Chimeric polypeptide system and gene modulation method

Abstract

Certain aspects of the present disclosure provide systems, compositions and methods for modulating expression or activity of a target protein in a cell. In some cases, the present disclosure provides a system comprising an actuator moiety capable of complexing with a target polynucleotide sequence to modulate the expression or activity of the target protein. The actuator portion may be heterologous with the cell. The actuator portion may be capable of being activated when the cell is exposed to an external stimulus. The actuator portion can be activated to modulate the expression or activity of the target protein when the cell is exposed to the external stimulus.

Description

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 357,728, filed on July 1, 2022, which is incorporated herein by reference in its entirety. Background Art

[0002] Cancer (e.g., neoplasm, tumor) is a large class of diseases involving abnormal cell growth in many body tissues. Cancer can invade or spread to other parts of the body. As the leading cause of death worldwide, cancer causes the death of about 10 million people each year. Non-limiting examples of body tissues attacked by cancer include lung, prostate, colorectal, stomach, liver, breast, colon, rectum, cervix and thyroid. In order to treat or control cancer, different therapies have been developed, for example, small molecules, antibodies and adoptive cell therapy (e.g., cellular immunotherapy). Summary of the invention

[0003] The present disclosure provides methods and systems for adoptive cell therapy to treat subjects suffering from or suspected of suffering from conditions such as cancer. The methods and systems of the present disclosure can be used, for example, to enhance the activity (e.g., anti-tumor activity) of cellular immunotherapy (e.g., cancer therapy using autologous or allogeneic immune cells).

[0004] In one aspect, the present disclosure provides a system for regulating the expression or activity of a target protein of a cell, the system comprising an actuator portion capable of complexing with a target polynucleotide sequence in the cell, wherein the actuator portion is heterologous to the cell, and wherein the target polynucleotide sequence is endogenous to the cell, wherein the complexing causes at least about a 10% change in the expression or the activity of the target protein as compared to a control cell, wherein the complexing is sufficient to cause the change without editing the target polynucleotide sequence, and wherein the target protein comprises one or more members selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), basic leucine zipper transcription factor ATF-like (BATF), inhibitor of DNA binding / differentiation (ID), T-box transcription factor (TBX), c-Jun.

[0005] In another aspect, the present disclosure provides a system for regulating the expression or activity of a target protein in a cell, the system comprising an actuator portion capable of complexing with a target polynucleotide sequence in the cell, wherein the actuator portion is heterologous to the cell and can be activated for the complexing when the cell is exposed to an external stimulus, and wherein the target polynucleotide sequence is endogenous to the cell, wherein upon the exposure, the actuator portion is activated for the complexing to cause a change in the expression or the activity of the target protein, wherein the complexing is sufficient to cause the change to occur without editing the target polynucleotide sequence, and wherein the target protein comprises an inositol phosphatase containing a Src homology 2 domain (SHIP) or β-2-microglobulin (B2M) and a TGFβ receptor (TGFbR).

[0006] In another aspect, the present disclosure provides a system comprising a guide nucleic acid molecule designed to bind to a target polynucleotide sequence for regulating the expression or activity of a target protein in a cell, wherein the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4,000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, or between about 2,500 bases and about 3,000 bases from the TSS of the gene encoding the target protein. The target protein is between about 1,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base, wherein the target protein is selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), inositol phosphatase containing Src homology 2 domain (SHIP), basic leucine zipper transcription factor ATF-like (BATF), beta-2-microglobulin (B2M), DNA binding / differentiation inhibitor (ID), T-box transcription factor (TBX), c-Jun and TGFβ receptor (TGFbR).

[0007] In another aspect, the present disclosure provides a system comprising an actuator portion capable of binding to a target polynucleotide sequence for regulating the expression or activity of a target protein of a cell, wherein the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4,000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, or between about 2,000 bases and about 3,000 bases from the TSS of the gene encoding the target protein. The target protein is between about 0 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base, wherein the target protein is selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), inositol phosphatase containing Src homology 2 domain (SHIP), basic leucine zipper transcription factor ATF-like (BATF), beta-2-microglobulin (B2M), DNA binding / differentiation inhibitor (ID), T-box transcription factor (TBX), c-Jun and TGFβ receptor (TGFbR).

[0008] On the other hand, the present disclosure provides a method for regulating the expression or activity of a target protein in a cell, the method comprising: (a) forming a complex comprising an actuator portion and a target polynucleotide sequence in the cell, wherein the actuator portion is heterologous to the cell and wherein the target polynucleotide sequence is endogenous to the cell; and (b) in response to the formation, inducing a change in the expression or activity of the target protein of at least about 10% as compared to the situation in the control cell, wherein the formation of the complex is sufficient to cause the change to occur without editing the target polynucleotide sequence, wherein the target protein comprises one or more members selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), basic leucine zipper transcription factor ATF-like (BATF), DNA binding / differentiation inhibitor (ID), T-box transcription factor (TBX), c-Jun and TGFβ receptor (TGFbR).

[0009] In another aspect, the present disclosure provides a method for regulating the expression or activity of a target protein in a cell, the method comprising: (a) exposing the cell to an external stimulus to activate an actuator portion to complex with a target polynucleotide sequence in the cell, wherein the actuator portion is heterologous to the cell, and wherein the target polynucleotide sequence is endogenous to the cell; and (b) in response to the complex, inducing a change in the expression or activity of the target protein, wherein the formation of the complex is sufficient to cause the change to occur without editing the target polynucleotide sequence, wherein the target protein comprises an inositol phosphatase containing a Src homology 2 domain (SHIP) or beta-2-microglobulin (B2M).

[0010] Additional aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only exemplary embodiments of the present disclosure are shown and described. It should be appreciated that the present disclosure is capable of other and different embodiments, and that its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Therefore, the drawings and descriptions are to be regarded as illustrative rather than restrictive in nature.

[0011] References

[0012] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and accompanying drawings (also referred to herein as "figures") which set forth exemplary embodiments in which the principles of the present invention are utilized, wherein:

[0014] Figure 1

[0015] [ Figure 1 ] Figure 1 A to Figure 1 D schematically illustrates the release of the actuator moiety from GMP in a system comprising a receptor undergoing phosphorylation; Figure 1 E- Figure 1 H schematically illustrates the release of the actuator moiety from GMP in a system comprising a receptor undergoing a conformational change.

[0016] Figure 2

[0017] [ Figure 2 ] Figure 2 A to Figure 2 D schematically shows the release of the actuator moiety from GMP in a different system comprising a receptor that undergoes phosphorylation upon ligand binding; Figure 2 E to Figure 2 H schematically illustrates the release of the actuator moiety from GMP in a system comprising a receptor undergoing a conformational change.

[0018] Figure 3

[0019] [ Figure 3 ] Figure 3 A to Figure 3 D schematically illustrates the release of the actuator moiety from GMP in a system comprising at least two adaptor polypeptides and a receptor undergoing phosphorylation; Figure 3 E to Figure 3 H schematically illustrates the release of the actuator moiety from a GMP in a system comprising at least two adaptor polypeptides and a receptor undergoing a conformational change.

[0020] Figure 4

[0021] [ Figure 4 ][ Figure 4 ] schematically illustrates conditionally inducible expression of the actuator portion via chimeric receptor signaling.

[0022] Figure 5

[0023] [Figure 5] Figure 5A and Figure 5B Examples of guide nucleic acid molecules for the ID3 gene and the relative expression levels of ID3 when modulated by the systems and methods of the present disclosure are shown.

[0024] Figure 6

[0025] [Figure 6] Fig. 6A and Figure 6B Examples of guide nucleic acid molecules targeting the c-Jun gene and the relative expression levels of c-Jun when regulated by the systems and methods of the present disclosure are shown.

[0026] Figure 7

[0027] [Figure 7] Fig. 7A and Figure 7B Examples of guide nucleic acid molecules for the TBX21 gene and the relative expression levels of TBX21 when modulated by the systems and methods of the present disclosure are shown.

[0028] Figure 8

[0029] [Figure 8] Fig. 8A and Figure 8B Examples of guide nucleic acid molecules for the IL-21 gene and the relative expression levels of IL-21 when modulated by the systems and methods of the present disclosure are shown.

[0030] Fig. 9

[0031] [Figure 9] Fig.9A and Fig. 9B Examples of guide nucleic acid molecules targeting the TOX1 gene and the relative expression levels of TOX1 when regulated by the systems and methods of the present disclosure are shown.

[0032] Fig.10

[0033] [Figure 10] Fig. 10A and Fig. 10B Examples of guide nucleic acid molecules targeting the TOX2 gene and the relative expression levels of TOX2 when regulated by the systems and methods of the present disclosure are shown.

[0034] Fig.11

[0035] [Figure 11] Fig.11A and Fig. 11B Examples of guide nucleic acid molecules for the SHIP1 gene and the relative expression levels of SHIP1 when regulated by the systems and methods of the present disclosure are shown.

[0036] Fig.12

[0037] [Figure 12] Fig. 12A and Fig. 12B Examples of guide nucleic acid molecules for B2M genes and relative expression levels of B2M when modulated by the systems and methods of the present disclosure are shown.

[0038] Fig.13

[0039] [Figure 13] Fig.13A and Fig. 13B Examples of guide nucleic acid molecules for the BATF gene and the relative expression levels of BATF when modulated by the systems and methods of the present disclosure are shown.

[0040] Fig.14

[0041] [Figure 14] Fig.14A and Fig. 14B Examples of guide nucleic acid molecules targeting the SOCS1 gene and the relative expression levels of SOCS1 when modulated by the systems and methods of the present disclosure are shown.

[0042] Fig.15

[0043] [Figure 15] Fig.15A An exemplary target polynucleotide sequence of a target gene encoding TGFbR2 that can be targeted by one or more guide RNAs is shown, and Fig. 15B Shown are the modified expression levels of target genes by the systems and methods of the present disclosure. DETAILED DESCRIPTION

[0044] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Various changes, modifications and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0045] As used in the specification and claims, the singular form "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0046] The term "about" or "approximately" generally means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" may 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 may mean within an order of magnitude of a value, preferably within 5 times and more preferably within 2 times. When describing a particular value in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for a particular value.

[0047] The use of alternatives (eg, "or") should be understood to mean one, both, or any combination of the alternatives. The term "and / or" should be understood to mean one or both of the alternatives.

[0048] The term "cell" generally refers 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 having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopodiella, hornwort, liverwort, mosses), algae cells (e.g., Brown Cells from mammals (e.g., C. elegans, C. reinhardtii, C. pseudochlosporum, C. pyrenoidosa, C. sphaeroides, C. sphaeroides, 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. Sometimes cells are not derived from natural organisms (e.g., cells can be synthetically prepared, sometimes referred to as artificial cells).

[0049] The terms "hematopoietic stem cell", "hematopoietic progenitor cell" or "hematopoietic precursor cell" as used interchangeably herein generally refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., differentiation into T cells), and include multipotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem cells (HSCs) are multipotent stem cells that produce all blood cell types, including myeloid cells (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells).

[0050] The term "immune cell" or "lymphocyte" generally refers to a differentiated hematopoietic cell. Non-limiting examples of immune cells may include T cells, NK cells, monocytes, innate lymphocytes, tumor infiltrating lymphocytes, macrophages, granulocytes, and the like.

[0051] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotide may include synthetic nucleotides. Nucleotide may include synthetic nucleotide analogs. Nucleotide may be a monomer unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphate adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or their derivatives. Such derivatives may 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 may refer to dideoxyribonucleoside triphosphates (ddNTP) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddtp, ddCTP, ddGTP, ddITP and ddTTP. By well-known technology, nucleotide can be unlabeled or detectably labeled.Also can be labeled with quantum dots.Detectable labeling can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.The fluorescent labeling of nucleotide can include, but is 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-carboxyl-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif., FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP; 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 Boehringer Mannheim, Indianapolis, Ind.; and fluorescein-15-dATP available from Molecular Probes, Eugene, Oreg., 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 by chemical modification. The chemically modified mononucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include biotin-dATP (e.g., b,io-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).

[0052] The terms "polynucleotide," "oligonucleotide," or "nucleic acid," as used interchangeably herein, generally refer to a polymeric form of nucleotides of any length, which may be deoxyribonucleotides or ribonucleotides or their analogs, in single-stranded, double-stranded, or multi-stranded form. The polynucleotide may be exogenous or endogenous to the cell. The polynucleotide may be present in a cell-free environment. The polynucleotide may be a gene or a fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The polynucleotide may contain one or more analogs (e.g., altered backbones, sugars, or nucleobases). If present, the nucleotide structure may be modified before or after polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xenologous nucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, quinoline, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes and primers. The nucleotide sequence may be interrupted by non-nucleotide components.

[0053] As used herein, the term "gene" refers to nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence involved in encoding RNA transcripts. The term with reference to genomic DNA as used herein includes the non-coding region in the middle and the regulatory region, and may include 5' and 3' ends. In some uses, the term covers transcribed sequences, including 5' and 3' non-translated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will include an "open reading frame" encoding a polypeptide. In some uses of the term, a "gene" only includes the coding sequence (e.g., "open reading frame" or "coding region") necessary for encoding a polypeptide. In some cases, a gene does not encode a polypeptide, for example, a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to a natural gene in its natural position in an "endogenous gene" or the genome of an organism. A gene may refer to an "exogenous gene" or a non-natural gene. A non-natural gene may refer to a gene that is not normally found in a host organism but is introduced into a host organism by gene transfer. A non-natural gene may also refer to a gene that is not in its natural location in the genome of an organism. A non-natural gene may also refer to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-natural sequence) that comprises a mutation, insertion, and / or deletion.

[0054] The term "transfection" or "transfected" refers 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 part thereof.

[0055] The term "expression" refers to one or more processes by which polynucleotides are transcribed from a DNA template (e.g., transcribed into mRNA transcripts or other RNA transcripts) and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing mRNA in eukaryotic cells. With regard to expression, "upregulation" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or a polypeptide sequence relative to its expression level in a wild-type state, while "downregulation" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or a polypeptide sequence relative to its expression level in a wild-type state. The expression of a transfected gene may occur transiently or stably in a cell. During "transient expression", the transfected gene is not transferred to daughter cells during cell division. Since its expression is limited to transfected cells, the expression of the gene is lost over time. In contrast, when a gene is co-transfected with another gene that gives a selective advantage to the transfected cell, stable expression of the transfected gene may occur. Such a selective advantage may be resistance to a toxin of the presenting cell.

[0056] The terms "expression cassette", "expression construct" or "expression vector" refer to a nucleic acid comprising nucleotide sequences, such as a coding sequence and a template sequence, as well as sequences necessary for the expression of the coding sequence. An expression cassette may be viral or non-viral. For example, an expression cassette comprises a nucleic acid construct that, when introduced into a host cell, results in the transcription and / or translation of an RNA or polypeptide, respectively. This definition explicitly includes antisense constructs or sense constructs that are not or cannot be translated. One skilled in the art will recognize that the inserted polynucleotide sequence need not be identical, but may only be substantially similar to the gene sequence from which it is derived.

[0057] As used herein, "plasmid" generally refers to a non-viral expression vector, such as a nucleic acid molecule encoding a gene and / or regulatory elements necessary for gene expression. As used herein, "viral vector" generally refers to a virus-derived nucleic acid capable of transporting another nucleic acid into a cell. When present in an appropriate environment, a viral vector is capable of directing the expression of one or more proteins encoded by one or more genes carried by the vector. Examples of viral vectors include, but are not limited to, retroviral, adenoviral, lentiviral, and adeno-associated viral vectors.

[0058] The term "promoter" as used herein refers to a polynucleotide sequence that can drive the transcription of a coding sequence in a cell. Therefore, the promoter used in the polynucleotide construct of the present disclosure includes cis-acting transcription control elements and regulatory sequences, which participate in regulating or regulating the timing and / or rate of gene transcription. For example, a promoter can be a cis-acting transcription control element, including an enhancer, a promoter, a transcription terminator, a replication origin, a chromosome integration sequence, a 5' and 3' non-translated region or an intron sequence that participate in transcriptional regulation. These cis-acting sequences usually interact with proteins or other biomolecules to perform (on / off, regulation, regulation, etc.) gene transcription. "Constitutive promoters" can initiate transcription in almost all tissue types, while "tissue-specific promoters" only initiate transcription in one or more specific tissue types. "Inducible promoters" are promoters that initiate transcription only under specific environmental conditions, developmental conditions, or drugs or chemical conditions.

[0059] As used herein, the terms "complement", "complementary" and "complementarity" generally refer to a sequence that is completely complementary to and hybridizable with a given sequence. In some cases, a sequence that hybridizes with a given nucleic acid is referred to as the "complement" or "reverse complement" of a given molecule if its base sequence above a given region is capable of complementary binding to the base sequence of its binding partner, such as to form AT, AU, GC and GU base pairs. Generally speaking, a first sequence capable of hybridizing with a second sequence can specifically or selectively hybridize with the second sequence, such that during a hybridization reaction, hybridization with the second sequence or a second sequence group is superior to hybridization with a non-target sequence (e.g., thermodynamically more stable under a given set of conditions, such as stringent conditions commonly used in the art). Typically, hybridizable sequences share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-100% complementarity, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence complementarity. Sequence identity, such as for the purpose of assessing percent complementarity, can be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, optionally with default settings). Optimal alignment can be assessed using any suitable parameters of the selected algorithm, including the default parameters.

[0060] Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids can mean that the two nucleic acids can form a duplex, wherein each base in the duplex is bound to a complementary base by Watson-Crick pairing. Substantial or sufficient complementarity can mean that the sequence in one chain is not completely and / or perfectly complementary to the sequence in the opposite chain, but sufficient binding occurs between the bases on the two chains, thereby forming a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequence and standard mathematical calculations to predict the Tm of the hybrid chain, or by empirical determination of the Tm using conventional methods.

[0061] The terms "peptide", "polypeptide" or "protein" as used interchangeably herein generally refer to a polymer of at least two amino acid residues connected by a peptide bond. The term does not mean the specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally present. The term is applicable 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 term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structures (e.g., domains). The term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operation, such as conjugation with a labeling component. The term "amino acid" as used herein generally refers to natural amino acids and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids may include natural amino acids and non-natural amino acids that have been chemically modified to include groups or chemical moieties that are not naturally present on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0062] As used herein, the term "derivative", "variant" or "fragment" with respect to a polypeptide generally refers to a polypeptide related to a wild-type polypeptide, for example by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Derivatives, variants and fragments of a polypeptide may comprise one or more amino acid changes (e.g., mutations, insertions and deletions), truncations, modifications or combinations thereof compared to a wild-type polypeptide.

[0063] As used herein, the term "gene regulatory polypeptide" or "GMP" refers to a polypeptide comprising at least one actuator portion capable of regulating the expression or activity of a gene and / or editing a nucleic acid sequence. A GMP may contain additional peptide sequences that are not involved in regulating gene expression, such as cleavage recognition sites, linker sequences, targeting sequences, etc.

[0064] As used herein, the terms "actuator portion", "actuator domain" and "gene regulatory domain" refer to portions that can regulate the expression or activity of a gene and / or edit a nucleic acid sequence, whether exogenous or endogenous. The actuator portion can regulate the expression of a gene at the transcriptional level and / or translational level. The actuator portion can regulate gene expression at the transcriptional level, for example, by regulating the production of mRNA from DNA (such as chromosomal DNA or cDNA). In some embodiments, the actuator portion recruits at least one transcription factor that binds to a specific DNA sequence, thereby controlling the transcription rate of genetic information from DNA to mRNA. The actuator portion itself can bind to DNA and regulate transcription by physical obstruction, for example, preventing proteins, such as RNA polymerase and other related proteins from assembling on a DNA template. The actuator portion can regulate the expression of a gene at the translational level, for example, by regulating the production of proteins by regulating the mRNA template. In some embodiments, the actuator portion regulates gene expression by affecting the stability of mRNA transcripts. In some embodiments, the actuator portion regulates the expression of a gene by editing a nucleic acid sequence (e.g., a region of a genome). In some embodiments, the actuator moiety regulates the expression of a gene by editing an mRNA template. In some cases, editing a nucleic acid sequence can change the underlying template for gene expression.

[0065] As used herein, the term "targeting sequence" refers to a nucleotide sequence and a corresponding amino acid sequence encoding a targeting polypeptide that mediates protein localization (or retention) to a subcellular location, such as the plasma membrane or membrane of a given organelle, nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi apparatus, chloroplast, apoplast, peroxisome, or other organelle. For example, a targeting sequence can direct a protein (e.g., a receptor polypeptide or an adapter polypeptide) to the nucleus using a nuclear localization signal (NLS); outside the nucleus, such as the cytoplasm, using a nuclear export signal (NES); mitochondria using a mitochondrial targeting signal; endoplasmic reticulum (ER) using an endoplasmic reticulum retention signal; peroxisomes using a peroxisomal targeting signal; plasma membrane using a membrane localization signal; or a combination thereof.

[0066] As used herein, "fusion" may refer to a protein and / or nucleic acid comprising one or more non-natural sequences (e.g., portions). A fusion may comprise one or more identical non-natural sequences in the same non-natural sequence. A fusion may comprise one or more different non-natural sequences in different non-natural sequences. A fusion may be a chimera. A fusion may comprise a nucleic acid affinity tag. A fusion may comprise a barcode. A fusion may comprise a peptide affinity tag. A fusion may provide subcellular localization of a site-directed polypeptide (e.g., a nuclear localization signal (NLS) for targeting the nucleus, a mitochondrial localization signal for targeting mitochondria, a chloroplast localization signal for targeting chloroplasts, an endoplasmic reticulum (ER) retention signal, etc.). A fusion may provide a non-natural sequence (e.g., an affinity tag) that can be used for tracking or purification. A fusion may be a small molecule, such as biotin or a dye, such as an alexa fluor dye, anthocyanin 3 dye, anthocyanin 5 dye.

[0067] Fusions may refer to any protein with a functional effect. For example, fusion proteins may include methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinase activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthase activity or demyristoylation activity. Effector proteins may modify genomic loci. Fusion proteins may be fusions in Cas proteins. Fusion proteins may be non-native sequences in Cas proteins.

[0068] As used herein, "non-natural" may refer to nucleic acid or polypeptide sequences that are not found in natural nucleic acids or proteins. Non-natural may refer to affinity tags. Non-natural may refer to fusions. Non-natural may refer to naturally occurring nucleic acids or polypeptide sequences that contain mutations, insertions and / or deletions. Non-natural sequences may display and / or encode activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) and may also be displayed by nucleic acids and / or polypeptide sequences fused to non-natural sequences. Non-natural nucleic acids or polypeptide sequences may be connected to naturally occurring nucleic acids or polypeptide sequences (or variants thereof) by genetic engineering to generate chimeric nucleic acids and / or polypeptide sequences encoding chimeric nucleic acids and / or polypeptides.

[0069] The term "antibody" generally refers to a protein binding molecule with an immunoglobulin-like function. The term antibody includes antibodies (e.g., monoclonal antibodies and polyclonal antibodies), as well as derivatives, variants and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) and subclasses (e.g., IgG1, IgG2, etc.) of different classes (i.e., IgA, IgG, IgM, IgD and IgE). Its derivatives, variants or fragments may refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), miniantibodies, double antibodies and single-domain antibodies ("sdAb" or "nanoantibodies" or "camel"). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of optimized antibodies include affinity-matured antibodies. Examples of antibodies that have been engineered include Fc-optimized antibodies (eg, antibodies optimized in the fragment crystallizable region) and multispecific antibodies (eg, bispecific antibodies).

[0070] The terms "antigen binding portion" or "antigen binding domain" as used interchangeably herein generally refer to constructs that exhibit preferential binding to a specific target antigen. An antigen binding domain may be a polypeptide construct, such as an antibody, a modification thereof, a fragment thereof, or a combination thereof. An antigen binding domain may be any antibody as disclosed herein, or a functional variant thereof. Non-limiting examples of antigen binding domains may include mouse antibodies, human antibodies, humanized antibodies, camel Ig, antibodies containing only shark heavy chains (VNAR), Ig NAR, chimeric antibodies, recombinant antibodies, or antibody fragments thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen binding fragments (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), miniantibodies, double antibodies, three antibodies, four antibodies, single domain antigen binding fragments (sdAb, nanoantibodies), antibodies containing only recombinant heavy chains (VHH), and other antibody fragments that retain the binding specificity of the entire antibody.

[0071] The terms "enhanced activity", "increased activity" or "up-regulated activity" generally refer to the activity of a portion of interest (e.g., a polynucleotide or polypeptide) that is modified to a level that is higher than the normal activity level of the portion of interest in a host strain (e.g., a host cell). The normal activity level may be substantially zero (or empty) or higher than zero. The portion of interest may comprise a polypeptide construct of the host strain. The portion of interest may comprise a heterologous polypeptide construct introduced or entered into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked into (KI) the genome of a host strain for enhanced activity of the polypeptide of interest in the host strain.

[0072] The term "reduced activity", "decreased activity" or "down-regulated activity" generally refers to an activity of a portion of interest (e.g., a polynucleotide or polypeptide) that is modified to a level below the normal activity level of the portion of interest in a host strain (e.g., a host cell). The normal activity level is above zero. The portion of interest may comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the portion of interest may be knocked out or knocked down in the host strain. In some examples, the reduced activity of the portion of interest may include complete inhibition of such activity in the host strain.

[0073] The terms "subject", "individual" or "patient" as used interchangeably herein generally refer to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, mice, apes, humans, farm animals, sports animals and pets. Tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro are also encompassed.

[0074] The term "treatment" generally refers to an approach for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and / or a preventive benefit. For example, treatment may include administration of a system or cell population disclosed herein. A therapeutic benefit means any treatment-related improvement or effect on one or more diseases, disorders, or symptoms in a treatment. For preventive benefit, a composition may be administered to a subject at risk of developing a particular disease, disorder, or symptom, or to a subject who reports one or more physiological symptoms of a disease, even though the disease, disorder, or symptom may not yet be manifested.

[0075] As used herein, "administering" and its derivatives refer to methods that can be used to deliver an agent or composition to a desired biological site of action. These methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intranasal, intravitreal, infusion, and local injection), transmucosal injection, oral administration, administration as a suppository, and topical administration. Administration can be by any route, including parenteral. Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposome preparations, intravenous infusion, transplantation, etc.

[0076] The term "effective amount" or "therapeutically effective amount" generally refers to an amount of a composition, such as a composition as disclosed herein (e.g., one or more unit doses), sufficient to produce the desired activity when administered to a subject in need thereof. Within the context of the present disclosure, the term "therapeutically effective" generally refers to an amount of a composition sufficient to delay manifestation, prevent progression, alleviate or mitigate at least one symptom of a disease treated by the methods of the present disclosure.

[0077] I. Introduction

[0078] Immune cells (e.g., T cells, NK cells) can be engineered to express specific affinity tags for one or more specific antigens (e.g., cancer or tumor antigens) for adoptive immunotherapy to treat cancer (e.g., solid tumors, lymphomas, etc.). In some cases, immune cells can be engineered to express heterologous receptors (e.g., chimeric antigen receptors or "CARs", engineered T cell receptors (TCRs), etc.) that can bind to one or more specific antigens, thereby targeting cancer cells in the subject.

[0079] However, the therapeutic efficacy of engineered immune cells may be limited by, for example, poor transport, limited persistence in subject serum, rapid exhaustion, or inhibitory activity of subject cancer cells or immune cells to engineered immune cells. In some cases, a variety of recombinant cytokines (e.g., interleukins or "IL") can be administered to subjects together with engineered immune cells to improve their efficacy (e.g., cytotoxic activity, persistence, proliferation). However, the co-administration of such recombinant cytokines also can show undesirable side effects, for example, reduced blood volume, nausea, liver function defects, systemic toxicity, and even death. In some cases, as disclosed herein, during engineered T cells to express heterologous receptors, the stemness of immune cells (e.g., characterized in that the ability of self-renewal, multipotency and / or the persistence of proliferation potential) can be reduced, thereby limiting the therapeutic efficacy of engineered immune cells (e.g., in vivo).

[0080] Therefore, there remains a significant unmet need for the use of allogeneic cytokines to enhance the efficacy of adoptive immunotherapy treatments, such as cancer, but to suppress or reduce the extent of side effects.

[0081] II. Gene Regulation System

[0082] On the one hand, the present disclosure provides a system for regulating the expression or activity of a target protein of a cell. The system may include an actuator portion that can be compounded with a target polynucleotide sequence in a cell. The actuator portion may be heterologous to the cell. In some examples, at least a portion of the amino acid sequence in the amino acid sequence of the actuator portion may be heterologous to the cell. At least a portion (e.g., all) of the target polynucleotide sequence may be endogenous to the cell. Compared to the expression or activity of the target protein in a control cell (or a comparable cell), compounding (e.g., forming a complex comprising at least an actuator portion and a target polynucleotide sequence) may cause a change in the expression or activity of the target protein (e.g., at least 10%). In some cases, compounding may be sufficient to cause a change in the expression or activity of the target protein without editing (e.g., gene editing, such as insertion, deletion, substitution, mutation, etc.) at least a portion of the target polynucleotide sequence.

[0083] In some cases, a change (eg, increase, decrease) in the expression or activity level of a target protein (eg, an endogenous protein) can occur (or can be observed) in vitro, ex vivo, or in vivo.

[0084] The target polynucleotide sequence can be operably coupled to a gene encoding a target protein. The target polynucleotide sequence can be a portion of a coding region (e.g., exon) of a gene encoding a target protein. The target polynucleotide sequence can be a portion of a non-coding region (e.g., intron, promoter, transcription start site (TSS)) of a gene encoding a target protein.

[0085] The target protein may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more proteins (e.g., different types of proteins). The target protein may comprise at most about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 protein (e.g., different types of proteins). Non-limiting examples of target proteins may include thymocyte selection-associated high-mobility group box proteins (TOXs, such as TOX1, TOX2, TOX3, TOX4), suppressors of cytokine signaling (SOCSs, such as SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, CISH), inositol phosphatases containing Src homology 2 domains (SHIPs, such as SHIP1, SHIP2, SHIP3), basic leucine zipper transcription factor ATF-like (BATF), beta-2-microglobulin (B2M), inhibitors of DNA binding / differentiation (IDs, such as ID1, ID2, ID3, ID4), c-Jun, T-box transcription factors (TBXs, such as TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX7, TBX8, TBX9, TBX10, TBX11, TBX12, TBX3, TBX4, TBX5, TBX6, TBX7, TBX8, TBX9, TBX10, TBX12, TBX13, TBX14, TBX15, TBX6, TBX7, TBX8, TBX9, TBX10, TBX15, TBX16, TBX17, TBX18, TBX19, TBX10, TBX11 1. TBX12, TBX13, TBX14, TBX15, TBX16, TBX17, TBX18, TBX19, TBX20, TBX21 or T-Bet, TBX22, TBR1 ), interleukin (IL, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13 , IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36) and transforming growth factor beta receptor (TGFbR). Non-limiting examples of TGFbR include type I TGFbR (e.g., ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7), type II TGFbR (e.g., TGFbR2, BMPR2, ACVR2A, ACVR2B, AMHR2) and type III TGFbR (e.g., TGFbR3).

[0086] In some cases, the target protein may not be a secretory protein. In some cases, the target protein may not be a cytokine. In some examples, the target protein may not be an IL protein. Alternatively, the target protein may include an IL protein.

[0087] In some embodiments, the target protein can be operably coupled to (e.g., participates in activating, maintaining or prolonging) the stemness of a cell (e.g., an immune cell, such as a T cell). For example, one or more proteins provided in Table 1 can be modulated (e.g., activated) to enhance or prolong the stemness of an engineered immune cell as disclosed herein, thereby enhancing or prolonging the therapeutic efficacy of the engineered immune cell.

[0088] Table 1. Target proteins involved in stemness of cells (eg, immune cells) .

[0089]

[0090] In some cases, changes in the expression or activity of a target protein as provided herein can promote one or more characteristics, including (i) maintaining stemness of a cell (e.g., an immune cell, such as a T cell), (ii) enhancing survival of the cell, and / or (iii) enhancing expansion of the cell.

[0091] In some cases, changes in the expression or activity of a target protein can activate stemness of a cell as disclosed herein (e.g., an immune cell, such as a T cell). In some cases, changes in the expression or activity of a target protein can maintain stemness of a cell such that stemness is prolonged (e.g., in terms of time) by at least or at most about 5%, at least or at most about 10%, at least or at most about 20%, at least or at most about 30%, at least or at most about 40%, at least or at most about 50%, at least or at most about 60%, at least or at most about 70%, at least or at most about 80%, at least or at most about 90% compared to a control cell without a system as disclosed herein. , at least or up to about 100%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, at least or up to about 500%, at least or up to about 600%, at least or up to about 700%, at least or up to about 800%, at least or up to about 900%, at least or up to about 1,000%, at least or up to about 2,000%, at least or up to about 3,000%, at least or up to about 4,000% or at least or up to about 5,000%.

[0092] In some cases, stemness of a cell as disclosed herein can be observed for at least or up to about 6 hours, at least or up to about 12 hours, at least or up to about 18 hours, at least or up to about 24 hours, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 2 weeks, at least or up to about 3 weeks, at least or up to about 4 weeks, at least or up to about 1 month, at least or up to about 2 months, at least or up to about 3 months, at least or up to about 4 months, at least or up to about 5 months, or at least or up to about 6 months after induction of complexing as disclosed herein (e.g., after activation of the actuator portion, or after binding of the ligand to the ligand binding domain of the chimeric receptor).

[0093] In some cases, the stemness of a cell as disclosed herein can be determined (or measured) in vitro, ex vivo, or in vivo.

[0094] In some cases, changes in the expression or activity of a target protein can increase the survival rate (e.g., in terms of how long a cell remains viable) of a cell as disclosed herein (e.g., an immune cell, such as a T cell) by at least or at most about 5%, at least or at most about 10%, at least or at most about 20%, at least or at most about 30%, at least or at most about 40%, at least or at most about 50%, at least or at most about 60%, at least or at most about 70%, at least or at most about 80%, as compared to a control cell without a system as disclosed herein. , at least or up to about 90%, at least or up to about 100%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, at least or up to about 500%, at least or up to about 600%, at least or up to about 700%, at least or up to about 800%, at least or up to about 900%, at least or up to about 1,000%, at least or up to about 2,000%, at least or up to about 3,000%, at least or up to about 4,000% or at least or up to about 5,000%.

[0095] In some cases, after induction of a complex as disclosed herein (e.g., after activation of the actuator portion, or after binding of a ligand to the ligand binding domain of a chimeric receptor), the viability of a cell as disclosed herein (e.g., an immune cell, such as a T cell) can be observed for at least or at most about 6 hours, at least or at most about 12 hours, at least or at most about 18 hours, at least or at most about 24 hours, at least or at most about 2 days, at least or at most about 3 days, at least or at most about 4 days, at least or at most about 5 days, at least or at most about 6 days, at least or at most about 7 days, at least or at most about 2 weeks, at least or at most about 3 weeks, at least or at most about 4 weeks, at least or at most about 1 month, at least or at most about 2 months, at least or at most about 3 months, at least or at most about 4 months, at least or at most about 5 months, or at least or at most about 6 months.

[0096] In some cases, the viability of a cell as disclosed herein may be determined (or measured) in vitro, ex vivo, or in vivo.

[0097] In some cases, changes in the expression or activity of a target protein can enhance the expansion (e.g., in terms of proliferation) of a cell as disclosed herein (e.g., an immune cell, such as a T cell) by at least or at most about 5%, at least or at most about 10%, at least or at most about 20%, at least or at most about 30%, at least or at most about 40%, at least or at most about 50%, at least or at most about 60%, at least or at most about 70%, at least or at most about 80%, at least or at most about 90%, at least or at most about 100%, at least or at most about 110%, at least or at most about 120%, at least or at most about 130%, at least or at most about 140%, at least or at most about 150%, at least or at most about 160%, at least or at most about 170%, at least or at most about 180%, at least or at most about 190%, at least or at most about 200%, at least or at most about 210%, at least or at most about 220%, at least or at most about 230%, at least or at most about 240%, at least or at most about 250%, at least or at most about 260%, at least or at most about 270%, at least or at most about 280%, at least or at most about 290%, at least or at most about 300%, at least or at most about 310%, at least or at most about 320%, at least or at most about 330%, at least or at most about 340%, at least or at most about 350%, at least or at most about 360%, at least or at most about 370%, at least or at most about 380%, at least or at most about 390%, at least or at most about 400%, at least or at most about 410%, at least or at most about 4 In some embodiments, the present invention relates to an aqueous solution of at least or up to about 90%, at least or up to about 100%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, at least or up to about 500%, at least or up to about 600%, at least or up to about 700%, at least or up to about 800%, at least or up to about 900%, at least or up to about 1,000%, at least or up to about 2,000%, at least or up to about 3,000%, at least or up to about 4,000% or at least or up to about 5,000%.

[0098] In some cases, after induction of expansion as disclosed herein (e.g., after activation of the actuator portion, or after binding of a ligand to the ligand binding domain of a chimeric receptor), the survival rate of a cell as disclosed herein (e.g., an immune cell, such as a T cell) can be observed for at least or at most about 6 hours, at least or at most about 12 hours, at least or at most about 18 hours, at least or at most about 24 hours, at least or at most about 2 days, at least or at most about 3 days, at least or at most about 4 days, at least or at most about 5 days, at least or at most about 6 days, at least or at most about 7 days, at least or at most about 2 weeks, at least or at most about 3 weeks, at least or at most about 4 weeks, at least or at most about 1 month, at least or at most about 2 months, at least or at most about 3 months, at least or at most about 4 months, at least or at most about 5 months, or at least or at most about 6 months.

[0099] In some cases, expansion of cells as disclosed herein can be determined (or measured) in vitro, ex vivo, or in vivo.

[0100] In some cases, a cell population (e.g., an immune cell population, such as a T cell population) treated by the systems and methods disclosed herein can produce at least or up to about 1%, at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 36%, at least or up to about 37%, at least or up to about 38%, at least or up to about 39%, at least or up to about 40%, at least or up to about 41%, at least or up to about 42%, at least or up to about 43%, at least or up to about 44%, at least or up to about 46%, at least or up to about 47%, at least or up to about 48%, at least or up to about 49%, at least or up to about 50%, at least or up to about 51%, at least or up to about 52%, at least or up to about 53%, at least or up to about 54%, at least or up to about 56%, at least or up to about 57%, at least or up to about 58%, at least or up to about 59%, at least or up to about 60%, at least or up to about 61%, at least or up to about 62%, at least or up to about 63%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 67%, at least or up to about 68%, at least or up to about 69%, at least or up to about 70%, at least or up to about 71%, at least or up to about At least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90% or at least or up to about 95% of a certain proportion (e.g., percentage) of memory T cells (e.g., as indicated by CD45RO positivity). For example, a CAR-T cell population treated by the systems and methods disclosed herein can produce 50% memory T cells, while a comparable CAR-T cell population that has not been treated by these systems and methods can produce 10% memory T cells, so the former is 40% higher than the latter.

[0101] In some cases, in a cell population (e.g., an immune cell population, such as a T cell population) treated by the systems and methods disclosed herein, central memory T cells (TCM) in the population may be enriched at least or at most about 1%, at least or at most about 2%, at least or at most about 5%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 40%, at least or at most about 45%, at least or at most about 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 90%, or at least or at most about 95%. TCM can be CD45RO positive and CD62L positive. TEM can be CD45RO positive and CD62L negative.

[0102] In some cases, a cell population (e.g., an immune cell population, such as a T cell population) treated by the systems and methods disclosed herein can produce a proportion (e.g., percentage) of central memory T cells (e.g., TCMs) that is at least or up to about 1%, at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, or at least or up to about 95% higher than a comparable cell population that has not been treated with these systems and methods. For example, a CAR-T cell population treated by the systems and methods disclosed herein may produce 25% TCM, while a comparable stem cell population that has not been treated by these systems and methods may produce 15% TCM, thus being 10% higher than the latter.

[0103] In some cases, a cell population (e.g., an immune cell population, such as a T cell population) treated by the systems and methods disclosed herein can produce a proportion (e.g., percentage) of stem cell memory T cells (TSCM, e.g., as indicated by CD45RO negativity and CD62L positivity) that is at least or at most about 5%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 40%, at least or at most about 45%, at least or at most about 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 90%, or at least or at most about 95%.

[0104] In some cases, a cell population (e.g., an immune cell population, such as a T cell population) treated by the systems and methods disclosed herein can produce at least or at most about 1%, at least or at most about 2%, at least or at most about 5%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 36%, at least or at most about 37%, at least or at most about 38%, at least or at most about 39%, at least or at most about 40%, at least or at most about 41%, at least or at most about 42%, at least or at most about 43%, at least or at most about 44%, at least or at most about 46%, at least or at most about 47%, at least or at most about 48%, at least or at most about 49%, at least or at most about 50%, at least or at most about 51%, at least or at most about 52%, at least or at most about 53%, at least or at most about 54%, at least or at most about 55%, at least or at most about 56%, at least or at most about 57%, at least or at most about 58%, at least or at most about 59%, at least or at most about 60%, at least or at most about 61%, at least or at most about 62%, at least or at most about 64%, at least or at most about 65%, at least or at most about 66%, at least or at most about 67%, at least or at most about 68%, at least or at most about 69%, at least or at most about 70%, at least or at most about 71%, at least or at most about 72%, at least or at most about 73%, at least or at most about 74%, at least or at most about 75%, at least or at most about 76 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90% or at least or up to about 95% of a certain proportion (e.g., percentage) of stem memory T cells (TSCM, e.g., as indicated by CD45RO negative and CD62L positive). For example, a CAR-T cell population treated by the systems and methods disclosed herein can produce 50% TSCM, while a comparable stem cell population that has not been treated by these systems and methods can produce 10% TSCM, so the former is 40% higher than the latter.

[0105] In some cases, a cell population (e.g., an immune cell population, such as a T cell population) treated by the systems and methods disclosed herein can produce a proportion (e.g., percentage) of stem cell memory T cells (TSCM, e.g., as indicated by CD45RO negativity and CD62L positivity) that is at least or at most about 5%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 40%, at least or at most about 45%, at least or at most about 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 90%, or at least or at most about 95%.

[0106] In some cases, after induction of a complex as disclosed herein (e.g., after activation of the actuator portion, or after binding of a ligand to the ligand binding domain of a chimeric receptor), a T cell type as disclosed herein (e.g., memory T cell, TCM, TEM, TSCM) or a proportion thereof can be observed at least or at most about 6 hours, at least or at most about 12 hours, at least or at most about 18 hours, at least or at most about 24 hours, at least or at most about 2 days, at least or at most about 3 days, at least or at most about 4 days, at least or at most about 5 days, at least or at most about 6 days, at least or at most about 7 days, at least or at most about 2 weeks, at least or at most about 3 weeks, at least or at most about 4 weeks, at least or at most about 1 month, at least or at most about 2 months, at least or at most about 3 months, at least or at most about 4 months, at least or at most about 5 months, or at least or at most about 6 months.

[0107] In some cases, the type of T cells or their proportion as disclosed herein can be determined (or measured) in vitro, ex vivo, or in vivo.

[0108] In some embodiments, the target protein can be operably coupled to the function of an engineered cell (e.g., CAR-T cell) as disclosed herein. For example, one or more proteins provided in Table 2 can be regulated (e.g., inhibited) to improve one or more functions of an engineered immune cell (e.g., CAR, engineered TCR) with a chimeric receptor.

[0109] Table 2. Target proteins involved in one or more functions of engineered immune cells .

[0110]

[0111] In some cases, changes in the expression or activity of a target protein as provided herein can promote one or more characteristics, including (i) reducing exhaustion of engineered immune cells, (ii) enhancing cytokine production by engineered immune cells, (iii) enhancing cytotoxicity of engineered immune cells against target cell populations, and / or (iv) enhancing differentiation of immune cells into immune cell subtypes (e.g., from naive T cells to T helper cells).

[0112] In some cases, the expression of target protein as provided herein or the change of activity can reduce the exhaustion of engineered immune cells.Exhaustion of immune cells (e.g., T cells) can be determined by detecting the presence of one or more exhaustion markers, such as CTLA-4, BTLA, PD-1, GITR, VISTA, TIGIT, LAG-3 and TIM-3. In some examples, a population of engineered immune cells (e.g., T cells) treated by the systems and methods disclosed herein may exhibit a proportion (e.g., percentage) of depleted immune cells (e.g., depleted T cells) that is at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, or at least or up to about 95% lower than a comparable population of immune cells that has not been treated with these systems and methods.

[0113] In some cases, an immune cell exhaustion marker as disclosed herein can be measured at least or up to about 6 hours, at least or up to about 12 hours, at least or up to about 18 hours, at least or up to about 24 hours, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 2 weeks, at least or up to about 3 weeks, at least or up to about 4 weeks, at least or up to about 1 month, at least or up to about 2 months, at least or up to about 3 months, at least or up to about 4 months, at least or up to about 5 months, or at least or up to about 6 months after induction of a complex as disclosed herein (e.g., after activation of the actuator portion, or after binding of a ligand to the ligand binding domain of a chimeric receptor).

[0114] In some cases, immune cell exhaustion markers as disclosed herein can be determined (or measured) in vitro, ex vivo, or in vivo.

[0115] In some cases, changes in the expression or activity of a target protein as provided herein can enhance the production (e.g., expression) of cytokines by engineered immune cells. Such cytokines may not be nor need to be proteins identical to the target protein, the expression or activity of which is directly regulated by the systems and methods disclosed herein. Such cytokines may be endogenous cytokines of cells. Such cytokines with increased production may be different from target proteins directly regulated by the systems and methods disclosed herein. In some examples, a cell population (e.g., engineered immune cells) treated by the systems and methods disclosed herein may exhibit a cell population that is at least or at most about 5%, at least or at most about 10%, at least or at most about 20%, at least or at most about 30%, at least or at most about 40%, at least or at most about 50%, at least or at most about 60%, at least or at most about 70%, at least or at most about 80%, at least or at most about 90%, at least or at most about 100%, at least or at most about 150%, at least or at most about 160%, at least or at most about 170%, at least or at most about 180%, at least or at most about 190%, at least or at most about 200%, at least or at most about 210%, at least or at most about 220%, at least or at most about 230%, at least or at most about 240%, at least or at most about 250%, at least or at most about 260%, at least or at most about 270%, at least or at most about 280%, at least or at most about 290%, at least or at most about 300%, at least or at most about 310%, at least or at most about 320%, at least or at most about 330%, at least or at most about 340%, at least or at most about 350%, at least or at most about 360%, at least 100%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, at least or up to about 500%, at least or up to about 600%, at least or up to about 700%, at least or up to about 800%, at least or up to about 900%, at least or up to about 1,000%, at least or up to about 2,000%, at least or up to about 3,000%, at least or up to about 4,000%, or at least or up to about 5,000% of the cytokine expression or activity level.

[0116] In some cases, after induction of a complex as disclosed herein (e.g., after activation of the actuator portion, or after binding of a ligand to the ligand binding domain of a chimeric receptor), cytokine production as disclosed herein can be measured at least or up to about 6 hours, at least or up to about 12 hours, at least or up to about 18 hours, at least or up to about 24 hours, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 2 weeks, at least or up to about 3 weeks, at least or up to about 4 weeks, at least or up to about 1 month, at least or up to about 2 months, at least or up to about 3 months, at least or up to about 4 months, at least or up to about 5 months, or at least or up to about 6 months.

[0117] In some cases, cytokine production as disclosed herein can be determined (or measured) in vitro, ex vivo, or in vivo.

[0118] In some cases, as disclosed herein, changes (e.g., increases, decreases) in the expression or activity levels of cytokines (e.g., endogenous cytokines) induced when the expression or activity levels of a target protein are modulated can occur (or can be observed) in vitro, ex vivo, or in vivo.

[0119] In some cases, changes in the expression or activity of a target protein as provided herein can enhance the differentiation of T cells (e.g., naive T cells expressing a chimeric receptor as disclosed herein) into immune cell subtypes (e.g., T helper (Th) cells expressing a chimeric receptor as disclosed herein). Non-limiting types of Th cells can include T follicular helper cells (Tfh cells, e.g., Bcl-6 positive), Th1 cells (e.g., T-bet positive), Th2 cells (e.g., Gata3 positive), Th17 cells (e.g., RAR-related orphan receptor gamma (e.g., RORγ2) positive) and induced regulatory T (iTreg) cells (e.g., Foxp3 positive). In some examples, a population of engineered immune cells (e.g., naive T cells) treated by the systems and methods disclosed herein may exhibit a proportion (e.g., percentage) of Th cells (e.g., Th1 cells) that is at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, or at least or up to about 95% higher than a comparable population of immune cells that has not been treated with these systems and methods.

[0120] In some examples, after induction of a complex as disclosed herein (e.g., after activation of the actuator portion, or after binding of a ligand to the ligand binding domain of a chimeric receptor), the proportion of an immune cell subtype as disclosed herein (e.g., Th1 cells) can be measured at least or at most about 6 hours, at least or at most about 12 hours, at least or at most about 18 hours, at least or at most about 24 hours, at least or at most about 2 days, at least or at most about 3 days, at least or at most about 4 days, at least or at most about 5 days, at least or at most about 6 days, at least or at most about 7 days, at least or at most about 2 weeks, at least or at most about 3 weeks, at least or at most about 4 weeks, at least or at most about 1 month, at least or at most about 2 months, at least or at most about 3 months, at least or at most about 4 months, at least or at most about 5 months, or at least or at most about 6 months.

[0121] In some cases, the proportion of immune cell subtypes (eg, Th1 cells) as disclosed herein can be determined (or measured) in vitro, ex vivo, or in vivo.

[0122] In some cases, cytokines (e.g., endogenous cytokines) may include IFN. In some cases, cytokines may be selected from the group consisting of IFN-α (α), IFN-β (β), IFN-κ (κ), IFN-δ (δ), IFN-ε (ε), IFN-τ (τ), IFN-ω (ω), IFN-ζ (ζ), IFN-γ (γ) and IFN-λ (λ). In some cases, cytokines may include IFN-γ (γ). In some cases, as disclosed herein, when regulating the expression or activity level of a target protein in a cell, the cell may be made to show an increase in the expression or activity level of IFN (e.g., IFN-γ).

[0123] In some cases, cytokines (e.g., endogenous cytokines) may include TNF proteins. In some cases, cytokines may be selected from the group consisting of the following: TNFβ, TNFα, TNFγ, CD252 (OX40 ligand), CD154 (CD40 ligand), CD178 (Fas ligand), CD70 (CD27 ligand), CD153 (CD30 ligand), 4-1BBL (CD137 ligand), CD253 (TRAIL), CD254 (RANKL), APO-3L (TWEAK), CD256 (APRIL), CD257 (BAFF), CD258 (LIGHT), TL1 (VEGI), GITRL (TNFSF18) and foreign protein A. In some cases, cytokines may include TNFα. In some cases, as disclosed herein, when regulating the expression or activity level of a target protein in a cell, cells may be made to show an increase in TNF (e.g., TNFα) expression or activity level.

[0124] In some cases, cytokine (for example, endogenous cytokine) can include IL.IL-c cytokine In some cases, IL can include one or more members of the group selected from the following: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35 and IL-36. In some cases, cytokine can be IL-2. In some cases, cells can be made to show the reduction of IL expression or activity level.

[0125] In some cases, changes in the expression or activity of a target protein as provided herein can promote enhanced cytotoxicity of the engineered immune cells against a target cell population.

[0126] In some cases, enhanced cytotoxicity against a target cell population can be determined by a reduction in the size of the target cell population by at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, or at least or up to about 95%.

[0127] In some cases, after induction of a complex as disclosed herein (e.g., after activation of the actuator portion, or after binding of a ligand to the ligand binding domain of a chimeric receptor), enhanced cytotoxicity against a target cell population as disclosed herein can be measured at least or at most about 6 hours, at least or at most about 12 hours, at least or at most about 18 hours, at least or at most about 24 hours, at least or at most about 2 days, at least or at most about 3 days, at least or at most about 4 days, at least or at most about 5 days, at least or at most about 6 days, at least or at most about 7 days, at least or at most about 2 weeks, at least or at most about 3 weeks, at least or at most about 4 weeks, at least or at most about 1 month, at least or at most about 2 months, at least or at most about 3 months, at least or at most about 4 months, at least or at most about 5 months, or at least or at most about 6 months.

[0128] In some cases, enhanced cytotoxicity against a target cell population as disclosed herein may occur (or may be observed) in vitro, ex vivo, or in vivo.

[0129] In some examples, the enhanced cytotoxicity against a target cell population as disclosed herein can be determined by measuring the size of a tumor (comprising a target cell population, such as a cancer cell) in a subject, e.g., after administering cells comprising the disclosed system to the subject. In some cases, upon administration of cells comprising the disclosed system, the size of a tumor (e.g., a solid tumor) in a subject can be reduced by at least or at most about 5%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 40%, at least or at most about 45%, at least or at most about 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 90%, or at least or at most about 95%.

[0130] In some cases, a reduction in tumor size can occur (or be observed) at least or at most about 24 hours, at least or at most about 2 days, at least or at most about 3 days, at least or at most about 4 days, at least or at most about 5 days, at least or at most about 6 days, at least or at most about 7 days, at least or at most about 2 weeks, at least or at most about 3 weeks, at least or at most about 4 weeks, at least or at most about 1 month, at least or at most about 2 months, at least or at most about 3 months, at least or at most about 4 months, at least or at most about 5 months, or at least or at most about 6 months after induction of a complex as disclosed herein (e.g., after activation of the actuator portion, or after binding of a ligand to the ligand binding domain of a chimeric receptor).

[0131] In some cases, the actuator portion can be heterologous to the cell. In some cases, the actuator portion can be activated for complexing (e.g., forming a complex comprising the actuator portion and a target polynucleotide sequence, as disclosed herein) when the cell is exposed to an external stimulus (e.g., an extracellular ligand, such as an antigen).

[0132] In some cases, activation of the actuator portion can include modification of the actuator portion (e.g., conformational change, chemical modification). In some cases, activation of the actuator portion can include release of the actuator portion from a substrate (e.g., a polypeptide substrate). In this case, the actuator portion may not be activated when bound to the substrate.

[0133] In some cases, the system may include a chimeric receptor polypeptide (receptor) that undergoes modification when bound to a ligand. The actuator portion may be activatable (e.g., to regulate the expression or activity level of a target protein) upon binding between the ligand and the chimeric receptor polypeptide, and / or upon receptor modification. In some cases, the receptor may include an antigen binding portion that is capable of specifically binding to at least one ligand (e.g., at least 1, 2, 3, 4, 5, or more ligands). The antigen binding portion may be (i) monovalent or multivalent, and (ii) monospecific or multispecific.

[0134] In some cases, the actuator portion can be activated in the absence of a signaling pathway involving one or more transcription factors (e.g., endogenous transcription factors). Alternatively, the actuator portion can be activated via a signaling pathway involving one or more transcription factors (e.g., endogenous transcription factors).

[0135] In some cases, the target polynucleotide sequence as disclosed herein can be an endogenous gene. Alternatively or in addition, the target polynucleotide sequence can be a heterologous gene encoding a target protein (e.g., an endogenous protein or a heterologous protein). For example, a heterologous gene can comprise the natural amino acid sequence of an endogenous protein.

[0136] In some cases, an actuator portion as disclosed herein (e.g., an actuator portion that is part of a gene regulatory polypeptide or a GMP) can be heterologous to the cell. The GMP can be a substrate, and activating the actuator portion can include releasing the actuator portion from the GMP upon receptor modification, as disclosed herein. The GMP can be part of a lager protein (e.g., a receptor polypeptide or an adaptor polypeptide as disclosed herein).

[0137] As disclosed herein, a target polynucleotide sequence may comprise at least a portion of a transcription start site (TSS) of a target gene encoding a target protein. The target polynucleotide sequence may comprise at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the TSS of the target gene. The target polynucleotide sequence may comprise up to about 100%, 99%, 98%, 87%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20%, 15%, 10%, 5% or less of the TSS of the target gene. Alternatively or in addition, the target polynucleotide sequence can be located up to about 20,000 bases, 10,000 bases, 9,000 bases, 8,000 bases, 7,000 bases, 6,000 bases, 5,000 bases, 4,000 bases, 3,000 bases, 2,500 bases, 2,000 bases, 1,900 bases, or more from the TSS of the target gene (e.g., up to about the disclosed number of bases upstream or downstream of the hub nucleobase of the TSS of the target gene). In some embodiments, the target polynucleotide sequence may be at least 1,000 bases, 1,800 bases, 1,700 bases, 1,600 bases, 1,500 bases, 1,400 bases, 1,300 bases, 1,200 bases, 1,100 bases, 1,000 bases, 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 450 bases, 400 bases, 350 bases, 300 bases, 250 bases, 200 bases, 150 bases, 100 bases or less. At least a portion of the target polynucleotide sequence may be downstream of the TSS of the target gene. Alternatively or in addition, at least a portion of the target polynucleotide sequence may be upstream of the TSS of the target gene. In some examples, the systems and methods as disclosed herein may utilize multiple target polynucleotide sequences of a target gene (e.g., complexed with an actuator portion as disclosed herein), and the multiple target polynucleotide sequences may include one or more members (e.g., 1 member, 2 members, or all 3 members) selected from the group consisting of: (1) a target polynucleotide sequence that is at least partially located downstream of the TSS of the target gene, (2) a target polynucleotide sequence that is at least partially located upstream of the TSS of the target gene, and (3) the TSS of the target gene.

[0138] In some cases, the distance between the target polynucleotide sequence (e.g., the central nucleobase of the target polynucleotide sequence) and the TSS of the target gene (e.g., the central nucleobase of the TSS) can be from about 1 base to about 10,000 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be at least about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene can be at most about 10,000 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be about 10,000 bases to about 9,000 bases, about 10,000 bases to about 8,000 bases, about 10,000 bases to about 7,000 bases, about 10,000 bases to about 6,000 bases, about 10,000 bases to about 5,000 bases, about 10,000 bases to about 4,000 bases, about 10,000 bases to about 3,000 bases, about 10,000 bases to about 2,000 bases, about 10,000 bases to about 1,000 bases, about 1 0,000 bases to about 500 bases, about 10,000 bases to about 1 base, about 9,000 bases to about 8,000 bases, about 9,000 bases to about 7,000 bases, about 9,000 bases to about 6,000 bases, about 9,000 bases to about 5,000 bases, about 9,000 bases to about 4,000 bases, about 9,000 bases to about 3,000 bases, about 9,000 bases to about 2,000 bases, about 9,000 bases to about 1,000 bases, about 9,000 bases to about 500 bases, about 9,000 bases to about 1 base, about 8,000 bases to about 7,000 bases, about 8,000 bases to about 6,000 bases, about 8,000 bases to about 5,000 bases, about 8,000 bases to about 4,000 bases, about 8,000 bases to about 3,000 bases, about 8,000 bases to about 2,000 bases, about 8,000 bases to about 1,000 bases, about 8,000 bases to about 500 bases, about 8,000 bases to about 1 base, about 7,000 bases to about 6,000 bases, about 7,000 bases to about 5,000 bases. bases to about 5,000 bases, about 7,000 bases to about 4,000 bases, about 7,000 bases to about 3,000 bases, about 7,000 bases to about 2,000 bases, about 7,000 bases to about 1,000 bases, about 7,000 bases to about 500 bases, about 7,000 bases to about 1 base, about 6,000 bases to about 5,000 bases, about 6,000 bases to about 4,000 bases, about 6,000 bases to about 3,000 bases, about 6,000 bases to about 2,000 bases, about 6,000 bases to about 1,000 bases.bases to about 1,000 bases, about 6,000 bases to about 500 bases, about 6,000 bases to about 1 base, about 5,000 bases to about 4,000 bases, about 5,000 bases to about 3,000 bases, about 5,000 bases to about 2,000 bases, about 5,000 bases to about 1,000 bases, about 5,000 bases to about 500 bases, about 5,000 bases to about 1 base, about 4,000 bases to about 3,000 bases, about 4,000 bases to about 2,000 bases, about 4,000 bases to about 1 ,000 bases, about 4,000 bases to about 500 bases, about 4,000 bases to about 1 base, about 3,000 bases to about 2,000 bases, about 3,000 bases to about 1,000 bases, about 3,000 bases to about 500 bases, about 3,000 bases to about 1 base, about 2,000 bases to about 1,000 bases, about 2,000 bases to about 500 bases, about 2,000 bases to about 1 base, about 1,000 bases to about 500 bases, about 1,000 bases to about 1 base, or about 500 bases to about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene may be about 10,000 bases, about 9,000 bases, about 8,000 bases, about 7,000 bases, about 6,000 bases, about 5,000 bases, about 4,000 bases, about 3,000 bases, about 2,000 bases, about 1,000 bases, about 500 bases, or about 1 base.

[0139] In some cases, the distance between the target polynucleotide sequence (e.g., the central nucleobase of the target polynucleotide sequence) and the TSS of the target gene (e.g., the central nucleobase of the TSS) can be from about 1 base to about 5,000 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be at least about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene can be at most about 5,000 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be about 5,000 bases to about 4,500 bases, about 5,000 bases to about 4,000 bases, about 5,000 bases to about 3,500 bases, about 5,000 bases to about 3,000 bases, about 5,000 bases to about 2,500 bases, about 5,000 bases to about 2,000 bases, about 5,000 bases to about 1,500 bases, about 5,000 bases to about 1,000 bases, about 5,000 bases to about 500 bases, about 5,000 bases to about 1,000 bases. 00 bases, about 5,000 bases to about 1 base, about 4,500 bases to about 4,000 bases, about 4,500 bases to about 3,500 bases, about 4,500 bases to about 3,000 bases, about 4,500 bases to about 2,500 bases, about 4,500 bases to about 2,000 bases, about 4,500 bases to about 1,500 bases, about 4,500 bases to about 1,000 bases, about 4,500 bases to about 500 bases, about 4,500 bases to about 100 bases, about 4,500 bases to about 1 base , about 4,000 bases to about 3,500 bases, about 4,000 bases to about 3,000 bases, about 4,000 bases to about 2,500 bases, about 4,000 bases to about 2,000 bases, about 4,000 bases to about 1,500 bases, about 4,000 bases to about 1,000 bases, about 4,000 bases to about 500 bases, about 4,000 bases to about 100 bases, about 4,000 bases to about 1 base, about 3,500 bases to about 3,000 bases, about 3,500 bases to about 2,500 bases, about 3,500 bases to about 2,000 bases, about 3,500 bases to about 1,500 bases, about 3,500 bases to about 1,000 bases, about 3,500 bases to about 500 bases, about 3,500 bases to about 100 bases, about 3,500 bases to about 1 base, about 3,000 bases to about 2,500 bases, about 3,000 bases to about 2,000 bases, about 3,000 bases to about 1,500 bases, about 3,000 bases to about 1,000 bases, about 3,000 bases to about 500 bases, about 3,500 bases to about 100 bases.bases to about 100 bases, about 3,000 bases to about 1 base, about 2,500 bases to about 2,000 bases, about 2,500 bases to about 1,500 bases, about 2,500 bases to about 1,000 bases, about 2,500 bases to about 500 bases, about 2,500 bases to about 100 bases, about 2,500 bases to about 1 base, about 2,000 bases to about 1,500 bases, about 2,000 bases to about 1,000 bases, about 2,000 bases to about 500 bases, about 2,000 bases to about 100 bases, about 2,000 bases to about 1 base, about 1,500 bases to about 1,000 bases, about 1,500 bases to about 500 bases, about 1,500 bases to about 100 bases, about 1,500 bases to about 1 base, about 1,000 bases to about 500 bases, about 1,000 bases to about 100 bases, about 1,000 bases to about 1 base, about 500 bases to about 100 bases, about 500 bases to about 1 base, or about 100 bases to about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene can be about 5,000 bases, about 4,500 bases, about 4,000 bases, about 3,500 bases, about 3,000 bases, about 2,500 bases, about 2,000 bases, about 1,500 bases, about 1,000 bases, about 500 bases, about 100 bases, or about 1 base. ,

[0140] In some cases, the distance between the target polynucleotide sequence (e.g., the central nucleobase of the target polynucleotide sequence) and the TSS of the target gene (e.g., the central nucleobase of the TSS) can be from about 1 base to about 2,000 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be at least about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene can be at most about 2,000 bases. The distance between the target polynucleotide sequence and the TSS of the target gene may be about 2,000 bases to about 1,800 bases, about 2,000 bases to about 1,600 bases, about 2,000 bases to about 1,400 bases, about 2,000 bases to about 1,200 bases, about 2,000 bases to about 1,000 bases, about 2,000 bases to about 800 bases, about 2,000 bases to about 600 bases, about 2,000 bases to about 400 bases, about 2,000 bases to about 200 bases, about 2,000 bases to about 1,000 bases. , about 1,800 bases to about 1,600 bases, about 1,800 bases to about 1,400 bases, about 1,800 bases to about 1,200 bases, about 1,800 bases to about 1,000 bases, about 1,800 bases to about 800 bases, about 1,800 bases to about 600 bases, about 1,800 bases to about 400 bases, about 1,800 bases to about 200 bases, about 1,800 bases to about 1 base, about 1,600 bases to about 1,400 bases, about 1,600 bases to about 1,200 bases, about 1,600 bases to about 1,000 bases, about 1,600 bases to about 800 bases, about 1,600 bases to about 600 bases, about 1,600 bases to about 400 bases, about 1,600 bases to about 200 bases, about 1,600 bases to about 1 base, about 1,400 bases to about 1,200 bases, about 1,400 bases to about 1,000 bases, about 1,400 bases to about 800 bases, about 1,400 bases to about 600 bases, about 1,400 bases to about 400 bases, about 1,400 bases to about 1,000 bases. bases to about 200 bases, about 1,400 bases to about 1 base, about 1,200 bases to about 1,000 bases, about 1,200 bases to about 800 bases, about 1,200 bases to about 600 bases, about 1,200 bases to about 400 bases, about 1,200 bases to about 200 bases, about 1,200 bases to about 1 base, about 1,000 bases to about 800 bases, about 1,000 bases to about 600 bases, about 1,000 bases to about 400 bases, about 1,000 bases to about 200 bases, about 1,In the present invention, the length of the nucleic acid sequence of the present invention is from about 1,000 bases to about 1 base, from about 800 bases to about 600 bases, from about 800 bases to about 400 bases, from about 800 bases to about 200 bases, from about 800 bases to about 1 base, from about 600 bases to about 400 bases, from about 600 bases to about 200 bases, from about 600 bases to about 1 base, from about 400 bases to about 200 bases, from about 400 bases to about 1 base, or from about 200 bases to about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene may be about 2,000 bases, about 1,800 bases, about 1,600 bases, about 1,400 bases, about 1,200 bases, about 1,000 bases, about 800 bases, about 600 bases, about 400 bases, about 200 bases, or about 1 base.

[0141] In some cases, the distance between the target polynucleotide sequence (e.g., the central nucleobase of the target polynucleotide sequence) and the TSS of the target gene (e.g., the central nucleobase of the TSS) can be from about 1 base to about 1,000 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be at least about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene can be at most about 1,000 bases.The distance between the target polynucleotide sequence and the TSS of the target gene can be about 1,000 bases to about 900 bases, about 1,000 bases to about 800 bases, about 1,000 bases to about 700 bases, about 1,000 bases to about 600 bases, about 1,000 bases to about 500 bases, about 1,000 bases to about 400 bases, about 1,000 bases to about 300 bases, about 1,000 bases to about 200 bases, about 1,000 bases to about 100 bases, about 1,000 bases to about 800 bases, about 1,000 bases to about 900 bases, about 1,000 bases to about 1 ... bases, about 900 bases to about 600 bases, about 900 bases to about 500 bases, about 900 bases to about 400 bases, about 900 bases to about 300 bases, about 900 bases to about 200 bases, about 900 bases to about 100 bases, about 900 bases to about 1 base, about 800 bases to about 700 bases, about 800 bases to about 600 bases, about 800 bases to about 500 bases, about 800 bases to about 400 bases, about 800 bases to about 300 bases, about 800 bases to about 200 bases, about 800 bases to about 100 bases. bases, about 800 bases to about 1 base, about 700 bases to about 600 bases, about 700 bases to about 500 bases, about 700 bases to about 400 bases, about 700 bases to about 300 bases, about 700 bases to about 200 bases, about 700 bases to about 100 bases, about 700 bases to about 1 base, about 600 bases to about 500 bases, about 600 bases to about 400 bases, about 600 bases to about 300 bases, about 600 bases to about 200 bases, about 600 bases to about 100 bases, about 600 bases to about 1 base, about 5 ... bases to about 400 bases, about 500 bases to about 300 bases, about 500 bases to about 200 bases, about 500 bases to about 100 bases, about 500 bases to about 1 base, about 400 bases to about 300 bases, about 400 bases to about 200 bases, about 400 bases to about 100 bases, about 400 bases to about 1 base, about 300 bases to about 200 bases, about 300 bases to about 100 bases, about 300 bases to about 1 base, about 200 bases to about 100 bases, about 200 bases to about 1 base, or about 100 bases to about 1 base.The distance between the target polynucleotide sequence and the TSS of the target gene can be about 1,000 bases, about 900 bases, about 800 bases, about 700 bases, about 600 bases, about 500 bases, about 400 bases, about 300 bases, about 200 bases, about 100 bases, or about 1 base.

[0142] In some cases, the distance between the target polynucleotide sequence (e.g., the central nucleobase of the target polynucleotide sequence) and the TSS of the target gene (e.g., the central nucleobase of the TSS) can be from about 1 base to about 500 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be at least about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene can be at most about 500 bases.The distance between the target polynucleotide sequence and the TSS of the target gene can be about 500 bases to about 450 bases, about 500 bases to about 400 bases, about 500 bases to about 350 bases, about 500 bases to about 300 bases, about 500 bases to about 250 bases, about 500 bases to about 200 bases, about 500 bases to about 150 bases, about 500 bases to about 100 bases, about 500 bases to about 50 bases, about 500 bases to about 1 base, about 450 bases to about 400 bases, about 450 bases to about 350 bases, about 450 bases to about 500 bases. to about 300 bases, about 450 bases to about 250 bases, about 450 bases to about 200 bases, about 450 bases to about 150 bases, about 450 bases to about 100 bases, about 450 bases to about 50 bases, about 450 bases to about 1 base, about 400 bases to about 350 bases, about 400 bases to about 300 bases, about 400 bases to about 250 bases, about 400 bases to about 200 bases, about 400 bases to about 150 bases, about 400 bases to about 100 bases, about 400 bases to about 50 bases, about 400 bases to about 1 base. bases to about 1 base, about 350 bases to about 300 bases, about 350 bases to about 250 bases, about 350 bases to about 200 bases, about 350 bases to about 150 bases, about 350 bases to about 100 bases, about 350 bases to about 50 bases, about 350 bases to about 1 base, about 300 bases to about 250 bases, about 300 bases to about 200 bases, about 300 bases to about 150 bases, about 300 bases to about 100 bases, about 300 bases to about 50 bases, about 300 bases to about 1 base, about 250 bases To about 200 bases, about 250 bases to about 150 bases, about 250 bases to about 100 bases, about 250 bases to about 50 bases, about 250 bases to about 1 base, about 200 bases to about 150 bases, about 200 bases to about 100 bases, about 200 bases to about 50 bases, about 200 bases to about 1 base, about 150 bases to about 100 bases, about 150 bases to about 50 bases, about 150 bases to about 1 base, about 100 bases to about 50 bases, about 100 bases to about 1 base, or about 50 bases to about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene can be about 500 bases, about 450 bases, about 400 bases, about 350 bases, about 300 bases, about 250 bases, about 200 bases, about 150 bases, about 100 bases, about 50 bases, or about 1 base.

[0143] In some cases, the distance between the target polynucleotide sequence (e.g., the central nucleobase of the target polynucleotide sequence) and the TSS of the target gene (e.g., the central nucleobase of the TSS) can be from about 1 base to about 250 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be at least about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene can be at most about 250 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be about 250 bases to about 225 bases, about 250 bases to about 200 bases, about 250 bases to about 175 bases, about 250 bases to about 150 bases, about 250 bases to about 125 bases, about 250 bases to about 100 bases, about 250 bases to about 75 bases, about 250 bases to about 50 bases, about 250 bases to about 25 bases, about 250 bases to about 1 base, about 225 bases to about 200 bases, about 225 bases to about 175 bases, about 225 bases to about 150 bases, about 250 bases to about 125 bases, about 250 bases to about 100 bases, about 250 bases to about 75 bases, about 250 bases to about 50 bases, about 250 bases to about 25 bases, about 250 bases to about 1 base, about 225 bases to about 200 bases, about 225 bases to about 175 bases, about 225 bases to about 150 bases. bases to about 150 bases, about 225 bases to about 125 bases, about 225 bases to about 100 bases, about 225 bases to about 75 bases, about 225 bases to about 50 bases, about 225 bases to about 25 bases, about 225 bases to about 1 base, about 200 bases to about 175 bases, about 200 bases to about 150 bases, about 200 bases to about 125 bases, about 200 bases to about 100 bases, about 200 bases to about 75 bases, about 200 bases to about 50 bases, about 200 bases to about 25 bases, about 200 bases to about 1 base, about 175 bases to about 150 bases, about 175 bases to about 125 bases, about 175 bases to about 100 bases, about 175 bases to about 75 bases, about 175 bases to about 50 bases, about 175 bases to about 25 bases, about 175 bases to about 1 base, about 150 bases to about 125 bases, about 150 bases to about 100 bases, about 150 bases to about 75 bases, about 150 bases to about 50 bases, about 150 bases to about 25 bases, about 150 bases to about 1 base, about 1 From about 25 bases to about 100 bases, from about 125 bases to about 75 bases, from about 125 bases to about 50 bases, from about 125 bases to about 25 bases, from about 125 bases to about 1 base, from about 100 bases to about 75 bases, from about 100 bases to about 50 bases, from about 100 bases to about 25 bases, from about 100 bases to about 1 base, from about 75 bases to about 50 bases, from about 75 bases to about 25 bases, from about 75 bases to about 1 base, from about 50 bases to about 25 bases, from about 50 bases to about 1 base, or from about 25 bases to about 1 base.The distance between the target polynucleotide sequence and the TSS of the target gene can be about 250 bases, about 225 bases, about 200 bases, about 175 bases, about 150 bases, about 125 bases, about 100 bases, about 75 bases, about 50 bases, about 25 bases, or about 1 base.

[0144] In some cases, the target protein may be a secreted protein. In some cases, the target protein may not be a secreted protein.

[0145] In some cases, the cells can be modified to exhibit at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 10 ... In some embodiments, the present invention relates to a change in the amount of at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, at least or up to about 500%, at least or up to about 600%, at least or up to about 700%, at least or up to about 800%, at least or up to about 900%, at least or up to about 1,000%, at least or up to about 2,000%, at least or up to about 3,000%, at least or up to about 4,000%, or at least or up to about 5,000%.

[0146] In some cases, cells can be made to exhibit at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, at least or up to about 500%, at least or up to about 600%, at least or up to about 700%, at least or up to about 800%, at least or up to about 900%, at least or up to about 1,000%, at least or up to about 2,000%, at least or up to about 3,000%, at least or up to about 4,000%, or at least or up to about 5,000% increase in the expression or activity level of a target protein compared to a control cell.

[0147] In some cases, the system comprises a cell that exhibits at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 95% reduction in the expression or activity level of a target protein.

[0148] In some cases, a change (e.g., increase, decrease) in the expression or activity level of a target protein as compared to a control cell can be observed when an actuator portion as disclosed herein is activated for at least or up to about 6 hours, at least or up to about 12 hours, at least or up to about 18 hours, at least or up to about 24 hours, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 2 weeks, at least or up to about 3 weeks, or at least or up to about 4 weeks (or when a receptor is modified, or when a ligand is bound to the ligand binding domain of a chimeric receptor).

[0149] In some cases, the actuator portion may include a nucleic acid-guided actuator portion. In some cases, the system may also include a guide nucleic acid compounded with the actuator portion. In some cases, the system also includes two or more guide nucleic acids (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more guide nucleic acids) with complementarity to different target polynucleotide sequences (e.g., different parts of a target gene encoding a target protein). In some cases, a guide nucleic acid as disclosed herein may include a guide ribonucleic acid (RNA). In some examples, a cell as disclosed herein may include (1) a first guide nucleic acid (e.g., a first guide RNA) capable of binding to a first target polynucleotide sequence operably coupled to a target protein, and (2) a second guide nucleic acid (e.g., a second guide RNA) capable of binding to a second target polynucleotide sequence operably coupled to a target protein.

[0150] In some cases, a ligand as disclosed herein may be an antigen of a diseased cell. In some cases, a target cell population as disclosed herein may include diseased cells. In some cases, a diseased cell as disclosed herein may include cancer cells or tumor cells.

[0151] In some cases, the cell can be a hematopoietic stem cell (HSC). In some cases, the cell can be an immune cell (lymphocyte). In some cases, the immune cell can be selected from the group consisting of: T cells, NK cells, monocytes, innate lymphocytes, tumor infiltrating lymphocytes, macrophages and granulocytes.

[0152] In some cases, a control as disclosed herein can be a control cell without one or more members comprising (i) a functional chimeric receptor polypeptide, (ii) a functional actuator portion, (iii) a functional guide nucleic acid sequence designed to target a target gene (e.g., a functional guide RNA), (iv) a chimeric adapter polypeptide operably coupled to the chimeric receptor polypeptide (as described below). In some cases, a cell can utilize a guide nucleic acid sequence, and a control cell can comprise a control nucleic acid sequence that is not designed to complex with a target polynucleotide sequence. In some cases, a cell can utilize two different guide nucleic acid sequences, and a control cell can comprise none of the two different guide nucleic acid sequences or only one of them.

[0153] In some cases, the disclosed system can enhance the immune response of the subject. The non-limiting enhancement of immune response can include the generation of increased CD4+ helper T cell activity and cytolytic T cells. The enhancement of immune response can be evaluated using a variety of in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release (e.g., IL-12, IL-2 or IFN-γ production), tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers and cytotoxicity.

[0154] In some cases, the regulated expression and / or activity of a protein as disclosed herein (e.g., an endogenous cytokine) can be determined by a variety of methods, including, but not limited to, (i) phosphorylation of downstream signaling proteins (e.g., (a) TYK2, JAK2, or STAT4 for IL-12 signaling; (b) JAK1, JAK2, STAT1, STAT2, or STAT3 for IL-21 signaling; (c) JAK1, JAK2, or STAT3 for IFN-γ signaling; (d) PI3K, Akt, IκB kinase, STAT5, etc. for TNFα signaling) or (ii) expression of downstream genes (e.g., IFN-γ or TNFα) via Western blotting or polymerase chain reaction (PCR) techniques.

[0155] On the one hand, the present disclosure provides a cell colony comprising any of the systems disclosed herein. In some cases, the cell colony may comprise an engineered immune cell. In some cases, the engineered immune cell comprises an engineered T cell. In some cases, the engineered immune cell comprises an engineered NK cell.

[0156] III. Chimeric Receptor Polypeptides

[0157] In some cases, a chimeric receptor polypeptide (receptor) as disclosed herein may be operably coupled to a chimeric adapter polypeptide (adapter). In some cases, the receptor and adapter may be configured to form a complex (e.g., a signaling complex) when a ligand binds to the receptor (e.g., when a cell comprising the receptor contacts the ligand) and / or when the receptor is modified. The adapter may be a transmembrane protein. Alternatively, the adapter may be an intracellular protein. In some cases, the adapter may be a signaling protein of a receptor signaling pathway that is recruited toward the receptor when the receptor is modified.

[0158] In some cases, the complex of the receptor and the adapter can be direct and / or indirect. In direct complex, one of the receptor and the adapter can be configured to directly bind (e.g., via covalent and / or non-covalent interactions) to the other of the receptor and the adapter. In some examples, one of the receptor and the adapter may include a binding domain (e.g., a polypeptide sequence) configured to bind at least a portion of the other of the receptor and the adapter (e.g., an intracellular portion). In indirect complex, relative to the case where there is no receptor modification, the receptor and the adapter may be configured to be closer to each other (e.g., one is raised toward the other) without any direct binding to the receptor modification. In some examples, the receptor may include a chimeric antigen receptor (CAR) or a modified immune cell receptor (e.g., a modified T cell receptor or "TCR"), and the adapter may include at least a portion of a connector for activating T cells (LAT), which is recruited as part of the signal transduction cascade of the receptor when the receptor is modified.

[0159] In some cases, one of the receptor and the adapter may include a gene regulatory polypeptide comprising an actuator portion connected to a cleavage recognition site, and the other of the receptor and the adapter may include a cleavage portion configured to cleave the cleavage recognition site to release the actuator portion from the GMP. In some examples, the cleavage of the cleavage recognition site by the cleavage portion may occur during direct recombination between the receptor and the adapter. In some examples, the cleavage of the cleavage recognition site by the cleavage portion may occur during indirect recombination between the receptor and the adapter. As disclosed herein, after receptor confirmation, the receptor and the adapter may be recruited toward each other so that the cleavage portion can cleave the actuator portion from the GMP, thereby activating the actuator portion to regulate the expression or activity of endogenous proteins (e.g., endogenous cytokines).

[0160] In some cases, a chimeric receptor polypeptide (receptor) as disclosed herein may be operably coupled to a first chimeric adapter polypeptide (first adapter) and a second chimeric adapter polypeptide (second adapter). In some cases, the first adapter and the second adapter may be signaling proteins of a receptor signaling pathway that are recruited toward the receptor or toward another signaling protein of the receptor signaling pathway when the receptor is modified. In some examples, the first adapter and the second adapter may be recruited toward each other when the receptor is modified. As disclosed herein, the first adapter and the second adapter may form a complex via direct binding. Alternatively, the first adapter and the second adapter may form a complex via indirect binding (e.g., near each other). In some cases, as disclosed herein, the first adapter may include a GMP (including an actuator portion connected to a cleavage recognition site), and the second adapter head may include a cleavage portion. Upon receptor confirmation, the first adapter and the second adapter may be recruited toward each other so that the cleavage portion may cleave the actuator portion from the GMP, thereby activating the actuator portion to regulate the expression or activity of an endogenous protein (e.g., an endogenous cytokine).

[0161] In some cases, one of the first adaptor and the second adaptor may comprise a gene regulatory polypeptide comprising an actuator portion linked to a cleavage recognition site, and the other of the first adaptor and the second adaptor may comprise a cleavage moiety configured to cleave the cleavage recognition site to release the actuator portion from the GMP. In some examples, cleavage of the cleavage recognition site by the cleavage moiety may occur upon direct recombination between the first linker and the second linker. In some examples, cleavage of the cleavage recognition site by the cleavage moiety may occur upon indirect recombination between the first linker and the second linker.

[0162] In some cases, a receptor as disclosed herein may undergo receptor modification upon binding of a ligand, including conformational changes or chemical modifications (eg, phosphorylation or dephosphorylation).

[0163] Figure 1 A to Figure 1 D schematically shows the release of the actuator part from the GMP. Figure 1 A shows the binding of an antigen to a transmembrane chimeric receptor polypeptide. The transmembrane chimeric receptor polypeptide comprises an extracellular region having an antigen interaction domain 101 and an intracellular region comprising a GMP. The GMP comprises an actuator portion 102a connected to a cleavage recognition site 102b. In response to antigen binding, the receptor is modified by phosphorylation 103 of the intracellular region of the receptor ( Figure 1 B) After receptor modification (e.g. phosphorylation), adaptor proteins containing receptor binding moieties are recruited to the receptor, e.g. Figure 1C. The receptor comprises a cleavage portion 104; the cleavage portion can be complexed with an adaptor, or connected to the receptor binding portion, for example, via a peptide bond and / or a peptide linker. Figure 1 As shown in D, when approaching the cleavage recognition site, the cleavage portion can cleave the recognition site to release the actuator portion from the GMP. Upon release, the actuator portion can enter the nucleus to regulate the expression and / or activity of the target gene (e.g., a target protein as disclosed herein) or edit the nucleic acid sequence. Figure 1 E to Figure 1 H shows a similar system, where the receptor modification comprises a conformational change. In some embodiments, the adaptor protein is tethered to a membrane (eg, as a membrane-bound protein).

[0164] Figure 2 A to Figure 2 D schematically shows the release of the actuator part from the GMP. Figure 2 A shows the binding of an antigen to a transmembrane chimeric receptor polypeptide. The transmembrane chimeric receptor polypeptide comprises an extracellular region having an antigen interaction domain 205 and an intracellular region comprising a cleavage moiety 206. The cleavage moiety can be complexed with the receptor or connected to the receptor, for example, by a peptide bond and / or a peptide linker. The GMP forms part of the chimeric adaptor polypeptide. The GMP connected to the receptor binding moiety 201 comprises an actuator portion 202a connected to a cleavage recognition site 202b. In response to antigen binding, the receptor is modified by phosphorylation 203 of the intracellular region of the receptor ( Figure 2 B) Upon receptor modification (e.g., phosphorylation), the chimeric adaptor polypeptide is recruited to the receptor, e.g. Figure 3 C. The receptor comprises a cleavage portion 206. Figure 2 As shown in D, when approaching the cleavage recognition site, the cleavage moiety can cleave the recognition site to release the actuator moiety from the GMP. When released, the actuator moiety can enter the cell nucleus to regulate the expression and / or activity of the target gene or edit the nucleic acid sequence. Figure 2 E to Figure 2 H shows a similar system, where the receptor modification comprises a conformational change. In some embodiments, the chimeric adaptor protein is tethered to a membrane (eg, as a membrane-bound protein).

[0165] Figure 3 A to Figure 3 D schematically shows the release of the actuator part from the GMP. Figure 3 A shows the binding of an antigen to a transmembrane chimeric receptor polypeptide. The transmembrane chimeric receptor polypeptide comprises an extracellular region having an antigen interaction domain 305 and an intracellular region. A GMP comprising an actuator portion linked to a cleavage recognition site forms part of the chimeric adaptor polypeptide. The cleavage recognition site 302b is flanked by a receptor binding portion 301 and an actuator portion 302a. In response to antigen binding, the receptor is modified by phosphorylation 303 in the intracellular region ( Figure 3 B) Upon receptor modification (e.g., phosphorylation), the chimeric adaptor polypeptide is recruited to the receptor, e.g. Figure 3 B. A second adaptor polypeptide 307 comprising a cleavage portion 306 is also recruited to the modified receptor ( Figure 3 C) The cleavage moiety may be complexed with a second adaptor polypeptide, or connected to the adaptor, for example, via a peptide bond and / or a peptide linker. Figure 3 As shown in D, when approaching the cleavage recognition site, the cleavage moiety can cleave the recognition site to release the actuator moiety from the GMP. When released, the actuator moiety can enter the cell nucleus to regulate the expression and / or activity of the target gene or edit the nucleic acid sequence. Figure 3 E to Figure 3 H shows a similar system, where the receptor modification comprises a conformational change. In some embodiments, the chimeric adaptor polypeptide is tethered to the membrane (e.g., as a membrane-bound protein). In some embodiments, the second adaptor polypeptide is tethered to the membrane (e.g., as a membrane-bound protein).

[0166] In some cases, the chimeric receptor polypeptide (receptor) may comprise a ligand binding domain, a transmembrane domain, and a signaling domain. When the ligand binds to the ligand binding domain, the signaling domain may activate the signaling pathway of the cell. The cell may also comprise an expression cassette comprising a polynucleotide sequence encoding an actuator portion as disclosed herein (e.g., a GMP comprising an actuator portion) placed under the control of a promoter. The actuator portion may comprise a heterologous endonuclease. When the ligand binds to the ligand binding domain, the promoter may be activated to drive the expression of the actuator portion. The expressed actuator portion may be complexed with a target gene encoding an endogenous protein as disclosed herein (e.g., an endogenous cytokine) to regulate the expression or activity of the endogenous protein. The promoter may comprise an endogenous promoter of the cell. When the ligand binds to the ligand binding domain of the receptor, the endogenous promoter may be activated.

[0167] [ Figure 4] shows an exemplary system comprising a transmembrane receptor for regulating the expression of at least one target gene. When the ligand binds to a chimeric receptor polypeptide (e.g., scFv-CAR), the intrinsic signal transduction pathway is activated, resulting in at least one cellular transcription factor (e.g., endogenous transcription factor) being recruited to the promoter region of an endogenous gene (signature gene) at its natural locus. The actuator portion coding sequence (e.g., a GMP coding sequence comprising an actuator portion coding sequence) is integrated into the genome and placed under the control of the signature gene promoter. Transcriptional activation of the promoter leads to expression of the actuator portion (e.g., comprising dCas connected to a transcriptional activator (e.g., VPR) or a transcriptional repressor (e.g., KRAB)). The expressed actuator portion, when compounded with a guide RNA (e.g., sgRNAa, sgRNAb) (e.g., constitutive or conditional expression), can regulate (activate or inhibit) the expression of an endogenous protein as disclosed herein (e.g., gene A such as IL-12A, gene B such as IL-12B).

[0168] In some cases, a chimeric receptor polypeptide (receptor) as disclosed herein may be a chimeric antigen receptor (CAR) and / or a modified T cell receptor (TCR).

[0169] In some cases, CAR as disclosed herein can be a first generation, second generation, third generation or fourth generation CAR system, its functional variant or any combination thereof. The first generation CAR (e.g., CD19R or CD19CAR) includes an antigen binding domain specific to a specific antigen (e.g., an antibody or its antigen binding fragment, such as scFv, Fab fragment, VHH domain or only the VH domain of a heavy chain antibody), a transmembrane domain derived from an adaptive immune receptor (e.g., a transmembrane domain from a CD28 receptor) and a signaling domain derived from an adaptive immune receptor (e.g., derived from one or more (e.g., three) ITAM domains of the intracellular region of a CD3ζ receptor or FcεRIγ). The second generation CAR modifies the first generation CAR by adding a costimulatory domain (e.g., derived from a costimulatory receptor acting together with a T cell receptor such as CD28, CD137 / 4-1BB and CD134 / OX40) to the intracellular signaling domain portion of the CAR, which eliminates the need to administer cofactors (e.g., IL-2) with the first generation CAR. The third generation CAR adds multiple costimulatory domains (e.g., CD3ζ-CD28-OX40, or CD3ζ-CD28-41BB) to the intracellular signaling domain portion of CAR. The fourth generation CAR modifies the second generation CAR or the third generation CAR by adding activation cytokines (e.g., IL-23 or IL-27) to the intracellular signaling portion of CAR (e.g., between one or more of the costimulatory domain and CD3ζITAM domain) or under the control of a promoter (e.g., NFAT / IL-2 minimal promoter) induced by CAR.

[0170] IV. Actuator Part

[0171] An actuator portion as disclosed herein (e.g., an actuator portion that is part of a GMP) may be able to edit (e.g., via insertion and / or deletion (indel), homology-directed repair (HDR), non-homologous end joining (NHEJ)) a target gene to modulate the expression or activity of a target protein (e.g., an endogenous protein). Alternatively, the actuator portion may not be able to edit the target gene, but still exhibit the ability to complex with the target gene (e.g., a deactivated or dead CRISPR / Cas protein, as provided herein).

[0172] As disclosed herein, an actuator portion (e.g., an actuator portion as part of a GMP) may be operably coupled to at least one effector domain. At least one effector domain may be configured to regulate the expression or activity of an endogenous protein (e.g., an endogenous cytokine), and in some cases, the actuator portion may be fused to at least one effector domain to form a fusion portion. In some cases, the actuator portion may include a first coupling portion (e.g., a polynucleotide), and at least one effector domain may include a second coupling portion (e.g., a second polynucleotide having complementarity with the first polynucleotide), so that the actuator portion and at least one effector domain may be coupled to each other. In some examples, at least one effector domain may be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain to regulate the expression or activity of an endogenous protein (e.g., an endogenous cytokine).

[0173] Non-limiting examples of the functions of at least one effector domain as disclosed herein can include methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinase activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthase activity, and demyristoylation activity.

[0174] Non-limiting examples of at least one effector domain as disclosed herein can include a methyltransferase, a demethylase, a dismutase, an alkylase, a depurinase, an oxidase, a pyrimidine dimer-forming enzyme, an integrase, a transposase, a recombinase, a polymerase, a ligase, a helicase, a photolyase or glycosylase, an acetyltransferase, a deacetylase, a kinase, a phosphatase, a ubiquitin ligase, a deubiquitinating enzyme, an adenylase, a deadenylase, a SUMOylating enzyme, a deSUMOylating enzyme, a ribosylating enzyme, a deribosylating enzyme, a myristoylases, a remodeling enzyme, a protease, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a synthase, a synthetase, and a demyristoylases.

[0175] The actuator moiety as disclosed herein may comprise a nuclease, such as an endonuclease (e.g., Cas). The endonuclease may be a heterologous enzyme to any cell disclosed herein.

[0176] The actuator portion as disclosed herein may include a Cas endonuclease, a zinc finger nuclease (ZFN), a zinc finger-related gene regulatory polypeptide, a transcription activator-like effector nuclease (TALEN), a transcription activator-like effector-related gene regulatory polypeptide, a large range of nucleases, a natural master transcription factor, an epigenetic modification enzyme, a recombinase, a flip enzyme, a transposase, an RNA binding protein (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof. In some embodiments, the actuator portion includes a Cas protein, and the system further includes a guide RNA (gRNA) complexed with the Cas protein. In some embodiments, the actuator portion includes an RBP complexed with a gRNA, which is capable of forming a complex with the Cas protein. In some embodiments, the gRNA includes a targeting segment that exhibits at least 80% sequence identity with a target polynucleotide. In some embodiments, the Cas protein substantially lacks DNA cleavage activity (ie, dead Cas, deactivated Cas, or dCas). For example, the Cas protein is mutated and / or modified to produce a nuclease-deficient protein or a protein with reduced nuclease activity relative to a wild-type Cas protein. A nuclease-deficient protein may retain the ability to bind DNA but may lack or have reduced nucleic acid cleavage activity.

[0177] In some cases, a suitable actuator portion comprises a CRISPR-associated (Cas) protein or Cas nuclease, including a type I CRISPR-associated (Cas) polypeptide, a type II CRISPR-associated (Cas) polypeptide, a type III CRISPR-associated (Cas) polypeptide, a type IV CRISPR-associated (Cas) polypeptide, a type V CRISPR-associated (Cas) polypeptide, and a type VI CRISPR-associated (Cas) polypeptide; a zinc finger nuclease (ZFN); a transcription activator-like effector nuclease (TALEN); a meganuclease; an RNA binding protein (RBP); a CRISPR-associated RNA binding protein; a recombinase; a flippase; a transposase; an Argonaute (Ago) protein (e.g., a prokaryotic Argonaute (pAgo), an archaeal Argonaute (aAgo), and a eukaryotic argonaute (eAgo)); any derivative thereof, any variant thereof; and any fragment thereof.

[0178] The Cas protein referred to herein may be a type of protein or polypeptide. The Cas protein may refer to a nuclease. The Cas protein may refer to an endoribonuclease. The Cas protein may refer to any modified (e.g., shortened, mutated, lengthened) polypeptide sequence or homolog of the Cas protein. The Cas protein may be codon-optimized. The Cas protein may be a codon-optimized homolog of the Cas protein. The Cas protein may be enzymatically inactive, partially active, constitutively active, fully active, inducibly active, and / or more active (e.g., more active than a wild-type homolog of a protein or polypeptide). The Cas protein may be Cas9. The Cas protein may be Cpf1. The Cas protein may be C2c2. The Cas protein (e.g., a variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive site-directed polypeptide) may bind to a target nucleic acid. The Cas protein (e.g., a variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endoribonuclease) may bind to a target RNA or DNA.

[0179] Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, Cas10, Cas10d, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Cse 1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cul966 and homologs or modified versions thereof.

[0180] In some cases, the nucleases disclosed herein (e.g., Cas) can be nucleic acid-guided nucleases (e.g., RNA-guided endonucleases). The term "guide nucleic acid" generally refers to a nucleic acid that can hybridize with another nucleic acid. The guide nucleic acid can be RNA. The guide nucleic acid can be DNA. The guide nucleic acid can be programmed to specifically bind to a nucleic acid sequence site. The nucleic acid or target nucleic acid to be targeted can contain nucleotides. The guide nucleic acid can contain nucleotides. A portion of the target nucleic acid can be complementary to a portion of the guide nucleic acid. A double-stranded target polynucleotide chain that is complementary to and hybridized with the guide nucleic acid can be referred to as a complementary chain. A double-stranded target polynucleotide chain that is complementary to the complementary chain, and therefore may not be complementary to the guide nucleic acid, can be referred to as a non-complementary chain. A guide nucleic acid can contain a polynucleotide chain and can be referred to as a "single guide nucleic acid". A guide nucleic acid can contain two polynucleotide chains and can be referred to as a "dual guide nucleic acid". If not otherwise specified, the term "guide nucleic acid" can be inclusive, referring to both a single guide nucleic acid and a dual guide nucleic acid.

[0181] The guide nucleic acid may comprise a segment referred to as a “nucleic acid targeting segment” or a “nucleic acid targeting sequence.” The nucleic acid targeting segment may comprise a subsegment, which may be referred to as a “protein binding segment” or a “protein binding sequence” or a “Cas protein binding segment.”

[0182] The guide nucleic acid may include two independent nucleic acid molecules, which may be referred to as dual guide nucleic acids. The guide nucleic acid may include a single nucleic acid molecule, which may be referred to as a single guide nucleic acid (e.g., sgRNA). In some cases, the guide nucleic acid is a single guide nucleic acid comprising fusion CRISPR RNA (crRNA) and trans-activated crRNA (tracrRNA). In some cases, the guide nucleic acid is a single guide nucleic acid comprising crRNA. In some cases, the guide nucleic acid is a single guide nucleic acid comprising crRNA but lacking tracrRNA. In some cases, the guide nucleic acid is a dual guide nucleic acid comprising non-fusion crRNA and tracrRNA. Exemplary dual guide nucleic acids may include crRNA-like molecules and tracrRNA-like molecules. Exemplary single guide nucleic acids may include crRNA-like molecules. Exemplary single guide nucleic acids may include fused crRNA-like molecules and tracrRNA-like molecules.

[0183] As used herein, the term "crRNA" generally refers to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% sequence identity and / or sequence similarity with a wild-type exemplary crRNA (e.g., crRNA from Streptococcus pyogenes). CrRNA generally refers to a nucleic acid having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% sequence identity and / or sequence similarity with a wild-type exemplary crRNA (e.g., crRNA from Streptococcus pyogenes). CrRNA may refer to a modified form of crRNA, which may include nucleotide changes, such as deletions, insertions or substitutions, variants, mutations or chimeras. In a segment of at least 6 consecutive nucleotides, crRNA may be a nucleic acid having at least about 60% sequence identity with a wild-type exemplary crRNA (e.g., crRNA from Streptococcus pyogenes) sequence. 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., a crRNA from Streptococcus pyogenes) over a stretch of at least 6 contiguous nucleotides.

[0184] The term "tracrRNA" as used herein generally refers to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes). TracrRNA may refer to a nucleic acid having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes). TracrRNA may refer to a modified form of tracrRNA, which may include nucleotide changes, such as deletions, insertions or substitutions, variants, mutations or chimeras. TracrRNA can refer to a nucleic acid that is at least about 60% identical to a wild-type exemplary tracrRNA (e.g., tracrRNA from Streptococcus pyogenes) sequence within a segment of at least 6 consecutive 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., tracrRNA from Streptococcus pyogenes) sequence within a segment of at least 6 consecutive nucleotides.

[0185] The crRNA may comprise a nucleic acid targeting segment (e.g., a spacer) of a guide nucleic acid and a stretch of nucleotides that may form one half of a double-stranded duplex of a Cas protein binding segment of the guide nucleic acid.

[0186] The tracrRNA may comprise a stretch of nucleotides that forms the other half of the double-stranded duplex of the Cas protein binding segment of the gRNA. A stretch of nucleotides of the crRNA may be complementary and hybridized with a stretch of nucleotides of the tracrRNA to form a double-stranded duplex of the Cas protein binding domain of the guide nucleic acid.

[0187] crRNA and tracrRNA can hybridize to form a guide nucleic acid. crRNA can also provide a single-stranded nucleic acid targeting segment (e.g., a spacer) hybridized with a target nucleic acid recognition sequence (e.g., a protospacer). The sequence of crRNA, including a spacer or tracrRNA molecule, can be designed to be specific to the species using the guide nucleic acid.

[0188] In some cases, the effector domain can be a transcriptional activation domain selected from the group consisting of GAL4, VP16, VP64, p65, Rta, VPR, and variants thereof (e.g., mini-VPR). In some examples, the actuator moiety can be a Cas protein (e.g., dCas, such as dCas9) fused to a transcriptional activation domain, as disclosed herein.

[0189] In some cases, the effector domain can be a transcriptional repressor domain selected from the group consisting of KRAB, SID, ERD, and variants thereof. In some examples, the actuator portion can be a Cas protein (e.g., dCas, such as dCas9) fused to a transcriptional repressor domain as disclosed herein.

[0190] In one aspect, the present disclosure provides a system comprising an actuator portion as disclosed herein, which is capable of binding to a target polynucleotide sequence in a cell to modulate the expression or activity of an endogenous cytokine (e.g., an interleukin (IL)) in the cell, as disclosed herein. In some cases, the actuator portion is heterologous to the cell. For example, the IL can be IL-12 (e.g., IL-12A and / or IL-12B) or IL-21.

[0191] V. Guide nucleic acid

[0192] On the one hand, the present disclosure provides a system comprising a guide nucleic acid molecule designed to bind to a target polynucleotide sequence in a cell to regulate the expression or activity of a target protein in the cell, as disclosed herein. In some cases, the guide nucleic acid molecule may be able to recruit an actuator portion to a target polynucleotide sequence in a cell to regulate the expression or activity of a target protein. In some cases, the system may comprise an actuator portion. For example, the target protein may comprise one or more proteins from Tables 1 and 2.

[0193] In some cases, the target polynucleotide sequence may be endogenous to the cell. In some cases, the TSS of a target gene encoding a target protein may be endogenous to the cell.

[0194] In some cases, the system can include at least or at most 2, at least or at most 3, at least or at most 3, at least or at most 4, at least or at most 5, at least or at most 6, at least or at most 7, at least or at most 8, at least or at most 9, or at least or at most 10 different guide nucleic acid molecules having different nucleic acid sequences. In some cases, the guide nucleic acid molecule can include a guide ribonucleic acid (RNA). In some examples, the system can include multiple guide nucleic acids (e.g., multiplex guide RNAs).

[0195] In some cases, the system may include (i) a first guide nucleic acid molecule designed to bind to a first target polynucleotide sequence as disclosed herein, and (ii) a second guide nucleic acid molecule designed to bind to a second target polynucleotide sequence as disclosed herein. In some examples, the system may include (i) a first guide nucleic acid molecule designed to bind to a first portion of a TSS of a target gene encoding a target protein, and (ii) a second guide nucleic acid molecule designed to bind to a second portion of a TSS of a target gene encoding a target protein. In some examples, the first target polynucleotide sequence and the second target polynucleotide sequence can be separated by at least or at most about 1 base, at least or at most 2 bases, at least or at most 3 bases, at least or at most 3 bases, at least or at most 4 bases, at least or at most 5 bases, at least or at most 6 bases, at least or at most 7 bases, at least or at most 8 bases, at least or at most 9 bases, at least or at most 10 bases, at least or at most 15 bases, at least or at most 20 bases, at least or at most 30 bases, at least or at most 40 bases, at least or at most 50 bases, at least or at most 60 bases. , at least or at most 70 bases, at least or at most 80 bases, at least or at most 90, at least or at most 100, at least or at most 200 bases, at least or at most 300 bases, at least or at most 400, at least or at most 500, at least or at most 600, at least or at most 700 bases, at least or at most 800 bases, at least or at most 900 bases, at least or at most 1,000 bases, at least or at most 2,000 bases, at least or at most 3,000 bases, at least or at most 4,000 bases, or at least or at most 5,000 bases. The first target polynucleotide sequence and the second target polynucleotide sequence can be on the same strand of the target nucleic acid molecule (e.g., the target genome of the cell). Alternatively, the first target polynucleotide sequence and the second target polynucleotide sequence can be on different strands of the target nucleic acid molecule.

[0196] In some cases, the target gene encoding the target protein may include multiple TSSs, including a first TSS and a second TSS. Each of the first TSS and the second TSS may encode a different part of the target gene. For example, the target protein may be a heterodimer, and the first TSS may be from a gene encoding a first monomer of the heterodimer, and the second TSS may be from a gene encoding a second monomer of the heterodimer. Therefore, in some examples, the first guide nucleic acid molecule may (1a) include at least a portion of the first TSS or (1b) a certain distance from the first TSS as provided herein, and the second guide nucleic acid may (2a) include at least a portion of the second TSS or (2b) a certain distance from the second TSS as provided herein.

[0197] In some cases, the TSS (e.g., the first TSS) can have at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% sequence identity to any of the polynucleotide sequences provided in Table 3.

[0198] Table 3 .

[0199]

[0200] VI. Delivery of the System Expressed in Cells

[0201] In one aspect, the disclosure provides cells (eg, immune cells) comprising (or expressing) any of the subject systems disclosed herein.

[0202] In one aspect, the disclosure provides a cell population (eg, an immune cell population) comprising (or expressing) any of the subject systems disclosed herein.

[0203] RNA or DNA virus-based systems can be used to deliver one or more genes encoding any polypeptide and / or polynucleotide disclosed herein (e.g., chimeric receptors, chimeric adapters, actuator moieties with or without effector domains, or genes encoding them) to cells of the present disclosure. Viral vectors can be used to treat cells in vitro, and modified cells can be optionally administered (ex vivo). Alternatively, viral vectors can be administered directly (in vivo) to a subject. Virus-based systems can include retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors for gene transfer. Integration in the host genome can be performed by retroviral, lentiviral, and adeno-associated viral gene transfer methods, which can result in long-term expression of the inserted transgene.

[0204] In some cases, non-viral delivery methods can be used to deliver any polypeptide and / or polynucleotide disclosed herein (e.g., chimeric receptors, chimeric adapters, actuator moieties with or without effector domains, or genes encoding them) to cells of the present disclosure. Such non-viral delivery methods for delivery may include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, exosomes, polycations or lipids: drug-enhanced uptake of carrier conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial viral particles, and polypeptide or DNA. Cationic lipids and neutral lipids suitable for effective receptor recognition lipid delivery of polynucleotides or polypeptides can be used.

[0205] VII. Methods and Compositions

[0206] In one aspect, the present disclosure provides methods for conditionally regulating the expression or activity of a target protein (eg, endogenous target protein 33) in a cell by introducing (or expressing) any subject system as disclosed herein.

[0207] In one aspect, the present disclosure provides a method for conditionally regulating the expression or activity of a target protein (e.g., an endogenous target protein) of a cell. The method may include (a) exposing a chimeric receptor polypeptide (receptor) to a ligand, wherein the receptor undergoes modification when bound to the ligand. The method may include (b) in response to the receptor modification, forming a complex between an actuator portion and a target polynucleotide sequence, as disclosed herein, to regulate the expression or activity of the target protein.

[0208] In some cases, the method further comprises administering a co-therapeutic agent.

[0209] In some cases, the cells administered to a subject can be autologous or allogeneic to the subject. For example, the cells administered to a subject can be autologous immune cells or allogeneic immune cells.

[0210] On the one hand, the present disclosure provides a composition comprising a cell or cell colony (e.g., an engineered immune cell colony), which comprises (or expresses) any subject system as disclosed herein. The composition can be administered to a subject to treat a condition (e.g., cancer, tumor) of the subject. The composition can comprise at least or up to about 1 dose, at least or up to about 2 doses, at least or up to about 3 doses, at least or up to about 4 doses, at least or up to about 5 doses, at least or up to about 6 doses, at least or up to about 7 doses, at least or up to about 8 doses, at least or up to about 9 doses, or at least or up to about 10 doses.

[0211] In some cases, the composition further comprises a co-therapeutic agent.

[0212] Compositions as disclosed herein can be pharmaceutical compositions. Pharmaceutical compositions can be in any suitable form (depending on the desired method of administration). Pharmaceutical compositions can be provided in unit dosage form, can be provided in a sealed container, and / or can be provided as part of a kit. Such kits can include instructions for use. The kit can include a plurality of such unit dosage forms.

[0213] Non-limiting examples of co-therapeutic agents can include cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, for example, anti-CD20 antibodies, anti-PD1 antibodies (e.g., pembrolizumab), platelet-derived growth factor inhibitors (e.g., Gleevec), TM(imatinib mesylate), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: PDGFR-β, BlyS, APRIL, BCMA receptor, TRAIL / Apo2, other biologically active and organic chemical agents, etc.

[0214] The term "cytotoxic agent" generally refers to a substance that inhibits or prevents cell function and / or causes cell destruction. Non-limiting examples of cytotoxic agents may include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu), chemotherapeutic agents such as methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof, such as nucleolytic enzymes, antibiotics, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin.

[0215] Non-limiting examples of chemotherapeutic agents may include alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodepa, carboquinone, metodepa and uredepa; ethyleneimines and methylamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; polyacetic acids (especially bratacin and bratacinone); delta-9-tetrahydrocannabinol (dronabinol, ); β-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including the synthetic analogue topotecan CPT-11 (irinotecan, ), acetylcamptothecin, scopolamine and 9-aminocamptothecin); bryostatin; bromostatin; CC-1065 (including its synthetic analogs of adolesin, carzelesin and biszelesin); podophyllotoxin; podophyllic acid; teniposide; Nostoc cyclic peptides (specifically Nostoc cyclic peptide 1 and Nostoc cyclic peptide 8); Aplysia caudatum; docamisin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, naphthyl mustard, cholephosphamide , estramustine, ifosfamide, dichloromethyl diethylamine, methoxychlor hydrochloride, melphalan, new nitrogen mustard, phenylephrine, prednimustine, trofosamide, uracil nitrogen mustard; nitrosoureas such as carmustine, chlorzoxazone, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics; danemycins, including danemycin A; espermycins; and new carcinogens and related pigment proteins enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin, calabikine, carminomicin, carmophorin, chromomycin, actinomycin D, daunorubicin, detoxibacine, 6-diazo-5-oxo-L-norleucine, Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicinol, marseromycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olive mycins, peplomycin, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozocin, streptozotocin, tuberculocidin, ubenimex, netastatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethylfolate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azacitidine, 6-nitropropene, pyrimidine ... uridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as caprotestosterone, drostanolone propionate, cyclothiodine, melastane, testolactone; antiadreners such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; busamustine ; bisantrene; edatrexate; defosfamide; colcemid; diazocone; eflornithine; elliptinium acetate; epothilone; etogluconate; gallium nitrate; hydroxyurea; lentinan; lonidanine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; diamine nitrazepam; pentostatin; methambucil; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizoran; spirogermanamine; tenuisporic acid; triazoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (specifically T-2 toxin, verracurin A, baculosporin A, and serpentin); urethane; vindesine Dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxanes, such as the taxanes, including Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ) without ABRAXANE TM Cremophor’s albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumburg, IL), and Docetaxel (Rhône-Poulenc Rorer, Antoni, France); chlorambucil; gemcitabine 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine Oxaliplatin; Folic acid; Vinorelbine Noxil; edatrexate; daunorubicin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine any of the above; and combinations of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, an abbreviation for the combination therapy of oxaliplatin (ELOXATIN TM ) in combination with 5-FU and leucovorin. Other chemotherapeutic agents include cytotoxic agents that can be used as antibody drug conjugates, such as, for example, maytansinoids (eg, DM1) and auristatins MMAE and MMAF.

[0216] Examples of chemotherapeutic agents may also include "antihormonal agents" or "endocrine therapeutic agents," which act to regulate, reduce, block or inhibit the effects of hormones that promote cancer growth, and are usually in the form of systemic or systemic treatments. They may themselves be hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Tamoxifen), Raloxifene, droloxifene, 4-hydroxytamoxifen, troloxifen, raloxifene hydrochloride e, LY117018, onapristone and toremifene; antiprogestins; estrogen receptor downregulators (ERDs); agents that inhibit or shut down the ovaries, for example, luteinizing hormone-releasing hormone (LHRH) agonists, such as and ELIGARD) leuprolide acetate, goserelin acetate, buserelin acetate, and triptolide; other antiandrogens, such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, Megestrol acetate, Exemestane, formestane, fadrozole, Vorozole, Letrozole and Anastrozole. In addition, this definition of chemotherapeutic agent includes bisphosphonates, such as clodronate (e.g., or ), etidronate, NE-58095, Zoledronic acid / zoledronate, Alendronate, Pamidronate, Tirucuronic acid or Risedronate; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways associated with antibody cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGFR); vaccines, such as Vaccines and gene therapy vaccines, e.g. vaccine, Vaccines and vaccine; Topoisomerase 1 inhibitors; rmRH; lapatinib ditosylate (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, also known as GW 572016); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0217] Examples of chemotherapeutic agents may also include antibodies such as alemtuzumab (Campass), bevacizumab ( Genentech; Cetuximab ( imclone); panitumumab ( Amgen), rituximab ( Genentech / Biogen Idec), pertuzumab (OMNITARG, 2C4, Genentech), trastuzumab ( Genentech), tositumomab (Bexxar, Corixia), and antibody-drug conjugates, gemtuzumab, gemtuzumab ozogamicin ( Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents for combination with the compounds of the invention include: apolizumab, aseluzumab, tocilizumab, bapinezumab, mobivalizumab maytansine, mecanizumab maytansine, cedizumab, certolizumab pegol, sidefocizumab, situtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, femuzumab, fentetumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovezumab, natalizumab, nimotuzumab, norvegicin, nivolumab ... ibizumab, numavuzumab, ocrelizumab, omalizumab, palivizumab, paxilumab, pefocizumab, pexetozumab, pexelizumab, lalivizumab, ranibizumab, leslivuzumab, reslizumab, resivuzumab, rovizumab, rupilizumab, sibutruzumab, siprilimab, sotuzumab, tacalizumab-tetraacetic acid conjugate, tadolizumab, talimumab, tefizumab, tocitumumab, toralizumab, tukavicuzumab-simoleukin conjugate, tucucizumab, umavizumab, udoxorumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), which is a recombinant, pure human sequence, full-length IgG1λ antibody that has been genetically modified to recognize the interleukin-12p40 protein.

[0218] Examples of chemotherapeutic agents may also include "tyrosine kinase inhibitors," such as EGFR-targeting agents (e.g., small molecules, antibodies, etc.); small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724,714, oral selective inhibitors of ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors, such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016 available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (CI-1033; pharmacia); Raf-1 inhibitors, such as the antisense agent ISIS-5132, available from ISIS Pharmaceuticals, inhibiting Raf-1 signaling; non-HER-targeted TK inhibitors, such as imatinib mesylate ( available from Glaxo SmithKline); multitargeted tyrosine kinase inhibitors, such as sunitinib ( available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines such as PD 153035, 4-(3-chloroanilino)oxazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidine, 4-(amino)7H-pyrrolo[2,3-d]pyrimidine; curcumin (difuroylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to a nucleic acid encoding a HER); quinoxaline (U.S. Pat. No. 5,804,396); tryptamines (U.S. Pat. No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors, such as CI-1033 (Pfizer); afenitak (ISIS 3521; Isis / Lilly); imatinib mesylate PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semacinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1c 11 (Imclone); and rapamycin (sirolimus, ).

[0219] Examples of chemotherapeutic agents may also include dexamethasone, interferon, colchicine, chlorpheniramine, cyclosporine, amphotericin, metronidazole, alemtuzumab, avermectin, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, ilotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, The invention relates to oxadiazine, levamisole, mesna, methoxsalen, nandrolone, nelarabine, thionomolgus, oprelvekin, palifermin, pamidronate sodium, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate and zoledronic acid and pharmaceutically acceptable salts thereof.

[0220] Examples of chemotherapeutic agents may also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tisocotorol pivalate, triamcinolone acetonide, triamcinolone acetonide alcohol, mometasone, amcinonide, budesonide, desonide, fluocinolone, fluocinolone acetate, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, alclomethasone dipropionate, betamethasone valerate, betamethasone dipropionate, betamethasone propionate, clobetasone-17-butyrate, clobetasol-17-propionate, flucortin hexanoate, flucortin pivalate, and fluprednisolone acetate: immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer form (FEG) (IMULAN BioTherapeutics, LLC); antirheumatic drugs, such as azathioprine, cyclosporine (CsA), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers, such as etanercept Infliximab Adalimumab Certolizumab polyethylene glycol Golimumab Interleukin-1 (IL-1) blockers, such as anakinra T cell costimulation blockers, such as abatacept Interleukin-6 (IL-6) blockers, such as tocilizumab Interleukin-13 (IL-13) blockers, such as levofloxacin; interferon-alpha (IFN) blockers, such as rotazumab; β7 integrin blockers, such as rhu mab β7; IgE pathway blockers, such as anti-M1 primers; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa / β2 blockers, such as anti-lymphotoxin α (LTa); miscellaneous investigational agents, such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or famylase inhibitors (L-739749, L-744832); polyphenols, such as quercetin, resveratrol, epigallocatechin gallate, theaflavins, flavanols, proanthocyanidins, betulinic acid, and their derivatives; autophagy inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, ); β-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopoletin, and 9-aminocamptothecin); podophyllotoxin; tegafur Besarotten Bisphosphonates, such as clodronate (e.g. or ), etidronate NE-58095, Zoledronic Acid / Zoledronate Alendronate Pamidronate Tiludronate Risedronate and epidermal growth factor receptor (EGF-R); vaccines, e.g. Vaccines; perifosine, COX-2 inhibitors (such as celecoxib or etoricoxib), proteosome inhibitors (for example, PS341); CCI-779; tepimib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as ablactam sodium Pixentron; farnesyl transferase inhibitors, such as lonafarnib (SCH 6636, SARASAR TM ); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

[0221] The term "growth inhibitor" generally refers to a compound or composition that inhibits the growth and / or proliferation of cells (e.g., cells whose growth depends on PD-L1 expression) in vitro or in vivo. A growth inhibitor can be a substance that significantly reduces the percentage of cells in the S phase. Non-limiting examples of growth inhibitors include agents that block cell cycle progression (at a position different from the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vinca alkaloids (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as anthracycline antibiotics doxorubicin ((8S-cis) 10-[(3-amino-2,3,6-trideoxy-α-L-toluene-hexapyranosyl) oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(acetyl) 1-methoxy-5,12-naphthalenedione), epirubicin, daunorubicin, etoposide, and bleomycin. Agents that arrest G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Taxanes (paclitaxel and docetaxel) are anticancer drugs that are derived from the yew tree. Docetaxel ( Rhone-Poulenc Rorer), derived from European yew, is a Paclitaxel and docetaxel promote the assembly of microtubules by tubulin dimers and stabilize microtubules by preventing depolymerization, thereby inhibiting cell mitosis.

[0222] VIII. Therapeutic Applications

[0223] The present system can be introduced into a variety of immune cells, including any cells involved in an immune response. In some embodiments, immune cells include granulocytes, such as eosinophils, eosinophils, and neutrophils; mast cells; monocytes that can develop into macrophages; antigen presenting cells, such as dendritic cells; and lymphocytes, such as natural killer cells (NK cells), B cells, and T cells. In some embodiments, immune cells are immune effector cells. Immune effector cells refer to immune cells that can perform specific functions in response to stimulation. In some embodiments, immune cells are immune effector cells that can induce cell death. In some embodiments, immune cells are lymphocytes. In some embodiments, lymphocytes are NK cells. In some embodiments, lymphocytes are T cells. In some embodiments, T cells are activated T cells. T cells include both naive cells and memory cells (e.g., central memory or T cells). CM , effector memory or T EM and effector memory RA or T EMRA), effector cells (e.g., cytotoxic T cells or CTL or Tc cells), helper cells (e.g., Th1, Th2, Th3, Th9, Th7, TFH), regulatory cells (e.g., Treg and Tr1 cells), natural killer T cells (NKT cells), tumor infiltrating lymphocytes (TIL), lymphocyte-activated killer cells (LAK), αβT cells, γδT cells, and similar unique categories of T cell lineages. T cells can be divided into two broad categories: CD8+T cells and CD4+T cells based on the proteins present on the cell surface. T cells expressing the subject system can perform a variety of functions, including killing infected cells and activating or recruiting other immune cells. CD8+T cells are called cytotoxic T cells or cytotoxic T lymphocytes (CTL). CTL expressing the subject system can participate in the recognition and removal of virus-infected cells and cancer cells. CTL has a special compartment or granule containing cytotoxins that cause apoptosis, such as programmed cell death. CD4+ T cells can be subdivided into four subgroups - Th1, Th2, Th17 and Treg, where "Th" refers to "T helper cells", although other subgroups may exist. Th1 cells can coordinate immune responses against intracellular microorganisms, especially bacteria. They can produce and secrete molecules that warn and activate other immune cells, such as macrophages that engulf bacteria. Th2 cells are involved in coordinating immune responses against extracellular pathogens such as helminths (parasites) by warning B cells, granulocytes and mast cells. Th17 cells can produce interleukin 17 (IL-17), which is a signaling molecule for activated immune cells and non-immune cells. Th17 cells are important for recruiting neutrophils.

[0224] The ligand or antigen (ie, target antigen) of the antigen binding moiety as disclosed herein may be a cell surface marker, a secreted marker, or an intracellular marker.

[0225] Non-limiting examples of antigens (i.e., target antigens) of antigen binding moieties as disclosed herein can include ADGRE2, carbonic anhydrase IX (CA1X), CCRI, CCR4, carcinoembryonic antigen (CEA), CD3ζ, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CD269 (BCMA), CD S, CLEC12A, antigens of cytomegalovirus (CMV) infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP), 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2,3,4, EGFR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), gp100, human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13rα2), kappa light chain, kinase insert domain receptor (KDR), Kappa, Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, MART-1, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, NY-ESO-2, carcinoembryonic antigen (h5T4), PRAIVIE, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and various pathogen antigens (e.g., pathogen antigens derived from viruses, bacteria, fungi, parasites, and protozoa that are capable of causing disease). In some examples, the pathogen antigen is derived from HIV, HBV, EBV, human papillomavirus, Lassa virus, influenza virus, or coronavirus.

[0226] Additional examples of antigens of antigen binding moieties as disclosed herein may include 1-40-β-amyloid, 4-1BB, 5AC, 5T4, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1), B7-H3, anthrax bacillus, BAFF, β-amyloid, B-lymphoma cells, C242 antigen, C5, CA-125, canine lupus family IL31, carbonic anhydrase 9 (CA-IX), myocardial globulin, CCL11 (eosinophilic granulocytes), CCL ...242 antigen, C5, CA-125, canine lupus family IL31, carbonic anhydrase 9 (CA-IX), myocardial globulin, CCL242 antigen, CCL242 antigen, C5, CA-125, canine lupus family IL31, carbonic anhydrase 9 (CA-IX), myocardial globulin, CCL242 antigen, CCL242 antigen, C5, CA-125, canine lupus family IL31, carbonic anhydrase 9 (CA-IX), myocardial globulin, CCL242 antigen, CCL242 antigen, C5, CA-125, canine lupus family IL31 chemokine-1), CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basic protein), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3ε, CD30, CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-related antigen, CFD, ch4D5, CLDN18.2, Clostridium difficile, clumping factor A, CSF1R, CSF2, CTLA-4, CXC chemokine receptor type 4, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2, EGFL7, EGFR, endotoxin, EpCAM, epithelial sialic acid protein, ERBB3, Escherichia coli, F protein of respiratory syncytial virus, FAP, fibrin II β chain, fibronectin additional domain-B, folate hydrolase, folate receptor 1, folate receptor α, frizzled receptor, ganglioside GD2, GD2, GD3 ganglioside, phosphatidylinositol proteoglycan 3, GMCSF receptor α chain, GPNMB, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, Hsp90, human scatter factor receptor kinase, human TNF, human β-amyloid protein, ICAM-1 (CD54), IFN-α, IFN-γ, IgE, IgEFc region, IGF-1 receptor, IGF-1, IGHE, IL17A, IL17F, IL20, IL-12, IL-13, IL-17, IL-1β, IL-22, IL-23, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, ILGF2, influenza A hemagglutinin, influenza A virus hemagglutinin, insulin-like growth factor I receptor, integrin α4β7, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, Integrin αvβ3, interferon α / β receptor, interferon γ-induced protein, ITGA2, ITGB2 (CD18), KIR2D, Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), LTA, MCP-1, mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen), neural apoptosis regulating protease 1. NGF, N-glycolylneuraminic acid, NOGO-A, Notch receptor, NRP1, European wild rabbit, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-Rα, sodium phosphate cotransporter, phosphatidylserine, platelet-derived growth factor receptor β, prostate cancer cells, Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, rhesus factor, RON, RTN4, sclerostin, SDC1, selectin P, SLAMF7, SOST , sphingosine-1-phosphate, Staphylococcus aureus, STEAP1, TAG-72, T cell receptor, TEM1, tenascin C, TFPI, TGF-β1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, tumor-specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA, VEGFR1, VEGFR2, vimentin, and VWF.

[0227] Additional examples of antigens of antigen binding moieties as disclosed herein may include 707-AP, biotinylated molecules, a-actin-4, abl-bcr alb-b3 (b2a2), abl-bcr alb-b4 (b3a2), adipose differentiation-related protein, 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, Eph A3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6 / AML, FBP, fetal acetylcholine receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2(FGF6), HST-2 / neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR / FUT, Lewis Y, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, malic enzyme, mammaglobin-A, MART-1 / Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, myosin, NA88-A, Neo-PAP, NKG2D, NPM / ALK, N-RAS,NY-ESO-1, OA1, OGT, tumor-like antigen (h5T4), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, survivin, survivin-2B, SYT / SSX, TAG-72, TEL / AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2 / INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ-light chain. ,

[0228] Additional examples of antigens of antigen-binding moieties as disclosed herein may include antibodies, fragments thereof, or variants thereof. Such antibodies may be natural antibodies (e.g., naturally secreted by immune cells of a subject, such as B cells), synthetic antibodies, or modified antibodies. In some cases, the antigens of antigen-binding moieties as disclosed herein may include Fc domains from antibodies comprising: 20-(74)-(74) (milatuzumab; veltuzumab), 20-2b-2b, 3F8, 74-(20)-(20) (milatuzumab; veltuzumab), 8H9, A33, AB-16B5, abagozumab, abciximab, abituzumab, zlintuzumab), a Kesuzumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, atikatuzumab, aducanumab, afelimomab, AFM13, aftozumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, asacetuzumab, ALD518, alemtuzumab, amorobumab, atumotuzumab triamine pentaacetic acid, ametuximab, AMG 228, AMG 820, maanumab, anetuzumab, rituximab, amifuzumab, anrulizumab, APN301, APN311, apolizumab, APX003 / SIM-BD0801 (sevacizumab), APX005M, acitumomab, ARX788, asciquinavax, aseluzumab, ASG-15ME, atezolizumab, antamidizumab, ATL101, tocilizumab (also known as tocilizumab) umab), atolitumumab, avelumab, B-701, bassinizumab, basiliximab, baviximab, BAY1129980, BAY1187982, betumomab, beglozine, belimumab, beralizumab, betitumumab, besozumab, betalutin (177Lu-tetraxetam-tertulocumab), bevacizumab, BEVZ92 (bevacizumab biosimilar), bevacizumab, BGB-A317, BHQ880, BI 836880, BI-505, bicizumab, bimoglobin, bimegizumab, mobivalmab maytansine, BIW-8962, blinatumomab, brolockizumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bokoximab, brentuximab, BrevaRex, brianuzumab, brodolizumab, brolucizumab, brontocizumab, C2-2b-2b, canakinumab, cantuzumab maytansine, cantuzumab rettansine, caprocuzumab, caprocuzumab pendetide, kaluzumab, catumaxomab, cBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab),CC-90002, CDX-014, CDX-1401, cedizumab, tocilizumab, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, cetaxeluzumab bogatoxin, cetaxeluzumab, clazazumab, clenoliximab, clevazumab tetraazacyclododecane tetraacetate, CM-24, cordrizumab, coltuizumab retansine, cannatuzumab, concizumab (iodine I-131 delotuximab biotin), cR6261, crenazumab, DA-3111 (trastuzumab biosimilar ), daritumomab, daclizumab, dalopizumab, dapiromab pegylated, daratumumab, (daratumumab): daratumumab boosted, dalurugin, declizumab, demsitumab, denitozumab mafodotin, denosumab, depertuzumab, depertuzumab mafodotin, delotuximab biotin, detumumab, DI-B4, denutucimab, diridavucumab, DKN-01, DMOT4039A, dolimumab alitoxin, derosizumab, DS-1123, DS-8895, duligomab, dupilumab, durvalumab, ducegizumab, ixekizumab, eculizumab, edopalatumumab, erecuzumab, efalizumab Monoclonal antibody, ifengolimab, eldiruumab, ergetumab, elotuzumab, elcilimab, emtansumab, embetuzumab, enavatumumab, envolizumab (enfototumab vedicitutumab), enlimomab pegylated, ennoblizumab, enoxitumab, enoxitumomab, encitumomab, epitumomab cetuxitatan, epratuzumab, erlizumab, ertusumab, ertacilizumab, ertusumab, ertracilizumab, efenazumab, eloumab, ecilinumab, fanolexomab, farametumomab, faretuzumab, fasinumab, FBTA05, felvimab, fezacitumomab, FF-21101, FGFR2 antibody-drug conjugate, fibromonas, feclatuzumab tozumab, fegetumomab, firivimumab, franvotumab, flecumumab, fontolimab, forulalumab, forlavirumab, FPA144, fresolizumab, FS102, fulalumab, fultuximab, galiximab, ganituzumab, gantena, gavilimumab, gemtuzumab ozogamicin, grilimumab, gevuzumab, glentozumab, glibatuzumab vedicituzumab, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hu14.18K322A monoclonal antibody, hu3S193,Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, imumotuzumab titracetate (ibrutinib titracetate), iclozumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (claudiximab), imalumab, IMC-CS4, IMC-D11, imciromax, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraazacyclododecane tetraacetate), IMMU-114, immunomodulatory IMP701 antagonist antibody, INCAGN1876, inclazumab, INCSHR1210, indatuximab retansine, indu Satuzumab vedicizumab, infliximab, inolizumab, inotuzumab ozogamicin, intetozumab, ipafrecept, IPH4102, ipilimumab, isatuzumab, isatuximab, istilatuzumab, itozumab, ixetuzumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2 / L19TNF, labetuzumab, labetuzumab govitecan, LAG525, lambolizumab, lambalizumab, L-DOS47, levoximab, lemasomumab, lenzilumab, lerdilimab, leukotuximab, levoxilimab, ribavirin, rifatuximab vedicizumab tozumab, ligelizumab, lilotuzumab satitrazine, lintuximab, lirelumab, LKZ145, lodesizumab, lovatorumab, lovatoruximab maytansine, rucatumumab, rulizumab pegylated, lumiliximab, lumirituzumab, LY3164530, mapatumumab, makituximab, masilimomab, matuzumab, mavulizumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (enelizumab), MEDI-565, MEDI6469, mepolizumab, metelizumab, MGB453, MGD006 / S80880, MGD007, MGD009, MGD011, miratumomab, miratumomab-SN-38, miratumomab, miratumomab soratumosin, miratumomab, MK-4166, MM-111, MM-151, MM-302, mogamozumab, MOR202, MOR208, MORAb-066, morolimumab, motavimab, mositumomab pasutoxin, morolimumab-CD3, naclolimumab tafenatoxin, nanomab, naputumomab estafenaxin, natutumomab, natalizumab, nebazumab, nesitutumomab, nemolimumab, nerelimumab, nesvastatin, nimotuzumab, nivolumab,Nofitumomab mepentate, NOV-10, obilitoxamab, obinutuzumab, ocalatumumab, ocrelizumab, odulizumab, ofatumumab, olaratumumab, ololituzumab, omalizumab, OMP-131R10, OMP-305B83, onatumumab, ontuzumab, opiximab, obotuzumab monatuximab, oregol, otecumab, otelizumab, ox002 / MEN1309, oxerizumab, ozanezumab, ozolavizumab, pagribuzumab, palivizumab, panitumumab, pancolizumab, pancolizumab-GE X, panobomab, palsatumumab, pascolimumab, pasotuzumab, patrickizumab, patrimonumab, PAT-SC1, PAT-SM6, pembrolizumab, pentotumomab, pelagizumab, pertuzumab, perximab, PF-05082566 (utumumab), PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatumumab vedicitumumab, pinatumumab, pracumumab, pelotonumab vedicitumumab, bonecumab, preliximab, pretoxan, pretoxan, PRO 140, proximal lumib, prostate-specific membrane antigen antibody drug conjugate (PSMA) ADC), quilimumab, lacotumomab, radrelimumab, ravivimab, ralpancitumomab, ramucirumab, ranibizumab, recibazumab, refelizumab, regavir, REGN1400, REGN2810 / SAR439684, reslizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7 841, RG7876, RG7888, RG7986, rellotuzumab, renulumab, rituximab, RM-1929, RO7009789, robatuzumab, roleumab, romosomal, lontalizumab, rovelimab, ruprilimab, certolizumab govitecan, samaritumumab, SAR408701, SAR566658, sarrelumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, ceribantumab, cetosazumab, sevimab, SGN-CD19A, SGN-CD19B, SGN-CD33A, SGN-CD70A, SGN-LIV1A, sibutumab, sifalimumab, cetuximab, simutumab, siplimab, silutumab, sofitumab vedicitutumab, solanumab, solituzumab, sonopimumab, soentuximab, stamlumumab, solesuzumab, sovereignumab, SYD985, SYM004 (fultuximab and modotuximab), Sym015, TAB08, tabarumab,Tacatumomab tetraazacyclododecane tetraacetate, tadocizumab, talimumab, tanezumab, tanibirumab, taplitumomab paptox, taretuzumab, TB-403, tefibazumab, tiligen, tilitumomab alitoxin, tenatumomab, teneliximab, teprotumumab, teprotuzumab, texidomab, tetulocumab, TG-1303, TGN1412, thorium -227-epazolizumab conjugate, tesilinumab, tegatumumab, tirazumab, tesutuzumab vedicitutumab, TNX-650, tocilizumab, tolarizumab, tosatumumab, tositumomab, toviromab, telaluzumab, trastuzumab, trastuzumab enmetuzumab, TRBS07, TRC105, troglitumab, tremelimumab, travoglumab, TRPH 011, TRX518, TSR-042, TTI-200.7, Tucotumomab Selmoleukin, Tuvelumab, U3-1565, U3-1784, Ubulituzumab, Ulocurumab, Urelumab, Utosamab, Ustekinumab, Vadatumomab Talilin, Vandotumomab Vedicituzumab, Vantikumab, Vannumab, Valpaliximab, Valilumab, Vatelimab, VB6-845, Vedolizumab, Veltuzumab, Vepalimumab, Visenumab, Vesilimumab, Voloximab, Vorsetumab Mafudotin, Vortotumomab, YYB-101, Zarutozumab, Zanolizumab, Zatuximab, Zilalimumab, Zolimumab Aritoxin. ,

[0229] Any system as disclosed herein can be used to modulate the expression or activity of endogenous proteins in a cell. Tables 4, 5 and 6 provide exemplary genes encoding endogenous proteins as disclosed herein. Tables 4 and 5 provide exemplary genes associated with certain diseases and conditions. Examples of genes and polynucleotides associated with signal transduction biochemical pathways are listed in Table 6.

[0230] [Table 4]

[0231]

[0232]

[0233] [Table 5]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240] [Table 6]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259] Any of the systems and methods disclosed herein can be used to treat a target cell, target tissue, target condition, or target disease in a subject.

[0260] The target disease may be a viral, bacterial and / or parasitic infection; an inflammatory and / or autoimmune disease; or a neoplasm such as a cancer and / or a tumor.

[0261] The target cell can be a diseased cell. The diseased cell may have altered metabolism, gene expression and / or morphological characteristics. The diseased cell can be a cancer cell, a diabetic cell, and an apoptotic cell. The diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood diseases, cancer, metabolic diseases, eye diseases, organ diseases, musculoskeletal diseases, heart diseases, etc.

[0262] A variety of target cells can be killed using any of the methods or compositions disclosed herein. The target cells can 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 inside an organism. The target cell can be an organism. The target cell can be a cell in a 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 can be 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.

[0263] The target cell can be a stem cell or a progenitor cell. The target cell can include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem cells (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.). The target cell can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Cloned cells can include the offspring of cells. The target cell can contain a target nucleic acid. The target cell can be present in a living organism. The target cell can be a genetically modified cell. The target cell can be a host cell.

[0264] The target cell can be a totipotent stem cell, however, 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, however, 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 cell lineage, but cannot differentiate into any other non-hematopoietic cells. The target cell may be able to develop into a complete organism. The target cell may or may not develop into a complete organism. The target cell can be a complete organism.

[0265] 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 a culture medium.

[0266] The target cell can be a diseased cell. The diseased cell may have altered metabolism, gene expression and / or morphological characteristics. The diseased cell can be a cancer cell, a diabetic cell, and an apoptotic cell. The diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood diseases, cancer, metabolic diseases, eye diseases, organ diseases, musculoskeletal diseases, heart diseases, etc.

[0267] If the target cell is a primary cell, it can be obtained from an individual by any method. For example, leukocytes can be obtained by single collection, leukocyte single collection, density gradient separation and other methods. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be obtained by biopsy. A suitable solution can be used to disperse or suspend the harvested cells. Such solutions can generally be balanced salt solutions (e.g., physiological saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc.), conveniently supplemented with fetal bovine serum or other naturally occurring factors, combined with acceptable low concentration buffers. Buffers may include HEPES, phosphate buffer, lactate buffer, etc. Cells can be used immediately, or can be stored (e.g., by freezing). Frozen cells can be thawed and reused. Cells can be frozen in DMSO, serum, culture medium buffer (e.g., 10% DMSO, 50% serum, 40% buffer) and / or some other such common solutions for preserving cells at freezing temperatures.

[0268] Non-limiting examples of cells that can be target cells include, but are not limited to, lymphocytes, such as B cells, T cells (cytotoxic T cells, natural black cells, regulatory T cells, T helper cells), natural killer cells, cytokine-induced killer (CIK) cells (see, e.g., US20080241194); myeloid cells, such as granulocytes (basophils, eosinophils, neutrophils / high-fragment neutrophils), monocytes / macrophages, red blood cells (reticulocytes) , mast cells, platelets / megakaryocytes, dendritic cells; cells from the endocrine system, including thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, eosinophils), adrenal (chromaffin cells), pineal (pinealocytes) cells; cells of the nervous system, including glial cells (astroglia, microglia), magnocellular neurosecretory cells, stellate cells, Boettcher cells, and pituitary (gonadotropins, adrenocorticotropic cells, thyrotropin-stimulating cells, growth hormone , prolactin); cells of the respiratory system, including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells; cells of the circulatory system, including cardiomyocytes, pericytes; cells of the digestive system, including stomach (gastric chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromatin 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 cells, keratinocytes, melanocytes (nevus cells); muscle cells, including myocytes; urinary system cells, including podocytes, juxtaglomerular cells, glomerular mesangial cells / extraglomerular mesangial cells, renal proximal tubule brush border cells, macula densa cells; reproductive system cells, including sperm, supporting cells, testicular interstitial cells, eggs; and other cells,Includes adipocytes, fibroblasts, tenocytes, epidermal keratinocytes (differentiating 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, hair epithelium, hair root sheath keratinocytes, Huxley's layer hair root sheath cells, Henle's layer hair root sheath cells, outer hair root sheath cells, hair matrix cells (stem cells), moist stratified barrier epithelial cells, stratified squamous surface epithelial cells of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina Cells, basal cells (stem cells) of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vaginal epithelium, urothelial cells (lining the bladder and urethra), exocrine secretory epithelial cells, salivary gland mucous cells (secreting polysaccharide-rich substances), salivary gland serous cells (secreting glycoprotein-rich substances), von Ebner gland cells in the tongue (rinsing taste buds), mammary gland cells (secreting milk), lacrimal gland cells (secreting tears), cerumen gland cells in the ear (secreting earwax), eccrine sweat gland dark cells (secreting glycoproteins), eccrine sweat gland light cells (secreting small molecules). Apocrine sweat gland cells (secreting odorous substances, sensitive to sex hormones), Morel gland cells of the eyelids (specialized sweat glands), sebaceous gland cells (secreting lipid-rich sebum), Bowman gland cells in the nasal cavity (rinsing the olfactory epithelium), Brunner gland cells in the duodenum (secreting enzymes and alkaline mucus), seminal vesicle cells (secreting semen components,Including fructose that provides energy for sperm swimming), prostate cells (secreting semen components), bulbourethral gland cells (secreting mucus), Bartholin's gland cells (secreting vaginal lubricant), paraurethral gland cells (secreting mucus), endometrial cells (secreting carbohydrates), solitary goblet cells of the respiratory and digestive tracts (secreting mucus), gastric mucosal mucous cells (secreting mucus), gastric gland chief cells (secreting pepsinogen), gastric gland parietal cells (secreting hydrochloric acid), pancreatic acinar cells (secreting bicarbonate and digestive enzymes), Paneth cells of the small intestine (secreting lysozyme), type II alveolar cells of the lungs (secreting surfactant), Clara cells of the lungs, hormone-secreting cells, anterior pituitary cells, growth hormone-secreting cells, prolactin-secreting cells, thyroid-stimulating hormone-secreting cells, gonadotropin-secreting cells, adrenocorticotropic hormone-secreting cells, pituitary Intermediate cells, magnocellular neurosecretory cells, gastrointestinal and respiratory tract cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophils, adrenal cells, chromaffin cells, interstitial cells of the testis, endometrial cells of ovarian follicles, luteal cells of ruptured follicles, granulosa-luteal cells, theca-luteal cells, juxtaglomerular cells (secreting renin), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lungs, intestines, exocrine glands and urogenital tract), kidneys, type I alveolar cells (lining the air cavities of the lungs), pancreatic duct cells (alveolar heart cells), duct cells (of sweat glands, salivary glands, mammary glands, etc.), duct cells (of seminal vesicles, prostate, etc.), epithelial cells lining closed body cavities in the body, ciliated cells with propulsion function, extracellular matrix-secreting cells, contractile cells. Skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (multiple types), Langerhans cells of the epidermis, osteoclasts (found in bones), dendritic cells (found in lymphoid tissue), microglia (found in the central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, blood and immune system Stem cells and committed progenitor cells of the system (multiple types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomous neuronal cells, sensory organ and peripheral neuronal supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, trophoblasts, follicular cells, supporting cells (present in the testes), thymic epithelial cells, interstitial cells and renal interstitial cells.

[0269] Of particular interest are cancer cells. In some embodiments, the target cell is a cancer cell. Non-limiting examples of cancer cells include cells from the following types of cancer: acanthoma, acinar cell carcinoma, acoustic neuroma, acral lentiginous melanoma, sweat gland spiradenoma, acute eosinophilic leukemia, acute lymphocytic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloid leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, ameloblastoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, ameloblastic fibroma, odontogenic adenoid tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive natural killer cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anal canal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, renal collecting duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, cancer of unknown primary site, carcinosarcoma, Castleman disease, embryonal tumor of the central nervous system, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, bile duct cancer, chondroma, chondrosarcoma tumor, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, 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 neuroepithelioma, embryonal carcinoma, endodermal sinus tumor, endometrial carcinoma, endometrial uterine carcinoma, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, olfactory neuroblastoma, Ewing tumor family, Ewing Ewing's sarcoma family, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, thyroid follicular cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, gangliocytoma, gastric cancer, 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, glioma cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer,Hemangioblastoma, hemangiopericytoma, angiosarcoma, hematological malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin 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, hilar cholangiocarcinoma, Krukenberg tumor, laryngeal cancer, laryngeal cancer, malignant lentigo melanoma, leukemia, leukemia, lip and oral cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoid leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone Histiocytoma, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant salamander tumor, mucosa-associated lymphoid tissue lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell carcinoma of the neck from occult primary, metastatic urothelial carcinoma, mixed mullerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasm syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic syndrome, myelodysplastic syndrome, myeloid Leukemia, Myeloid sarcoma, Myeloproliferative disorders, Myxoma, Nasal cancer, Nasopharyngeal carcinoma, Nasopharyngeal carcinoma, Tumor, Schwannoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin lymphoma, Non-Hodgkin lymphoma, Non-melanoma skin cancer, Non-small cell lung cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Large oncocytoma, Optic nerve sheath meningioma, Oral cancer, Oral cancer, Oropharyngeal cancer, Osteosarcoma, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor, Ovarian low malignant potential tumor, Paget's disease of the breast, Superior sulcus tumor, Pancreatic cancer, Pancreatic cancer, Thyroid papillary carcinoma, Papillomatosis, Paraganglioma, Paranasal sinus cancer, Parathyroid cancer, Penile cancer , perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pinealoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polyembryony, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory cancer involving the NUT gene of chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary tumors, seminoma, serous tumor,Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestinal cancer, soft tissue sarcoma, somatostatinoma, sooty warts, spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric 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, terminal lymphangiogenic carcinoma, testicular cancer, theca cell tumor, laryngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal carcinoma, urethral cancer, genitourinary tumors, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-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 in a cancer cell population, such as a cancer stem cell. In some embodiments, the cancer is of the hematopoietic lineage, such as a lymphoma. The antigen may be a tumor-associated antigen. ,

[0270] In some cases, the subject may have or may be suspected of having an autoimmune disease. Non-limiting examples of autoimmune diseases include acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease (adrenocortical insufficiency), agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplant rejection, anti-glomerular basement membrane / anti-tubular basement membrane nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune Epidemiological retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy, Barlow's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), allergic granulomatous vasculitis (Charg-Strauss syndrome), cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, coxsackievirus myocarditis, CREST syndrome (calcification, renal cell carcinoma), Noah's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia syndrome), essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, neuromyelitis optica (Devic's disease), discoid lupus erythematosus, Dressler syndrome (post-myocardial infarction syndrome), endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, pulmonary fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease (toxic diffuse goiter), Guillain-Barré syndrome, bridge This encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes mellitus (type 1 diabetes), interstitial cystitis, juvenile idiopathic arthritis, juvenile diabetes, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, wood-like conjunctivitis, linear IgA disease (LAD), systemic lupus erythematosus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD),Monoclonal gammopathy of undetermined significance (MGUS), Moren's ulcer, acute pityriasis lichenoides (Mucha-Habermann disease), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatic disease, pediatric autoimmune neuropsychiatric disorder with streptococcal infection (PANDAS), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry's Romberg syndrome (progressive hemifacial atrophy), Parsonnage-Turner syndrome (brachial plexus neuritis), peripheral uveitis (pars planitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes syndrome), polyarteritis nodosa, autoimmune polyglandular syndromes type I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome, progestogen dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reynolds syndrome Syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome (autoimmune polyendocrine disease syndrome), scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-man syndrome, subacute infective endocarditis (SBE), Susac syndrome, sympathetic ophthalmia, Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, bullous dermatosis, vitiligo, Waldenstrom's macroglobulinemia (WM), and Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)).

[0271] In some cases, the autoimmune disease comprises one or more selected from the following group: rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE), myasthenia gravis, multiple sclerosis, scleroderma, Addison's disease (adrenocortical insufficiency), bullous pemphigoid, pemphigus vulgaris, Guillain-Barre syndrome, Sjögren's syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia (WM), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Hashimoto's encephalopathy (HE), Hashimoto's thyroiditis, Graves' disease (toxic diffuse goiter), Wegener's granulomatosis, and antibody-mediated transplant rejection (e.g., for tissue transplants such as kidney transplants). In an example, the autoimmune disease can be type 1 diabetes, lupus or rheumatoid arthritis.

[0272] In some cases, the target cells form tumors (i.e., solid tumors). Tumors treated with the methods herein can result in stable tumor growth (e.g., one or more tumors increase in size by no more than 1%, 5%, 10%, 15%, or 20%, and / or do not metastasize). In some cases, the tumor is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some cases, the tumor is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some cases, the tumor is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more years. In some cases, the size of the tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some cases, the tumor is completely eliminated, or reduced to below the detection level. In some cases, 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 after treatment. In some cases, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months after treatment. In some cases, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more after treatment.

[0273] Example

[0274] Example 1: Regulation of endogenous target proteins

[0275] Jurkat cells are transduced with a lentivirus containing ef1a-dCas9-VPR-Q8 (hereinafter referred to as "Q8"), and Q8-positive cells are subsequently sorted (e.g., about 1 week later). The sorted Q8-positive cells are then sorted again (e.g., about 2 weeks later) to establish a cell line expressing dCas9-VPR. These cells (e.g., about 200,000 cells per reaction) are then transfected with sgRNA (e.g., about 250ng-500ng of sgRNA) using a transfection agent. Prior to gene expression analysis, the cells are plated in culture medium (e.g., RPMI1640+10% FCS) (e.g., in a 96-well plate) and incubated at 37°C for a period of time (e.g., 48 hours to 72 hours).

[0276] Gene expression of the target protein was measured with SYBR green qPCR using the delta delta Ct method using the primers provided in Table 7 (eg, forward (F) primer, reverse (R) primer).

[0277] Table 7 .

[0278]

[0279] a.ID3

[0280] The positions of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding ID3 are shown in Figure 5A and multiple guide RNA sequences targeting ID3 are provided in Figure 5B (Top) Middle.

[0281] Enhanced expression of endogenous ID3 in Jurkat cells, after activation by the system disclosed herein, comprising Q8 and one of a variety of guide RNAs for ID3, such as Figure 5B (Bottom). In some cases, the use of such a system promotes the expression level of endogenous ID3 to be enhanced by about 25-fold (e.g., ID3_UP_gR61r), about 23-fold (e.g., ID3_UP_gR31r), or about 5-fold (e.g., ID3_UP_gR62r) compared to a control Jurket cell having a control (e.g., a control gRNA that binds to a different position of the ID3 gene or does not exhibit specific binding affinity to the ID3 gene).

[0282] bc-Jun

[0283] The target polynucleotide sequences of the multiple guide RNAs are located at Fig. 6A and the sequences of multiple guide RNAs targeting c-Jun are shown in Figure 6B (top) is provided.

[0284] Enhanced expression of endogenous c-Jun in Jurkat cells, after activation by the system disclosed herein, comprising Q8 and one of a plurality of guide RNAs for c-Jun, such as Figure 6B As shown (bottom). In some cases, the expression level of endogenous c-Jun is promoted by about 19-fold (e.g., JUN_UP_gR94f), about 13-fold (e.g., JUN_UP_gR31f or Jun_gR53f), or about 4-fold (e.g., JUN_UP_gR53f) using this system compared to a control Jurket cell having a control (e.g., a control gRNA that binds to a different location of the c-Jun gene or does not exhibit specific binding affinity to the c-Jun gene).

[0285] c.TBX21

[0286] The positions of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding TBX21 are shown in Fig. 7A and the sequences of multiple guide RNAs targeting TBX21 are provided in Figure 7B (Top) Middle.

[0287] Enhanced expression of endogenous TBX21 in Jurkat cells, after activation by the system disclosed herein, comprising Q8 and one of a plurality of guide RNAs for TBX21, such as Figure 7B In some cases, use of such a system can promote expression levels of endogenous TBX21 by about 400-fold (e.g., TBX21_UP_gR32r), about 250-fold (e.g., TBX21_UP_gR8r), or about 140-fold (e.g., TBX21_UP_gR77f), or about 120-fold (e.g., TBX21_UP_gR64r), relative to Jurket cells with a control, e.g., a control gRNA that binds to a different location of the TBX21 gene or does not exhibit specific binding affinity to the TBX21 gene.

[0288] d.IL-21

[0289] The positions of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding IL-21 are shown in Fig. 8A and the sequences of multiple guide RNAs for IL-21 are provided in Figure 8B (Top) Middle.

[0290] Enhanced expression of endogenous IL-21 in Jurkat cells, after activation by the system disclosed herein, comprising Q8 and one of a variety of guide RNAs against IL-21, such as Figure 8BAs shown (bottom). In some cases, use of such a system can promote the expression level of endogenous IL-21 by about 10 times (e.g., IL-21_UP_gR8r), about 100 times (e.g., IL-21_UP_gR16r), or about 1,000 times (e.g., IL-21_UP_gR42f), relative to control Jurket cells with a control gRNA (e.g., IL21_UP_gR92f), which binds to a different position of the IL-21 gene or does not exhibit specific binding affinity to the IL-21 gene.

[0291] e.TOX1

[0292] The positions of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding TOX1 are shown in Fig.9A In the present invention, the sequences of multiple guide RNAs targeting TOX1 are provided in Fig. 9B Middle (top).

[0293] The expression of endogenous TOX1 in Jurkat cells, after being inhibited by the system disclosed herein, comprises Q8 and one of a plurality of guide RNAs for TOX1, such as Fig. 9B In some cases, use of such a system promotes a reduction in endogenous TOX1 expression levels, such as an endogenous TOX1 expression level of about 0.4 (e.g., TOX_1), about 0.5 (e.g., TOX_2), or about 0.7 to about 0.8 (e.g., TOX_3 and TOX_4) relative to a control Jurket cell having a control gRNA that binds to a different location of the TOX1 gene or does not exhibit specific binding affinity for the TOX1 gene.

[0294] f.TOX2

[0295] The positions of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding TOX2 are shown in Fig. 10A and the sequences of multiple guide RNAs targeting TOX2 are provided in Fig. 10B (Top) Middle.

[0296] The expression of endogenous TOX2 in Jurkat cells, after being inhibited by the system disclosed herein, comprises Q8 and one of a plurality of guide RNAs against TOX2, such as Fig. 10B(Bottom). In some cases, use of such a system promotes a reduction in endogenous TOX2 expression levels, as shown by an expression level of endogenous TOX2 relative to the expression level in a control Jurket cell with a control gRNA that binds to a different location of the TOX2 gene or does not exhibit specific binding affinity to the TOX2 gene.

[0297] g.SHIP1

[0298] The positions of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding SHIP1 are shown in Fig.11A and the sequences of multiple guide RNAs targeting SHIP1 are provided in Fig. 11B (Top) Middle.

[0299] The expression of endogenous SHIP1 in Jurkat cells, after being inhibited by the system disclosed herein comprising Q8 and one of multiple guide RNAs for SHIP1, is shown in Fig. 11B (Bottom). In some cases, use of such a system promotes a reduction in endogenous SHIP1 expression levels, such as an expression level of endogenous SHIP1 of about 0.2 (e.g., SHIPgRr7, SHIPgR32f, or SHIPgR49r) or about 0.3 (e.g., SHIPgR60f) relative to a control Jurket cell having a control gRNA that binds to a different location of the SHIP1 gene or does not exhibit specific binding affinity to the SHIP1 gene.

[0300] h.B2M

[0301] The locations of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding B2M are shown in Fig. 12A and the sequences of multiple guide RNAs for B2M are provided in Fig. 12B (Top) Middle.

[0302] The expression of endogenous B2M in Jurkat cells, after being inhibited by the system disclosed herein, comprises Q8 and one of a plurality of guide RNAs against B2M, such as Fig. 12B(Bottom). In some cases, using such a system to promote a reduction in endogenous B2M expression levels indicates that the expression level of endogenous B2M is about 0.7 (e.g., B2M_d_gR56f), about 0.6 (e.g., B2M_d_gR21r), or about 0.4 (e.g., B2M_gR21r), relative to a control Jurket cell that binds to a different position of the B2M gene (e.g., B2M_d_gR248f) or a control gRNA that does not exhibit specific binding affinity to the B2M gene.

[0303] i.BATF

[0304] The locations of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding BATF are shown in Fig.13A and the sequences of multiple guide RNAs for BATF are provided in Fig. 13B (Top) Middle.

[0305] The expression of endogenous BATF in Jurkat cells, after being inhibited by the system disclosed herein, comprises Q8 and one of a plurality of guide RNAs against BATF, such as Fig. 13B As shown (bottom). In some cases, the use of such a system promotes the reduction of endogenous BATF expression levels, as shown, the expression level of endogenous BATF relative to the expression level in control Jurket cells with a control gRNA is about 0.6 (e.g., BATF_d_gR41f or BATF_d_gR56f), about 0.5 (e.g., BATF_d_gR62f), or about 0.3 (e.g., BATF_d_gR22f), which controls bind to different locations of the BATF gene or do not exhibit specific binding affinity to the BATF gene.

[0306] j.SOCS1

[0307] The positions of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding SOCS1 are shown in Fig.14A and the sequences of multiple guide RNAs targeting SOCS1 are provided in Fig. 14B (Top) Middle.

[0308] Expression of endogenous SOCS1 in Jurkat cells, after being inhibited by the system disclosed herein, comprising Q8 and one of multiple guide RNAs for SOCS1, is shown in Fig. 14B(Bottom). In some cases, use of such a system promotes a reduction in endogenous SOCS1 expression levels, as shown by an expression level of endogenous SOCS1 relative to the expression level in a control Jurket cell with a control gRNA of about 0.9 (e.g., SOCS1_d_gR20f), about 0.6 (e.g., SOCS1_d_gR52r), about 0.4 (e.g., SOCS1_d_gR43f), or about 0.3 (e.g., SOCS1_d_gR53f), which controls bind to a different location of the SOCS1 gene or do not exhibit specific binding affinity to the SOCS1 gene.

[0309] k.TGFbR2

[0310] TGFβ (TGFb) can be a pleiotropic cytokine that can be secreted by tumor cells in the tumor microenvironment (TME). T cells can express receptors for TGFb, such as TGFβ2. After TGFb binds to TGFbR (e.g., TGFbR2), downstream signaling of TGFbR can prevent T cells from differentiating into T cell subsets (e.g., Th1 cells) and reduce anti-tumor responses (e.g., IFNg secretion) and / or tumor cell toxicity.

[0311] In order to screen for guide nucleic acid molecules that can be used to inhibit TGFbR2 signaling or activity, Jurkat cells were engineered to express dCAS9-KRAB and transfected with plasmids encoding different TGFbR2 targeting gRNAs. After transfection (e.g., 48-72 hours after transfection), cells were collected and stained with anti-TGFbR2-PE antibodies, and expression was determined by flow cytometry. The screening was repeated (e.g., 3 times) and the most effective gRNA was selected. The most dominant gRNA was mainly located in the region 50-100 base pairs (bp) downstream of the TSS, or in the region 30-70 base pairs upstream of the TSS. The position of the target polynucleotide sequences of multiple guide RNAs relative to the gene encoding TGFbR2 is as shown in FIG. Fig.15A shown.

[0312] Fig. 15B The expression of endogenous TGFbR2 in Jurkat cells is shown, as inhibited by a system disclosed herein, the system comprising dCAS9-KRAB and one of a plurality of guide RNAs for TGFbR2. In some cases, the use of such a system promotes a reduction in the expression level of endogenous TGFbR2, which indicates that the expression level of endogenous TGFbR2 is about 35% to about 20% lower than the expression level in a control Jurkat cell with a control gRNA that binds to a different location of the TGFbR2 gene or does not exhibit specific binding affinity to the TGFbR2 gene.

[0313] Implementation

[0314] The following non-limiting embodiments provide illustrative examples of the present invention but do not limit the scope of the present invention.

[0315] Embodiment 1. A system for regulating the expression or activity of a target protein in a cell, the system comprising:

[0316] an actuator moiety capable of complexing with a target polynucleotide sequence in the cell, wherein the actuator moiety is heterologous to the cell, and wherein the target polynucleotide sequence is endogenous to the cell,

[0317] wherein said complexing causes a change in said expression or said activity of said target protein of at least about 10% as compared to a control cell, wherein said complexing is sufficient to cause said change without editing said target polynucleotide sequence, and

[0318] wherein the target protein comprises one or more members selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), basic leucine zipper transcription factor ATF-like (BATF), inhibitor of DNA binding / differentiation (ID), T-box transcription factor (TBX), c-Jun,

[0319] Optionally among them:

[0320] (1) the target protein is TOX, optionally wherein the TOX comprises TOX1 or TOX2; and / or

[0321] (2) the target protein is the SOCS, optionally wherein the SOCS comprises SOCS1; and / or

[0322] (3) the target protein is BATF; and / or

[0323] (4) the target protein is the ID, optionally wherein the ID comprises ID3; and / or

[0324] (5) the target protein is the TBX, optionally wherein the TBX is TBX21 (T-Bet); and / or

[0325] (6) the target protein is c-Jun; and / or

[0326] (7) The actuator portion can be activated for the complexing when the cell is exposed to an external stimulus.

[0327] Embodiment 2. A system for regulating the expression or activity of a target protein in a cell, the system comprising:

[0328] an actuator moiety capable of complexing with a target polynucleotide sequence in the cell, wherein the actuator moiety is heterologous to the cell and is activated for complexing when the cell is exposed to an external stimulus, and wherein the target polynucleotide sequence is endogenous to the cell,

[0329] wherein upon said exposure, said actuator moiety is activated to effect said complexing, such that said expression or said activity of said target protein changes, wherein said complexing is sufficient to effect said change without editing said target polynucleotide sequence, and

[0330] wherein the target protein comprises inositol phosphatase containing Src homology 2 domain (SHIP) or beta-2-microglobulin (B2M) and TGFβ receptor (TGFbR),

[0331] Optionally among them:

[0332] (1) upon said exposure, activating said actuator moiety to perform said complexing so as to cause said expression or said activity of said target protein to change by at least about 10% compared to that in a control cell; and / or

[0333] (2) the target protein is the SHIP, optionally wherein the SHIP is SHIP1; and / or

[0334] (3) the target protein is the B2M; and / or

[0335] (4) The target protein is the TGFbR, optionally wherein the TGFbR is TGFbR2.

[0336] Embodiment 3. The system according to embodiment 1 or embodiment 2, wherein the system is optionally:

[0337] (1) the target protein is not a secretory protein; and / or (2) the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4,000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases from the TSS of the gene encoding the target protein. and / or (3) the target polynucleotide sequence is located between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4,000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base upstream of the TSS of the gene encoding the target protein; and / or

[0338] (4) the target polynucleotide sequence is located between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4,000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base downstream of the TSS of the gene encoding the target protein; and / or

[0339] (5) the change is that the expression or activity of the target protein is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more compared to that in the control cell; and / or

[0340] (6) the change is an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more in the expression or activity of the target protein as compared to that in the control cell; and / or (7) the cell is an immune cell, and the change in the expression or activity of the target protein promotes the maintenance of the stemness of the immune cell, the survival of the immune cell and / or the expansion of the immune cell; and / or or (8) the cell is an immune cell, and the change in the expression or activity of the target protein promotes enhanced cytokine production by the engineered immune cell, enhanced cytotoxicity of the engineered immune cell against a target cell population, and / or reduced exhaustion of the immune cell; and / or (9) the external stimulus is a ligand, and the system comprises a chimeric receptor polypeptide (receptor) that undergoes modification upon binding to the ligand, wherein the actuator portion is capable of activation after the receptor is modified; and / or

[0341] (10) the activation of the actuator moiety comprises (1) release of the actuator moiety from a substrate, or (2) modification of the actuator moiety; and / or

[0342] (11) the actuator moiety comprises a nucleic acid-guided actuator moiety, and wherein the system further comprises a guide nucleic acid complexed with the actuator moiety; and / or

[0343] (12) the guide nucleic acid comprises guide ribonucleic acid (RNA); and / or

[0344] (13) the system comprises two or more guide nucleic acids having complementarity to different target polynucleotide sequences; and / or (14) the actuator portion comprises an effector domain configured to modulate the expression or activity of the target protein; and / or (15) the effector domain is selected from the group consisting of a cleavage domain, an epigenetic modification domain, a transcriptional activation domain or a transcriptional repressor domain, and optionally wherein:

[0345] (a) the effector domain is a transcriptional activation domain; and / or

[0346] (b) the effector domain is a transcriptional repressor domain; and / or

[0347] (16) The actuator portion comprises a heterologous endonuclease or a variant thereof; and / or

[0348] (17) the modification is a conformational change or a chemical modification; and / or

[0349] (18) The cells are immune cells; and / or (19) The cells are T cells or NK cells.

[0350] Embodiment 4. An engineered cell population, wherein each engineered cell of the population comprises a system according to any one of embodiments 1 to 3.

[0351] Embodiment 5. A composition comprising the engineered cell population according to embodiment 4, optionally wherein the composition further comprises a co-therapeutic agent.

[0352] Embodiment 6. A system comprising a guide nucleic acid molecule, wherein the guide nucleic acid molecule is designed to bind to a target polynucleotide sequence for regulating the expression or activity of a target protein in a cell, wherein the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases away from the TSS of the gene encoding the target protein, between about 5,000 and about 400 bases away from the TSS of the gene encoding the target protein. bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base,

[0353] wherein the target protein is selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), inositol phosphatase containing Src homology 2 domain (SHIP), basic leucine zipper transcription factor ATF-like (BATF), beta-2-microglobulin (B2M), DNA binding / differentiation inhibitor (ID), T-box transcription factor (TBX), c-Jun and TGFβ receptor (TGFbR),

[0354] Optionally among them:

[0355] (1) the guide nucleic acid molecule is capable of recruiting the actuator portion to the target polynucleotide sequence to regulate the expression or activity of the target protein, and wherein the system further comprises the actuator portion; and / or

[0356] (2) the actuator portion comprises a heterologous endonuclease or a variant thereof; and / or

[0357] (3) the gene encoding the target protein is endogenous to the cell; and / or

[0358] (4) The TSS is endogenous to the cell.

[0359] Embodiment 7. A system comprising an actuator portion capable of binding to a target polynucleotide sequence for regulating the expression or activity of a target protein of a cell, wherein the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases, between about 5,000 and about 4,000 bases, from the TSS of the gene encoding the target protein. bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base,

[0360] wherein the target protein is selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), inositol phosphatase containing Src homology 2 domain (SHIP), basic leucine zipper transcription factor ATF-like (BATF), beta-2-microglobulin (B2M), DNA binding / differentiation inhibitor (ID), T-box transcription factor (TBX), c-Jun and TGFβ receptor (TGFbR),

[0361] Optionally among them:

[0362] (1) the actuator portion comprises a heterologous endonuclease or a variant thereof; and / or

[0363] (2) the gene encoding the target protein is endogenous to the cell; and / or

[0364] (3) The TSS is endogenous to the cell.

[0365] Embodiment 8. A method for regulating the expression or activity of a target protein in a cell, the method comprising:

[0366] (a) forming in the cell a complex comprising an actuator moiety and a target polynucleotide sequence, wherein the actuator moiety is heterologous to the cell, and wherein the target polynucleotide sequence is endogenous to the cell,

[0367] (b) in response to said forming, inducing a change in said expression or said activity of said target protein by at least about 10% as compared to that in said control cell, wherein said forming of said complex is sufficient to cause said change to occur without editing said target polynucleotide sequence,

[0368] wherein the target protein comprises one or more members selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), basic leucine zipper transcription factor ATF-like (BATF), inhibitor of DNA binding / differentiation (ID), T-box transcription factor (TBX), c-Jun and TGFβ receptor (TGFbR),

[0369] Optionally among them:

[0370] (1) the target protein is TOX, optionally wherein the TOX comprises TOX1 or TOX2; and / or (2) the target protein is SOCS, optionally wherein the SOCS comprises SOCS1; and / or

[0371] (3) the target protein is BATF; and / or

[0372] (4) the target protein is the ID, optionally wherein the ID comprises ID3; and / or

[0373] (5) the target protein is the TBX, optionally wherein the TBX is TBX21 (T-Bet); and / or (6) the target protein is the c-Jun; and / or

[0374] (7) the target protein is the TGFbR, optionally wherein the TGFbR is TGFbR2; and / or

[0375] (8) the target protein is not a cytokine, optionally wherein the target protein is not a secreted protein; and / or

[0376] (9) The actuator portion can be activated for the complexing when the cell is exposed to an external stimulus.

[0377] Embodiment 9. A method for regulating the expression or activity of a target protein in a cell, the method comprising:

[0378] (a) exposing the cell to an external stimulus to activate the actuator moiety to complex with a target polynucleotide sequence in the cell, wherein the actuator moiety is heterologous to the cell, and wherein the target polynucleotide sequence is endogenous to the cell; and

[0379] (b) in response to said complexing, inducing said expression or said change in activity of said target protein, wherein said formation of said complex is sufficient to cause said change to occur without editing said target polynucleotide sequence,

[0380] wherein the target protein comprises Src homology 2 domain-containing inositol phosphatase (SHIP) or beta-2-microglobulin (B2M),

[0381] Optionally among them:

[0382] (1) in response to the complexing, the induced change in the expression or activity of the target protein is at least about 10% compared to that in a control cell; and / or

[0383] (2) the target protein is the SHIP, optionally wherein the SHIP is SHIP1; and / or

[0384] (3) the target protein is the B2M; and / or

[0385] (4) the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4,000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, or between about 2,500 bases and about 2,000 bases from the TSS of the gene encoding the target protein. ,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base; and / or (5) the target polynucleotide sequence is located between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4000 bases, between about 4,000 bases and about 3,000 bases upstream of the TSS of the gene encoding the target protein, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base; and / or (6) the target polynucleotide sequence is located between about 10,000 and about 5,000 bases downstream of the TSS of the gene encoding the target protein. bases, between about 5,000 bases and about 4,000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base; and / or

[0386] (7) the change is a decrease in the expression or activity of the target protein by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more compared to the control cell; and / or (8) the change is an increase in the expression or activity of the target protein by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to the control cell; and / or

[0387] (9) the cell is an immune cell, and the expression of the target protein or the change in the activity promotes the maintenance of the stemness of the immune cell, the survival of the immune cell and / or the expansion of the immune cell; and / or (10) the cell is an immune cell, and the expression of the target protein or the change in the activity promotes enhanced cytokine production by the engineered immune cell, enhanced cytotoxicity of the engineered immune cell against a target cell population, and / or reduced exhaustion of the immune cell; and / or

[0388] (11) the external stimulus is a ligand, and the cell comprises a chimeric receptor polypeptide (receptor) that undergoes modification upon binding to the ligand, wherein the actuator portion is capable of activation after modification of the receptor; and / or

[0389] (12) the activation of the actuator moiety comprises (1) release of the actuator moiety from a substrate, or (2) modification of the actuator moiety; and / or

[0390] (13) the actuator moiety comprises a nucleic acid-guided actuator moiety, and wherein the method further comprises contacting the cell with a guide nucleic acid complexed with the actuator moiety; and / or (14) the guide nucleic acid comprises a guide ribonucleic acid (RNA); and / or

[0391] (15) the guide nucleic acid comprises two or more guide nucleic acids that are complementary to different target polynucleotide sequences; and / or

[0392] (16) the actuator portion comprises an effector domain configured to regulate the expression or activity of the target protein; and / or

[0393] (17) The effector domain is selected from the group consisting of a cleavage domain, an epigenetic modification domain, a transcriptional activation domain or a transcriptional repressor domain, and optionally wherein:

[0394] (a) the effector domain is a transcriptional activation domain; and / or

[0395] (b) the effector domain is a transcriptional repressor domain; and / or

[0396] (18) The actuator portion comprises a heterologous endonuclease or a variant thereof; and / or

[0397] (19) the modification is a conformational change or a chemical modification; and / or

[0398] (20) The cell is an immune cell; and / or

[0399] (21) The cell is a T cell or a NK cell; and / or

[0400] (22) further comprising administering the cell to a subject in need thereof; and / or

[0401] (23) the cells are autologous or allogeneic to the subject; and / or

[0402] (24) further comprising administering a co-therapeutic agent to the subject; and / or

[0403] (25) The subject is a mammal; and / or

[0404] (26) The subject is a human.

[0405] Although preferred embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that these embodiments are provided as examples only. This does not mean that the present invention is limited by the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not meant to be interpreted in a limiting sense. Without departing from the present invention, those skilled in the art will think of many variations, changes and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that in implementing the present invention, various alternatives to the embodiments of the present invention described herein may be adopted. Therefore, it is envisioned that the present invention will also cover any such substitutions, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention, and thus cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A system for regulating the expression or activity of a target protein in a cell, the system comprising: an actuator moiety capable of complexing with a target polynucleotide sequence in the cell, wherein the actuator moiety is heterologous to the cell, and wherein the target polynucleotide sequence is endogenous to the cell, wherein said complexing causes a change in said expression or said activity of said target protein of at least about 10% as compared to a control cell, wherein said complexing is sufficient to cause said change without editing said target polynucleotide sequence, and The target protein comprises one or more members selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), basic leucine zipper transcription factor ATF-like (BATF), DNA binding / differentiation inhibitor (ID), T-box transcription factor (TBX), and c-Jun.

2. The system according to claim 1, wherein the target protein is TOX.

3. The system of claim 2, wherein the TOX comprises TOX1 or TOX2.

4. The system of claim 1, wherein the target protein is the SOCS. The system of claim 3 , wherein the SOCS comprises SOCS1. The system of claim 1 , wherein the target protein is the BATF.

7. The system of claim 1, wherein the target protein is the ID. The system according to claim 7 , wherein the ID comprises ID3.

9. The system of claim 1, wherein the target protein is the TBX.

10. The system of claim 9, wherein the TBX is TBX21 (T-Bet).

11. The system of claim 1, wherein the target protein is the c-Jun.

12. The system of any of the preceding claims, wherein the actuator portion is capable of being activated for said recombination when the cell is exposed to an external stimulus.

13. A system for regulating the expression or activity of a target protein in a cell, the system comprising: an actuator moiety capable of complexing with a target polynucleotide sequence in the cell, wherein the actuator moiety is heterologous to the cell and is capable of being activated for said complexing when the cell is exposed to an external stimulus, and wherein the target polynucleotide sequence is endogenous to the cell, wherein upon said exposure, said actuator moiety is activated to effect said complexing, such that said expression or said activity of said target protein changes, wherein said complexing is sufficient to effect said change without editing said target polynucleotide sequence, and The target protein comprises Src homology 2 domain-containing inositol phosphatase (SHIP) or beta-2-microglobulin (B2M) and TGF beta receptor (TGFbR).

14. The system of claim 13, wherein upon said exposing, said actuator moiety is activated to effect said complexing to cause at least about a 10% change in said expression or said activity of said target protein as compared to that in a control cell.

15. The system of claim 13, wherein the target protein is the SHIP.

16. The system of claim 15, wherein the SHIP is SHIP1.

17. The system of claim 13, wherein the target protein is the B2M.

18. The system of claim 13, wherein the target protein is the TGFbR.

19. The system of claim 18, wherein the TGFBR is TGFbR2.

20. The system according to any one of the preceding claims, wherein the target protein is not a cytokine.

21. The system of any of the preceding claims, wherein the target protein is not a secreted protein.

22. The system of any of the preceding claims, wherein the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base from the TSS of the gene encoding the target protein.

23. The system of any of the preceding claims, wherein the target polynucleotide sequence is located between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base upstream of the TSS of the gene encoding the target protein.

24. The system of any of the preceding claims, wherein the target polynucleotide sequence is located between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base downstream of the TSS of the gene encoding the target protein.

25. The system of any of the preceding claims, wherein the change is a decrease in the expression or activity of the target protein by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more compared to that in the control cell.

26. The system of any of the preceding claims, wherein the change is an increase in the expression or activity of the target protein by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, 15000%, 20000%, 30000%, 40000%, 50000%, 60000%, 70000%, 80000%, 90000%, 100000%, 150000%, 30 ... 200%, 300%, 400%, 500% or more.

27. The system of any of the preceding claims, wherein the cell is an immune cell, and the change in the expression or the activity of the target protein promotes the maintenance of the stemness of the immune cell, the survival of the immune cell, and / or the expansion of the immune cell.

28. The system of any of the preceding claims, wherein the cell is an immune cell and the change in the expression or the activity of the target protein promotes enhanced cytokine production by the engineered immune cell, enhanced cytotoxicity of the engineered immune cell against a target cell population, and / or reduced depletion of the immune cell.

29. The system according to any one of the preceding claims, wherein the external stimulus is a ligand and the system comprises: A chimeric receptor polypeptide (receptor) that undergoes modification upon binding to the ligand, wherein the actuator portion is capable of activation upon receptor modification.

30. The system of any of the preceding claims, wherein the activation of the actuator moiety comprises (1) release of the actuator moiety from a substrate, or (2) modification of the actuator moiety.

31. The system of any of the preceding claims, wherein the actuator moiety comprises a nucleic acid-guided actuator moiety, and wherein the system further comprises a guide nucleic acid complexed with the actuator moiety.

32. The system of any of the preceding claims, wherein the guide nucleic acid comprises a guide ribonucleic acid (RNA).

33. The system of any preceding claim, comprising two or more guide nucleic acids having complementarity to different target polynucleotide sequences.

34. The system of any of the preceding claims, wherein the actuator moiety comprises an effector domain configured to modulate the expression or the activity of the target protein.

35. The system of claim 34, wherein the effector domain is selected from the group consisting of a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.

36. The system of claim 35, wherein the effector domain is a transcriptional activation domain.

37. The system of claim 35, wherein the effector domain is a transcriptional repressor domain.

38. The system of any of the preceding claims, wherein the actuator portion comprises a heterologous endonuclease or a variant thereof.

39. The system of any of the preceding claims, wherein the modification is a conformational change or a chemical modification.

40. The system of any preceding claim, wherein the cell is an immune cell.

41. The system of any of the preceding claims, wherein the cell is a T cell or a NK cell.

42. A population of engineered cells, wherein each engineered cell of the population comprises a system according to any one of the preceding claims.

43. A composition comprising the engineered cell population of claim 42.

44. The composition of claim 43, further comprising a co-therapeutic agent.

45. A system comprising a guide nucleic acid molecule, the guide nucleic acid molecule being designed to bind to a target polynucleotide sequence for regulating the expression or activity of a target protein in a cell, wherein the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases, between about 5,000 and about 4,000 bases, from the TSS of the gene encoding the target protein. bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base, The target protein is selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), inositol phosphatase containing Src homology 2 domain (SHIP), basic leucine zipper transcription factor ATF-like (BATF), β-2-microglobulin (B2M), DNA binding / differentiation inhibitor (ID), T-box transcription factor (TBX), c-Jun and TGFβ receptor (TGFbR).

46. ​​The system of claim 45, wherein the guide nucleic acid molecule is capable of recruiting an actuator moiety to the target polynucleotide sequence to modulate the expression or activity of the target protein, and wherein the system further comprises the actuator moiety.

47. A system comprising an actuator portion capable of binding to a target polynucleotide sequence for regulating the expression or activity of a target protein in a cell, wherein the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases, between about 5,000 and about 4,000 bases from the TSS of the gene encoding the target protein. bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base, The target protein is selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), inositol phosphatase containing Src homology 2 domain (SHIP), basic leucine zipper transcription factor ATF-like (BATF), β-2-microglobulin (B2M), DNA binding / differentiation inhibitor (ID), T-box transcription factor (TBX), c-Jun and TGFβ receptor (TGFbR).

48. A system according to any of the preceding claims, wherein the actuator portion comprises a heterologous nuclease or a variant thereof.

49. The system of any of the preceding claims, wherein the gene encoding the target protein is endogenous to the cell.

50. The system of any preceding claim, wherein the TSS is endogenous to the cell.

51. A method for regulating the expression or activity of a target protein in a cell, the method comprising: (a) forming in the cell a complex comprising an actuator moiety and a target polynucleotide sequence, wherein the actuator moiety is heterologous to the cell, and wherein the target polynucleotide sequence is endogenous to the cell, (b) in response to said forming, inducing a change in said expression or said activity of said target protein by at least about 10% as compared to that in said control cell, wherein said forming of said complex is sufficient to cause said change to occur without editing said target polynucleotide sequence, The target protein comprises one or more members selected from the group consisting of: thymocyte selection-associated high-mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), basic leucine zipper transcription factor ATF-like (BATF), DNA binding / differentiation inhibitor (ID), T-box transcription factor (TBX), c-Jun and TGFβ receptor (TGFbR).

52. The method of claim 51, wherein the target protein is TOX.

53. The method of claim 52, wherein the TOX comprises TOX1 or TOX2.

54. The method of claim 51, wherein the target protein is the SOCS.

55. The method of claim 54, wherein the SOCS comprises SOCS1.

56. The method of claim 51, wherein the target protein is the BATF.

57. The method of claim 51, wherein the target protein is the ID.

58. The method of claim 57, wherein the ID comprises ID3.

59. The method of claim 51, wherein the target protein is the TBX.

60. The method of claim 59, wherein the TBX is TBX21 (T-Bet).

61. The method of claim 51, wherein the target protein is c-Jun.

62. The method of claim 51, wherein the target protein is the TGFbR.

63. The method of claim 62, wherein the TGFbR is TGFbR2.

64. The method of claim 51, wherein the target protein is not a cytokine.

65. The method of claim 64, wherein the target protein is not a secreted protein.

66. A method according to any of the preceding claims, wherein the actuator portion is capable of being activated for said complexing when the cell is exposed to an external stimulus.

67. A method for regulating the expression or activity of a target protein in a cell, the method comprising: (a) exposing the cell to an external stimulus to activate the actuator moiety to complex with a target polynucleotide sequence in the cell, wherein the actuator moiety is heterologous to the cell, and wherein the target polynucleotide sequence is endogenous to the cell; and (b) in response to said complexing, inducing said expression or said change in activity of said target protein, wherein said formation of said complex is sufficient to cause said change to occur without editing said target polynucleotide sequence, The target protein comprises Src homology 2 domain-containing inositol phosphatase (SHIP) or beta-2-microglobulin (B2M).

68. The method of claim 67, wherein in response to said complexing, the induced change in said expression or said activity of said target protein is at least about 10% as compared to that in a control cell.

69. The method of claim 67, wherein the target protein is the SHIP.

70. The method of claim 69, wherein the SHIP is SHIP1.

71. The method of claim 67, wherein the target protein is the B2M.

72. The method of any of the preceding claims, wherein the target polynucleotide sequence (i) comprises at least a portion of a transcription start site (TSS) of a gene encoding the target protein, or (ii) is between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base from the TSS of the gene encoding the target protein.

73. The method of any of the preceding claims, wherein the target polynucleotide sequence is located between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base upstream of the TSS of the gene encoding the target protein.

74. The method of any of the preceding claims, wherein the target polynucleotide sequence is located between about 10,000 and about 5,000 bases, between about 5,000 bases and about 4000 bases, between about 4,000 bases and about 3,000 bases, between about 3,000 bases and about 2,000 bases, between about 2,500 bases and about 2,000 bases, between about 2,000 bases and about 1,500 bases, between about 1,500 bases and about 1,000 bases, between about 1,000 bases and about 500 bases, or between about 500 bases and 1 base downstream of the TSS of the gene encoding the target protein.

75. The method of any of the preceding claims, wherein the change is a decrease in the expression or activity of the target protein by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more compared to that in the control cell.

76. The method of any of the preceding claims, wherein the change is an increase in the expression or activity of the target protein by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, 15000%, 20000%, 30000%, 40000%, 50000%, 60000%, 70000%, 80000%, 90000%, 100000%, 150000%, 30 ... 200%, 300%, 400%, 500% or more.

77. The method of any of the preceding claims, wherein the cell is an immune cell, and the expression of the target protein or the change in the activity promotes the maintenance of the stemness of the immune cell, the survival of the immune cell, and / or the expansion of the immune cell.

78. The method of any of the preceding claims, wherein the cell is an immune cell and the change in the expression or activity of the target protein promotes enhanced cytokine production by the engineered immune cell, enhanced cytotoxicity of the engineered immune cell against a target cell population, and / or reduced depletion of the immune cell.

79. The method of any one of the preceding claims, wherein the external stimulus is a ligand and the cell comprises: A chimeric receptor polypeptide (receptor) that undergoes modification upon binding to the ligand, wherein the actuator portion is capable of activation upon modification of the receptor.

80. The method of any of the preceding claims, wherein the activation of the actuator moiety comprises (1) release of the actuator moiety from a substrate, or (2) modification of the actuator moiety.

81. A method according to any of the preceding claims, wherein the actuator portion comprises a nucleic acid-guided actuator portion, and wherein the method further comprises contacting the cell with a guide nucleic acid complexed with the actuator portion.

82. The method of any of the preceding claims, wherein the guide nucleic acid comprises a guide ribonucleic acid (RNA).

83. The method of any of the preceding claims, wherein the guide nucleic acid comprises two or more guide nucleic acids having complementarity to different target polynucleotide sequences.

84. The method of any of the preceding claims, wherein the actuator moiety comprises an effector domain configured to modulate the expression or the activity of the target protein.

85. The method of claim 84, wherein the effector domain is selected from the group consisting of a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.

86. The method of claim 85, wherein the effector domain is a transcriptional activation domain.

87. The method of claim 85, wherein the effector domain is a transcriptional repressor domain.

88. A method according to any of the preceding claims, wherein the actuator portion comprises a heterologous nuclease or a variant thereof.

89. The method of any one of the preceding claims, wherein the modification is a conformational change or a chemical modification.

90. The method of any of the preceding claims, wherein the cell is an immune cell.

91. The method of any of the preceding claims, wherein the cell is a T cell or a NK cell.

92. The method of any of the preceding claims, further comprising administering the cells to a subject in need thereof.

93. The method of any of the preceding claims, wherein the cells are autologous or allogeneic to the subject.

94. The method of any of the preceding claims, further comprising administering to the subject a co-therapeutic agent.

95. The method of any of the preceding claims, wherein the subject is a mammal.

96. The method of any of the preceding claims, wherein the subject is a human.

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