB, and the RAET1 / ULBP family (e.g., RAET1 E / ULBP4, RAET1G / ULBP5, RAET1 H / ULBP2, RAET1 / ULBP1 , RAET1 L / ULBP6, and RAET1 N / ULBP3).

[0184] In some embodiments, the system of the γδ cell (e.g., the γδ T cell) can comprise the chimeric polypeptide sequence comprising a CAR. The CAR may comprise a ligand binding domain, a transmembrane domain, and a signaling domain (e.g., an intracellular signaling domain). The signaling domain may activate a signaling pathway of the immune cell upon binding of a ligand to the ligand binding domain. The γδ cell that is administered with such system may further comprise an expression cassette comprising a polynucleotide sequence encoding a GMP placed under control of a promoter. The GMP may comprise an actuator moiety. The promoter may be activated to drive expression of the GMP upon binding of the ligand to the ligand binding domain. The expressed GMP may regulate expression of a target polynucleotide in the γδ cell. The promoter may comprise an endogenous promoter of the γδ cell. The endogenous promoter may be activated upon binding of the ligand to the ligand binding domain of the CAR.

[0185] In some embodiments, the system of the γδ cell (e.g., the γδ T cell) can comprise a chimeric polypeptide sequence and an additional polypeptide sequence. The additional polypeptide sequence can comprise a chimeric receptor polypeptide that activates a cellular signaling pathway upon binding a ligand. The chimeric polypeptide sequence can comprise a GMP fused in-frame with a heterologous nuclear localization domain, wherein the heterologous nuclear localization domain is operable to translocate the chimeric polypeptide to a nucleus of the γδ cell upon induction by a cellular signaling pathway. Upon binding of the ligand to the chimeric receptor polypeptide, the chimeric receptor polypeptide can localize to the nucleus of the γδ cell via the induced heterologous nuclear localization domain and the gene modulating polypeptide can regulate expression of a target polynucleotide in the cell nucleus. In some cases, activation of the nuclear localization domain upon induction by the cellular signaling pathway can comprise a chemical modification of at least one nuclear localization sequence of the nuclear localization domain.

[0186] Further details of design and application of systems comprising the chimeric polypeptide (e.g., chimeric receptor polypeptide, the chimeric adaptor polypeptide, etc.), CAR, GMP, ligands (e.g., antigens), modifications thereof, and expression cassettes comprising thereof are disclosed in PCT Patent Application No. PCT / US17 / 012885, PCT Patent Application No. PCT / US17 / 012881, PCT Patent Application No. PCT / US18 / 041704, U.S. Patent No. 9,856,497, U.S. Non-Provisional Application No. 15 / 806,756, U.S. Non-Provisional Application No. 16 / 029,299, U.S. Non-Provisional Application No. 16 / 029,299, U.S. Provisional Application No. 62 / 639,427, U.S. Provisional Application No. 62 / 639,386, U.S. Provisional Application No. 62 / 647,543, and U.S. Provisional Application No. 62 / 675,134, each of which is incorporated in its entirety herein by reference.

[0187] In some cases, the γδ T cell may be contacted with a stimulant (e.g., a small molecule, polynucleotide, polypeptide, protein, antibody, ligand and / or receptor from another cell, etc.) to activate (or conditionally activate) the GMP comprising the actuator moiety. The activation of the GMP may comprise expressing the GMP and / or the chimeric polypeptide comprising the GMP. Alternatively or in addition to, the activation of the GMP may comprise release of the GMP from a chimeric polypeptide (e.g., a CAR, a chimeric transmembrane protein, a chimeric intracellular protein, etc.) comprising the GMP. In another alternative, or additionally, the activation of the GMP may comprise a chemical (e.g, phosphorylation) or physical (e.g., secondary structure) modification of one or more additional portions of the chimeric polypeptide comprising the GMP (e.g., a nuclear localization signal (NLS) domain of the chimeric polypeptide). In an example, such modified NLS may be operable to translocate at least the GMP (and an effector and / or an activator) to a nucleus of the γδ T cell in response to the stimulant. The activated GMP may be operable to regulate the expression of the target polynucleotide in the γδ T cell, thereby regulating the activity related to the target polynucleotide or the polypeptide encoded by the target polynucleotide.

[0188] The stimulant may activate one or more intrinsic signaling pathways in the γδ T cell to promote the activation (e.g., conditional activation, conditional expression, etc.) of the GMP or the chimeric polypeptide comprising the GMP. In some cases, the stimulant may bind to a cell surface receptor on the γδ T cell. In an example, the cell surface receptor may be an endogenous cell surface receptor of the γδ T cell. In another example, the cell surface receptor may be a heterologous cell surface receptor (e.g., a CAR) of the γδ T cell. The cell surface receptor may or may not be the chimeric polypeptide that comprises the GMP. In some cases, the stimulant may permeate through the membrane and bind an intracellular protein (e.g., in the cytoplasm or the nucleus, etc.) in the γδ T cell. The intracellular protein may or may not be the chimeric polypeptide that comprises the GMP. In an example, the intracellular protein may be an endogenous intracellular protein of the γδ T cell. In another example, the intracellular protein may be a heterologous intracellular protein in the γδ T cell.

[0189] Contacting the γδ T cell with a stimulant can occur directly and / or indirectly. Direct stimulation may occur when the stimulant binds the γδ T cell. In some cases, the stimulant may bind to a transmembrane receptor of the γδ T cell, an intracellular protein (e.g., in the cytoplasm or the nucleus of the cell) of the γδ T cell, or both. In an example, the stimulant may bind to a stimulant binding domain (or a ligand binding domain) of the chimeric polypeptide comprising the GMP. In another example, the stimulant may bind to a stimulant binding domain (or a ligand binding domain) of a different polypeptide (e.g., a different cell surface receptor or a different CAR) that does not comprise the GMP. Indirect stimulation can occur when the stimulant activates or deactivates a different cell, which different cell is operable to activate the γδ T cell by using its cell surface marker (e.g., a cell surface ligand) to bind a receptor of the γδ T cell. Consequentially, the γδ T cell may be activated to initiate expression of the GMP or the chimeric polypeptide comprising the GMP. The different cell may be of the same (e.g., another γδ T cell) or different cell type than the γδ T cell (e.g., a different type of lymphocyte, or a cancer / tumor cell).

[0190] Contacting the γδ T cell with the stimulant may occur prior to, during, and / or subsequent to administration of the GMP comprising the actuator moiety to the γδ T cell. The γδ T cell may be ex vivo and / or in vivo during the contact with the stimulant and conditional activation of the GMP comprising the actuator moiety.

[0191] Contacting the γδ T cell with the stimulant may occur prior to, during, and / or subsequent to administration of the γδ T cell (e.g., the engineered γδ T cell) to a subject. The γδ T cell may be contacted with the stimulant prior to, during, and / or subsequent to administration of the γδ T cell to the subject for a duration of time of at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 days, or more. The γδ T cell may be contacted with the stimulant prior to, during, and / or subsequent to administration of the γδ T cell to the subject for a duration of time of at most about 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5 days, or less. The γδ T cell may be contacted with the stimulant for a duration of time of at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 days, or more prior to administration of the γδ T cell to the subject. The γδ T cell may be contacted with the stimulant for a duration of time of at most about 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5 days, or less prior to administration of the γδ T cell to the subject. The γδ T cell may be contacted with the stimulant for a duration of time of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400 days, or more subsequent to administration of the γδ T cell to the subject. The γδ T cell may be contacted with the stimulant for a duration of time of at most about 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 days, or less subsequent to administration of the γδ T cell to the subject. In some cases, the γδ T cell may be contacted with the stimulant at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. In some cases, the γδ T cell may be contacted with the stimulant at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some cases, the γδ T cell may be contacted with the stimulant at a dose concentration of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 900, 1000 international units per millilitre (IU / mL), or more. In other cases, the γδ T cell may be contacted with the stimulant at a dose concentration of at most about 1000, 900, 800, 790, 780, 770, 760, 750, 740, 730, 720, 710, 700, 690, 680, 670, 660, 650, 640, 630, 620, 610, 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 IU / mL, or less.

[0192] In some cases, the stimulant of the γδ T cell may be selected from the group consisting of interleukins (e.g., IL-2), interferons, transforming growth factors (TGFs), ligands for cluster of differentiation (CD) receptors, and variants thereof. The stimulant may be an antigen described in the subject disclosure. In some examples, the antigen may induce migration, survival, proliferation, and / or differentiation of an immune cell (e.g., the γδ T cell). In some cases, the stimulant may comprise a vaccine (e.g., an immune cell vaccine). A vaccine may be a pharmaceutical composition comprising at least one immunologically protective molecule that induces an immunological and / or protective response in a cell (e.g., an immune cell) or an animal. A vaccine may further comprise one or more additional components (e.g., adjuvants) that enhance the immunological activity. In an example, the immune cell vaccine may be a peptide vaccine (e.g., p-27L) or a viral vaccine (e.g., p-210M, rFP-210M).

[0193] In some cases, the stimulant binding domain binds an antigen that is not membrane bound (e.g., non-membrane-bound), for example an extracellular antigen that is secreted by a cell (e.g., a target cell) or an antigen located in the cytoplasm of a cell (e.g., a target cell). Antigens (e.g., membrane bound and non-membrane bound) can be associated with a disease such as a viral, bacterial, and / or parasitic infection; inflammatory and / or autoimmune disease; or neoplasm such as a cancer and / or tumor. Non-limiting examples of antigens which can be bound by a ligand binding domain of a chimeric transmembrane receptor polypeptide of a subject system include, but are not limited to, 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A kinase anchor protein 4 (AKAP-4), activin receptor type-2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenoceptor β 3 (ADRB3), AGS-22M6, α folate receptor, α-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie 2), anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis anthrax, B-cell activating factor (BAFF), B-lymphoma cell, bone marrow stromal cell antigen 2 (BST2), Brother of the Regulator of Imprinted Sites (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 (eotaxin-1), CCR4, CCR5, 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, CD3 ε, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-related antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6), Clostridium difficile, clumping 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), C-X-C chemokine receptor type 4, cyclin B1, cytochrome P4501B1 (CYP1B1), cyp-B, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli shiga toxin type-1, E. coli shiga toxin type-2, ecto-ADP- ribosyltransferase 4 (ART4), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), EGF-like-domain multiple 7 (EGFL7), elongation factor 2 mutated (ELF2M), endotoxin, Ephrin A2, Ephrin B2, ephrin type-A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), episialin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML), F protein of respiratory syncytial virus, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II β chain, fibroblast activation protein α (FAP), fibronectin extra domain-B, FGF-5, Fms-Like Tyrosine Kinase 3 (FLT3), folate binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, Frizzled receptor, Fucosyl GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), glypican-3 (GPC3), GMCSF receptor α-chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, heat shock protein 70-2 mutated (mut hsp70-2), hemagglutinin, Hepatitis A virus cellular receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, hexasaccharide portion 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 papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), human scatter 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, IGHE, 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 lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor, interferon γ-induced protein, Interleukin 11 receptor α (IL-11Rα), Interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxyl esterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex, locus K 9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or Tumor necrosis factor-β (TNF-β), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary gland differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma inhibitor of apoptosis (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, mucin 1, cell surface associated (MUC1), MUC-2, mucin CanAg, myelin-associated glycoprotein, myostatin, N-Acetyl glucosaminyl-transferase V (NA17), NCA-90 (granulocyte antigen), nerve growth factor (NGF), neural apoptosis-regulated proteinase 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitor (e.g., NOGO-A, NOGO-B, NOGO-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), oncofetal antigen (h5T4), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), Oryctolagus cuniculus, OX-40, oxLDL, p53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), phosphate-sodium co-transporter, phosphatidylserine, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PDGF-R α), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, proacrosin binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostatic carcinoma cells, prostein, Protease Serine 21 (Testisin or PRSS21), Proteasome (Prosome, Macropain) Subunit, β Type, 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras Homolog Family Member C (RhoC), receptor activator of nuclear factor kappa-B ligand (RANKL), Receptor for Advanced Glycation Endproducts (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, Rhesus factor, sarcoma translocation breakpoints, sclerostin (SOST), selectin P, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine-1-phosphate, squamous cell carcinoma antigen recognized by T Cells 1, 2, and 3 (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, surviving, syndecan 1 (SDC1)+A314, SOX10, survivin, surviving-2B, synovial sarcoma, X breakpoint 2 (SSX2), T-cell receptor, TCR Γ Alternate Reading Frame Protein (TARP), telomerase, TEM1, tenascin C, TGF-β (e.g., TGF-β 1, TGF-β 2, TGF-β 3), 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 signal transducer 2, tumor-associated glycoprotein 72 (TAG72), tumor-associated glycoprotein 72 (TAG-72)+A327, TWEAK receptor, tyrosinase, tyrosinase-related protein 1 (TYRP1 or glycoprotein 75), tyrosinase-related protein 2 (TYRP2), uroplakin 2 (UPK2), vascular endothelial growth factor (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 myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), von Willebrand factor (VWF), Wilms tumor protein (WT1), X Antigen Family, Member 1A (XAGE1), β-amyloid, and κ-light chain, and variants thereof.

[0194] In some cases, the stimulant binding domain (or the ligand binding domain).

[0195] In some embodiments, the ligand binding domain binds an antigen selected from the group consisting of: 707-AP, a biotinylated molecule, a-Actinin-4, abl-bcr alb-b3 (b2a2), abl-bcr alb-b4 (b3a2), adipophilin, AFP, AIM-2, Annexin II, ART-4, BAGE, b-Catenin, 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, CD44v7 / 8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, 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, Lewis Y, 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 enzyme, Mammaglobin-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, oncofetal antigen (h5T4), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, Survivin, Survivin-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 binding domain binds to a tumor associated antigen.

[0196] In some embodiments, the target polynucleotide encodes for a cytokine. Non-limiting examples of cytokines include 4-1BBL, activin βA, activin βB, activin βC, activin βE, artemin (ARTN), BAFF / BLyS / TNFSF138, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, bone morphogenetic protein 1 (BMP1), 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, CD153 / CD30L / TNFSF8, CD40L / CD154 / TNFSF5, CD40LG, CD70, CD70 / CD27L / TNFSF7, CLCF1, c-MPL / CD110 / TPOR, CNTF, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, EDA-A1, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, Fas Ligand / FASLG / CD95L / CD178, GDF10, GDF11, GDF15, GDF2, GDF3, GDF4, GDF5, GDF6, GDF7, GDF8, GDF9, glial cell line-derived neurotrophic factor (GDNF), growth differentiation factor 1 (GDF1), IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA5 / IFNaG, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNZ, IFNω / IFNW1, IL11, IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1RL2, IL31, IL33, IL6, IL8 / CXCL8, inhibin-A, inhibin-B, Leptin, LIF, LTA / TNFB / TNFSF1, LTB / TNFC, neurturin (NRTN), OSM, OX-40L / TNFSF4 / CD252, persephin (PSPN), RANKL / OPGL / TNFSF11(CD254), TL1A / TNFSF15, TNFA, TNF-alpha / TNFA, TNFSF10 / TRAIL / APO-2L(CD253), TNFSF12, TNFSF13, TNFSF14 / LIGHT / CD258, XCL1, and XCL2. In some embodiments, the target gene encodes for an immune checkpoint inhibitor. Non-limiting examples of such immune checkpoint inhibitors include PD-1, CTLA-4, LAG3, TIM-3, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, and VISTA. In some embodiments, the target gene encodes for a T cell receptor (TCR) alpha, beta, gamma, and / or delta chain.

[0197] A subject system can be introduced in a variety of immune cells, including any cell that is involved in an immune response. A variety of immune cells can be engineered to express the γδ TCR complex and / or transdifferentiate into a cell that expresses the γδ TCR complex. In some embodiments, immune cells comprise granulocytes such as asophils, eosinophils, and neutrophils; mast cells; monocytes which 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, an immune cell is an immune effector cell. An immune effector cell refers to an immune cell that can perform a specific function in response to a stimulus. In some embodiments, an immune cell is an immune effector cell which can induce cell death. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a 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 both naive and memory cells (e.g. central memory or TCM, effector memory or TEMand effector memory RA or TEMRA), effector cells (e.g. cytotoxic T cells or CTLs or Tc cells), helper cells (e.g. Thl, 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 (LAKs), αβ Τ cells, γδ Τ cells, and similar unique classes of the T cell lineage. T cells can be divided into two broad categories: CD8+ T cells and CD4+ T cells, based on which protein is present on the cell's surface. T cells expressing a subject system can carry out 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 a subject system can be involved in recognizing and removing virus-infected cells and cancer cells. CTLs have specialized compartments, or granules, containing cytotoxins that cause apoptosis, e.g., programmed cell death. CD4+ T cells can be subdivided into four sub-sets – Th1, Th2, Th17, and Treg, with “Th” referring to “T helper cell,” although additional sub-sets may exist. Th1 cells can coordinate immune responses against intracellular microbes, especially bacteria. They can produce and secrete molecules that alert and activate other immune cells, like bacteria-ingesting macrophages. Th2 cells are involved in coordinating immune responses against extracellular pathogens, like helminths (parasitic worms), by alerting B cells, granulocytes, and mast cells. Th17 cells can produce interleukin 17 (IL-17), a signaling molecule that activates immune and non-immune cells. Th17 cells are important for recruiting neutrophils.

[0198] A variety of cells can be used as a host cell to realize the systems and methods of the subject disclosure. A host cell to which any of the embodiments (e.g., a cell comprising or expressing the γδ TCR complex) disclosed herein can be applied (e.g., transduced) includes a wide variety of cell types. A host cell can bein vitro. A host cell can bein vivo. A host cell can beex vivo. A host cell can be an isolated cell. A host cell can be a cell inside of an organism. A host cell can be an organism. A host cell can be a cell in a cell culture. A host cell can be one of a collection of cells. A host cell can be a mammalian cell or derived from a mammalian cell. A host cell can be a rodent cell or derived from a rodent cell. A host cell can be a human cell or derived from a human cell. A host cell can be a prokaryotic cell or derived from a prokaryotic cell. A host cell can be a bacterial cell or can be derived from a bacterial cell. A host cell can be an archaeal cell or derived from an archaeal cell. A host cell can be a eukaryotic cell or derived from a eukaryotic cell. A host cell can be a pluripotent stem cell. A host cell can be a plant cell or derived from a plant cell. A host cell can be an animal cell or derived from an animal cell. A host cell can be an invertebrate cell or derived from an invertebrate cell. A host cell can be a vertebrate cell or derived from a vertebrate cell. A host cell can be a microbe cell or derived from a microbe cell. A host cell can be a fungi cell or derived from a fungi cell. A host cell can be from a specific organ or tissue.

[0199] A host cell can be an immune cell, as abovementioned in the subject disclosure.

[0200] A host cell can be a stem cell or progenitor cell. Host cells can include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.). Host cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Clonal cells can comprise the progeny of a cell. A host cell can be in a living organism. A host cell can be a genetically modified cell.

[0201] A host cell can be a totipotent stem cell, however, in some embodiments of this disclosure, the term “cell” may be used but may not refer to a totipotent stem cell. A host cell can be a plant cell, but in some embodiments of this disclosure, the term “cell” may be used but may not refer to a plant cell. A host cell can be a pluripotent cell. For example, a host cell can be a pluripotent hematopoietic cell that can differentiate into other cells in the hematopoietic cell lineage but may not be able to differentiate into any other non-hematopoietic cell. A host cell may be able to develop into a whole organism. A host cell may or may not be able to develop into a whole organism. A host cell may be a whole organism.

[0202] A variety of one or more intrinsic signaling pathways (e.g. NFkB) of a cell are available for embodiments provided herein. Table 1 provides exemplary signaling pathways and genes associated with the signaling pathway. A signaling pathway activated by stimulant binding to a cell (e.g., an immune cell, a stem cell, etc.) and / or a ligand binding to a transmembrane receptor in embodiments provided herein can be any one of those provided in Table 1. In an example, a promoter activated to drive expression of the GMP upon binding of a stimulant to the stimulant binding domain of a transmembrane receptor in embodiments provided can comprise the promoter sequence driving any of the genes provided in Table 1, any variant of the promoter sequence, or any partial promoter sequence (e.g., a minimal promoter sequence).CELLULAR FUNCTIONGENESPI3K / AKT SignalingPRKCE; 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; PRKCD; 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; RPS6KB1ERK / MAPK SignalingPRKCE; 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; ETS1; 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; SGKGlucocorticoid Receptor SignalingRAC1; 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; JAK1; IL8; NCOA2; AKT1; JAK2;PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1;ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1;STAT1; IL6; HSP90AA1Axonal Guidance SignalingPRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12;IGF1; RAC1; RAP1A; E1F4E; PRKCZ; NRP1; NTRK2;ARHGEF7; SMO; ROCK2; MAPK1; PGF; RAC2;PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2;CFL1; GNAQ; PIK3CB; CXCL12; PIK3C3; WNT11;PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA;PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1;FYN; ITGB1; MAP2K2; PAK4; ADAM17; AKT1; PIK3R1;GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3;CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B;AKT3; PRKCAEphrin Receptor SignalingPRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1;PRKAA2; EIF2AK2; RAC1; RAP1A; GRK6; ROCK2;MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2;DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14;CXCL12; MAPK8; GNB2L1; ABL1; MAPK3; ITGA1;KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2;PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1;MAP2K2; PAK4, AKT1; JAK2; STAT3; ADAM10;MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2;EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13; ATF4;AKT3; SGKActin Cytoskeleton SignalingACTN4; PRKCE; ITGAM; ROCK1; ITGA5; IRAK1;PRKAA2; EIF2AK2; RAC1; INS; ARHGEF7; GRK6;ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8;PTK2; CFL1; PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8;F2R; MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD;PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7;PPP1CC; PXN; VIL2; RAF1; GSN; DYRK1A; ITGB1;MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1; PAK3;ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL;BRAF; VAV3; SGKHuntington's Disease SignalingPRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2;MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2;PIK3CA; HDAC5; CREB1; PRKC1; HSPA5; REST;GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1;GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2;HDAC7A; PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A;HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1;PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN; BAX;ATF4; AKT3; PRKCA; CLTC; SGK; HDAC6; CASP3Apoptosis SignalingPRKCE; ROCK1; BID; IRAK1; PRKAA2; EIF2AK2; BAK1;BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB;CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8;BCL2L1; CAPN1; MAPK3; CASP8; KRAS; RELA;PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF;RAF1; IKBKG; RELB; CASP9; DYRK1A; MAP2K2;CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA; CASP2;BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK;CASP3; BIRC3; PARP1B Cell Receptor SignalingRAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11;AKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A;MAP3K14; PIK3CB; PIK3C3; MAPK8; BCL2L1; ABL1;MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9;EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB;MAP3K7; MAP2K2; AKT1; PIK3R1; CHUK; MAP2K1;NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN;GSK3B; ATF4; AKT3; VAV3; RPS6KB1Leukocyte Extravasation SignalingACTN4; CD44; PRKCE; ITGAM; ROCK1; CXCR4; CYBA;RAC1; RAP1A; PRKCZ; ROCK2; RAC2; PTPN11;MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12;PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; CYBB;MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A; BTK;MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2;CTNND1; PIK3R1; CTNNB1; CLDN1; CDC42; F11R; ITK;CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9Integrin SignalingACTN4; 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; AKT3Acute Phase Response SignalingIRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11;AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14;PIK3CB; MAPK8; RIPK1; MAPK3; IL6ST; KRAS;MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1;TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1;IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1;CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN;AKT3; IL1R1; IL6PTEN SignalingITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11;MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA;CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1;MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR;RAF1; IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2;AKT1; PIK3R1; CHUK; PDGFRA; PDPK1; MAP2K1;NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2;GSK3B; AKT3; FOXO1; CASP3; RPS6KB1p53 SignalingPTEN; EP300; BBC3; PCAF; FASN; BRCA1; GADD45A;BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2;PIK3CB; PIK3C3; MAPK8; THBS1; ATR; BCL2L1; E2F1;PMAIP1; CHEK2; TNFRSF10B; TP73; RB1; HDAC9;CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A;HIPK2; AKT1; PIK3R1; RRM2B; APAF1; CTNNB1;SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN;SNAI2; GSK3B; BAX; AKT3Aryl Hydrocarbon Receptor SignalingHSPB1; EP300; FASN; TGM2; RXRA; MAPK1; NQO1;NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1;SMARCA4; NFKB2; MAPK8; ALDH1A1; ATR; E2F1;MAPK3; NRIP1; CHEK2; RELA; TP73; GSTP1; RB1;SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF;CDKN1A; NCOA2; APAF1; NFKB1; CCND1; ATM; ESR1;CDKN2A; MYC; JUN; ESR2; BAX; IL6; CYP1B1;HSP90AA1Xenobiotic Metabolism SignalingPRKCE; EP300; PRKCZ; RXRA; MAPK1; NQO1;NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A;PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1;ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13; PRKCD;GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL;NFE2L2; PIK3C2A; PPARGC1A; MAPK14; TNF; RAF1;CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1;NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1;HSP90AA1SAPK / JNK SignalingPRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1;GRK6; MAPK1; GADD45A; RAC2; PLK1; AKT2; PIK3CA;FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1;GNB2L1; IRS1; MAPK3; MAPK10; DAXX; KRAS;PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A;TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2;PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTK; CSNK1A1;CRKL; BRAF; SGKPPAr / RXR SignalingPRKAA2; EP300; INS; SMAD2; TRAF6; PPARA; FASN;RXRA; MAPK1; SMAD3; GNAS; IKBKB; NCOR2;ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8;IRS1; MAPK3; KRAS; RELA; PRKAA1; PPARGC1A;NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7;CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1;TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1;ADIPOQNF-KB SignalingIRAK1; EIF2AK2; EP300; INS; MYD88; PRKCZ: TRAF6;TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2;MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2;KRAS; RELA; PIK3C2A; TRAF2; TLR4: PDGFRB; TNF;INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1;PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10;GSK3B; AKT3; TNFAIP3; IL1R1Neuregulin SignalingERBB4; PRKCE; ITGAM; ITGA5: PTEN; PRKCZ; ELK1;MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI;CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KRAS;PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2;ADAM17; AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3;EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL;AKT3; PRKCA; HSP90AA1; RPS6KB1Wnt & Beta catenin SignalingCD44; EP300; LRP6; DVL3; CSNK1E; GJA1; SMO;AKT2; PIN1; CDH1; BTRC; GNAQ; MARK2; PPP2R1A;WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2: ILK;LEF1; SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1;PPP2R5C; WNT5A; LRP5; CTNNB1; TGFBR1; CCND1;GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B;AKT3; SOX2Insulin ReceptorPTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1;PTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3;MAPK8; IRS1; MAPK3; TSC2; KRAS; EIF4EBP1;SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN;MAP2K2; JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1;GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXO1; SGK;RPS6KB1IL-6 SignalingHSPB1; TRAF6; MAPKAPK2; ELK1; MAPK1; PTPN11;IKBKB; FOS; NFKB2: MAP3K14; MAPK8; MAPK3;MAPK10; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1;MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1; IKBKG;RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3;MAP2K1; NFKB1; CEBPB; JUN; IL1R1; SRF; IL6Hepatic CholestasisPRKCE; IRAK1; INS; MYD88; PRKCZ; TRAF6; PPARA;RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8;PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1;TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7; IL8;CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4;JUN; IL1R1; PRKCA; IL6IGF-1 SignalingIGF1; PRKCZ; ELK1; MAPK1; PTPN11; NEDD4; AKT2;PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8;IGF1R; IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A;YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1;PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3;FOXO1; SRF; CTGF; RPS6KB1NRF2-Mediated Oxidative Stress ResponsePRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1;NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8;PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL;NFE2L2; PIK3C2A; MAPK14; RAF1; MAP3K7; CREBBP;MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN; KEAP1;GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1Hepatic Fibrosis / Hepatic Stellate Cell ActivationEDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF;SMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9;IGF1R; IL6R; RELA; TLR4; PDGFRB; TNF; RELB; IL8;PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX;IL1R1; CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9PPAR SignalingEP300; INS; TRAF6; PPARA; RXRA; MAPK1; IKBKB;NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3;NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2;PPARGC1A; PDGFRB; TNF; INSR; RAF1; IKBKG;RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA;MAP2K1; NFKB1; JUN; IL1R1; HSP90AA1Fc Epsilon RI SignalingPRKCE; RAC1; PRKCZ; LYN; MAPK1; RAC2; PTPN11;AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; MAPK8;PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD;MAPK9; PIK3C2A; BTK; MAPK14; TNF; RAF1; FYN;MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3;VAV3; PRKCAG-Protein Coupled Receptor SignalingPRKCE; 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;PRKCAInositol Phosphate MetabolismPRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6;MAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3;MAPK8; MAPK3; PRKCD; PRKAA1; MAPK9; CDK2;PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1;MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGKPDGF SignalingEIF2AK2; ELK1; ABL2; MAPK1; PIK3CA; FOS; PIK3CB;PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS; SRC;PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2;PIK3R1; PDGFRA; STAT3; SPHK1; MAP2K1; MYC;JUN; CRKL; PRKCA; SRF; STAT1; SPHK2VEGF SignalingACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF;AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB; PIK3C3;BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN;RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN;VEGFA; AKT3; FOXO1; PRKCANatural Killer Cell SignalingPRKCE; RAC1; PRKCZ; MAPK1; RAC2; PTPN11;KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB;PIK3C3; PRKD1; MAPK3; KRAS; PRKCD; PTPN6;PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1;PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCACell Cycle: G1 / S Checkpoint RegulationHDAC4; SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC;ATR; ABL1; E2F1; HDAC2; HDAC7A; RB1; HDAC11;HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1;E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1;GSK3B; RBL1; HDAC6T Cell Receptor SignalingRAC1; 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; VAV3Death Receptor SignalingCRADD; HSPB1; BID; BIRC4; TBK1; IKBKB; FADD;FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8;DAXX; TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB;CASP9; CHUK; APAF1; NFKB1; CASP2; BIRC2; CASP3;BIRC3FGF SignalingRAC1; FGFR1; MET; MAPKAPK2; MAPK1; PTPN11;AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8;MAPK3; MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1;AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL; ATF4;AKT3; PRKCA; HGFGM-CSF SignalingLYN; ELK1; MAPK1; PTPN11; AKT2; PIK3CA; CAMK2A;STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3;ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2;AKT1; JAK2; PIK3R1; STAT3; MAP2K1; CCND1; AKT3;STAT1Amyotrophic Lateral Sclerosis SignalingBID; IGF1; RAC1; BIRC4; PGF; CAPNS1; CAPN2;PIK3CA; BCL2; PIK3CB; PIK3C3; BCL2L1; CAPN1;PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1;APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3JAK / Stat SignalingPTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B;PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1; STAT5A;PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1;AKT1; JAK2; PIK3R1; STAT3; MAP2K1; FRAP1; AKT3;STAT1Nicotinate and Nicotinamide MetabolismPRKCE; IRAK1; PRKAA2; EIF2AK2; GRK6; MAPK1;PLK1; AKT2; CDK8; MAPK8; MAPK3; PRKCD; PRKAA1;PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2;MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGKChemokine SignalingCXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ;CAMK2A; CXCL12; MAPK8; MAPK3; KRAS; MAPK13;RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1;MAP2K2; MAP2K1; JUN; CCL2; PRKCAIL-2 SignalingELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK; FOS;STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS;SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2;JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3Synaptic Long Term DepressionPRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1; GNAS;PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3;KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA;YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCAEstrogen Receptor SignalingTAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2;SMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1;HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP;MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2Protein Ubiquitination PathwayTRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4;CBL; UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7;USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8;USP1; VHL; HSP90AA1; BIRC3IL-10 SignalingTRAF6; CCR1; ELK1; IKBKB; SP1; FOS; NFKB2;MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF;IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1;JUN; IL1R1; IL6VDR / RXR ActivationPRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1;NCOR2; SP1; PRKC1; CDKN1B; PRKD1; PRKCD;RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1;LRP5; CEBPB; FOXO1; PRKCATGF-beta SignalingEP300; SMAD2; SMURF1; MAPK1; SMAD3; SMAD1;FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2;SERPINE1; RAF1; MAP3K7; CREBBP; MAP2K2;MAP2K1; TGFBR1; SMAD4; JUN; SMAD5Toll-like Receptor SignalingIRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1;IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13;RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK;NFKB1; TLR2; JUNp38 MAPK SignalingHSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1; FADD; FAS;CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2;MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1;SRF; STAT1Neurotrophin / TRK SignalingNTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS;PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A;RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1;CDC42; JUN; ATF4FXR / RXR ActivationINS; PPARA; FASN; RXRA; AKT2; SDC1; MAPK8;APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A;TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1Synaptic Long Term PotentiationPRKCE; RAP1A; EP300; PRKCZ; MAPK1; CREB1;PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS;PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1;ATF4; PRKCACalcium SignalingRAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1;CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11;HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4;HDAC6EGF SignalingELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3;MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1;STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1Hypoxia Signaling in the Cardiovascular SystemEDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT;HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM;VEGFA; JUN; ATF4; VHL; HSP90AA1LPS / IL-1 Mediated Inhibition of RXR FunctionIRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1,MAPK8; ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2;TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; IL1R1LXR / RXR ActivationFASN; RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA;NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1;SREBF1; IL1R1; CCL2; IL6; MMP9Amyloid ProcessingPRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CAPN2;CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1;PSEN1; CSNK1A1; GSK3B; AKT3; APPIL-4 SignalingAKT2; PIK3CA; PIK3CB; PIK3C3; IRS1; KRAS; SOCS1;PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1;FRAP1; AKT3; RPS6KB1Cell Cycle: G2 / M DNA Damage Checkpoint RegulationEP300; PCAF; BRCA1; GADD45A; PLK1; BTRC;CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A;PRKDC; ATM; SFN; CDKN2ANitric Oxide Signaling in the Cardiovascular SystemKDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3;CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1;VEGFA; AKT3; HSP90AA1Purine MetabolismNME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4;PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C;NT5E; POLD1; NME1cAMP-mediated SignalingRAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3;SRC; RAF1; MAP2K2; STAT3; MAP2K1; BRAF; ATF4Mitochondrial DysfunctionSOD2; MAPK8; CASP8; MAPK10; MAPK9; CASP9;PARK7; PSEN1; PARK2; APP; CASP3Notch SignalingHES1; JAG1; NUMB; NOTCH4; ADAM17; NOTCH2;PSEN1; NOTCH3; NOTCH1; DLL4Endoplasmic Reticulum Stress PathwayHSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4;EIF2AK3; CASP3Pyrimidine MetabolismNME2; AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B;NT5E; POLD1; NME1Parkinson's SignalingUCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7;PARK2; CASP3Cardiac & BetaAdrenergic SignalingGNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC;PPP2R5CGlycolysis / GluconeogenesisHK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1Interferon SignalingIRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; IFIT3Sonic Hedgehog SignalingARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B; DYRKIBGlycerophospholipid MetabolismPLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2Phospholipid DegradationPRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2Tryptophan MetabolismSIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; SIAH1Lysine DegradationSUV39H1; EHMT2; NSD1; SETD7; PPP2R5CNucleotide Excision Repair PathwayERCC5; ERCC4; XPA; XPC; ERCC1Starch and Sucrose MetabolismUCHL1; HK2; GCK; GPI; HK1Aminosugars MetabolismNQO1; HK2; GCK; HK1Arachidonic Acid MetabolismPRDX6; GRN; YWHAZ; CYP1B1Circadian Rhythm SignalingCSNK1E; CREB1; ATF4; NR1D1Coagulation SystemBDKRB1; F2R; SERPINE1; F3Dopamine Receptor SignalingPPP2R1A; PPP2CA; PPP1CC; PPP2R5CGlutathione MetabolismIDH2; GSTP1; ANPEP; IDH1Glycerolipid MetabolismALDH1A1; GPAM; SPHK1; SPHK2Linoleic Acid MetabolismPRDX6; GRN; YWHAZ; CYP1B1Methionine MetabolismDNMT1; DNMT3B; AHCY; DNMT3APyruvate MetabolismGLO1; ALDH1A1; PKM2; LDHAArginine and Proline MetabolismALDH1A1; NOS3; NOS2AEicosanoid SignalingPRDX6; GRN; YWHAZFructose and Mannose MetabolismHK2; GCK; HK1Galactose MetabolismHK2; GCK; HK1Stilbene, Coumarine and Lignin BiosynthesisPRDX6; PRDX1; TYRAntigen Presentation PathwayCALR; B2MBiosynthesis of SteroidsNQO1; DHCR7Butanoate MetabolismALDH1A1; NLGN1Citrate CycleIDH2; IDH1Fatty Acid MetabolismALDH1A1; CYP1B1Glycerophospholipid MetabolismPRDX6; CHKAHistidine MetabolismPRMT5; ALDH1A1Inositol MetabolismERO1L; APEX1Metabolism of Xenobiotics by Cytochrome p450GSTP1; CYP1B1Methane MetabolismPRDX6; PRDX1Phenylalanine MetabolismPRDX6; PRDX1Propanoate MetabolismALDH1A1; LDHASelenoamino Acid MetabolismPRMT5; AHCYSphingolipid MetabolismSPHK1; SPHK2Aminophosphonate MetabolismPRMT5Androgen and Estrogen MetabolismPRMT5Ascorbate and Aldarate MetabolismALDH1A1Bile Acid BiosynthesisALDH1A1Cysteine MetabolismLDHAFatty Acid BiosynthesisFASNGlutamate Receptor SignalingGNB2L1NRF2-mediated Oxidative Stress ResponsePRDX1Pentose Phosphate PathwayGPIPentose and Glucuronate InterconversionsUCHL1Retinol MetabolismALDH1A1Riboflavin MetabolismTYRTyrosine MetabolismPRMT5, TYRUbiquinone BiosynthesisPRMT5Valine, Leucine and Isoleucine DegradationALDH1A1Glycine, Serine and Threonine MetabolismCHKALysine DegradationALDH1A1Pain / TasteTRPM5; TRPA1PainTRPM7; TRPC5; TRPC6; TRPC1; Cnr1; cnr2; Grk2;Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5; Prkaca;Prkacb; Prkar1a; Prkar2aMitochondrial FunctionAIF; CytC; SMAC (Diablo); Aifm-1; Aifm-2Developmental NeurologyBMP-4; Chordin (Chrd); Noggin (Nog); WNT (Wnt2;Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b; Wnt8b;Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin;Dkk-1; Frizzled related proteins; Otx-2; Gbx2; FGF-8;Reelin; Dab1; unc-86 (Pou4fl or Brn3a); Numb; Reln

[0203] Systems and compositions of the present disclosure are useful for a variety of applications. For example, systems and methods of the present disclosure are useful in methods of regulating gene expression and / or cellular activity. In an aspect, the systems and compositions disclosed herein are utilized in methods of regulating gene expression and / or cellular activity in an immune cell. Immune cells regulated using a subject system can be useful in a variety of applications, including, but not limited to, immunotherapy to treat diseases and disorders. Diseases and disorders that can be treated using modified immune cells of the present disclosure include inflammatory conditions, cancer, and infectious diseases. In some embodiments, immunotherapy is used to treat cancer.

[0204] A variety of target cells can be killed using the systems and methods of the subject disclosure. A target cell to which this method can be applied includes a wide variety of cell types. A target cell can bein vitro. A target cell can bein vivo. A target cell can beex vivo. A target cell can be an isolated cell. A target cell can be a cell inside of an organism. A target cell can be an organism. A target cell can be a cell in a cell culture. A target cell can be one of a collection of cells. A target cell can be a mammalian cell or derived from a mammalian cell. A target cell can be a rodent cell or derived from a rodent cell. A target cell can be a human cell or derived from a human cell. A target cell can be a prokaryotic cell or derived from a prokaryotic cell. A target cell can be a bacterial cell or can be derived from a bacterial cell. A target cell can be an archaeal cell or derived from an archaeal cell. A target cell can be a eukaryotic cell or derived from a eukaryotic cell. A target cell can be a pluripotent stem cell. A target cell can be a plant cell or derived from a plant cell. A target cell can be an animal cell or derived from an animal cell. A target cell can be an invertebrate cell or derived from an invertebrate cell. A target cell can be a vertebrate cell or derived from a vertebrate cell. A target cell can be a microbe cell or derived from a microbe cell. A target cell can be a fungi cell or derived from a fungi cell. A target cell can be from a specific organ or tissue.

[0205] A target cell can be a stem cell or progenitor cell. Target cells can include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.). Target cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Clonal cells can comprise the progeny of a cell. A target cell can comprise a target nucleic acid. A target cell can be in a living organism. A target cell can be a genetically modified cell. A target cell can be a host cell.

[0206] A target cell can be a totipotent stem cell, however, in some embodiments of this disclosure, the term “cell” may be used but may not refer to a totipotent stem cell. A target cell can be a plant cell, but in some embodiments of this disclosure, the term “cell” may be used but may not refer to a plant cell. A target cell can be a pluripotent cell. For example, a target cell can be a pluripotent hematopoietic cell that can differentiate into other cells in the hematopoietic cell lineage but may not be able to differentiate into any other non-hematopoietic cell. A target cell may be able to develop into a whole organism. A target cell may or may not be able to develop into a whole organism. A target cell may be a whole organism.

[0207] A target cell can be a primary cell. For example, cultures of primary cells can be passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15 times or more. Cells can be unicellular organisms. Cells can be grown in culture.

[0208] A target cell can be a diseased cell. A diseased cell can have altered metabolic, gene expression, and / or morphologic features. A diseased cell can be a cancer cell, a diabetic cell, and a apoptotic cell. A diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.

[0209] If the target cells are primary cells, they may be harvested from an individual by any method. For example, leukocytes may be harvested by apheresis, leukocytapheresis, density gradient separation, etc. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. An appropriate solution may be used for dispersion or suspension of the harvested cells. Such solution can generally be a balanced salt solution, (e.g. normal saline, phosphate-buffered saline (PBS), Hank’s balanced salt solution, etc.), conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration. Buffers can include HEPES, phosphate buffers, lactate buffers, etc. Cells may be used immediately, or they may be stored (e.g., by freezing). Frozen cells can be thawed and can be capable of being reused. Cells can be frozen in a DMSO, serum, medium buffer (e.g., 10% DMSO, 50% serum, 40% buffered medium), and / or some other such common solution used to preserve cells at freezing temperatures.

[0210] Non-limiting examples of cells which can be target cells include, but are not limited to, lymphoid cells, such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell), Natural killer cell, cytokine induced killer (CIK) cells (see e.g. US20080241194); myeloid cells, such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte / Hypersegmented neutrophil), Monocyte / Macrophage, Red blood cell (Reticulocyte), Mast cell, Thrombocyte / Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell), parathyroid (Parathyroid chief cell, Oxyphil cell), adrenal (Chromaffin cell), pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia), Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope, Corticotrope, Thyrotrope, Somatotrope, Lactotroph ); cells of the Respiratory system, including Pneumocyte (Type I pneumocyte, Type II pneumocyte), Clara cell, Goblet cell, Dust cell; cells of the circulatory system, including Myocardiocyte, Pericyte; cells of the digestive system, including stomach (Gastric chief cell, Parietal cell), Goblet cell, Paneth cell, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffm cell, APUD cell, liver (Hepatocyte, Kupffer cell), Cartilage / bone / muscle; bone cells, including Osteoblast, Osteocyte, Osteoclast, teeth (Cementoblast, Ameloblast); cartilage cells, including Chondroblast, Chondrocyte; skin cells, including Trichocyte, Keratinocyte, Melanocyte (Nevus cell); muscle cells, including Myocyte; urinary system cells, including Podocyte, Juxtaglomerular cell, Intraglomerular mesangial cell / Extraglomerular mesangial cell, Kidney proximal tubule brush border cell, Macula densa cell; reproductive system cells, including Spermatozoon, Sertoli cell, Leydig cell, Ovum; and other cells, including Adipocyte, Fibroblast, Tendon cell, Epidermal keratinocyte (differentiating epidermal cell), Epidermal basal cell (stem cell), Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell), Medullary hair shaft cell, Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair root sheath cell, Hair root sheath cell of Huxley's layer, Hair root sheath cell of Henle's layer, External hair root sheath cell, Hair matrix cell (stem cell), Wet stratified barrier epithelial cells, Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Urinary epithelium cell (lining urinary bladder and urinary ducts), Exocrine secretory epithelial cells, Salivary gland mucous cell (polysaccharide-rich secretion), Salivary gland serous cell (glycoprotein enzyme -rich secretion), Von Ebner's gland cell in tongue (washes taste buds), Mammary gland cell (milk secretion), Lacrimal gland cell (tear secretion), Ceruminous gland cell in ear (wax secretion), Eccrine sweat gland dark cell (glycoprotein secretion), Eccrine sweat gland clear cell (small molecule secretion). Apocrine sweat gland cell (odoriferous secretion, sex -hormone sensitive), Gland of Moll cell in eyelid (specialized sweat gland), Sebaceous gland cell (lipid-rich sebum secretion), Bowman's gland cell in nose (washes olfactory epithelium), Brunner's gland cell in duodenum (enzymes and alkaline mucus), Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm), Prostate gland cell (secretes seminal fluid components), Bulbourethral gland cell (mucus secretion), Bartholin's gland cell (vaginal lubricant secretion), Gland of Littre cell (mucus secretion), Uterus endometrium cell (carbohydrate secretion), Isolated goblet cell of respiratory and digestive tracts (mucus secretion), Stomach lining mucous cell (mucus secretion), Gastric gland zymogenic cell (pepsinogen secretion), Gastric gland oxyntic cell (hydrochloric acid secretion), Pancreatic acinar cell (bicarbonate and digestive enzyme secretion), Paneth cell of small intestine (lysozyme secretion), Type II pneumocyte of lung (surfactant secretion), Clara cell of lung, Hormone secreting cells, Anterior pituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary cell, Magnocellular neurosecretory cells, Gut and respiratory tract cells, Thyroid gland cells, thyroid epithelial cell, parafollicular cell, Parathyroid gland cells, Parathyroid chief cell, Oxyphil cell, Adrenal gland cells, chromaffin cells, Ley dig cell of testes, Theca interna cell of ovarian follicle, Corpus luteum cell of ruptured ovarian follicle, Granulosa lutein cells, Theca lutein cells, Juxtaglomerular cell (renin secretion), Macula densa cell of kidney, Metabolism and storage cells, Barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract), Kidney, Type I pneumocyte (lining air space of lung), Pancreatic duct cell (centroacinar cell), Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc.), Duct cell (of seminal vesicle, prostate gland, etc.), Epithelial cells lining closed internal body cavities, Ciliated cells with propulsive function, Extracellular matrix secretion cells, Contractile cells; Skeletal muscle cells, stem cell, Heart muscle cells, Blood and immune system cells, Erythrocyte (red blood cell), Megakaryocyte (platelet precursor), Monocyte, Connective tissue macrophage (various types), Epidermal Langerhans cell, Osteoclast (in bone), Dendritic cell (in lymphoid tissues), Microglial cell (in central nervous system), Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural Killer T cell, B cell, Natural killer cell, Reticulocyte, Stem cells and committed progenitors for the blood and immune system (various types), Pluripotent stem cells, Totipotent stem cells, Induced pluripotent stem cells, adult stem cells, Sensory transducer cells, Autonomic neuron cells, Sense organ and peripheral neuron supporting cells, Central nervous system neurons and glial cells, Lens cells, Pigment cells, Melanocyte, Retinal pigmented epithelial cell, Germ cells, Oogonium / Oocyte, Spermatid, Spermatocyte, Spermatogonium cell (stem cell for spermatocyte), Spermatozoon, Nurse cells, Ovarian follicle cell, Sertoli cell (in testis), Thymus epithelial cell, Interstitial cells, and Interstitial kidney cells.

[0211] Of particular interest are cancer cells. In some embodiments, the target cell is a cancer cell. Non-limiting examples of cancer cells include cells of cancers including Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia,Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the targeted cancer cell represents a subpopulation within a cancer cell population, such as a cancer stem cell. In some embodiments, the cancer is of a hematopoietic lineage, such as a lymphoma. The antigen can be a tumor associated antigen.

[0212] In some embodiments, the target cells form a tumor. A tumor treated with the methods herein can result in stabilized tumor growth (e.g., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20% in size, and / or do not metastasize). In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. In some embodiments, the size of a tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the tumor is completely eliminated, or reduced below a level of detection. In some embodiments, a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.

[0213] Death of target cells can be determined by any suitable method, including, but not limited to, counting cells before and after treatment, or measuring the level of a marker associated with live or dead cells (e.g. live or dead target cells). Degree of cell death can be determined by any suitable method. In some embodiments, degree of cell death is determined with respect to a starting condition. For example, an individual can have a known starting amount of target cells, such as a starting cell mass of known size or circulating target cells at a known concentration. In such cases, degree of cell death can be expressed as a ratio of surviving cells after treatment to the starting cell population. In some embodiments, degree of cell death can be determined by a suitable cell death assay. A variety of cell death assays are available, and can utilize a variety of detection methodologies. Examples of detection methodologies include, without limitation, the use of cell staining, microscopy, flow cytometry, cell sorting, and combinations of these.

[0214] When a tumor is subject to surgical resection following completion of a therapeutic period, the efficacy of treatment in reducing tumor size can be determined by measuring the percentage of resected tissue that is necrotic (i.e., dead). In some embodiments, a treatment is therapeutically effective if the necrosis percentage of the resected tissue is greater than about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the necrosis percentage of the resected tissue is 100%, that is, no living tumor tissue is present or detectable.

[0215] Exposing a target cell to an immune cell or population of immune cells disclosed herein can be conducted eitherin vitroorin vivo. Exposing a target cell to an immune cell or population of immune cells generally refers to bringing the target cell in contact with the immune cell and / or in sufficient proximity such that an antigen of a target cell (e.g., membrane bound or non-membrane bound) can bind to the ligand interacting domain of the chimeric transmembrane receptor polypeptide expressed in the immune cell. Exposing a target cell to an immune cell or population of immune cellsin vitrocan be accomplished by co-culturing the target cells and the immune cells. Target cells and immune cells can be co-cultured, for example, as adherent cells or alternatively in suspension. Target cells and immune cells can be co-cultured in various suitable types of cell culture media, for example with supplements, growth factors, ions, etc. Exposing a target cell to an immune cell or population of immune cellsin vivocan be accomplished, in some cases, by administering the immune cells to a subject, for example a human subject, and allowing the immune cells to localize to the target cell via the circulatory system. In some cases, an immune cell can be delivered to the immediate area where a target cell is localized, for example, by direct injection.

[0216] Exposing can be performed for any suitable length of time, for example at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or longer.

[0217] In some embodiments, cells expressing a system provided herein induce death of a target cell in anin vitrocell death assay. The cells expressing a system provided herein may exhibit enhanced ability to induce death of the target cell compared to control cells not expressing a system of the present disclosure. In some cases, the enhanced ability to induce death of the target cell is at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 5-fold, 10-fold, 100-fold, or 1000-fold increase in induced cell death. The degree of induced cell death can be determined at any suitable time point, for example, at least 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 hours, or 52 hours after contacting the cell to the target cell.

[0218] In some embodiments, a target polynucleotide can comprise one or more disease-associated genes and polynucleotides as well as signaling biochemical pathway-associated genes and polynucleotides. Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissue compared with tissue(s) or cells of a non-disease control. In some embodiments, it is a gene that becomes expressed at an abnormally high level. In some embodiments, it is a gene that becomes expressed at an abnormally low level. The altered expression can correlate with the occurrence and / or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is response for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level.

[0219] Examples of disease-associated genes and polynucleotides are available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web. Exemplary genes associated with certain diseases and disorders are provided in Tables 2 and 3.

[0220] Mutations in these genes and pathways can result in production of improper proteins or proteins in improper amounts which affect function.

[0221] Promoters that can be used with the methods and compositions of the disclosure include, for example, promoters active in a eukaryotic, mammalian, non-human mammalian or human cell. The promoter can be an inducible or constitutively active promoter. Alternatively or additionally, the promoter can be tissue or cell specific. The promoter can be native or composite promoter.

[0222] Non-limiting examples of suitable eukaryotic promoters (i.e. promoters functional in a eukaryotic cell) can include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-1 promoter (EF1), ubiquitin B promoter (UB), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-active promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK) and mouse metallothionein-I. The promoter can be cell, tissue or tumor specific, such as CD45 promoter, AFP promoter, human Albumin promoter (Alb), MUC1 promoter, COX2 promoter, SP-B promoter, OG-2 promoter. The promoter can be a fungi promoter. The promoter can be a plant promoter. A database of plant promoters can be found (e.g., PlantProm). The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. Another example of a promoter for the expression vector may include myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter. A promoter for driving RNA can include RNA Pol III promoters (e.g., U6 or H1), Pol II promoters, and / or tRNA(val) promoter.DISEASE / DISORDERSGENE(S)NeoplasiaPTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4;Notch1; Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF;HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPARgamma; WT1 (Wilms Tumor); FGF Receptor Familymembers (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB(retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR(Androgen Receptor); TSG101; IGF; IGF Receptor; Igf1 (4variants); Igf2 (3 variants); Igf 1 Receptor; Igf 2 Receptor;Bax; Bcl2; caspases family (9 members:1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; ApcAge-related Macular DegenerationAbcr; Ccl2; Cc2; cp (ceruloplasmin); Timp3; cathepsinD;Vldlr; Ccr2SchizophreniaNeuregulin1 (Nrg1); Erb4 (receptor for Neuregulin);Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a;GSK3bDisorders5-HTT (Slc6a4); COMT; DRD (Drd1a); SLC6A3; DAOA;DTNBP1; Dao (Dao1)Trinucleotide Repeat DisordersHTT (Huntington's Dx); SBMA / SMAX1 / AR (Kennedy'sDx); FXN / X25 (Friedrich's Ataxia); ATX3 (Machado-Joseph's Dx); ATXN1 and ATXN2 (spinocerebellarataxias); DMPK (myotonic dystrophy); Atrophin-1 and Atn1(DRPLA Dx); CBP (Creb-BP - global instability); VLDLR(Alzheimer's); Atxn7; Atxn10Fragile X SyndromeFMR2; FXR1; FXR2; mGLUR5Secretase Related DisordersAPH-1 (alpha and beta); Presenilin (Psen1); nicastrin(Ncstn); PEN-2OthersNos1; Parp1; Nat1; Nat2Prion - related disordersPrpALSSOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a;VEGF-b; VEGF-c)Drug addictionPrkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2;Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol)AutismMecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X(FMR2 (AFF2); FXR1; FXR2; Mglur5)Alzheimer's DiseaseE1; CHIP; UCH; UBB; Tau; LRP; PICALM; Clusterin; PS1;SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28 (Aqp1,Aquaporin 1); Uchl1; Uchl3; APPInflammationIL-10; IL-1 (IL-1a; IL-1b); IL-13; IL-17 (IL-17a (CTLA8); IL-17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1; ptpn22; TNFa;NOD2 / CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b);CTLA4; Cx3cl1Parkinson's Diseasex-Synuclein; DJ-1; LRRK2; Parkin; PINK1Blood and coagulation diseases and disordersAnemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1,PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB,ABCB7, ABC7, ASAT); Bare lymphocyte syndrome (TAPBP, TPSN,TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP,RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor H andfactor H-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2);Factor VII deficiency (F7); Factor X deficiency (F10); Factor XIdeficiency (F11); Factor XII deficiency (F12, HAF); Factor XIIIAdeficiency (F13A1, F13A); Factor XIIIB deficiency (F13B); Fanconianemia (FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064,FANCB, FANCC, FACC, BRCA2, FANCD1, FANCD2, FANCD,FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1,BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596);Hemophagocytic lymphohistiocytosis disorders (PRF1, HPLH2,UNC13D, MUNC13-4, HPLH3, HLH3, FHL3); Hemophilia A (F8, F8C,HEMA); Hemophilia B (F9, HEMB), Hemorrhagic disorders (PI, ATT,F5); Leukocyde deficiencies and disorders (ITGB2, CD18, LCAMB,LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH,CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia (HBA2, HBB,HBD, LCRB, HBA1).Cell dysregulation and oncology diseases and disordersB-cell non-Hodgkin lymphoma (BCL7A, BCL7); Leukemia (TAL1TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1,HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2,GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, CLTH,CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214,D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3,FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B,AF10, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML,PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11, PTP2C, SHP2,NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1,NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN).Inflammation and immune related diseases and disordersAIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12,SDF1); Autoimmune lymphoproliferative syndrome (TNFRSF6, APT1,FAS, CD95, ALPS1A); Combined immunodeficiency, (IL2RG,SCIDX1, SCIDX, IMD4); HIV-1 (CCL5, SCYA5, D17S136E, TCP228),HIV susceptibility or infection (IL10, CSIF, CMKBR2, CCR2,CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G,AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4,TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX,TNFRSF14B, TACI); Inflammation (IL-10, IL-1 (IL-1a, IL-1b), IL-13,IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1,ptpn22, TNFa, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b),CTLA4, Cx3cl1); Severe combined immunodeficiencies (SCIDs)(JAK3,JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC,CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4).Metabolic, liver, kidney and protein diseases and disordersAmyloid neuropathy (TTR, PALB); Amyloidosis (APOA1, APP, AAA,CVAP, AD1, GSN, FGA, LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8,CIRH1A, NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7,CF, MRP7); Glycogen storage diseases (SLC2A2, GLUT2, G6PC,G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2,PYGL, PFKM); Hepatic adenoma, 142330 (TCF1, HNF1A, MODY3),Hepatic failure, early onset, and neurologic disorder (SCOD1, SCO1),Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancer andcarcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN,CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5;Medullary cystic kidney disease (UMOD, HNFJ, FJHN, MCKD2,ADMCKD2); Phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS);Polycystic kidney and hepatic disease (FCYT, PKHD1, ARPKD, PKD1,PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63).Muscular / Skeletal diseases and disordersBecker muscular dystrophy (DMD, BMD, MYF6), Duchenne MuscularDystrophy (DMD, BMD); Emery-Dreifuss muscular dystrophy (LMNA,LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA, LMN1,EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy(FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C,LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD,TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C,DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB,LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G,CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN,CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN,RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1, LRP7,LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1,TIRC7, OC116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8,SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1,CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1).Neurological and neuronal diseases and disordersALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b,VEGF-c); Alzheimer disease (APP, AAA, CVAP, AD1, APOE, AD2,PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE,DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH, BMH,PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4,KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2,mGLUR5); Huntington's disease and disease like disorders (HD, IT15,PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease(NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA,NACP, PARK1, PARK4, DJ1, PARK7, LRRK2, PARK8, PINK1,PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN,PARK2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2, RTT, PPMX,MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16,MRX79, x-Synuclein, DJ-1); Schizophrenia (Neuregulin1 (Nrg1), Erb4(receptor for Neuregulin), Complexin1 (Cplx1), Tph1 Tryptophanhydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3,GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3,DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders (APH-1(alpha and beta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2, Nos1,Parp1, Nat1, Nat2); Trinucleotide Repeat Disorders (HTT (Huntington'sDx), SBMA / SMAX1 / AR (Kennedy's Dx), FXN / X25 (Friedrich'sAtaxia), ATX3 (Machado- Joseph's Dx), ATXN1 and ATXN2(spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-1 andAtn1 (DRPLA Dx), CBP (Creb-BP - global instability), VLDLR(Alzheimer's), Atxn7, Atxn10).Ocular diseases and disordersAge-related macular degeneration (Abcr, Ccl2, Cc2, cp (ceruloplasmin),Timp3, cathepsinD, Vldlr, Ccr2); Cataract (CRYAA, CRYA1, CRYBB2,CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1, PAX6, AN2,MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19,CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM,MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4,CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYA1, GJA8,CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1);Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1,CSD, BIGH3, CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD,PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD); Cornea planacongenital (KERA, CNA2); Glaucoma (MYOC, TIGR, GLC1A, JOAG,GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1,NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1,RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20,AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3);Macular dystrophy (ELOVL4, ADMD, STGD2, STGD3, RDS, RP7,PRPH2, PRPH, AVMD, AOFMD, VMD2).

[0223] Systems and compositions of the present disclosure are useful for other varieties of applications. For example, systems and methods of the present disclosure are useful in methods of regulating gene expression and / or cellular activity critical for cell proliferation, differentiation, trans-differentiation, and / or de-differentiation during tissue (e.g., an organ) growth, repair, regeneration, and or engineering. Examples of the tissue include epithelial, connective, nerve, muscle, organ, and other tissues. Other exemplary tissues include artery, ligament, skin, tendon, kidney, nerve, liver, pancreas, bladder, bone, lung, blood vessels, heart valve, cartilage, eyes, etc.

[0224] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0225] Various aspects of the disclosure are further illustrated by the following non-limiting examples.Examples

[0226] Example 1:Constitutive expression of a system comprising a GMP in γδ T cells.

[0227] FIGs. 1A-1B schematically illustrate expression of a system comprising a GMP in γδ T cells. One or more components of the system may be expressed constitutively in the γδ T cells. The one or more components of the system comprising the GMP may be under the control of an exogenous promoter. The system may be expressed in the γδ T cells for a cancer / tumor antigen mediated regulation of gene regulation of the γδ T cells.

[0228] FIG. 1A schematically illustrates example vectors (e.g., viral vectors) to be used for the subject systems and methods. Each vector may comprise polynucleotides encoding a chimeric polypeptide (e.g., a chimeric receptor polypeptide and / or a chimeric adaptor polypeptide) and / or a guide RNA (e.g., sgRNA) that is configured to bind a target gene (e.g., a target DNA sequence) in a cell. Vector 1 may comprise a chimeric receptor polypeptide operatively linked to MND promoter. The chimeric receptor polypeptide of vector 1 may comprise a CAR linked a protease (e.g., TEV). The chimeric receptor polypeptide of vector 1 may also comprise a posttranscriptional regulatory element (PRE) (e.g., Woodchuck hepatitis virus PRE (WPRE)) linked to the protease.

[0229] Vector 2a may comprise a chimeric adaptor polypeptide operatively linked to an exogenous MND promoter. The chimeric adaptor polypeptide of may comprise an adaptor (e.g., linker for activation of T cells (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds a portion (e.g., a extracellular, transmembrane, and / or intracellular domain) of the chimeric receptor polypeptide, and / or (ii) moves in proximity to the chimeric receptor polypeptide. The adaptor is linked to a cleavage recognition site (e.g., TEV cutting site (TCS)). The chimeric adaptor polypeptide may also comprise a GMP linked to the cleavage recognition site, wherein the GMP comprises an actuator moiety (e.g., a dCas9 fused with a gene repressor, such as KRAB). The chimeric adaptor polypeptide may also comprise a PRE (e.g., WPRE) linked to the GMP. Furthermore, vector 2a may comprise a guide RNA (e.g., a sgRNA operable to target a portion of a gene encoding for CD39) operatively linked to U6 promoter. The U6 promoter may be disposed downstream of the MND promoter and the genes of interest that are operatively linked to the MND promoter.

[0230] Vector 2b may comprise a chimeric adaptor polypeptide operatively linked to an exogenous MND promoter. The chimeric adaptor polypeptide of may comprise an adaptor (e.g., linker for activation of T cells (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds a portion (e.g., a extracellular, transmembrane, and / or intracellular domain) of the chimeric receptor polypeptide, and / or (ii) moves in proximity to the chimeric receptor polypeptide. The adaptor is linked to a cleavage recognition site (e.g., TEV cutting site (TCS)). The chimeric adaptor polypeptide may also comprise a GMP linked to the cleavage recognition site, wherein the GMP comprises an actuator moiety (e.g., a dCas9 fused with a gene activator, such as VP64, p65, SAM, VPR, etc). The chimeric adaptor polypeptide may also comprise a PRE (e.g., WPRE) linked to the GMP. Furthermore, vector 2b may comprise a guide RNA (e.g., a sgRNA operable to target a portion of a gene encoding for Serpin B4 and / or Serpin B9) operatively linked to U6 promoter. The U6 promoter may be disposed downstream of the MND promoter and the genes of interest that are operatively linked to the MND promoter.

[0231] Vector 2c may comprise a chimeric adaptor polypeptide operatively linked to an exogenous MND promoter. The chimeric adaptor polypeptide of may comprise an adaptor (e.g., linker for activation of T cells (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds a portion (e.g., a extracellular, transmembrane, and / or intracellular domain) of the chimeric receptor polypeptide, and / or (ii) moves in proximity to the chimeric receptor polypeptide. The adaptor is linked to a cleavage recognition site (e.g., TEV cutting site (TCS)). The chimeric adaptor polypeptide may also comprise a GMP linked to the cleavage recognition site, wherein the GMP comprises an actuator moiety (e.g., a dCas9 fused with a gene activator, such as VP64, p65, SAM, VPR, etc). The chimeric adaptor polypeptide may also comprise a PRE (e.g., WPRE) linked to the GMP. Furthermore, vector 2c may comprise a guide RNA (e.g., a sgRNA operable to target a portion of a gene encoding for c-FLIP) operatively linked to U6 promoter. The U6 promoter may be disposed downstream of the MND promoter and the genes of interest that are operatively linked to the MND promoter.

[0232] Referring to FIG. 1B, the γδ T cell may express the γδ TCR complex. In some cases, the γδ T cell may also express NKG2D and its associating adaptor DAP10. The γδ T cell may be administered with a chimeric receptor polypeptide comprising a cancer / tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a γδ T cell signaling domain and TEV. The γδ T cell may also be administered with a chimeric adaptor polypeptide comprising an adaptor that binds and / or moves in proximity to the chimeric receptor polypeptide (e.g., to the cellular signaling domain of the chimeric receptor polypeptide) and a GMP comprising an actuator moiety. The chimeric receptor and adaptor may be administered using a viral vector. The chimeric receptor and adaptor may be expressed constitutively in the γδ T cell. Upon binding of the tumor antigen to the antigen binding domain of the chimeric receptor polypeptide, the chimeric adaptor polypeptide may move in proximity to the chimeric receptor polypeptide, wherein the TEV of the chimeric receptor polypeptide can cleave the actuator moiety from the GMP at a cleavage recognition site of the chimeric receptor polypeptide. The released actuator moiety may translocate to the nucleus of the γδ T cell to (i) downregulate expression of CD39 and / or (ii) upregulate expression of SerpinB9 / 4 and / or cFLIP.

[0233] Example 2:Bisphosphonate-induced expression of a system comprising a GMP in γδ T cells.

[0234] FIGs. 2A-2B schematically illustrate bisphosphonate (e.g., zolendronate)-induced expression of a system comprising a GMP in γδ T cells. One or more components of the system may be expressed constitutively in the γδ T cells. One or more components of the system may be conditionally expressed, either directly or indirectly by the bisphosphonate. One or more components of the system comprising the GMP may be under the control of an exogenous promoter. The system may be expressed in the γδ T cells for a cancer / tumor antigen mediated regulation of gene regulation of the γδ T cells.

[0235] FIG. 2A schematically illustrates example vectors (e.g., viral vectors) to be used for the subject systems and methods. Each vector may comprise polynucleotides encoding a chimeric polypeptide (e.g., a chimeric receptor polypeptide and / or a chimeric adaptor polypeptide) and / or a guide RNA (e.g., sgRNA) that is configured to bind a target gene (e.g., a target DNA sequence) in a cell. Vector 1 may comprise a chimeric receptor polypeptide operatively linked to MND promoter. The chimeric receptor polypeptide of vector 1 may comprise a CAR linked a protease (e.g., TEV). The chimeric receptor polypeptide of vector 1 may also comprise a posttranscriptional regulatory element (PRE) (e.g., Woodchuck hepatitis virus PRE (WPRE)) linked to the protease.

[0236] Vector 2a may comprise a chimeric adaptor polypeptide operatively linked to an exogenous promoter, such asLAG3promoter orFASpromoter. The exogenous promoter may be an immune checkpoint of the γδ T cells. The chimeric adaptor polypeptide of may comprise an adaptor (e.g., linker for activation of T cells (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds a portion (e.g., a extracellular, transmembrane, and / or intracellular domain) of the chimeric receptor polypeptide, and / or (ii) moves in proximity to the chimeric receptor polypeptide. The adaptor is linked to a cleavage recognition site (e.g., TEV cutting site (TCS)). The chimeric adaptor polypeptide may also comprise a GMP linked to the cleavage recognition site, wherein the GMP comprises an actuator moiety (e.g., a dCas9 fused with a gene repressor, such as KRAB). The chimeric adaptor polypeptide may also comprise a PRE (e.g., WPRE) linked to the GMP. Furthermore, vector 2a may comprise a guide RNA (e.g., a sgRNA operable to target a portion of a gene encoding for CD39) operatively linked to U6 promoter. The U6 promoter may be disposed downstream of theLAG3promoter orFASpromoter and the genes of interest that are operatively linked to theLAG3promoter orFASpromoter.

[0237] Vector 2b may comprise a chimeric adaptor polypeptide operatively linked to an exogenous promoter, such asLAG3promoter orFASpromoter. The exogenous promoter may be an immune checkpoint of the γδ T cells. The chimeric adaptor polypeptide of may comprise an adaptor (e.g., linker for activation of T cells (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds a portion (e.g., a extracellular, transmembrane, and / or intracellular domain) of the chimeric receptor polypeptide, and / or (ii) moves in proximity to the chimeric receptor polypeptide. The adaptor is linked to a cleavage recognition site (e.g., TEV cutting site (TCS)). The chimeric adaptor polypeptide may also comprise a GMP linked to the cleavage recognition site, wherein the GMP comprises an actuator moiety (e.g., a dCas9 fused with a gene activator, such as VP64, p65, SAM, VPR, etc). The chimeric adaptor polypeptide may also comprise a PRE (e.g., WPRE) linked to the GMP. Furthermore, vector 2b may comprise a guide RNA (e.g., a sgRNA operable to target a portion of a gene encoding for Serpin B4 and / or Serpin B9) operatively linked to U6 promoter. The U6 promoter may be disposed downstream of theLAG3promoter orFASpromoter and the genes of interest that are operatively linked to theLAG3promoter orFASpromoter.

[0238] Vector 2c may comprise a chimeric adaptor polypeptide operatively linked to an exogenous promoter, such asLAG3promoter orFASpromoter. The exogenous promoter may be an immune checkpoint of the γδ T cells. The chimeric adaptor polypeptide of may comprise an adaptor (e.g., linker for activation of T cells (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds a portion (e.g., a extracellular, transmembrane, and / or intracellular domain) of the chimeric receptor polypeptide, and / or (ii) moves in proximity to the chimeric receptor polypeptide. The adaptor is linked to a cleavage recognition site (e.g., TEV cutting site (TCS)). The chimeric adaptor polypeptide may also comprise a GMP linked to the cleavage recognition site, wherein the GMP comprises an actuator moiety (e.g., a dCas9 fused with a gene activator, such as VP64, p65, SAM, VPR, etc). The chimeric adaptor polypeptide may also comprise a PRE (e.g., WPRE) linked to the GMP. Furthermore, vector 2c may comprise a guide RNA (e.g., a sgRNA operable to target a portion of a gene encoding for c-FLIP) operatively linked to U6 promoter. The U6 promoter may be disposed downstream of theLAG3promoter orFASpromoter and the genes of interest that are operatively linked to theLAG3promoter orFASpromoter.

[0239] Referring to FIG. 2B, the γδ T cell may express the γδ TCR complex. In some cases, the γδ T cell may also express NKG2D and its associating adaptor DAP10. The γδ T cell may be administered with a chimeric receptor polypeptide comprising a cancer / tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a γδ T cell signaling domain and TEV. The γδ T cell may also be administered with a chimeric adaptor polypeptide comprising an adaptor that binds and / or moves in proximity to the chimeric receptor polypeptide (e.g., to the cellular signaling domain of the chimeric receptor polypeptide) and a GMP comprising an actuator moiety. The chimeric receptor and adaptor may be administered using a viral vector. The chimeric receptor may be expressed constitutively in the γδ T cell. The chimeric adaptor may be expressed conditionally under the treatment of a bisphosphonate (e.g., zolendronate). Zolendronate may be metabolized by a cancer / tumor cell into at least an activator (e.g., IPP) of the γδ T cell, which activator may be secreted or presented on a surface of the cancer / tumor cell. Subsequently, the activator may bind to a receptor of the γδ T cell (e.g., the γδ TCR)_and activate the γδ T cell. Activation of the γδ T cell may lead to activation of one or more intracellular signaling pathways that promote expression of a gene under the control of theLAG3promoter orFASpromoter, whether such promoter(s) is endogenous or exogenous to the γδ T cell. Since the chimeric adaptor polypeptide is under the control of the exogenousLAG3promoter orFASpromoter, activation of the γδ T cell by the bi-product of the bisphosphonate then induces expression of the chimeric adaptor polypeptide. Additionally, upon binding of the tumor antigen to the antigen binding domain of the chimeric receptor polypeptide, the chimeric adaptor polypeptide may move in proximity to the chimeric receptor polypeptide, wherein the TEV of the chimeric receptor polypeptide can cleave the actuator moiety from the GMP at a cleavage recognition site of the chimeric receptor polypeptide. The released actuator moiety may translocate to the nucleus of the γδ T cell to (i) downregulate expression of CD39 and / or (ii) upregulate expression of SerpinB9 / 4 and / or cFLIP.

[0240] Example 3:Knock-in of a system comprising a GMP under the control of an endogenous promoter of γδ T cells.

[0241] FIGs. 3A-3B schematically illustrate administration of a system comprising a GMP in γδ T cells. A polynucleotide encoding for one or more components of the system (i.e., an exogenous expression cassette) may be inserted (e.g., knocked-in) to a genome of the γδ T cells using various gene editing approaches (e.g., CRISPR / Cas9 or a functional variant thereof). Such expression cassette may be inserted under the control of an endogenous promoter of the γδ T cells. Expression of an endogenous polynucleotide of the γδ T cells that is under the control of the same endogenous promoter may or may not be disrupted by the insertion of the exogenous expression cassette. In some cases, various gene editing approaches may be used to delete (e.g., knock-out) the endogenous polynucleotide of the γδ T cells that is under the control of the same endogenous promoter.

[0242] schematically illustrates an exogenous expression cassette that is knocked-in under the control of an endogenous promoter of the γδ T cell. Exogenous expression cassette 1 may comprise a first polynucleotide sequence that encodes for a chimeric receptor polypeptide comprising at least a portion of NKG2D that is linked to a TEV. The first polynucleotide sequence may additionally comprise a terminator (e.g., SV40, hGH, BGH, or rbGlob). The exogenous expression cassette 1 may further comprise a second polynucleotide sequence. The second polynucleotide sequence may comprise an exogenous promoter MND that controls expression of a chimeric adaptor polypeptide. The chimeric adaptor polypeptide may comprise an adaptor moiety (e.g., LAT) that is linked to a GMP comprising dCas9 and VP64. Additionally, the second polynucleotide sequence may comprise an exogenous promoter U6 that controls expression of a nucleotide (e.g., SerpinB9 / 4 sgRNA) sequence to target a specific gene (e.g., SerpinB9 / 4) of the γδ T cell. In some cases, the endogenous promoter of the γδ T cell used to control expression of the exogenous expression cassette 1 may be an endogenousNKG2Dpromoter. In some cases, the endogenous NKG2D gene under the control of the endogenousNKG2Dpromoter may be knocked-out.

[0243] Exogenous expression cassette 2 may comprise a first polynucleotide sequence that encodes for a chimeric receptor polypeptide comprising at least a portion of Vγ9 / Vδ2 TCR that is linked to a TEV. The first polynucleotide sequence may additionally comprise a terminator (e.g., SV40, hGH, BGH, or rbGlob). The exogenous expression cassette 2 may further comprise a second polynucleotide sequence. The second polynucleotide sequence may comprise an exogenous promoter MND that controls expression of a chimeric adaptor polypeptide. The chimeric adaptor polypeptide may comprise an adaptor moiety (e.g., LAT) that is linked to a GMP comprising dCas9 and VP64. Additionally, the second polynucleotide sequence may comprise an exogenous promoter U6 that controls expression of a nucleotide (e.g., SerpinB9 / 4 sgRNA) sequence to target a specific gene (e.g., SerpinB9 / 4) of the γδ T cell. In some cases, the endogenous promoter of the γδ T cell used to control expression of the exogenous expression cassette 2 may be an endogenousVγ9 / Vδ2 TCRpromoter. In some cases, the endogenous Vγ9 / Vδ2 TCR gene under the control of the endogenousVγ9 / Vδ2 TCRpromoter may be knocked-out.

[0244] Exogenous expression cassette 3 may comprise a first polynucleotide sequence that encodes for a chimeric receptor polypeptide comprising a CAR that is linked to a TEV. The first polynucleotide sequence may additionally comprise a terminator (e.g., SV40, hGH, BGH, or rbGlob). The exogenous expression cassette 3 may further comprise a second polynucleotide sequence. The second polynucleotide sequence may comprise an exogenous promoter MND that controls expression of a chimeric adaptor polypeptide. The chimeric adaptor polypeptide may comprise an adaptor moiety (e.g., LAT) that is linked to a GMP comprising dCas9 and VP64. Additionally, the second polynucleotide sequence may comprise an exogenous promoter U6 that controls expression of a nucleotide (e.g., SerpinB9 / 4 sgRNA) sequence to target a specific gene (e.g., SerpinB9 / 4) of the γδ T cell. In some cases, the endogenous promoter of the γδ T cell used to control expression of the exogenous expression cassette 3 may be an endogenous promoter (e.g.,LAG,FAS, and / orKIR2DSpromoter). In some cases, the endogenous LAG, FAS, and / or KIR2DS gene under the control of the endogenous promoter may be knocked-out.

[0245] schematically illustrates regulation of a target polynucleotide by the system described in. In some cases, as shown in(top), once the actuator moiety of the GMP is released from the chimeric adaptor polypeptide, the actuator moiety which comprise KRAB may bind to or adjacent to a polynucleotide encoding for CD39 to downregulate or inhibit expression of CD39. In some cases, as shown in(below), once the actuator moiety of the GMP is released from the chimeric adaptor polypeptide, the actuator moiety which comprise a gene activator (e.g., VPR, VP64, p300, SunT, MS2-p65-HSF1, etc.) may bind to or adjacent to a polynucleotide encoding for SerpinB9 / 4 or c-FLIP to upregulate or initiate expression of SerpinB9 / 4 or c-FLIP.

[0246] In some cases, the exogenous expression cassettes as shown inmay be knocked-in under the control of an endogenous beta-2 microglobulin (B2M) promoter of the γδ T cell, and the endogenous polynucleotide encoding for the endogenous B2M may be knocked-out. In such a case, the γδ T cell may have an alternative escape mechanism against NK cells.

[0247] Example 4: Treatment of engineered γδ T cells with bisphosphonates.

[0248] An activity of the experimental γδ T cells engineered to express (e.g., constitutively and / or conditionally) any one of the systems described herein in the present disclosure may be compared to one or more controls, including, but not limited to, (1) non-engineered γδ T cells and / or (2) engineered γδ T cells with a control gRNA which does not bind to a target polynucleotide of the engineered γδ T cells. In some cases, since alpha (α)-beta (β) T cells do not respond (e.g., directly or indirectly) to Zolendronate, the αβ T cells may be used as a negative control in experiments where Zolendronate is used.

[0249] The γδ T cells may be engineered to express any one of the systems described herein in the present disclosure by utilizing viral vectors (e.g., gamma retroviral vectors) or gene editing techniques (e.g., CRISPR / Cas9).

[0250] For in vitro experiments, the engineered γδ T cells and the control cells may be treated with or without zolendronate. Subsequently, expression of SerpinB9 / 4, c-FLIP, and / or CD39 in the engineered γδ T cells and the control cells may be assessed (e.g., Western blot, polymerase chain reaction (PCR), etc.).

[0251] For another set of in vitro experiments, the engineered γδ T cells and the control cells may be treated with or without zolendronate. Subsequently, expression of SerpinB9 / 4, c-FLIP, and / or CD39 in the engineered γδ T cells and the control cells may be assessed (e.g., Western blot, polymerase chain reaction (PCR), etc.). Following, the engineered γδ T cells and the control cells may be treated with a death-inducing compound (e.g., a recombinant Fas ligand) at one or more different time points. After the one or more different time points, survival and / or proliferation of the engineered γδ T cells and the control cells may be measured (e.g., cell counting, DNA and / or protein amount counting, etc.).

[0252] For a different set of in vitro experiments, the engineered γδ T cells and the control cells may be treated with or without zolendronate. Subsequently, expression of SerpinB9 / 4, c-FLIP, and / or CD39 in the engineered γδ T cells and the control cells may be assessed (e.g., Western blot, polymerase chain reaction (PCR), etc.). Following, the engineered γδ T cells and the control cells may be exposed to one or more cells that express a ligand (e.g., an antigen) that bind to the ligand-binding domain of the chimeric polypeptide receptor (e.g. CAR) of the system of the engineered γδ T cells. The one or more cells may be cancer / tumor cells that express the ligand or model cells that express the ligand (e.g., Raji cells). Afterwards, tumor cytotoxicity of the engineered γδ T cells and the control cells may be measured. In addition, proliferation and / or survival of the engineered γδ T cells and the control cells may be measured.

[0253] For in vivo experiments, each of the engineered γδ T cells and the control cells may be transplanted into NOD scid gamma (NSG) mice. Following, the NSG mice may be treated with zolendronate. The NSG mice may be challenged with a model tumor (e.g., by transplanting tumor cells to create a model subcutaneous tumor) prior to, during, and / or subsequent to the zolendronate treatment. In some cases, the NSG mice may be challenged with the model tumor subsequent to the zolendronate treatment. At one or more time points, tumor burden in the NSG mice and their survival may be assessed, along with expansion, persistence, and / or activity of the engineered γδ T cells and the control cells in the NSG mice.

[0254] For another set of in vivo experiments, the NSG mice may be challenged with a model tumor (e.g., by transplanting tumor cells to create a model subcutaneous tumor). Following, each of the engineered γδ T cells and the control cells may be transplanted into the NSG mice. In some cases, the NSG mice may be treated with zolendronate prior to, during, and / or subsequent to the transplantation of each of the engineered γδ T cells and the control cells. In some cases, the NSG mice may be treated with zolendronate subsequent to the transplantation of each of the engineered γδ T cells and the control cells. At one or more time points, tumor burden in the NSG mice and their survival may be assessed, along with expansion, persistence, and / or activity of the engineered γδ T cells and the control cells in the NSG mice. In some cases, when the tumor is assessed to be cleared from one or more of the NSG mice, the NSG mice may be challenged with the same or different model tumor one or more times in the absence of any further zolendronate treatment.

Claims

A method for regulating an activity of a gamma delta T cell, comprising:administering to said gamma delta T cell a gene modulating polypeptide (GMP) comprising an actuator moiety configured to regulate expression of a target polynucleotide in said gamma delta T cell, wherein said target polynucleotide encodes a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolase.The method of claim 1, wherein regulating said activity of said gamma delta T cell comprises decreasing fratricide of gamma delta T cells.The method of claim 1, wherein regulating said activity of said gamma delta T cell comprises prolonging activation of said gamma delta T cell.The method of claim 1, wherein said actuator moiety is an RNA-guided actuator moiety or a variant thereof, which RNA-guided actuator moiety forms a complex with said target polynucleotide.The method of claim 2, wherein said actuator moiety is a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity.The method of claim 2, wherein said actuator moiety is Cas9 and / or Cpf1.The method of claim 1, wherein said actuator moiety comprises an activator effective to increase expression of said target polynucleotide.The method of claim 1, wherein said actuator moiety comprises a repressor effective to decrease expression of said target polynucleotide.The method of claim 1, wherein said polypeptide is said death-inducing protein.The method of claim 9, wherein said death-inducing protein comprises Fas, Fas ligand, granzyme B, granzyme M, and / or perforin.The method of claim 9, wherein the regulation of expression of the target polynucleotide comprises decreasing the expression of the target polynucleotide.The method of claim 1, wherein said polypeptide is said inhibitor of said death-inducing protein.The method of claim 12, wherein said inhibitor comprises serpin B9,serpin B4, and / or c-FLIP.The method of claim 12, wherein the regulation of expression of the target polynucleotide comprises increasing the expression of the target polynucleotide.The method of claim 1, wherein said polypeptide is said hydrolase.The method of claim 15, wherein said polypeptide comprises CD39.The method of claim 15, wherein the regulation of expression of the target polynucleotide comprises decreasing the expression of the target polynucleotide.The method of claim 1, further comprising treating said gamma delta T cell with a virus to administer said GMP to said gamma delta T cell.The method of claim 18, further comprising integrating into a genome of said gamma delta T cell a nucleic acid sequence encoding said GMP by using said virus.The method of claim 18, wherein said virus is a retrovirus.The method of claim 21, wherein said retrovirus is a gamma-retrovirus selected from the group consisting of: Moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), and spleen focus-forming virus (SFFV), and / or variants thereof.The method of claim 1, further comprising treating said gamma delta T cell with a nuclease to administer said GMP to said gamma delta T cell.The method of claim 22, further comprising inserting into a genome of said gamma delta T cell a nucleic acid sequence encoding said GMP by using said nuclease.The method of claim 23, wherein the nuclease comprises a CRISPR- associated polypeptide (Cas), zinc finger nuclease (ZFN), zinc finger associate gene regulation polypeptides, transcription activator-like effector nuclease (TALEN), transcription activator-like effector associated gene regulation polypeptides, meganuclease, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.The method of claim 1, further comprising administering to said gamma delta T cell a chimeric polypeptide comprising said GMP, wherein said chimeric polypeptide is operable to release said GMP from said chimeric polypeptide in response to a stimulant, and wherein said released GMP is operable to regulate expression of said target polynucleotide in said gamma delta T cell.The method of claim 1, further comprising administering to said gamma delta T cell a chimeric polypeptide comprising said GMP and a nuclear localization domain, wherein said nuclear localization domain is operable to translocate said chimeric polypeptide to a nucleus of said gamma delta T cell in response to a stimulant, and wherein said translocated GMP is operable to regulate expression of said target polynucleotide in said gamma delta T cell.A system for regulating an activity of a gamma delta T cell, comprising:a gene modulating polypeptide (GMP) comprising an actuator moiety configured to regulate expression of a target polynucleotide in said gamma delta T cell, wherein said target polynucleotide encodes a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolase.The system of claim 27, wherein said GMP is operable to decrease fratricide of gamma delta T cells.The system of claim 27, wherein said GMP is operable to prolong activation of said gamma delta T cell.The system of claim 27, wherein said actuator moiety is an RNA-guided actuator moiety or a variant thereof, which RNA-guided actuator moiety forms a complex with said target polynucleotide.The system of claim 30, wherein said actuator moiety is a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity.The system of claim 30, wherein said actuator moiety is Cas9 and / or Cpf1.The system of claim 27, wherein said actuator moiety comprises an activator effective to increase expression of said target polynucleotide.The system of claim 27, wherein said actuator moiety comprises a repressor effective to decrease expression of said target polynucleotide.The system of claim 27, wherein said polypeptide is said death-inducing protein.The system of claim 35, wherein said death-inducing protein comprises Fas, Fas ligand, granzyme B, granzyme M, and / or perforin.The system of claim 35, wherein the regulation of expression of the target polynucleotide comprises decreasing the expression of the target polynucleotide.The system of claim 27, wherein said polypeptide is said inhibitor of said death-inducing protein.The system of claim 38, wherein said inhibitor comprises serpin B9, serpin B4, and / or c-FLIP.The system of claim 38, wherein the regulation of expression of the target polynucleotide comprises increasing the expression of the target polynucleotide.The system of claim 27, wherein said polypeptide is said hydrolase.The system of claim 41, wherein said polypeptide comprises CD39.The system of claim 41, wherein the regulation of expression of the target polynucleotide comprises decreasing the expression of the target polynucleotide.The system of claim 27, wherein a nucleic acid sequence encoding said GMP is integrated into a genome of said gamma delta T cell by a virus.The system of claim 44, wherein said virus is a retrovirus.The system of claim 45, wherein said retrovirus is a gamma-retrovirus selected from the group consisting of: Moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), and spleen focus-forming virus (SFFV), and / or variants thereof.The system of claim 27, a nucleic acid sequence encoding said GMP is integrated into a genome of said gamma delta T cell by a nuclease.The system of claim 47, wherein the nuclease comprises a CRISPR- associated polypeptide (Cas), zinc finger nuclease (ZFN), zinc finger associate gene regulation polypeptides, transcription activator-like effector nuclease (TALEN), transcription activator-like effector associated gene regulation polypeptides, meganuclease, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.The system of claim 27, further comprising a chimeric polypeptide comprising said GMP, wherein said chimeric polypeptide is operable to release said GMP from said chimeric polypeptide in response to a stimulant, and wherein said released GMP is operable to regulate expression of said target polynucleotide in said gamma delta T cell.The system of claim 27, further comprising a chimeric polypeptide comprising said GMP and a nuclear localization domain, wherein said nuclear localization domain is operable to translocate said chimeric polypeptide to a nucleus of said gamma delta T cell in response to a stimulant, and wherein said translocated GMP is operable to regulate expression of said target polynucleotide in said gamma delta T cell.

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