Regulation of gene expression in cells expressing the gamma delta T cell receptor.

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to a method for modulating the activity of a gamma delta T cell, comprising administering to the gamma delta T cell a gene modulating polypeptide (GMP) comprising an actuator portion configured to modulate expression of a target polynucleotide in the gamma delta T cell, wherein the target polynucleotide encodes a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolytic enzyme. [Background technology]

[0002] Cells expressing gamma (γ) and / or delta (δ) T cell receptors (e.g., gamma-delta (γδ) T cells) may participate in one or more immune responses during the progression of disease, e.g., cancer. In some cases, γδ T cells may play a direct and / or indirect anti-tumor role (e.g., anti-tumor cytotoxicity, cytokine production, activation of one or more immune cell types, etc.). Thus, γδ T cells can be used in cancer immunotherapy. In some cases, γδ T cells can be activated (e.g., by stimulation) to enhance and / or prolong their anti-tumor activity. Alternatively, or in addition, γδ T cells can be isolated, expanded, and administered for cancer immunotherapy.

[0003] γδ T cells can be activated through one or more pathways (e.g., one or more cellular receptors). In some cases, cellular receptor-ligand interactions may play a role in sensing stimuli (e.g., one or more environmental cues) and translating such extracellular stimuli into intracellular signaling. Intracellular signaling may result in the regulation of biochemical processes, including the transcriptional activation of gene expression and the synthesis of new proteins to control cellular activity, e.g., antitumor activity. However, the effectiveness of γδ T cells in immunotherapy may be reduced by, for example, insufficient activation from insufficient receptor-ligand interactions and / or cognate immune checkpoint pathways (e.g., apoptotic pathways). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent Application Serial No. 12 / 601,628 [Patent Document 2] PCT / US17 / 012885 [Patent Document 3] No. PCT / US17 / 012881 [Patent Document 4] No. PCT / US18 / 041704 [Patent Document 5] U.S. Patent No. 9,856,497 [Patent Document 6] U.S. Nonprovisional Application No. 15 / 806,756 [Patent Document 7] U.S. Nonprovisional Application No. 16 / 029,299 [Patent Document 8] U.S. Provisional Application No. 62 / 639,427 [Patent Document 9] U.S. Provisional Application No. 62 / 639,386 [Patent Document 10] U.S. Provisional Application No. 62 / 647,543 [Patent Document 11] U.S. Provisional Application No. 62 / 675,134 [Patent Document 12] No. US20080241194 [Non-patent literature]

[0005] [Non-Patent Document 1] Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012) [Non-patent document 2] Current Protocols in Molecular Biology series (ed. FM Ausubel et al.) [Non-patent document 3] Methods in Enzymology series (Academic Press, Inc.) [Non-patent document 4] PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)) [Non-Patent Document 5] Harlow and Lane (eds.) (1988) Antibodies, A Laboratory Manual [Non-patent document 6] Culture of Animal Cells: A Manual of Basic Techniques and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)) [Non-Patent Document 7] Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1~18.88 [Non-patent document 8] www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html [Non-Patent Document 9] blast.ncbi.nlm.nih.gov / Blast.cgi [Non-Patent Document 10] www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the foregoing, there is a considerable need for alternative methods and systems for controlling the activity (eg, anti-tumor activity) of γδ T cells. [Means for solving the problem]

[0007] One aspect of the present disclosure provides a method for modulating the activity of a gamma delta T cell, the method comprising administering to the gamma delta T cell a gene modulating polypeptide (GMP) comprising an actuator portion configured to modulate expression of a target polynucleotide in the gamma delta T cell, wherein the target polynucleotide encodes a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of the death-inducing protein, and / or (iii) a hydrolase.

[0008] In some embodiments, modulating gamma delta T cell activity comprises reducing gamma delta T cell fratricide.

[0009] In some embodiments, modulating the activity of gamma delta T cells comprises prolonging activation of gamma delta T cells.

[0010] In some embodiments, the actuator moiety is an RNA-guided actuator moiety or a variant thereof, and the RNA-guided actuator moiety forms a complex with a target polynucleotide. In some embodiments, the actuator moiety is a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity. In some embodiments, the actuator moiety is Cas9 and / or Cpf1.

[0011] In some embodiments, the actuator portion comprises an activator effective to increase expression of the target polynucleotide.

[0012] In some embodiments, the actuator portion comprises a repressor effective to reduce expression of the target polynucleotide.

[0013] In some embodiments, the polypeptide is a death-inducing protein. In some embodiments, the death-inducing protein comprises Fas, Fas ligand, granzyme B, granzyme M, and / or perforin. In some embodiments, modulating expression of the target polynucleotide comprises reducing expression of the target polynucleotide.

[0014] In some embodiments, the polypeptide is an inhibitor of a death-inducing protein. In some embodiments, the inhibitor comprises serpin B9, serpin B4, and / or c-FLIP. In some embodiments, modulating expression of the target polynucleotide comprises increasing expression of the target polynucleotide.

[0015] In some embodiments, the polypeptide is a hydrolase. In some embodiments, the polypeptide comprises CD39. In some embodiments, modulating expression of the target polynucleotide comprises decreasing expression of the target polynucleotide.

[0016] In some embodiments, the method further comprises treating the gamma delta T cells with a virus to administer GMP to the gamma delta T cells. In some embodiments, the method further comprises using the virus to integrate a nucleic acid sequence encoding GMP into the genome of the gamma delta T cells. In some embodiments, the virus is a retrovirus. In some embodiments, the retrovirus is a gamma-retrovirus selected from the group consisting of Moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), and spleen focus forming virus (SFFV), and / or variants thereof.

[0017] In some embodiments, the method further comprises treating the gamma delta T cells with a nuclease and administering GMP to the gamma delta T cells. In some embodiments, the method further comprises inserting a nucleic acid sequence encoding GMP into the genome of the gamma delta T cells by using the nuclease. In some embodiments, the nuclease comprises a CRISPR-associated polypeptide (Cas), a zinc finger nuclease (ZFN), a zinc finger-associated gene-regulating polypeptide, a transcription activator-like effector nuclease (TALEN), a transcription activator-like effector-associated gene-regulating polypeptide, a meganuclease, a natural master transcription factor, an epigenetic modification enzyme, a recombinase, a flippase, a transposase, an RNA-binding protein (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0018] In some embodiments, the method further comprises administering to the gamma delta T cell a chimeric polypeptide comprising GMP, wherein the chimeric polypeptide is operable to release GMP from the chimeric polypeptide in response to a stimulus, and the released GMP is operable to regulate expression of a target polynucleotide in the gamma delta T cell.

[0019] In some embodiments, the method further comprises administering to the gamma delta T cell a chimeric polypeptide comprising a GMP and a nuclear translocation domain, wherein the nuclear translocation domain is operable to translocate the chimeric polypeptide to the nucleus of the gamma delta T cell in response to a stimulus, and wherein the translocated GMP is operable to regulate expression of a target polynucleotide in the gamma delta T cell.

[0020] Another aspect of the present disclosure provides a system for modulating the activity of a gamma delta T cell, the system comprising a gene modulating polypeptide (GMP) comprising an actuator moiety configured to modulate expression of a target polynucleotide in the gamma delta T cell, wherein the target polynucleotide encodes a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of the death-inducing protein, and / or (iii) a hydrolase.

[0021] In some embodiments, the GMP is operable to reduce gamma delta T cell fratricide.

[0022] In some embodiments, the GMP is operable to prolong the activation of gamma delta T cells.

[0023] In some embodiments, the actuator moiety is an RNA-guided actuator moiety or a variant thereof, and the RNA-guided actuator moiety forms a complex with a target polynucleotide. In some embodiments, the actuator moiety is a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity. In some embodiments, the actuator moiety is Cas9 and / or Cpf1.

[0024] In some embodiments, the actuator portion comprises an activator effective to increase expression of the target polynucleotide.

[0025] In some embodiments, the actuator portion comprises a repressor effective to reduce expression of the target polynucleotide.

[0026] In some embodiments, the polypeptide is a death-inducing protein. In some embodiments, the death-inducing protein comprises Fas, Fas ligand, granzyme B, granzyme M, and / or perforin. In some embodiments, modulating expression of the target polynucleotide comprises reducing expression of the target polynucleotide.

[0027] In some embodiments, the polypeptide is an inhibitor of a death-inducing protein. In some embodiments, the inhibitor comprises serpin B9, serpin B4, and / or c-FLIP. In some embodiments, modulating expression of the target polynucleotide comprises increasing expression of the target polynucleotide.

[0028] In some embodiments, the polypeptide is a hydrolase. In some embodiments, the polypeptide comprises CD39. In some embodiments, modulating expression of the target polynucleotide comprises decreasing expression of the target polynucleotide.

[0029] In some embodiments, the nucleic acid sequence encoding GMP is integrated into the genome of the gamma delta T cell by a virus. In some embodiments, the virus is a retrovirus. In some embodiments, the retrovirus is a gamma-retrovirus selected from the group consisting of Moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), and spleen focus forming virus (SFFV), and / or variants thereof.

[0030] In some embodiments, the nucleic acid sequence encoding the GMP is integrated into the genome of the gamma delta T cell by a nuclease, hi some embodiments, the nuclease comprises a CRISPR-associated polypeptide (Cas), a zinc finger nuclease (ZFN), a zinc finger-associated gene regulatory polypeptide, a transcription activator-like effector nuclease (TALEN), a transcription activator-like effector-associated gene regulatory polypeptide, a meganuclease, a natural master transcription factor, an epigenetic modification enzyme, a recombinase, a flippase, a transposase, an RNA-binding protein (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0031] In some embodiments, the nucleic acid sequence encoding the GMP is integrated into the genome of the gamma delta T cell by an endonuclease. In some embodiments, the endonuclease comprises a CRISPR-associated polypeptide (Cas), a zinc finger nuclease (ZFN), a zinc finger-associated gene regulatory polypeptide, a transcription activator-like effector nuclease (TALEN), a transcription activator-like effector-associated gene regulatory polypeptide, a meganuclease, a natural master transcription factor, an epigenetic modification enzyme, a recombinase, a flippase, a transposase, an RNA-binding protein (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0032] In some embodiments, the system further includes a chimeric polypeptide comprising GMP, wherein the chimeric polypeptide is operable to release GMP from the chimeric polypeptide in response to a stimulus, and the released GMP is operable to regulate expression of a target polynucleotide in a gamma delta T cell.

[0033] In some embodiments, the system further comprises a chimeric polypeptide comprising a GMP and a nuclear translocation domain, wherein the nuclear translocation domain is operable to translocate the chimeric polypeptide to the nucleus of a gamma delta T cell in response to a stimulus, and wherein the translocated GMP is operable to regulate expression of a target polynucleotide in the gamma delta T cell.

[0034] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the 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 present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0035] Incorporated by reference All publications, patents, and patent applications cited herein are 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 that the publications and patents or patent applications incorporated by reference conflict with the present disclosure contained herein, the present specification is intended to supersede and / or supersede any such conflicting material.

[0036] 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 referred to herein as "Figure" and "FIG."). [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 10 is a schematic illustrating an example of a vector and its use for expression of a system comprising a chimeric receptor polypeptide and a chimeric adaptor polypeptide in γδ T cells. The chimeric receptor polypeptide comprises a CAR, and the chimeric adaptor polypeptide comprises a GMP comprising a LAT-dCas9-effector. Upon exogenous expression, tumor antigen recognition by the system and the CAR of the chimeric receptor polypeptide in γδ T cells can (1) upregulate serpin B9, serpin B4, and / or c-Flip, thereby enhancing γδ T cell survival, and / or (2) downregulate CD39, maintaining the proliferation and / or activation responsiveness of γδ T cells to pyrophosphate, pyrophosphate by-products, and / or other γδ TCR ligands. Pyrophosphate, pyrophosphate by-products, and / or other γδ TCR ligands can be administered after tumor-chimeric receptor polypeptide interaction. [Figure 2]

[0023] Figure 10 schematically illustrates an example of a vector and its use for pyrophosphate-induced expression of a system comprising a chimeric adapter polypeptide in γδ T cells. The chimeric adapter polypeptide (1) comprises a GMP containing an LAT-dCas9-effector; (2) is under the control of an exogenous promoter, e.g., the LAG3 and / or Fas promoter; and (3) is compatible with a chimeric receptor polypeptide comprising a CAR. The exogenous LAG3 and / or Fas promoter can be identical to or a functional variant of the endogenous LAG3 and / or Fas promoter of the γδ T cells. In these examples, the chimeric receptor polypeptide comprising a CAR is constitutively expressed in the γδ T cells. During one or more treatments with pyrophosphate, pyrophosphate by-products, and / or other γδ TCR ligands, the activity of the LAG3 and / or Fas promoter (e.g., endogenous and / or exogenous) is induced and / or upregulated in γδ T cells, thereby inducing expression of the chimeric adaptor polypeptide in the γδ T cells. The expressed chimeric adaptor polypeptide and chimeric receptor polypeptide work in conjunction to activate a dCas9-effector that upregulates expression of serpin B9, serpin B4, and / or c-Flip, thereby enhancing the survival and activation state of the γδ T cells. Alternatively, or in addition, the expressed chimeric adaptor polypeptide and chimeric receptor polypeptide work in conjunction to activate a dCas9-effector that downregulates expression of CD39. In these examples, the γδ T cells can function with or without the chimeric antigen polypeptide. [Figure 3]

[0023] Figure 10 is a schematic illustrating gene knock-in in a system comprising GMP under the control of an endogenous promoter in a γδ T cell. One or more endogenous genes under the control of an endogenous promoter in a γδ T cell can be knocked out. One or more gene editing endonucleases can be used to knock in and / or knock out genes. DETAILED DESCRIPTION OF THE INVENTION

[0038] While various 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. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0039] The practice of some of the methods disclosed herein will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), Current Protocols in Molecular Biology series (F.M. Ausubel et al., eds.), Methods in Enzymology series (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.R. Taylor, eds. (1995)), Antibodies, A Laboratory Manual (Harlow and Lane, eds. (1988)), and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0040] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a transmembrane receptor" may include multiple transmembrane receptors.

[0041] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends 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, according to 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 specific values ​​are described in this application and claims, unless otherwise stated, the term "about" means that an acceptable error range for the particular value should be assumed.

[0042] As used herein, "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 be derived from any organism that has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopersicons, hornworts, liverworts, and mosses), algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. In some cases, the cells are not derived from a natural organism (e.g., cells can be synthetically produced, also called artificial cells).

[0043] The terms "gamma delta cells," "γδ cells," and "GD cells," used interchangeably herein, can refer to cells having a unique T-cell receptor (TCR) consisting of one gamma (γ) TCR chain and one delta (δ) TCR chain. In some cases, γδ cells can include lymphocytes having a γδ TCR, such as γδ T cells or T lymphocytes. The terms "gamma delta T cells," "γδ T cells," and "GD T cells," used interchangeably herein, can refer to T cells (T lymphocytes) that contain a unique TCR consisting of a γ TCR chain and a δ TCR chain. A γδ T cell can contain at least a portion of a γδ TCR expressed on the cell membrane. The γ TCR chain can be any one of Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, Vγ10, functional variants thereof, or combinations thereof. The delta TCR chain can be any one of delta 1, delta 2, delta 3, a functional variant thereof, or a combination thereof. In some examples, the delta TCR can be a Vg9 / V52 TCR, a Vg10 / V52 TCR, and / or a Vg2 / V52 TCR.

[0044] γδ T cells can be activated by γδ T cell activators. As used herein, the term "γδ T cell activator" can refer to a molecule (natural or synthetic) that can activate or induce the biological activity of a γδ T cell (e.g., migration, proliferation, synthesis of one or more polypeptides, etc.). In some cases, a γδ T cell activator can be a ligand of a γδ TCR and / or a distinct receptor. Alternatively, or in addition, a γδ T cell activator can be an endogenous ligand. A γδ T cell activator can be of various natures, e.g., a peptide, lipid, small molecule, etc. A γδ T cell activator can be purified or otherwise artificially produced (e.g., by chemical synthesis or by a microbiological process). In some cases, a γδ T cell activator can include an antibody with functional antigen specificity like another γδ T cell activator.

[0045] A γδ T cell activator may be administered to a subject (e.g., a patient with cancer or a tumor) in an amount and / or under conditions sufficient to increase the activity of γδ T cells in the subject. Increasing the activity of γδ T cells in the subject may increase cytokine secretion by the γδ T cells and / or increase the cytolytic activity of the γδ T cells (e.g., cytolytic activity against one or more tumor or cancer cells).

[0046] The γδ T cell activator can comprise a phosphoantigen. The phosphoantigen can comprise one or more phosphates. The phosphoantigen can comprise an organic pyrophosphate and / or functional modifications thereof. The phosphoantigen can be a non-peptide antigen. Alternatively, or in addition, the phosphoantigen can be linked (e.g., covalently or non-covalently) to one or more amino acids and / or one or more polynucleotides. Examples of such phosphate-containing compounds that act as phosphoantigens can include a nucleotide (or polynucleotide), a nucleotide analog (e.g., 7-deaza-dGTP, 7-deaza-dATP, etc.), or a pyrophosphonate (e.g., a bisphosphonate, e.g., zoledronate or zoledronic acid).

[0047] As used herein, the term "bisphosphonate" can refer to any compound that is an analog of pyrophosphate (e.g., endogenous pyrophosphate). Examples of pyrophosphate (PP) and / or functional derivatives thereof can include, for example, isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), farnesyl pyrophosphate (FPP), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), hexaprenyl pyrophosphate (HPP), neryl pyrophosphate (NPP), octaprenyl pyrophosphate (OPP), solanesyl pyrophosphate (SPP), functional derivatives thereof, or combinations thereof. Further examples of phosphoantigens may include BrHPP, BrIAPP, CBrHPP, EpoxPP, HDMAPP, CHDMAPP, NHDMAPP, H-angelylPP, H-tiglylPP, functional derivatives thereof, or combinations thereof. Examples of bisphosphonates may include aminobisphosphonates. Examples of bisphosphonates may include, but are not limited to, zoledronic acid, risedronate, alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, piridronate, or pamidronate, and functional analogs thereof.

[0048] A phosphoantigen can activate a γδ T cell, thereby, for example, increasing the biological activity of the γδ T cell (e.g., migration, proliferation, synthesis of one or more polypeptides, etc.). A phosphoantigen can cause proliferation of a γδ T cell. A phosphoantigen can increase cytokine secretion from a γδ T cell. A phosphoantigen can increase the cytolytic activity of a γδ T cell. A phosphoantigen can directly and / or indirectly activate a γδ T cell. In some cases, a phosphoantigen (e.g., IPP) can directly interact with a receptor (e.g., Vγ9Vδ2 TCR) of a γδ T cell to activate the γδ T cell. In some cases, a phosphoantigen can interact with a target cell (e.g., a cancer / tumor cell) of the γδ T cell, and the target cell can metabolize the phosphoantigen into a γδ T cell activator. The target cell can then secrete and / or present the γδ T cell activator to activate neighboring γδ T cells. Alternatively, phosphoantigens can be taken up by target cells, causing them to release γδ T cell activators. In one example, zoledronate can be an indirect γδ T cell activator. When zoledronate is taken up by cancer / tumor cells, it can inhibit FPP synthase in the cancer / tumor cells, causing IPP to accumulate. Cancer / tumor cells can secrete or display IPP on their surface. IPP can be recognized by γδ T cells (e.g., Vγ9Vδ2 T cells) and activate γδ T cells to respond to cancer / tumor cells.

[0049] Further details of the design and application of phosphoantigens and modifications thereof in the context of treating cancer or tumors are disclosed in U.S. Patent Application No. 12 / 601,628, which is incorporated herein by reference in its entirety.

[0050] The biological function of a γδ T cell activator can be prevented and / or inhibited by a γδ T cell activator inhibitor. The γδ T cell activator inhibitor may bind to a target (e.g., a target protein) of the γδ T cell activator and block γδ T cell activator-target binding. Alternatively, or in addition, the γδ T cell activator inhibitor may bind to a molecule (e.g., a polypeptide or polynucleotide) involved in the signaling cascade of γδ T cell activator-target binding, thereby blocking the signaling activity of the γδ T cell activator. Alternatively, or in addition, the γδ T cell activator inhibitor may degrade or metabolize the γδ T cell activator and / or the target of the γδ T cell activator. The γδ T cell activator inhibitor may or may not be a transmembrane protein. The γδ T cell activator inhibitor may be an extracellular and / or intracellular molecule.

[0051] In some cases, the γδ T cell activator can be a phosphoantigen, and the inhibitor of the γδ T cell activator can be a phosphoantigen inhibitor. The phosphoantigen inhibitor can include a hydrolase. As used herein, the term "hydrolase" can refer to a catalyst (e.g., an enzyme) configured to break a chemical bond. A hydrolase can split a molecule into two or more smaller molecules. Examples of hydrolases can include lipases, phosphatases, glycosidases, peptidases, nucleosidases, nucleotidases, functional modifications thereof, or combinations thereof.

[0052] Phosphoantigen inhibitor (e.g., hydrolase) hydrolase can include a nucleotidase. The nucleotidase can be configured to catalyze the hydrolysis of nucleotides in the presence and / or absence of another enzyme. The nucleotidase can include a 3'-nucleotidase and a 5'-nucleotidase. The 5'-nucleotidase can be configured to cleave the phosphate from the 5'-terminus of a phosphate-containing molecule (e.g., a nucleotide), while the 3'-nucleotidase can be configured to cleave the phosphate from the 3'-terminus of a phosphate-containing molecule (e.g., a nucleotide).

[0053] The phosphoantigen inhibitor (e.g., hydrolase) can include an ectonucleotidase. In some cases, the ectonucleotidase can be expressed on the plasma membrane of a cell. At least a portion of the active site (e.g., catalytic site) of the ectonucleotidase can be on the extracellular domain of such a transmembrane ectonucleotidase. The phosphoantigen inhibitor can include a hydrolase (e.g., an ectonucleoside triphosphate diphosphohydrolase (NTPDase), e.g., NTPDase1 / ENTPD1 / CD39), a phosphodiesterase (e.g., a nucleotide pyrophosphatase / phosphodiesterase (NPP)), an alkaline phosphatase, a functional variant thereof, or a combination thereof. Examples of NTPDases include NTPDase1 / ENTPD1 / CD39, NTPDase2 / ENTPD2 / CD39L1, NTPDase3 / ENTPD3 / CD39L3, NTPDase4 / ENTPD4 / LALP70, NTPDase5 / ENTPD5 / CD39L4, NTPDase6 / ENTPD6 / CD39L2, NTPDase7 / ENTPD7 / LALP1, NTPDase8 / ENTPD8, functional modifications thereof, or combinations thereof.

[0054] The terms "cell death" or "cell death," as used interchangeably herein, can refer to a process or event that causes a cell to cease and / or attenuate 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 or different type). In some cases, cell death can include, but is not limited to, programmed cell death (i.e., apoptosis), gradual death of a cell such as occurs in disease states (i.e., necrosis), and more immediate cell death, such as toxicity (e.g., cytotoxicity, e.g., acute cytotoxicity). In some cases, cell apoptosis can be extrinsic (e.g., via signaling through cell surface receptors, e.g., death receptors) or intrinsic (e.g., via the mitochondrial pathway).

[0055] In some cases, cell death may be caused, initiated, and / or prolonged by a compound. In some cases, such compounds may be referred to as death-inducing compounds (e.g., death-inducing polypeptides or proteins). In some cases, death-inducing compounds may include apoptosis-inducing compounds and / or cytotoxic compounds. Death-inducing compounds may include small molecules (e.g., lipids, sugars, etc.), one or more nucleic acids (e.g., single nucleic acids, polynucleotides, etc.), one or more amino acids (e.g., single amino acids, peptides, proteins, polypeptides, etc.), and / or combinations thereof. Death-inducing compounds may be endogenous and / or exogenous to the cell undergoing cell death. Death-inducing compounds may be synthetic or natural. Death-inducing compounds may be intracellular proteins, transmembrane proteins (e.g., cell surface receptors), and / or extracellular proteins. Death-inducing compounds may be at least one receptor (e.g., an individual receptor or a complex of multiple receptors), at least one ligand of at least one receptor, and / or a combination of at least one receptor and at least one ligand. In some cases, the death-inducing compound may activate the cell to express the same and / or a different compound that can cause or prolong death of that cell or another cell. Alternatively, or in addition, the death-inducing compound may activate or promote death-related (e.g., apoptotic) intracellular signaling, such as a cellular apoptosis pathway. The death-inducing compound can include an apoptosis regulator, e.g., a pro-apoptotic regulator.

[0056] In some cases, cell death can be slowed, stopped, and / or prevented by an inhibitor. Such an inhibitor can be referred to as a cell death inhibitor. A cell death inhibitor can include a small molecule (e.g., a lipid, a sugar, etc.), one or more nucleic acids (e.g., a single nucleic acid, a polynucleotide, etc.), one or more amino acids (e.g., a single amino acid, a peptide, a protein, a polypeptide, etc.), and / or a combination thereof. In some cases, a cell death inhibitor can be an inhibitor of a death-inducing compound (e.g., a death-inducing protein). A cell death inhibitor can interfere with the production (e.g., synthesis, metabolism, etc.) of a death-inducing compound. A cell death inhibitor can bind to, transform, and / or degrade a death-inducing compound, thereby interfering with the function of the death-inducing compound. In some cases, a cell death inhibitor can bind to or degrade a cell death activator (e.g., a protease), thereby slowing, stopping, and / or preventing cell death.

[0057] The cell death inhibitor can include an apoptosis regulator, e.g., an anti-apoptosis regulator. In some cases, the cell death inhibitor can reduce or prevent the formation of the death-inducing signaling complex (DISC) and subsequent activation of the pro-caspase and / or caspase cascade. In some cases, the cell death inhibitor can suppress tumor necrosis factor-α (TNF-α), Fas / Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and / or apoptosis triggered by a death inducer (e.g., a chemotherapeutic agent) in tumor / cancer cells. In some cases, the cell death inhibitor can include cellular FLICE (FADD-like interleukin (IL)-1β-converting enzyme)-inhibitory protein (c-FLIP / CFLAR / CASH), or a functional variant thereof. c-FLIP can be expressed in cells (e.g., human cells) as long (c-FLIP(L)), short (c-FLIP(S)), and / or c-FLIP(R) splice variants.

[0058] In one example, the cell death inhibitor can be a serpin superfamily polypeptide that (i) binds to a protease (e.g., a cell death-inducing serine protease) and (ii) induces a large conformational change in the target protease, disrupting its active site. Examples of the serpin superfamily can include, for example, serpin B1, serpin B2, serpin B3, serpin B4, serpin B6, serpin B8, serpin B9, alpha 1-antitrypsin, angiotensinogen, ovalbumin, antiplasmin, alpha 1-antichymotrypsin, thyroxine-binding protein, complement 1 inactivator, antithrombin III, heparin cofactor II, plasminogen inactivator, gene Y protein, placental plasminogen activator inhibitor, barley Z protein, functional variations thereof, or combinations thereof. In some cases, one or more members of the serpin family may be substrates rather than inhibitors of serine endopeptidases.

[0059] In some cases, the cell death inhibitor can upregulate one or more cytoprotective and / or pro-survival signaling proteins, such as Akt, ERK, NF-kB, etc. In one example, the cell death inhibitor can include c-Flip or a functional variant thereof.

[0060] The death-inducing compound (e.g., a death-inducing protein) can include the tumor necrosis factor receptor superfamily (TNFRSF) and / or one or more respective ligands of the TNFRSF, such as one or more tumor necrosis factors (TNFs). The TNFRSF can be a transmembrane protein or a soluble protein (e.g., a cleaved transmembrane protein, e.g., TNFR1, or one that naturally lacks a transmembrane domain, e.g., DcR3). In some cases, the TNFRSF can include one or more TNFRSF adaptor proteins, such as TNF receptor type 1-associated death domain protein (TRADD), TNF receptor-associated factors (TRAFs), receptor-interacting protein kinases (RIPs) (e.g., RIPK1, RIPK2, RIPK3, RIPK4, RIPK5, etc.), and Fas-associated protein with a death domain (FADD / MORT1).

[0061] In some cases, TNFRSF can include a death domain (DD). The death domain can be an interaction module (e.g., a protein interaction module) consisting of one or more secondary structures (e.g., a bundle of six alpha helices). TNFRSFs that include a death domain can be referred to as death receptors. Examples of death receptors include TNFR1, Fas receptor, DR4, and / or DR5.

[0062] TNFRSFs can include tumor necrosis factor receptor 1 (i.e., CD120a), whose ligand can include TNF-alpha (cachectin). TNFRSFs can include tumor necrosis factor receptor 2 (i.e., CD120b), whose ligand can include TNF-alpha (cachectin). TNFRSFs can include lymphotoxin beta receptor (i.e., CD18), whose ligand can include lymphotoxin beta (TNF-C). TNFRSFs can include OX40 (i.e., CD134), whose ligand can include OX40L. TNFRSFs can include CD40 (i.e., Bp50), whose ligand can include CD154. TNFRSFs can include Fas receptors (i.e., Apo-1, CD95), whose ligand can include Fas ligand (FasL). TNFRSFs can include decoy receptor 3 (i.e., TR6, M68), and their ligands can include FasL, LIGHT, and / or TL1A. TNFRSFs can include CD27 (i.e., S152, Tp55), and their ligands can include CD70 and / or Siva. TNFRSFs can include CD30 (i.e., Ki-1), and their ligands can include CD153. TNFRSFs can include 4-1BB (i.e., CD137), and their ligands can include 4-1BB ligand. TNFRSFs can include death receptor 4 (i.e., TRAILR1, Apo-2, CD261), and their ligands can include TRAIL. TNFRSFs can include death receptor 5 (i.e., TRAILR2, CD262), and their ligands can include TRAIL. TNFRSFs can include decoy receptor 1 (ie, TRAILR3, LIT, TRID, CD263), and their ligands can include TRAIL.TNFRSFs can include decoy receptor 2 (i.e., TRAILR4, TRUNDD, CD264), and their ligands can include TRAIL. TNFRSFs can include RANK (i.e., CD265), and their ligands can include RANKL. TNFRSFs can include osteoprotegerin (i.e., OCIF, TR1), and their ligands can include RANKL. TNFRSFs can include TWEAK receptors (i.e., Fn14, CD266), and their ligands can include TWEAK. TNFRSFs can include TACI (i.e., IGAD2, CD267), and their ligands can include APRIL, BAFF, and / or CAMLG. TNFRSFs can include BAFF receptors (i.e., CD268), and their ligands can include BAFF. TNFRSFs can include herpesvirus entry mediators (i.e., ATAR, TR2, CD270), and their ligands can include LIGHT. TNFRSFs can include nerve growth factor receptors (i.e., p75NTR, CD271), and their ligands can include NGF, BDNF, NT-3, and / or NT-4. TNFRSFs can include B cell maturation antigens (i.e., TNFRSF13A, CD269), and their ligands can include BAFF. TNFRSFs can include glucocorticoid-inducible TNFR-related (i.e., AITR, CD357), and their ligands can include GITR ligands. TNFRSFs can include TROY (i.e., TAJ, TRADE). TNFRSFs can include death receptor 6 (i.e., CD358). TNFRSFs can include death receptor 3 (ie, Apo-3, TRAMP, LARD, WS-1), and their ligands can include TL1A.The TNFRSF can include the ectodysplasin A2 receptor (ie, XEDAR), and its ligand can include EDA-A2.

[0063] Death-inducing compounds (e.g., apoptosis-inducing compounds) can include granzymes, perforin, defensins, one or more components of the membrane invasion complex (MAC) or terminal complement complex (TCC), Bcl-2 family members, cytochrome C, caspases, human leukocyte antigen (HLA) complexes, or combinations thereof. Granzymes can be proteases (e.g., serine proteases) that induce programmed cell death (e.g., apoptosis) in target cells. In some cases, granzymes can activate one or more proteins (e.g., caspases, Bid, etc.) that are configured to execute cell death. Examples of granzymes can include granzyme A, granzyme B, granzyme C, granzyme D, granzyme E, granzyme F, granzyme G, granzyme H, granzyme I, granzyme J, granzyme K, granzyme L, granzyme M, granzyme N, or combinations thereof. Perforin can be a pore-forming polypeptide that binds to and oligomerizes (e.g., in a calcium-dependent manner) with the plasma membrane of a target cell to form a pore on the plasma membrane of the target cell. Examples of perforins can include perforin-1, perforin-2, or a combination thereof. Defensins can be cationic polypeptides (e.g., cysteine-rich cationic polypeptides) that bind to and disrupt the plasma membrane of a target cell. Examples of defensins can include alpha-defensins, beta-defensins, theta-defensins, or combinations thereof. The Bcl-2 family can include proteins that share a Bcl-2 homology (BH) domain. Examples of Bcl-2 family members can include Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Hrk, Bok, or combinations thereof. Examples of caspases include caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, their respective procaspases, or combinations thereof.An HLA complex can encode one or more major histocompatibility complex (MHC) proteins. An HLA complex can include HLA class I, HLA class II, HLA class III, functional variants thereof, or combinations thereof. HLA class I can include HLA-A, HLA-B, HLA-C, functional variants thereof, or combinations thereof. HLA class II can include HLA-DP, HLA-DM, HLA-DO, HLA-DQ, HLA-DR, functional variants thereof, or combinations thereof. HLA class III can include other immune components, such as complement components (e.g., C2, C4, factor B, etc.), cytokines (e.g., TNF-α), heat shot proteins (hsp), functional variants thereof, or combinations thereof.

[0064] In some cases, a family of compounds (e.g., a family of proteins) can include both pro-cell death (e.g., pro-apoptotic) and anti-cell death (e.g., anti-apoptotic) proteins. In one example, the Bcl-2 family can include both pro-apoptotic proteins (e.g., Bik, Bid, Bim, Bad, Bak, Bax, Bcl-Xs, Diva, Egl-1, Noxa, etc.) and anti-apoptotic proteins (e.g., Bcl-2, BC1-XL, Mcl-1, CED-9, A1, Bfl-1, etc.).

[0065] As used herein, the term "fratricide" can refer to the observation that antigens and / or receptors associated with a disease (e.g., cancer or tumor) can be present on effector cells (e.g., lymphocytes, e.g., T lymphocytes) configured to target one or more diseased cells, in addition to diseased cells (e.g., cancer or tumor cells). In such cases, the effector cells can target each other in addition to, or instead of, targeting the diseased cells. In some cases, an effector cell can target additional effector cells of the same or different type and induce a biological response, e.g., cell death, in the additional effector cells. Such a side effect of inducing a biological response in additional effector cells can also be referred to as fratricide. In some cases, the terms "fratricide," "self-directed injury," and "killing of a cell by another cell of the same type" can be used interchangeably herein. In one example, the effector cell can be a γδ T cell and the diseased cell can be a cancer / tumor cell.

[0066] In one example, the effector cells may be γδ T cells and the disease cells may be cancer / tumor cells. The cancer / tumor cells may express cell surface receptors (e.g., Fas / CD95 / APO-1 / APT1), and the γδ T cells may express respective cell surface ligands (e.g., FasL / CD95L / APTL) that are configured to bind to and target the cell surface receptors of the cancer / tumor cells, resulting in cell death (e.g., apoptosis) of the cancer / tumor cells. However, γδ T cells may also express both receptors and ligands (e.g., Fas and FasL), which may result in interaction between one γδ T cell and another γδ T cell via receptor-ligand binding, resulting in cell death (e.g., apoptosis) of one or both of the γδ T cells.

[0067] As used herein, the term "antigen" refers to a molecule or fragment thereof (e.g., a ligand) that can be bound by a selective binding agent. As one 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). Antigen also refers to a molecule or fragment thereof that can be used in an animal to produce antibodies that can bind to that antigen.

[0068] As used herein, the term "antibody" refers to a proteinaceous binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies) and variants thereof. Antibodies include, but are not limited to, the various immunoglobulin (Ig) classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). Variants can refer to functional derivatives or fragments that retain the binding specificity (e.g., fully and / or partially) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), minibodies, diabodies, and single-domain antibodies ("sdAb" or "nanobody" or "camelid"). The term antibody includes optimized, engineered, or chemically conjugated antibodies and antigen-binding fragments of antibodies. Examples of optimized antibodies include affinity-matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (eg, antibodies optimized in fragment crystallizable regions) and multispecific antibodies (eg, bispecific antibodies).

[0069] As used herein, the term "Fc receptor" or "FcR" generally refers to a receptor or any variant thereof that can bind to the Fc region of an antibody. In certain embodiments, FcRs bind IgG antibodies (gamma receptors, FcgammaR) and include receptors of the FcgammaRI (CD64), FcgammaRII (CD32), and FcgammaRIII (CD16) subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcgammaRII receptors include FcgammaRIIA (an "activating receptor") and FcγRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The term "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus.

[0070] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include the ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term "nucleotide" can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Labeling can also be performed using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, California; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Illinois; Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP available from Mannheim, Indianapolis, Ind.; and Molecular Examples of chromosomally labeled nucleotides available from Probes, Inc., Eugene, Oregon include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides can also be labeled or marked by chemical modification.The chemically modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotin-labeled dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0071] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in either 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 a 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 contain one or more analogs (e.g., altered backbones, sugars, or nucleobases). 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 acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., sugar-linked rhodamine or fluorescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queusine, and wiosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, multiple loci (locuses) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers.The sequence of nucleotides may be interrupted by non-nucleotide components.

[0072] As used herein, the term "gene" refers to nucleic acids (e.g., DNA, e.g., genomic DNA and cDNA) and their corresponding nucleotide sequences that are involved in encoding an RNA transcript. As used herein with reference to genomic DNA, the term includes intervening, non-coding, and regulatory regions, and can include the 5' and 3' ends. In some uses, the term encompasses the transcribed sequence, including the 5' and 3' untranslated regions (5'-UTP and 3'-UTP), exons, and introns. In some genes, the transcribed region contains an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only the coding sequence (e.g., an "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, a gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" not only includes the transcribed sequence, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene" or native gene in its natural location in the genome of an organism. A gene can refer to an "exogenous gene" or non-native gene. A non-native gene can refer to a gene not normally found in a host organism but that has been introduced into the host organism by gene transfer (e.g., a transgene). A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-native sequence) that contains mutations, insertions, and / or deletions.

[0073] As used herein, the terms "target polynucleotide" and "target nucleic acid" refer to a nucleic acid or polynucleotide targeted by an actuator moiety of the present disclosure. A target polynucleotide can be DNA (e.g., endogenous or exogenous). DNA can refer to a template for generating an mRNA transcript and / or various regulatory regions that regulate transcription of mRNA from a DNA template. A target polynucleotide can be a portion of a larger polynucleotide, e.g., 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. The RNA can be, for example, an mRNA that can serve as a template encoding a protein. Target polynucleotides comprising RNA can include various regulatory regions that regulate protein translation from an mRNA template. A target polynucleotide can encode a gene product (e.g., DNA encoding an RNA transcript or RNA encoding a protein product) or can include regulatory sequences that regulate expression of a gene product. Generally, 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, etc. When targeted by an actuator portion, the target polynucleotide can result in altered gene expression and / or activity. When targeted by an actuator portion, the target polynucleotide can result in an edited nucleic acid sequence. The target nucleic acid can include a nucleic acid sequence that may not be related to any other sequence in a nucleic acid sample by a single nucleotide substitution. The target nucleic acid can include a nucleic acid sequence that may not be related to any other sequence in a nucleic acid sample by 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 the 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 the target nucleic acid.

[0074] The terms "transfection" or "transfected" refer to the introduction of nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecule can be a gene sequence encoding a complete protein or a functional portion thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0075] The term "expression" refers to the process or processes by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. "Up-regulated," when referring to expression, generally refers to an increase in the level of expression of a polynucleotide (e.g., RNA, e.g., mRNA) and / or polypeptide sequence compared to its expression level in the wild-type state, while "down-regulated" generally refers to a decrease in the level of expression of a polynucleotide (e.g., RNA, e.g., mRNA) and / or polypeptide sequence compared to its expression in the wild-type state.

[0076] As used herein, the term "vector" can refer to a nucleic acid molecule capable of transferring or transporting a payload nucleic acid molecule. The payload nucleic acid molecule is generally linked to, e.g., inserted into, a vector nucleic acid molecule. A vector can include sequences that direct autonomous replication in a cell or can include sequences sufficient to allow integration into host cell genomes (e.g., host cell DNA). Examples of vectors include, but are not limited to, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0077] As used herein, "plasmid" generally refers to a non-viral expression vector, e.g., a nucleic acid molecule encoding a gene and / or regulatory elements necessary for the expression of a gene. As used herein, "viral vector" generally refers to a nucleic acid derived from a virus that is capable of transporting another nucleic acid into a cell. When present under the appropriate circumstances, a viral vector is capable of directing the expression of a protein or proteins encoded by one or more genes carried by the vector. Examples of viral vectors include, but are not limited to, gamma-retrovirus, alpha-retrovirus, foamy virus, lentivirus, adenovirus, or adeno-associated virus vectors.

[0078] The vectors of any of the embodiments of the present disclosure can include exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An "endogenous" regulatory sequence is one that is naturally linked to a given gene in the genome. An "exogenous" regulatory sequence is one that has been placed in proximity to a gene by genetic engineering (i.e., molecular biological techniques) so that transcription of the gene is directed by the linked enhancer / promoter. A "heterologous" regulatory sequence is an exogenous sequence that is from a different species than the cell being genetically engineered. A "synthetic" regulatory sequence may further include elements of one endogenous and / or exogenous sequence and / or sequences determined in vitro or in silico to provide optimal promoter and / or enhancer activity for a particular gene therapy.

[0079] As used herein, the terms "complement," "multiple complements," "complementary," and "complementarity" generally refer to a sequence that is perfectly complementary to and hybridizable with a given sequence. In some cases, a sequence hybridized to a given nucleic acid is said to be the "complement" or "reverse complement" of the given molecule when its base sequence over a given region is capable of complementary binding with that of its binding partner, e.g., so that AT, AU, GC, and GU base pairs are formed. Generally, a first sequence capable of hybridizing to a second sequence is capable of specifically or selectively hybridizing to the second sequence such that hybridization to the second sequence or set of second sequences during a hybridization reaction is more favorable (e.g., thermodynamically more stable under a given set of conditions, e.g., stringent conditions commonly used in the art) than hybridization to a non-target sequence. Typically, hybridizable sequences share a degree of sequence complementarity over all or a portion of their respective lengths, e.g., 25% to 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, for example for purposes 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 using default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally using 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 using default settings). Optimal alignment can be assessed using any suitable parameters, including default parameters, of the selected algorithm.

[0080] 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 all bases in the duplex are bound to complementary bases by Watson-Crick pairing. Substantial or sufficient complementarity can mean that the sequence in one strand is not completely and / or perfectly complementary to the sequence in the opposite strand, but that sufficient binding occurs between bases on the two strands under a set of hybridization conditions (e.g., salt concentration and temperature) to form a stable hybrid complex. Such conditions can be predicted by predicting the Tm of the hybridized strands using the sequences and standard mathematical calculations, or by empirical determination of the Tm using routine methods.

[0081] As used herein, the term "modulating" in reference to expression or activity refers to altering expression levels or activity. Modulation can occur at the transcriptional level, post-transcriptional level, translational level, and / or post-translational level.

[0082] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymer of at least two amino acid residues joined by peptide bonds. The terms do not imply a specific length of the polymer, nor are they 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 and amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may 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 amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation, such as conjugation with a labeling moiety. As used herein, the terms "amino acid" and "amino acids" generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include chemically modified natural and unnatural amino acids, including groups or chemical moieties that do not naturally occur on amino acids. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0083] The term "variant," as used herein with reference to a polypeptide, refers to a polypeptide that is related to a wild-type polypeptide but is not identical, for example, by any of amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Variants include polypeptides that contain one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide. Variants also include derivatives of wild-type polypeptides and fragments of wild-type polypeptides.

[0084] As used herein, the term "percent (%) identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in 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 ignored for comparison purposes). Alignment for purposes of determining percent identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. The 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 at the same positions in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0085] As used herein, the term "gene-modulating polypeptide" or "GMP" refers to a polypeptide that includes at least an actuator portion that is capable of regulating the expression or activity of a gene and / or editing a nucleic acid sequence. A GMP can include additional peptide sequences that are not directly involved in modulating gene expression, such as targeting sequences, polypeptide folding domains, etc.

[0086] As used herein, the term "actuator moiety" refers to a moiety, whether exogenous or endogenous, that can regulate gene expression or activity and / or edit a nucleic acid sequence. An actuator moiety can regulate gene expression at the transcriptional, post-transcriptional, translational, and / or post-translational levels. An actuator moiety can regulate gene expression at the transcriptional level, for example, by regulating the production of mRNA from DNA, e.g., 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 regulate transcription by binding to DNA and physically interfering, e.g., preventing proteins, e.g., RNA polymerase and other associated proteins, from assembling on a DNA template. An actuator moiety can regulate gene expression at the translational level, for example, by regulating the production of a protein from an mRNA template. In some embodiments, an actuator moiety regulates gene expression at the post-transcriptional level by affecting the stability of mRNA transcripts. In some embodiments, the actuator moiety regulates gene expression at a post-translational level by modifying a polypeptide, e.g., altering the glycosylation of a newly synthesized protein. In some embodiments, the actuator moiety regulates gene expression by editing a nucleic acid sequence (e.g., a region of a genome). In some embodiments, the actuator moiety regulates gene expression by editing an mRNA template. In some cases, editing a nucleic acid sequence can alter the underlying template for gene expression.

[0087] A Cas protein, as 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 homolog of a Cas protein. A Cas protein can be codon-optimized. A Cas protein can be a codon-optimized homolog of a Cas protein. A Cas protein can be enzymatically inactive, partially active, constitutively active, fully active, inducibly active, and / or more active (e.g., than a wild-type homolog 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 Cas13a. A Cas protein (e.g., a variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive site-directed polypeptide) can bind to a target nucleic acid. Cas proteins (e.g., variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endoribonucleases) can bind to target RNA or DNA.

[0088] The terms "deactivated nuclease" or "dead nuclease," as used interchangeably herein, can refer to a nuclease in which the nuclease function is fully or partially deactivated. In cases where the nuclease is a Cas protein, the deactivated / dead Cas nuclease can be referred to as "dCas" (e.g., dCas9).

[0089] As used herein, the term "crRNA" can generally refer to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary crRNA (e.g., crRNA from S. pyogenes, S. aureus, etc.). A crRNA can generally refer to a nucleic acid having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary crRNA (e.g., crRNA from S. pyogenes, S. aureus, etc.). A crRNA can also refer to modified forms, variants, mutations, or chimeras of crRNA, which can include nucleotide changes, e.g., deletions, insertions, or substitutions. The crRNA can be a nucleic acid having at least about 60% sequence identity to a wild-type exemplary crRNA sequence (e.g., crRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over a stretch of at least six contiguous nucleotides. For example, the 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., crRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over a stretch of at least six contiguous nucleotides.

[0090] As used herein, the term "tracrRNA" generally refers to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.). A tracrRNA can refer to a nucleic acid having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.). A tracrRNA can also refer to modified forms, variants, mutations, or chimeras of tracrRNA, which may include nucleotide changes, e.g., deletions, insertions, or substitutions. 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 Streptococcus pyogenes, Staphylococcus aureus, etc.) sequence over a stretch of at least six 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 Streptococcus pyogenes, Staphylococcus aureus, etc.) sequence over a stretch of at least six contiguous nucleotides.

[0091] As used herein, a "guide nucleic acid" can refer to a nucleic acid that can hybridize with another nucleic acid. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. A guide nucleic acid can be programmed to site-specifically bind to a nucleic acid sequence. A targeted nucleic acid, or target nucleic acid, can include nucleotides. A guide nucleic acid can include nucleotides. A portion of a target nucleic acid can be complementary to a portion of a guide nucleic acid. A strand of a double-stranded target polynucleotide that is complementary to and hybridizes with a guide nucleic acid can be referred to as a complementary strand. A strand of a double-stranded target polynucleotide that is complementary to a complementary strand and therefore may not be complementary to the guide nucleic acid can be referred to as a non-complementary strand. A guide nucleic acid can include one polynucleotide strand and can be referred to as a "single guide nucleic acid." A guide nucleic acid can include two polynucleotide strands and can be referred to as a "dual guide nucleic acid." Unless otherwise specified, the term "guide nucleic acid" is inclusive and can refer to both single and dual guide nucleic acids.

[0092] A guide nucleic acid can include 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 include a sub-segment that can be referred to as a "protein binding segment" or a "protein binding sequence" or a "Cas protein binding segment."

[0093] The term "cleavage recognition sequence" or "cleavage recognition site," as used herein in reference to a peptide, refers to a site in a peptide that can cleave a chemical bond, e.g., a peptide bond or a disulfide bond. Cleavage can be achieved by a variety of methods. Cleavage of a peptide bond can be facilitated, for example, by an enzyme, e.g., a protease.

[0094] As used herein, the term "targeting sequence" refers to a nucleotide sequence and corresponding amino acid sequence that encodes a targeting polypeptide that mediates the localization (or retention) of a protein to a subcellular location, such as the plasma membrane or membrane of a given organelle, the nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi, chloroplast, apoplast, peroxisome, or other organelle. For example, a targeting sequence can target a protein (e.g., GMP) to the nucleus using a nuclear localization signal (NLS), out of the nucleus of a cell, e.g., to the cytoplasm, using a nuclear export signal (NES), to mitochondria using a mitochondrial targeting signal, to the endoplasmic reticulum (ER) using an ER retention signal, to peroxisomes using a peroxisomal targeting signal, to the plasma membrane using a membrane localization signal, or a combination thereof.

[0095] As used herein, a "fusion" can refer to a protein and / or nucleic acid that includes one or more non-native sequences (e.g., moieties). A fusion can include one or more of the same non-native sequences. A fusion can include one or more different non-native sequences. A fusion can be chimeric. A fusion can include a nucleic acid affinity tag. A fusion can include a barcode. A fusion can include a peptide affinity tag. A fusion can provide subcellular localization of a site-specific polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, an endoplasmic reticulum (ER) retention signal, etc.). A fusion can provide a non-native sequence (e.g., an affinity tag) that can be used for tracking or purification. A fusion can be to a small molecule, such as biotin, or a dye, such as an Alexa fluor dye, a cyanine 3 dye, or a cyanine 5 dye.

[0096] A fusion can refer to any protein that has a functional effect. For example, the fusion protein can comprise a methyltransferase activity, a demethylase activity, a dismutase activity, an alkylating activity, a depurinating activity, an oxidizing activity, a pyrimidine dimer-forming activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity, or a glycosylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, a remodeling activity, a protease activity, an oxidoreductase activity, a transferase activity, a hydrolase activity, a lyase activity, an isomerase activity, a synthetase activity, a synthetase activity, or a demyristoylating activity. The effector protein can modify a genomic locus. The fusion protein can be a fusion in a Cas protein. The fusion protein can be a non-native sequence in a Cas protein.

[0097] Thus, in some embodiments, the actuator portion may comprise a fusion polypeptide. The fusion polypeptide can comprise two or more fragments, each conferring at least one activity selected from the group consisting of: nuclease activity, methyltransferase activity, demethylase activity, dismutase activity, alkylating activity, depurinating activity, oxidizing 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, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthetase activity, synthetase activity, and demyristoylating activity.

[0098] In some cases, the actuator portion may comprise a fusion polypeptide, which may include two fragments that confer (i) nuclease activity (or a modification thereof, e.g., Cas activity or reduced Cas activity), and (ii) hydrolase activity (e.g., cytidine deaminase activity), respectively. In some cases, the actuator portion comprising the fusion polypeptide may be a nucleobase editor. The terms "nucleobase editor" or "base editor," as used interchangeably herein, may refer to an agent that includes a polypeptide that can make modifications to nucleobases (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some cases, a base editor (e.g., a deaminase) may be capable of deaminating a base within a nucleic acid. In some cases, a base editor may be capable of deaminating a base within a DNA molecule. In some cases, a base editor may be capable of deaminating cytosine (C) in DNA. In some cases, a base editor may be capable of excising a base within a DNA molecule. In some cases, the base editor may be capable of excising 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 provided herein, e.g., Cas or dCas) fused to a cytidine deaminase. In some cases, the base editor may be fused to a uracil binding protein (UBP), e.g., 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).

[0099] In some cases, base editors can introduce one or more transition mutations (e.g., C to T, G to A, A to G, or T to C) without the need for a double-strand break in many cell types and organisms, including mammals.

[0100] In some cases, the actuator portion may comprise a fusion polypeptide, which may comprise two fragments that confer, respectively, (i) nuclease activity (or a modification thereof, e.g., Cas activity or reduced Cas activity), and (ii) polymerase activity (e.g., DNA or RNA polymerase activity). As used herein, the term "polymerase" may refer to a polypeptide that can catalyze the 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 one example, a DNA insert sequence encoded by a template RNA molecule may be added to the 3' end of a target DNA molecule by the action of a polymerase (e.g., reverse transcriptase). Examples of polymerases 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, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, (iii) T7, T3, SP6 RNA polymerase, and (iv) AMV, M-MLV, and HIV reverse transcriptase.

[0101] In some examples, the actuator moiety may comprise a fusion polypeptide, which may comprise (i) a Cas protein or a modification thereof (e.g., a non-activated Cas or a Cas nickase) coupled (e.g., covalently coupled) with (ii) a reverse transcriptase. The Cas protein may be configured to nick only 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 strand) by coping from a nucleic acid template (e.g., an RNA template). Such an actuator moiety may function in concert with an engineered gRNA (i.e., a prime editing gRNA, or pegRNA). The pegRNA may comprise multiple segments. The multiple segments can include (i) a nucleic acid targeting segment (e.g., a 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 the desired nucleic acid edit, and (iv) a binding segment that binds to the nicked strand of the target nucleic acid. In one example, the reverse transcriptase template segment of the pegRNA can encode a desired DNA sequence. Alternatively, the reverse transcriptase template segment of the pegRNA can encode a complementary DNA sequence that has complementarity to a desired DNA sequence, such that when the complementary DNA sequence is introduced into the first strand of the target gene, the desired DNA sequence can subsequently be added to the second, opposite strand of the target gene (e.g., via one or more DNA repair mechanisms).

[0102] In one example, a fusion complex of (i) an actuator moiety comprising a Cas protein and a reverse transcriptase and (ii) a pegRNA can introduce one or more transition mutations (e.g., C to T, G to A, A to G, or T to C) without the need for a double-strand break in many cell types and organisms, including mammals. Alternatively, or in addition, such a fusion complex can make 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) without the need for a double-strand break in many cell types and organisms, including mammals, for example, for a TA to AT mutation required to correct sickle cell disease. Alternatively, or in addition, such a fusion complex can introduce indels (e.g., insertions and / or deletions) into a target nucleic acid or target gene. The fusion complex may introduce 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 nucleotide additions into the target gene. The fusion complex may introduce up to 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 additions into 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 into the target gene. The fusion complex may introduce a deletion of up to 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 nucleotides into the target gene. The fusion complex may or may not introduce a frameshift into the gene.

[0103] In some cases, the engineered gRNA (e.g., 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: a nuclease activity, a methyltransferase activity, a demethylase activity, a dismutase activity, an alkylating activity, a depurinating activity, an oxidizing activity, a pyrimidine dimer forming activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, or a helicase activity. activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthetase activity, synthetase activity, and demyristoylating activity. In one example, a pegRNA can be operably coupled to a nucleic acid polymerase (e.g., reverse transcriptase) by the action of the nucleic acid polymerase recognizing and non-covalently binding to a fragment (e.g., a loop structure) of the pegRNA. In such cases, the nucleic acid polymerase may or may not be covalently coupled to a nuclease (e.g., a Cas protein or dCas protein).

[0104] As used herein, "non-native" can refer to a nucleic acid or polypeptide sequence not found in a native nucleic acid or protein. Non-native can refer to an affinity tag. Non-native can refer to a fusion. Non-native can refer to a naturally occurring nucleic acid or polypeptide sequence that includes mutations, insertions, and / or deletions. A non-native sequence may exhibit and / or encode an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) that may 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 can be linked by genetic engineering to a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) to produce a chimeric nucleic acid and / or polypeptide sequence that encodes a chimeric nucleic acid and / or polypeptide.

[0105] The terms "subject," "individual," and "patient" are used interchangeably herein and 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. Also included are tissues, cells, and the progeny thereof of a biological entity obtained in vivo or cultured in vitro.

[0106] As used herein, the terms "treatment" and "treating" refer to an approach for obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. For example, treatment can include administering a system or cell population disclosed herein. A therapeutic benefit refers to any therapeutically relevant improvement in, or effect on, one or more diseases, conditions, or symptoms under treatment. A prophylactic benefit includes administering a composition to a subject at risk of developing a particular disease, condition, or symptom, or reporting one or more physiological symptoms of a disease, even when the disease, condition, or symptom may not yet be manifest.

[0107] The term "effective amount" or "therapeutically effective amount" refers to the amount of a composition comprising a system of the present disclosure, e.g., a composition comprising immune cells, e.g., lymphocytes (e.g., T lymphocytes and / or NK cells), that is sufficient to produce a desired activity when administered to a subject in need thereof. Within the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition that is sufficient to delay the onset of, arrest the progression of, relieve or alleviate at least one symptom of a disorder treated by a method of the present disclosure.

[0108] The term "chimeric antigen receptor" or "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 endogenous signaling domain") comprising a functional signaling domain derived from a stimulatory molecule. In some cases, the stimulatory molecule may be a 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-terminus) 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, where the leader sequence is optionally cleaved from the antigen-recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. In some cases, the CAR may further comprise a GMP as described in this disclosure.

[0109] As used herein, a CAR may be a first-, second-, third-, or fourth-generation CAR system, a functional variant thereof, or any combination thereof. A first-generation CAR (e.g., CD19R or CD19CAR) includes an antigen-binding domain (e.g., an antibody or antigen-binding fragment thereof, e.g., an scFv, Fab fragment, VHH domain, or the VH domain of a heavy chain-only antibody) with specificity for a particular antigen, a transmembrane domain derived from an adaptive immune receptor (e.g., a transmembrane domain from the CD28 receptor), and a signaling domain derived from an adaptive immune receptor (e.g., one or more (e.g., three) ITAM domains derived from the intracellular region of the CD3ζ receptor or FcεRIγ). A second-generation CAR modifies a first-generation CAR by adding a costimulatory domain to the intracellular signaling domain portion of the CAR (e.g., derived from a costimulatory receptor that acts with a T-cell receptor, e.g., CD28, CD137 / 4-1BB, and CD134 / OX40), which eliminates the need to administer a cofactor (e.g., IL-2) with the first-generation CAR. Third-generation CARs add multiple costimulatory domains to the intracellular signaling domain portion of the CAR (e.g., CD3ζ-CD28-OX40 or CD3ζ-CD28-41BB). Fourth-generation CARs modify second- or third-generation CARs by adding an activating cytokine (e.g., IL-12, IL-23, or IL-27) to the intracellular signaling portion of the CAR (e.g., between one or more of the costimulatory domains and the CD3ζ ITAM domain) or by placing it under the control of a CAR-inducible promoter (e.g., the NFAT / IL-2 minimal promoter).

[0110] The term "conditionally enhanced expression" refers to expression of a polypeptide sequence (e.g., an endogenous polypeptide sequence, a chimeric polypeptide sequence, etc.) that occurs under one or more conditions rather than continuously. When increasing, maintaining, and / or decreasing expression of a polypeptide sequence in a cell (e.g., an immune cell, a stem cell, etc.), the cell may be contacted with a stimulator (e.g., a ligand or an antigen) to initiate conditional enhancement of expression of the polypeptide sequence in the cell. In some cases, the cell may not have begun to express the polypeptide sequence prior to at least initial contact with the stimulator. In some cases, the cell may have begun to express the polypeptide sequence, and after expression of the polypeptide sequence has plateaued or decreased, the cell may be contacted with a stimulator to initiate conditional enhancement of expression of 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 expression of the polypeptide sequence in the cell may be temporary or permanent. In some cases, the cells may be contacted with the stimulatory agent at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some cases, the cells may be contacted with the stimulatory agent up to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time.

[0111] In some cases, continuous expression of a polypeptide sequence (e.g., Cas or dCas) may have off-target effects, e.g., cytotoxicity, on host cells. In such cases, conditionally promoted and / or enhanced expression of the polypeptide sequence (e.g., via contacting cells with a stimulant) may be beneficial, at least because the cytotoxicity may be controlled (e.g., attenuated or prevented). Alternatively, or in addition, conditionally promoted and / or enhanced expression of the polypeptide sequence may be beneficial in that the continuous metabolic burden of the host cell for synthesizing the polypeptide sequence may be controlled (e.g., attenuated or prevented). Without wishing to be bound by theory, controlling the metabolic burden of the host cell may improve the viability, growth, and / or function of the host cell.

[0112] The terms "operably linked" and "under operable control" are used interchangeably herein and can 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 functionally linked such that at least one of the sequences can affect 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 operably linked when the expression (e.g., transcription and translation) of the additional nucleotide sequence can be directed by the gene regulatory sequence. Thus, the gene regulatory sequence and the additional nucleotide sequence to be expressed can be physically linked to each other, for example, by inserting the gene regulatory sequence at or adjacent to the 5' end of the additional nucleotide sequence to be expressed. Alternatively, the gene regulatory sequence and the additional nucleotide sequence to be expressed can simply be physically proximate such that the gene regulatory sequence is functionally linked to the additional nucleotide sequence to be expressed. In some cases, two operably linked sequences may be separated by at least 5, 10, 20, 40, 60, 80, 100, 300, 500, 1500 bp, or more. In some cases, two operably linked sequences may be separated by at most 1500, 500, 300, 100, 80, 60, 40, 20, 10, 5 bp, or less.

[0113] As used herein, the term "promoter" may refer to a regulatory DNA region that controls the transcription or expression of a gene and may be located adjacent to or overlapping the nucleotide or nucleotide region at which RNA transcription is initiated. A promoter may contain specific DNA sequences that bind protein factors, often called transcription factors, which facilitate the binding of RNA polymerase to DNA, resulting in gene transcription. A "basal promoter," also known as a "core promoter," generally refers to a promoter that contains all the basic necessary elements to promote the transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters typically, but not necessarily, contain a TATA box and / or a CAAT box.

[0114] In one aspect, the present disclosure provides methods for modulating the activity of gamma-delta (γδ) cells, such as γδ T cells. In some cases, the method for modulating the activity of a γδ T cell can include administering to the γδ T cell a gene-modulating polypeptide (GMP) comprising an actuator portion configured to modulate expression of a target polynucleotide in the γδ T cell, wherein the target polynucleotide can encode a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of the death-inducing protein, and / or (iii) a hydrolase.

[0115] In some cases, administration of GMP to a γδ T cell can increase or decrease the activity of a γδ T cell by at least 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, compared to the absence of administration of GMP to the γδ T cell. In some cases, administration of GMP to a γδ T cell can increase or decrease the activity of the γδ T cell by up to 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, compared to the absence of administration of GMP to the γδ T cell.

[0116] In some cases, administration of GMP to a γδ T cell can increase or decrease the activity of a γδ T cell for at least 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, 1 year, or longer, compared to the absence of administration of GMP to the γδ T cell. In some cases, administration of GMP to a γδ T cell can increase or decrease the activity of a γδ T cell for up to 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, compared to the absence of administration of GMP to the γδ T cell.

[0117] Modulating the expression of a target polypeptide in a γδ T cell can comprise decreasing, increasing, inhibiting, and / or prolonging the expression of the target polypeptide in the γδ T cell. Modulating the expression of a target polypeptide in a γδ T cell can be decreasing the expression of the target polypeptide in the γδ T cell. Modulating the expression of a target polypeptide in a γδ T cell can be increasing the expression of the target polypeptide in the γδ T cell.

[0118] Modulating the expression of a target polypeptide in a γδ T cell can directly and / or indirectly allow for modulating the activity of a γδ T cell. In some cases, modulating the activity of a γδ T cell can include reducing and / or inhibiting one or more γδ T cell fratricide, γδ T cell self-harm, killing of a γδ T cell by another γδ T cell, and / or killing of a γδ T cell by another γδ T cell, thereby improving (directly and / or indirectly) the viability, proliferation, and / or function of the cell.

[0119] In some cases, the target polynucleotide can encode a polypeptide, and the polypeptide can include a death-inducing protein. The polypeptide can be a death-inducing protein. The death-inducing protein can include one or more of the death-inducing compounds described herein in this disclosure. Examples of death-inducing proteins can include Fas, Fas ligand, granzyme B, granzyme M, and / or perforin.

[0120] In some cases, GMP-containing γδ T cells may be operable to target diseased cells (e.g., cancer / tumor cells) and induce cell death (e.g., apoptosis) of the targeted diseased cells. The diseased cells may express cell surface receptors (e.g., Fas / CD95 / APO-1 / APT1), and the γδ T cells may express respective cell surface ligands (e.g., FasL / CD95L / APTL) that are operable to bind to and target the cell surface receptors of the diseased cells.

[0121] In some cases, upon γδ T cell-disease cell binding (e.g., via FasL-Fas binding), the γδ T cell may induce one or more intracellular signaling cascades (e.g., one or more apoptotic pathways) to induce cell death of the diseased cell. In one example, the intracellular signaling cascade may be induced via a cellular receptor (e.g., TNFR, Fas, TRAILR, etc.) of the diseased cell. Alternatively, or in addition, the γδ T cell may release one or more first compounds (e.g., proteases, e.g., granzymes), which are operable to enter the cytosol of the diseased cell (e.g., via endosomal uptake and escape) and activate one or more death-inducing compounds (e.g., caspases) within the diseased cell, thereby inducing diseased cell death. Alternatively, or in addition, γδ T cells may release one or more second compounds that are operable to bind to and form one or more pores on the plasma membrane of diseased cells, thereby lysing and killing the diseased cells. In some cases, γδ T cells may express both a first compound and a second compound, and the second compound (e.g., perforin) may form pores on the diseased cell membrane, allowing the first compound (e.g., granzyme) to pass (e.g., diffuse) into the diseased cells. The first compound and the second compound may be the same or different. The first compound and / or the second compound may be expressed and / or released by the γδ T cells before, during, and following binding of the γδ T cells to the diseased cells (e.g., via FasL-Fas binding).

[0122] In some cases, γδ T cells can express both a disease-cell-targeting cell surface ligand (e.g., FasL / CD95L / APTL) and its respective cell surface receptor (e.g., Fas / CD95 / APO-1 / APT1). Thus, γδ T cells can be operable to target and induce cell death of disease cells and / or cell death (e.g., fratricide) of other γδ T cells. In some cases, the cell surface ligand of a γδ T cell can bind to the respective cell surface receptor of the same γδ T cell and promote self-induced death of the γδ T cell. Thus, a GMP comprising an actuator moiety can be configured to modulate (e.g., reduce or inhibit) expression of a death-inducing protein (e.g., Fas / CD95 / APO-1 / APT1) in a γδ T cell, thereby reducing or inhibiting fratricide of the γδ T cell, while maintaining or increasing its cytotoxic (and / or cytolytic) activity against disease cells.

[0123] In some cases, γδ T cells can express and release death-inducing compounds operable to induce the death of diseased cells. However, the death-inducing compounds may also induce the death of the γδ T cells releasing such compounds. In one example, the concentration of the death-inducing compound in the extracellular environment of the γδ T cells and diseased cells may be sufficient to diffuse into both the diseased cells and the γδ T cells. Thus, GMPs containing actuator moieties can be configured to modulate (e.g., reduce or inhibit) the expression of death-inducing proteins (e.g., granzymes, perforins, etc.) in γδ T cells, thereby reducing or inhibiting γδ T cell fratricide.

[0124] In some cases, the target polynucleotide can encode a polypeptide, and the polypeptide can include a death-inducing protein inhibitor. The polypeptide can be a death-inducing protein inhibitor. The death-inducing protein inhibitor can include one or more of the cell death inhibitors described herein (e.g., the serpin superfamily, including serpin B1 and / or serpin B4). In some cases, the death-inducing protein inhibitor can prevent the production of, alter, and / or degrade one or more death-inducing compounds. In such cases, expression of a death-inducing protein inhibitor by a γδ T cell can protect the γδ T cell from cell death (e.g., apoptosis) caused by one or more death-inducing proteins expressed by the same or different γδ T cells. Thus, a GMP comprising an actuator moiety can be configured to regulate (e.g., induce or increase) expression of a death-inducing protein inhibitor in a γδ T cell, thereby reducing or inhibiting γδ T cell fratricide.

[0125] In some cases, modulating the activity of γδ T cells can include inducing and / or prolonging the activation of γδ T cells. Activation of γδ T cells can include activation of one or more biological activities described herein (e.g., migration, proliferation, synthesis of one or more polypeptides, etc.). Inducing / prolonging the activation of γδ T cells can include inducing and / or prolonging the expression of (1) one or more γδ T cell activators and / or (2) one or more targets of a γδ T cell activator. In one example, a γδ T cell activator can be a ligand, and the target of a γδ T cell activator can be a different polypeptide (e.g., a receptor, an intracellular protein, etc.). Alternatively, or in addition, inducing / prolonging the activation of γδ T cells can include (1) reducing and / or inhibiting the expression of one or more inhibitors of a γδ T cell activator and / or (2) degrading and / or metabolizing an inhibitor of a γδ T cell activator.

[0126] In some cases, the γδ T cell activator can be provided (e.g., synthesized) by the γδ T cell or a different type of cell (e.g., other immune cell or non-immune cell). In some cases, the γδ T cell can be exogenous to a subject (e.g., a patient) that includes the γδ T cell.

[0127] In some cases, the target polynucleotide can encode a polypeptide, and the polypeptide can comprise a hydrolase. The polypeptide can be a hydrolase. The hydrolase can include any of the hydrolases described herein in this disclosure. In some cases, the γδ T cell activator can be a phosphoantigen, and the hydrolase can include any of the phosphoantigen inhibitors described herein in this disclosure. In one example, the hydrolase can be an ectonucleoside triphosphate diphosphohydrolase (e.g., NTPDase1 / ENTPD1 / CD39), which is operable to cleave one or more phosphates in a phosphoantigen, thereby reducing or inhibiting phosphoantigen-induced activation of γδ T cells. As a result, the hydrolase, when unperturbed by a GMP of the present disclosure, can indirectly reduce the activity (e.g., cytotoxic activity) of γδ T cells against diseased cells, e.g., cancer / tumor cells.

[0128] Thus, in some cases, a GMP comprising an actuator moiety can be configured to induce and / or prolong expression of: (1) one or more γδ T cell activators; (2) one or more substrates or proforms (e.g., prodrugs) of a γδ T cell activator; and / or (3) one or more regulators (e.g., catalysts, e.g., enzymes) of the synthesis of a γδ T cell activator.

[0129] In some cases, a GMP comprising an actuator moiety can be configured to induce and / or prolong expression of (1) one or more targets of a γδ T cell activator, (2) one or more substrates or proforms of a γδ T cell activator, and / or (3) one or more regulators (e.g., catalysts, e.g., enzymes) of the synthesis of a target of a γδ T cell activator.

[0130] In some cases, a GMP comprising an actuator moiety can be configured to reduce and / or inhibit expression of one or more inhibitors (e.g., enzymes) of a γδ T cell activator, hi some cases, a GMP comprising an actuator moiety can be configured to degrade and / or metabolize an inhibitor of a γδ T cell activator.

[0131] In some cases, modulating the activity of γδ T cells can include reducing and / or preventing activation of γδ T cells.

[0132] The GMP may comprise an actuator moiety that regulates expression of a target polynucleotide in a γδ T cell. The target polynucleotide in a γδ T cell may encode a target polypeptide. In some cases, the target polypeptide may induce or inhibit immune cell proliferation, differentiation, and / or survival. In some cases, the target polypeptide may comprise (i) a death-inducing protein, (ii) an inhibitor of a death-inducing protein, and / or (iii) a hydrolase. The actuator moiety can bind to the target polynucleotide to regulate the expression and / or activity of a target gene. In some embodiments, the target polynucleotide comprises genomic DNA. In some embodiments, the target polynucleotide comprises a plasmid, e.g., a region of a plasmid carrying an exogenous gene. In some embodiments, the target polynucleotide comprises RNA, e.g., mRNA. In some embodiments, the target polynucleotide comprises an endogenous gene or gene product. An actuator moiety can comprise a nuclease (e.g., a DNA nuclease and / or an RNA nuclease), a modified nuclease (e.g., a DNA nuclease and / or an RNA nuclease) that is nuclease-deficient or has reduced nuclease activity compared to a wild-type nuclease, or a variant thereof. The actuator moiety can regulate gene expression or activity 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, e.g., 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 an RNA nuclease, e.g., an engineered (e.g., programmable or targetable) RNA nuclease, to induce editing of a target RNA sequence. In some embodiments, the actuator moiety has reduced or minimal nuclease activity.An actuator portion with reduced or minimal nuclease activity can regulate gene expression and / or activity by physically obstructing a target polynucleotide or recruiting additional factors effective to suppress or enhance expression of the target polynucleotide. The actuator portion can physically obstruct a target polynucleotide or recruit additional factors effective to suppress or enhance expression of the target polynucleotide. In some cases, the actuator portion comprises an activator effective to increase expression of the target polynucleotide. In some embodiments, the actuator portion comprises a transcriptional activator effective to increase expression of the target polynucleotide. In other cases, the actuator portion comprises a repressor effective to decrease expression of the target polynucleotide. Non-limiting examples of transcriptional activators include GAL4, VP16, VP64, p65 subdomain (NF-kappaB), and VP64-p65-Rta (VPR). In some embodiments, the actuator portion comprises a transcriptional repressor effective to decrease expression of the target polynucleotide. Non-limiting examples of transcriptional repressors include Kruippel-associated box (KRAB or SKD), Mad mSIN3-interacting domain (SID), and ERF repressor domain (ERD). In some embodiments, the actuator moiety comprises a nuclease-null DNA-binding protein derived from a DNA nuclease capable of inducing transcriptional activation or repression of a target DNA sequence. In some embodiments, the actuator moiety comprises a nuclease-null RNA-binding protein derived from an RNA nuclease capable of inducing 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, wherein the RNA-guided actuator moiety forms a complex with a target polynucleotide.Actuator moieties, whether exogenous or endogenous, can regulate gene expression or activity and / or edit nucleic acid sequences.

[0133] Any suitable nuclease can be used in the two receptor system. Suitable nucleases include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases (including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, RNA-binding proteins (RBPs), 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 variants thereof. In some cases, the actuator moiety is a CRISPR-associated (Cas) protein, or a fragment thereof that substantially lacks DNA cleavage activity (dCas). In some cases, the actuator moiety can be Cas9 and / or Cpf1.

[0134] Any target gene can be regulated by including an actuator moiety. It is contemplated that genetic homologs of the genes described herein are encompassed. For example, the gene can exhibit a particular identity and / or homology to the genes disclosed herein. Thus, it is contemplated that expression of genes exhibiting 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 genes exhibiting, or exhibiting 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 modulated.

[0135] In some cases, administering GMP to a γδ T cell can include treating the γδ T cell with a delivery vehicle, wherein the delivery vehicle comprises at least a portion of the GMP and / or a polynucleotide encoding at least a portion of the GMP. The delivery vehicle can be viral or non-viral. The at least a portion of the GMP and / or the polynucleotide encoding at least a portion of the GMP can be covalently and / or non-covalently (e.g., ionically, via hydrogen bonding, etc.) attached to the delivery vehicle. Alternatively, or in addition, the at least a portion of the GMP and / or the polynucleotide encoding at least a portion of the GMP can be encapsulated by the delivery vehicle without being physically attached to the delivery vehicle.

[0136] In some cases, the delivery vehicle may include a targeting moiety that has affinity for 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 a γδ T cell. The targeting moiety may enhance targeting and binding of the delivery vehicle to a γδ T cell. The targeting moiety may enhance intracellular entry, uptake, and / or penetration of the delivery vehicle into a γδ T cell. The targeting moiety may be linked to the external surface of the delivery vehicle (e.g., via a covalent and / or non-covalent bond). 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 protein), an antibody, or a functional fragment thereof, a functional derivative thereof, or a combination thereof.

[0137] In some cases, the delivery vehicle may not include such a targeting moiety for γδ T cells.

[0138] Examples of viral delivery vehicles may include adenovirus, retrovirus, lentivirus (e.g., human immunodeficiency virus (HIV)), adeno-associated virus (AAV), and / or herpes simplex virus (HSV). In one 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 viruses (MLV) (e.g., Friend murine leukemia virus (FMLV), Moloney murine leukemia virus (MMLV), murine type C retrovirus (TCR)), 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 (MPV). Provirus) (MMMEPP), PreXMRV-1, RD114 retrovirus, spleen focus-forming virus (SFFV), Abelson murine leukemia virus (AMLV), murine stem cell virus (MSCV), and their variants.

[0139] A delivery vehicle may comprise 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 one example, a delivery vehicle may comprise a diamond nanoparticle ("nanodiamond"), a gold nanoparticle, a silver nanoparticle, a calcium phosphate nanoparticle, or the like. A delivery vehicle may or may not comprise a fluid (e.g., a liquid or a gas). A delivery vehicle may have a variety of shapes and sizes. For example, a delivery vehicle may be in the shape of a sphere, a cube, or a disk, or any sub-shape or combination of shapes thereof. A delivery vehicle may have a cross-section that is circular, triangular, square, rectangular, pentagonal, hexagonal, or any sub-shape or combination of shapes thereof.

[0140] Examples of non-viral delivery vehicles may include nanoparticles, nanospheres, nanocapsules, microparticulates, microspheres, microcapsules, liposomes, nanoemulsions, solid lipid nanoparticles, modifications thereof, or combinations thereof. The non-viral delivery vehicles of the present invention may be prepared by, for example, but not limited to, nanoprecipitation, emulsion solvent evaporation method, emulsion cross-linking method, emulsion solvent diffusion method, microemulsion method, gas antisolvent precipitation method, ionic gelation method, milling or size reduction method, PEGylation method, salting out method, dialysis method, single or double emulsion method, nano spray drying method, layer by layer method, desolvation method, supercritical fluid technique, supramolecular assembly, or combinations thereof.

[0141] In some cases, the method may further include integrating a nucleic acid sequence (e.g., a polynucleotide) encoding GMP into the genome of the γδ T cell. In some cases, the nucleic acid sequence (e.g., a polynucleotide) encoding GMP may be integrated into the genome of the γδ T cell. Upon administration of a polynucleotide encoding at least a portion of GMP (e.g., with or without a delivery vehicle), at least a portion of the polynucleotide may be integrated into the genome of the γδ T cell. At least a portion of the integrated polynucleotide may be under the control of an autologous promoter of the γδ T cell. Alternatively, or in addition, at least a portion of the integrated polynucleotide may further comprise a promoter heterologous to the γδ T cell (heterologous promoter). The heterologous promoter may be configured to bind one or more molecules (e.g., RNA polymerase, transcription factor, etc.) that are homologous or heterologous to the γδ T cell.

[0142] The γδ T cells can be in vivo and / or ex vivo (e.g., in vitro) during treatment with a delivery vehicle comprising a payload (e.g., at least a portion of GMP and / or a polynucleotide encoding at least a portion of GMP).

[0143] In some cases, a delivery vehicle containing a payload may be injected into a body part of a subject (e.g., into a patient's vein, bone marrow, etc.), and the delivery vehicle may interact with (e.g., enter) a γδ T cell in vivo. Other examples of injection methods may include intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavernosal, and / or intravitreal.

[0144] In some cases, a subject may be injected with a dose of a delivery vehicle comprising a payload at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some cases, a subject may be injected with a dose of a delivery vehicle comprising a payload at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some cases, a subject may be injected with a dose of a delivery vehicle comprising a payload 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, a subject may be injected with a dose of a delivery vehicle comprising a payload at most once every 360, 180, 90, 60, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day.

[0145] In some cases, γδ T cells may be isolated from a subject, and the isolated γδ T cells may be treated (e.g., cultured in culture medium) with a delivery vehicle comprising a payload. The isolated γδ T cells may be allowed to proliferate or stimulated to proliferate before, during, and / or following treatment with a delivery vehicle comprising a payload. Alternatively, or in addition, cells that are not γδ T cells (e.g., stem cells, skin cells, blood cells, etc.) may be isolated from a subject, and the isolated cells may be induced to differentiate into γδ T cells, transdifferentiate into γδ T cells, and / or express a γδ TCR complex before treatment with a delivery vehicle comprising a payload. In some cases, the non-γδ T cells may first be dedifferentiated into induced pluripotent stem cells (iPSCs) before differentiation into γδ T cells and / or induction of expression of a γδ TCR complex. The isolated and treated γδ T cells may then be injected (transplanted) into the subject.

[0146] Any of the cells provided herein that have been treated with at least a payload (ex vivo and / or in vivo) and administered a GMR that includes an actuator moiety may be referred to as an engineered γδ T cell.

[0147] In some cases, such engineered γδ T cells may be injected into a body part of a subject (e.g., into a patient's vein, bone marrow, etc.), and the delivery vehicle may interact with (e.g., enter) the γδ T cells in vivo. Other examples of injection methods may include intradermal, subcutaneous, intramuscular, intravenous, intraosseous, and intraperitoneal.

[0148] In some cases, a subject may be injected with a dose of treated γδ T cells at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some cases, a subject may be injected with a dose of treated γδ T cells at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some cases, a subject may be injected with a dose of treated γδ T cells 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, a subject may be injected with a dose of treated γδ T cells at most once every 360, 180, 90, 60, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day.

[0149] In some cases, the subject receives 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(×10 9) treated γδ T cells, or more. In other cases, a subject may be injected with up to 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(×10 9 ) treated γδ T cells, or fewer, may be injected.

[0150] In some cases, GMP can be a part of chimeric polypeptide.Chimeric polypeptide can be transmembrane protein or not.In one example, chimeric polypeptide can be CAR, and GMP can be at least a part of the intracellular domain of CAR.In another example, chimeric polypeptide can be chimeric transmembrane protein, and GMP can be at least a part of the intracellular domain of chimeric transmembrane protein.In another example, chimeric polypeptide comprising GMP can be intracellular protein.

[0151] In some cases, administering GMP to a γδ T cell can include treating the γδ T cell with at least a portion of a chimeric polypeptide comprising GMP and / or a polynucleotide encoding at least a portion of the chimeric polypeptide comprising GMP. Such treatment can occur in the presence or absence of one or more delivery vehicles described herein in this disclosure. In some cases, the method can further include administering to the γδ T cell a chimeric polypeptide comprising GMP, wherein the chimeric polypeptide is operable to release GMP from the chimeric polypeptide in response to a stimulus, and the released GMP is operable to regulate expression of a target polynucleotide in the γδ T cell. In some cases, the method can further include administering to the γδ T cell a chimeric polypeptide comprising GMP and a nuclear translocation domain, wherein the nuclear translocation domain is operable to translocate the chimeric polypeptide to the nucleus of the γδ T cell in response to a stimulus, and the translocated GMP is operable to regulate expression of a target polynucleotide in the γδ T cell.

[0152] In some cases, the nuclear translocation domain can be derived from a transcription factor, as described above. The transcription factor can be a regulatable transcription factor that is active only in response to a signal or signal transduction pathway and can be translocated into the nucleus. The transcription factor can be a regulatable transcription factor that is active primarily in response to a signal or signal transduction pathway and can be translocated into the nucleus. The transcription factor can be a regulatable transcription factor that is generally active in response to a signal or signal transduction pathway and can be translocated into the nucleus.

[0153] In some examples, the nuclear localization domain can be derived from an NFAT family member (e.g., NFATp, NFAT1, NFATc1, NFATc2, NFATc3, NFAT4, NFATx, NFATc4, NFAT3, and NFAT5), nuclear factor kappa B (NF-κB), NFKB1 p50, activator protein 1 (AP-1), a family member of signal transducers and activators of transcription (e.g., STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6), a sterol response element binding protein (e.g., SREBP-1 and SREBF1), a light- or circadian- or electromagnetic-sensing protein, such as cryptochrome (e.g., CRY1, CRY2), timeless (TIM), the PAS domain of a PER protein (e.g., PER1, PER2, and PER3), or another transcription factor or signal transducer.

[0154] In one aspect, the present disclosure provides a system for modulating the activity of gamma-delta (γδ) cells, such as γδ T cells. In some cases, the system for modulating the activity of γδ T cells can include a gene-modulating polypeptide (GMP) comprising an actuator portion configured to modulate expression of a target polynucleotide in a γδ T cell, where the target polynucleotide can encode a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of the death-inducing protein, and / or (iii) a hydrolase. The system disclosed herein can utilize all of the components and configurations described in the methods for modulating the activity of γδ cells of the present disclosure.

[0155] In some cases, the system (e.g., GMP) can be expressed by a γδ T cell. In some cases, the system (e.g., GMP) can be administered to a γδ T cell.

[0156] In some cases, GMP can increase or decrease the activity of a γδ T cell by at least 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, compared to the activity of a γδ T cell in the absence of GMP. In some cases, GMP can increase or decrease the activity of a γδ T cell by up to 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, compared to a γδ T cell in the absence of GMP.

[0157] In some cases, GMP can increase or decrease the activity of a γδ T cell for at least 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, 1 year, or longer, compared to a γδ T cell in the absence of GMP. In some cases, GMP can increase or decrease the activity of a γδ T cell for up to 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, compared to a γδ T cell in the absence of GMP.

[0158] Modulating the expression of a target polypeptide in a γδ T cell can comprise decreasing, increasing, inhibiting, and / or prolonging the expression of the target polypeptide in the γδ T cell. Modulating the expression of a target polypeptide in a γδ T cell can be decreasing the expression of the target polypeptide in the γδ T cell. Modulating the expression of a target polypeptide in a γδ T cell can be increasing the expression of the target polypeptide in the γδ T cell.

[0159] Modulating the expression of a target polypeptide in a γδ T cell can directly and / or indirectly modulate the activity of a γδ T cell. In some cases, modulating the activity of a γδ T cell can include reducing and / or inhibiting one or more γδ T cell fratricide, γδ T cell self-injury, killing of a γδ T cell by another γδ T cell, and / or killing of a γδ T cell by another γδ T cell, thereby improving (directly and / or indirectly) cell viability, proliferation, and / or function. In some cases, a GMP can be operable to reduce γδ T cell fratricide.

[0160] In some cases, the target polynucleotide can encode a polypeptide, and the polypeptide can include a death-inducing protein. The polypeptide can be a death-inducing protein. The death-inducing protein can include one or more of the death-inducing compounds described herein in this disclosure. Examples of death-inducing proteins can include Fas, Fas ligand, granzyme B, granzyme M, and / or perforin.

[0161] In some cases, GMP-containing γδ T cells may be operable to target diseased cells (e.g., cancer / tumor cells) and induce cell death (e.g., apoptosis) of the targeted diseased cells. The diseased cells may express cell surface receptors (e.g., Fas / CD95 / APO-1 / APT1), and the γδ T cells may express respective cell surface ligands (e.g., FasL / CD95L / APTL) that are operable to bind to and target the cell surface receptors of the diseased cells.

[0162] In some cases, the target polynucleotide can encode a polypeptide, and the polypeptide can include a death-inducing protein inhibitor. The polypeptide can be a death-inducing protein inhibitor. The death-inducing protein inhibitor can include one or more of the cell death inhibitors described herein (e.g., the serpin superfamily, including serpin B1 and / or serpin B4). In some cases, the death-inducing protein inhibitor can prevent the production of, alter, and / or degrade one or more death-inducing compounds. In such cases, expression of a death-inducing protein inhibitor by a γδ T cell can protect the γδ T cell from cell death (e.g., apoptosis) caused by one or more death-inducing proteins expressed by the same or different γδ T cells. Thus, a GMP comprising an actuator moiety can be configured to regulate (e.g., induce or increase) expression of a death-inducing protein inhibitor in a γδ T cell, thereby reducing or inhibiting γδ T cell fratricide.

[0163] In some cases, modulating the activity of γδ T cells can include inducing and / or prolonging the activation of γδ T cells. Activation of γδ T cells can include activation of one or more biological activities described herein (e.g., migration, proliferation, synthesis of one or more polypeptides, etc.). Inducing / prolonging the activation of γδ T cells can include inducing and / or prolonging the expression of (1) one or more γδ T cell activators and / or (2) one or more targets of a γδ T cell activator. In one example, a γδ T cell activator can be a ligand, and the target of a γδ T cell activator can be a different polypeptide (e.g., a receptor, an intracellular protein, etc.). Alternatively, or in addition, inducing / prolonging the activation of γδ T cells can include (1) reducing and / or inhibiting the expression of one or more inhibitors of a γδ T cell activator and / or (2) degrading and / or metabolizing an inhibitor of a γδ T cell activator.

[0164] In some cases, the target polynucleotide can encode a polypeptide, and the polypeptide can comprise a hydrolase. The polypeptide can be a hydrolase. The hydrolase can include any of the hydrolases described herein in this disclosure. In some cases, the γδ T cell activator can be a phosphoantigen, and the hydrolase can include any of the phosphoantigen inhibitors described herein in this disclosure. In one example, the hydrolase can be an ectonucleoside triphosphate diphosphohydrolase (e.g., NTPDase1 / ENTPD1 / CD39) that is operable to cleave one or more phosphates in a phosphoantigen, thereby reducing or inhibiting phosphoantigen-induced activation of γδ T cells. As a result, the hydrolase, when unperturbed by a GMP of the present disclosure, can indirectly reduce the activity (e.g., cytotoxic activity) of γδ T cells against diseased cells, e.g., cancer / tumor cells.

[0165] Thus, in some cases, a GMP comprising an actuator moiety can be configured to induce and / or prolong expression of: (1) one or more γδ T cell activators; (2) one or more substrates or proforms (e.g., prodrugs) of a γδ T cell activator; and / or (3) one or more regulators (e.g., catalysts, e.g., enzymes) of the synthesis of a γδ T cell activator.

[0166] In some cases, a GMP comprising an actuator moiety can be configured to induce and / or prolong expression of (1) one or more targets of a γδ T cell activator, (2) one or more substrates or proforms of a γδ T cell activator, and / or (3) one or more regulators (e.g., catalysts, e.g., enzymes) of the synthesis of a target of a γδ T cell activator.

[0167] In some cases, a GMP comprising an actuator moiety can be configured to reduce and / or inhibit expression of one or more inhibitors (e.g., enzymes) of a γδ T cell activator, hi some cases, a GMP comprising an actuator moiety can be configured to degrade and / or metabolize an inhibitor of a γδ T cell activator.

[0168] In some cases, GMPs may be configured to reduce and / or prevent activation of γδ T cells.

[0169] In some cases, a GMP comprising an actuator portion can be configured to increase or decrease the expression of one or more angiogenic factors in a γδ T cell. In some cases, a GMP comprising an actuator portion can be configured to decrease the expression of one or more angiogenic factors in a γδ T cell. In some cases, a GMP comprising an actuator portion can be configured to decrease the expression of one or more angiogenic factors in a γδ T cell. A GMP comprising an actuator portion can be expressed with a guide RNA (e.g., sgRNA) directed to one or more polynucleotide sequences encoding one or more angiogenic factors in a γδ T cell. The actuator portion of the GMP can be configured to cooperate with the guide RNA to increase or decrease the expression of one or more angiogenic factors in a γδ T cell.

[0170] The one or more angiogenic factors can include pro-angiogenic and / or anti-angiogenic factors. Examples of pro-angiogenic factors include, but are not limited to, FGF, VEGF, VEGFR, NRP-1, Ang1, Ang2, PDGF (BB-homodimer), PDGFR, TGF-β, endoglin, TGF-β receptor, MCP-1, and integrin α. V β3, α V β3, α5β1, VE-cadherin, CD31, ephrin, plasminogen activator, plasminogen activator inhibitor-1, eNOS, COX-2, AC133, Id1 / Id3, angiogenin, HGF, Vegf, IL-17, IL-1alpha, IL-8, IL-6, Cxcl5, Fgfα, Fgfβ, Tgfα, Tgfβ, MMPs (including mmp9), plasminogen activator inhibitor-1, thrombospondin, angiopoietin 1, angiopoietin 2, amphiregulin, leptin, endothelin-1, AAMP, AGGF1, AMOT, ANGLPTL3, ANGPTL4, BTG1, IL-1β, NOS3, TNFSF12, and / or VASH2.

[0171] In some cases, the nucleic acid sequence encoding the GMP may be integrated into the genome of a γδ T cell.

[0172] In some cases, GMP can be a part of chimeric polypeptide.Chimeric polypeptide can be transmembrane protein or not.In one example, chimeric polypeptide can be CAR, and GMP can be at least a part of the intracellular domain of CAR.In another example, chimeric polypeptide can be chimeric transmembrane protein, and GMP can be at least a part of the intracellular domain of chimeric transmembrane protein.In another example, chimeric polypeptide comprising GMP can be intracellular protein.

[0173] In some cases, the system may include: (a) a chimeric receptor polypeptide that is modified upon binding to an antigen, where the receptor modification comprises a conformational change or a chemical modification; (b) a chimeric adaptor polypeptide that binds to a receptor in response to the receptor modification; (c) a genetically modulated polypeptide (GMP) that includes an actuator portion linked to a cleavage recognition site, where upon cleavage of the cleavage recognition site, the actuator portion is activated and complexes with a target polynucleotide; and (d) a cleavage portion that cleaves the cleavage recognition site when in proximity to the cleavage recognition site. In some cases, the GMP may form a portion of the intracellular region of the chimeric receptor polypeptide, and the cleavage portion may form a portion of the chimeric adaptor polypeptide. In another example, the GMP may form a portion of the chimeric adaptor polypeptide, and the cleavage portion may form a portion of the intracellular region of the chimeric receptor polypeptide. In a different example, the cleavage portion may be complexed with a second adaptor polypeptide that binds to the chimeric receptor polypeptide in response to receptor modification, and the GMP may form a portion of the chimeric adaptor polypeptide.

[0174] In some cases, the cleavage recognition site may comprise a polypeptide sequence and the cleavage moiety may comprise a protease activity. In some cases, the cleavage recognition site may comprise a disulfide bond and the cleavage moiety may comprise an 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 an actuator moiety.

[0175] In some cases, a cleavage moiety can cleave a recognition site only when it is in close proximity to the cleavage recognition site. The cleavage recognition site can comprise a polypeptide sequence that is a recognition sequence for a protease. The cleavage moiety can comprise a protease activity that recognizes a polypeptide sequence. A cleavage moiety comprising a 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 a polypeptide is 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 hydrolyzed. Some proteases are highly specific and can cleave only substrates having a specific sequence, such as a cleavage recognition sequence or peptide cleavage domain. In some cases, a cleavage recognition site can comprise multiple cleavage recognition sequences, each of which can be recognized by the same or different cleavage moieties (e.g., proteases) with protease activity. Sequence-specific proteases that can be used as cleavage moieties include, but are not limited to, superfamily CA proteases, such as those from family C1, C2, C6, C10, C12, C16, C19, C28, C31, C32, C33, C39, C47, C51, C54, C58, C64, C65, C66, C67, C70, C71, C76, C78, ​​C83, C85, C86, C87, C93, C96, C98, and C101 [papain (Carica papaya), bromelain (pineapple (Ananas comosus)), cathepsin K (Marchantia polymorpha), and calpain (Homo sapiens)]. sapiens)], superfamily CD proteases, e.g., families C11, C13, C14, C25, C50, C80, and C84, e.g., caspase-1 (Rattus norvegicus) and separase (Saccharomyces cerevisiae),cerevisiae), superfamily CE proteases, such as families C5, C48, C55, C57, C63, and C79 [including adenine (human adenovirus type 2)], superfamily CF proteases, such as family C15 [including pyroglutamyl-peptidase I (Bacillus amyloliquefaciens)], superfamily CL proteases, such as families C60 and C82 [including sortase A (Staphylococcus aureus)], aureus)], superfamily CM proteases, such as family C18 [including hepatitis C virus peptidase 2 (hepatitis C virus)], superfamily CN proteases, such as family C9 [including Sindbis virus nsP2-type peptidase (Sindbis virus)], superfamily CO proteases, such as family C40 [dipeptidyl-peptidase VI (Lysinibacillus sphaericus)], sphaericus)], superfamily CP proteases, e.g., family C97 [including DeSI-1 peptidase (house mouse)], superfamily PA proteases, e.g., families C3, C4, C24, C30, C37, C62, C74, and C99 [including TEV protease (tobacco etch virus)], superfamily PB proteases, e.g., families C44, C45, C59, C69, C89, and C95 [including amidophosphoribosyltransferase precursor (Homo sapiens)], superfamily PC proteases, families C26 and C56 [including U-glutamyl hydrolase (Rattus norvegicus)], superfamily PD proteases, e.g., family C46 [including hedgehog protein (Drosophila melanogaster)], superfamily PE proteases, such as family P1 [DmpA aminopeptidase (Ochrobactrum anthropi)],anthropi)], other proteases, e.g., families C7, C8, C21, C23, C27, C36, C42, C53, and C75. Additional proteases include serine proteases, e.g., those in superfamily SB, e.g., families S8 and S53, including subtilisins (Bacillus licheniformis), superfamily SC, e.g., those in families S9, S10, S15, S28, S33, and S37, including prolyl oligopeptidases (Sus scrofa), superfamily SE, e.g., those in families S11, S12, and S13, including d-Ala-d-Ala peptidase C (Escherichia coli), superfamily SF, e.g., those of families S24 and S26 [including signal peptidase I (E. coli)]; superfamily SJ, e.g., those of families S16, S50, and S69 [including lon-A peptidase (E. coli)]; superfamily SK, e.g., those of families S14, S41, and S49 [including Clp protease (E. coli)]; superfamily SO, e.g., those of family S74 [including phage K1F endosialidase CIMCD self-cleaving protein (Enterobacteriaceae phage K1F)]; superfamily SP, e.g., those of family S59 [including nucleoporin 145 (Homo sapiens)]; superfamily SR, e.g., those of family S60 [including lactoferrin (Homo sapiens)]; superfamily SS, those of family S66 [including murein tetrapeptidase LD-carboxypeptidase (Pseudomonas aeruginosa)]; aeruginosa)], superfamily ST, such as those in family S54 [including rhomboid-1 (Drosophila melanogaster)], superfamily PA, such as those in families S1, S3, S6, S7, S29, S30, S31, S32, S39, S46, S55, S64, S65, and S75 [chymotrypsin A (bovinetaurus)], superfamily PB, e.g., those in families S45 and S63 [including penicillin G acylase precursor (Escherichia coli)], superfamily PC, e.g., those in family S51 [including dipeptidase E (Escherichia coli)], superfamily PE, e.g., those in family P1 [including DmpA aminopeptidase (Ochrobacterium anthropi)], unassigned ones, e.g., threonine proteases of families S48, S62, S68, S71, S72, S79, and S81, superfamily PB clans, e.g., those in families T1, T2, T3, and T6 [archaeal proteasome, u component (Thermoplasma acidophilum)], Examples of proteases include those from the superfamily PE clan, such as those from family T5 (including ornithine acetyltransferase (Saccharomyces cerevisiae)), aspartic proteases, such as BACE1, BACE2, cathepsin D, cathepsin E, chymosin, napsin A, nepenthesin, pepsin, plasmepsin, presenilin, renin, and HIV-1 protease, and metalloproteinases, such as exopeptidases, metalloexopeptidases, endopeptidases, and metalloendopeptidases. The cleavage recognition sequence (e.g., polypeptide sequence) can be recognized by any of the proteases disclosed herein.

[0176] In some cases, the cleavage recognition site can comprise a cleavage recognition sequence (e.g., a polypeptide sequence or peptide cleavage domain) recognized by a protease selected from the group consisting of achromopeptidase, aminopeptidase, ancrod, angiotensin-converting enzyme, bromelain, calpain, calpain I, calpain II, carboxypeptidase A, carboxypeptidase B, carboxypeptidase G, Carboxypeptidase P, Carboxypeptidase W, Carboxypeptidase Y, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 11, Caspase 12, Caspase 13, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin H, Cathepsin L, Chymopapain, Chymase, Chymotrypsin, clostripain, collagenase, complement C1r, complement C1s, complement factor D, complement factor I, cucumisin, dipeptidyl peptidase IV, elastase (leukocyte), elastase (pancreas), endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, enterokinase, factor Xa, ficin, furin, granzyme A, granzyme Mucosal B, HIV protease, IGase, tissue kallikrein, leucine aminopeptidase (general), leucine aminopeptidase (cytosolic), leucine aminopeptidase (microsomal), matrix metalloproteinase, methionine aminopeptidase, neutrase, papain, pepsin, plasmin, prolidase, pronase E, prostate-specific antigen, alkalophilic protease from Streptomyces griseus, protease from Aspergillus, protease from Aspergillus saitoi, protease from Aspergillus sojae, protease (B. licheniformis).licheniformis) (alkaline or alcalase), protease from Bacillus polymyxa, protease from Bacillus sp., protease from Rhizopus sp., protease S, proteasome, proteinase from Aspergillus oryzae, proteinase 3, proteinase A, proteinase K, protein C, pyroglutamic acid aminopeptidase, rennin, streptokinase, subtilisin, thermolysin, thrombin, tissue plasminogen activator, trypsin, tryptase, and urokinase.

[0177] Further details of proteases and associated recognition sequences that can be used in the systems and methods of the present disclosure are disclosed in Patent Cooperation Treaty (PCT) Patent Application No. PCT / US17 / 012885 and PCT Patent Application No. PCT / US17 / 012881, each of which is incorporated herein by reference in its entirety.

[0178] In some cases, the system administered to a γδ cell (e.g., a γδ T cell) can include a chimeric polypeptide comprising GMP and / or a polynucleotide encoding at least a portion of the chimeric polypeptide comprising GMP. The GMP-containing system can be administered to a γδ T cell in the presence or absence of one or more delivery vehicles described herein. In some cases, the system can further include a chimeric polypeptide comprising GMP, wherein the chimeric polypeptide can be operable to release GMP from the chimeric polypeptide in response to a stimulus, and the released GMP can be operable to regulate expression of a target polynucleotide in a gamma delta T cell. In some cases, the system can further include a chimeric polypeptide comprising GMP and a nuclear translocation domain, wherein the nuclear translocation domain can be operable to translocate the chimeric polypeptide to the nucleus of the gamma delta T cell in response to a stimulus, and the translocated GMP can be operable to regulate expression of a target polynucleotide in the gamma delta T cell. The chimeric polypeptide may or may not be a transmembrane receptor. The chimeric polypeptide may or may not be an intracellular protein. The chimeric polypeptide may or may not bind to a ligand (e.g., an antigen on a target cell).

[0179] In some cases, the actuator portion can be an RNA-guided actuator portion or a variant thereof, and the RNA-guided actuator portion forms a complex with the target polynucleotide. In some cases, the actuator portion can be a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity. In some cases, the actuator portion can be Cas9 and / or Cpf1. In some cases, the actuator portion can comprise an activator effective to increase expression of the target polynucleotide. In some cases, the actuator portion can comprise a repressor effective to decrease expression of the target polynucleotide.

[0180] The GMP of the system can include any of the GMPs described herein in this disclosure. The actuator portion of the system can include any of the actuator portions described herein in this disclosure.

[0181] In some embodiments, the system can include: (a) a chimeric transmembrane receptor polypeptide comprising: (i) an extracellular region comprising a ligand (e.g., antigen)-interacting domain that binds to a ligand (e.g., an antigen); and (ii) an intracellular region comprising a signaling domain (e.g., an immune cell signaling domain); (b) a chimeric adaptor polypeptide comprising a receptor-associating portion (e.g., receptor-binding portion) that (i) binds to the receptor polypeptide when the receptor polypeptide undergoes receptor modification upon binding to the ligand and / or (ii) moves into proximity with the receptor polypeptide when the receptor polypeptide undergoes receptor modification upon binding to the ligand; (c) a GMP comprising an actuator portion linked to a cleavage recognition site; and (d) a cleavage portion that cleaves the cleavage recognition site when in proximity to the cleavage recognition site, releasing the actuator portion from the GMP. In some cases, the cleavage portion forms part of the intracellular region of the receptor polypeptide, and the GMP forms part of the chimeric adaptor polypeptide. In some cases, the cleavage portion complexes with a second adaptor polypeptide that binds to and / or migrates in proximity to the receptor-modified receptor polypeptide upon antigen binding, and the GMP forms a portion of the chimeric adaptor polypeptide. In some cases, the cleavage portion forms a portion of the chimeric adaptor polypeptide and the GMP forms a portion of the intracellular region of the receptor polypeptide.

[0182] In some cases, the proximity between the chimeric adaptor polypeptide and the chimeric receptor polypeptide may be sufficient to allow the cleavage moiety to cleave the cleavage recognition site and release the actuator moiety from the GMP.

[0183] In some cases, the chimeric transmembrane receptor polypeptide can include at least a portion of a TCR. In such cases, the chimeric adaptor polypeptide can include at least a portion of a TCR signaling adaptor (e.g., a transmembrane signaling adaptor). When a chimeric receptor polypeptide including at least a portion of a TCR undergoes receptor modification upon binding to a ligand, at least a portion of the TCR signaling adaptor can be activated and bind to and / or move into proximity with the chimeric receptor polypeptide, thereby enabling the cleavage moiety to cleave the cleavage recognition site and release the actuator moiety from the GMP. In one example, the TCR signaling adaptor can be a linker for T cell activation (LAT).

[0184] In some cases, the chimeric transmembrane receptor polypeptide can include at least a portion of a C-type lectin-like receptor, such as a CD94 family receptor. Examples of CD95 family receptors include NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2G. In such cases, the chimeric adaptor polypeptide can include at least a portion of a signaling adaptor (e.g., a transmembrane signaling adaptor) of the C-type lectin-like receptor. When a chimeric receptor polypeptide including at least a portion of a C-type lectin-like receptor undergoes receptor modification upon binding to a ligand, at least a portion of the signaling adaptor of the C-type lectin-like receptor can be activated and bind to and / or move into proximity with the chimeric receptor polypeptide, thereby enabling the cleavage moiety to cleave the cleavage recognition site and release the actuator moiety from GMP. In one example, the C-type lectin-like receptor can be NKG2D, and the signaling adaptor of NKG2D can be DAP10. In some cases, a chimeric receptor polypeptide comprising at least a portion of an NKG2D receptor can be activated upon binding of an NKG2D ligand (NKG2DL) to the NKG2DL-binding domain of at least a portion of the NKG2D receptor of the chimeric receptor polypeptide, inducing release of an actuator moiety from the GMP of the system. Examples of NKG2DLs can include, but are not limited to, MICA, MICB, and the RAET1 / ULBP family (e.g., RAET1 E / ULBP4, RAET1 G / ULBP5, RAET1 H / ULBP2, RAET1 / ULBP1, RAET1 L / ULBP6, and RAET1 N / ULBP3).

[0185] In some embodiments, a γδ cell (e.g., γδ T cell) system can comprise a chimeric polypeptide sequence comprising a CAR. The CAR can comprise a ligand-binding domain, a transmembrane domain, and a signaling domain (e.g., an intracellular signaling domain). The signaling domain can activate a signaling pathway in an immune cell upon binding of a ligand to the ligand-binding domain. γδ cells administered using such a system can further comprise an expression cassette comprising a polynucleotide sequence encoding a GMP under the control of a promoter. The GMP can comprise an actuator portion. The promoter can be activated to drive expression of the GMP upon binding of a ligand to the ligand-binding domain. The expressed GMP can regulate expression of a target polynucleotide in the γδ cell. The promoter can comprise an endogenous promoter of the γδ cell. The endogenous promoter can be activated upon binding of a ligand to the ligand-binding domain of the CAR.

[0186] In some embodiments, a γδ cell (e.g., γδ T cell) system can comprise a chimeric polypeptide sequence and an additional polypeptide sequence. The additional polypeptide sequence can comprise a chimeric receptor polypeptide that activates a cell signaling pathway upon binding to a ligand. The chimeric polypeptide sequence can comprise a GMP fused in-frame with a heterologous nuclear translocation domain, where the heterologous nuclear translocation domain is operable to translocate the chimeric polypeptide to the nucleus of the γδ cell upon induction by the cell signaling pathway. Upon binding of a ligand to the chimeric receptor polypeptide, the chimeric receptor polypeptide can localize to the nucleus of the γδ cell via the induced heterologous nuclear translocation domain, and the gene modulating polypeptide can regulate expression of a target polynucleotide in the cell nucleus. In some cases, activation of the nuclear translocation domain upon induction by the cell signaling pathway can comprise chemical modification of at least one nuclear translocation sequence of the nuclear translocation domain.

[0187] Further details regarding the design and application of systems comprising chimeric polypeptides (e.g., chimeric receptor polypeptides, chimeric adaptor polypeptides, etc.), CARs, GMPs, ligands (e.g., antigens), modifications thereof, and expression cassettes containing them can be found in PCT Patent Application Nos. PCT / US17 / 012885 and PCT Patent Application No. PCT / US17 / 01288, each of which is incorporated herein by reference in its entirety. 1, PCT Patent Application No. PCT / US18 / 041704, U.S. Patent No. 9,856,497, U.S. Non-Provisional Application No. 15 / 806,756, U.S. Non-Provisional Application No. 16 / 029,299, U.S. Non-Provisional Application No. 16 / 029,299, U.S. Provisional Application No. 62 / 639,427, U.S. Provisional Application No. 62 / 639,386, U.S. Provisional Application No. 62 / 647,543, and U.S. Provisional Application No. 62 / 675,134.

[0188] In some cases, a γδ T cell can be contacted with a stimuli (e.g., a small molecule, polynucleotide, polypeptide, protein, antibody, ligand, and / or receptor from another cell, etc.) to activate (or conditionally activate) a GMP comprising an actuator moiety. Activation of the GMP can include expressing the GMP and / or a chimeric polypeptide comprising the GMP. Alternatively, or in addition, activation of the GMP can include release of the GMP from a chimeric polypeptide comprising the GMP (e.g., a CAR, a chimeric transmembrane protein, a chimeric intracellular protein, etc.). Alternatively, or in addition, activation of the GMP can include chemical (e.g., phosphorylation) or physical (e.g., secondary structure) modification of one or more additional portions of the chimeric polypeptide comprising the GMP (e.g., a nuclear localization signal (NLS) domain of the chimeric polypeptide). In one example, such a modified NLS can be operable to translocate at least the GMP (and effector and / or activator) to the nucleus of the γδ T cell in response to the stimuli. Activated GMP can operate to regulate the expression of a target polynucleotide in a γδ T cell, thereby regulating an activity associated with the target polynucleotide or a polypeptide encoded by the target polynucleotide.

[0189] The stimulator may activate one or more endogenous signaling pathways in the γδ T cell to promote activation (e.g., conditional activation, conditional expression, etc.) of GMP or a chimeric polypeptide comprising GMP. In some cases, the stimulator may bind to a cell surface receptor on the γδ T cell. In one example, the cell surface receptor may be an endogenous cell surface receptor of the γδ T cell. In another example, the cell surface receptor may be a heterologous cell surface receptor (e.g., a CAR) of the γδ T cell. The cell surface receptor may or may not be a chimeric polypeptide comprising GMP. In some cases, the stimulator may permeate the membrane and bind to an intracellular protein in the γδ T cell (e.g., in the cytoplasm or nucleus, etc.). The intracellular protein may or may not be a chimeric polypeptide comprising GMP. In one example, the intracellular protein may be an endogenous intracellular protein of the γδ T cell. In another example, the intracellular protein may be a heterologous intracellular protein in the γδ T cell.

[0190] Contacting a γδ T cell with a stimulator can occur directly and / or indirectly. Direct stimulation can occur when a stimulator binds to a γδ T cell. In some cases, a stimulator can bind to a transmembrane receptor of a γδ T cell, an intracellular protein of the γδ T cell (e.g., in the cytoplasm or nucleus of the cell), or both. In one example, a stimulator can bind to the stimulator-binding domain (or ligand-binding domain) of a chimeric polypeptide containing GMP. In another example, a stimulator can bind to the stimulator-binding domain (or ligand-binding domain) of a different polypeptide (e.g., a different cell surface receptor or a different CAR) that does not contain GMP. Indirect stimulation can occur when a stimulator activates or deactivates a different cell, which is operable to activate the γδ T cell by binding to the receptor of the γδ T cell using its cell surface marker (e.g., cell surface ligand). As a result, the γδ T cell can be activated and initiate expression of GMP or a chimeric polypeptide containing GMP. The different cell can be of the same cell type as the γδ T cell (eg, another γδ T cell) or a different cell type than the γδ T cell (eg, a different type of lymphocyte or cancer / tumor cell).

[0191] Contacting the γδ T cells with a stimulatory agent can occur before, during, and / or following administration of a GMP comprising an actuator moiety to the γδ T cells. The γδ T cells can be ex vivo and / or in vivo during contact with the stimulatory agent and conditional activation of the GMP comprising the actuator moiety.

[0192] Contacting the γδ T cells with the stimulatory agent can occur before, during, and / or following administration of the γδ T cells (e.g., engineered γδ T cells) to a subject. The γδ T cells can be contacted with the stimulatory agent for 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 longer before, during, and / or following administration of the γδ T cells to a subject. The γδ T cells can be contacted with the stimulatory agent for up to about 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5 days or shorter before, during, and / or following administration of the γδ T cells to a subject. The γδ T cells may be contacted with a stimulatory agent for 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 longer prior to administration of the γδ T cells to a subject. The γδ T cells may be contacted with a stimulatory agent for up to about 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5 days or shorter prior to administration of the γδ T cells to a subject. The γδ T cells may be contacted with a stimulatory agent for 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 longer following administration of the γδ T cells to the subject. The γδ T cells may be contacted with a stimulatory agent for up to 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, following administration of the γδ T cells to the subject. In some cases, the γδ T cells may be contacted with a stimulatory agent at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.In some cases, the γδ T cells may be contacted with a stimulator up to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some cases, the γδ T cells may be contacted with a stimulator 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, 810, 820, 830, 840, 850, 860, 870, 880, In other cases, the stimulator may be contacted at a dosage concentration of 70, 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 milliliter (IU / mL), or more. T cells are up to approximately 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, 410, 420, 430, 440, 450, 460, 470, 480, 490, 480, 470, 460, 450, 440, 430, 420, 410, 420, 430, 440, 450, 450, 460, 470, 480, 49 ... The stimulant may be contacted at a dosage concentration of 0, 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.

[0193] In some cases, the stimulator of γδ T cells may be selected from the group consisting of interleukins (e.g., IL-2), interferons, transforming growth factors (TGFs), ligands of cluster of differentiation (CD) receptors, and variants thereof. The stimulator may be an antigen described in the subject disclosure. In some examples, the antigen may induce migration, survival, proliferation, and / or differentiation of immune cells (e.g., γδ T cells). In some cases, the stimulator may include a vaccine (e.g., an immune cell vaccine). The 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. The vaccine may further include one or more additional components (e.g., an adjuvant) that enhance immunological activity. In one example, the immune cell vaccine may be a peptide vaccine (e.g., p-27L) or a viral vaccine (e.g., p-210M, rFP-210M).

[0194] In some cases, the stimulator binding domain binds to an antigen that is not membrane-bound (e.g., non-membrane-bound), e.g., an extracellular antigen 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) may be associated with a disease, e.g., a viral, bacterial, and / or parasitic infection, an inflammatory and / or autoimmune disease, or a neoplasm, e.g., a cancer and / or a tumor. Non-limiting examples of antigens that may be bound by the ligand binding domain of a chimeric transmembrane receptor polypeptide of a subject system include, but are not limited to, 1-40-beta-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A-kinase anchor protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenoceptor beta 3 (ADRB3), AGS-22M6, alpha folate receptor, alpha-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie2), anthrax toxin, AOC3 (VAP-1), B-cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis (Bacillus anthracis anthrax, B-cell activating factor (BAFF), B-lymphoma cells, bone marrow stromal cell antigen 2 (BST2), Brother of the Regulator of Imprinted Sites (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCCTC-binding factor (CTCF), CCL11 (eotaxin-1), C CR4, CCR5, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (α chain of IL-2 receptor), CD27,CD274, CD28, CD3, CD3ε, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-related antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6), Clostridium difficile 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), CXC chemokine receptor type 4, cyclin B1, cytochrome P4501B1 (CYP1B1), cyp-B, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, Escherichia coli Shiga toxin type 1, Escherichia 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 (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 transition-variant gene 6, located on chromosome 12p (ETV6-AML), respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II beta chain, fibroblast activation protein alpha (FAP),Fibronectin extradomain B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, frizzled receptor, fucosyl-GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), glypican-3 (GPC3), GMCSF receptor α-chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, heat shock protein 70-2 mutant (mutated) hsp70-2), hemagglutinin, hepatitis A virus cellular receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, globoH glycoceramide hexasaccharide moiety (GloboH), HGF, HHGFR, high molecular weight melanoma-associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), human TNF, ICAM-1 (CD54), iCE, IFN-α, IFN-β, IFN-γ, IgE, IgE Fc region, IGF-1, IGF-1 receptor, IGHE, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1β, IL-20, IL-22, IL-23, IL-31, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor Interleukin-11 receptor alpha (IL-11Rα), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), interferon α / β receptor, interferon gamma-inducible protein, interleukin-11 receptor alpha (IL-11Rα), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2),Intestinal carboxylesterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex combination, locus K9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or tumor necrosis factor-β (TNF-β), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary gland differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma inhibitor of apoptosis Interleukin-1-associated protein (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, mucin 1, cell surface associated (MUC1), MUC-2, mucin CanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyltransferase V (NA17), NCA-90 (granulocyte antigen), nerve growth factor (NGF), neuronal apoptosis-regulating proteinase 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitor (e.g., NOGO-A, NOG) O-B, NOGO-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), carcinoembryonic antigen (h5T4), oncogene fusion protein (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl), and Oryctolagus cuniculus), OX-40, oxLDL, p53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), phosphate-sodium cotransporter, phosphatidylserine, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PDGF-Rα), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid,Proacrosin-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostate cancer cells, prostein, protease serine 21 (testisin or PRSS21), proteasome (prosome, macropain) subunit, beta type, 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor kappa-B RANKL, receptor for advanced glycation end products (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 transition breakpoint, sclerostin (SOST), selectin P, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine-1-phosphate, squamous cell carcinoma antigens 1, 2, and 3 recognized by T cells (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, survivin, syndecan 1 (SDC1)+A314, SOX10, survivin, survivin-2B, synovial sarcoma, X breakpoint 2 (SSX2), T-cell receptor, TCR gamma alternate reading frame protein (TARP), telomerase, TEM1, tenascin-C, TGF-β (e.g., TGF-β1, TGF-β2, TGF-β3), thyroid-stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), TNF-α, TRAIL-R1, TRAIL-R2, TRG, transglutaminase 5 (TGS5), tumor antigen CTAA16.88, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R),Tumor protein p53 (p53), tumor-specific glycosylation of MUC1, tumor-associated calcium signaling factor 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 (VEGF-A, V), These include EGF-B, VEGF-C, VEGF-D, and PIGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), vimentin, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), von Willebrand factor (VWF), Wilms tumor protein (WT1), X antigen family, member 1A (XAGE1), β-amyloid, and κ-light chain, as well as variants thereof.

[0195] In some cases, a stimulator binding domain (or ligand binding domain)

[0196] In some embodiments, the ligand binding domain binds to an antigen selected from the group consisting of: 707-AP, biotinylated molecule, a-actinin-4, abl-bcr alb-b3(b2a2), abl-bcr alb-b4(b3a2), adipophilin, AFP, AIM-2, Annexin II, ART-4, BAGE, b-catenin, bcr-abl, bcr-abl p190(e1a2), bcr-abl p210(b2a2), bcr-abl p210(b3a2), BING-4, CAG-3, CAIX, CAMEL, caspase-8, CD171, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v7 / 8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3 , erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6 / AML, FBP, fetal acetylcholine receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2(FGF6), HST-2 / neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR / FUT, LewisY, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, malic enzyme, mammaglobin-A, MART-1 / Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, myosin, NA88-A, Neo-PAP, NKG2D, NPM / ALK, N-RAS, NY-ESO-1, OA1, OGT , carcinoembryonic antigen (h5T4), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, survivin, survivin-2B, SYT / SSX, TAG-72, TEL / AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2 / INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, alpha-folate receptor, and kappa-light chain. In some embodiments, the ligand-binding domain binds to a tumor-associated antigen.

[0197] In some embodiments, the target polynucleotide encodes a cytokine, including, but not limited to, 4-1BBL, activin βA, activin βB, activin βC, activin βE, artemin (ARTN), BAFF / BLyS / TNFSF138, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, bone morphogenetic protein 1 (BMP1), CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, and CCL20. L2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9 , CD153 / CD30L / TNFSF8, CD40L / CD154 / TNFSF5, CD40LG, CD70, CD70 / CD27L / TNFSF7, CLCF1, c-MPL / CD110 / TPOR, CNTF, CX3CL1, CXCL1, CXCL10, CXCL 11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, EDA-A1, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, Fas ligand / FASLG / CD95L / CD178, GDF10, GDF11, GDF15, GDF2, GDF3, GDF4, GDF5, GDF6, GDF7, GDF8, GDF9, glial cell line-derived neurotrophic factors Nutrient factor (GDNF), growth differentiation factor 1 (GDF1), IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA5 / IFNaG, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNZ, IFNω / IFNW1, IL 11, IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1RL2, IL31, IL33, IL6, IL8 / CXCL8, inhibin-A, inhibin-B,These include leptin, LIF, LTA / TNFB / TNFSF1, LTB / TNFC, neurturin (NRTN), OSM, OX-40L / TNFSF4 / CD252, persephin (PSPN), RANKL / OPGL / TNFSF11 (CD254), TL1A / TNFSF15, TNFA, TNF-alpha / TNFA, TNFSF10 / TRAIL / APO-2L (CD253), TNFSF12, TNFSF13, TNFSF14 / LIGHT / CD258, XCL1, and XCL2. In some embodiments, the target gene encodes 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 the alpha, beta, gamma, and / or delta chain of a T cell receptor (TCR).

[0198] The subject system can be introduced into a variety of immune cells, including any cells involved in an immune response. Various immune cells can be engineered to express a γδ TCR complex and / or transdifferentiate into cells that express a γδ TCR complex. In some embodiments, immune cells include granulocytes, e.g., asophils, eosinophils, and neutrophils, mast cells, monocytes that can develop into macrophages, antigen-presenting cells, e.g., dendritic cells, and lymphocytes, e.g., natural killer cells (NK cells), B cells, and T cells. In some embodiments, the immune cells are immune effector cells. Immune effector cells are immune cells that can perform a specific function in response to a stimulus. In some embodiments, the immune cells are immune effector cells that can induce cell death. In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are NK cells. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are activated T cells. T cells include both naive and memory cells (e.g., central memory or T cells). CM , Effector Memory or T EM, and effector memory RA or T EMRA T cells include CD8+ T cells, effector cells (e.g., cytotoxic T cells or CTLs or Tc cells), helper cells (e.g., Th1, Th2, Th3, Th9, Th7, TfH), regulatory cells (e.g., Treg and Trl cells), natural killer T cells (NKT cells), tumor-infiltrating lymphocytes (TILs), lymphocyte-activated killer cells (LAKs), αβ T cells, γδ T cells, and similar unique T cell lineage classes. T cells can be divided into two broad categories, CD8+ T cells and CD4+ T cells, based on which proteins are present on the cell surface. T cells expressing the target system can perform multiple functions, including killing infected cells and activating or recruiting other immune cells. CD8+ T cells are called cytotoxic T cells or cytotoxic T lymphocytes (CTLs). CTLs expressing the target system can be involved in recognizing and eliminating virus-infected and cancer cells. CTLs possess specialized compartments, or granules, that contain cytotoxins that trigger apoptosis, i.e., programmed cell death. CD4+ T cells can be further divided into four subsets: Th1, Th2, Th17, and Treg. "Th" refers to "T helper cells," although additional subsets may exist. Th1 cells can orchestrate immune responses to intracellular microorganisms, particularly bacteria. They can produce and secrete molecules that alert and activate other immune cells, such as bacteria-ingesting macrophages. Th2 cells are involved in orchestrating immune responses to extracellular pathogens, such as helminths (parasites), by alerting B cells, granulocytes, and mast cells. Th17 cells can produce interleukin-17 (IL-17), a signaling molecule that activates immune and non-immune cells. Th17 cells are important for recruiting neutrophils.

[0199] A variety of cells can be used as host cells to implement the subject disclosed systems and methods. Host cells to which any of the embodiments disclosed herein (e.g., cells comprising or expressing a γδ TCR complex) can be applied (e.g., transduced) include a wide variety of cell types. The host cell can be in vitro. The host cell can be in vivo. The host cell can be ex vivo. The host cell can be an isolated cell. The host cell can be a cell within an organism. The host cell can be an organism. The host cell can be a cell in cell culture. The host cell can be one of a collection of cells. The host cell can be a mammalian cell or derived from a mammalian cell. The host cell can be a rodent cell or derived from a rodent cell. The host cell can be a human cell or derived from a human cell. The host cell can be a prokaryotic cell or derived from a prokaryotic cell. The host cell can be a bacterial cell or derived from a bacterial cell. The host cell can be an archaeal cell or derived from an archaeal cell. The host cell can be a eukaryotic cell or derived from a eukaryotic cell. The host cell can be a pluripotent stem cell. The host cell can be a plant cell or derived from a plant cell. The host cell can be an animal cell or derived from an animal cell. The host cell can be an invertebrate cell or derived from an invertebrate cell. The host cell can be a vertebrate cell or derived from a vertebrate cell. The host cell can be a microbial cell or derived from a microbial cell. The host cell can be a fungal cell or derived from a fungal cell. The host cell can be from a specific organ or tissue.

[0200] The host cell can be an immune cell, as described above in the subject disclosure.

[0201] The host cell can be a stem or progenitor cell. The host cell 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.). The host cell can include mammalian stem and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. A clonal cell can include the progeny of a cell. The host cell can be in a living organism. The host cell can be a genetically modified cell.

[0202] The host cell can be a totipotent stem cell, although in some embodiments of the present disclosure the term "cell" may be used but may not refer to a totipotent stem cell. The host cell can be a plant cell, although in some embodiments of the present disclosure the term "cell" may be used but may not refer to a plant cell. The host cell can be a pluripotent cell. For example, the host cell can be a pluripotent hematopoietic cell that can differentiate into other cells in the hematopoietic lineage but may not be able to differentiate into any other non-hematopoietic cells. The host cell may be capable of developing into a whole organism. The host cell may or may not be capable of developing into a whole organism. The host cell can be a whole organism.

[0203] A variety of one or more endogenous signaling pathways of a cell (e.g., NFkB) are available for use in the embodiments provided herein. Table 1 provides exemplary signaling pathways and genes associated with the signaling pathways. In the embodiments provided herein, the signaling pathway activated by binding of a stimuli to a cell (e.g., an immune cell, a stem cell, etc.) and / or ligand binding to a transmembrane receptor can be any one of those provided in Table 1. In one example, in the provided embodiments, the promoter activated to drive expression of GMP upon binding of a stimuli to the stimuli-binding domain of a transmembrane receptor can include a promoter sequence, any variant of a promoter sequence, or any partial promoter sequence (e.g., a minimal promoter sequence) driving any of the genes provided in Table 1.

[0204] [Table 1A]

[0205] [Table 1B]

[0206] [Table 1C]

[0207] [Table 1D]

[0208] [Table 1E]

[0209] [Table 1F]

[0210]

Table 1G

[0211] Table 1H

[0212]

Table 1I

[0213]

Table 1J

[0214] Table 1K

[0215]

Table 1L

[0216]

Table 1M

[0217]

Table 1N

[0218]

Table 10

[0219] [Table 1P]

[0220] [Table 1Q]

[0221] [Table 1R]

[0222] The disclosed systems and compositions are useful in a variety of applications. For example, the disclosed systems and methods are useful in methods for modulating gene expression and / or cellular activity. In one aspect, the systems and compositions disclosed herein are utilized in methods for modulating gene expression and / or cellular activity in immune cells. Immune cells modulated using the subject system may be useful in a variety of applications, including, but not limited to, immunotherapy for treating diseases and disorders. Diseases and disorders that can be treated using the modified immune cells of the present disclosure include inflammatory conditions, cancer, and infectious diseases. In some embodiments, immunotherapy is used to treat cancer.

[0223] A variety of target cells can be killed using the subject disclosed systems and methods. Target cells to which the methods can be applied include a wide variety of cell types. The target cell can be in vitro. The target cell can be in vivo. The target cell can be ex vivo. The target cell can be an isolated cell. The target cell can be a cell within an organism. The target cell can be an organism. The target cell can be a cell in cell culture. The target cell can be one of a collection of cells. The target cell can be a mammalian cell or derived from a mammalian cell. The target cell can be a rodent cell or derived from a rodent cell. The target cell can be a human cell or derived from a human cell. The target cell can be a prokaryotic cell or derived from a prokaryotic cell. The target cell can be a bacterial cell or derived from a bacterial cell. The target cell can be an archaeal cell or derived from an archaeal cell. The target cell can be a eukaryotic cell or derived from a eukaryotic cell. The target cell can be a pluripotent stem cell. The target cell can be a plant cell or derived from a plant cell. The target cell can be an animal cell or derived from an animal cell. The target cell can be an invertebrate cell or derived from an invertebrate cell. The target cell can be a vertebrate cell or derived from a vertebrate cell. The target cell can be a microbial cell or derived from a microbial cell. The target cell can be a fungal cell or derived from a fungal cell. The target cell can be from a specific organ or tissue.

[0224] The target cell can be a stem cell or progenitor cell. The target cell 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.). The target cell can include mammalian stem and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. A clonal cell can comprise the progeny of a cell. The target cell can comprise the target nucleic acid. The target cell can be in a living organism. The target cell can be a genetically modified cell. The target cell can be a host cell.

[0225] The target cell can be a totipotent stem cell, although in some embodiments of the present disclosure the term "cell" may be used but may not refer to a totipotent stem cell. The target cell can be a plant cell, although in some embodiments of the present disclosure the term "cell" may be used but may not refer to a plant cell. The target cell can be a pluripotent cell. For example, the target cell can be a pluripotent hematopoietic cell that can differentiate into other cells in the hematopoietic lineage but may not be able to differentiate into any other non-hematopoietic cells. The target cell may be capable of developing into a whole organism. The target cell may or may not be capable of developing into a whole organism. The target cell can be a whole organism.

[0226] The target cell can be a primary cell. For example, a culture of primary cells can be passaged 0, 1, 2, 4, 5, 10, 15, or more times. The cell can be a unicellular organism. The cell can be grown in culture.

[0227] The target cells can be diseased cells. Diseased cells can have altered metabolism, gene expression, and / or morphology. Diseased cells can be cancer cells, diabetic cells, and apoptotic cells. Diseased cells can be cells from a diseased subject. Exemplary diseases can include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart diseases, etc.

[0228] If the target cells are primary cells, they may be collected from an individual by any method. For example, leukocytes may be collected by apheresis, leukapheresis, density gradient separation, etc. Cells from tissues, such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestinal tract, stomach, etc., may be collected by biopsy. An appropriate solution may be used to disperse or suspend the collected cells. Such solutions may generally be balanced salt solutions (e.g., normal saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.), conveniently supplemented with fetal bovine serum or other naturally occurring factors, along with a low concentration of an acceptable buffer. Buffers may include HEPES, phosphate buffer, lactate buffer, etc. The cells may be used immediately or may be stored (e.g., by freezing). Frozen cells may be thawed and may be usable again. Cells can be frozen in DMSO, serum, media buffer (e.g., 10% DMSO, 50% serum, 40% buffered media), and / or any other such common solution used to store cells at freezing temperatures.

[0229] Non-limiting examples of cells that can be target cells include, but are not limited to, lymphoid cells, e.g., B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, cytokine-induced killer (CIK) cells (see, e.g., US20080241194), myeloid cells, e.g., granulocytes (basophilic granulocytes, eosinophilic granulocytes, neutrophilic granulocytes / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, dendritic cells, cells from the endocrine system (thyroid (including thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, eosinophilic cells), adrenal (chromaffin cells), and pineal (pinealocyte) cells), cells of the nervous system (including glial cells (stellate cells, microglia), magnocellular neurosecretory cells, stellate cells, Boettcher cells, and pituitary (gonadotropes, adrenocorticotropes, thyrotropes, growth hormones, and lactotropes)), cells of the respiratory system (including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, and dust cells), cells of the circulatory system (cardiac muscle cells) , including pericytes], cells of the digestive system [including stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, and S cells], enteroendocrine cells [including enterochromaffm cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage / bone / muscle], bone cells [including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts)], chondrocytes [including chondroblasts, chondrocytes], skin cells [including hair follicles, keratinocytes, and melanocytes (nevus cells)], muscle cells [including myocytes] ], urinary system cells [including podocytes, juxtaglomerular cells, intraglomerular mesangial cells / extraglomerular mesangial cells, renal proximal tubule brush border cells, macula densa cells], reproductive system cells [including sperm, Sertoli cells, Leydig cells, egg cells], and other cells [adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, keratinocyte hair stem cells, keratinocyte root sheath cells, root sheath cells of Huxley's layer, root sheath cells of Henle's layer, outer root sheath cells,Hair matrix cells (stem cells), moist stratified barrier epithelial cells, surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, urinary epithelial cells (lining the bladder and ureters), exocrine epithelial cells, salivary gland mucosal cells (secretion rich in polysaccharides), salivary gland serous cells (secretion rich in glycoprotein enzymes), von Ebner's gland cells in the tongue (cleansing the taste buds), mammary gland cells (secreting milk), lacrimal gland cells (secreting tears), auditory canal gland cells in the ear (secreting earwax), eccrine sweat gland dark cells (secreting glycoproteins) secrete substances), eccrine clear sweat gland cells (secrete small molecules), apocrine sweat gland cells (aromatic secretions, sex hormone sensitivity), Mollus cell glands in the eyelids (specialized sweat glands), sebaceous gland cells (secrete lipid-rich sebum), Bowman's gland cells in the nose (cleansing the olfactory epithelium), Brunner's gland cells in the duodenum (secrete enzymes and alkaline mucus), seminal vesicle cells (secrete seminal fluid components including fructose to allow sperm to swim), prostate cells (secrete seminal fluid components), bulbourethral gland cells (secrete mucus), Bartholin's gland cells (secrete vaginal lubrication), Little cell glands (secrete mucus), endometrial cells (secrete carbohydrates), respiratory Isolated goblet cells of the rectum and digestive tract (secreting mucus), gastric lining cells (secreting mucus), gastric zymogen cells (secreting pepsinogen), gastric oxyntic cells (secreting hydrochloric acid), pancreatic acinar cells (secreting bicarbonate and digestive enzymes), Paneth cells of the small intestine (secreting lysozyme), type II pneumocytes of the lung (secreting surfactant), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, growth hormone-producing cells, lactotrophs, thyrotropes, gonadotropes, adrenocorticotrophs, intermediate pituitary cells, giant cell neurosecretory cells , intestinal and respiratory tract cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophilic cells, adrenal cells, chromaffin cells, Leydig cells of the testes, theca interna cells of the follicle, lutein cells of ruptured follicles, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (secreting renin), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands, and urogenital tract), kidney, type I pneumocytes (lining the air spaces of the lung), pancreatic duct cells (central acinar cells), smooth muscle duct cells (those in sweat glands, salivary glands, mammary glands, etc.), duct cells (those in seminal vesicles, prostate, etc.),Epithelial cells lining closed body cavities, ciliated cells with propulsive functions, extracellular matrix-secreting cells, contractile cells, skeletal muscle cells, stem cells, cardiac muscle cells, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglial cells (in the central nervous system), neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, mast cells, helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes Blood cells, stem cells and committed progenitors (various types) of the blood and immune systems, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic neuron cells, sensory organ and peripheral neuron support cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (stem cells of spermatocytes), spermatozoa, nurse cells, follicle cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, and interstitial kidney cells.

[0230] Of particular interest are cancer cells. In some embodiments, the target cells are cancer cells. Non-limiting examples of cancer cells include acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acral hidradenoma, 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, adenomatous odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, enamelled leukemia, and leukemia. leukocyte fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, hematologic immunoblastic 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 ductal carcinoma, bile duct cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, cancer, carcinoma in situ, cancer of the penis, primary Cancer of unknown location, carcinosarcoma, Castleman's disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, bile duct carcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid 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 tumor, 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 tumors, Ewing family of sarcomas, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, inclusion malformation, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancercancer), gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granular cell tumor, hairy cell leukemia, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin's lymphoma Lymphoma), hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Klatzkin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, lentigo maligna melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphatic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant adenocarcinoma 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 carcinoma, medulloblastoma, medulloepithelioma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer of unknown primary, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer cancer, mucinous tumors, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cavity cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular oncology, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancercancer), oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, epithelial ovarian 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, sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, pituitary adenoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, polyembryomatous tumor, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary abdominal Membrane cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasms, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, signet ring cell carcinoma, skin cancer, small ovarian tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatin-secreting tumor, sooty wart, spinal cord tumor, vertebral tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer cancer), superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoid cancer, testicular cancer, thecoma, pharyngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, genitourinary neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Werner-Morrison syndrome, verrucous carcinoma, optic pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation within a cancer cell population, e.g., cancer stem cells. In some embodiments, the cancer is hematopoietic, e.g., lymphoma. The antigen can be a tumor-associated antigen.

[0231] In some embodiments, the target cells form tumors. Tumors treated using the methods herein may result in stabilized tumor growth (e.g., one or more tumors do not increase in size by more than 1%, 5%, 10%, 15%, or 20% and / or do not metastasize). In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or more. In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or more. In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or more. In some embodiments, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years, or more. In some embodiments, tumor size or tumor cell count 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 to below detection levels. In some embodiments, the 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, the 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, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years following treatment.

[0232] Target cell death 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). The degree of cell death can be determined by any suitable method. In some embodiments, the degree of cell death is determined relative to a starting state. For example, an individual can have a known starting amount of target cells, e.g., a starting cell mass of known size or a known concentration of circulating target cells. In such cases, the degree of cell death can be expressed as the ratio of viable cells to the starting cell population after treatment. In some embodiments, the degree of cell death can be determined by a suitable cell death assay. A variety of cell death assays are available, and a variety of detection methodologies can be utilized. Examples of detection methodologies include, but are not limited to, the use of cell stains, microscopy, flow cytometry, cell sorting, and combinations thereof.

[0233] When the tumor is subjected to surgical resection after completion of the treatment period, the effectiveness of the treatment in reducing tumor size can be determined by measuring the percentage of resected tissue that is necrotic (i.e., dead). In some embodiments, the treatment is therapeutically effective if the percentage of necrosis 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 percentage of necrosis of the resected tissue is 100%, i.e., no viable tumor tissue is present or detectable.

[0234] Exposing a target cell to an immune cell or population of immune cells disclosed herein can be performed either in vitro or in vivo. Exposing a target cell to an immune cell or population of immune cells generally refers to contacting and / or bringing the target cell into sufficient proximity with the immune cell such that an antigen (e.g., membrane-bound or non-membrane-bound) on the target cell can bind to the ligand-interacting domain of a 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 achieved by co-culturing the target cell and the immune cell. The target cell and the immune cell can be co-cultured, for example, as adherent cells or in suspension. The target cell and the immune cell can be co-cultured in various suitable types of cell culture media, for example, with additives, growth factors, ions, etc. Exposing a target cell to an immune cell or population of immune cells in vivo can, in some cases, be achieved by administering the immune cell to a subject, e.g., a human subject, and allowing the immune cell to localize to the target cell via the circulatory system. In some cases, immune cells can be delivered to an area adjacent to where the target cells are localized, for example, by direct injection.

[0235] Exposure can be 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.

[0236] In some embodiments, cells expressing the systems provided herein induce target cell death in an in vitro cell death assay. Cells expressing the systems provided herein may exhibit enhanced ability to induce target cell death compared to control cells not expressing the disclosed system. In some cases, the enhanced ability to induce target cell death 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 appropriate 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 cells with the target cells.

[0237] In some embodiments, the target polynucleotide can include one or more disease-associated genes and polynucleotides and signal transduction biochemical pathway-associated genes and polynucleotides. Examples of target polynucleotides include sequences associated with signal transduction biochemical pathways, such as signal transduction biochemical pathway-associated genes or polynucleotides. Examples of target polynucleotides include disease-associated genes or polynucleotides. A "disease-associated" gene or polynucleotide refers to any gene or polynucleotide that provides a transcription or translation product at an abnormal level or in an abnormal form in cells derived from disease-affected tissue compared to non-disease control tissue or cells. In some embodiments, this is a gene that is expressed at an abnormally high level. In some embodiments, this is a gene that is expressed at an abnormally low level. Altered expression may be correlated with the development and / or progression of a disease. A disease-associated gene also refers to a gene harboring a mutation or genetic variation that is directly responsible for or in linkage disequilibrium with a gene that is responsible for the etiology of a disease. The transcription or translation product may be known or unknown, and may be at a normal or abnormal level.

[0238] Examples of disease-associated genes and polynucleotides are available on the World Wide Web from the Johns Hopkins University, McKusick-Nathans Institute of Genetic Medicine (Baltimore, MD) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD). Exemplary genes associated with specific diseases and disorders are provided in Tables 2 and 3.

[0239] Mutations in these genes and pathways can result in the production of inappropriate proteins or inappropriate amounts of proteins, which affects function.

[0240] Promoters that can be used with the methods and compositions of the present disclosure include, for example, promoters that are active in eukaryotic, mammalian, non-human mammalian, or human cells. 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 a native or composite promoter.

[0241] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, human elongation factor-1 promoter (EF1), ubiquitin B promoter (UB), a hybrid construct containing the cytomegalovirus (CMV) enhancer fused to the chicken beta-active promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I. Promoters can be cell-, tissue-, or tumor-specific, such as the CD45 promoter, AFP promoter, human albumin promoter (Alb), MUC1 promoter, COX2 promoter, SP-B promoter, and OG-2 promoter. Promoters can be fungal promoters. Promoters can be plant promoters. Databases 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 contain appropriate sequences for amplifying expression. Other examples of promoters for expression vectors may include the myeloproliferative sarcoma virus enhancer, negative control region deletion, and dl587rev primer-binding site substitution (MND) promoter. Promoters for driving RNA may include RNA Pol III promoters (e.g., U6 or H1), Pol II promoters, and / or tRNA(val) promoters.

[0242] [Table 2A]

[0243] [Table 2B]

[0244] [Table 3A]

[0245] [Table 3B]

[0246] [Table 3C]

[0247] [Table 3D]

[0248] [Table 3E]

[0249] The disclosed systems and compositions are useful in a variety of other applications. For example, the disclosed systems and methods are useful in methods for modulating gene expression and / or cellular activity critical to cell proliferation, differentiation, transdifferentiation, and / or dedifferentiation during tissue (e.g., organ) growth, repair, regeneration, and / or engineering. Examples of tissues include epithelial, connective, nerve, muscle, organ, and other tissues. Other exemplary tissues include arteries, ligaments, skin, tendons, kidneys, nerves, liver, pancreas, bladder, bone, lungs, blood vessels, heart valves, cartilage, eyes, etc.

[0250] 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 present invention be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not intended 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 is to be understood that all aspects of the present 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 is to be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. Accordingly, it is contemplated that the present invention shall also cover any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. [Example]

[0251] Various aspects of the present disclosure are further illustrated by the following non-limiting examples.

[0252] Example 1 Constitutive expression of the GMP-containing system in γδ T cells. 1A-1B schematically illustrate the expression of the GMP-containing system in γδ T cells. One or more components of the system can be constitutively expressed in γδ T cells. One or more components of the GMP-containing system can be under the control of an exogenous promoter. The system can be expressed in γδ T cells for cancer / tumor antigen-mediated modulation of gene regulation in γδ T cells.

[0253] Figure 1A schematically illustrates examples of vectors (e.g., viral vectors) for use in the subject systems and methods. Each vector can include a polynucleotide encoding a chimeric polypeptide (e.g., a chimeric receptor polypeptide and / or a chimeric adapter polypeptide) and / or a guide RNA (e.g., sgRNA) configured to bind to a target gene (e.g., a target DNA sequence) in a cell. Vector 1 can include a chimeric receptor polypeptide operably linked to an MND promoter. The chimeric receptor polypeptide of Vector 1 can include a CAR-linked protease (e.g., TEV). The chimeric receptor polypeptide of Vector 1 can also include a post-transcriptional regulatory element (PRE) (e.g., a woodchuck hepatitis virus PRE (WPRE)) linked to the protease.

[0254] Vector 2a may comprise a chimeric adapter polypeptide operably linked to an exogenous MND promoter. The chimeric adapter polypeptide may comprise an adapter (e.g., a linker for T cell activation (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds to a portion of the chimeric receptor polypeptide (e.g., extracellular, transmembrane, and / or intracellular domains) and / or (ii) translocates in proximity to the chimeric receptor polypeptide. The adapter is linked to a cleavage recognition site (e.g., a TEV cleavage site (TCS)). The chimeric adapter polypeptide may also comprise a GMP linked to the cleavage recognition site, which includes an actuator portion (e.g., dCas9 fused to a gene repressor, e.g., KRAB). The chimeric adapter polypeptide may also comprise a PRE (e.g., a WPRE) linked to the GMP. Furthermore, Vector 2a may comprise a guide RNA (e.g., an sgRNA operable to target a portion of a gene encoding CD39) operably linked to a U6 promoter. The U6 promoter may be positioned downstream of the MND promoter and the gene of interest operably linked to the MND promoter.

[0255] Vector 2b may comprise a chimeric adapter polypeptide operably linked to an exogenous MND promoter. The chimeric adapter polypeptide may comprise an adapter (e.g., a linker for T cell activation (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds to a portion of the chimeric receptor polypeptide (e.g., extracellular, transmembrane, and / or intracellular domains) and / or (ii) translocates in proximity to the chimeric receptor polypeptide. The adapter is linked to a cleavage recognition site (e.g., a TEV cleavage site (TCS)). The chimeric adapter polypeptide may also comprise a GMP linked to the cleavage recognition site, which includes an actuator portion (e.g., dCas9 fused to a gene activator, e.g., VP64, p65, SAM, VPR, etc.). The chimeric adapter polypeptide may also comprise a PRE (e.g., a WPRE) linked to the GMP. Additionally, vector 2b can include a guide RNA (e.g., an sgRNA operable to target a portion of a gene encoding serpin B4 and / or serpin B9) operably linked to a U6 promoter. The U6 promoter can be positioned downstream of the MND promoter and a gene of interest operably linked to the MND promoter.

[0256] Vector 2c may comprise a chimeric adapter polypeptide operably linked to an exogenous MND promoter. The chimeric adapter polypeptide may comprise an adapter (e.g., a linker for T cell activation (LAT) in the case of TCR signaling, a DAP10 in the case of NKG2D signaling, etc.) that (i) binds to a portion of the chimeric receptor polypeptide (e.g., an extracellular, transmembrane, and / or intracellular domain) and / or (ii) translocates in proximity to the chimeric receptor polypeptide. The adapter is linked to a cleavage recognition site (e.g., a TEV cleavage site (TCS)). The chimeric adapter polypeptide may also comprise a GMP linked to the cleavage recognition site, which includes an actuator portion (e.g., dCas9 fused to a gene activator, e.g., VP64, p65, SAM, VPR, etc.). The chimeric adapter polypeptide may also comprise a PRE (e.g., a WPRE) linked to the GMP. Additionally, vector 2c can include a guide RNA (e.g., an sgRNA operable to target a portion of the gene encoding c-FLIP) operably linked to a U6 promoter. The U6 promoter can be positioned downstream of the MND promoter and the gene of interest operably linked to the MND promoter.

[0257] Referring to Figure 1B, γδ T cells can express the γδ TCR complex. In some cases, γδ T cells can also express NKG2D and its associated adaptor DAP10. γδ T cells can be administered with a chimeric receptor polypeptide comprising a cancer / tumor antigen-binding domain, a transmembrane domain, and an intracellular domain comprising a γδ T cell signaling domain and a TEV. γδ T cells can also be administered with a chimeric adapter polypeptide comprising an adapter that binds to and / or migrates in proximity to the chimeric receptor polypeptide (e.g., the cell signaling domain of the chimeric receptor polypeptide) and a GMP comprising an actuator portion. The chimeric receptor and adapter can be administered using a viral vector. The chimeric receptor and adapter can be constitutively expressed in γδ T cells. Upon binding of a tumor antigen to the antigen-binding domain of the chimeric receptor polypeptide, the chimeric adapter polypeptide can migrate in proximity to the chimeric receptor polypeptide, and the TEV of the chimeric receptor polypeptide can cleave the actuator portion from the GMP at the cleavage recognition site of the chimeric receptor polypeptide. The released actuator moieties may translocate to the nuclei of γδ T cells and (i) downregulate the expression of CD39 and / or (ii) upregulate the expression of serpin B9 / 4 and / or cFLIP.

[0258] Example 2 Bisphosphonate-induced expression of GMP-containing systems in γδ T cells. 2A-2B schematically illustrate bisphosphonate (e.g., zoledronate)-induced expression of the GMP-containing system in γδ T cells. One or more components of the system can be constitutively expressed in γδ T cells. One or more components of the system can be conditionally expressed, either directly or indirectly by bisphosphonates. One or more components of the GMP-containing system can be under the control of an exogenous promoter. The system can be expressed in γδ T cells for cancer / tumor antigen-mediated modulation of γδ T cell gene regulation.

[0259] Figure 2A schematically illustrates examples of vectors (e.g., viral vectors) for use in the subject systems and methods. Each vector can include a polynucleotide encoding a chimeric polypeptide (e.g., a chimeric receptor polypeptide and / or a chimeric adapter polypeptide) and / or a guide RNA (e.g., sgRNA) configured to bind to a target gene (e.g., a target DNA sequence) in a cell. Vector 1 can include a chimeric receptor polypeptide operably linked to an MND promoter. The chimeric receptor polypeptide of Vector 1 can include a CAR-linked protease (e.g., TEV). The chimeric receptor polypeptide of Vector 1 can also include a post-transcriptional regulatory element (PRE) (e.g., a woodchuck hepatitis virus PRE (WPRE)) linked to the protease.

[0260] Vector 2a can include a chimeric adapter polypeptide operably linked to an exogenous promoter, such as a LAG3 promoter or a FAS promoter. The exogenous promoter can be an immune checkpoint for γδ T cells. The chimeric adapter polypeptide can include an adapter (e.g., linker for T cell activation (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds to a portion of the chimeric receptor polypeptide (e.g., extracellular, transmembrane, and / or intracellular domains) and / or (ii) moves in close proximity to the chimeric receptor polypeptide. The adapter is linked to a cleavage recognition site (e.g., a TEV cleavage site (TCS)). The chimeric adapter polypeptide can also include a GMP linked to the cleavage recognition site, which includes an actuator portion (e.g., dCas9 fused to a gene repressor, e.g., KRAB). The chimeric adapter polypeptide can also include a PRE (e.g., a WPRE) linked to the GMP. Additionally, vector 2a may include a guide RNA (e.g., an sgRNA operable to target a portion of the gene encoding CD39) operably linked to a U6 promoter. The U6 promoter may be positioned downstream of the LAG3 promoter or FAS promoter and a gene of interest operably linked to the LAG3 promoter or FAS promoter.

[0261] Vector 2b can include a chimeric adapter polypeptide operably linked to an exogenous promoter, such as the LAG3 promoter or the FAS promoter. The exogenous promoter can be an immune checkpoint for γδ T cells. The chimeric adapter polypeptide can include an adapter (e.g., linker for T cell activation (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds to a portion of the chimeric receptor polypeptide (e.g., extracellular, transmembrane, and / or intracellular domains) and / or (ii) translocates in close proximity to the chimeric receptor polypeptide. The adapter is linked to a cleavage recognition site (e.g., a TEV cleavage site (TCS)). The chimeric adapter polypeptide can also include a GMP linked to the cleavage recognition site, which includes an actuator portion (e.g., dCas9 fused to a gene activator, e.g., VP64, p65, SAM, VPR, etc.). The chimeric adapter polypeptide can also include a PRE (e.g., a WPRE) linked to the GMP. Additionally, vector 2b can include a guide RNA (e.g., an sgRNA operable to target a portion of a gene encoding serpin B4 and / or serpin B9) operably linked to a U6 promoter. The U6 promoter can be positioned downstream of the LAG3 promoter or FAS promoter and a gene of interest operably linked to the LAG3 promoter or FAS promoter.

[0262] Vector 2c can include a chimeric adapter polypeptide operably linked to an exogenous promoter, such as the LAG3 promoter or the FAS promoter. The exogenous promoter can be an immune checkpoint for γδ T cells. The chimeric adapter polypeptide can include an adapter (e.g., linker for T cell activation (LAT) in the case of TCR signaling, DAP10 in the case of NKG2D signaling, etc.) that (i) binds to a portion of the chimeric receptor polypeptide (e.g., extracellular, transmembrane, and / or intracellular domains) and / or (ii) translocates in close proximity to the chimeric receptor polypeptide. The adapter is linked to a cleavage recognition site (e.g., a TEV cleavage site (TCS)). The chimeric adapter polypeptide can also include a GMP linked to the cleavage recognition site, which includes an actuator portion (e.g., dCas9 fused to a gene activator, e.g., VP64, p65, SAM, VPR, etc.). The chimeric adapter polypeptide can also include a PRE (e.g., a WPRE) linked to the GMP. Additionally, vector 2c can include a guide RNA (e.g., an sgRNA operable to target a portion of the gene encoding c-FLIP) operably linked to a U6 promoter. The U6 promoter can be positioned downstream of the LAG3 promoter or FAS promoter and the gene of interest operably linked to the LAG3 promoter or FAS promoter.

[0263] Referring to Figure 2B, γδ T cells can express the γδ TCR complex. In some cases, γδ T cells can also express NKG2D and its associated adaptor DAP10. γδ T cells can be administered with a chimeric receptor polypeptide comprising a cancer / tumor antigen-binding domain, a transmembrane domain, and an intracellular domain comprising a γδ T cell signaling domain and TEV. γδ T cells can also be administered with a chimeric adapter polypeptide comprising an adaptor that binds to and / or moves in proximity to the chimeric receptor polypeptide (e.g., the cell signaling domain of the chimeric receptor polypeptide) and a GMP comprising an actuator portion. The chimeric receptor and adaptor can be administered using a viral vector. The chimeric receptor can be constitutively expressed in γδ T cells. The chimeric adaptor can be conditionally expressed under treatment with a bisphosphonate (e.g., zoledronate). Zolendronate may be metabolized by cancer / tumor cells into at least a γδ T cell activator (e.g., IPP), which may be secreted or displayed on the surface of the cancer / tumor cells. The activator may then bind to a receptor (e.g., γδ TCR) on the γδ T cell to activate the γδ T cell. Activation of the γδ T cell may result in activation of one or more intracellular signaling pathways that promote expression of genes under the control of the LAG3 promoter or FAS promoter, whether such promoter is endogenous or exogenous to the γδ T cell. Because the chimeric adapter polypeptide is under the control of the exogenous LAG3 promoter or FAS promoter, activation of the γδ T cell by a by-product of the bisphosphonate subsequently induces expression of the chimeric adapter polypeptide. Furthermore, upon binding of a tumor antigen to the antigen-binding domain of the chimeric receptor polypeptide, the chimeric adaptor polypeptide may move into proximity with the chimeric receptor polypeptide, and the TEV of the chimeric receptor polypeptide may cleave the actuator moiety from GMP at the cleavage recognition site of the chimeric receptor polypeptide. The released actuator moiety may translocate to the nucleus of γδ T cells and (i) downregulate expression of CD39 and / or (ii) upregulate expression of serpin B9 / 4 and / or cFLIP.

[0264] Example 3 Knock-in of the GMP-containing system under the control of an endogenous promoter in γδ T cells. 3A-3B schematically illustrate administration of a GMP-containing system in a γδ T cell. Polynucleotides encoding one or more components of the system (i.e., exogenous expression cassettes) can be inserted (e.g., knocked in) into the genome of a γδ T cell using various gene editing techniques (e.g., CRISPR / Cas9 or functional variants thereof). Such expression cassettes can be inserted under the control of an endogenous promoter in the γδ T cell. Expression of an endogenous polynucleotide in the γδ T cell under the control of the same endogenous promoter may or may not be disrupted by the insertion of the exogenous expression cassette. In some cases, various gene editing techniques can be used to delete (e.g., knock out) an endogenous polynucleotide in the γδ T cell under the control of the same endogenous promoter.

[0265] Figure 3A illustrates a schematic diagram of an exogenous expression cassette knocked into a γδ T cell under the control of an endogenous promoter. Exogenous expression cassette 1 may include a first polynucleotide sequence encoding a chimeric receptor polypeptide comprising at least a portion of NKG2D linked to TEV. The first polynucleotide sequence may further include a terminator (e.g., SV40, hGH, BGH, or rbGlob). Exogenous expression cassette 1 may further include a second polynucleotide sequence. The second polynucleotide sequence may include an exogenous promoter MND that controls expression of a chimeric adapter polypeptide. The chimeric adapter polypeptide may include an adapter portion (e.g., LAT) linked to a GMP comprising dCas9 and VP64. Furthermore, the second polynucleotide sequence may include an exogenous promoter U6 that controls expression of a nucleotide sequence (e.g., serpinB9 / 4 sgRNA) to target a specific gene (e.g., serpinB9 / 4) in a γδ T cell. In some cases, the endogenous promoter of the γδ T cells used to control expression of exogenous expression cassette 1 can be the endogenous NKG2D promoter. In some cases, the endogenous NKG2D gene under the control of the endogenous NKG2D promoter can be knocked out.

[0266] Exogenous expression cassette 2 may comprise a first polynucleotide sequence encoding a chimeric receptor polypeptide comprising at least a portion of a Vy9 / V52 TCR linked to TEV. The first polynucleotide sequence may further comprise a terminator (e.g., SV40, hGH, BGH, or rbGlob). Exogenous expression cassette 2 may further comprise a second polynucleotide sequence. The second polynucleotide sequence may comprise an exogenous promoter MND that controls expression of a chimeric adapter polypeptide. The chimeric adapter polypeptide may comprise an adapter portion (e.g., LAT) linked to a GMP comprising dCas9 and VP64. Furthermore, the second polynucleotide sequence may comprise an exogenous promoter U6 that controls expression of a nucleotide sequence (e.g., serpinB9 / 4 sgRNA) to target a specific gene (e.g., serpinB9 / 4) in γδ T cells. In some cases, the endogenous promoter of the γδ T cell used to control expression of exogenous expression cassette 2 can be the endogenous Vγ9 / Vδ2 TCR promoter. In some cases, the endogenous Vγ9 / Vδ2 TCR gene under the control of the endogenous Vγ9 / Vδ2 TCR promoter can be knocked out.

[0267] Exogenous expression cassette 3 may comprise a first polynucleotide sequence encoding a chimeric receptor polypeptide comprising a CAR linked to TEV. The first polynucleotide sequence may further comprise a terminator (e.g., SV40, hGH, BGH, or rbGlob). Exogenous expression cassette 3 may further comprise a second polynucleotide sequence. The second polynucleotide sequence may comprise an exogenous promoter MND that controls expression of a chimeric adapter polypeptide. The chimeric adapter polypeptide may comprise an adapter portion (e.g., LAT) linked to a GMP comprising dCas9 and VP64. Furthermore, the second polynucleotide sequence may comprise an exogenous promoter U6 that controls expression of a nucleotide sequence (e.g., serpinB9 / 4 sgRNA) to target a specific gene (e.g., serpinB9 / 4) in γδ T cells. In some cases, the endogenous promoter of the γδ T cell used to control expression of exogenous expression cassette 3 can be an endogenous promoter (e.g., a LAG, FAS, and / or KIR2DS promoter). In some cases, the endogenous LAG, FAS, and / or KIR2DS gene under the control of the endogenous promoter can be knocked out.

[0268] Figure 3B schematically illustrates regulation of a target polynucleotide by the system described in Figure 3A. In some cases, as shown in Figure 3B (top), after the actuator portion of the GMP is released from the chimeric adaptor polypeptide, the actuator portion comprising KRAB may bind to or be adjacent to a polynucleotide encoding CD39, thereby downregulating or inhibiting expression of CD39. In some cases, as shown in Figure 3B (bottom), after the actuator portion of the GMP is released from the chimeric adaptor polypeptide, the actuator portion comprising a gene activator (e.g., VPR, VP64, p300, SunT, MS2-p65-HSF1, etc.) may bind to or be adjacent to a polynucleotide encoding serpin B9 / 4 or c-FLIP, thereby upregulating or initiating expression of serpin B9 / 4 or c-FLIP.

[0269] In some cases, the exogenous expression cassette shown in Figure 3A may be knocked in under the control of the endogenous beta-2 microglobulin (B2M) promoter in γδ T cells, knocking out the endogenous polynucleotide encoding endogenous B2M. In such cases, γδ T cells may have an alternative escape mechanism against NK cells.

[0270] Example 4 Treatment of engineered γδ T cells with bisphosphonates. In the present disclosure, the activity of experimental γδ T cells engineered to express (e.g., constitutively and / or conditionally) any one of the systems described herein may be compared to one or more controls, including, but not limited to, (1) unengineered γδ T cells and / or (2) engineered γδ T cells with a control gRNA that does not bind to the target polynucleotide of the engineered γδ T cells. In some cases, alpha (α)-beta (β) T cells may be used as a negative control in experiments using zoledronate, because alpha (α)-beta (β) T cells do not respond (e.g., directly or indirectly) to zoledronate.

[0271] γδ T cells can be engineered to express any one of the systems described herein in this disclosure by utilizing viral vectors (e.g., gammaretroviral vectors) or gene editing techniques (e.g., CRISPR / Cas9).

[0272] In in vitro experiments, engineered γδ T cells and control cells can be treated with or without zoledronate, followed by assessment of serpin B9 / 4, c-FLIP, and / or CD39 expression in the engineered γδ T cells and control cells (e.g., Western blot, polymerase chain reaction (PCR), etc.).

[0273] In another set of in vitro experiments, engineered γδ T cells and control cells can be treated with or without zoledronate. Subsequently, expression of serpin B9 / 4, c-FLIP, and / or CD39 in the engineered γδ T cells and control cells can be assessed (e.g., Western blot, polymerase chain reaction (PCR), etc.). The engineered γδ T cells and control cells can then be treated with a death-inducing compound (e.g., recombinant Fas ligand) for one or more different time points. After one or more different time points, survival and / or proliferation of the engineered γδ T cells and control cells can be measured (e.g., cell count, DNA and / or protein amount enumeration, etc.).

[0274] In a different set of in vitro experiments, engineered γδ T cells and control cells can be treated with or without zoledronate. Subsequently, the expression of serpin B9 / 4, c-FLIP, and / or CD39 in the engineered γδ T cells and control cells can be assessed (e.g., Western blot, polymerase chain reaction (PCR), etc.). The engineered γδ T cells and control cells can then be exposed to one or more cells expressing a ligand (e.g., an antigen) that binds to the ligand-binding domain of a chimeric polypeptide receptor (e.g., CAR) of the engineered γδ T cell line. The one or more cells can be cancer / tumor cells expressing the ligand or model cells expressing the ligand (e.g., Raji cells). The tumor cytotoxicity of the engineered γδ T cells and control cells can then be measured. Furthermore, the proliferation and / or survival of the engineered γδ T cells and control cells can be measured.

[0275] For in vivo experiments, engineered γδ T cells and control cells may each be implanted into NOD scid gamma (NSG) mice. The NSG mice may then be treated with zoledronate. The NSG mice may be challenged with a model tumor (e.g., by implanting tumor cells to create a model subcutaneous tumor) before, during, and / or following zoledronate treatment. In some cases, the NSG mice may be challenged with a model tumor following zoledronate treatment. At one or more time points, tumor burden and survival in the NSG mice may be assessed, along with the expansion, persistence, and / or activity of the engineered γδ T cells and control cells in the NSG mice.

[0276] In another set of in vivo experiments, NSG mice may be loaded with model tumors (e.g., by implanting tumor cells to create model subcutaneous tumors). Engineered γδ T cells and control cells may then be implanted into the NSG mice. In some cases, the NSG mice may be treated with zoledronate before, during, and / or following implantation of the engineered γδ T cells and control cells, respectively. In some cases, the NSG mice may be treated with zoledronate following implantation of the engineered γδ T cells and control cells, respectively. At one or more time points, tumor burden and survival in the NSG mice may be assessed, along with the expansion, persistence, and / or activity of the engineered γδ T cells and control cells in the NSG mice. In some cases, when tumors are assessed to have cleared from one or more of the NSG mice, the NSG mice may be challenged one or more times with the same or different model tumors, in the absence of any further zoledronate treatment.

Claims

1. 1. A method for modulating the activity of a gamma delta T cell, comprising administering to the gamma delta T cell a gene modulating polypeptide (GMP) comprising an actuator portion configured to modulate expression of a target polynucleotide in the gamma delta T cell, wherein the target polynucleotide encodes a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of the death-inducing protein, and / or (iii) a hydrolase.

2. 2. The method of claim 1, wherein modulating the activity of the gamma delta T cells comprises reducing gamma delta T cell fratricide.

3. 2. The method of claim 1, wherein modulating the activity of the gamma delta T cells comprises prolonging activation of the gamma delta T cells.

4. 10. The method of claim 1, wherein the actuator moiety is an RNA-guided actuator moiety or a variant thereof, and the RNA-guided actuator moiety forms a complex with the target polynucleotide.

5. 3. The method of claim 2, wherein the actuator moiety is a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity.

6. 3. The method of claim 2, wherein the actuator moiety is Cas9 and / or Cpf1.

7. 10. The method of claim 1, wherein the actuator portion comprises an activator effective to increase expression of the target polynucleotide.

8. 10. The method of claim 1, wherein the actuator portion comprises a repressor effective to reduce expression of the target polynucleotide.

9. The method of claim 1, wherein the polypeptide is the death-inducing protein.

10. 10. The method of claim 9, wherein the death-inducing protein comprises Fas, Fas ligand, granzyme B, granzyme M, and / or perforin.

11. 10. The method of claim 9, wherein said modulating expression of said target polynucleotide comprises decreasing said expression of said target polynucleotide.

12. The method of claim 1, wherein the polypeptide is the inhibitor of the death-inducing protein.

13. The method of claim 12, wherein the inhibitor comprises serpin B9, serpin B4, and / or c-FLIP.

14. 13. The method of claim 12, wherein said modulating expression of said target polynucleotide comprises increasing said expression of said target polynucleotide.

15. The method of claim 1 , wherein the polypeptide is the hydrolase.

16. 16. The method of claim 15, wherein the polypeptide comprises CD39.

17. 16. The method of claim 15, wherein said modulating expression of said target polynucleotide comprises decreasing said expression of said target polynucleotide.

18. 2. The method of claim 1, further comprising treating the gamma delta T cells with a virus and administering the GMP to the gamma delta T cells.

19. 19. The method of claim 18, further comprising integrating a nucleic acid sequence encoding the GMP into the genome of the gamma delta T cell by using the virus.

20. 19. The method of claim 18, wherein the virus is a retrovirus.

21. 22. The method of claim 21, wherein the retrovirus is a gamma-retrovirus selected from the group consisting of Moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), and spleen focus forming virus (SFFV), and / or variants thereof.

22. 2. The method of claim 1, further comprising treating the gamma delta T cells with a nuclease and administering the GMP to the gamma delta T cells.

23. 23. The method of claim 22, further comprising inserting a nucleic acid sequence encoding said GMP into the genome of said gamma delta T cell by using said nuclease.

24. 24. The method of claim 23, wherein the nuclease comprises a CRISPR-associated polypeptide (Cas), a zinc finger nuclease (ZFN), a zinc finger-associated gene regulatory polypeptide, a transcription activator-like effector nuclease (TALEN), a transcription activator-like effector-associated gene regulatory polypeptide, a meganuclease, a natural master transcription factor, an epigenetic modification enzyme, a recombinase, a flippase, a transposase, an RNA-binding protein (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

25. 2. The method of claim 1, further comprising administering to the gamma delta T cell a chimeric polypeptide comprising the GMP, wherein the chimeric polypeptide is operable to release the GMP from the chimeric polypeptide in response to a stimulus, and the released GMP is operable to regulate expression of the target polynucleotide in the gamma delta T cell.

26. 2. The method of claim 1, further comprising administering to the gamma delta T cell a chimeric polypeptide comprising the GMP and a nuclear translocation domain, wherein the nuclear translocation domain is operable to translocate the chimeric polypeptide to the nucleus of the gamma delta T cell in response to a stimulus, and the translocated GMP is operable to regulate expression of the target polynucleotide in the gamma delta T cell.

27. 1. A system for modulating the activity of a gamma delta T cell, comprising a gene modulating polypeptide (GMP) comprising an actuator portion configured to modulate expression of a target polynucleotide in the gamma delta T cell, wherein the target polynucleotide encodes a polypeptide comprising (i) a death-inducing protein, (ii) an inhibitor of the death-inducing protein, and / or (iii) a hydrolase.

28. 28. The system of claim 27, wherein the GMP is operable to reduce gamma delta T cell fratricide.

29. 28. The system of claim 27, wherein the GMP is operable to prolong activation of the gamma delta T cells.

30. 28. The system of claim 27, wherein the actuator moiety is an RNA-guided actuator moiety or a variant thereof, and the RNA-guided actuator moiety forms a complex with the target polynucleotide.

31. 31. The system of Claim 30, wherein the actuator moiety is a CRISPR-associated (Cas) protein or a fragment thereof that substantially lacks DNA cleavage activity.

32. 31. The system of claim 30, wherein the actuator moiety is Cas9 and / or Cpf1.

33. 28. The system of claim 27, wherein the actuator portion comprises an activator effective to increase expression of the target polynucleotide.

34. 28. The system of claim 27, wherein the actuator moiety comprises a repressor effective to reduce expression of the target polynucleotide.

35. 28. The system of claim 27, wherein the polypeptide is the death-inducing protein.

36. 36. The system of claim 35, wherein the death-inducing protein comprises Fas, Fas ligand, granzyme B, granzyme M, and / or perforin.

37. 36. The system of claim 35, wherein said modulating expression of said target polynucleotide comprises decreasing said expression of said target polynucleotide.

38. 28. The system of claim 27, wherein the polypeptide is the inhibitor of the death-inducing protein.

39. 39. The system of claim 38, wherein the inhibitor comprises serpin B9, serpin B4, and / or c-FLIP.

40. 39. The system of claim 38, wherein said modulating expression of said target polynucleotide comprises increasing said expression of said target polynucleotide.

41. 28. The system of claim 27, wherein the polypeptide is the hydrolase.

42. 42. The system of claim 41, wherein the polypeptide comprises CD39.

43. 42. The system of claim 41 , wherein said modulating expression of said target polynucleotide comprises decreasing said expression of said target polynucleotide.

44. 28. The system of claim 27, wherein the nucleic acid sequence encoding the GMP is integrated into the genome of the gamma delta T cell by a virus.

45. 45. The system of claim 44, wherein the virus is a retrovirus.

46. 46. ​​The system of claim 45, wherein the retrovirus is a gamma-retrovirus selected from the group consisting of Moloney murine leukemia virus (MMLV), murine stem cell virus (MSCV), and spleen focus forming virus (SFFV), and / or variants thereof.

47. 28. The system of claim 27, wherein the nucleic acid sequence encoding the GMP is integrated into the genome of the gamma delta T cell by a nuclease.

48. 48. The system of claim 47, wherein the nuclease comprises a CRISPR-associated polypeptide (Cas), a zinc finger nuclease (ZFN), a zinc finger-associated gene regulatory polypeptide, a transcription activator-like effector nuclease (TALEN), a transcription activator-like effector-associated gene regulatory polypeptide, a meganuclease, a natural master transcription factor, an epigenetic modification enzyme, a recombinase, a flippase, a transposase, an RNA-binding protein (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

49. 28. The system of claim 27, further comprising a chimeric polypeptide comprising the GMP, operable to release the GMP from the chimeric polypeptide in response to a stimulus, wherein the released GMP is operable to regulate expression of the target polynucleotide in the gamma delta T cell.

50. 28. The system of claim 27, further comprising a chimeric polypeptide comprising the GMP and a nuclear translocation domain, wherein the nuclear translocation domain is operable to translocate the chimeric polypeptide to the nucleus of the gamma delta T cell in response to a stimulus, and the translocated GMP is operable to regulate expression of the target polynucleotide in the gamma delta T cell.

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