SYSTEMS AND METHODS FOR NUCLEAR LOCALIZATION OF

GENE MODULATING POLYPEPTIDES

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Patent Application No. 62/844,600, filed on May 7, 2019, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Regulation of cell activities can involve binding of a ligand to a membrane-bound receptor comprising an extracellular ligand binding domain and an intracellular (e.g., cytoplasmic) signaling domain. The formation of a complex between a ligand and the ligand binding domain can result in a conformational and/or chemical modification in the receptor which can result in a signal transduced within the cell. In some situations, the signal transduced within the cell results in modification (e.g., phosphorylation) of a downstream target, resulting in a change in its activity. These downstream targets can be activated and then carry out various functions within a cell.

[0003] In some cases, attachment of an extracellular domain (e.g., a ligand binding domain) of one protein to an intracellular domain of another protein involved in signal transduction (e.g., a signaling domain) can create a molecule (e.g., a chimeric receptor) that combines the ligand recognition of the former to the signal transduction of the latter. Such chimeric molecules (e.g., chimeric receptors or chimeric antigen receptors) can be useful for various purposes, for example for regulating immune cells in immunotherapy.

Immunotherapy can involve modifying a patient’s own immune cells to express a chimeric receptor in which arbitrary ligand specificity is grafted onto an immune cell signaling domain. The immune cell signaling domain can be involved in activating and/or de-activating an immune cell to respond to a disease such as cancer.

[0004] Conventional methods of immunotherapy can suffer from various deficiencies. Such deficiencies can include insufficient signaling from co-stimulatory receptors for persistent and/or adequate immune responses for therapeutic effects, inadequate specificity of modified immune cells for diseased cells such as cancer cells (e.g., on-target off-tumor effects and toxicities), and side-effects such as cytokine-release syndrome (CRS). Signaling in immune cells can involve various receptors, including co-stimulatory receptors.

Insufficient signals from co-stimulatory receptors may result in decreased immune cell responses and reduced effectiveness of immunotherapy. Off-target effects and side-effects such as cytokine-release syndrome can result in further medical complications including inflammatory responses, organ failure, and, in extreme cases, death.

SUMMARY

[0005] In an aspect, the present disclosure provides a system for regulating expression of a target polynucleotide in a cell, comprising: a first chimeric polypeptide comprising a gene modulating polypeptide (GMP) that comprises an actuator moiety linked to a cleavage recognition site, wherein the actuator moiety regulates the expression of the target polynucleotide in the cell; and a second chimeric polypeptide comprising a sensing moiety exhibiting specific binding to a trigger molecule, wherein the sensing moiety is linked to a cleavage moiety that cleaves the cleavage recognition site, wherein upon binding of the trigger molecule to the sensing moiety or upon releasing of the trigger molecule from the sensing moiety, the sensing moiety undergoes a modification to activate the cleavage moiety, wherein, upon introducing the system into the cell and contacting of the cell by a ligand, the second chimeric polypeptide is induced to activate the cleavage moiety, such that the activated cleavage moiety releases the actuator moiety from the GMP of the first chimeric polypeptide to effect regulating expression of the target polynucleotide in the cell.

[0006] In some embodiments, the activating of the cleavage moiety comprises exposing an active domain of the cleavage moiety.

[0007] In some embodiments, the first chimeric polypeptide further comprises at least one nuclear export signal (NES) configured to reduce translocation of the first chimeric polypeptide to nucleus of the cell. In some embodiments, the cleavage recognition site is flanked by the at least one NES and the actuator moiety. In some embodiments, the second chimeric polypeptide further comprises at least one nuclear export signal (NES) configured to reduce translocation of the second chimeric polypeptide to nucleus of the cell. In some embodiments, (i) the sensing moiety is flanked by the at least one NES and the cleavage moiety, (ii) the cleavage moiety is flanked by the at least one NES and the sensing moiety, or (iii) the at least one NES is flanked by the sensing moiety and the cleavage moiety.

[0008] In some embodiments, the system further comprises a chimeric receptor having a ligand binding domain specific for the ligand. In some embodiments, upon the contacting of the ligand binding domain by the ligand, the second chimeric polypeptide is induced to activate the cleavage moiety. [0009] In some embodiments, the first chimeric polypeptide is fused in-frame with the chimeric receptor. In some embodiments, the cleavage recognition site is flanked by the chimeric receptor and the actuator moiety. In some embodiments, the second chimeric polypeptide further comprises an adaptor polypeptide configured to bind the chimeric receptor or a downstream signaling moiety of the chimeric receptor in response to the contacting of the ligand binding domain by the ligand. In some embodiments, (i) the sensing moiety is flanked by the adaptor polypeptide and the cleavage moiety, (ii) the cleavage moiety is flanked by the adaptor polypeptide and the sensing moiety, or (iii) the adaptor polypeptide is flanked by the sensing moiety and the cleavage moiety.

[0010] In some embodiments, the second chimeric polypeptide is fused in-frame with the chimeric receptor. In some embodiments, the first chimeric polypeptide further comprises an adaptor polypeptide configured to bind the chimeric receptor or a downstream signaling moiety of the chimeric receptor in response to the contacting of the ligand binding domain by the ligand. In some embodiments, the cleavage recognition site is flanked by the adaptor polypeptide and the actuator moiety.

[0011] In another aspect, the present disclosure provides a system for regulating expression of a target polynucleotide in a cell, comprising: a first chimeric polypeptide comprising (i) a gene modulating polypeptide (GMP) that comprises an actuator moiety linked to a cleavage recognition site, wherein the actuator moiety regulates the expression of the target polynucleotide in the cell, and (ii) a sensing moiety exhibiting specific binding to a trigger molecule and linked to the GMP, wherein upon binding of the trigger molecule to the sensing moiety or upon releasing of the trigger molecule from the sensing moiety, the sensing moiety undergoes a modification to activate the cleavage recognition site; and a second chimeric polypeptide comprising a cleavage moiety that cleaves the activated cleavage recognition site, wherein, upon introducing the system into the cell and contacting of the cell by a ligand, the first chimeric polypeptide is induced to activate the cleavage recognition site, such that the cleavage moiety releases the actuator moiety from the GMP of the first chimeric polypeptide to effect regulating expression of the target polynucleotide in the cell.

[0012] In some embodiments, the activating of the cleavage recognition site comprises exposing an active domain of the cleavage recognition site.

[0013] In some embodiments, the first chimeric polypeptide further comprises at least one nuclear export signal (NES) configured to reduce translocation of the first chimeric polypeptide to nucleus of the cell. In some embodiments, (i) the at least one NES is flanked by the sensing moiety and the cleavage recognition site, or (ii) the sensing moiety is flanked by the at least one NES and the cleavage recognition site. In some embodiments, In some embodiments, the second chimeric polypeptide further comprises at least one nuclear export signal (NES) configured to reduce translocation of the second chimeric polypeptide to nucleus of the cell.

[0014] In some embodiments, the system further comprises a chimeric receptor having a ligand binding domain specific for the ligand. In some embodiments, upon the contacting of the ligand binding domain by the ligand, the first chimeric polypeptide is induced to activate the cleavage recognition site.

[0015] In some embodiments, the first chimeric polypeptide is fused in-frame with the chimeric receptor. In some embodiments, the cleavage recognition site is flanked by the chimeric receptor and the actuator moiety. In some embodiments, the sensing moiety is flanked by the chimeric receptor and the cleavage recognition site. In some embodiments, the second chimeric polypeptide further comprises an adaptor polypeptide configured to bind the chimeric receptor or a downstream signaling moiety of the chimeric receptor in response to the contacting of the ligand binding domain by the ligand.

[0016] In some embodiments, the second chimeric polypeptide is fused in-frame with the chimeric receptor. In some embodiments, the first chimeric polypeptide further comprises an adaptor polypeptide configured to bind the chimeric receptor or a downstream signaling moiety of the chimeric receptor in response to the contacting of the ligand binding domain by the ligand. In some embodiments, the cleavage recognition site is flanked by the adaptor polypeptide and the actuator moiety. In some embodiments, (i) the adaptor polypeptide is flanked by the sensing moiety and the cleavage recognition site, or (ii) the sensing moiety is flanked by the adaptor polypeptide and the cleavage recognition site.

[0017] In some embodiments of any one of the subject systems, a cytosolic concentration of the trigger molecule is not activated by light.

[0018] In some embodiments of any one of the subject systems, the ligand is an extracellular ligand. In some embodiments of any one of the subject systems, the ligand is an antigen presented on a target cell of the cell. In some embodiments of any one of the subject systems, the contacting of the cell by the ligand increases a cytosolic concentration of the trigger molecule in the cell. In some embodiments of any one of the subject systems, the contacting of the cell by the ligand decreases a cytosolic concentration of the trigger molecule in the cell.

[0019] In some embodiments of any one of the subject systems, the chimeric receptor is configured to undergo a modification including a conformational change or chemical modification upon the contacting of the ligand binding domain by the ligand. In some embodiments, the chemical modification is selected from the group consisting of:

dephosphorylation, phosphorylation, acetylation, methylation, ubiquitination, proteolytic processing, or combination thereof.

[0020] In some embodiments of any one of the subject systems, the adaptor polypeptide comprises an adaptor protein of the chimeric receptor, kinase, phosphatase, nucleotide exchange factor, another adaptor protein thereof, a fragment thereof, or a combination thereof.

[0021] In some embodiments of any one of the subject systems, the ligand binding domain of the chimeric receptor is heterologous to the cell.

[0022] In some embodiments of any one of the subject systems, the trigger molecule is selected from the group consisting of: an ion, a lipid, a small molecule, a polynucleotide, a polypeptide, a modification thereof, and a combination thereof. In some embodiments, the trigger molecule is the ion. In some embodiments, the trigger molecule is calcium,

magnesium, and/or zinc.

[0023] In some embodiments of any one of the subject systems, the modification of the sensing moiety comprises a conformational change or chemical modification. In some embodiments, the chemical modification is selected from the group consisting of:

dephosphorylation, phosphorylation, acetylation, methylation, ubiquitination, proteolytic processing, or a combination thereof.

[0024] In some embodiments of any one of the subject systems, upon the contacting of the cell by the ligand, the chimeric receptor activates a transmembrane protein capable of altering or regulating a cytosolic concentration of the trigger molecule in the cell. In some embodiments, the transmembrane protein is capable of increasing the cytosolic concentration of the trigger molecule. In some embodiments, the transmembrane protein is capable of decreasing the cytosolic concentration of the trigger molecule. In some embodiments, the transmembrane protein comprises a calcium channel. In some embodiments, the calcium channel comprises a ligand-gated calcium channel and/or a voltage-gated calcium channel.

[0025] In some embodiments of any one of the subject systems, the sensing moiety comprises at least a portion of a protein selected from the group consisting of: calmodulins, troponin C, calcineurin, parvalbumin, S100, calpain, myosin, neuronal Ca2+ sensor (NCS)-l, calsequestrin, calreticulin, annexing (AXA1, AXA2, AXA3, AXA4, AXA5, AXA6, AXA7, AXA8, AXA9, AXA10, AXA11, AXA13), PKC, synaptotagmins (synaptotagmin I, II, III,

IX, VI, Vn, V, X), phospholipase C (PLC), phospholipase A (PLA), Fam62a, Fam62b, Fam62c, dysferlin, otoferlin, myoferlin, mctpl, pctp2, Rph3A, Doc2a, Doc2b, sytll, Sytl2, Sytl3, Sytl4, Sytl5, Unci 3a, Cpne6, Rcn2, osteonectin, hevin, QR1, testicans 1-3, tsc 36, SMOC-1, SMOC-2, coagulation factors VII, IX and X, protein C, protein S, fibrillin, notch and delta receptors, LDL receptors, and a combination thereof.

[0026] In some embodiments of any one of the subject systems, the actuator moiety further comprises a Cas protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a recombinases, a flippase, a transposase, or an Argonaute (Ago) protein (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)). In some embodiments, the actuator moiety comprises a Cas protein. In some embodiments, the Cas protein is complexed with a guide RNA. In some embodiments, the Cas protein substantially lacks DNA cleavage activity.

[0027] In some embodiments of any one of the subject systems, the actuator moiety further comprises a heterologous functional domain. In some embodiments, the heterologous functional domain comprises a transcription activator. In some embodiments, the

heterologous functional domain comprises a transcription repressor. In some embodiments, the heterologous functional domain comprises a chromosome modification enzyme.

[0028] In some embodiments of any one of the subject systems, the target polynucleotide is genomic DNA. In some embodiments of any one of the subject systems, the target polynucleotide is RNA. In some embodiments of any one of the subject systems, the target polynucleotide encodes a protein involved in immune cell regulation. In some embodiments, the encoded protein comprises PD-1, CTLA-4, LAG3, TIM-3, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, VISTA, a fragment thereof, a modification thereof, or a combination thereof.

[0029] In some embodiments of any one of the subject systems, the chimeric receptor comprises a chimeric antigen receptor (CAR). In some embodiments of any one of the subject systems, the chimeric receptor comprises at least a portion of a Notch receptor, a G- protein coupled receptor (GPCR), an integrin receptor, a cadherin receptor, a receptor tyrosine kinase, a death receptor, or an immune receptor. In some embodiments, the immune receptor comprises a T cell receptor.

[0030] In some embodiments of any one of the subject systems, regulating the expression of the target polynucleotide in the cell enhances and/or prolongs cytotoxicity of the cell against a tumor or cancer cell, as compared to without the regulating. In some embodiments of any one of the subject systems, regulating the expression of the target polynucleotide in the cell obliterates a tumor or reduces a size of the tumor, as compared to without the regulating. [0031] In a different aspect, the present disclosure provides a composition comprising one or more polynucleotides that encode any one of the subject systems. In a different aspect, the present disclosure provides a kit comprising the subject composition.

[0032] In a different aspect, the present disclosure provides an isolated host cell expressing any one of the subject systems. In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the lymphocyte is a natural killer (NK) cell. In some embodiments, the host cell is a hematopoietic stem cell or an Induced pluripotent stem cell (iPSC).

[0033] In another aspect, the present disclosure provides a method of regulating expression of a target polynucleotide in a cell, comprising: (a) expressing a system in the cell; and (b) contacting the cell by a ligand, wherein the system expressed in the cell comprises: a first chimeric polypeptide comprising a gene modulating polypeptide (GMP) that comprises an actuator moiety linked to a cleavage recognition site, wherein the actuator moiety regulates the expression of the target polynucleotide in the cell; and a second chimeric polypeptide comprising a sensing moiety exhibiting specific binding to a trigger molecule, wherein the sensing moiety is linked to a cleavage moiety that cleaves the cleavage recognition site, wherein upon binding of the trigger molecule to the sensing moiety or upon releasing of the trigger molecule from the sensing moiety, the sensing moiety undergoes a modification to activate the cleavage moiety, and wherein, upon contacting of the cell by the ligand, the second chimeric polypeptide is induced to activate the cleavage moiety, such that the activated cleavage moiety releases the actuator moiety from the GMP of the first chimeric polypeptide to effect regulating expression of the target polynucleotide in the cell.

[0034] In some embodiments, the activating of the cleavage moiety comprises exposing an active domain of the cleavage moiety.

[0035] In some embodiments, the first chimeric polypeptide further comprises at least one nuclear export signal (NES) configured to reduce translocation of the first chimeric polypeptide to nucleus of the cell. In some embodiments, the cleavage recognition site is flanked by the at least one NES and the actuator moiety. In some embodiments, the second chimeric polypeptide further comprises at least one nuclear export signal (NES) configured to reduce translocation of the second chimeric polypeptide to nucleus of the cell. In some embodiments, (i) the sensing moiety is flanked by the at least one NES and the cleavage moiety, (ii) the cleavage moiety is flanked by the at least one NES and the sensing moiety, or (iii) the at least one NES is flanked by the sensing moiety and the cleavage moiety.

[0036] In some embodiments, the system further comprises a chimeric receptor having a ligand binding domain specific for the ligand. In some embodiments, upon the contacting of the ligand binding domain by the ligand, the second chimeric polypeptide is induced to activate the cleavage moiety.

[0037] In some embodiments, the first chimeric polypeptide is fused in-frame with the chimeric receptor. In some embodiments, the cleavage recognition site is flanked by the chimeric receptor and the actuator moiety. In some embodiments, the second chimeric polypeptide further comprises an adaptor polypeptide configured to bind the chimeric receptor or a downstream signaling moiety of the chimeric receptor in response to the contacting of the ligand binding domain by the ligand. In some embodiments, (i) the sensing moiety is flanked by the adaptor polypeptide and the cleavage moiety, (ii) the cleavage moiety is flanked by the adaptor polypeptide and the sensing moiety, or (iii) the adaptor polypeptide is flanked by the sensing moiety and the cleavage moiety.

[0038] In some embodiments, the second chimeric polypeptide is fused in-frame with the chimeric receptor. In some embodiments, the first chimeric polypeptide further comprises an adaptor polypeptide configured to bind the chimeric receptor or a downstream signaling moiety of the chimeric receptor in response to the contacting of the ligand binding domain by the ligand. In some embodiments, the cleavage recognition site is flanked by the adaptor polypeptide and the actuator moiety.

[0039] In a different aspect, the present disclosure provides a method of regulating expression of a target polynucleotide in a cell, comprising: (a) expressing a system in the cell; and (b) contacting the cell by a ligand, wherein the system expressed in the cell comprises: a first chimeric polypeptide comprising (i) a gene modulating polypeptide (GMP) that comprises an actuator moiety linked to a cleavage recognition site, wherein the actuator moiety regulates the expression of the target polynucleotide in the cell, and (ii) a sensing moiety exhibiting specific binding to a trigger molecule and linked to the GMP, wherein upon binding of the trigger molecule to the sensing moiety or upon releasing of the trigger molecule from the sensing moiety, the sensing moiety undergoes a modification to activate the cleavage recognition site; and a second chimeric polypeptide comprising a cleavage moiety that cleaves the activated cleavage recognition site, and wherein, upon contacting of the cell by a ligand, the first chimeric polypeptide is induced to activate the cleavage recognition site, such that the cleavage moiety releases the actuator moiety from the GMP of the first chimeric polypeptide to effect regulating expression of the target polynucleotide in the cell.

[0040] In some embodiments, the activating of the cleavage recognition site comprises exposing an active domain of the cleavage recognition site.

[0041] In some embodiments, the first chimeric polypeptide further comprises at least one nuclear export signal (NES) configured to reduce translocation of the first chimeric polypeptide to nucleus of the cell. In some embodiments, (i) the at least one NES is flanked by the sensing moiety and the cleavage recognition site, or (ii) the sensing moiety is flanked by the at least one NES and the cleavage recognition site. In some embodiments, the second chimeric polypeptide further comprises at least one nuclear export signal (NES) configured to reduce translocation of the second chimeric polypeptide to nucleus of the cell.

[0042] In some embodiments, the system further comprises a chimeric receptor having a ligand binding domain specific for the ligand. In some embodiments, upon the contacting of the ligand binding domain by the ligand, the first chimeric polypeptide is induced to activate the cleavage recognition site.

[0043] In some embodiments, the first chimeric polypeptide is fused in-frame with the chimeric receptor. In some embodiments, the cleavage recognition site is flanked by the chimeric receptor and the actuator moiety. In some embodiments, the sensing moiety is flanked by the chimeric receptor and the cleavage recognition site. In some embodiments, the second chimeric polypeptide further comprises an adaptor polypeptide configured to bind the chimeric receptor or a downstream signaling moiety of the chimeric receptor in response to the contacting of the ligand binding domain by the ligand.

[0044] In some embodiments, the second chimeric polypeptide is fused in-frame with the chimeric receptor. In some embodiments, the first chimeric polypeptide further comprises an adaptor polypeptide configured to bind the chimeric receptor or a downstream signaling moiety of the chimeric receptor in response to the contacting of the ligand binding domain by the ligand. In some embodiments, the cleavage recognition site is flanked by the adaptor polypeptide and the actuator moiety. In some embodiments, (i) the adaptor polypeptide is flanked by the sensing moiety and the cleavage recognition site, or (ii) the sensing moiety is flanked by the adaptor polypeptide and the cleavage recognition site.

[0045] In some embodiments of any one of the subject methods, a cytosolic concentration of the trigger molecule is not activated by light. In some embodiments of any one of the subject methods, the ligand is an extracellular ligand. In some embodiments of any one of the subject methods, the ligand is an antigen presented on a target cell of the cell. In some embodiments of any one of the subject methods, the contacting of the cell by the ligand increases a cytosolic concentration of the trigger molecule in the cell. In some embodiments of any one of the subject methods, the contacting of the cell by the ligand decreases a cytosolic concentration of the trigger molecule in the cell.

[0046] In some embodiments of any one of the subject methods, the chimeric receptor is configured to undergo a modification including a conformational change or chemical modification upon the contacting of the ligand binding domain by the ligand. In some embodiments, the chemical modification is selected from the group consisting of:

dephosphorylation, phosphorylation, acetylation, methylation, ubiquitination, proteolytic processing, or combination thereof.

[0047] In some embodiments of any one of the subject methods, the adaptor polypeptide comprises an adaptor protein of the chimeric receptor, kinase, phosphatase, nucleotide exchange factor, another adaptor protein thereof, a fragment thereof, or a combination thereof.

[0048] In some embodiments of any one of the subject methods, the ligand binding domain of the chimeric receptor is heterologous to the cell.

[0049] In some embodiments of any one of the subject methods, the trigger molecule is selected from the group consisting of: an ion, a lipid, a small molecule, a polynucleotide, a polypeptide, a modification thereof, and a combination thereof. In some embodiments, the trigger molecule is the ion. In some embodiments, the trigger molecule is calcium,

magnesium, and/or zinc.

[0050] In some embodiments of any one of the subject methods, the modification of the sensing moiety comprises a conformational change or chemical modification. In some embodiments, the chemical modification is selected from the group consisting of:

dephosphorylation, phosphorylation, acetylation, methylation, ubiquitination, proteolytic processing, or a combination thereof.

[0051] In some embodiments of any one of the subject methods, upon the contacting of the cell by the ligand, the chimeric receptor activates a transmembrane protein capable of altering or regulating a cytosolic concentration of the trigger molecule in the cell. In some embodiments, the transmembrane protein is capable of increasing the cytosolic concentration of the trigger molecule. In some embodiments, the transmembrane protein is capable of decreasing the cytosolic concentration of the trigger molecule. In some embodiments, the transmembrane protein comprises a calcium channel. In some embodiments, the calcium channel comprises a ligand-gated calcium channel and/or a voltage-gated calcium channel. [0052] In some embodiments of any one of the subject methods, the sensing moiety comprises at least a portion of a protein selected from the group consisting of: calmodulins, troponin C, calcineurin, parvalbumin, S100, calpain, myosin, neuronal Ca2+ sensor (NCS)-l, calsequestrin, calreticulin, annexing (AXA1, AXA2, AXA3, AXA4, AXA5, AXA6, AXA7, AXA8, AXA9, AXA10, AXA11, AXA13), PKC, synaptotagmins (synaptotagmin I, II, III,

IX, VI, VII, V, X), phospholipase C (PLC), phospholipase A (PLA), Fam62a, Fam62b, Fam62c, dysferlin, otoferlin, myoferlin, mctpl, pctp2, Rph3A, Doc2a, Doc2b, sytll, Sytl2, Sytl3, Sytl4, Sytl5, Unc13a, Cpne6, Rcn2, osteonectin, hevin, QR1, testicans 1-3, tsc 36, SMOC-1, SMOC-2, coagulation factors VII, IX and X, protein C, protein S, fibrillin, notch and delta receptors, LDL receptors, and a combination thereof.

[0053] In some embodiments of any one of the subject methods, the actuator moiety further comprises a Cas protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a recombinases, a flippase, a transposase, or an Argonaute (Ago) protein (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)). In some embodiments, the actuator moiety comprises a Cas protein. In some embodiments, the Cas protein is complexed with a guide RNA. In some embodiments, the Cas protein substantially lacks DNA cleavage activity.

[0054] In some embodiments of any one of the subject methods, the actuator moiety further comprises a heterologous functional domain. In some embodiments, the heterologous functional domain comprises a transcription activator. In some embodiments, the

heterologous functional domain comprises a transcription repressor. In some embodiments, the heterologous functional domain comprises a chromosome modification enzyme.

[0055] In some embodiments of any one of the subject methods, the target polynucleotide is genomic DNA. In some embodiments of any one of the subject methods, the target polynucleotide is RNA. In some embodiments of any one of the subject methods, the target polynucleotide encodes a protein involved in immune cell regulation. In some embodiments, the encoded protein comprises PD-1, CTLA-4, LAG3, TIM-3, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, VISTA, a fragment thereof, a modification thereof, or a combination thereof.

[0056] In some embodiments of any one of the subject methods, the chimeric receptor comprises a chimeric antigen receptor (CAR). In some embodiments of any one of the subject methods, the chimeric receptor comprises at least a portion of a Notch receptor, a G- protein coupled receptor (GPCR), an integrin receptor, a cadherin receptor, a receptor tyrosine kinase, a death receptor, or an immune receptor. In some embodiments of any one of the subject methods, the immune receptor comprises a T cell receptor. [0057] In some embodiments of any one of the subject methods, regulating the expression of the target polynucleotide in the cell enhances and/or prolongs cytotoxicity of the cell against a tumor or cancer cell, as compared to without the regulating. In some embodiments of any one of the subject methods, regulating the expression of the target polynucleotide in the cell obliterates a tumor or reduces a size of the tumor, as compared to without the regulating.

[0058] 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

[0059] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] 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:

[0061] FIGs. 1-2 schematically illustrate various embodiments of a system for regulating expression of a target polynucleotide in a cell. The system comprises a chimeric polypeptide comprising an ion sensor linked to a protease, wherein the ion sensor“hides” an active site of the protease when the ion is not bound the ion sensor. The system comprises an additional chimeric polypeptide comprising a nuclear export signal (NES) linked to a protease cleavage site and an actuator configured to regulate expression of the target polynucleotide. Activation of a receptor of the cell and a subsequent activation of an ion channel increases cytosolic concentration of the ion (i.e., [ion]) in the cell, and binding of the ion to the ion sensor activates the ion sensor, which in turn exposes the active site of the protease. Following, the exposed protease cleaves the protease cleavage site and releases the actuator from the NES, thereby allowing the actuator to enter nucleus of the cell to regulate expression of the target polynucleotide in the cell. FIG. 1 illustrates a receptor comprising an extracellular domain (ECD), transmembrane domain (TMD), and intracellular domain (ICD). FIG. 2 illustrates a chimeric antigen receptor (CAR) comprising an antigen binding domain and at least one intracellular signaling domain (e.g., two intracellular signaling domains);

[0062] FIG. 3 schematically illustrates another system for regulating expression of a target polynucleotide in a cell. The system comprises a chimeric polypeptide comprising an ion sensor linked to a protease, wherein the ion sensor“hides” an active site of the protease when the ion is not bound the ion sensor. The system comprises a CAR comprising a protease cleavage site and an actuator configured to regulate expression of the target polynucleotide. Activation of the CAR and a subsequent activation of an ion channel increases cytosolic [ion] in the cell, and binding of the ion to the ion sensor activates the ion sensor, which in turn exposes the active site of the protease. Following, the exposed protease cleaves the protease cleavage site and releases the actuator from the CAR, thereby allowing the actuator to enter nucleus of the cell to regulate expression of the target polynucleotide in the cell;

[0063] FIG. 4 schematically illustrates a different system for regulating expression of a target polynucleotide in a cell. The system comprises a chimeric polypeptide comprising a NES linked to a protease cleavage site and an actuator configured to regulate expression of the target polynucleotide. The system comprises a CAR comprising an ion sensor linked to a protease, wherein the ion sensor“hides” an active site of the protease when the ion is not bound the ion sensor. Activation of the CAR and a subsequent activation of an ion channel increases cytosolic [ion] in the cell, and binding of the ion to the ion sensor activates the ion sensor, which in turn exposes the active site of the protease. Following, the exposed protease cleaves the protease cleavage site and releases the actuator from the NES, thereby allowing the actuator to enter nucleus of the cell to regulate expression of the target polynucleotide in the cell;

[0064] FIG. 5 schematically illustrates a different system for regulating expression of a target polynucleotide in a cell. The system comprises a CAR comprising a protease cleavage site and an actuator configured to regulate expression of the target polynucleotide. The system comprises a chimeric polypeptide comprising an ion sensor linked to a protease, wherein the ion sensor“hides” an active site of the protease when the ion is not bound the ion sensor. The chimeric polypeptide further comprises an adaptor configured to bind to an intracellular signaling domain of the CAR. Activation of the CAR and a subsequent activation of an ion channel increases cytosolic [ion] in the cell, and binding of the ion to the ion sensor activates the ion sensor, which in turn exposes the active site of the protease. Activation of the CAR also induces a change (e.g., conformational and/or chemical) in the intracellular signaling domain of the CAR, which change recruits and binds the adaptor of the chimeric polypeptide. Following, the exposed protease cleaves the protease cleavage site and releases the actuator from the CAR, thereby allowing the actuator to enter nucleus of the cell to regulate expression of the target polynucleotide in the cell;

[0065] FIG. 6 schematically illustrates a different system for regulating expression of a target polynucleotide in a cell. The system comprises a CAR comprising an ion sensor linked to a protease, wherein the ion sensor“hides” an active site of the protease when the ion is not bound the ion sensor. The system comprises a chimeric polypeptide comprising a NES linked to a protease cleavage site and an actuator configured to regulate expression of the target polynucleotide. The chimeric polypeptide further comprises an adaptor configured to bind to an intracellular signaling domain of the CAR. Activation of the CAR and a subsequent activation of an ion channel increases cytosolic [ion] in the cell, and binding of the ion to the ion sensor activates the ion sensor, which in turn exposes the active site of the protease. Activation of the CAR also induces a change (e.g., conformational and/or chemical) in the intracellular signaling domain of the CAR, which change recruits and binds the adaptor of the chimeric polypeptide. Following, the exposed protease cleaves the protease cleavage site and releases the actuator from the NES of the chimeric polypeptide, thereby allowing the actuator to enter nucleus of the cell to regulate expression of the target polynucleotide in the cell;

[0066] FIG. 7 schematically illustrates a different system for regulating expression of a target polynucleotide in a cell. The system comprises a chimeric polypeptide comprising an ion sensor linked to a NES, which NES is linked to a protease cleavage site and an actuator configured to regulate expression of the target polynucleotide. The ion sensor“hides” an active site of the protease when the ion is not bound the ion sensor. The system comprises an intracellular protease configured to cleave the protease cleavage site. Activation of a receptor of the cell and a subsequent activation of an ion channel increases cytosolic [ion] in the cell, and binding of the ion to the ion sensor activates the ion sensor, which in turn exposes the active site of the protease cleavage site. Following, the protease cleaves the exposed protease cleavage site and releases the actuator from the ion sensor and the NES of the chimeric polypeptide, thereby allowing the actuator to enter nucleus of the cell to regulate expression of the target polynucleotide in the cell; and

[0067] FIG. 8 schematically illustrates a different system for regulating expression of a target polynucleotide in a cell. The system comprises a chimeric polypeptide comprising a NES linked to an ion sensor, which ion sensor is linked to a protease cleavage site and an actuator configured to regulate expression of the target polynucleotide. The ion sensor“hides” an active site of the protease when the ion is not bound the ion sensor. The system comprises an intracellular protease configured to cleave the protease cleavage site. Activation of a receptor of the cell and a subsequent activation of an ion channel increases cytosolic [ion] in the cell, and binding of the ion to the ion sensor activates the ion sensor, which in turn exposes the active site of the protease cleavage site. Following, the protease cleaves the exposed protease cleavage site and releases the actuator from the NES and the ion sensor of the chimeric polypeptide, thereby allowing the actuator to enter nucleus of the cell to regulate expression of the target polynucleotide in the cell.

DETAILED DESCRIPTION

[0068] 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.

[0069] 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)).

[0070] Background [0071] As used in the specification and claims, the singular forms“a, 99 U 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.

[0072] 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.

[0073] 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, com, 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 originating from a natural organism (e.g. a cell can be a synthetically made, sometimes termed an artificial cell).

[0074] 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).

[0075] The term“receptor,” as used herein, refers to a molecule (e.g., a polypeptide) that has an affinity for a given ligand. Receptors can be naturally occurring or synthetic molecules. The given ligand (or ligand) can be naturally occurring or synthetic molecules. Receptors can be employed in an unaltered state or as aggregates with other species (e.g., with one or more co-receptors, one or more adaptors, lipid rafts, etc.). Examples of receptors may include, but are not limited to, cell membrane receptors, soluble receptors, cloned receptors, recombinant receptors, complex carbohydrates and glycoproteins hormone receptors, drug receptors, transmitter receptors, autocoid receptors, cytokine receptors, antibodies, antibody fragments, engineered antibodies, antibody mimics, molecular recognition units, adhesion molecules, agglutinins, integrins, selectins, nucleic acids and synthetic heteropolymers comprising amino acids, nucleotides, carbohydrates or nonbiologic monomers, including analogs and derivatives thereof, and conjugates or complexes formed by attaching or binding any of these molecules to a second molecule. In some cases, activation of the receptor directly or indirectly induces activation of one or more ion channels, such as, for example, a calcium channel receptor. In some cases, an activated calcium channel receptor on a cell membrane may induce an influx of calcium ions into the cell, thereby increasing a cytosolic concentration of calcium (i.e., [Ca ²⁺ ]) in the cell.

[0076] The term“ion channel,” as used herein, refers to a protein or polypeptide capable of regulating a flow of ions such as calcium cations through a channel. The flow may comprise (i) from the extracellular space and into the intracellular space of the cell, and/or (ii) from the intracellular space of the cell and towards the extracellular space. The ion channel may regulate the flow of a particular cation and/or anion, or may be less discriminating and allow multiple types of cations and/or anions to flow through it. The flow of ions may be regulated by the presence or absence of a ligand bound to the ion channel, or may be regulated by activation (e.g., phosphorylation or dephosphorylation) of the ion channel by another protein associated with the channel (e.g., another receptor or a chimeric antigen receptor (CAR)). In some cases, the ion channel protein may undergo a conformational change based on the binding of a ligand, chemical modification (e.g., phosphorylation) of a particular residue, and/or the binding or activation of another protein or biomolecule. [0077] In some cases, ion channel family proteins can form water-filled pores across cell membranes. Ion channel proteins can be located in the plasma membrane of cells (e.g., mammalian and/or plant cells) and are further characterized by small highly selective pores that participate in ion transport. Typically, more than 10 ⁶ ions can pass through such a channel each second. Many ion channels can allow specific ions, such as Na ⁺ , K ⁺ , Ca ²⁺ , or Cl , to diffuse down their electrochemical gradients across the lipid bilayer. The ion channel proteins can show ion selectivity, permitting some ions to pass but not others. Another feature of ion channel proteins is that they may not continuously open, in contrast to simple aqueous pores. Instead, they have“gates,” which may open briefly and then close again. This opening and closing may be in response to a specific perturbation of the membrane, such as a change in voltage across the membrane (voltage-gated channels), mechanical stimulation

(mechanically-gated channels), the binding of a signaling molecule (ligand-gated channels) to the ion channel protein, or signaling from another activated receptor protein. In the case of ligand-gated channels, the signaling ligand can be either an extracellular mediator, such as a neurotransmitter (transmitter-gated channels), an intracellular mediator, such as an ion (ion- gated channels), a nucleotide (nucleotide-gated channels), or a GTP-binding regulatory protein (G-protein-gated channels). Other examples include non-gated ion channels, viral ion channels, etc.

[0078] In some cases, the ion channel can comprise a calcium channel. The term “calcium channel,” as used herein, can refer to a protein or polypeptide on a lipid bilayer, exhibiting selective permeability to calcium ions. The amino acid sequences of calcium channel subunits in different organisms (e.g., human, rabbit, and rat) and in different tissues of the same organism (e.g., neuronal tissue, cardiovascular tissue) are known in the art. Ellis et al., Science 241(4873): 1661-4 (1988); Williams et al., Neuron 8(l):71-84 (1992); Ellis et al. U.S. Patent No. 5,686,241 (hereby incorporated by reference); and Harpold et al., U.S. Patent No. 5,792, 846 (hereby incorporated by reference).

[0079] In some cases, calcium ion (Ca ²⁺ ) can be a key and pivotal second messenger involved in one or more cellular functions, such as, for example, cell migration, muscle contraction/relaxation, neurotransmission, cell-cycle regulation, differentiation, and programmed cell death. To utilize signaling by Ca ²⁺ , i.e., a change in cytosolic Ca ²⁺ concentration (i.e., [Ca ²⁺ ]), to regulate one or more cellular processes, cells may be equipped with one or more mechanisms or cellular machineries to control [Ca ²⁺ ] in a localization- specific and time-dependent manner within the cells. For example, a cell can have a plurality of types of calcium channels with different response mechanisms and functions, such as, for example, receptor-operated, voltage-operated, and store-operated channels. Additionally, a cell can have a plurality of different calcium binding proteins which may have different isoforms, cell type-specific distribution, and/or diverse associated proteins.

[0080] The term“sensing moiety,” as used herein, can refer to a polypeptide, protein, or macromolecule (e.g., natural or synthetic) that can exhibit a specific binding (e.g., covalent or non-covalent) to one or more trigger molecules (e.g., ions, small molecule, lipid, nucleotide, polynucleotide, amino acid, peptide, polypeptide, protein, etc.). In some cases, the sensing moiety may bind to the trigger molecule(s) or release the trigger molecule(s) upon one or more stimuli. In some cases, the one or more stimuli may be one or more intracellular signaling pathways of a cell (e.g., via activation of one more receptors, e.g., endogenous or exogenous receptors, of the cell). In some cases, the binding of trigger molecule(s) by the sensing moiety or the releasing of the trigger molecule(s) from the sensing moiety may induce a change or modification (e.g., a conformational and/or chemical) at one or more sites (or domains) of the sensing moiety. In some cases, the sensing moiety may comprise a polypeptide, and the detecting of the specific molecule(s) may induce one or more

conformational changes (e.g., unfolding or folding to a different secondary, tertiary, and/or quaternary structure), one or more chemical changes (e.g., phosphorylation,

dephosphorylation, phosphorylation, acetylation, methylation, ubiquitination, proteolytic processing, etc.), or both. In some cases, the sensing moiety may comprise a calcium binding polypeptide, and the specific trigger molecule may comprise one or more calcium ions. In some cases, the sensing moiety may comprise a magnesium binding polypeptide (e.g., ZAK), and the specific trigger molecule may comprise one or more magnesium ions. In some cases, the sensing moiety may comprise a zinc binding polypeptide (e.g., zinc finger proteins, e.g., ZFP36, ZFN74, ZnuABC, etc.), and the specific trigger molecule may comprise one or more zinc ions.

[0081] The term“calcium binding polypeptide,”“calcium binding protein,” and“calcium sensor,” as used interchangeably herein, can refer to a polypeptide or a macromolecule that binds one or more calcium ions and which undergoes a conformational change upon calcium binding. Examples of a calcium binding polypeptide include, for example, Calmodulin (CaM) as well as proteins known as Calmodulin-like proteins including Aequorin, Calcium-binding protein (CABP), Calcineurin (e.g., Calcineurin B subunit isoform 1), Calmodulin-related protein NB-1 (CLP), Calcium vector protein (CAVP), Guanylyl cyclase activating protein 3 (GCAP 3), Calcium and integrin-binding protein 1 (Calmyrin; KIP; CIB), Myosin-2 light chain, Myosin essential light chain striated adductor muscle (E-LC), Myosin regulatory light chain striated adductor muscle (R-LC), Myosin regulatory light chain cdc4, Neurocalcin delta, Neuronal calcium sensor 1 (Frequenin), Sarcoplasmic calcium-binding protein (SCP), Troponin C, calbindins, calretinin, caltractin (e.g., CETN1, CETN2, CETN3), functional fragments thereof, functional variations thereof, and modifications thereof.

[0082] Other examples of a calcium binding polypeptide can include parvalbumin, S100, calpain, neuronal Ca2+ sensor (NCS)-1, calsequestrin, calreticulin, calnexin, gelsolin, annexins (e.g., AXA1, AXA2, AXA3, AXA4, AXA5, AXA6, AXA7, AXA8, AXA9, AXA10, AXA11, AXA13), PKC, synaptotagmins (e.g., synaptotagmin I, II, III, IX, VI, VII, V, X), phospholipase C (PLC), phospholipase A (PLA), Fam62a, Fam62b, Fam62c, dysferlin, otoferlin, myoferlin, mctpl, pctp2, Rph3A, Doc2a, Doc2b, sytll, Sytl2, Sytl3,

Sytl4, Sytl5, Uncl3a, Cpne6, Rcn2, osteonectin, hevin, QR1, testicans 1-3, tsc 36, SMOC-1, SMOC-2, coagulation factors VII, IX and X, protein C, protein S, fibrillin, notch and delta receptors, LDL receptors, functional fragments thereof, functional variations thereof, and modifications thereof. Examples of genes encoding S100 family proteins can include, but are not limited to, S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A7 (psoriasin), S100A8 (calgranulin A), S100A9 (calgranulin B), S100A10, S100A11, S100A12

(calgranulin C), S100A13, S100A14, S100A15 (koebnerisin), S100A16, S100B, S100P, S100Z, CRNN, FLG, FLG2, HRNR, RPTN, S100G, TCHH, and THHLl.

[0083] In some cases, the calcium binding polypeptide can be linked (e.g., covalently or non-covalently attached) to a cargo (e.g., a small molecule, a polypeptide, a polynucleotide, etc.) to form a fusion complex (e.g., a chimeric polypeptide). In some examples, in the absence of calcium ions (or insufficient calcium ion concentration to permit binding of one or more calcium ions to the calcium binding polypeptide), the calcium binding polypeptide may be configured in a first state that“hides” one or more active sites of the cargo. On the other hand, in the presence of calcium ions and upon binding of one or more calcium ions to the calcium binding polypeptide, the calcium binding polypeptide may transform to a second state (e.g., via a conformational and/or chemical change, such as different folding and/or phosphorylation), thereby to“expose” the one or more active sites of the cargo. In some examples, in the presence of calcium ions (or sufficient calcium ion concentration to permit binding of one or more calcium ions to the calcium binding polypeptide), the calcium binding polypeptide may be configured in a first state that“hides” one or more active sites of the cargo. On the other hand, in the absence of calcium ions and upon release of one or more calcium ions from the calcium binding polypeptide, the calcium binding polypeptide may transform to a second state (e.g., via a conformational and/or chemical change, such as different folding and/or phosphorylation), thereby to“expose” the one or more active sites of the cargo.

[0084] In some cases, the calcium binding polypeptide may be linked to the cargo via a linker, such as, for example, a linker peptide. The term“linker peptide” or“linker,” as used interchangeably herein, may refer to amino acid sequences (e.g., synthetic amino acid sequences) that connect or link two moieties (e.g., two polypeptide sequences). In some examples, the linker peptide may comprise (GGGGS)n, where n may be 0, 1, 2, 3, 4, 5 or more (e.g., n may be 2, 3, or 4, preferably). Such linker may allow flexibility of positioning of the calcium binding polypeptide relative to the cargo of the fusion complex.

[0085] In some cases, the fusion complex (e.g., a chimeric polypeptide) may comprise at least 1, 2, 3, 4, 5, or more calcium binding polypeptides. In some cases, fusion complex (e.g., a chimeric polypeptide) may comprise at most 5, 4, 3, 2, or 1 calcium binding polypeptide. In some cases, the fusion complex (e.g., a chimeric polypeptide) may comprise at least 1, 2, 3, 4, 5, or more cargoes. In some cases, fusion complex (e.g., a chimeric polypeptide) may comprise at most 5, 4, 3, 2, or 1 cargo. In some cases, a calcium binding polypeptide may be capable of“hiding” and“exposing” one or more active sites of at least 1, 2, 3, 4, 5, or more cargoes. In some cases, a calcium binding polypeptide may be capable of“hiding” and “exposing” one or more active sites of at most 5, 4, 3, 2, or 1 cargo.

[0086] In some cases, one or more calcium binding polypeptides may be activated (e.g., direcdy or indirectly) by light. In some cases, the one or more calcium binding polypeptides may not be activated (e.g., directly or indirectly) by light. In some cases, one or more calcium channels may be activated (e.g., directly or indirectly) by light. In some cases, the one or more calcium channels may not be activated (e.g., directly or indirectly) by light. In some cases, the one or more calcium channels may be activated by at least 1, 2, 3, 4, 5, or more intracellular signaling pathways. In some cases, the one or more calcium channels may be activated by at most 5, 4, 3, 2, or 1 intracellular signaling pathway. The intracellular signaling pathway may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more intracellular and/or extracellular ligands to activate the calcium channel(s). The intracellular signaling pathway may comprise at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 intracellular and/or extracellular ligand to activate the calcium channel(s).

[0087] The term“cell membrane,” as used herein, refers to the boundary membrane, external membrane, interfacial membrane, protoplasmic membrane, or cell wall that separates the protoplasm of the cell from the outside. Thus, the term“cell membrane receptor” or “transmembrane receptor,” as used here, refers to a receptor in the boundary membrane, external membrane, interfacial membrane, protoplasmic membrane, or cell wall that separates the protoplasm of the cell from the outside.

[0088] 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.

[0089] 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 IgGl, 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).

[0090] 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 Rin (CD 16) subclasses, including allelic variants and alternatively spliced forms of these receptors.

Fcgamma RII receptors include Fcgamma RIIA (an“activating receptor”) and Fcgamma RUB (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.

[0091] 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, dlTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [<xS]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 delectably 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-l -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]ddGIP, [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,

BODIP Y -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, tetram ethyl rhodami ne-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-ll-dCTP, biotin- 14- dCTP), and biotin-dUTP (e.g. biotin- 11-dUTP, biotin- 16-dUTP, biotin-20-dUTP).

[0092] 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, morpholines, 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.

[0093] 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 S’ 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).

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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. [0099] 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.

[0100] 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 complementarity 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.

[0101] 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.

[0102] 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.

[0103] 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. [0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] The actuator moiety may comprise a Cas protein or a modification thereof. 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 Cpfl . A Cas protein can be C2c2. A Cas protein can be Casl 3a. A cas protein can be Casl2, or a functional variant thereof. A Casl2 protein can be Casl2a, Casl2b,

Casl 2c, C2c4, C2c8, C2c5, C2cl0, C2c9, CasX (Casl2e), CasY (Casl2d), or a modification thereof. 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.

[0109] 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).

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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”.

[0114] 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

[0115] 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.

[0116] The term“nuclear export signal (NES),” as used herein, can refer to an amino acid sequence capable of direct a polypeptide containing it (such as aNES-containing chimeric polypeptide) to be exported from the nucleus of a cell. In some cases, such export may be mostly mediated by one or more proteins (e.g., one or more exportin proteins, such as chromosomal region maintenance 1 (Crml)). In some cases, the NES may be rich in hydrophobic amino acid residues, such as leucine (Leu). Other examples of hydrophobic residues can include one or more of: glycine (Gly), alanine (Ala), valine (Val), isoleucine (He), proline (Pro), phenylalanine (Phe), methionine (Met), tryptophan (Trp), modifications thereof, and combinations thereof. In some examples, a leucine-rich NES may be a motif (e.g., a conservative or non-conservative motif) comprising 3 or 4 hydrophobic residues. In an example, a NES motif may comprise a polynucleotide pattern LxxLxL, LxxxLxL, or LxxxLxxLxL, wherein each L is independently selected from the hydrophobic resides (e.g., leucine, isoleucine, valine, phenylalanine and methionine), and each x is independently selected from any amino acid (see La Cour et al., Protein Engineering, Design and Selection 17(6):527-536, 2004). The NES may have a net positive charge. As an alternative, the NES may have a net negative charge. In a different alternative, the NES may have a net neutral charge.

[0117] 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.

[0118] 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, remodeling 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.

[0119] 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, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, and demyristoylation activity.

[0120] 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).

[0121] 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.

[0122] 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 HTV reverse transcriptase.

[0123] 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).

[0124] 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.

[0125] 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, remodeling 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).

[0126] 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.

[0127] 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.

[0128] 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.

[0129] The term“effective amounf’ 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.

[0130] 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.

[0131] 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) GGAM domains derived from the intracellular region of the CD3 z receptor or FceRIy). 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., CD3z- CD28-OX40, or CD3z-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 CD3z IT AM domain) or under the control of a CAR-induced promoter (e.g., the NFAT/IL-2 minimal promoter).

[0132] 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.

[0133] In some cases, a continual expression of a polypeptide sequence (e.g., Cas, dCas, or a different protein that is endogenous or exogenous to the cell) 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.

[0134] 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.

[0135] 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.

[0136] The term“2A peptide” may refer to a class of viral oligopeptides (e.g., 18-22 amino-acid (aa)-long viral oligopeptides) that mediate“cleavage” of polypeptides during translation in cells (e.g., eukaryotic cells). The designation“2A” refers to a specific region of the viral genome and different viral 2As have generally been named after the virus they were derived from. The first discovered 2A was F2A (foot-and-mouth disease virus), after which E2A (equine rhinitis A vims), P2A (porcine teschovirus-1 2 A), and T2A (thosea asigna vims 2A) were also identified. The mechanism of 2A-mediated“self-cleavage” is believed to be ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A sequence.

[0137] Systems and methods for regulating expression of a target polynucleotide

[0138] In an aspect, the present disclosure provides a system for regulating expression of a target polynucleotide in a cell. The system may comprise a first chimeric polypeptide and a second chimeric polypeptide. The first chimeric polypeptide may comprise a gene modulating polypeptide (GMP) that comprises an actuator moiety linked to a cleavage recognition site. The actuator moiety may regulate the expression of the target polynucleotide in the cell. The second chimeric polypeptide may comprise a sensing moiety exhibiting specific binding to a trigger molecule. The sensing moiety may be linked to a cleavage moiety that cleaves the cleavage recognition site. Upon binding of the trigger molecule to the sensing moiety or upon releasing of the trigger molecule from the sensing moiety, the sensing moiety may undergo a modification to activate the cleavage moiety. Upon introducing the system into the cell and contacting of the cell by a ligand, the second chimeric polypeptide may be induced to activate the cleavage moiety, such that the activated cleavage moiety releases the actuator moiety from the GMP of the first chimeric polypeptide to effect regulating expression of the target polynucleotide in the cell.

[0139] By controlling (e.g., activating or de-activating) the interaction between the cleavage moiety and the cleavage recognition site (e.g., by controlling the cleavage activity of the cleavage moiety), and thereby controlling the activity of the actuator to regulating expression of the target polynucleotide in the nucleus of the cell, the system provided in the present disclosure can be advantageous in comparison to conventional actuator systems (e.g., Cas, dCas coupled to a transcription factor, transcription factor, etc.). For example, controlling when to allow the actuator to enter the nucleus and regulate expression of the target polynucleotide in the nucleus (e.g., by providing the ligand specific for inducing the sensing moiety), undesired or background activity of the actuator may be reduced or prevented. In another example, an amount of the actuator needed to yield a sufficient regulation of the target polynucleotide may be reduced.

[0140] Upon contacting of the cell by the ligand, the sensing moiety may be induced to bind at least one trigger molecule. Alternatively or in addition to, the sensing moiety may already be bound to at least one trigger molecule, and, upon contacting of the cell by the ligand, the sensing moiety may be induced to release the at least one trigger molecule. In some cases, upon contacting of the cell by the ligand, a first portion of the sensing moiety may be induced to bind a first trigger molecule, while a second portion of the sensing moiety may be induced to bind a second trigger molecule that is different than the first trigger molecule. In some cases, upon contacting of the cell by the ligand, a first portion of the sensing moiety may be induced to bind a trigger molecule, while a second portion of the sensing moiety may be induced to release another previously-bound trigger molecule.

[0141] The binding of the trigger molecule by the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety may induce at least 1, 2, 3, 4, 5, or more modifications of the sensing moiety. The binding of the trigger molecule by the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety may induce at most 5, 4, 3, 2, or 1 more modification of the sensing moiety.

[0142] In some cases, without the binding of the trigger molecule to the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety, and thus without (or prior to) the conformational change of the sensing moiety, the sensing moiety may be configured to hide, cover, block, or shield an active site of the cleavage moiety, such that the cleavage moiety is inactive or non-functional. In such a case, the binding of the trigger molecule to the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety may induce a transformation (e.g., conformational and/or chemical modification) in the sensing moiety, thereby exposing the active site of the cleavage moiety, such that the cleavage moiety becomes active and functional.

[0143] In some cases, the cleavage moiety may (i) lack a chemical modification (e.g., one or more phosphates, one or more amino acids, etc.) that is necessary for the cleavage moiety to become active, and/or (ii) comprise an additional chemical functionality (e.g., one or more phosphates, one or more amino acids, etc.) that needs to be altered or removed for the cleavage moiety to become active. In such cases, the binding of the trigger molecule to the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety may activate the sensing moiety, which in return activates the cleavage moiety by (i) inducing the necessary chemical modification on the cleavage moiety, and/or (ii) altering or removing the additional chemical functionality from the cleavage moiety.

[0144] In some cases, upon the binding of the trigger molecule or releasing of the trigger molecule, the sensing moiety may undergo a modification, and at least a portion of the modified sensing moiety may fuse (e.g., via covalent or non-covalent complexation) with at least a portion of the cleavage moiety to form a functional cleavage moiety. [0145] In some cases, the first chimeric polypeptide and the second chimeric polypeptide may be in contact with one another prior to the activation of the cleavage moiety by the sensing moiety. In some cases, the first chimeric polypeptide and the second chimeric polypeptide may not be in contact with one another prior to the activation of the cleavage moiety by the sensing moiety.

[0146] In some cases, the second chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more sensing moieties. In some cases, the second chimeric polypeptide may comprise at most 5, 4, 3, 2, or 1 sensing moiety. In some cases, at least 1, 2, 3, 4, 5, or more sensing moieties may be operatively coupled to at least one cleavage moiety to regulate activity of the at least one cleavage moiety. In some cases, at most 5, 4, 3, 2, or 1 sensing moiety may be operatively coupled to at least one cleavage moiety to regulate activity of the at least one cleavage moiety. In some cases, each sensing moiety may be activatable upon binding of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more trigger molecules. In some cases, each sensing moiety may be activatable upon binding of at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 trigger molecule. The sensing moiety may exhibit specific binding to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more trigger molecules. The sensing moiety may exhibit specific binding to a plurality of trigger molecules that are the same or different.

[0147] In some cases, the first chimeric polypeptide may further comprise at least one nuclear export signal (NES) sequence configured to reduce translocation of the first chimeric polypeptide to nucleus of the cell. Once the first chimeric polypeptide is in the cytoplasm of the cell (e.g., exported from the nucleus out into the cytoplasm), the at least one NES sequence may be configured to prevent the first chimeric polypeptide from translocating to the nucleus of the cell. In some cases, the at least one NES sequence may need to be removed or altered (e.g., chemically altered an overall charge of the at least one NES sequence) to improve translocation of at least a portion (e.g., the actuator) of the first chimeric polypeptide to the cell nucleus. In some cases, the at least one NES sequence may need to be removed or altered (e.g., chemically altered an overall charge of the at least one NES sequence) to allow at least a portion (e.g., the actuator) of the first chimeric polypeptide to translocate to the cell nucleus. In some cases, the first chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more NES sequences. In some cases, the first chimeric polypeptide may comprise at most 5,

4, 3, 2, or 1 NES sequence. In some cases, the at least one NES sequence may be linked to the C-terminus and/or N-terminus of the GMP. In some cases, the cleavage recognition site is flanked by the at least one NES and the actuator moiety. [0148] In some cases, the second chimeric polypeptide further comprises at least one NES sequence configured to reduce translocation of the second chimeric polypeptide to the nucleus of the cell. Once the second chimeric polypeptide is in the cytoplasm of the cell (e.g., exported from the nucleus out into the cytoplasm), the at least one NES sequence may be configured to prevent the second chimeric polypeptide from translocating to the nucleus of the cell. In some cases, the at least one NES sequence may need to be removed or altered (e.g., chemically altered an overall charge of the at least one NES sequence) to improve translocation of at least a portion (e.g., the actuator) of the second chimeric polypeptide to the cell nucleus. In some cases, the at least one NES sequence may need to be removed or altered (e.g., chemically altered an overall charge of the at least one NES sequence) to allow at least a portion (e.g., the actuator) of the second chimeric polypeptide to translocate to the cell nucleus. In some cases, the second chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more NES sequences. In some cases, the second chimeric polypeptide may comprise at most 5, 4, 3, 2, or 1 NES sequence. In some cases, the at least one NES sequence may be linked to the C-terminus and/or N-terminus of the second chimeric polypeptide. In some cases, (i) the sensing moiety may be flanked by the at least one NES and the cleavage moiety, (ii) the cleavage moiety may be flanked by the at least one NES and the sensing moiety, or (iii) the at least one NES may be flanked by the sensing moiety and the cleavage moiety.

[0149] In some cases, the system may further comprise a receptor (e.g., a chimeric receptor) having a ligand binding domain specific for the ligand. In an example, the system may further comprise a chimeric receptor having the ligand binding domain specific for the ligand. In some cases, upon the contacting of the ligand binding domain by the ligand, the second chimeric polypeptide may be induced to activate the cleavage moiety. In some cases, the receptor (e.g., the chimeric receptor) may comprise at least 1, 2, 3, 4, 5, or more ligand binding domains specific for the ligand. In some cases, the receptor (e.g., the chimeric receptor) may comprise at most 5, 4, 3, 2, or 1 ligand binding domains specific for the ligand. In some cases, the receptor may comprise at least 2, 3, 4, 5, or more ligand binding domains, each specific for a different ligand. In some cases, the receptor may comprise at most 5, 4, 3, or 2 ligand binding domains, each specific for a different ligand.

[0150] In some cases, the first chimeric polypeptide may be fused in-frame with the receptor (e.g., the chimeric receptor). In some cases, the cleavage recognition site may be flanked by the receptor (e.g., the chimeric receptor) and the actuator moiety. In some cases, the second chimeric polypeptide may further comprise an adaptor polypeptide configured to bind (e.g., directly or indirectly) the receptor (e.g., the chimeric receptor) or a downstream signaling moiety of the receptor (e.g., the chimeric receptor) in response to the contacting of the ligand binding domain by the ligand. In some cases, the sensing moiety may be flanked by the adaptor polypeptide and the cleavage moiety. In some cases, the cleavage moiety may be flanked by the adaptor polypeptide and the sensing moiety. In some cases, the adaptor polypeptide may be flanked by the sensing moiety and the cleavage moiety. In some cases, the second chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more adaptor polypeptides. In some cases, the second chimeric polypeptide may comprise at most 5, 4, 3, 2, or 1 adaptor polypeptide.

[0151] In some cases, the second chimeric polypeptide may be fused in-frame with the receptor (e.g., the chimeric receptor). In some cases, the first chimeric polypeptide may further comprise an adaptor polypeptide configured to bind (e.g., directly or indirectly) the receptor (e.g., the chimeric receptor) or a downstream signaling moiety of the receptor (e.g., the chimeric receptor) in response to the contacting of the ligand binding domain by the ligand. In some cases, the cleavage recognition site may be flanked by the adaptor

polypeptide and the actuator moiety. In some cases, the first chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more adaptor polypeptides. In some cases, the first chimeric polypeptide may comprise at most 5, 4, 3, 2, or 1 adaptor polypeptide.

[0152] In some cases, one or more components (e.g., all components) of any one of the subject systems may not be activated by light (e.g., one or more wavelengths of the electromagnetic spectrum). In some cases, the chimeric receptor may not be activated by light. In some cases, the first chimeric polypeptide (e.g., comprising the GMP) may not be activated by light. In some cases, the second chimeric polypeptide (e.g., comprising the sensing moiety and the cleavage moiety) may not be activated by light. In some cases, the sensing moiety may not be activated by light. In some cases, the cytosolic concentration of the trigger molecule may not be activated, upregulated, and/or downregulated by light.

[0153] In some cases, one or more components (e.g., all components) of any one of the subject systems may be activated by light (e.g., one or more wavelengths of the

electromagnetic spectrum). In some cases, the chimeric receptor may be activated by light. In some cases, the first chimeric polypeptide (e.g., comprising the GMP) may be activated by light. In some cases, the second chimeric polypeptide (e.g., comprising the sensing moiety and the cleavage moiety) may be activated by light. In some cases, the sensing moiety may be activated by light. In some cases, the cytosolic concentration of the trigger molecule may be activated, upregulated, and/or downregulated by light. [0154] In some cases, or more components (e.g., all components) of any one of the subject systems may be activated by at least one ligand (e.g., an extracellular ligand) and light.

[0155] In another aspect, the present disclosure provides a system for regulating expression of a target polynucleotide in a cell. The system may comprise a first chimeric polypeptide and a second chimeric polypeptide. The first chimeric polypeptide may comprise a gene modulating polypeptide (GMP) that comprises an actuator moiety linked to a cleavage recognition site. The actuator moiety may regulate the expression of the target polynucleotide in the cell. The first chimeric polypeptide may further comprise a sensing moiety exhibiting specific binding to a trigger molecule and linked to the GMP. Upon binding of the trigger molecule to the sensing moiety or upon releasing of the trigger molecule from the sensing moiety, the sensing moiety may undergo a modification to activate the cleavage recognition site. The second chimeric polypeptide may comprise a cleavage moiety that cleaves the activated cleavage recognition site. Upon introducing the system into the cell and contacting of the cell by a ligand, the first chimeric polypeptide may be induced to activate the cleavage recognition site, such that the cleavage moiety releases the actuator moiety from the GMP of the first chimeric polypeptide to effect regulating expression of the target polynucleotide in the cell.

[0156] By controlling (e.g., activating or de-activating) the interaction between the cleavage moiety and the cleavage recognition site (e.g., by controlling the activity of the cleavage recognition site), and thereby controlling the activity of the actuator to regulating expression of the target polynucleotide in the nucleus of the cell, the system provided in the present disclosure can be advantageous in comparison to conventional actuator systems (e.g., Cas, dCas coupled to a transcription factor, transcription factor, etc.). For example, controlling when to allow the actuator to enter the nucleus and regulate expression of the target polynucleotide in the nucleus (e.g., by providing the ligand specific for inducing the sensing moiety), undesired or background activity of the actuator may be reduced or prevented. In another example, an amount of the actuator needed to yield a sufficient regulation of the target polynucleotide may be reduced.

[0157] Upon contacting of the cell by the ligand, the sensing moiety may be induced to bind at least one trigger molecule. Alternatively or in addition to, the sensing moiety may already be bound to at least one trigger molecule, and, upon contacting of the cell by the ligand, the sensing moiety may be induced to release the at least one trigger molecule. In some cases, upon contacting of the cell by the ligand, a first portion of the sensing moiety may be induced to bind a first trigger molecule, while a second portion of the sensing moiety may be induced to bind a second trigger molecule that is different than the first trigger molecule. In some cases, upon contacting of the cell by the ligand, a first portion of the sensing moiety may be induced to bind a trigger molecule, while a second portion of the sensing moiety may be induced to release another previously-bound trigger molecule.

[0158] The binding of the trigger molecule by the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety may induce at least 1, 2, 3, 4, 5, or more modifications of the sensing moiety. The binding of the trigger molecule by the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety may induce at most 5, 4, 3, 2, or 1 more modification of the sensing moiety.

[0159] In some cases, without the binding of the trigger molecule to the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety, and thus without (or prior to) the conformational change of the sensing moiety, the sensing moiety may be configured to hide, cover, block, or shield an active site of the cleavage recognition site, such that the cleavage recognition site is inactive or non-functional. In such a case, the binding of the trigger molecule to the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety may induce a transformation (e.g., conformational and/or chemical modification) in the sensing moiety, thereby exposing the active site of the cleavage recognition site, such that the cleavage recognition site becomes active and functional to be recognized by the cleavage moiety.

[0160] In some cases, the cleavage recognition site may (i) lack a chemical modification (e.g., one or more phosphates, one or more amino acids, etc.) that is necessary for the cleavage recognition site to become active, and/or (ii) comprise an additional chemical functionality (e.g., one or more phosphates, one or more amino acids, etc.) that needs to be altered or removed for the cleavage recognition site to become active. In such cases, the binding of the trigger molecule to the sensing moiety and/or the releasing of the trigger molecule from the sensing moiety may activate the sensing recognition site, which in return activates the cleavage recognition site by (i) inducing the necessary chemical modification on the cleavage recognition site, and/or (ii) altering or removing the additional chemical functionality from the cleavage recognition site.

[0161] In some cases, upon the binding of the trigger molecule or releasing of the trigger molecule, the sensing moiety may undergo a modification, and at least a portion of the modified sensing moiety may fuse (e.g., via covalent or non-covalent complexation) with at least a portion of the cleavage recognition site to form a functional cleavage recognition site. [0162] In some cases, the first chimeric polypeptide and the second chimeric polypeptide may be in contact with one another prior to the activation of the cleavage recognition site by the sensing moiety. In some cases, the first chimeric polypeptide and the second chimeric polypeptide may not be in contact with one another prior to the activation of the cleavage recognition site by the sensing moiety.

[0163] In some cases, the first chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more sensing moieties. In some cases, the first chimeric polypeptide may comprise at most 5, 4, 3, 2, or 1 sensing moiety. In some cases, at least 1, 2, 3, 4, 5, or more sensing moieties may be operatively coupled to at least one cleavage recognition site to regulate activity of the at least one cleavage recognition site. In some cases, at most 5, 4, 3, 2, or 1 sensing moiety may be operatively coupled to at least one cleavage recognition site to regulate activity of the at least one cleavage recognition site. In some cases, each sensing moiety may be activatable upon binding of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more trigger molecules. In some cases, each sensing moiety may be activatable upon binding of at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 trigger molecule. The sensing moiety may exhibit specific binding to at least 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, or more trigger molecules. The sensing moiety may exhibit specific binding to a plurality of trigger molecules that are the same or different.

[0164] In some cases, the first chimeric polypeptide may further comprise at least one nuclear export signal (NES) sequence configured to reduce translocation of the first chimeric polypeptide to nucleus of the cell. Once the first chimeric polypeptide is in the cytoplasm of the cell (e.g., exported from the nucleus out into the cytoplasm), the at least one NES sequence may be configured to prevent the first chimeric polypeptide from translocating to the nucleus of the cell. In some cases, the at least one NES sequence may need to be removed or altered (e.g., chemically altered an overall charge of the at least one NES sequence) to improve translocation of at least a portion (e.g., the actuator) of the first chimeric polypeptide to the cell nucleus. In some cases, the at least one NES sequence may need to be removed or altered (e.g., chemically altered an overall charge of the at least one NES sequence) to allow at least a portion (e.g., the actuator) of the first chimeric polypeptide to translocate to the cell nucleus. In some cases, the first chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more NES sequences. In some cases, the first chimeric polypeptide may comprise at most 5, 4, 3, 2, or 1 NES sequence. In some cases, the at least one NES sequence may be linked to the C-terminus and/or N-terminus of the GMP. In some cases, the at least one NES may be flanked by the sensing moiety and the cleavage recognition site. Alternatively, the sensing moiety may be flanked by the at least one NES and the cleavage recognition site. [0165] In some cases, the second chimeric polypeptide further comprises at least NES sequence configured to reduce translocation of the second chimeric polypeptide to the nucleus of the cell. Once the second chimeric polypeptide is in the cytoplasm of the cell (e.g., exported from the nucleus out into the cytoplasm), the at least one NES sequence may be configured to prevent the second chimeric polypeptide from translocating to the nucleus of the cell. In some cases, the at least one NES sequence may need to be removed or altered (e.g., chemically altered an overall charge of the at least one NES sequence) to improve translocation of at least a portion (e.g., the actuator) of the second chimeric polypeptide to the cell nucleus. In some cases, the at least one NES sequence may need to be removed or altered (e.g., chemically altered an overall charge of the at least one NES sequence) to allow at least a portion (e.g., the actuator) of the second chimeric polypeptide to translocate to the cell nucleus. In some cases, the second chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more NES sequences. In some cases, the second chimeric polypeptide may comprise at most 5, 4, 3, 2, or 1 NES sequence. In some cases, the at least one NES sequence may be linked to the C-terminus and/or N-terminus of the second chimeric polypeptide.

[0166] In some cases, the system may further comprise a receptor (e.g., a chimeric receptor) having a ligand binding domain specific for the ligand. In an example, the system may further comprise a chimeric receptor having the ligand binding domain specific for the ligand. In some cases, upon the contacting of the ligand binding domain by the ligand, the first chimeric polypeptide is induced to activate the cleavage recognition site (e.g., by action of the sensing moiety). In some cases, the receptor (e.g., the chimeric receptor) may comprise at least 1, 2, 3, 4, 5, or more ligand binding domains specific for the ligand. In some cases, the receptor (e.g., the chimeric receptor) may comprise at most 5, 4, 3, 2, or 1 ligand binding domains specific for the ligand. In some cases, the receptor may comprise at least 2, 3, 4, 5, or more ligand binding domains, each specific for a different ligand. In some cases, the receptor may comprise at most 5, 4, 3, or 2 ligand binding domains, each specific for a different ligand.

[0167] In some cases, the first chimeric polypeptide may be fused in-frame with the receptor (e.g., the chimeric receptor). In some cases, the cleavage recognition site may be flanked by the chimeric receptor and the actuator moiety. In addition, the sensing moiety may be flanked by the chimeric receptor and the cleavage recognition site. In some cases, the second chimeric polypeptide may further comprise an adaptor polypeptide configured to bind (e.g., directly or indirectly) the receptor (e.g., the chimeric receptor) or a downstream signaling moiety of the receptor (e.g., the chimeric receptor) in response to the contacting of the ligand binding domain by the ligand. In some cases, the second chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more adaptor polypeptides. In some cases, the second chimeric polypeptide may comprise at most 5, 4, 3, 2, or 1 adaptor polypeptide.

[0168] In some cases, the second chimeric polypeptide may be fused in-frame with the receptor (e.g., the chimeric receptor). In some cases, the first chimeric polypeptide may further comprise an adaptor polypeptide configured to bind (e.g., directly or indirectly) the receptor (e.g., the chimeric receptor) or a downstream signaling moiety of the receptor (e.g., the chimeric receptor) in response to the contacting of the ligand binding domain by the ligand. In some cases, the cleavage recognition site may be flanked by the adaptor polypeptide and the actuator moiety. In addition, the adaptor polypeptide may be flanked by the sensing moiety and the cleavage recognition site, or, alternatively, the sensing moiety nay be flanked by the adaptor polypeptide and the cleavage recognition site. In some cases, the first chimeric polypeptide may comprise at least 1, 2, 3, 4, 5, or more adaptor polypeptides. In some cases, the first chimeric polypeptide may comprise at most 5, 4, 3, 2, or 1 adaptor polypeptide.

[0169] In some cases, one or more components (e.g., all components) of any one of the subject systems may not be activated by light (e.g., one or more wavelengths of the electromagnetic spectrum). In some cases, the chimeric receptor may not be activated by light. In some cases, the first chimeric polypeptide (e.g., comprising the GMP) may not be activated by light. In some cases, the second chimeric polypeptide (e.g., comprising the sensing moiety and the cleavage moiety) may not be activated by light. In some cases, the sensing moiety may not be activated by light. In some cases, the cytosolic concentration of the trigger molecule may not be activated, upregulated, and/or downregulated by light.

[0170] In some cases, one or more components (e.g., all components) of any one of the subject systems may be activated by light (e.g., one or more wavelengths of the

electromagnetic spectrum). In some cases, the chimeric receptor may be activated by light. In some cases, the first chimeric polypeptide (e.g., comprising the GMP) may be activated by light. In some cases, the second chimeric polypeptide (e.g., comprising the sensing moiety and the cleavage moiety) may be activated by light. In some cases, the sensing moiety may be activated by light. In some cases, the cytosolic concentration of the trigger molecule may be activated, upregulated, and/or downregulated by light.

[0171] In some cases, or more components (e.g., all components) of any one of the subject systems may be activated by at least one ligand (e.g., an extracellular ligand) and light. [0172] In some cases of any one of the subject systems, the ligand comprises an extracellular ligand. In some cases, the extracellular ligand may be an antigen presented on a target cell of the cell. In some cases, the ligand may be an antigen presented on a target cell (e.g., a tumor or cancer cell) of the cell (e.g., a lymphocyte or immune cell, such as a T cell).

[0173] In some cases of any one of the subject systems, the contacting of the cell by the ligand may increase a cytosolic concentration of the trigger molecule in the cell. In some cases, the increase in the cytosolic concentration of the trigger molecule may induce the binding of the trigger molecule to the sensing moiety. In some cases of any one of the subject systems, the contacting of the cell by the ligand may decrease a cytosolic concentration of the trigger molecule in the cell. In some cases, the decrease in the cytosolic concentration of the trigger molecule may induce the releasing of the trigger molecule from the sensing moiety.

[0174] In some cases of any one of the subject systems, the adaptor polypeptide may be configured to bind at least one intracellular signaling domain of the receptor, e.g., the chimeric receptor. In some cases, the receptor may comprise the at least one intracellular signaling domain. In some examples, the chimeric receptor may be configured to undergo a modification (e.g., a conformational change and/or chemical modification) upon binding of the ligand to the ligand binding domain of the chimeric receptor. Such modification may comprise one or more modifications in the at least one intracellular signaling domain. Upon the one or more modifications in the at least one intracellular signaling domain of the chimeric receptor, the adaptor polypeptide may bind the at least one modified intracellular signaling domain of the chimeric receptor. As such, a chimeric polypeptide (e.g., the first chimeric polypeptide and/or the second chimeric polypeptide) comprising the adaptor polypeptide may be recruited to the chimeric receptor upon activation of the chimeric receptor. In some cases, the adaptor polypeptide may be configured to bind the downstream signaling moiety (e.g., a co-receptor, an adaptor protein of the chimeric polypeptide, etc.) upon activation of the chimeric receptor. In some cases, the downstream signaling moiety may undergo a modification (e.g., conformational and/or chemical modification) to recruit the adaptor polypeptide to the downstream signaling moiety, thereby, in some examples, indirectly recruiting the first chimeric polypeptide and/or the second chimeric polypeptide to the chimeric receptor.

[0175] In some cases of any one of the subject systems, the chemical modification of the receptor (e.g., the chimeric receptor) and/or the downstream signaling moiety of the receptor (e.g., the chimeric receptor) may be selected from the group consisting of: dephosphorylation, phosphorylation, acetylation, methylation, ubiquitination, proteolytic processing, or combination thereof. In some cases, the adaptor polypeptide may comprise one or more adaptor proteins of the receptor (e.g., the chimeric receptor), kinase, hydrolase, nucleotide exchange factor, an adaptor protein thereof, a fragment thereof, or a combination thereof Examples of the hydrolase may comprise lipases, phosphatases, glycosidases, peptidases, and nucleosidases.

[0176] In some cases of any one of the subject systems, the first chimeric polypeptide may comprise the adaptor polypeptide, and the second chimeric polypeptide may not comprise the adaptor polypeptide. In some cases, the second chimeric polypeptide may comprise the adaptor polypeptide, and the first chimeric polypeptide may not comprise the adaptor polypeptide. In some cases, the first chimeric polypeptide may comprise a first adaptor polypeptide, and the second chimeric polypeptide may comprise a second adaptor polypeptide. Such first and second adaptor polypeptides may be the same or different. The first and second adaptor polypeptides may be configured to bind the same target (e.g., the same intracellular signaling domain of the chimeric receptor) or different targets (e.g., different intracellular signaling domains of the chimeric receptor).

[0177] In some cases of any one of the subject systems, the receptor (e.g., the chimeric receptor) may comprise at least 1, 2, 3, 4, 5, or more intracellular signaling domains. In some cases, the receptor (e.g., the chimeric receptor) may comprise at most 5, 4, 3, 2, or 1 intracellular signaling domain. In some cases, the receptor (e.g., the chimeric receptor) may comprise a plurality of intracellular signaling domains that have the same or different cellular functions.

[0178] In some cases of any one of the subject systems, one or more peptide linkers may be flanked by the receptor (e.g., the chimeric receptor) and (i) the first chimeric polypeptide or (ii) the second chimeric polypeptide.

[0179] In some cases of any one of the subject systems, the trigger molecule may be selected from the group consisting of: ion, lipid, small molecule, polynucleotide, polypeptide, a modification thereof, and a combination thereof. In some cases, the trigger molecule may be at least one ion. In some cases, the trigger molecule may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the same or different ions. In some cases, the trigger molecule may be at most 10, 9, 8, 7, 6, 5, 4, 3, or 2 of the same or different ions. In some cases, the trigger molecule may be calcium. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more calcium ions may need to bind to the sensing moiety to activate the sensing moiety. In some cases, at most 10, 9, 8,

7, 6, 5, 4, 3, 2, or 1 calcium ion may need to bind to the sensing moiety to activate the sensing moiety. [0180] In some cases of any one of the subject systems, activation of the sensing moiety upon binding the at least one trigger molecule and/or releasing of the at least one trigger molecule may comprise modification of the sensing moiety. In some cases, the modification of the sensing moiety may comprise a conformational change and/or chemical modification, thereby exposing the active site of the cleavage moiety and/or the cleavage recognition site.

In some cases, the chemical modification may be selected from the group consisting of: dephosphorylation, phosphorylation, acetylation, methylation, ubiquitination, proteolytic processing, or a combination thereof.

[0181] In some cases of any one of the subject systems, the sensing moiety may be a calcium sensor (or calcium binding polypeptide). In some cases, the sensing moiety may be a sensor for an ion that is not calcium (e.g., sodium, potassium, etc.). In some cases, the sensing moiety may comprise at least a portion of a protein selected from the group consisting of: calmodulins, troponin C, calcineurin, parvalbumin, S100, calpain, myosin, neuronal Ca2+ sensor (NCS)-l, calsequestrin, calreticulin, annexins (AXA1, AXA2, AXA3, AXA4, AXA5, AXA6, AXA7, AXA8, AXA9, AXA10, AXA11, AXA13), PKC, synaptotagmins

(synaptotagmin I, II, III, IX, VI, VII, V, X), phospholipase C (PLC), phospholipase A (PLA), F am 62a, Fam62b, Fam62c, dysferlin, otoferlin, myoferlin, mctpl, pctp2, Rph3A, Doc2a, Doc2b, sytll, Sytl2, Sytl3, Sytl4, Sytl5, Uncl3a, Cpne6, Rcn2, osteonectin, hevin, QR1, testicans 1-3, tsc 36, SMOC-1, SMOC-2, coagulation factors VII, IX and X, protein C, protein S, fibrillin, notch and delta receptors, and LDL receptors.

[0182] In some cases of any one of the subject systems, upon the contacting of the cell by the ligand and the binding of the ligand to the ligand binding domain of the receptor, the receptor may be configured to activate at least one additional protein (e.g., a transmembrane protein, an intracellular protein, etc.) capable of regulating the cytosolic concentration of the trigger molecule. In some cases, upon the contacting of the cell by the ligand and the binding of the ligand to the ligand binding domain of the chimeric receptor, the chimeric receptor may be configured to activate at least one additional protein (e.g., a transmembrane protein, an intracellular protein, etc.) capable of altering or regulating the cytosolic concentration of the trigger molecule. In some cases, upon activation, the chimeric receptor may be configured to activate a transmembrane protein that is capable of regulating the cytosolic concentration of the trigger molecule. In some cases, the regulating the cytosolic concentration of the trigger molecule may comprise increasing, decreasing, and/or maintaining the cytosolic concentration of the trigger molecule. In some cases, the transmembrane protein may be capable of increasing the cytosolic concentration of the trigger molecule. In some cases, the transmembrane protein may comprise an ion channel. In some cases, the transmembrane protein may comprise a calcium channel. In some cases, the calcium channel may comprise a ligand-gated calcium channel and/or a voltage-gated calcium channel.

[0183] In some cases, the transmembrane protein may be capable of increasing the cytosolic concentration of the trigger molecule by at least about 2 fold, 3 fold, 4 fold, 5 fold,

6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, or greater, as compared to without the binding of the ligand to the chimeric receptor. In some cases, the transmembrane protein may be capable of decreasing the cytosolic concentration of the trigger molecule by at least about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold,

150 fold, 200 fold, 300 fold, 400 fold, 500 fold, or greater, as compared to without the binding of the ligand to the chimeric receptor. Such change in the cytosolic concentration of the trigger molecule may be temporary or permanent.

[0184] In another aspect, the present disclosure provides a composition comprising one or more polynucleotides that encode one or more components (e.g., the first chimeric polypeptide, the second chimeric polypeptide, the chimeric receptor, etc.) of any one of the subject systems provided herein. In some cases, the one or more polynucleotides of the composition may be in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be generally safe, non-toxic, and neither biologically nor otherwise undesirable, and may include a carrier acceptable for veterinary use as well as human pharmaceutical use. In some cases, the pharmaceutically acceptable carrier may comprise a pharmaceutically acceptable salt. The pharmaceutically acceptable salt may be derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. In another aspect, the present disclosure provides a kit comprising the composition provided herein.

[0185] Methods of use

[0186] In another aspect, the present disclosure provides a method of regulating expression of a target polynucleotide in a cell, comprising (a) expressing a system in the cell; and (b) contacting the cell by a ligand. The system expressed in the cell may comprise a first chimeric polypeptide and a second chimeric polypeptide. The first chimeric polypeptide may comprise a gene modulating polypeptide (GMP) that comprises an actuator moiety linked to a cleavage recognition site. The actuator moiety may regulate the expression of the target polynucleotide in the cell. The second chimeric polypeptide may comprise a sensing moiety exhibiting specific binding to a trigger molecule. The sensing moiety may be linked to a cleavage moiety that cleaves the cleavage recognition site. Upon binding of the trigger molecule to the sensing moiety or upon releasing of the trigger molecule from the sensing moiety, the sensing moiety may undeigo a modification to activate the cleavage moiety.

Upon contacting of the cell by the ligand, the second chimeric polypeptide may be induced to activate the cleavage moiety, such that the activated cleavage moiety releases the actuator moiety from the GMP of the first chimeric polypeptide to effect regulating expression of the target polynucleotide in the cell. The method of regulating expression of the target

polynucleotide in the cell may utilize one or more components (or one or more embodiments) of any one of the subject systems for regulating expression of the target polynucleotide in the cell, as provided herein.

[0187] In another aspect, the present disclosure provides a method of regulating expression of a target polynucleotide in a cell, comprising (a) expressing a system in the cell; and (b) contacting the cell by a ligand. The system expressed in the cell may comprise a first chimeric polypeptide and a second chimeric polypeptide. The first chimeric polypeptide may comprise a gene modulating polypeptide (GMP) that comprises an actuator moiety linked to a cleavage recognition site. The actuator moiety may regulate the expression of the target polynucleotide in the cell. The first chimeric polypeptide may further comprise a sensing moiety exhibiting specific binding to a trigger molecule and linked to the GMP. Upon binding of the trigger molecule to the sensing moiety or upon releasing of the trigger molecule from the sensing moiety, the sensing moiety may undergo a modification to activate the cleavage recognition site. The second chimeric polypeptide may comprise a cleavage moiety that cleaves the activated cleavage recognition site. Upon contacting of the cell by a ligand, the first chimeric polypeptide may be induced to activate the cleavage recognition site, such that the cleavage moiety releases the actuator moiety from the GMP of the first chimeric polypeptide to effect regulating expression of the target polynucleotide in the cell. The method of regulating expression of the target polynucleotide in the cell may utilize one or more components (or one or more embodiments) of any one of the subject systems for regulating expression of the target polynucleotide in the cell, as provided herein.

[0188] Chimeric receptor [0189] In some cases, the receptor may be a chimeric receptor. In some cases, the ligand binding domain of the chimeric receptor may be heterologous to the cell. In some cases, the chimeric receptor may comprise a CAR. The CAR may comprise at least a portion of a Notch receptor, a G-protein coupled receptor (GPCR), an integrin receptor, a cadherin receptor, a receptor tyrosine kinase, a death receptor, an immune receptor. The immune receptor may comprise a T cell receptor (TCR). The TCR may comprise TCRA, TCRB, TCRG, and/or TCRD. The TCR may comprise a co-receptor of TCR, such as, CD3, CD4, and/or CDS. CDS may comprise CD3E, CD3D, CD3G, and/or CD3Z. The CAR may comprise at least a portion of an intracellular portion of a TCR complex. As an alternative, the CAR may not comprise any portion of an intracellular portion of the TCR complex. The CAR may comprise one or more signaling capabilities of the TCR complex. As an alternative, the CAR may not comprise any signaling capability of the TCR complex.

[0190] In some cases, the chimeric receptor of a subject system can comprise at least a portion of an endogenous receptor, or any derivative, variant or fragment thereof. The chimeric receptor can bind specifically to at least one antigen (e.g., at least one ligand), for example via an antigen interacting domain (also referred to herein as an“extracellular sensor domain”). The chimeric receptor can, in response to ligand binding, undergo a modification such as a conformational change and/or chemical modification. Such modification(s) can recruit to the chimeric receptor binding partners (e.g., partners such as proteins) including, but not limited to, signaling proteins involved in signaling events and various cellular processes. Signaling proteins, for example, can be involved in regulating (e.g., activating and/or de-activating) a cellular response such as programmed changes in gene expression via translational regulation; transcriptional regulation; and epigenetic modification including the regulation of methylation, acetylation, phosphorylation, ubiquitylation, sumoylation, ribosylation, and citrullination. Conformational changes of the chimeric receptor can expose one or more regions of the chimeric receptor which was previously not exposed, and the exposed region can recruit and/or bind signaling protein(s). Chemical modifications on a receptor, for example phosphorylation and/or dephosphorylation (e.g., at tyrosine, serine, threonine, and/or any other suitable amino acid residue), can also recruit signaling proteins involved in regulating intracellular processes. Signaling proteins can bind directly to a receptor or indirectly to a receptor, for example as part of a larger complex.

[0191] In some cases, the chimeric receptor polypeptide can comprise at least a portion of a transmembrane receptor. The transmembrane receptor may detect at least one signal (i.e., ligand), such as a small molecule, ion, or protein, from the surrounding environment (e.g., extracellular and/or intracellular environment) and can initiate a cellular response via at least one signaling cascade involving additional proteins and signaling molecules. The

transmembrane receptor may translocate from one region of a cell to another, for example from the plasma membrane or cytoplasm to the nucleus and vice versa. Such translocation can be conditional upon ligand binding to the transmembrane receptor. Examples of the transmembrane receptor may include, but are not limited to, Notch receptors; G-protein coupled receptors (GPCRs); integrin receptors; cadherin receptors; catalytic receptors including receptors possessing enzymatic activity and receptors, which, rather than possessing intrinsic enzymatic activity, act by stimulating non-covalently associated enzymes (e.g., kinases); death receptors such as members of the tumor necrosis factor receptor (TNFR) superfamily; and immune receptors.

[0192] In some cases, the chimeric receptor polypeptide may comprise a Notch, or any derivative, variant or fragment thereof, selected from Notchl, Notch2, Notch3, and Notch4 or any homolog thereof.

[0193] In some cases, the chimeric receptor polypeptide may comprise a GPCR, or any derivative, variant or fragment thereof, selected from Class A Orphans; Class B Orphans; Class C Orphans; taste receptors, type 1; taste receptors, type 2; 5-hydroxytryptamine receptors; acetylcholine receptors (muscarinic); adenosine receptors; adhesion class GPCRs; adrenoceptors; angiotensin receptors; apelin receptor; bile acid receptor; bombesin receptors; bradykinin receptors; calcitonin receptors; calcium-sensing receptors; cannabinoid receptors; chemerin receptor; chemokine receptors; cholecystokinin receptors; class Frizzled GPCRs (e.g., Wnt receptors); complement peptide receptors; corticotropin-releasing factor receptors; dopamine receptors; endothelin receptors; G protein-coupled estrogen receptor;

formylpeptide receptors; free fatty acid receptors; GABAB receptors; galanin receptors; ghrelin receptor; glucagon receptor family; glycoprotein hormone receptors; gonadotrophin- releasing hormone receptors; GPR18, GPR55 and GPR119; histamine receptors;

hydroxycarboxylic acid receptors; kisspeptin receptor; leukotriene receptors;

lysophospholipid (LPA) receptors; lysophospholipid (SIP) receptors; melanin-concentrating hormone receptors; melanocortin receptors; melatonin receptors; metabotropic glutamate receptors; motilin receptor; neuromedin U receptors; neuropeptide FF/neuropeptide AF receptors; neuropeptide S receptor; neuropeptide W/neuropeptide B receptors; neuropeptide Y receptors; neurotensin receptors; opioid receptors; orexin receptors; oxoglutarate receptor; P2Y receptors; parathyroid hormone receptors; platelet-activating factor receptor;

prokineticin receptors; prolactin-releasing peptide receptor; prostanoid receptors; proteinase- activated receptors; QRFP receptor; relaxin family peptide receptors; somatostatin receptors; succinate receptor; tachykinin receptors; thyrotropin-releasing hormone receptors; trace amine receptor; urotensin receptor; vasopressin and oxytocin receptors; VIP and PACAP receptors.

[0194] In some cases, the chimeric receptor polypeptide may comprise a GPCR selected from the group consisting of: 5-hydroxytryptamine (serotonin) receptor 1 A (HTR1A), 5- hydroxytryptamine (serotonin) receptor IB (HTR1B), 5-hydroxytryptamine (serotonin) receptor ID (HTR1D), 5-hydroxytryptamine (serotonin) receptor IE (HTR1E), 5- hydroxytryptamine (serotonin) receptor IF (HTR1F), 5-hydroxytiyptamine (serotonin) receptor 2A (HTR2A), 5-hydroxytryptamine (serotonin) receptor 2B (HTR2B), 5- hydroxytryptamine (serotonin) receptor 2C (HTR2C), 5-hydroxytryptamine (serotonin) receptor 4 (HTR4), 5-hydroxytryptamine (serotonin) receptor 5A (HTR5A), 5- hydroxytiyptamine (serotonin) receptor 5B (HTR5BP), 5-hydroxytryptamine (serotonin) receptor 6 (HTR6), 5-hydroxytryptamine (serotonin) receptor 7, adenylate cyclase-coupled (HTR7), cholinergic receptor, muscarinic 1 (CHRM1), cholinergic receptor, muscarinic 2 (CHRM2), cholinergic receptor, muscarinic 3 (CHRM3), cholinergic receptor, muscarinic 4 (CHRM4), cholinergic receptor, muscarinic 5 (CHRM5), adenosine A1 receptor (ADORAl), adenosine A2a receptor (ADORA2A), adenosine A2b receptor (ADORA2B), adenosine A3 receptor (ADORA3), adhesion G protein-coupled receptor A1 (ADGRAl), adhesion G protein-coupled receptor A2 (ADGRA2), adhesion G protein-coupled receptor A3

(ADGRA3), adhesion G protein-coupled receptor B1 (ADGRB1), adhesion G protein- coupled receptor B2 (ADGRB2), adhesion G protein-coupled receptor B3 (ADGRB3), cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2), cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3), adhesion G protein-coupled receptor D1 (ADGRDl), adhesion G protein-coupled receptor D2 (ADGRD2), adhesion G protein-coupled receptor El (ADGREl), adhesion G protein- coupled receptor E2 (ADGRE2), adhesion G protein-coupled receptor E3 (ADGRE3), adhesion G protein-coupled receptor E4 (ADGRE4P), adhesion G protein-coupled receptor E5 (ADGRE5), adhesion G protein-coupled receptor FI (ADGRFl), adhesion G protein- coupled receptor F2 (ADGRF2), adhesion G protein-coupled receptor F3 (ADGRF3), adhesion G protein-coupled receptor F4 (ADGRF4), adhesion G protein-coupled receptor F 5 (ADGRF5), adhesion G protein-coupled receptor G1 (ADGRGl), adhesion G protein- coupled receptor G2 (ADGRG2), adhesion G protein-coupled receptor G3 (ADGRG3), adhesion G protein-coupled receptor G4 (ADGRG4), adhesion G protein-coupled receptor G5 (ADGRG5), adhesion G protein-coupled receptor G6 (ADGRG6), adhesion G protein- coupled receptor G7 (ADGRG7), adhesion G protein-coupled receptor LI (ADGRLl), adhesion G protein-coupled receptor L2 (ADGRL2), adhesion G protein-coupled receptor L3 (ADGRL3), adhesion G protein-coupled receptor L4 (ADGRL4), adhesion G protein-coupled receptor VI (ADGRV1), adrenoceptor alpha 1A (ADRA1A), adrenoceptor alpha IB

(ADRA1B), adrenoceptor alpha ID (ADRAID), adrenoceptor alpha 2A (ADRA2A), adrenoceptor alpha 2B (ADRA2B), adrenoceptor alpha 2C (ADRA2C), adrenoceptor beta 1 (ADRBl), adrenoceptor beta 2 (ADRB2), adrenoceptor beta 3 (ADRB3), angiotensin II receptor type 1 (AGTR1), angiotensin II receptor type 2 (AGTR2), apelin receptor (APLNR), G protein-coupled bile acid receptor 1 (GPBARl), neuromedin B receptor (NMBR), gastrin releasing peptide receptor (GRPR), bombesin like receptor 3 (BRS3), bradykinin receptor B1 (BDKRB1), bradykinin receptor B2 (BDKRB2), calcitonin receptor (CALCR), calcitonin receptor like receptor (CALCRL), calcium sensing receptor (CASR), G protein-coupled receptor, class C (GPRC6A), cannabinoid receptor 1 (brain) (CNR1), cannabinoid receptor 2 (CNR2), chemerin chemokine-like receptor 1 (CMKLR1), chemokine (C-C motif) receptor 1 (CCR1), chemokine (C-C motif) receptor 2 (CCR2), chemokine (C-C motif) receptor 3 (CCR3), chemokine (C-C motif) receptor 4 (CCR4), chemokine (C-C motif) receptor 5 (gene/pseudogene) (CCRS), chemokine (C-C motif) receptor 6 (CCR6), chemokine (C-C motif) receptor 7 (CCR7), chemokine (C-C motif) receptor 8 (CCRS), chemokine (C-C motif) receptor 9 (CCR9), chemokine (C-C motif) receptor 10 (CCR10), chemokine (C-X-C motif) receptor 1 (CXCR1), chemokine (C-X-C motif) receptor 2 (CXCR2), chemokine (C- X-C motif) receptor 3 (CXCR3), chemokine (C-X-C motif) receptor 4 (CXCR4), chemokine (C-X-C motif) receptor 5 (CXCR5), chemokine (C-X-C motif) receptor 6 (CXCR6), chemokine (C-X3-C motif) receptor 1 (CX3CR1), chemokine (C motif) receptor 1 (XCR1), atypical chemokine receptor 1 (Duffy blood group) (ACKR1), atypical chemokine receptor 2 (ACKR2), atypical chemokine receptor 3 (ACKR3), atypical chemokine receptor 4

(ACKR4), chemokine (C-C motif) receptor-like 2 (CCRL2), cholecystokinin A receptor (CCKAR), cholecystokinin B receptor (CCKBR), G protein-coupled receptor 1 (GPR1), bombesin like receptor 3 (BRS3), G protein-coupled receptor 3 (GPR3), G protein-coupled receptor 4 (GPR4), G protein-coupled receptor 6 (GPR6), G protein-coupled receptor 12 (GPR12), G protein-coupled receptor 15 (GPR15), G protein-coupled receptor 17 (GPR17),

G protein-coupled receptor 18 (GPR18), G protein-coupled receptor 19 (GPR19), G protein- coupled receptor 20 (GPR20), G protein-coupled receptor 21 (GPR21), G protein-coupled receptor 22 (GPR22), G protein-coupled receptor 25 (GPR25), G protein-coupled receptor 26 (GPR26), G protein-coupled receptor 27 (GPR27), G protein-coupled receptor 31 (GPR31),

G protein-coupled receptor 32 (GPR32), G protein-coupled receptor 33 (gene/pseudogene) (GPR33), G protein-coupled receptor 34 (GPR34), G protein-coupled receptor 35 (GPR35),

G protein-coupled receptor 37 (endothelin receptor type B-like) (GPR37), G protein-coupled receptor 37 like 1 (GPR37L1), G protein-coupled receptor 39 (GPR39), G protein-coupled receptor 42 (gene/pseudogene) (GPR42), G protein-coupled receptor 45 (GPR45), G protein- coupled receptor 50 (GPR50), G protein-coupled receptor 52 (GPR52), G protein-coupled receptor 55 (GPRS 5), G protein-coupled receptor 61 (GPR61), G protein-coupled receptor 62 (GPR62), G protein-coupled receptor 63 (GPR63), G protein-coupled receptor 65 (GPR65),

G protein-coupled receptor 68 (GPR68), G protein-coupled receptor 75 (GPR75), G protein- coupled receptor 78 (GPR78), G protein-coupled receptor 79 (GPR79), G protein-coupled receptor 82 (GPR82), G protein-coupled receptor 83 (GPR83), G protein-coupled receptor 84 (GPR84), G protein-coupled receptor 85 (GPR85), G protein-coupled receptor 87 (GPR87),

G protein-coupled receptor 88 (GPR88), G protein-coupled receptor 101 (GPR101), G protein-coupled receptor 119 (GPR119), G protein-coupled receptor 132 (GPR132), G protein-coupled receptor 135 (GPR135), G protein-coupled receptor 139 (GPR139), G protein-coupled receptor 141 (GPR141), G protein-coupled receptor 142 (GPR142), G protein-coupled receptor 146 (GPR146), G protein-coupled receptor 148 (GPR148), G protein-coupled receptor 149 (GPR149), G protein-coupled receptor 150 (GPR150), G protein-coupled receptor 151 (GPR151), G protein-coupled receptor 152 (GPR152), G protein-coupled receptor 153 (GPR153), G protein-coupled receptor 160 (GPR160), G protein-coupled receptor 161 (GPR161), G protein-coupled receptor 162 (GPR162), G protein-coupled receptor 171 (GPR171), G protein-coupled receptor 173 (GPR173), G protein-coupled receptor 174 (GPR174), G protein-coupled receptor 176 (GPR176), G protein-coupled receptor 182 (GPR182), G protein-coupled receptor 183 (GPR183), leucine- rich repeat containing G protein-coupled receptor 4 (LGR4), leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), leucine-rich repeat containing G protein-coupled receptor 6 (LGR6), MASI proto-oncogene (MAS 1), MASI proto-oncogene like (MAS1L), MAS related GPR family member D (MRGPRD), MAS related GPR family member E

(MRGPRE), MAS related GPR family member F (MRGPRF), MAS related GPR family member G (MRGPRG), MAS related GPR family member XI (MRGPRX1), MAS related GPR family member X2 (MRGPRX2), MAS related GPR family member X3 (MRGPRX3), MAS related GPR family member X4 (MRGPRX4), opsin 3 (OPN3), opsin 4 (OPN4), opsin 5 (OPNS), purinergic receptor P2Y (P2RY8), purinergic receptor P2Y (P2RY10), trace amine associated receptor 2 (TAAR2), trace amine associated receptor 3 (gene/pseudogene) (TAAR3), trace amine associated receptor 4 (TAAR4P), trace amine associated receptor 5 (TAAR5), trace amine associated receptor 6 (TAAR6), trace amine associated receptor 8 (TAAR8), trace amine associated receptor 9 (gene/pseudogene) (TAAR9), G protein-coupled receptor 156 (GPR156), G protein-coupled receptor 158 (GPR158), G protein-coupled receptor 179 (GPR179), G protein-coupled receptor, class C (GPRC5A), G protein-coupled receptor, class C (GPRC5B), G protein-coupled receptor, class C (GPRC5C), G protein- coupled receptor, class C (GPRC5D), frizzled class receptor 1 (FZD1), frizzled class receptor

2 (FZD2), frizzled class receptor 3 (FZD3), frizzled class receptor 4 (FZD4), frizzled class receptor 5 (FZD5), frizzled class receptor 6 (FZD6), frizzled class receptor 7 (FZD7), frizzled class receptor 8 (FZD8), frizzled class receptor 9 (FZD9), frizzled class receptor 10 (FZD10), smoothened, frizzled class receptor (SMO), complement component 3a receptor 1 (C3AR1), complement component 5a receptor 1 (C5AR1), complement component 5a receptor 2 (C5AR2), corticotropin releasing hormone receptor 1 (CRHR1), corticotropin releasing hormone receptor 2 (CRHR2), dopamine receptor D1 (DRD1), dopamine receptor D2 (DRD2), dopamine receptor D3 (DRD3), dopamine receptor D4 (DRD4), dopamine receptor D5 (DRD5), endothelin receptor type A (EDNRA), endothelin receptor type B (EDNRB), formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (FPR2), formyl peptide receptor

3 (FPR3), free fatty acid receptor 1 (FFARl), free fatty acid receptor 2 (FFAR2), free fatty acid receptor 3 (FFAR3), free fatty acid receptor 4 (FFAR4), G protein-coupled receptor 42 (gene/pseudogene) (GPR42), gamma-aminobutyric acid (GABA) B receptor, 1 (GABBR1), gamma-aminobutyric acid (GABA) B receptor, 2 (GABBR2), galanin receptor 1 (GALR1), galanin receptor 2 (GALR2), galanin receptor 3 (GALR3), growth hormone secretagogue receptor (GHSR), growth hormone releasing hormone receptor (GHRHR), gastric inhibitory polypeptide receptor (GIPR), glucagon like peptide 1 receptor (GLP1R), glucagon-like peptide 2 receptor (GLP2R), glucagon receptor (GCGR), secretin receptor (SCTR), follicle stimulating hormone receptor (FSHR), luteinizing hormone/choriogonadotropin receptor (LHCGR), thyroid stimulating hormone receptor (TSHR), gonadotropin releasing hormone receptor (GNRHR), gonadotropin releasing hormone receptor 2 (pseudogene) (GNRHR2), G protein-coupled receptor 18 (GPR18), G protein-coupled receptor 55 (GPR55), G protein- coupled receptor 119 (GPR119), G protein-coupled estrogen receptor 1 (GPER1), histamine receptor HI (HRH1), histamine receptor H2 (HRH2), histamine receptor H3 (HRH3), histamine receptor H4 (HRH4), hydroxycarboxylic acid receptor 1 (HCAR1),

hydroxycarboxylic acid receptor 2 (HCAR2), hydroxycarboxylic acid receptor 3 (HCAR3), KISS1 receptor (KISS1R), leukotriene B4 receptor (LTB4R), leukotriene B4 receptor 2 (LTB4R2), cysteinyl leukotriene receptor 1 (CYSLTR1), cysteinyl leukotriene receptor 2 (CYSLIR2), oxoeicosanoid (OXE) receptor 1 (OXER1), formyl peptide receptor 2 (FPR2), lysophosphatidic acid receptor 1 (LPAR1), lysophosphatidic acid receptor 2 (LPAR2), lysophosphatidic acid receptor 3 (LPAR3), lysophosphatidic acid receptor 4 (LPAR4), lysophosphatidic acid receptor 5 (LPAR5), lysophosphatidic acid receptor 6 (LPAR6), sphingosine-1 -phosphate receptor 1 (S1PR1), sphingosine- 1 -phosphate receptor 2 (S1PR2), sphingosine-1 -phosphate receptor 3 (S1PR3), sphingosine-1 -phosphate receptor 4 (S1PR4), sphingosine-1 -phosphate receptor 5 (S1PR5), melanin concentrating hormone receptor 1 (MCHR1), melanin concentrating hormone receptor 2 (MCHR2), melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R), melanocortin 2 receptor

(adrenocorticotropic hormone) (MC2R), melanocortin 3 receptor (MC3R), melanocortin 4 receptor (MC4R), melanocortin 5 receptor (MC5R), melatonin receptor 1 A (MTNR1 A), melatonin receptor IB (MTNRIB), glutamate receptor, metabotropic 1 (GRM1), glutamate receptor, metabotropic 2 (GRM2), glutamate receptor, metabotropic 3 (GRM3), glutamate receptor, metabotropic 4 (GRM4), glutamate receptor, metabotropic 5 (GRM5), glutamate receptor, metabotropic 6 (GRM6), glutamate receptor, metabotropic 7 (GRM7), glutamate receptor, metabotropic 8 (GRM8), motilin receptor (MLNR), neuromedin U receptor 1 (NMURl), neuromedin U receptor 2 (NMUR2), neuropeptide FF receptor 1 (NPFFR1), neuropeptide FF receptor 2 (NPFFR2), neuropeptide S receptor 1 (NPSR1), neuropeptides B/W receptor 1 (NPBWR1), neuropeptides B/W receptor 2 (NPBWR2), neuropeptide Y receptor Y1 (NPY1R), neuropeptide Y receptor Y2 (NPY2R), neuropeptide Y receptor Y4 (NPY4R), neuropeptide Y receptor Y5 (NPY5R), neuropeptide Y receptor Y6 (pseudogene) (NPY6R), neurotensin receptor 1 (high affinity) (NTSR1), neurotensin receptor 2 (NTSR2), opioid receptor, delta 1 (OPRDl), opioid receptor, kappa 1 (OPRK1), opioid receptor, mu 1 (OPRM1), opiate receptor-like 1 (OPRL1), hypocretin (orexin) receptor 1 (HCRTRl), hypocretin (orexin) receptor 2 (HCRTR2), G protein-coupled receptor 107 (GPR107), G protein-coupled receptor 137 (GPR137), olfactory receptor family 51 subfamily E member 1 (OR51E1), transmembrane protein, adipocyte associated 1 (TPRA1), G protein-coupled receptor 143 (GPR143), G protein-coupled receptor 157 (GPR157), oxoglutarate (alpha- ketoglutarate) receptor 1 (OXGR1), purinergic receptor P2Y (P2RY1), purinergic receptor P2Y (P2RY2), pyrimidinergic receptor P2Y (P2RY4), pyrimidinergic receptor P2Y (P2RY6), purinergic receptor P2Y (P2RY11), purinergic receptor P2Y (P2RY12), purinergic receptor P2Y (P2RY13), purinergic receptor P2Y (P2RY14), parathyroid hormone 1 receptor (PTH1R), parathyroid hormone 2 receptor (PTH2R), platelet-activating factor receptor (PTAFR), prokineticin receptor 1 (PROKR1), prokineticin receptor 2 (PROKR2), prolactin releasing hormone receptor (PRLHR), prostaglandin D2 receptor (DP) (PTGDR),

prostaglandin D2 receptor 2 (PTGDR2), prostaglandin E receptor 1 (PTGER1), prostaglandin E receptor 2 (PTGER2), prostaglandin E receptor 3 (PTGER3), prostaglandin E receptor 4 (PTGER4), prostaglandin F receptor (PTGFR), prostaglandin 12 (prostacyclin) receptor (IP) (PTGIR), thromboxane A2 receptor (TBXA2R), coagulation factor II thrombin receptor (F2R), F2R like trypsin receptor 1 (F2RL1), coagulation factor II thrombin receptor like 2 (F2RL2), F2R like thrombin/trypsin receptor 3 (F2RL3), pyroglutamylated RFamide peptide receptor (QRFPR), relaxin/insulin-like family peptide receptor 1 (RXFP1), relaxin/insulin- like family peptide receptor 2 (RXFP2), relaxin/insulin-like family peptide receptor 3 (RXFP3), relaxin/insulin-like family peptide receptor 4 (RXFP4), somatostatin receptor 1 (SSTR1), somatostatin receptor 2 (SSTR2), somatostatin receptor 3 (SSTR3), somatostatin receptor 4 (SSIR4), somatostatin receptor 5 (SSTR5), succinate receptor 1 (SUCNR1), tachykinin receptor 1 (TACR1), tachykinin receptor 2 (TACR2), tachykinin receptor 3 (TACR3), taste 1 receptor member 1 (TAS1R1), taste 1 receptor member 2 (TAS1R2), taste

1 receptor member 3 (TAS1R3), taste 2 receptor member 1 (TAS2R1), taste 2 receptor member 3 (TAS2R3), taste 2 receptor member 4 (TAS2R4), taste 2 receptor member 5 (TAS2R5), taste 2 receptor member 7 (TAS2R7), taste 2 receptor member 8 (TAS2R8), taste

2 receptor member 9 (TAS2R9), taste 2 receptor member 10 (TAS2R10), taste 2 receptor member 13 (TAS2R13), taste 2 receptor member 14 (TAS2R14), taste 2 receptor member 16 (TAS2R16), taste 2 receptor member 19 (TAS2R19), taste 2 receptor member 20

(TAS2R20), taste 2 receptor member 30 (TAS2R30), taste 2 receptor member 31

(TAS2R31), taste 2 receptor member 38 (TAS2R38), taste 2 receptor member 39

(TAS2R39), taste 2 receptor member 40 (TAS2R40), taste 2 receptor member 41

(TAS2R41), taste 2 receptor member 42 (TAS2R42), taste 2 receptor member 43

(TAS2R43), taste 2 receptor member 45 (TAS2R45), taste 2 receptor member 46

(TAS2R46), taste 2 receptor member 50 (TAS2R50), taste 2 receptor member 60

(TAS2R60), thyrotropin-releasing hormone receptor (TRHR), trace amine associated receptor 1 (TAAR1), urotensin 2 receptor (UTS2R), arginine vasopressin receptor 1A (AVPR1A), arginine vasopressin receptor IB (AVPR1B), arginine vasopressin receptor 2 (AVPR2), oxytocin receptor (OXTR), adenylate cyclase activating polypeptide 1 (pituitary) receptor type I (ADCYAPIRI), vasoactive intestinal peptide receptor 1 (VIPR1), vasoactive intestinal peptide receptor 2 (VIPR2), any derivative thereof, any variant thereof, and any fragment thereof.

[0195] The chimeric receptor comprising a GPCR, or any derivative, variant or fragment thereof, may bind an antigen comprising any suitable GPCR ligand, or any derivative, variant or fragment thereof. Non-limiting examples of ligands which can be bound by a GPCR include (-)-adrenaline, (-)-noradrenaline, (lyso)phospholipid mediators, [des-ArglO]kallidin, [des-Arg9]bradykinin, [des-Glnl4]ghrelin, [Hyp3]bradykinin, [Leu]enkephalin,

[Met]enkephalin, 12-hydroxyheptadecatrienoic acid, 12R-HETE, 12S-HETE, 12S-HPETE, 15S-HETE, 17b-estradiol, 20-hydroxy-LTB4, 2-arachidonoylglycerol, 2-oleoyl-LPA, 3- hydroxyoctanoic acid, 5-hydroxytryptamine, 5-oxo-15-HETE, 5-oxo-ETE, 5-oxo-ETrE, 5- oxo-ODE, 5S-HETE, 5S-HPETE, 7a,25-dihydroxycholesterol, acetylcholine, ACTH, adenosine diphosphate, adenosine, adrenomedullin 2/intermedin, adrenomedullin, amylin, anandamide, angiotensin II, angiotensin III, annexin I, apelin receptor early endogenous ligand, apelin-13, apelin-17, apelin-36, aspirin triggered lipoxin A4, aspirin-triggered resolvin Dl, ATP, beta-defensin 4 A, big dynorphin, bovine adrenal medulla peptide 8-22, bradykinin, C3a, C5a, Ca2+, calcitonin gene related peptide, calcitonin, cathepsin G, CCK-33, CCK-4, CCK-8, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL7, CCL8, chemerin, chenodeoxycholic acid, cholic acid, corticotrophin-releasing hormone, CST-17, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12a, CXCL12b, CXCL13, CXCL16, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, cysteinyl- leukotrienes (CysLTs), uracil nucleotides, deoxycholic acid, dihydrosphingosine-1- phosphate, dioleoylphosphatidic acid, dopamine, dynorphin A, dynorphin A-(l-13), dynorphin A-(l-8), dynorphin B, endomorphin-1, endothelin-1, endothelin-2, endothelin-3, F2L, Free fatty acids, FSH, GABA, galanin, galanin-like peptide, gastric inhibitory polypeptide, gastrin- 17, gastrin-releasing peptide, ghrelin, GHRH, glucagon, glucagon-like peptide l-(7-36) amide, glucagon-like peptide l-(7-37), glucagon-like peptide 2, glucagon- like peptide 2-(3-33), GnRH I, GnRH II, GRP-(18-27), hCG, histamine, humanin, INSL3, INSL5, kallidin, kisspeptin-10, kisspeptin-13, kisspeptin-14, kisspeptin-54, kynurenic acid, large neuromedin N, large neurotensin, L-glutamic acid, LH, lithocholic acid, L-lactic acid, long chain carboxylic acids, LPA, LTB4, LTC4, LTD4, LTE4, LXA4, Lys-[Hyp3]- bradykinin, lysophosphatidylinositol, lysophosphatidyl serine, Medium-chain-length fatty acids, melanin-concentrating hormone, melatonin, methylcarbamyl PAF, Mg2+, motilin, N- arachidonoylglycine, neurokinin A, neurokinin B, neuromedin B, neuromedin N, neuromedin S-33, neuromedin U-25, neuronostatin, neuropeptide AF, neuropeptide B-23, neuropeptide B-

29, neuropeptide FF, neuropeptide S, neuropeptide SF, neuropeptide W-23, neuropeptide W-

30, neuropeptide Y, neuropeptide Y-(3-36), neurotensin, nociceptin/orphanin FQ, N- oleoylethanolamide, obestatin, octopamine, orexin-A, orexin-B, Oxysterols, oxytocin, PACAP-27, PACAP-38, PAF, pancreatic polypeptide, peptide YY, PGD2, PGE2, PGF2a, PGI2, PGJ2, PHM, phosphatidylserine, PHV, prokineticin-1, prokineticin-2, prokineticin-2b, prosaposin, PrRP-20, PrRP-31, PTH, PTHrP, PTHrP-(l-36), QRFP43, relaxin, relaxin-1, relaxin-3, resolvin Dl, resolvin El, RFRP-1, RFRP-3, R-spondins, secretin, serine proteases, sphingosine 1-phosphate, sphingosylphosphorylcholine, SRIF-14, SRIF-28, substance P, succinic acid, thrombin, thromboxane A2, TIP39, T-kinin, TRH, TSH, tyramine, UDP- glucose, uridine diphosphate, urocortin 1, urocortin 2, urocortin 3, urotensin II-related peptide, urotensin-Il, vasopressin, VIP, Wnt, Wnt-1, Wnt-lOa, Wnt-lOb, Wnt-11, Wnt-16, Wnt-2, Wnt-2b, Wnt-3, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, XCL1, XCL2, Zn2+, a-CGRP, a-ketoglutaric acid, a-MSH, a- neoendorphin, b-alanine, b-CGRP, b-D-hydroxybutyric acid, b-endorphin, b-MSH, b- neoendorphin, b-phenylethylamine, and g-MSH.

[0196] In some cases, the chimeric receptor may comprise an integrin receptor a subunit, or any derivative, variant or fragment thereof, selected from the group consisting of: al, a2, o3, a4, a5, a6, a7, a8, a9, alO, al 1, aV, aL, aM, aX, aD, aE, and allb. In some

embodiments, a chimeric receptor polypeptide comprises an integrin receptor b subunit, or any derivative, variant or fragment thereof, selected from the group consisting of: bΐ, b2, b3, b4, b5, b6, b7, and b8. Chimeric receptor polypeptides comprising an a subunit, a b subunit, or any derivative, variant or fragment thereof, can heterodimerize (e.g., a subunit dimerizing with a b subunit) to form an integrin receptor, or any derivative, variant or fragment thereof. Non-limiting examples of integrin receptors include an a1b1, a2b1, a3b1, a4b1, a5b1, a6b1, a7b1, a8b1, a9b1, a10b1, aUbI, aLbI, aMbI, aCbI, aDb1, aIIbb1, aEbI, a1b2, a2b2, a3b2, a4b2, a5b2, a6b2, a7b2, a8b2, a9b2, a10b2, aUb2, aLb2, aMb2, aCb2, aDb2, aIIbb2, aEb2, a1b3, a2b3, a3b3, a4b3, a5b3, a6b3, a7b3, a8b3, a9b3, a10b3, aVb3, aLb3, aMb3, aCb3, aDb3, aIIbb3, aEb3, a1b4, a2b4, a3b4, a4b4, a5b4, a6b4, a7b4, a8b4, a9b4, a10b4, aVb4, aLb4, aMb4, aCb4, aBb4, aIIbb4, aEb4, a1b5, a2b5, a3b5, a4b5, a5b5, a6b5, a7b5, a8b5, a9b5, a10b5, aVb5, aLb5, aMb5, aCb5, aDb5, aIIbb5, aEb5, a1b6, a2b6, a3b6, a4b6, a5b6, a6b6, a7b6, a8b6, a9b6, a10b6, aVb6, aLb6, aMb6, aCb6, aDb6, aIIbb6, aEb6, a1b7, a2b7, a3b7, a4b7, a5b7, a6b7, a7b7, a8b7, a9b7, a10b7, aVb7, aLb7, aMb7, aCb7, aDb7, aIIbb7, aEb7, a1b8, a2b8, a3b8, a4b8, a5b8, a6b8, a7b8, a8b8, a9b8, a10b8, aVb8, aLb8, aMb8, aCb8, aDb8, aIIbb8, and aEb8 receptor. The chimeric receptor comprising an integrin subunit, or any derivative, variant or fragment thereof, may dimerize with an endogenous integrin subunit (e.g., wild-type integrin subunit).

[0197] In some cases, the chimeric receptor may comprise an integrin subunit, or any derivative, variant or fragment thereof, can bind an antigen comprising any suitable integrin ligand, or any derivative, variant or fragment thereof. Non-limiting examples of ligands which can be bound by an integrin receptor may include adenovirus penton base protein, beta-glucan, bone sialoprotein (BSP), Borrelia burgdorferi, Candida albicans, collagens (CN, e.g., CNI-IV), cytotactin/tenascin-C, decorsin, denatured collagen, disintegrins, E-cadherin, echovirus 1 receptor, epiligrin, Factor X Fc epsilon RII (CD23), fibrin (Fb), fibrinogen (Fg), fibronectin (Fn), heparin, HIV Tat protein, iC3b, intercellular adhesion molecule (e.g., ICAM-1,2,3,4,5), invasin, LI cell adhesion molecule (Ll-CAM), laminin, lipopolysaccharide (LPS), MAdCAM-1, matrix metalloproteinase-2 (MMPe), neutrophil inhibitory factor (NIF), osteopontin (OP or OPN), plasminogen, prothrombin, sperm fertilin, thrombospondin (TSP), vascular cell adhesion molecule 1 (VCAM-1), vitronectin (VN or VTN), and von Willebrand factor (vWF).

[0198] In some cases, the chimeric receptor can comprise a cadherin, or any derivative, variant or fragment thereof, selected from a classical cadherin, a desmosoma cadherin, a protocadherin, and an unconventional cadherin. In some embodiments, a chimeric receptor polypeptide comprises a classical cadherin, or any derivative, variant or fragment thereof, selected from CDH1 (E-cadherin, epithelial), CDH2 (N-cadherin, neural), CDH12 (cadherin 12, type 2, N-cadherin 2), and CDH3 (P-cadherin, placental). In some embodiments, a chimeric receptor polypeptide comprises a desmosoma cadherin, or any derivative, variant or fragment thereof, selected from desmoglein (DSG1, DSG2, DSG3, DSG4) and desmocollin (DSC1, DSC2, DSC3). In some embodiments, a chimeric receptor polypeptide comprises a protocadherin, or any derivative, variant or fragment thereof, selected from PCDH1,

PCDH10, PCDH11X PCDH11 Y, PCDH12, PCDH15, PCDH17, PCDH18, PCDH19, PCDH20, PCDH7, PCDH8, PCDH9, PCDHA1, PCDHA10, PCDHA11, PCDHA12, PCDHA13, PCDHA2, PCDHA3, PCDHA4, PCDHA5, PCDHA6, PCDHA7, PCDHA8, PCDHA9, PCDHAC1, PCDHAC2, PCDHB1, PCDHB10, PCDHB11, PCDHB12,

PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB17, PCDHB18, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHB7, PCDHB8, PCDHB9, PCDHGA1, PCDHGA10, PCDHGA11, PCDHGA12, PCDHGA2, PCDHGA3, PCDHGA4, PCDHGA5, PCDHGA6, PCDHGA7, PCDHGA8, PCDHGA9, PCDHGB1, PCDHGB2, PCDHGB3, PCDHGB4, PCDHGB5, PCDHGB6, PCDHGB7, PCDHGC3, PCDHGC4, PCDHGC5, FAT, FAT2, and FAT). In some embodiments, a chimeric receptor polypeptide comprises an unconventional cadherin selected from CDH4 (R-cadherin, retinal), CDH5 (VE-cadherin, vascular endothelial), CDH6 (K-cadherin, kidney), CDH7 (cadherin 7, type 2), CDH8 (cadherin 8, type 2), CDH9 (cadherin 9, type 2, Tl-cadherin), CDH10 (cadherin 10, type 2, T2-cadherin), CDH11 (OB-cadherin, osteoblast), CDH13 (T-cadheiin, H-cadherin, heart), CDH15 (M- cadherin, myotubule), CDH16 (KSP-cadherin), CDH17 (LI cadherin, liver-intestine), CDH18 (cadherin 18, type 2), CDH19 (cadherin 19, type 2), CDH20 (cadherin 20, type 2), CDH23 (cadherin 23, neurosensory epithelium), CDH24, CDH26, CDH28, CELSR1, CELSR2, CELSR3, CLSTN1, CLSTN2, CLSTN3, DCHS1, DCHS2, LOC389118, PCLKC, RESDAl, and RET.

[0199] In some cases, the chimeric receptor may comprise at least an extracellular region (e.g., ligand binding domain) of a catalytic receptor such as a RTK, or any derivative, variant or fragment thereof. The chimeric receptor may comprise at least a membrane spanning region of a catalytic receptor such as a RTK, or any derivative, variant or fragment thereof. The chimeric receptor may comprise at least an intracellular region (e.g., cytosolic domain) of a catalytic receptor such as a RTK, or any derivative, variant or fragment thereof. The chimeric receptor may comprise an RTK, or any derivative, variant or fragment thereof, can recruit a binding partner. In some cases, ligand binding to a chimeric receptor comprising an RTK, or any derivative, variant or fragment thereof, results in a conformational change, chemical modification, or combination thereof, which recruits a binding partner to the chimeric receptor.

[0200] In some cases, the chimeric receptor may comprise a class I RTK (e.g., the epidermal growth factor (EGF) receptor family including EGFR; the ErbB family including ErbB-2, ErbB-3, and ErbB-4), a class II RTK (e.g., the insulin receptor family including INSR, IGF-1R, and IRR), a class III RTK (e.g., the platelet-derived growth factor (PDGF) receptor family including PDGFR-a, PDGFR-b, CSF-1R, KIT/SCFR, and FLK2/FLT3), a class IV RTK (e.g., the fibroblast growth factor (FGF) receptor family including FGFR-1, FGFR-2, FGFR-3, and FGFR-4), a class V RTK (e.g., the vascular endothelial growth factor (VEGF) receptor family including VEGFRl, VEGFR2, and VEGFR3), a class VI RTK (e.g., the hepatocyte growth factor (HGF) receptor family including hepatocyte growth factor receptor (HGFR/MET) and RON), a class VII RTK (e.g., the tropomyosin receptor kinase (Trk) receptor family including TRKA, TRKB, and TRKC), a class VIII RTK (e.g., the ephrin (Eph) receptor family including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6), a class IX RTK (e.g., AXL receptor family such as AXL, MER, and TRY03), a class X RTK (e.g., LTK receptor family such as LTK and ALK), a class XI RTK (e.g., TIE receptor family such as TIE and TEK), a class CII RTK (e.g., ROR receptor family ROR1 and ROR2), a class CIII RTK (e.g., the discoidin domain receptor (DDR) family such as DDR1 and DDR2), a class XIV RTK (e.g., RET receptor family such as RET), a class XV RTK (e.g., KLG receptor family including PTK7), a class XVI RTK (e.g., RYK receptor family including Ryk), a class XVII RTK (e.g., MuSK receptor family such as MuSK), or any derivative, variant or fragment thereof.

[0201] The chimeric receptor comprising a RTK, or any derivative, variant or fragment thereof, may bind an antigen comprising any suitable RTK ligand, or any derivative, variant or fragment thereof. Non limiting examples of RTK ligands include growth factors, cytokines, and hormones. Growth factors include, for example, members of the epidermal growth factor family (e.g., epidermal growth factor or EGF, heparin-binding EGF-like growth factor or HB-EGF, transforming growth factor-a or TGF-a, amphiregulin or AR, epiregulin or EPR, epigen, betacellulin or BTC, neuregulin-1 orNRGl, neuregulin-2 or NRG2, neuregulin-3 or NRG3, and neuregulin-4 or NRG4), the fibroblast growth factor family (e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15/19, FGF16, FGF17, FGF18, FGF20, FGF21, and FGF23), the vascular endothelial growth factor family (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF), and the platelet-derived growth factor family (e.g., PDGFA, PDGFB, PDGFC, and PDGFD). Hormones include, for example, members of the insulin/IGF/relaxin family (e.g., insulin, insulin-like growth factors, relaxin family peptides including relaxinl, relaxin2, relaxin3, Leydig cell-specific insulin-like peptide (gene INSL3), early placenta insulin-like peptide (BLIP) (gene INSL4), insulin-like peptide 5 (gene INSL5), and insulin-like peptide

6).

[0202] In some cases, the chimeric receptor may comprise at least an extracellular region (e.g., ligand binding domain) of a catalytic receptor such as an RTSK, or any derivative, variant or fragment thereof. The chimeric receptor may comprise at least a membrane spanning region of a catalytic receptor such as an RTSK, or any derivative, variant or fragment thereof. The chimeric receptor may comprise at least an intracellular region (e.g., cytosolic domain) of a catalytic receptor such as an RTSK, or any derivative, variant or fragment thereof. The chimeric receptor polypeptide comprising an RTSK, or any derivative, variant or fragment thereof, may recruit a binding partner. In some cases, ligand binding to the chimeric receptor comprising an RTSK, or any derivative, variant or fragment thereof, may result in a conformational change, chemical modification, or combination thereof, which recruits a binding partner to the chimeric receptor.

[0203] The chimeric receptor comprising an RTSK, or any derivative, variant or fragment thereof, may phosphorylate a substrate at serine and/or threonine residues, and may select specific residues based on a consensus sequence. The chimeric receptor may comprise a type I RTSK, type II RTSK, or any derivative, variant or fragment thereof. The chimeric receptor comprising a type I receptor serine/threonine kinase may be inactive unless complexed with a type II receptor. In some cases, the chimeric receptor comprising a type II receptor serine/threonine may comprise a constitutively active kinase domain that can phosphorylate and activate a type I receptor when complexed with the type I receptor. A type II receptor serine/threonine kinase can phosphorylate the kinase domain of the type I partner, causing displacement of protein partners. Displacement of protein partners can allow binding and phosphorylation of other proteins, for example certain members of the SMAD family. The chimeric receptor can comprise a type I receptor, or any derivative, variant or fragment thereof, selected from the group consisting of: ALK1 (ACVRL1), ALK2 (ACVR1 A), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFbRl), ALK6 (BMPR1B), and ALK7

(ACVR1C). The chimeric receptor can comprise a type II receptor, or any derivative, variant or fragment thereof, selected from the group consisting of: TGFbR2, BMPR2, ACVR2A, ACVR2B, and AMHR2 (AMHR). The chimeric receptor can comprise a TGF-b receptor, or any derivative, variant or fragment thereof.

[0204] In some cases, the chimeric receptor can comprise a receptor which stimulates non-covalently associated intracellular kinases, such as a Src kinase (e.g., c-Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn, and Frk) or a JAK kinase (e.g., JAK1, JAK2, JAK3, and TYK2) rather than possessing intrinsic enzymatic activity, or any derivative, variant or fragment thereof. These include the cytokine receptor superfamily such as receptors for cytokines and polypeptide hormones. Cytokine receptors generally contain an N-terminal extracellular ligand-binding domain, transmembrane a helices, and a C -terminal cytosolic domain. The cytosolic domains of cytokine receptors are generally devoid of any known catalytic activity. Cytokine receptors instead can function in association with non-receptor kinases (e.g., tyrosine kinases or threonine/serine kinases), which can be activated as a result of ligand binding to the receptor. The chimeric receptor can comprise at least an extracellular region (e.g., ligand binding domain) of a catalytic receptor that non-covalently associates with an intracellular kinase (e.g., a cytokine receptor), or any derivative, variant or fragment thereof. The chimeric receptor can comprise at least a membrane spanning region of a catalytic receptor that non-covalently associates with an intracellular kinase (e.g., a cytokine receptor), or any derivative, variant or fragment thereof. The chimeric receptor can comprise at least an intracellular region (e.g., cytosolic domain) of a catalytic receptor that non-covalently associates with an intracellular kinase (e.g., a cytokine receptor), or any derivative, variant or fragment thereof. The chimeric receptor comprising a catalytic receptor that non-covalently associates with an intracellular kinase, or any derivative, variant or fragment thereof, can recruit a binding partner. In some cases, ligand binding to the chimeric receptor comprising a catalytic receptor that non-covalently associates with an intracellular kinase, or any derivative, variant or fragment thereof, may result in a conformational change, chemical modification, or combination thereof, which recruits a binding partner to the receptor.

[0205] In some cases, the chimeric receptor can comprise a cytokine receptor, for example a type I cytokine receptor or a type II cytokine receptor, or any derivative, variant or fragment thereof. The chimeric receptor can comprise an interleukin receptor (e.g., IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-11R, IL-12R, IL-13R, IL-15R, IL-21R, IL- 23R, IL-27R, and IL-31R), a colony stimulating factor receptor (e.g., erythropoietin receptor, CSF-1R, CSF-2R, GM-CSFR, and G-CSFR), a hormone receptor/neuropeptide receptor (e.g., growth hormone receptor, prolactin receptor, and leptin receptor), or any derivative, variant or fragment thereof. The chimeric receptor can comprise a type II cytokine receptor, or any derivative, variant or fragment thereof. The chimeric receptor can comprise an interferon receptor (e.g., IFNAR1, IFNAR2, and IFNGR), an interleukin receptor (e.g., IL-10R, IL-20R, IL-22R, and IL-28R), a tissue factor receptor (also called platelet tissue factor), or any derivative, variant or fragment thereof.

[0206] In some cases, the chimeric receptor comprising a cytokine receptor can bind an antigen comprising any suitable cytokine receptor ligand, or any derivative, variant or fragment thereof. Non-limiting examples of cytokine receptor ligands include interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-20, IL-21, IL-22, IL-23, IL-27, IL-28, and IL-31), interferons (e.g., IFN-a, IFN-b, IFN-g), colony stimulating factors (e.g., erythropoietin, macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factors or GM-CSFs, and granulocyte colony-stimulating factors or G-CSFs), and hormones (e.g., prolactin and leptin).

[0207] In some cases, the chimeric receptor can comprise a death receptor, a receptor containing a death domain, or any derivative, variant or fragment thereof. Death receptors are often involved in regulating apoptosis and inflammation. Death receptors include members of the TNF receptor family such as TNFR1, Fas receptor, DR4 (also known as TRAIL receptor 1 or TRAILRl) and DR5 (also known as TRAIL receptor 2 or TRAILR2). The chimeric receptor can comprise at least an extracellular region (e.g., ligand binding domain) of a death receptor, or any derivative, variant or fragment thereof. The chimeric receptor can comprise at least a membrane spanning region of a death receptor, or any derivative, variant or fragment thereof. The chimeric receptor can comprise at least an intracellular region (e.g., cytosolic) domain of a death receptor, or any derivative, variant or fragment thereof. The chimeric receptor polypeptide comprising a death receptor, or any derivative, variant or fragment thereof, can undergo receptor oligomerization in response to ligand binding, which in turn can result in the recruitment of specialized adaptor proteins and activation of signaling cascades, such as caspase cascades. The chimeric receptor can comprise a death receptor, or any derivative, variant or fragment thereof, results in a conformational change, chemical modification, or combination thereof, which recruits a binding partner to the chimeric receptor.

[0208] The chimeric receptor comprising a death receptor can bind an antigen comprising any suitable ligand of a death receptor, or any derivative, variant or fragment thereof. Non- limiting examples of ligands bound by death receptors include TNFo, Fas ligand, and TNF- related apoptosis-inducing ligand (TRAD.,).

[0209] In some cases, the chimeric receptor can comprise an immune receptor, or any derivative, variant or fragment thereof. Immune receptors can include members of the immunoglobulin superfamily (IgSF) which share structural features with immunoglobulins, e.g., a domain known as an immunoglobulin domain or fold. IgSF members include, but are not limited to, cell surface antigen receptors, co-receptors and costimulatory molecules of the immune system, and molecules involved in antigen presentation to lymphocytes. The chimeric receptor can comprise at least an extracellular region (e.g., ligand binding domain) of an immune receptor, or any derivative, variant or fragment thereof. The chimeric receptor can comprise at least a region spanning a membrane of an immune receptor, or any derivative, variant or fragment thereof. The chimeric receptor can comprise at least an intracellular region (e.g., cytoplasmic domain) of an immune receptor, or any derivative, variant or fragment thereof. The chimeric receptor comprising an immune receptor, or any derivative, variant or fragment thereof, can recruit a binding partner. Ligand binding to a chimeric receptor comprising an immune receptor, or any derivative, variant or fragment thereof, can result in a conformational change, chemical modification, or combination thereof, which recruits a binding partner to the chimeric receptor. [0210] In some cases, the chimeric receptor can comprise a cell surface antigen receptor such as a T cell receptor (TCR), a B cell receptor (BCR), or any derivative, variant or fragment thereof. T cell receptors generally comprise two chains, either the TCR-alpha and - beta chains or the TCR-delta and -gamma chains. A chimeric polypeptide comprising a TCR, or any derivative, variant or fragment thereof, can bind a major histocompatibility complex (MHC) protein. B cell receptors generally comprises a membrane bound immunoglobulin and a signal transduction moiety. A chimeric polypeptide comprising a BCR, or any derivative, variant or fragment thereof, can bind a cognate BCR antigen. A chimeric polypeptide comprising at least an immunoreceptor tyrosine-based activation motif (ITAM) found in the cytoplasmic domain of certain immune receptors. A chimeric polypeptide may comprise at least an immunoreceptor tyrosine-based inhibition motif (ITIM) found in the cytoplasmic domain of certain immune receptors. A chimeric polypeptide comprising ITAM and/or ITIM domains can be phosphorylated following ligand binding to an antigen interacting domain. The phosphorylated regions can serve as docking sites for other proteins involved in immune cell signaling.

[0211] The antigen interacting domain of a chimeric receptor can bind a membrane bound antigen, for example an antigen bound to the extracellular surface of a cell (e.g., a target cell). The antigen interacting domain may bind a non-membrane bound antigen, 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. 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. Cancer antigens, for example, may be proteins produced by tumor cells that can elicit an immune response, particularly a T-cell mediated immune response. The selection of the antigen binding portions of a chimeric receptor can depend on the particular type of cancer antigen to be targeted. In some cases, the tumor antigen may comprise one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors can express a number of proteins that can serve as target antigens for an immune attack. The antigen interaction domains can bind to cell surface signals, extracellular matrix (ECM), paracrine signals, juxtacrine signals, endocrine signals, autocrine signals, signals that can trigger or control genetic programs in cells, or any combination thereof. In some cases, interactions between the cell signals that bind to the chimeric receptor involve a cell-cell interaction, cell-soluble chemical interaction, and cell-matrix or microenvironment interaction.

[0212] Binding: affinity [0213] In some cases, one or more characteristics of a binding affinity (e.g., equilibrium dissociation constant (K _D ), equilibrium association constant (K _A ), etc.) between two molecules of interest (e.g., between the adaptor and the at least one intracellular signaling domain of the chimeric receptor, between the sensing moiety and the cleavage moiety, between the sensing moiety and the cleavage recognition site, between the sensing moiety and at least one trigger molecule, etc.) provided herein in the present disclosure may be assessed by techniques such as, for example, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), fluorescence depolarization, one or more computer simulations, etc.

[0214] In some cases, the two molecules of interest may directly complex (directly bind) with each other, with a K _D (wherein K _D = K _off (i.e.,“k _a ”)/Kon (i.e.,“k _a ”)) of about 10 ^-15 molar (M) to about 10 ^-5 M. In some cases, two molecules of interest may directly bind to each other, with a K _D of at least about 10 ^-15 M, 10 ^-14 M, 10 ^-13 M, 10 ^-12 M, 10 ^-11 M, 10 ^-10 M, 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M, 10 ^-5 M, or more. In some cases, two molecules of interest may directly bind to each other, with a K _D of at most about 10 ^-5 M, 10 ^-6 M, 10 ^-7 M, lO ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M, 10 ^-13 M, 10 ^-14 M, 10 ^-15 M, or less.

[0215] GMP and actuator moietv

[0216] The GMP may comprise an actuator moiety that regulates expression of a target polynucleotide in the cell. The target polynucleotide in the cell may encode a target polypeptide. In some cases, the target polypeptide may induce or inhibit proliferation, differentiation, and/or survival of the cell. The actuator moiety can bind to a target

polynucleotide to regulate expression and/or activity of a target gene encoded by the target polynucleotide. 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 (e.g., dCas). 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.

[0217] In some cases, the actuator moiety may comprise a heterologous functional domain (e.g., a transcription activator, a transcription repressor, a chromosome modification enzyme, etc.). 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, VP 16, 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.

[0218] Any suitable nuclease can be used. 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 Cpfl.

[0219] 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.

[0220] The target polypeptide may encode a peptide or a protein that is immune related (e.g., related to survival, proliferation, differentiation, activity, identification, etc. or an immune cell, such as a T cell). The target polypeptide may encode a peptide or protein involved in immune cell regulation. In some cases, the target polypeptide may be PD-1, PD- Ll, and/or CTLA-4.

[0221] Administration of GMP

[0222] In some cases, administration of the GMP to the cell can comprise treating the cell with a delivery vehicle, which delivery vehicle comprises 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.

[0223] 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 cell. The targeting moiety may enhance targeting and binding of the delivery vehicle to the cell. The targeting moiety may enhance intracellular entrance, uptake, and/or penetration of the delivery vehicle into the 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.

[0224] In some cases, the delivery vehicle may not comprise such targeting moiety against the cell.

[0225] 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, RDl 14 Retrovirus, Spleen Focus-Forming Virus (SFFV), Abelson murine leukemia virus (AMLV), Murine Stem Cell Virus (MSCV), and variants thereof.

[0226] 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.

[0227] 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.

[0228] In some cases, the method can further comprise integrating into the genome of the cell a nucleic acid sequence (e.g., a polynucleotide) encoding at least a portion of the first chimeric polypeptide and/or the second chimeric polypeptide, as provided herein in the present disclosure. In some cases, the nucleic acid sequence may encode at least a portion of the GMP. In some cases, the nucleic acid sequence (e.g., a polynucleotide) encoding the at least the portion of the first and/or second chimeric polypeptides may be integrated into the genome of the cell. Upon administration of the nucleic acid encoding the at least the portion of the first and/or second chimeric polypepyides (e.g., with or without the delivery vehicle), at least a portion of the nucleic acid may be integrated into the genome of the cell. The at least the portion of the integrated nucleic acid may be placed under the control of an autologous promoter of the cell. Alternatively or in addition to, the at least a portion of the integrated nucleic acid may further comprise a promoter that is autologous or heterologous (e.g., a heterologous promoter) to the 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 cell.

[0229] The 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 first and/or second chimeric polypepyides, the nucleic acid that encodes the at least the portion of the first and/or second chimeric polypeptides, etc.).

[0230] 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 cell in vivo. Other examples of the injection method may include intraderm al, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavemous, and/or intravitreal.

[0231] 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.

[0232] In some cases, the cell may be isolated from the subject, and the isolated cell may be treated (e.g., cultured in a culture media) with the delivery vehicle comprising the payload. The isolated cell may be allowed or stimulated to proliferate prior to, during, and/or subsequent to the treatment with the delivery vehicle comprising the payload. In some cases, the cell of interest may be an immune cell. In such a case, the immune cell (e.g., a T cell) may be isolated from the subject. Alternatively or in addition to, a cell that is not the immune cell (e.g., a stem cell, a skin cell, etc.) may be isolated from the subject, and the isolated cell may be induced to differentiate into the immune cell, trans-differentiate into the immune cell, and/or express one or more markers (e.g., one or more TCR complexes) indicative of the immune cell prior to the treatment with the delivery vehicle comprising a payload. In some cases, the cell that is not the immune cell may first be de-differentiated into an induced pluripotent stem cell (iPSC) prior to differentiation into the immune cell (e.g., the T cell) and/or inducing expression of the one or more TCR complexes. Following, the isolated and treated cell may be injected (transplanted) into the subject.

[0233] 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 cell (e.g., an engineered immune cell, such as an engineered T cell).

[0234] In some cases, such engineered cell 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 cell in vivo. Other examples of the injection method may include intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal.

[0235] In some cases, the subject may be injected with a dose of the engineered cells (e.g., cells administered with the GMP) 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 engineered cells 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 engineered 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 engineered 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.

[0236] 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 10 ⁹ ) of the engineered 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 10 ⁹ ) of the engineered cells, or less.

[0237] 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.

[0238] In some cases, the administration of the GMP to the cell can comprise treating the 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 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 (e.g., the ligand of the receptor provided herein in the present disclosure), and wherein the released GMP is operable to regulate expression of the target polynucleotide in the cell. In some cases, the method can further comprise administering to the 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 cell in response to a stimulant, and wherein the translocated GMP is operable to regulate expression of the target polynucleotide in the cell.

[0239] 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.

[0240] In some examples, the nuclear localization domain can be derived from the NFAT family members (e.g., NFATp, NFAT1, NFATcl, NFATc2, NFATc3, NFAT4, NFATx, NFATc4, NFAT3, and NFAT5), nuclear factor kappa B (NF-KB), NFKBl p50, activator protein 1 (AP-1), signal transducer and activator of transcription family members (e.g., STAT1, STAT2, STAT3, STAT4, STAT5 A, 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., PERI, PER2, and PER3), or other transcription factors or signal transducers.

[0241] In some cases, the GMP can regulate expression of the target polynucleotide in the 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 cell in the absence of the GMP. In some cases, the GMP can regulate expression of the target polynucleotide in the 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 cell in the absence of the GMP.

[0242] In some cases, the GMP can regulate expression of the target polynucleotide in the 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 cell in the absence of the GMP. In some cases, the GMP can regulate expression of the target polynucleotide in the 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 cell in the absence of the GMP.

[0243] The regulating the expression of the target polynucleotide in the cell can comprise decreasing, increasing, inhibiting, and/or prolonging the expression of the target

polynucleotide in the cell. The regulating the expression of the target polynucleotide in the cell can be decreasing the expression of the target polynucleotide in the cell. The regulating the expression of the target polynucleotide in the cell can be increasing the expression of the target polynucleotide in the cell.

[0244] The regulating the expression of the target polynucleotide in the cell may directly and/or indirectly allow the regulating the activity of the cell. In some cases, the regulating the activity of the cell can comprise decreasing and/or inhibiting self-inflicted injury of the cell, death of the cell by another cell, and/or death of another cell by the cell, thereby improving (directly and/or indirectly) viability, proliferation, and/or function of the cell.

[0245] In some cases, the regulating the activity of the cell can comprise inducing and/or prolonging activation of the cell (eg., activation of the immune cell, such as the T cell). The activation of the cell can comprise activation of one or more biological activities (eg., migration, proliferation, synthesis of one or more polypeptides, etc.) of the cell.

[0246] In some cases, the GMP may be configured to reduce and/or prevent activation of the cell.

[0247] 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 cell. In some cases, the GMP comprising the actuator moiety may be configured to decrease expression of one or more angiogenic factors in the cell. In some cases, the GMP comprising the actuator moiety may be configured to decrease expression of one or more angiogenic factors in the cell. The GMP comprising the actuator moiety may be expressed along with a guide RNA (eg., sgRNA) against one or more polynucleotide sequences encoding for the one or more angiogenic factors in the 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 cell.

[0248] 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, Angl, Ang2, PDGF (BB-homodimer), PDGFR, TGF-b, endoglin, TGF-breceptors, MCP-1, Integrins anb3, anb3, anb1, 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, Fgfa, Fgfb, Tgfa,

Tgfb, MMPs (including mmp9), Plasminogen activator inhibitor-1, Thrombospondin, Angiopoietin 1, Angiopoietin 2, Amphiregulin, Leptin, Endothelin-1, AAMP, AGGF1, AMOT, ANGLPTL3, ANGPTL4, BTG1, IL-Ib, NOS3, TNFSF12, and/or VASH2.

[0249] In some cases, a nucleic acid sequence encoding the GMP may be integrated into a genome of the cell.

[0250] 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.

[0251] In some cases, the cleavage moiety can 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 Cl, C2, C6, CIO, 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 Cl 1, 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 Cl 8 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 ϋ-glutamyl hydrolase (Rattus norvegicus); superfamily PD proteases, e.g., family C46 including Hedgehog protein (Drosophila melanogaster);

superfamily PE proteases, e.g., family PI 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 SI 1, 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 SI, 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 PI including DmpA aminopeptidase (Ochrobactirum 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 Tl, T2, T3, and T6 including archaean proteasome, u 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.

[0252] 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: achromopepti dase, 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 Cl r, complement Cls, 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.

[0253] 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/US 17/012885 and PCT Patent Application No.

PCT/US17/012881, each of which is incorporated in its entirety herein by reference.

[0254] In some cases, the actuator moiety of the GMP 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 CRI SPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity. In some cases, the actuator moiety can be Cas9 and/or Cpfl. 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.

[0255] 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/US 17/012885, PCT Patent Application No. PCT/US17/012881, PCT Patent Application No. PCT/US 18/041704, 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, PCT Patent Application No. PCT/US19/023721, and U.S. Provisional Application No. 62/799,456, each of which is incorporated in its entirety herein by reference.

[0256] Contacting the cell with a ligand (e.g., an exogenous ligand) can occur directly and/or indirectly. Direct stimulation may occur when the ligand binds a portion of the cell. In some cases, the ligand may bind to the receptor of the cell. In an example, the ligand may bind to a ligand binding domain of the receptor. Indirect stimulation can occur when the ligand activates or deactivates a different cell, which different cell is operable to activate the cell by using its cell surface marker (e.g., a cell surface ligand) to bind the receptor of the cell. Consequentially, the cell may be activated to regulate expression of the target polynucleotide in the cell. The different cell may be of the same (e.g., another cell of the same type) or different cell type than the cell.

[0257] Contacting the cell with the ligand may occur prior to, during, and/or subsequent to administration of the GMP comprising the actuator moiety to the cell. The cell may be ex vivo and/or in vivo during the contacting of the cell (e.g., the receptor of the cell) with the ligand. [0258] Contacting the cell with the ligand may occur prior to, during, and/or subsequent to administration of the cell (e.g., the engineered cell) to a subject. The cell may be contacted with the ligand prior to, during, and/or subsequent to administration of the 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 cell may be contacted with the ligand prior to, during, and/or subsequent to administration of the 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 cell may be contacted with the ligand 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 cell to the subject. The cell may be contacted with the ligand 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 cell to the subject. The cell may be contacted with the ligand 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 cell to the subject. The cell may be contacted with the ligand 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 cell to the subject. In some cases, the cell may be contacted with the ligand 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 ligand at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some cases, the cell may be contacted with the ligand 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 cell may be contacted with the ligand 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

[0259] In some cases, the ligand (i.e., the stimulant) of the receptor of the 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., a 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).

[0260] In some cases, the ligand binding domain (e.g., the stimulant binding domain) of the cell 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, l-40-b-amyloid, 4-1BB, SAC, 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 b 3 (ADRB3), AGS-22M6, a folate receptor, a-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 orMUC16), Cancer/testis antigen 1 (NY-ESO-1), Cancer/testis antigen 2 (LAGE-la), 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), GDIS, CD152, CD154 (CD40L), CD171, CD 179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (a chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3 e, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44 v6, CDS, 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 Bl, 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 b chain, fibroblast activation protein a (FAP), fibronectin extra domain-B, FGF-5, Fms-Like Tyrosine Kinase 3 (FLT3), folate binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor a, folate receptor b, 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 (gplOO), glypican-3 (GPC3), GMCSF receptor a-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, HERl, 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-a, IFN-b, IFN-g, IgE, IgE Fc region, IGF-1, IGF-1 receptor, IGHE, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-Ib, 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 a4b7, integrin b2, integrin a2, integrin a4, integrin a5b1, integrin a7b7, integrin aIIbb3, integrin anb3, interferon a/b receptor, interferon g-induced protein, Interleukin 11 receptor a (IL-1 IRa), Interieukin-13 receptor subunit a-2 (IL-13Ra2 or CD213A2), intestinal carboxyl esterase, kinase domain region (K _D R), KIR2D, KIT (CD 117), LI -cell adhesion molecule (LI -CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis-Y antigen, LFA-1 (CD 11a), 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-a (LT-a) or Tumor necrosis factor-b (TNF-b), 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 (NRPl), 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 a (PDGF-R a), platelet-derived growth factor receptor b (PDGFR-b), 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 MARTI), P15, P53, FRAME, 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, b 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 (SPA 17), sphingosine-1 -phosphate, squamous cell carcinoma antigen recognized by T Cells 1, 2, and 3 (SARTl, SART2, and SART3), stage- specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, surviving, syndecan 1 (SDC1)+A314, SOXIO, survivin, surviving-2B, synovial sarcoma, X breakpoint 2 (SSX2), T-cell receptor, TCR G Alternate Reading Frame Protein (TARP), telomerase, TEM1, tenascin C, TGF-b (e.g., TGF-b 1, TGF-b 2, TGF-b 3), thyroid stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen ((Tn Ag) or (GalNAca- Ser/Thr)), TNF receptor family member B cell maturation (BCMA), INF -a, 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 1 A (XAGE1), b-amyloid, and k-light chain, and variants thereof.

[0261] 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 pl90 (ela2), 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,

EGFRvin, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES- ESO-la, 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, GplOO, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6), HST- 2/neu, hTERT, iCE, IL-llRa, IL-13Ra2, KDR, KIAA0205, K-RAS, Ll-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-Bl, MAGE-B2, Malic enzyme, Mammaglobin-A, MART-l/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, FRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2,

S ART-1, S ART-2, SART-3, SOXIO, SSX-2, Survivin, Survivin-2B, SYT/SSX, TAG-72, TEL/AMLl, TGFaRII, TGFbRIl, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2/INT2, TRP-2- 6b, Tyrosinase, VEGF-R2, WT1, a-folate receptor, and k-light chain. In some embodiments, the ligand binding domain binds to a tumor associated antigen.

[0262] In some embodiments, the target polynucleotide encodes for a cytokine. Non- limiting examples of cytokines include 4-1BBL, activin bA, activin bB, activin bC, activin bE, artemin (ARTN), BAFF/BLyS/TNFSF 138, BMP10, BMP15, BMP2, BMP3, BMP4, BMPS, 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/CD 154/TNF SF5, CD40LG, CD70, CD70/CD27L/TNFSF7, CLCF1, c-MPL/CDl lO/ 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/F ASLG/CD95L/CD 178, 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, IFNw/IFNWl, IL11, IL18, IL18BP, ILIA, IL1B, IL1F10, IL1F3/IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1RL2, IL31, IL33, IL6, IL8/CXCL8, inhibin-A, inhibin-B, Leptin, LIF, LTA/TNFB/TNFSFl, LTB/TNFC, neurturin (NRTN), OSM, OX-40L/TNFSF4/CD252, persephin (PSPN), RANKL/OPGL/TNFSFl 1(CD254), TL1A/TNFSF15, TNFA, TNF-alpha/TNFA,

TNFSF 10/TRAIL/APO-2L(CD253), TNFSF12, TNFSF13, TNF SF 14/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. [0263] A subject system can be introduced in a variety of immune cells, including any cell that is involved in an immune response. 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 TEM and 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), ab T cells, gd T 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 cany 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 - Thl, Th2, Thl 7, and Treg, with“Th” referring to “T helper cell,” although additional sub-sets may exist. Thl 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. Thl7 cells can produce interieukin 17 (IL-17), a signaling molecule that activates immune and non-immune cells. Thl7 cells are important for recruiting neutrophils.

[0264] 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 gd TCR complex) disclosed herein can be applied (e.g., transduced) includes a wide variety of cell types. A host cell can be in vitro. A host cell can be in vivo. A host cell can be ex 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.

[0265] A host cell can be an immune cell, as abovementioned in the subject disclosure.

[0266] 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.

[0267] 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.

[0268] 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).

[0269] Table 1

[0270] Therapeutic use(s)

[0271] 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.

[0272] In some cases, regulating the expression of the target polynucleotide in the cell may enhance and/or prolong cytotoxicity of the cell against the tumor cell or cancer cell, as compared to without the regulating (or without binding of the ligand to the chimeric receptor). In some cases, regulating the expression of the target polynucleotide in the cell may be evidenced by at least about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, or greater cytotoxicity against the tumor/cancer cell, as compared to without the regulating (or without binding of the ligand to the chimeric receptor). In some cases, regulating the expression of the target polynucleotide in the cell may be evidenced by prolonging cytotoxicity of the cell against the tumor/cancer cell by at least about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, or longer, as compared to without the regulating (or without binding of the ligand to the chimeric receptor).

[0273] In some cases, regulating the expression of the target polynucleotide in the cell may reduce a size of a tumor as compared to without the regulating (or without binding of the ligand to the chimeric receptor), or obliterates the tumor. In some cases, regulating the expression of the target polynucleotide in the cell may be evidenced by at least about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, or greater reduction in the size of the tumor, as compared to without the regulating (or without binding of the ligand to the chimeric receptor).

[0274] In some cases, regulating the expression of the target polynucleotide in the cell may increase expression of one or more cytokines and/or one or more cell surface receptors by the cell, as compared to without the regulating (or without binding of the ligand to the chimeric receptor). In some cases, regulating the expression of the target polynucleotide in the cell may be evidenced by at least about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, or greater expression of the one or more cytokines and/or the one or more cell surface receptors by the cell, as compared to without the regulating (or without binding of the ligand to the chimeric receptor).

[0275] In some cases, regulating the expression of the target polynucleotide in the cell may decrease expression of one or more cytokines and/or one or more cell surface receptors by the cell, as compared to without the regulating (or without binding of the ligand to the chimeric receptor). In some cases, regulating the expression of the target polynucleotide in the cell may be evidenced by at least about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, or greater reduction in the expression of the one or more cytokines and/or the one or more cell surface receptors by the cell, as compared to without the regulating (or without binding of the ligand to the chimeric receptor).

[0276] 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 be in vitro. A target cell can be in vivo. A target cell can be ex 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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 (CDC) 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 Ebneris 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, Corti cotropes, 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.

[0283] 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 blastema, 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, Vemer 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] In some cases, exposing a target cell to an immune cell or population of immune cells disclosed herein can be conducted either in vitro or in 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 cells in vitro can 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 cells in vivo can 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.

[0288] 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.

[0289] In some embodiments, cells expressing a system provided herein induce death of a target cell in an in vitro cell 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.

[0290] 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. [0291] 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.

[0292] Mutations in these genes and pathways can result in production of improper proteins or proteins in improper amounts which affect function.

[0293] 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.

[0294] 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), phosphogly cerate 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 HI), Pol II promoters, and/or tRNA(val) promoter.

[0295] Table 2

[0296] Table 3

[0297] 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, regenerative medicine, 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.

[0298] Systems and methods of the present disclosure may be combined with or modified by other systems and methods, such as, for example, those described in U.S. Patent No. 9,856,497 (“CHIMERIC PROTEINS AND METHODS OF REGULATING GENE

EXPRESSION”), Patent Cooperation Treaty Patent Publication No. 2017/123559

(“CHIMERIC PROTEINS AND METHODS OF REGULATING GENE EXPRESSION”), Patent Cooperation Treaty Patent Publication No. 2017/123556 (“CHIMERIC PROTEINS AND METHODS OF IMMUNOTHERAPY”), Patent Cooperation Treaty Patent Publication No. 2019/014390 (“METHODS AND SYSTEMS FOR CONDITIONALLY REGULATING GENE EXPRESSION”), and Patent Cooperation Treaty Patent Application No.

PCT/US2019/023721 (“GENE REGULATION VIA CONDITIONAL NUCLEAR

LOCALIZATION OF GENE MODULATING POLYPEPTIDES”), each of which is entirely incorporated herein by reference.

[0299] 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.

EXAMPLES

[0300] Various aspects of the disclosure are further illustrated by the following non- limiting examples.

Example 1: Regulating inducible gene expression by receptor activation and an ion sensor- controlled protease.

[0301] FIG. 1 schematically illustrates a system for regulating expression of a target polynucleotide in a cell. In this system, inducible gene expression is regulated by receptor 100 activation and a calcium sensor (CaSensor) 120-controlled Tobacco Etch Virus (TEV) proteinase 130. Interaction (e.g., binding) of a ligand 110 with its corresponding receptor 100 (e.g., comprised of extracellular domain (ECD) 100a, transmembrane domain (TMD) 100b, and intracellular domain (ICD)) 100c activates at least a calcium signaling in the cell. The calcium signaling causes a rise in cytosolic calcium concentration (i.e., [Ca ²⁺ ]) 122 due to either a release of Ca ²⁺ from cellular organelles (not shown) or Ca ²⁺ influx from extracellular microenvironment, or both. In an example, upon binding of the ligand 110, the receptor 100 activates a calcium channel receptor 124 on the cell membrane to induce the Ca ²⁺ influx from the extracellular microenvironment. The rise in cytosolic [Ca ²⁺ ] 122 leads to biochemical and/or structural changes of a chimeric polypeptide 200 comprising the CaSensor 120 linked to the TEV 130, wherein the enzymatic activity of the TEV 130 is blocked by the CaSensor 120 in the absence of calcium and is activated in the presence of calcium. After activation by binding of one or more Ca ^2÷ , the CaSensor 120 exposes an activate site of the TEV 130, which then cleaves a TEV cleavage site (TCS) 150 to remove at least one nuclear export signal (NES) 160 from an additional chimeric polypeptide 300 comprising the at least one NES 160 linked to the TCS 150 and an actuator 140. When coupled to one another, the at least one NES 160 prevents the actuator 140 from translocating into nucleus of the cell. The actuator 140 can comprise a deactivated or dead endonuclease (e.g., dCas9) linked to a functional domain (e.g., transcriptional effector domain, such as VP64-p65-Rta (VPR) or Kriippel associated box (KRAB)). As a result, the actuator 140 is released from the at least one NES 160, and translocates into nucleus of the cell to regulate the expression of one or more target genes 400. Depending on the transcriptional effector domain (e.g., VPR or KRAB), expression of the one or more target genes 400 can be either upregulated (e.g., by

VPR) or downregulated (e.g., by KRAB).

[0302] FIG. 2 schematically illustrates another system for regulating expression of a target polynucleotide in a cell. In this system, a chimeric antigen receptor (CAR) 102 activation-dependent target gene expression is regulated by a CaSensor 120-controlled TEV proteinase 130. The CAR 102 comprises a ligand binding domain 102a (e.g., a single chain antibody variable fragment (scFv) domain), a spacer 102b, a transmembrane domain 102c, and at least two intracellular signal domains 102d. Binding of a specific antigen 112 to the ligand binding domain 102a of the CAR 102 activates one or more intracellular signaling pathways (e.g., intrinsic TCR signal transduction pathways) that include or further induce calcium signaling. The calcium signaling and a resulting rise in cytosolic [Ca ²⁺ ] 122 (e.g., upon activation of a calcium channel receptor 124 by the one or more intracellular signaling pathways activated by the CAR 102) leads to biochemical and/or structural changes of a chimeric polypeptide 200 comprising the CaSensor 120 linked to the TEV 130, wherein the enzymatic activity of the TEV 130 is blocked by the CaSensor 120 in the absence of calcium and is activated in the presence of calcium. After activation by binding of one or more Ca ²⁺ , the CaSensor 120 exposes an active site of the TEV 130, which then cleaves a TCS 150 to remove at least one NES 160 from an additional chimeric polypeptide 300 comprising the at least one NES 160 linked to the TCS 150 and an actuator 140. The actuator 140 can comprise a deactivated or dead endonuclease (e.g., dCas9) linked to a functional domain (e.g., transcriptional effector domain, such as VPR or KRAB). As a result, the actuator 140 is released from the at least one NES 160, and translocates into nucleus of the cell to regulate the expression of one or more target genes 400.

[0001] FIG. 3 schematically illustrates a different system for regulating expression of a target polynucleotide in a cell. In this system, a CAR 102 activation-dependent target gene expression is regulated by a CaSensor 120-controlled TEV proteinase 130. The CAR 102 comprises an actuator 140 that is linked to the CAR 102 via a TCS linker 150. The actuator 140 can comprise a deactivated or dead endonuclease (e.g., dCas9) linked to a functional domain (e.g., transcriptional effector domain, such as VPR or KRAB). Binding of a specific antigen 112 to an antigen binding domain 102a of the CAR 102 activates one or more intracellular signaling pathways (e.g., intrinsic TCR signal transduction pathways) that include or further induce calcium signaling. The calcium signaling and a resulting rise in cytosolic [Ca ²⁺ ] 122 (e.g., upon activation of a calcium channel receptor 124 by the one or more intracellular signaling pathways activated by the CAR 102) leads to biochemical and/or structural changes of a chimeric polypeptide 200 comprising the CaSensor 120 linked to the TEV 130, wherein the enzymatic activity of the TEV 130 is blocked by the CaSensor 120 in the absence of calcium and is activated in the presence of calcium. After activation by binding of one or more Ca ^2÷ , the CaSensor 120 exposes an active site of the TEV 130, which then cleaves the TCS 150 to release the actuator 140 from the CAR 102. As a result, the released actuator 140 translocates into nucleus of the cell to regulate the expression of one or more target genes 400.

[0002] FIG. 4 schematically illustrates a different system for regulating expression of a target polynucleotide in a cell. In this system, a CAR 102 activation-dependent target gene expression is regulated by a CaSensor 120-controlled TEV proteinase 130. The CAR 102 comprises the TEV 130 linked to the CaSensor 120. Binding of a specific antigen 112 to an antigen binding domain 102a of the CAR 102 activates one or more intracellular signaling pathways (e.g., intrinsic TCR signal transduction pathways) that include or further induce calcium signaling. The calcium signaling and the resulting rise in cytosolic [Ca ²⁺ ] 122 (e.g., upon activation of a calcium channel receptor 124 by the one or more intracellular signaling pathways activated by the CAR) leads to biochemical and/or structural changes of at least the CaSensor 120 of the CAR 102-TEV 130-CaSensor 120 chimeric receptor polypeptide, wherein the enzymatic activity of the TEV 130 is blocked by the CaSensor in the absence of calcium and is activated in the presence of calcium. After activation by binding of one or more Ca ²⁺ , the CaSensor 120 exposes an active site of the TEV 130, which then cleaves a TCS ISO to remove at least one NES 160 from a chimeric polypeptide 300 comprising the at least one NES 160 linked to the TCS ISO and an actuator 140. The actuator 140 can comprise a deactivated or dead endonuclease (e.g., dCas9) linked to a functional domain (e.g., transcriptional effector domain, such as VPR or KRAB). As a result, the actuator 140 is released from the at least one NES 160, and translocates into nucleus of the cell to regulate the expression of one or more target genes 400.

[0003] FIG. 5 schematically illustrates a different system for regulating expression of a target polynucleotide in a cell. In this system, a CAR 102 activation-dependent target gene expression is regulated by a CaSensor 120-controlled TEV proteinase 130. The CAR 102 comprises at least one intracellular signaling domain 102d. The CAR 102 further comprises an actuator 140 that is linked to the CAR 102 via a TCS linker 150. The actuator 140 can comprise a deactivated or dead endonuclease (e.g., dCas9) linked to a functional domain (e.g., transcriptional effector domain, such as VPR or KRAB). The system further comprises a chimeric polypeptide 200 comprising the CaSensor 120 linked to the TEV 130, as well as an adaptor moiety 170 configured to bind to the at least one intracellular signaling domain 102d of the CAR 102 (e.g., upon activation of the CAR 102). Binding of a specific antigen 112 to an antigen binding domain 102a of the CAR 102 induces a change (e.g., conformational and/or chemical) in the at least one intracellular domain 102d of the CAR 102, which change allows the adaptor 170 of the chimeric polypeptide 200 to be recruited to the CAR 102 and bind the at least one intracellular domain 102d of the CAR 102. Activation of the CAR 102 also activates one or more intracellular signaling pathways (e.g., intrinsic TCR signal transduction pathways) that include or further induce calcium signaling. The calcium signaling and the resulting rise in cytosolic [Ca ²⁺ ] 122 (e.g., upon activation of a calcium channel receptor 124 by the one or more intracellular signaling pathways activated by the CAR 102) leads to biochemical and/or structural changes of at least the CaSensor 120 of the chimeric polypeptide 200, wherein the enzymatic activity of the TEV 130 is blocked by the CaSensor 120 in the absence of calcium and is activated in the presence of calcium. After activation by binding of one or more Ca ²⁺ , the CaSensor 120 exposes an active site of the TEV 130, which then cleaves the TCS 150 to release the actuator 140 from the CAR 102. As a result, the released actuator 140 translocates into nucleus of the cell to regulate the expression of one or more target genes 400. [0004] FIG. 6 schematically illustrates a different system for regulating expression of a target polynucleotide in a cell. In this system, a CAR 102 activation-dependent target gene expression is regulated by a CaSensor 120-controlled TEV proteinase 130. An intracellular portion of the CAR 102 comprises at least one intracellular signaling domain 102d, as well as the TEV 130 linked to the CaSensor 120. The system also comprises a chimeric polypeptide 300 comprising an adaptor 170 configured to bind the at least one intracellular signaling domain 102d of the CAR 102, which adaptor 170 is linked to at least one NES 160 and an actuator 140 via a TCS ISO. The actuator 140 can comprise a deactivated or dead

endonuclease (e.g., dCas9) linked to a functional domain (e.g., transcriptional effector domain, such as VPR or KRAB). Binding of a specific antigen 112 to an antigen binding domain 102a of the CAR 102 induces a change (e.g., conformational and/or chemical) in the at least one intracellular domain 102d of the CAR 102, which change allows the adaptor 170 of the chimeric polypeptide 300 to be recruited to the CAR 102 and bind the at least one intracellular domain I02d of the CAR 102. Activation of the CAR 102 also activates one or more intracellular signaling pathways (e.g., intrinsic TCR signal transduction pathways) that include or further induce calcium signaling. The calcium signaling and the resulting rise in cytosolic [Ca ²⁺ ] 122 (e.g., upon activation of a calcium channel receptor 124 by the one or more intracellular signaling pathways activated by the CAR 102) leads to biochemical and/or structural changes of at least the CaSensor 120, wherein the enzymatic activity of the TEV 130 is blocked by the CaSensor 120 in the absence of calcium and is activated in the presence of calcium. After activation by binding of one or more Ca ²⁺ , the CaSensor 120 exposes an active site of the TEV 130, which then cleaves the TCS 150 to release the actuator 140 from the NES 160 and the adaptor 170 of the chimeric polypeptide 300. As a result, the released actuator 140 translocates into nucleus of the cell to regulate the expression of one or more target genes 400.

[0005] FIG. 7 schematically illustrates a system for regulating expression of a target polynucleotide in a cell. In this system, inducible gene expression is regulated by receptor 100 activation and a CaSensor 120-controlled TCS 150 cleavage. The system comprises a chimeric polypeptide 300 comprising the CaSensor 120 linked to at least one NES 160, the TCS 150, and an actuator 140. The at least one NES 160 is flanked by the CaSensor 120 and the TCS 150, while the TCS 150 is flanked by the at least one NES 160 and the actuator 140. The actuator 140 can comprise a deactivated or dead endonuclease (e.g., dCas9) linked to a functional domain (e.g., transcriptional effector domain, such as VPR or KRAB). Interaction (e.g., binding) of a ligand 110 with its corresponding receptor 100 activates at least a calcium signaling in the cell. The calcium signaling and a resulting rise in cytosolic [Ca ²⁺ ] 122 (e.g., upon activation of a calcium channel receptor 124 by the one or more intracellular signaling pathways activated by the receptor 100) leads to biochemical and/or structural changes of the CaSensor 120 of the chimeric polypeptide 300, wherein the TCS 150 of the chimeric polypeptide 300 is blocked by the CaSensor 120 in the absence of calcium and is exposed in the presence of calcium. After activation by binding of one or more Ca ²⁺ , the CaSensor 120 exposes the TCS 150, which can be cleaved by a TEV 130 to release the actuator 140 from the chimeric polypeptide 300. As a result, the released actuator 300 translocates into nucleus of the cell to regulate the expression of one or more target genes 400.

[0303] FIG. 8 schematically illustrates a system for regulating expression of a target polynucleotide in a cell. In this system, inducible gene expression is regulated by receptor 100 activation and a CaSensor 120-controlled TCS 150 cleavage. The system comprises a chimeric polypeptide 300 comprising at least one NES 160 linked to the CaSensor 120, the TCS 150, and an actuator 140. The CaSensor 120 is flanked by the at least one NES 160 and the TCS 150, while the TCS 150 is flanked by the CaSensor 120 and the actuator 140. The actuator 140 can comprise a deactivated or dead endonuclease (e.g., dCas9) linked to a functional domain (e.g., transcriptional effector domain, such as VPR or KRAB). Interaction (e.g., binding) of a ligand 110 with its corresponding receptor 100 activates at least a calcium signaling in the cell. The calcium signaling and a resulting rise in cytosolic [Ca ²⁺ ] 122 (e.g., upon activation of a calcium channel receptor 124 by the one or more intracellular signaling pathways activated by the receptor 100) leads to biochemical and/or structural changes of the CaSensor 120 of the chimeric polypeptide 300, wherein the TCS 150 of the chimeric polypeptide 300 is blocked by the CaSensor 120 in the absence of calcium and is exposed in the presence of calcium. After activation by binding of one or more Ca ²⁺ , the CaSensor 120 exposes the TCS 150, which can be cleaved by a TEV 130 to release the actuator 140 from the chimeric polypeptide 300. As a result, the released actuator 140 translocates into nucleus of the cell to regulate the expression of one or more target genes 400.

Example 2: Regulating inducible gene expression by receptor activation and an ion sensor- controlled protease in a CD4+ Jurkat cell line.

[0304] A stable Jurkat reporter cell line 2sg-CAR may be generated to contain one or more of the following transgenes: (1) an anti-CD19 CAR expression cassette; (2) a IRE3G promoter-driven GFP expression cassette (e.g., the promoter has 7 sgRNA binding sites); (3) a sgRNA targeting the TRE3G promoter; and (4) a sgRNA taigeting the CXCR4 promoter (negative control). The 2sg-CAR cell line may be transfected with one or more plasmids, e.g., (i) a NES-TCS-dCas9VPR plasmid and/or (ii) a CaSensor-TEV or TEV-CaSensor variant plasmid. Subsequently (e.g., a day later), the engineered Jurkat cells may be split for one or more assays (e.g., split into 2 equal parts for two different assays).

[0305] In some cases, the engineered Jurkat cells may be used for calcium influx assay using a BD calcium assay kit (BD, Cat#640176). Briefly, the engineered Jurkat cells may be incubated with 50% IX loading dye solution at 37°C for 1 hr, then cooled down (e.g., for 20 minutes) at room temperature. Data may be acquired in a flow cytometer for a time period (e.g., 1 min), after which data acquisition may be stopped to add Raji cells. Subsequently, additional data may be acquired (e.g., for 3 minutes) as an addition to the previous data acquired in the absence of the Raji cells. An increased calcium influx in 2sg-CAR cell line may be observed due to the activation of CD 19 CAR of the engineered Jurkat cells by interaction with CD19 expressed on Raji cells.

[0306] In some cases, the engineered Jurkat cells may be used for a marker (e.g., green fluorescent protein (GFP)) upregulation assay. Briefly, Raji cells may be added into the engineered Jurkat cells, as provided herein. Subsequently (e.g., two days later), cells may be collected, stained with anti-CD22-PE, and analyzed by using a flow cytometer. CD22- negative cells (i.e., the engineered Jurkat cells expressiong the 2sg-CAR) may be gated and analyzed for GFP expression. A higher level of GFP expression (compared to control samples) may be observed due to the conditional gene upregulation through the mechanism as illustrated in FIG. 2.