Chimeric polypeptide system and method of gene regulation

Abstract

Certain aspects of the present disclosure provide systems, compositions, and methods for regulating the expression or activity of a target protein in a cell. In some cases, the present disclosure provides a system comprising an actuator moiety that can complex with a target polynucleotide sequence to regulate the expression or activity of the target protein. The actuator moiety can be heterologous to the cell. The actuator moiety can be activated upon exposure of the cell to an external stimulus. Exposure of the cell to the external stimulus activates the actuator moiety to regulate the expression or activity of the target protein.

Description

Technical Field

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

Background Art

[0002] Cancer (e.g., neoplasm, tumor) is a large family of diseases associated with abnormal cell growth in many body tissues. Cancer can invade or spread to other parts of the body. As a major cause of death worldwide, cancer accounts for approximately 10 million deaths each year. Non-limiting examples of body tissues infiltrated by cancer include the lung, prostate, colorectal, stomach, liver, breast, colon, rectum, cervix, and thyroid. For the purpose of treating or controlling cancer, different therapies, such as small molecules, antibodies, and adoptive cell therapies (e.g., cellular immunotherapies), have been developed.

Summary of the Invention

[0003] The present disclosure provides methods and systems for adoptive cell therapy to treat a subject having or suspected of having a condition 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 immunotherapies (e.g., cancer therapies using autologous or allogeneic immune cells).

[0004] In one aspect, the present disclosure provides a system for modulating the expression or activity of a target protein in a cell, the system comprising an effector portion capable of complexing with a target polynucleotide sequence in the cell, the effector portion being heterologous to the cell, the target polynucleotide sequence being endogenous to the cell, the complexing resulting in at least about a 10% change in the expression or activity of the target protein as compared to that in a control cell, the complexing being sufficient to effect the change without editing the target polynucleotide sequence, and the target protein including 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 / inhibitor of differentiation (ID), T-box transcription factor (TBX), and c-Jun.

[0005] In another aspect, the present disclosure provides a system for modulating the expression or activity of a target protein in a cell, the system comprising an effector portion capable of complexing with a target polynucleotide sequence in the cell, the effector portion being heterologous to the cell and activatable for complexing upon exposure of the cell to an external stimulus, the target polynucleotide sequence being endogenous to the cell, and the exposure causing the effector portion to be activated for complexing to effect a change in the expression or activity of the target protein, the complexing being sufficient to effect the change without editing the target polynucleotide sequence, and the target protein including Src homology 2 domain-containing inositol phosphatase (SHIP) or beta-2-microglobulin (B2M), and TGF beta 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 modulating the expression or activity of a target protein in a cell, wherein the target polynucleotide sequence comprises (i) at least a portion of the transcription start site (TSS) of a gene encoding the target protein, or (ii) is about 10,000 to about 5,000 bases, about 5,000 to about 4,000 bases, about 4,000 to about 3,000 bases, about 3,000 to about 2,000 bases, about 2,500 to about 2,000 bases, about 2,000 to about 1,500 bases, about 1,500 to about 1,000 bases, about 1,000 to about 500 bases, or about 500 to 1 base away from the TSS of a gene encoding the target protein, and the target protein is selected from the group consisting of thymocyte selection associated high mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), Src homology 2 domain-containing inositol phosphatase (SHIP), basic leucine zipper transcription factor ATF-like (BATF), beta-2-microglobulin (B2M), DNA binding / inhibitor of differentiation (ID), T-box transcription factor (TBX), c-Jun, and TGF-beta receptor (TGFbR).

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

[0008] In another aspect, the present disclosure provides a method for regulating the expression or activity of a target protein in a cell, comprising: (a) forming a complex comprising an effector moiety and a target polynucleotide sequence in the cell, wherein the effector moiety is heterologous to the cell and the target polynucleotide sequence is endogenous to the cell; and (b) inducing at least a about 10% change in the expression or activity of the target protein in response to the forming, as compared to that in a control cell, wherein forming the complex is sufficient to effect the change without editing the 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 / inhibitor of differentiation (ID), T-box transcription factor (TBX), c-Jun, and TGF beta 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, comprising: (a) exposing the cell to an external stimulus to activate an effector moiety to complex with a target polynucleotide sequence in the cell, wherein the effector moiety is heterologous to the cell and the target polynucleotide sequence is endogenous to the cell; and (b) inducing a change in the expression or activity of the target protein in response to the complexing, wherein forming the complex is sufficient to effect the change without editing the target polynucleotide sequence, and the target protein comprises Src homology 2 domain-containing inositol phosphatase (SHIP) or beta-2-microglobulin (B2M).

[0010] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which only shows and describes exemplary embodiments of the present disclosure. As will be understood, the present disclosure is capable of other and different embodiments, and some of the details thereof are capable of modification in various obvious respects without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0011] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the incorporated publications and patents or patent applications conflict with the disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material.

Brief Description of the Drawings

[0012] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which shows exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings (or "Figure" and "FIG." in this specification). Figure 1

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[0028] Although various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, numerous variations, changes, and substitutions may occur to those skilled in the art. It should be understood that various alternative forms of the embodiments of the present invention described herein may be used.

[0029] As used in this specification and the claims, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise.

[0030] The terms "about" or "approximately" generally mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or beyond one standard deviation, in accordance with the conventions of the relevant art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within one order of magnitude, preferably within five times the value, more preferably within two times the value. Where a particular value is recited in the present application and claims, unless otherwise specified, the term "about" should be assumed to mean within the acceptable error range of the particular value.

[0031] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination of them. The term "and / or" should be understood to mean either one or both of the alternatives.

[0032] The term "cell" generally refers to biological cells. 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 eukaryotes, protozoan cells, cells from plants (e.g., plant crops, fruits, vegetables, grains, soybeans, corn, corn kernels, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, flax, tobacco, flowering plants, conifers, gymnosperms, ferns, bryophytes, mosses, liverworts, lichens), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc.), seaweeds (e.g., kelp), fungal cells (e.g., yeast, cells from mushrooms), animal cells, cells from invertebrates (e.g., Drosophila, 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, a cell is not derived from a natural organism (e.g., a cell can be synthetically produced and is sometimes called an artificial cell).

[0033] The terms "hematopoietic stem cell", "hematopoietic progenitor cell", or "hematopoietic precursor cell" are used interchangeably herein and generally refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., into T cells), and include pluripotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. A hematopoietic stem cell (HSC) is a pluripotent stem cell that gives rise to all blood cell types including the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells).

[0034] The terms "immune cell" or "lymphocyte" generally refer to differentiated hematopoietic cells. Non-limiting examples of immune cells can include T cells, NK cells, monocytes, natural lymphocytes, tumor-infiltrating lymphocytes, macrophages, granulocytes, and the like.

[0035] The term "nucleotide", as used herein, generally refers to a base - sugar - phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be the monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term "nucleotide" can include adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), which are ribonucleoside triphosphates, and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS]dATP, 7 - deaza - dGTP, and 7 - deaza - dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term "nucleotide" as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Exemplary examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well - known techniques. Labeling can also be performed using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.Examples of fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2’7’-dimethoxy-4’5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N’,N’-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4’-dimethylaminophenylazo)benzoic acid (DABCYL), cascade blue, Oregon green, Texas red, cyanine, and 5-(2’-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, available from Amersham, Arlington Heights, Ill.; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Boehringer Mannheim, Indianapolis, Ind.; and chromosome-labeling nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, cascade blue-7-UTP, cascade blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, rhodamine green-5-UTP, green rhodamine-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP, available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification.The chemically modified single nucleotide can be a biotin-dNTP. Some non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0036] The terms "polynucleotide", "oligonucleotide", or "nucleic acid" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in any of single-stranded, double-stranded, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can be present in a cell-free environment. A polynucleotide can be a gene or a fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. A polynucleotide can contain one or more analogs (e.g., altered backbone, sugar, or nucleobase). When present, modifications to the nucleotide structure can be imparted before or after construction of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to a sugar), thiol-containing nucleotides, biotin-binding nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.Non-limiting examples of polynucleotides include the coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The nucleotide sequence can be interrupted by non-nucleotide components.

[0037] As used herein, the term "gene" refers to nucleic acids (e.g., DNA such as genomic DNA and cDNA) involved in encoding RNA transcripts and their corresponding nucleotide sequences. When used herein in connection with genomic DNA, the term can include intervening, non-coding regions as well as regulatory regions, and can include 5' and 3' termini. In some uses, the term encompasses transcribed sequences that include 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region includes an "open reading frame" that encodes a polypeptide. In some uses of the term, "gene" includes only the coding sequence (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, genes do not encode polypeptides, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions including upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene" or a native gene in its natural position in the genome of an organism. A gene can refer to an "exogenous gene" or a non-native gene. A non-native gene can refer to a gene that is not normally found in the host organism but is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene that is not in its natural position in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-native sequence) that includes mutations, insertions, and / or deletions.

[0038] The term "transfection" or "transfected" refers to the introduction of nucleic acids into cells by non-viral or virus-based methods. The nucleic acid molecule can be a gene sequence that encodes a complete protein or a functional portion thereof.

[0039] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can together be referred to as the "gene product." When the polynucleotide is derived from genomic DNA, expression can include the splicing of mRNA in eukaryotic cells. "Upregulated" refers to the level of expression of a polynucleotide (e.g., an RNA such as mRNA) and / or polypeptide sequence that is increased, generally in relation to its level of expression in the wild-type state, while "downregulated" refers to the level of expression of a polynucleotide (e.g., an RNA such as mRNA) and / or polypeptide sequence that is decreased, generally in comparison to its expression in the wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression," the transfected gene is not transferred to daughter cells upon cell division. Since its expression is limited to the transfected cells, the expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selective advantage to the transfected cells. Such a selective advantage can be resistance to a particular toxin presented to the cells.

[0040] The terms "expression cassette," "expression construct," or "expression vector" refer to a nucleic acid that includes nucleotide sequences such as coding sequences and template sequences, and sequences necessary for the expression of the coding sequences. An expression cassette can be viral or non-viral. For example, an expression cassette includes a nucleic acid construct that, when introduced into a host cell, results in the transcription and / or translation of an RNA or polypeptide, respectively. Antisense constructs or sense constructs that are not translated or cannot be translated are explicitly included by this definition. One of ordinary skill in the art will recognize that the inserted polynucleotide sequences need not be identical, but only substantially similar to the sequences of the gene from which they are derived.

[0041] As used herein, "plasmid" generally refers to a non-viral expression vector, e.g., a nucleic acid molecule encoding a gene and / or regulatory elements necessary for gene expression. "Viral vector" as used herein generally refers to a virus-derived nucleic acid capable of transporting another nucleic acid into a cell. A viral vector, when present in an appropriate environment, can direct 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, retrovirus, adenovirus, lentivirus, and adeno-associated virus vectors.

[0042] As used herein, the term "promoter" refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, the promoters used in the polynucleotide constructs of the present disclosure include cis-acting transcriptional control elements and regulatory sequences involved in regulating or modulating the timing and / or rate of gene transcription. For example, a promoter can be a cis-acting transcriptional control element that includes enhancers, promoters, transcription terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences and is involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to effect (turn on / off, regulate, modulate, etc.) gene transcription. A "constitutive promoter" is one that can initiate transcription in almost all tissue types, while a "tissue-specific promoter" initiates transcription only in one or a few specific tissue types. An "inducible promoter" initiates transcription only under specific environmental, developmental, or drug or chemical conditions.

[0043] The terms "complement", "complements", "complementary", and "complementarity", as used herein, generally refer to a sequence that is fully complementary to and hybridizable with a given sequence. In some cases, a sequence that hybridizes to a given nucleic acid is called the "complement" or "reverse complement" of the given molecule if its base sequence over a given region can bind complementarily to that of its binding partner such that, for example, A-T, A-U, G-C, and G-U base pairs are formed. Generally, a first sequence that is hybridizable to a second sequence is specifically or selectively hybridizable to the second sequence such that hybridization of the first sequence to the second sequence or set of second sequences is preferred over hybridization to non-target sequences during the hybridization reaction (e.g., is more thermodynamically stable under certain conditions such as stringent conditions commonly used in the art). Typically, hybridizable sequences share a degree of sequence complementarity over all or part of their respective lengths, such as 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 can be measured by any suitable alignment algorithm including, but not limited to, the Needleman-Wunsch algorithm (e.g., refer to the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally with default settings), the BLAST algorithm (e.g., refer to the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (e.g., refer to the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, optionally with default settings). The optimal alignment can be evaluated using any suitable parameters of the selected algorithm, including default parameters.

[0044] Complementarity can be complete or substantial / adequate. Complete complementarity between two nucleic acids can mean that the two nucleic acids can form a double strand and all bases in the double strand are bound to complementary bases by Watson-Crick pairing. Substantially or adequately complementary means that the sequence in one strand is not all and / or completely complementary to the sequence in the opposing strand, but sufficient binding occurs between the bases on the two strands to form 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 for predicting the Tm of the hybridized strands, or by the empirical determination of Tm using conventional methods.

[0045] The terms "peptide", "polypeptide", or "protein" are used interchangeably herein and generally refer to a polymer of at least two amino acid residues linked by peptide bonds. This term is not meant to denote a polymer of a specific length, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The term applies to both naturally occurring amino acid polymers and amino acid polymers containing at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. This term includes amino acid chains of any length, including full-length proteins, as well as proteins that do or do not have secondary and / or tertiary structures (e.g., domains). This term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation, such as conjugation with a labeling component. The terms "amino acid" and "amino acids" as used herein generally refer to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural and non-natural amino acids that have been chemically modified to include groups or chemical moieties not naturally present in the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0046] The terms "derivative", "variant", or "fragment" as used herein in relation to a polypeptide generally refer to a polypeptide related to a wild-type polypeptide by, for example, any of 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 can include one or more amino acid changes (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof as compared to the wild-type polypeptide.

[0047] The term "gene regulatory polypeptide" or "GMP", as used herein, refers to a polypeptide that can regulate gene expression or activity and / or can edit nucleic acid sequences and includes at least an effector portion. A GMP can include additional peptide sequences not involved in regulating gene expression, such as cleavage recognition sites, linker sequences, targeting sequences, and the like.

[0048] The terms "effector portion", "effector domain", and "gene regulatory domain", as used herein, refer to a portion that can regulate gene expression or activity and / or can edit nucleic acid sequences, whether exogenous or endogenous. The effector portion can regulate gene expression at the transcriptional level and / or the translational level. The effector 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 effector portion recruits at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. The effector portion can itself bind to DNA and regulate transcription by physical obstruction, for example, preventing proteins such as RNA polymerase and other associated proteins from assembling on the DNA template. The effector portion can regulate gene expression at the translational level, for example, by regulating the production of protein from the mRNA template. In some embodiments, the effector portion regulates gene expression by affecting the stability of the mRNA transcript. In some embodiments, the effector portion regulates gene expression by editing a nucleic acid sequence (e.g., a region of the genome). In some embodiments, the effector portion regulates gene expression by editing the mRNA template. Editing the nucleic acid sequence can, in some cases, change the template underlying gene expression.

[0049] As used herein, the term "targeting sequence" refers to a nucleotide sequence encoding a targeting polypeptide that mediates the localization (or retention) of a protein to an intracellular location, such as the plasma membrane or the membrane of a given organelle, the nucleus, the cytosol, mitochondria, the endoplasmic reticulum (ER), Golgi, chloroplasts, apoplast, peroxisomes, or other organelles, and the corresponding amino acids. 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 of the cell nucleus, e.g., to the cytoplasm, using a nuclear export signal (NES), to mitochondria using a mitochondrial targeting signal, to the ER using an ER retention signal, to peroxisomes using a peroxisomal targeting signal, to the plasma membrane using a membrane localization signal, or combinations thereof.

[0050] As used herein, "fusion" can refer to a protein and / or nucleic acid that includes one or more non-native sequences (e.g., moieties). A fusion can include one or more of the same non-native sequences. A fusion can include one or more different non-native sequences. A fusion can be chimeric. A fusion can include a nucleic acid affinity tag. A fusion can include a barcode. A fusion can include a peptide affinity tag. A fusion can provide for intracellular localization of a site-specific polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, an ER retention signal, etc.). A fusion can provide a non-native sequence (e.g., an affinity tag) that can be used for tracking or purification. A fusion can be a small molecule such as biotin, or a dye such as an alexa fluor dye, a cyanine 3 dye, a cyanine 5 dye, etc.

[0051] Fusion can refer to any protein having a functional effect. For example, the fusion protein can have 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, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, or demyristoylation activity. The effector protein can modify a genomic locus. The fusion protein can be a fusion in a Cas protein. The fusion protein can be a non-native sequence in a Cas protein.

[0052] As used herein, "non-native" can refer to a nucleic acid or polypeptide sequence not found in a native nucleic acid or protein. Non-native can refer to an affinity tag. Non-native can refer to a fusion. Non-native can refer to a naturally occurring nucleic acid or polypeptide sequence containing mutations, insertions, and / or deletions. The non-native sequence can also exhibit and / or encode an activity (e.g., enzyme activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) indicated by the nucleic acid and / or polypeptide sequence to which the non-native sequence is fused. The non-native nucleic acid or polypeptide sequence can be linked by genetic manipulation to a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) to generate a chimeric nucleic acid and / or polypeptide sequence encoding a chimeric nucleic acid and / or polypeptide.

[0053] The term "antibody" generally refers to a proteinaceous binding molecule having immunoglobulin-like functions. 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) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). The derivative, variant, or fragment can refer to a functional derivative or fragment that retains (e.g., completely and / or partially) the binding specificity of the corresponding antibody. Antigen-binding fragments include Fab, Fab’, F(ab’)2, variable fragment (Fv), single chain variable fragment (scFv), minibody, diabody, and single domain antibodies ("sdAb" or "nanobody" or "camelid"). The term antibody includes antibodies and antigen-binding fragments of antibodies that are optimized, engineered, or chemically conjugated. Examples of optimized antibodies include affinity matured antibodies. Examples of engineered antibodies include Fc optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies).

[0054] The terms "antigen-binding portion" or "antigen-binding domain" are used interchangeably herein and generally refer to a construct that exhibits preferential binding to a specific target antigen. The antigen-binding domain can be a polypeptide construct such as an antibody, a modification thereof, a fragment thereof, or a combination thereof. The antigen-binding domain can be any antibody disclosed herein, or a functional variant thereof. Non-limiting examples of antigen-binding domains include murine antibodies, human antibodies, humanized antibodies, camel Ig, shark heavy chain antibodies (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 fragment (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain antibody (VHH), and other antibody fragments that maintain the binding specificity of a whole antibody.

[0055] The terms "enhanced activity", "increased activity", or "upregulated activity" generally refer to the activity of a moiety of interest that has been modified to a level above the normal level of activity of the moiety of interest in a host strain (e.g., a host cell). The normal level of activity can be substantially zero (or null), or higher than zero. The moiety of interest can include a polypeptide construct of the host strain. The moiety of interest can include a heterologous polypeptide construct that is introduced into the host strain or therein. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) into the genome of the host strain for enhanced activity of the polypeptide of interest in the host strain.

[0056] The terms "reduced activity", "decreased activity", or "downregulated activity" generally refer to the activity of a moiety of interest (e.g., a polynucleotide or polypeptide) that has been modified to a level below the normal level of activity of the moiety of interest in a host strain (e.g., a host cell). The normal level of activity is greater than zero. The moiety of interest can include an endogenous gene or polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked out or knocked down in the host strain. In some examples, the reduced activity of the moiety of interest can include complete inhibition of such activity in the host strain.

[0057] The terms "subject", "individual", or "patient" are used interchangeably herein and generally refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, mice, monkeys, humans, domestic animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0058] The term "treatment" or "treating" generally refers to an approach for obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. For example, treatment can include administering a system or cell population disclosed herein. A therapeutic benefit means any therapeutically relevant improvement or effect in one or more diseases, conditions, or symptoms under treatment. For a prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease even if the disease, condition, or symptom has not yet appeared.

[0059] As used herein, "administer", "administering", "administration", and derivatives thereof refer to methods that can be used to effect delivery of an agent or composition to a desired site of biological action. These methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intracascular, intrathecal, intranasal, intravitreal injection and topical 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, arteriolar, intradermal, subcutaneous, intraperitoneal, intracerebroventricular, and intracranial. Other delivery modalities include, but are not limited to, the use of liposomal formulations, intravenous infusion, transplantation, etc.

[0060] The terms "effective amount" or "therapeutically effective amount" generally refer to an amount (e.g., one or more unit doses) of a composition, such as a composition disclosed herein, that is sufficient to effect the desired activity upon administration to a subject in need thereof. In the context of the present disclosure, the term "therapeutically effective" generally refers to an amount of a composition that is sufficient to delay the onset, halt the progression, or alleviate or mitigate at least one symptom of a disorder being treated by the methods of the present disclosure.

[0061] I. Introduction

[0062] Immune cells (e.g., T cells, NK cells) can be engineered to exhibit specific affinity for one or more specific antigens (e.g., cancer antigens or tumor antigens) for adoptive immunotherapy for the treatment of cancer (e.g., solid tumors, lymphomas, etc.). In some cases, immune cells can be engineered to express a heterologous receptor (e.g., a chimeric antigen receptor or "CAR", an engineered T cell receptor (TCR), etc.) that can bind to one or more specific antigens, thereby targeting cancer cells in a subject.

[0063] However, the therapeutic efficacy of engineered immune cells can be limited, for example, by insufficient trafficking, limited persistence in the subject's serum, rapid exhaustion, or inhibitory activity of the subject's cancer cells or immune cells against the engineered immune cells. In some cases, some recombinant cytokines (e.g., interleukins or "ILs") can be administered to the subject together with the engineered immune cells to improve their efficacy (e.g., cytotoxic activity, persistence, proliferation). However, such co - administration of recombinant cytokines can also exhibit undesirable adverse effects, such as anemia, nausea, liver dysfunction, systemic toxicity, and even death. In some cases, immune cell stemness (e.g., characterized by the ability to self - renew, multipotency, and / or persistence of proliferative capacity) can be reduced when engineering T cells to express the heterologous receptors disclosed herein, thereby limiting the therapeutic effect (e.g., in vivo) of the engineered immune cells.

[0064] Accordingly, there remains an unaddressed critical need for using heterologous cytokines that enhance the efficacy of adoptive immunotherapy, for example, for treating cancer, but have a reduced or suppressed degree of adverse effects.

[0065] II. Systems for Gene Regulation

[0066] In one aspect, the present disclosure provides a system for regulating the expression or activity of a target protein in a cell. The system can include an actuator portion that can complex with a target polynucleotide sequence in the cell. The actuator portion can be heterologous to the cell. In some examples, at least a portion of the amino acid sequence of the actuator portion can be heterologous to the cell. At least a portion (e.g., all) of the target polynucleotide sequence can be endogenous to the cell. Complexation (e.g., formation of a complex including at least the actuator portion and the target polynucleotide sequence) can result in a change (e.g., at least 10%) in the expression or activity of the target protein as compared to the expression or activity of the target protein in a control cell (or comparable cell). In some cases, complexation can be sufficient to result in a change in the expression or activity of the target protein without editing at least a portion of the target polynucleotide sequence (e.g., gene editing such as insertion, deletion, substitution, mutation, etc.).

[0067] In some cases, a change (e.g., an increase, a decrease) in the expression or activity level of a target protein (e.g., an endogenous protein) can occur (or can be observed) in vitro, ex vivo, or in vivo.

[0068] The target polynucleotide sequence can be operably linked to a gene encoding the target protein. The target polynucleotide sequence can be a portion of the coding region (e.g., exon) of the gene encoding the target protein. The target polynucleotide sequence can be a portion of the non-coding region (e.g., intron, promoter, transcription start site (TSS), etc.) of the gene encoding the target protein.

[0069] The target protein can include 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 can include 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 include thymocyte selection-associated high-mobility group box proteins (TOX, e.g., TOX1, TOX2, TOX3, TOX4), suppressors of cytokine signaling (SOCS, e.g., SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, CISH), Src homology 2 domain-containing inositol phosphatases (SHIP, e.g., SHIP1, SHIP2, SHIP3), basic leucine zipper transcription factor ATF-like (BATF), beta-2-microglobulin (B2M), DNA-binding / inhibitor of differentiation factors (ID, e.g., ID1, ID2, ID3, ID4), c-Jun, T-box transcription factors (TBX, e.g., TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX7, TBX8, TBX9, TBX10, TBX11, TBX12, TBX13, TBX14, TBX15, TBX16, TBX17, TBX18, TBX19, TBX20, TBX21, or T-Bet, TBX22, TBR1), interleukins (IL, e.g., 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 receptors (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).

[0070] In some cases, the target protein cannot be a secreted protein. In some cases, the target protein cannot be a cytokine. In some examples, the target protein cannot be an IL protein. Alternatively, the target protein can include an IL protein.

[0071] In some embodiments, the target protein can be operably linked to the stemness of a cell (e.g., an immune cell such as a T cell) (e.g., involved in activating, maintaining, or extending stemness). For example, one or more of the proteins provided in Table 1 can be adjusted (e.g., activated) to enhance or extend the stemness of engineered immune cells, as disclosed herein, thereby enhancing or extending the therapeutic efficacy of the engineered immune cells.

[0072] [Table 1]

[0073] In some cases, changes in the expression or activity of the target proteins provided herein can promote one or more characteristics including (i) maintenance of the stemness of a cell (e.g., an immune cell such as a T cell), (ii) enhancing cell survival, and / or (iii) enhancing cell proliferation.

[0074] In some cases, a change in the expression or activity of a target protein can activate the stemness of the cells disclosed herein (e.g., immune cells such as T cells). In some cases, a change in the expression or activity of a target protein can maintain the stemness of the cells such that the stemness is extended by at least or up to about 5%, at least or up to about 10%, 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% (e.g., with respect to duration) as compared to control cells that do not have the system disclosed herein.

[0075] In some cases, the stem cell properties of the cells disclosed herein can be observed 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 inducing the complexation disclosed herein (e.g., after activating the actuator portion or after binding of a ligand to the ligand-binding domain of the chimeric receptor).

[0076] In some cases, the stem cell properties of the cells disclosed herein can be confirmed (or measured) in vitro, ex vivo, or in vivo.

[0077] In some cases, a change in the expression or activity of a target protein enhances the survival of the cells disclosed herein (e.g., immune cells such as T cells) (e.g., with respect to the period during which the cells remain viable) by at least or up to about 5%, at least or up to about 10%, 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% as compared to control cells that do not have the system disclosed herein.

[0078] In some cases, the survival of the cells disclosed herein (e.g., immune cells such as T cells) can be observed 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 inducing the complex formation disclosed herein (e.g., after activating the actuator portion or after binding of a ligand to the ligand-binding domain of the chimeric receptor).

[0079] In some cases, the survival of the cells disclosed herein can be confirmed (or measured) in vitro, ex vivo, or in vivo.

[0080] In some cases, the change in the expression or activity of the target protein enhances the proliferation (e.g., with respect to proliferation) of the cells disclosed herein (e.g., immune cells such as T cells) by at least or up to about 5%, at least or up to about 10%, 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% as compared to control cells without the system disclosed herein.

[0081] In some cases, the proliferation of the cells disclosed herein (e.g., immune cells such as T cells) can be observed 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 the complexation disclosed herein (e.g., after activation of the actuator moiety or after binding of a ligand to the ligand binding domain of the chimeric receptor).

[0082] In some cases, the proliferation of the cells disclosed herein can be confirmed (or measured) in vitro, ex vivo, or in vivo.

[0083] In some cases, a population of cells (e.g., a population of immune cells such as a population of T cells) treated by the systems and methods disclosed herein has 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 proportion (e.g., percentage) of memory T cells (e.g., as indicated by being CD45RO positive) than a comparable population of cells not treated by the systems and methods. For example, a population of CAR-T cells treated by the systems and methods disclosed herein can yield 50% memory T cells, while a comparable population of CAR-T cells not treated by the systems and methods can yield 10% memory T cells, and thus the former is 40% higher than the latter.

[0084] In some cases, in a population of cells treated by the systems and methods disclosed herein (e.g., a population of immune cells such as a population of T cells), central memory T cells (TCM) in the population can be 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% more abundant than effector memory T cells (TEM). TCM can be CD45RO positive and CD62L positive. TEM can be CD45RO positive and CD62L negative.

[0085] In some cases, a population of cells (e.g., a population of immune cells such as a population of T cells) treated by the systems and methods disclosed herein can have a higher percentage (e.g., percentage) of central memory T cells (TCM) by 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% than a comparable population of stem cells not treated by the systems and methods. For example, a population of CAR-T cells treated by the systems and methods disclosed herein can yield 25% TCM, while a comparable population of stem cells not treated by the systems and methods can yield 15% TCM, and thus the former is 10% higher than the latter.

[0086] In some cases, a population of cells (e.g., a population of immune cells such as a population of T cells) treated by the systems and methods disclosed herein can be 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% (e.g., percentage) of stem memory T cells (TSCM, e.g., as indicated by being CD45RO negative and CD62L positive).

[0087] In some cases, a population of cells (e.g., a population of immune cells such as a population of T cells) treated by the systems and methods disclosed herein can have a higher percentage (e.g., percentage) of stem memory T cells (TSCM, e.g., as indicated by being CD45RO negative and CD62L positive) 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 population of stem cells not treated by the systems and methods. For example, a population of CAR-T cells treated by the systems and methods disclosed herein can yield 50% TSCM, while a comparable population of stem cells not treated by the systems and methods can yield 10% TSCM, and thus the former is 40% higher than the latter.

[0088] In some cases, a population of cells (e.g., a population of immune cells such as a population of T cells) treated by the systems and methods disclosed herein can produce a proportion (e.g., a percentage) of stem memory T cells (TSCM, e.g., as indicated by being CD45RO negative and CD62L positive) 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%.

[0089] In some cases, the types of T cells (e.g., memory T cells, TCM, TEM, TSCM) or their proportions disclosed herein can be observed 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 inducing the complexation 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).

[0090] In some cases, the types or populations of T cells disclosed herein can be identified (or measured) in vitro, ex vivo, or in vivo.

[0091] In some embodiments, the target protein can be operably linked to the function of the engineered cells (e.g., CAR-T cells) disclosed herein. For example, one or more of the proteins provided in Table 2 can modulate (e.g., inhibit) the function of engineered immune cells having a chimeric receptor (e.g., CAR, engineered TCR) to improve one or more functions.

[0092] [Table 2]

[0093] In some cases, changes in the expression or activity of the target proteins provided herein can promote one or more characteristics including (i) reduced exhaustion of engineered immune cells, (ii) enhanced cytokine production by engineered immune cells, (iii) enhanced cytotoxicity of engineered immune cells against a population of target cells, and / or (iv) enhanced differentiation of immune cells into immune cell subtypes (e.g., from naive T cells to T helper cells).

[0094] In some cases, the changes in the expression or activity of the target protein provided herein can reduce the exhaustion of engineered immune cells. Exhaustion of immune cells (e.g., T cells) can be confirmed, for example, 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 has a lower percentage of exhausted immune cells (e.g., exhausted T cells) by 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% than that in a comparable population of immune cells not treated by the systems and methods.

[0095] In some cases, the immune cell exhaustion markers 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 the complexation disclosed herein (e.g., after activation of the actuator portion or after binding of a ligand to the ligand-binding domain of the chimeric receptor).

[0096] In some cases, the immune cell exhaustion markers disclosed herein can be confirmed (or measured) in vitro, ex vivo, or in vivo.

[0097] In some cases, the changes in the expression or activity of the target proteins provided herein can enhance cytokine production (e.g., expression) by the engineered immune cells. Such cytokines may not be the same protein as the target protein whose expression or activity is directly regulated by the systems and methods disclosed herein, and need not be the same protein. Such cytokines can be endogenous cytokines of the cells. Such cytokines whose production is enhanced may not be the same as the target protein directly regulated by the systems and methods disclosed herein. In some examples, a population of cells (e.g., engineered immune cells) treated by the systems and methods disclosed herein can have a cytokine expression or activity level that is at least or up to about 5%, at least or up to about 10%, 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% higher than that in a comparable population of cells not treated by the systems and methods.

[0098] In some cases, cytokine production 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 complex formation 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).

[0099] In some cases, cytokine production disclosed herein can be confirmed (or measured) in vitro, ex vivo, or in vivo.

[0100] In some cases, changes (e.g., increases, decreases) in the expression or activity level of a cytokine (e.g., an endogenous cytokine) induced upon modulating the expression or activity level of a target protein disclosed herein can occur (or be observed) in vitro, ex vivo, or in vivo.

[0101] In some cases, the changes in the expression or activity of the target proteins provided herein can enhance the differentiation of T cells (e.g., naive T cells expressing the chimeric receptors disclosed herein) into immune cell subtypes (e.g., T helper (Th) cells expressing the chimeric receptors 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., retinoic acid-related orphan receptor gamma (RORγ2, etc.) 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 can have 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 that in a comparable population of immune cells not treated by the systems and methods.

[0102] In some cases, a population of immune cell subtypes (e.g., Th1 cells) disclosed herein, after induction of complexation 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), 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.

[0103] In some cases, the proportion of immune cell subtypes (e.g., Th1 cells) disclosed herein can be confirmed (or measured) in vitro, ex vivo, or in vivo.

[0104] In some cases, cytokines (e.g., endogenous cytokines) can include IFN. In some cases, cytokines can be selected from the group consisting of IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), IFN-ζ (zeta), IFN-γ (gamma), and IFN-λ (lambda). In some cases, cytokines can include IFN-γ (gamma). In some cases, when modulating the expression or activity level of a target protein in a cell as disclosed herein, the cell can be affected to show an increase in the expression or activity level of IFN (e.g., IFN-γ).

[0105] In some cases, a cytokine (e.g., an endogenous cytokine) can include a TNF protein. In some cases, the cytokine can be selected from the group consisting of 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 ectodysplasin A. In some cases, the cytokine can include TNFα. In some cases, as disclosed herein, when modulating the expression or activity level of a target protein in a cell, the cell can be affected to show an increase in the expression or activity level of TNF (e.g., TNFα).

[0106] In some cases, a cytokine (e.g., an endogenous cytokine) can include an IL. In some cases of ILc cytokines, the IL can include one or more members selected from the group consisting of 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, the cytokine can be IL-2. In some cases, the cell can be affected to show a decrease in the expression or activity level of the IL.

[0107] In some cases, the changes in the expression or activity of the target protein provided herein can promote the enhanced cytotoxicity of engineered immune cells against a population of target cells.

[0108] In some cases, the enhanced cytotoxicity against a population of target cells can be confirmed by a decrease of 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% in the size of the population of target cells.

[0109] In some cases, the enhanced cytotoxicity against a population of target cells 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 inducing the complexation disclosed herein (e.g., after activating the effector portion or after binding of a ligand to the ligand-binding domain of a chimeric receptor).

[0110] In some cases, the enhanced cytotoxicity against a population of target cells disclosed herein can occur (or be observed) in vitro, ex vivo, or in vivo.

[0111] In some examples, the enhanced cytotoxicity against a population of target cells disclosed herein can be confirmed, for example, by measuring the size of a tumor (including a population of target cells such as cancer cells) in a subject after administering cells comprising the system of the present disclosure to the subject. In some cases, the size of a subject's tumor (e.g., a solid tumor) can be reduced by 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% by administration of cells comprising the system of the present disclosure.

[0112] In some cases, a decrease in tumor size can occur (or can be observed) 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 inducing the complex formation disclosed herein (for example, after activating the actuator portion or after binding of a ligand to the ligand binding domain of the chimeric receptor).

[0113] In some cases, the actuator portion can be heterologous to the cell. In some cases, the actuator portion can be activated for complex formation (for example, forming a complex comprising the actuator portion and the target polynucleotide sequence disclosed herein) upon exposure of the cell to an external stimulus (such as an extracellular ligand like an antigen).

[0114] In some cases, activation of the actuator portion can include modification of the actuator portion (such as conformational change, chemical modification). In some cases, activation of the actuator portion can include release of the actuator portion from a substrate (such as a polypeptide substrate). In such cases, the actuator portion cannot be activated when bound to the substrate.

[0115] In some cases, the system can include a chimeric receptor polypeptide (receptor) that is modified by binding to a ligand. The effector portion can be activated by the binding between the ligand and the chimeric receptor polypeptide and / or by receptor modification (e.g., to regulate the expression or activity level of a target protein). In some cases, the receptor can include an antigen-binding portion that can specifically bind to at least one ligand (e.g., at least one, two, three, four, five, or more ligands). The antigen-binding portion can be (i) monovalent or multivalent and (ii) monospecific or multispecific.

[0116] In some cases, the effector portion can be activated in the absence of a signaling pathway in which one or more transcription factors (e.g., endogenous transcription factors) are involved. Alternatively, the effector portion can be activated via a signaling pathway in which one or more transcription factors (e.g., endogenous transcription factors) are involved.

[0117] In some cases, the target polynucleotide sequences disclosed herein can be endogenous genes. Alternatively or in addition, the target polynucleotide sequences can be heterologous genes that encode a target protein (e.g., an endogenous or heterologous protein). For example, the heterologous gene can include the native amino acid sequence of an endogenous protein.

[0118] In some cases, the effector portions disclosed herein (e.g., effector portions that are part of a gene regulatory polypeptide or GMP) can be heterologous to the cell. The GMP can be a substrate, and the activation of the effector portion can include the release of the effector portion from the GMP by receptor modification, as disclosed herein. The GMP can be part of a ligation protein (e.g., a receptor polypeptide or an adapter polypeptide disclosed herein).

[0119] The target polynucleotide sequences disclosed herein can include at least a portion of the transcription start site (TSS) of a target gene encoding a target protein. The target polynucleotide sequence can include 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 can include 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 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, 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 (e.g., up to approximately the disclosed number of bases upstream or downstream of the central nucleobase of the TSS of the target gene) away from the TSS of the target gene. At least a portion of the target polynucleotide sequence can be downstream of the TSS of the target gene. Alternatively or in addition, at least a portion of the target polynucleotide sequence can be upstream of the TSS of the target gene.In some examples, multiple target polynucleotide sequences of a target gene can be utilized by the systems and methods disclosed herein (e.g., complexed with an actuator portion disclosed herein), and the multiple target polynucleotide sequences can include one or more members selected from the group consisting of (1) a target polynucleotide sequence that is at least partially downstream of the TSS of the target gene, (2) a target polynucleotide sequence that is at least partially upstream of the TSS of the target gene, and (3) the TSS of the target gene (e.g., one member, two members, or all three members).

[0120] 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 from about 10,000 bases to about 9,000 bases, from about 10,000 bases to about 8,000 bases, from about 10,000 bases to about 7,000 bases, from about 10,000 bases to about 6,000 bases, from about 10,000 bases to about 5,000 bases, from about 10,000 bases to about 4,000 bases, from about 10,000 bases to about 3,000 bases, from about 10,000 bases to about 2,000 bases, from about 10,000 bases to about 1,000 bases, from about 10,000 bases to about 500 bases, from about 10,000 bases to about 1 base, from about 9,000 bases to about 8,000 bases, from about 9,000 bases to about 7,000 bases, from about 9,000 bases to about 6,000 bases, from about 9,000 bases to about 5,000 bases, from about 9,000 bases to about 4,000 bases, from about 9,000 bases to about 3,000 bases, from about 9,000 bases to about 2,000 bases, from about 9,000 bases to about 1,000 bases, from about 9,000 bases to about 500 bases, from about 9,000 bases to about 1 base, from about 8,000 bases to about 7,000 bases, from about 8,000 bases to about 6,000 bases, from about 8,000 bases to about 5,000 bases, from about 8,000 bases to about 4,000 bases, from about 8,000 bases to about 3,000 bases, from about 8,000 bases to about 2,000 bases, from about 8,000 bases to about 1,000 bases, from about 8,000 bases to about 500 bases, from about 8,000 bases to about 1 base, from about 7,000 bases to about 6,000 bases, from about 7,000 bases to about 5,000 bases, from about 7,000 bases to about 4,000 bases, from about 7,000 bases to about 3,000 bases, from about 7,000 bases to about 2,000 bases, from about 7,000 bases to about 1,000 bases, from about 7,000 bases to about 500 bases, from about 7,000 bases to about 1 base, from about 6,000 bases to about 5,000 bases, from about 6,000 bases to about 4,000 bases, from about 6,000 bases to about 3,000 bases, from about 6,000 bases to about 2,000 bases, from about 6,000 bases to about 1,000 bases, from about 6,It can be from about 0 bases to about 500 bases, from about 6,000 bases to about 1 base, from about 5,000 bases to about 4,000 bases, from about 5,000 bases to about 3,000 bases, from about 5,000 bases to about 2,000 bases, from about 5,000 bases to about 1,000 bases, from about 5,000 bases to about 500 bases, from about 5,000 bases to about 1 base, from about 4,000 bases to about 3,000 bases, from about 4,000 bases to about 2,000 bases, from about 4,000 bases to about 1,000 bases, from about 4,000 bases to about 500 bases, from about 4,000 bases to about 1 base, from about 3,000 bases to about 2,000 bases, from about 3,000 bases to about 1,000 bases, from about 3,000 bases to about 500 bases, from about 3,000 bases to about 1 base, from about 2,000 bases to about 1,000 bases, from about 2,000 bases to about 500 bases, from about 2,000 bases to about 1 base, from about 1,000 bases to about 500 bases, from about 1,000 bases to about 1 base, or from about 500 bases to about 1 base. The distance between the target polynucleotide sequence and the TSS of the target gene can 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.,

[0121] 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 from about 5,000 bases to about 4,500 bases, from about 5,000 bases to about 4,000 bases, from about 5,000 bases to about 3,500 bases, from about 5,000 bases to about 3,000 bases, from about 5,000 bases to about 2,500 bases, from about 5,000 bases to about 2,000 bases, from about 5,000 bases to about 1,500 bases, from about 5,000 bases to about 1,000 bases, from about 5,000 bases to about 500 bases, from about 5,000 bases to about 100 bases, from about 5,000 bases to about 1 base, from about 4,500 bases to about 4,000 bases, from about 4,500 bases to about 3,500 bases, from about 4,500 bases to about 3,000 bases, from about 4,500 bases to about 2,500 bases, from about 4,500 bases to about 2,000 bases, from about 4,500 bases to about 1,500 bases, from about 4,500 bases to about 1,000 bases, from about 4,500 bases to about 500 bases, from about 4,500 bases to about 100 bases, from about 4,500 bases to about 1 base, from about 4,000 bases to about 3,500 bases, from about 4,000 bases to about 3,000 bases, from about 4,000 bases to about 2,500 bases, from about 4,000 bases to about 2,000 bases, from about 4,000 bases to about 1,500 bases, from about 4,000 bases to about 1,000 bases, from about 4,000 bases to about 500 bases, from about 4,000 bases to about 100 bases, from about 4,000 bases to about 1 base, from about 3,500 bases to about 3,000 bases, from about 3,500 bases to about 2,500 bases, from about 3,500 bases to about 2,000 bases, from about 3,500 bases to about 1,500 bases, from about 3,500 bases to about 1,000 bases, from about 3,500 bases to about 500 bases, from about 3,500 bases to about 100 bases, from about 3,500 bases to about 1 base, from about 3,000 bases to about 2,500 bases, from about 3,000 bases to about 2,000 bases, from about 3,000 bases to about 1,500 bases, from about 3,000 bases to about 1,000 bases, from about 3,000 bases to about 500 bases, from about 3,000 bases to about 100 bases, from about 3,from about 1 base to about 100 bases, from about 500 bases to about 100 bases, from about 1,000 bases to about 100 bases, from about 1,500 bases to about 100 bases, from about 2,000 bases to about 100 bases, from about 2,500 bases to about 100 bases, from about 1 base to about 500 bases, from about 1 base to about 1,000 bases, from about 1 base to about 1,500 bases, from about 1 base to about 2,000 bases, from about 1 base to about 2,500 bases, from about 100 bases to about 500 bases, from about 100 bases to about 1,000 bases, from about 100 bases to about 1,500 bases, from about 100 bases to about 2,000 bases, from about 100 bases to about 2,500 bases, from about 500 bases to about 1,000 bases, from about 500 bases to about 1,500 bases, from about 500 bases to about 2,000 bases, from about 500 bases to about 2,500 bases, from about 1,000 bases to about 1,500 bases, from about 1,000 bases to about 2,000 bases, from about 1,000 bases to about 2,500 bases, from about 1,500 bases to about 2,000 bases, from about 1,500 bases to about 2,500 bases, from about 2,000 bases to about 2,500 bases, or from about 2,500 bases to about 5,000 bases. The distance between the target polynucleotide sequence and the TSS of the target gene can be about 1 base, about 100 bases, about 500 bases, about 1,000 bases, about 1,500 bases, about 2,000 bases, about 2,500 bases, about 3,000 bases, about 3,500 bases, about 4,000 bases, about 4,500 bases, or about 5,000 bases.,

[0122] 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 up to about 2,000 bases.The distance between the target polynucleotide sequence and the TSS of the target gene can 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 base, 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 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 1,000 bases to about 1 base, about 800 bases to about 600 bases, about 800 bases to about 400 bases, about 800 bases to about 200 bases, about 800 bases to about 1 base, about 600 bases to about 400 bases, about 600 bases to about 200 bases, about 600 bases to about 1 base, about 400 bases to about 200 bases, about 400 bases to about 1 base, or about 200 bases to about 1 base.The distance between the target polynucleotide sequence and the TSS of the target gene can 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.

[0123] 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 up to about 1,000 bases.The distance between the target polynucleotide sequence and the TSS of the target gene can be from about 1,000 bases to about 900 bases, from about 1,000 bases to about 800 bases, from about 1,000 bases to about 700 bases, from about 1,000 bases to about 600 bases, from about 1,000 bases to about 500 bases, from about 1,000 bases to about 400 bases, from about 1,000 bases to about 300 bases, from about 1,000 bases to about 200 bases, from about 1,000 bases to about 100 bases, from about 1,000 bases to about 1 base, from about 900 bases to about 800 bases, from about 900 bases to about 700 bases, from about 900 bases to about 600 bases, from about 900 bases to about 500 bases, from about 900 bases to about 400 bases, from about 900 bases to about 300 bases, from about 900 bases to about 200 bases, from about 900 bases to about 100 bases, from about 900 bases to about 1 base, from about 800 bases to about 700 bases, from about 800 bases to about 600 bases, from about 800 bases to about 500 bases, from about 800 bases to about 400 bases, from about 800 bases to about 300 bases, from about 800 bases to about 200 bases, from about 800 bases to about 100 bases, from about 800 bases to about 1 base, from about 700 bases to about 600 bases, from about 700 bases to about 500 bases, from about 700 bases to about 400 bases, from about 700 bases to about 300 bases, from about 700 bases to about 200 bases, from about 700 bases to about 100 bases, from about 700 bases to about 1 base, from about 600 bases to about 500 bases, from about 600 bases to about 400 bases, from about 600 bases to about 300 bases, from about 600 bases to about 200 bases, from about 600 bases to about 100 bases, from about 600 bases to about 1 base, from about 500 bases to about 400 bases, from about 500 bases to about 300 bases, from about 500 bases to about 200 bases, from about 500 bases to about 100 bases, from about 500 bases to about 1 base, from about 400 bases to about 300 bases, from about 400 bases to about 200 bases, from about 400 bases to about 100 bases, from about 400 bases to about 1 base, from about 300 bases to about 200 bases, from about 300 bases to about 100 bases, from about 300 bases to about 1 base, from about 200 bases to about 100 bases, from about 200 bases to about 1 base, or from 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.

[0124] 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 from about 500 bases to about 450 bases, from about 500 bases to about 400 bases, from about 500 bases to about 350 bases, from about 500 bases to about 300 bases, from about 500 bases to about 250 bases, from about 500 bases to about 200 bases, from about 500 bases to about 150 bases, from about 500 bases to about 100 bases, from about 500 bases to about 50 bases, from about 500 bases to about 1 base, from about 450 bases to about 400 bases, from about 450 bases to about 350 bases, from about 450 bases to about 300 bases, from about 450 bases to about 250 bases, from about 450 bases to about 200 bases, from about 450 bases to about 150 bases, from about 450 bases to about 100 bases, from about 450 bases to about 50 bases, from about 450 bases to about 1 base, from about 400 bases to about 350 bases, from about 400 bases to about 300 bases, from about 400 bases to about 250 bases, from about 400 bases to about 200 bases, from about 400 bases to about 150 bases, from about 400 bases to about 100 bases, from about 400 bases to about 50 bases, from about 400 bases to about 1 base, from about 350 bases to about 300 bases, from about 350 bases to about 250 bases, from about 350 bases to about 200 bases, from about 350 bases to about 150 bases, from about 350 bases to about 100 bases, from about 350 bases to about 50 bases, from about 350 bases to about 1 base, from about 300 bases to about 250 bases, from about 300 bases to about 200 bases, from about 300 bases to about 150 bases, from about 300 bases to about 100 bases, from about 300 bases to about 50 bases, from about 300 bases to about 1 base, from about 250 bases to about 200 bases, from about 250 bases to about 150 bases, from about 250 bases to about 100 bases, from about 250 bases to about 50 bases, from about 250 bases to about 1 base, from about 200 bases to about 150 bases, from about 200 bases to about 100 bases, from about 200 bases to about 50 bases, from about 200 bases to about 1 base, from about 150 bases to about 100 bases, from about 150 bases to about 50 bases, from about 150 bases to about 1 base, from about 100 bases to about 50 bases, from about 100 bases to about 1 base, or from 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.

[0125] 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 from about 250 bases to about 225 bases, from about 250 bases to about 200 bases, from about 250 bases to about 175 bases, from about 250 bases to about 150 bases, from about 250 bases to about 125 bases, from about 250 bases to about 100 bases, from about 250 bases to about 75 bases, from about 250 bases to about 50 bases, from about 250 bases to about 25 bases, from about 250 bases to about 1 base, from about 225 bases to about 200 bases, from about 225 bases to about 175 bases, from about 225 bases to about 150 bases, from about 225 bases to about 125 bases, from about 225 bases to about 100 bases, from about 225 bases to about 75 bases, from about 225 bases to about 50 bases, from about 225 bases to about 25 bases, from about 225 bases to about 1 base, from about 200 bases to about 175 bases, from about 200 bases to about 150 bases, from about 200 bases to about 125 bases, from about 200 bases to about 100 bases, from about 200 bases to about 75 bases, from about 200 bases to about 50 bases, from about 200 bases to about 25 bases, from about 200 bases to about 1 base, from about 175 bases to about 150 bases, from about 175 bases to about 125 bases, from about 175 bases to about 100 bases, from about 175 bases to about 75 bases, from about 175 bases to about 50 bases, from about 175 bases to about 25 bases, from about 175 bases to about 1 base, from about 150 bases to about 125 bases, from about 150 bases to about 100 bases, from about 150 bases to about 75 bases, from about 150 bases to about 50 bases, from about 150 bases to about 25 bases, from about 150 bases to about 1 base, from about 125 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.

[0126] In some cases, the target protein can be a secreted protein. In some cases, the target protein cannot be a secreted protein.

[0127] In some cases, the cell is affected such that it shows 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% change in the expression or activity level of the target protein as compared to comparable cells not treated with the systems and methods disclosed herein.

[0128] In some cases, the cells can be affected such that they show an increase in the expression or activity level of the target protein of 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% compared to the control cells.

[0129] In some cases, the cells can be affected such that they show a decrease in the expression or activity level of the target protein of 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% compared to the control cells.

[0130] In some cases, changes (e.g., increases, decreases) in the expression or activity levels of a target protein when compared to control cells can be observed 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 after activation of the actuator portion disclosed herein (or receptor modification, or binding of a ligand to the ligand-binding domain of a chimeric receptor).

[0131] In some cases, the actuator portion can include a nucleic acid-inducible actuator portion. In some cases, the system can further include a guide nucleic acid that complexes with the actuator portion. In some cases, the system further 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) having complementarity to different target polynucleotide sequences (e.g., different portions of a target gene encoding a target protein). In some cases, the guide nucleic acids disclosed herein can include guide ribonucleic acid (RNA). In some examples, the cells disclosed herein can include (1) a first guide nucleic acid (e.g., a first guide RNA) capable of binding to a first target polynucleotide sequence operably linked 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 linked to the target protein.

[0132] In some cases, the ligands disclosed herein can be antigens of diseased cells. In some cases, the population of target cells disclosed herein can include diseased cells. In some cases, the diseased cells disclosed herein can include cancer cells or tumor cells.

[0133] In some cases, the cells can be hematopoietic stem cells (HSCs). In some cases, the cells can be immune cells (lymphocytes). In some cases, the immune cells can be selected from the group consisting of T cells, NK cells, monocytes, innate lymphocytes, tumor-infiltrating lymphocytes, macrophages, and granulocytes.

[0134] In some cases, the controls disclosed herein can be control cells that do not have one or more members including (i) a functional chimeric receptor polypeptide, (ii) a functional effector moiety, (iii) a functional guide nucleic acid sequence (e.g., a functional guide RNA) designed to target a target gene, and (iv) a chimeric adapter polypeptide operably linked to the chimeric receptor polypeptide (discussed below). In some cases, the cells can utilize a guide nucleic acid sequence, and the control cells can include a control nucleic acid sequence that is not designed to complex with the target polynucleotide sequence. In some cases, the cells can utilize two different guide nucleic acid sequences, and the control cells can either not include either of the two different guide nucleic acid sequences or can include only one of them.

[0135] In some cases, the systems of the disclosure can enhance an immune response in a subject. Non-limiting enhancements of the immune response can include increased CD4+ helper T cell activity and the generation of cytolytic T cells. Enhancement of the immune response can be evaluated using some in vitro or in vivo measurements known to those of skill 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.

[0136] In some cases, the regulated expression and / or activity of the proteins disclosed herein (e.g., endogenous cytokines) can be confirmed by several 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.

[0137] In one aspect, the disclosure provides a population of cells comprising any one of the systems disclosed herein. In some cases, the population of cells can comprise engineered immune cells. In some examples, the engineered immune cells can comprise engineered T cells. In some cases, the engineered immune cells can comprise engineered NK cells.

[0138] III. Chimeric Receptor Polypeptides

[0139] In some cases, the chimeric receptor polypeptides (receptors) disclosed herein can be operably linked to a chimeric adapter polypeptide (adapter). In some cases, the receptor and the adapter can be configured to form a complex (e.g., a signaling complex) upon binding of a ligand to the receptor (e.g., by contacting a cell comprising the receptor with the ligand) and / or upon receptor modification. The adapter can be a transmembrane protein. Alternatively, the adapter can be an intracellular protein. In some cases, the adapter can be a signaling protein of a receptor signaling pathway that is mobilized towards the receptor upon receptor modification.

[0140] In some cases, the formation of the complex between the receptor and the adapter can be direct and / or indirect. In direct complex formation, one of the receptor and the adapter can be configured to directly bind to the other of the receptor and the adapter (e.g., via covalent and / or non-covalent interactions). In some examples, one of the receptor and the adapter can include a binding domain (e.g., a polypeptide sequence) configured to bind to at least a portion (e.g., the intracellular portion) of the other of the receptor and the adapter. In indirect complex formation, the receptor and the adapter can be configured to be closer to each other without directly binding via receptor modification as compared to the case without receptor modification (e.g., one is mobilized towards the other). In some examples, the receptor can include a chimeric antigen receptor (CAR) or a modified immune cell receptor (e.g., a modified T cell receptor or “TCR”), and the adapter can include at least a portion of a T cell activation linker (LAT) that is mobilized as part of the receptor's signaling cascade upon receptor modification.

[0141] In some cases, one of the receptor and the adapter can include a gene regulatory polypeptide that includes an actuator portion linked to a cleavage recognition site, and the other of the receptor and the adapter can include a cleavage portion 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 portion can occur upon direct complex formation between the receptor and the adapter. In some examples, cleavage of the cleavage recognition site by the cleavage portion can occur upon indirect complex formation between the receptor and the adapter. In receptor engagement, the receptor and the adapter can mobilize towards each other such that, as disclosed herein, the cleavage portion cleaves 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).

[0142] In some cases, the chimeric receptor polypeptides (receptors) disclosed herein can be operably linked 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 can be signaling proteins of a receptor signaling pathway that are mobilized by receptor modification, directed toward the receptor, or directed toward another signaling protein of the receptor signaling pathway. In some examples, the first adapter and the second adapter can be mobilized toward each other by receptor modification. As disclosed herein, the first adapter and the second adapter can form a complex via direct binding. Alternatively, the first adapter and the second adapter can form a complex via indirect binding (e.g., in proximity to each other). In some cases, as disclosed herein, the first adapter can include a GMP (including an actuator portion linked to a cleavage recognition site), and the second adapter can include a cleavage portion. Upon receptor recognition, the first adapter and the second adapter can be mobilized toward each other such that the cleavage portion cleaves 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).

[0143] In some cases, one of the first adapter and the second adapter can include a gene regulatory polypeptide that includes an actuator portion linked to a cleavage recognition site, and the other of the first adapter and the second adapter can include a cleavage portion 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 portion can occur upon direct complex formation between the first adapter and the second adapter. In some examples, cleavage of the cleavage recognition site by the cleavage portion can occur upon indirect complex formation between the first adapter and the second adapter.

[0144] In some cases, the receptors disclosed herein can undergo receptor modifications, including conformational changes or chemical modifications (e.g., phosphorylation or dephosphorylation), upon binding to a ligand.

[0145] Figures 1A-1D schematically show the release of the effector portion from GMP. Figure 1A shows the binding of an antigen to a transmembrane chimeric receptor polypeptide. The transmembrane chimeric receptor polypeptide includes an extracellular region having an antigen interaction domain 101 and an intracellular region containing GMP. GMP includes an effector portion 102a linked to a cleavage recognition site 102b. In response to antigen binding, the receptor is modified by phosphorylation 103 in the intracellular region of the receptor (Figure 1B). Following receptor modification (e.g., phosphorylation), an adapter protein containing a receptor binding portion is recruited to the receptor as shown in Figure 1C. The receptor includes a cleavage portion 104, which can complex with the adapter or be linked to the receptor binding portion, e.g., by a peptide bond and / or a peptide linker. When proximal to the cleavage recognition site, the cleavage portion can cleave the recognition site to release the effector portion from GMP as shown in Figure 1D. Upon release, the effector portion can enter the nucleus and regulate the expression and / or activity of a target gene (e.g., a target protein disclosed herein) or edit a nucleic acid sequence. Figures 1E-1H show a similar system where receptor modification includes a conformational change. In some embodiments, the adapter protein is membrane-tethered (e.g., as a membrane-bound protein).

[0146] Figures 2A-2D schematically show the release of the actuator portion from the GMP. Figure 2A shows the binding of the antigen to the transmembrane chimeric receptor polypeptide. The transmembrane chimeric receptor polypeptide includes an extracellular region having an antigen interaction domain 205 and an intracellular region including a cleavage portion 206. The cleavage portion can complex with the receptor or be linked to the receptor, for example, by a peptide bond and / or a peptide linker. The GMP forms part of the chimeric adapter polypeptide. The GMP linked to the receptor binding portion 201 includes an actuator portion 202a linked to a cleavage recognition site 202b. In response to antigen binding, the receptor is modified by phosphorylation 203 in the intracellular region of the receptor (Figure 2B). Following receptor modification (e.g., phosphorylation), the chimeric adapter polypeptide is recruited to the receptor as shown in Figure 3C. The receptor includes a cleavage portion 206. When proximal to the cleavage recognition site, the cleavage portion can cleave the recognition site to release the actuator portion from the GMP as shown in Figure 2D. Upon release, the actuator portion can enter the nucleus to regulate gene expression and / or activity or edit nucleic acid sequences. Figures 2E-2H show a similar system in which receptor modification includes a conformational change. In some embodiments, the chimeric adapter protein is membrane-tethered (e.g., as a membrane-bound protein).

[0147] Figures 3A-3D schematically show the release of the actuator portion from the GMP. Figure 3A shows the binding of an antigen to a transmembrane chimeric receptor polypeptide. The transmembrane chimeric receptor polypeptide includes an extracellular region having an antigen interaction domain 305 and an intracellular region. The GMP, which includes an actuator portion linked to a cleavage recognition site, forms part of a chimeric adapter polypeptide. The cleavage recognition site 302b is adjacent to the receptor binding portion 301 and the actuator portion 302a. In response to antigen binding, the receptor is modified by phosphorylation 303 in the intracellular region (Figure 3B). Following receptor modification (e.g., phosphorylation), the chimeric adapter polypeptide is recruited to the receptor as shown in Figure 3B. A second adapter polypeptide 307, which includes a cleavage portion 306, is also recruited to the modified receptor (Figure 3C). The cleavage portion can complex with the second adapter polypeptide or be linked to the adapter, for example, by a peptide bond and / or a peptide linker. When proximal to the cleavage recognition site, the cleavage portion can cleave the recognition site to release the actuator portion from the GMP as shown in Figure 3D. Upon release, the actuator portion can enter the nucleus to regulate gene expression and / or activity or edit a nucleic acid sequence. Figures 3E-3H show a similar system where receptor modification includes a conformational change. In some embodiments, the chimeric adapter polypeptide is tethered to the membrane (e.g., as a membrane-bound protein). In some embodiments, the second adapter polypeptide is tethered to the membrane (e.g., as a membrane-bound protein).

[0148] In some cases, the chimeric receptor polypeptide (receptor) can include a ligand-binding domain, a transmembrane domain, and a signaling domain. The signaling domain can activate a cell's signal transduction pathway upon binding of the ligand to its ligand-binding domain. The cell can further include an expression cassette comprising a polynucleotide sequence encoding an actuator moiety (e.g., a GMP comprising the actuator moiety) placed under the control of a promoter. The actuator moiety can include a heterologous endonuclease. The promoter can be activated to drive expression of the actuator moiety upon binding of the ligand to its ligand-binding domain. The expressed actuator moiety can complex with a target gene encoding an endogenous protein (e.g., an endogenous cytokine) disclosed herein to regulate the expression or activity of the endogenous protein. The promoter can include an endogenous promoter of the cell. The endogenous promoter can be activated upon binding of the ligand to the ligand-binding domain of the receptor.

[0149] [Figure 4] shows an exemplary system that includes a transmembrane receptor useful for modulating the expression of at least one target gene. Binding of a ligand to a chimeric receptor polypeptide (e.g., scFv-CAR) activates an endogenous signaling pathway, resulting in recruitment of at least one cellular transcription factor (e.g., an endogenous transcription factor) to the promoter region of an endogenous gene (signature gene) at its native locus. An actuator moiety coding sequence (e.g., a GMP coding sequence that includes an actuator moiety coding sequence) is integrated into the genome and placed under the control of the promoter of the signature gene. Transcriptional activation of the promoter results in expression of the actuator moiety (e.g., including dCas linked to a transcriptional activator (e.g., VPR) or a transcriptional repressor (e.g., KRAB)). The expressed actuator moiety, upon complexing with a guide RNA (e.g., sgRNAa, sgRNAb) (e.g., constitutively or conditionally expressed), can modulate (activate or repress) the expression of an endogenous protein (e.g., gene A such as IL-12A, gene B such as IL-12B) disclosed herein.

[0150] In some cases, the chimeric receptor polypeptides (receptors) disclosed herein can be chimeric antigen receptors (CARs) and / or engineered T cell receptors (TCRs).

[0151] In some cases, the CARs disclosed herein can be of the first, second, third, or fourth generation CAR systems, functional variants thereof, or any combination thereof. First-generation CARs (e.g., CD19R or CD19CAR) include an antigen-binding domain having specificity for a particular antigen (e.g., an antibody, or an antigen-binding fragment thereof such as an scFv, Fab fragment, VHH domain, or VH domain of a heavy-chain antibody), a transmembrane domain from an adaptive immune receptor (e.g., the transmembrane domain from the CD28 receptor), and a signaling domain from an adaptive immune receptor (e.g., one or more (e.g., three) ITAM domains derived from the intracellular region of the CD3ζ receptor or FcεRIγ). Second-generation CARs improve upon first-generation CARs by the addition of a co-stimulatory domain to the intracellular signaling domain portion of the CAR (e.g., derived from a co-stimulatory receptor that acts with a T-cell receptor such as CD28, CD137 / 4-1BB, and CD134 / OX40), eliminating the need to administer co-factors (e.g., IL-2) together with the first-generation CAR. Third-generation CARs add multiple co-stimulatory domains to the intracellular signaling domain portion of the CAR (e.g., CD3ζ-CD28-OX40, or CD3ζ-CD28-41BB). Fourth-generation CARs improve second-generation or third-generation CARs by the addition of an activating cytokine (e.g., IL-23 or IL-27) to the intracellular signaling portion of the CAR (e.g., between one or more of the co-stimulatory domains and the CD3ζ ITAM domain), or under the control of a CAR-inducible promoter (e.g., the NFAT / IL-2 minimal promoter).

[0152] IV. Actuator portion

[0153] The actuator portion disclosed herein (e.g., the actuator portion that is part of GMP) can edit a target gene (e.g., via insertion and / or deletion (indel), homology directed repair (HDR), non-homologous end joining (NHEJ)) and can regulate 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 exhibits the ability to complex with a target gene (e.g., an inactivated or dead CRISPR / Cas protein provided herein).

[0154] The actuator portion disclosed herein (e.g., the actuator portion that is part of GMP) can be operably linked to at least one effector domain. The at least one effector domain can be configured to regulate the expression or activity of an endogenous protein (e.g., an endogenous cytokine). In some cases, the actuator portion can fuse with at least one effector domain to form a fusion portion. In some cases, the actuator portion can include a first binding portion (e.g., a polynucleotide) such that the actuator portion and the at least one effector domain can be bound to each other, and the at least one effector domain can include a second binding portion (e.g., a second polynucleotide complementary to the first polynucleotide). In some examples, the at least one effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain for regulating the expression or activity of an endogenous protein (e.g., an endogenous cytokine).

[0155] Non-limiting examples of the function of at least one effector domain disclosed herein 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, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthetase activity, synthase activity, or demyristoylation activity.

[0156] Non-limiting examples of at least one effector domain disclosed herein include methyltransferase, demethylase, dismutase, alkylating enzyme, depurinase, oxidase, pyrimidine dimer-forming enzyme, integrase, transposase, recombinase, polymerase, ligase, helicase, photolyase or glycosylase, acetyltransferase, deacetylase, kinase, phosphatase, ubiquitin ligase, deubiquitinating enzyme, adenylating enzyme, deadenylating enzyme, SUMOylating enzyme, desumoylating enzyme, ribosylating enzyme, deribosylating enzyme, myristoylating enzyme, remodeling enzyme, protease, oxidoreductase, transferase, hydrolase, lyase, isomerase, synthetase, synthase, and demyristoylating enzyme.

[0157] The actuator portion disclosed herein can include a nuclease such as an endonuclease (e.g., Cas). The endonuclease can be heterologous to any of the cells disclosed herein.

[0158] The actuator portion disclosed in this specification can 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 meganuclease, a native master transcription factor, an epigenetic modification enzyme, a recombinase, a flippase, a transposase, an RNA-binding protein (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof. In some embodiments, the actuator portion includes a Cas protein, and the system further includes a guide RNA (gRNA) that complexes with the Cas protein. In some embodiments, the actuator portion includes an RBP complexed with a gRNA that can form a complex with a Cas protein. In some embodiments, the gRNA includes a targeting segment that exhibits at least 80% sequence identity to the target polynucleotide. In some embodiments, the Cas protein is substantially lacking in DNA cleavage activity (i.e., dead Cas, inactivated Cas, or dCas). For example, the Cas protein is mutated and / or modified to result in reduced nuclease activity compared to a nuclease-deficient protein or a wild-type Cas protein. The nuclease-deficient protein can retain the ability to bind to DNA but lacks nucleic acid cleavage activity or can have reduced nucleic acid cleavage activity.

[0159] In some cases, suitable actuator moieties include CRISPR-associated (Cas) proteins or Cas nucleases, such as type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); any derivatives thereof; any variants thereof; and any fragments thereof.

[0160] The Cas protein referred to in this specification can be of the type of protein or polypeptide. The Cas protein can refer to a nuclease. The Cas protein can refer to an endoribonuclease. The Cas protein can refer to any modified (e.g., shortened, mutated, elongated) polypeptide sequence or homolog of the Cas protein. The Cas protein can be codon-optimized. The Cas protein can be a codon-optimized homolog of the Cas protein. The Cas protein can be enzymatically inactive, partially active, constitutively active, fully active, inducibly active, and / or more active (e.g., than the wild-type homolog of the protein or polypeptide). The Cas protein can be Cas9. The Cas protein can be Cpf1. The Cas protein can be C2c2. The Cas protein (e.g., variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive site-specific polypeptide) can bind to a target nucleic acid. The Cas protein (e.g., variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endoribonuclease) can bind to a target RNA or DNA.

[0161] 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, Cas1O, Cas1Od, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Cse1 (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, as well as homologs or modified forms thereof.

[0162] In some cases, the nucleases (e.g., Cas) disclosed herein 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 to 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 bind site-specifically to a nucleic acid sequence. The nucleic acid to be targeted, or target nucleic acid, 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. The strand of the double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid can be referred to as the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand and thus may not be complementary to the guide nucleic acid can be referred to as the non-complementary strand. The guide nucleic acid can contain a polynucleotide strand and can be referred to as a "single guide nucleic acid". The guide nucleic acid can contain two polynucleotide strands and can be referred to as a "dual guide nucleic acid". Unless otherwise specified, the term "guide nucleic acid" can be inclusive and refer to both single guide nucleic acids and dual guide nucleic acids.

[0163] The guide nucleic acid can contain a segment that can be referred to as a "nucleic acid targeting segment" or "nucleic acid targeting sequence". The nucleic acid targeting segment can contain a sub-segment that can be referred to as a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment".

[0164] The guide nucleic acid can comprise two separate nucleic acid molecules and can be referred to as a dual guide nucleic acid. The guide nucleic acid can comprise a single nucleic acid molecule and can 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 a fusion CRISPR RNA (CRISPR RNA, crRNA) and a transactivating crRNA (tracrRNA). In some cases, the guide nucleic acid is a single guide nucleic acid comprising a crRNA. In some cases, the guide nucleic acid is a single guide nucleic acid that comprises a crRNA but lacks a tracrRNA. In some cases, the guide nucleic acid is a dual guide nucleic acid comprising a non-fusion crRNA and a tracrRNA. An exemplary dual guide nucleic acid can comprise a crRNA-like molecule and a tracrRNA-like molecule. An exemplary single guide nucleic acid can comprise a crRNA-like molecule. An exemplary single guide nucleic acid can comprise a fusion crRNA-like molecule and a tracrRNA-like molecule.

[0165] As used herein, the term “crRNA” generally refers to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to an exemplary wild-type crRNA (e.g., a crRNA from S. pyogenes). A crRNA can generally refer to a nucleic acid having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to an exemplary wild-type crRNA (e.g., a crRNA from S. pyogenes). A crRNA can refer to a modified form of a crRNA that can include nucleotide changes, variants, mutations, or chimeras such as deletions, insertions, or substitutions. A crRNA can be a nucleic acid having at least about 60% sequence identity over a continuous stretch of at least 6 consecutive nucleotides to an exemplary wild-type crRNA (e.g., a crRNA from S. pyogenes) sequence. For example, a crRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to an exemplary wild-type crRNA sequence (e.g., a crRNA from S. pyogenes) over a continuous stretch of at least 6 consecutive nucleotides.

[0166] As used herein, the term “tracrRNA” generally refers to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to an exemplary wild-type tracrRNA sequence (e.g., tracrRNA from S. pyogenes). A tracrRNA can refer to a nucleic acid having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to an exemplary wild-type tracrRNA sequence (e.g., tracrRNA from S. pyogenes). A tracrRNA can refer to a modified form of tracrRNA that can include nucleotide changes such as deletions, insertions, or substitutions, variants, mutations, or chimeras. A tracrRNA can refer to a nucleic acid that can be at least about 60% identical over a continuous stretch of at least 6 contiguous nucleotides to an exemplary wild-type tracrRNA (e.g., tracrRNA from S. pyogenes) sequence. For example, a tracrRNA sequence can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical over a continuous stretch of at least 6 contiguous nucleotides to an exemplary wild-type tracrRNA sequence (e.g., tracrRNA from S. pyogenes).

[0167] A crRNA can include a nucleic acid targeting segment of the guide nucleic acid (e.g., the spacer region) and a stretch of nucleotides that can form half of a duplex of the Cas protein binding segment of the guide nucleic acid.

[0168] The tracrRNA can include a continuous stretch of nucleotides that form the other half of the duplex of the Cas protein-binding segment of the gRNA. The continuous stretch of nucleotides of the crRNA is complementary to the continuous stretch of nucleotides of the tracrRNA and can hybridize to form the duplex of the Cas protein-binding domain of the guide nucleic acid.

[0169] The crRNA and tracrRNA can hybridize to form a guide nucleic acid. The crRNA can also provide a single-stranded nucleic acid targeting segment (e.g., spacer region) that hybridizes to a target nucleic acid recognition sequence (e.g., protospacer). The sequence of the crRNA, or the sequence of the tracrRNA molecule, including the spacer region, can be designed to be species-specific for the species in which the guide nucleic acid is used.

[0170] 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 portion can be a Cas protein (e.g., dCas such as dCas9) fused to a transcriptional activation domain as disclosed herein.

[0171] 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 suppressor domain as disclosed herein.

[0172] In one aspect, the present disclosure provides a system comprising an effector portion that binds to a target polynucleotide sequence in a cell and modulates the expression or activity of an endogenous cytokine (e.g., interleukin (IL)) in the cell as disclosed herein. In some cases, the effector 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.

[0173] V. Guide Nucleic Acid

[0174] In one aspect, the present disclosure provides a system comprising a guide nucleic acid molecule designed to bind to a target polynucleotide sequence in a cell and modulate the expression or activity of a target protein in the cell as disclosed herein. In some cases, the guide nucleic acid molecule can recruit an effector portion to the target polynucleotide sequence in the cell to modulate the expression or activity of the target protein. In some cases, the system can comprise an effector portion. For example, the target protein can comprise one or more proteins from Tables 1 and 2.

[0175] In some cases, the target polynucleotide sequence can be endogenous to the cell. In some cases, the transcription start site (TSS) of the target gene encoding the target protein can be endogenous to the cell.

[0176] In some cases, the system can comprise at least or up to 2, at least or up to 3, at least or up to 3, at least or up to 4, at least or up to 5, at least or up to 6, at least or up to 7, at least or up to 8, at least or up to 9, or at least or up to 10 different guide nucleic acid molecules having different nucleic acid sequences. In some cases, the guide nucleic acid molecule can comprise a guide ribonucleic acid (RNA). In some examples, the system can comprise a multiplex guide nucleic acid (e.g., multiplex guide RNA).

[0177] In some cases, the system can include (i) a first guide nucleic acid molecule designed to bind to a first target polynucleotide sequence of the target polynucleotide sequences disclosed herein, and (ii) a second guide nucleic acid molecule designed to bind to a second target polynucleotide sequence of the target polynucleotide sequences disclosed herein. In some examples, the system can include (i) a first guide nucleic acid molecule designed to bind to a first portion of the 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 the 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 up to about 1, at least or up to 2, at least or up to 3, at least or up to 3, at least or up to 4, at least or up to 5, at least or up to 6, at least or up to 7, at least or up to 8, at least or up to 9, at least or up to 10, at least or up to 15, at least or up to 20, at least or up to 30, at least or up to 40, at least or up to 50, at least or up to 60, at least or up to 70, at least or up to 80, at least or up to 90, at least or up to 100, at least or up to 200, at least or up to 300, at least or up to 400, at least or up to 500, at least or up to 600, at least or up to 700, at least or up to 800, at least or up to 900, at least or up to 1,000, at least or up to 2,000, at least or up to 3,000, at least or up to 4,000, or at least or up to 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 a cell). Alternatively, the first target polynucleotide sequence and the second target polynucleotide sequence can be on different strands of the target nucleic acid molecule.

[0178] In some cases, the target gene encoding the target protein can include multiple transcription start sites (TSSs) including a first TSS and a second TSS. Each of the first TSS and the second TSS can encode different portions of the target gene. For example, the target protein can be a heterodimer, the first TSS can be from a gene encoding the first monomer of the heterodimer, and the second TSS can be from a gene encoding the second monomer of the heterodimer. Thus, in some examples, the first guide nucleic acid molecule can (1a) include at least a portion of the first TSS or (1b) be at a specific distance from the first TSS provided herein, and the second guide nucleic acid can (2a) include at least a portion of the second TSS or (2b) be at a specific distance from the second TSS provided herein.

[0179] In some cases, a 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 one of the polynucleotide sequences provided in Table 3.

[0180] [Table 3]

[0181] VI. Delivery of System Expression in Cells

[0182] In one aspect, the disclosure provides a cell (e.g., an immune cell) that includes (or expresses) any of the subject systems disclosed herein.

[0183] In one aspect, the disclosure provides a population of cells (e.g., a population of immune cells) that includes (or expresses) any of the subject systems disclosed herein.

[0184] Using an RNA or DNA virus-based system, one or more genes encoding any of the polypeptides and / or polynucleotides disclosed herein (e.g., chimeric receptors, chimeric adapters, actuator moieties (with or without effector domains), or genes encoding them) can be delivered to the cells of the present disclosure. Viral vectors can be used to treat cells in vitro, and the modified cells can optionally be administered (ex vivo). Alternatively, the viral vector can be administered directly to a subject (in vivo). Virus-based systems can include retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors for gene transfer. Integration into the host genome can occur using retroviral, lentiviral, and adeno-associated viral gene transfer methods and can result in long-term expression of the inserted transgene.

[0185] In some cases, non-viral delivery methods can be used to deliver any of the polypeptides and / or polynucleotides disclosed herein (e.g., chimeric receptors, chimeric adapters, actuator moieties (with or without effector domains), or genes encoding them) to the cells of the present disclosure. Methods of non-viral delivery of such cargo can include lipofection, nucleofection, microinjection, biolistic particle delivery, virosomes, liposomes, immunoliposomes, exosomes, polycations or lipid:cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virions, and drug-enhanced uptake of polypeptides or DNA. Cationic and neutral lipids suitable for efficient receptor-recognition liposomal delivery of polynucleotides or polypeptides can be used.

[0186] VII. Methods and Compositions

[0187] In one aspect, the present disclosure provides a method for conditionally regulating the expression or activity of a target protein (e.g., endogenous target protein 33) in a cell by introducing (or expressing) any of the subject systems disclosed herein.

[0188] 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) in a cell. The method can include (a) exposing a chimeric receptor polypeptide (receptor) to a ligand, wherein the receptor is modified upon binding to the ligand. The method can include (b) forming a complex between an effector moiety and a target polynucleotide sequence, as disclosed herein, in response to the receptor modification to regulate the expression or activity of the target protein.

[0189] In some cases, the method further includes administering a co-therapeutic agent.

[0190] In some cases, the cells administered to the subject can be autologous or allogeneic to the subject. For example, the cells administered to the subject can be autologous immune cells or allogeneic immune cells.

[0191] In one aspect, the present disclosure provides a composition comprising a cell or population of cells (e.g., a population of engineered immune cells) comprising any of the subject systems disclosed herein (or expressing the same). The composition can be administered to a subject for treating a condition of the subject (e.g., cancer, tumor). The composition can include 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.

[0192] In some cases, the composition further includes a co-therapeutic agent.

[0193] The compositions disclosed herein can be pharmaceutical compositions. The pharmaceutical compositions can be in any suitable form (depending on the desired method of administration). The 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 a kit can include instructions for use. The kit can include a plurality of such unit dosage forms.

[0194] Non-limiting examples of co-therapeutic agents include cytotoxic agents, chemotherapeutic agents, growth inhibitors, agents used in radiation therapy, anti-angiogenic agents, apoptosis agents, anti-tubulin agents, and other agents for treating cancer, such as anti-CD20 antibodies, anti-PD1 antibodies (e.g., pembrolizumab), platelet-derived growth factor inhibitors (e.g., GLEEVEC™ (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, and other bioactive and organic chemical agents, etc.

[0195] The term "cytotoxic agent" generally refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. Non-limiting examples of cytotoxic agents include radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioisotopes 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 nuclease, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin.

[0196] Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylene imines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially, bradycidin and bradycidinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopollectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (including adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllinic acid, teniposide; cryptophycin (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); erythrobicin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics; dynemicin including dynemicin A; esperamicin;and neocarzinostatin chromophore and related chromoprotein engyin antibiotic chromophores, actinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardinophyllin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodrubicin, streptomycin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyruridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epitostanol, mepitiostane, testolactone; antiadrenalines such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aidophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demeclocycline; diaziquone; ellorolnitin; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea, lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol;Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Rizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triazicone; 2,2’,2’’-Trichlorotriethylamine; Trichothecene (especially, T-2 toxin, Verrucarin A, Lolitrem A, and Anguizine); Urethane; Vinblastine (ELDISINE (registered trademark), FILDESIN (registered trademark)); Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacitabine; Arabinoside (“Ara-C”); Thiotepa; Taxoid, for example, TAXOL (registered trademark) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE (trademark) which is an albumin-engineered nanoparticle formulation without Cremophor of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE (registered trademark) docetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine (GEMZAR (registered trademark)); 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, for example, cisplatin and carboplatin; Vinblastine (VELBAN (registered trademark)); Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine (ONCOVIN (registered trademark)); Oxaliplatin; Leucovorin; Vinorelbine (NAVELBINE (registered trademark)); Novantrone; Edatrexate; Daunomycin; Aminopterin; Ibandronate; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoid, for example, retinoic acid; Capecitabine (XELODA (registered trademark)); Any pharmaceutically acceptable salt, acid, or derivative of the above;And combinations of two or more of the above, such as CHOP (abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisone), and FOLFOX (abbreviation for a treatment regimen using oxaliplatin (ELOXATIN (trademark)) combined with 5-FU and leucovorin), can be mentioned. Additional chemotherapeutic agents include maytansinoids (e.g., DM1), and cytotoxic agents useful as antibody-drug conjugates, such as auristatin MMAE and MMAF.;

[0197] Examples of chemotherapeutic agents can also include "anti-hormonal agents" or "endocrine therapeutic agents" which act to modulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth and which are often in the form of systemic or whole body treatment. They can themselves be hormones. Examples include, for example, anti-estrogens and selective estrogen receptor modulators (SERM) such as tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene; anti-progesterones; estrogen receptor down-regulators (ERD); agents that function to suppress or halt the ovaries, such as luteinizing hormone-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD leuprolide acetate, goserelin acetate, buserelin acetate, and tripterelin; other anti-androgens such as flutamide, nilutamide, and bicalutamide; aromatase inhibitors that inhibit the enzyme aromatase to regulate estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole, and the like.In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate disodium (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (LURTOTECAN®); rmRH (ABARELIX®); lapatinib ditosylate (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases also known as GW572016); and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0198] Examples of chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Further humanized monoclonal antibodies having therapeutic potential as agents in combination with the compounds of the present invention include apolizumab, aselizumab, atorizumab, bapineuzumab, bevacizumab mertansine, canertinib mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eclizumab, efalizumab, epratuzumab, elotuzumab, femuzumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labelizumab, lintuzumab, matuzumab, mapolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, norovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pekfusituzumab, pekizumab, peksizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, recevizumab, rovelizumab, rupizumab, sibrotuzumab, siprilizumab, sonozumab, takatsuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, tralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, ultoxazumab, ustekinumab, visilizumab, and the recombinant exclusive human sequence, full-length IgG1λ antibody anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories) genetically modified to recognize interleukin-12 p40 protein.

[0199] Examples of chemotherapeutic agents 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 (Pfizer and OSI), an oral selective inhibitor of ErbB2 receptor tyrosine kinase; dual HER inhibitors such as EKB-569 (available from Wyeth) that 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 that inhibits Raf-1 signaling; non-HER targeting TK inhibitors such as imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as bevacizumab (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines such as PD153035 which is 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP59326, CGP60261, and CGP62706; pyrazolopyrimidine which is 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostine containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to HER coding nucleic acids); quinoxaline (U.S. Patent No. 5,804,396); tyrphostine (U.S. Patent No. 5,804,396);ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors, e.g., CI-1033 (Pfizer); Affinitac (ISIS3521, Isis / Lilly); imatinib mesylate (GLEEVEC (R)); PKI166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); and rapamycin (sirolimus, RAPAMUNE (R)) can be mentioned.

[0200] Examples of chemotherapeutic agents also include dexamethasone, interferon, cortisone, methotrexate, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erlotinib, filgrastim, histrelin acetate, ibritumomab, interferon alpha-2a, interferon alpha-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, paricalcitol, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, tamoxifen, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and their pharmaceutically acceptable salts can be mentioned.

[0201] Examples of chemotherapeutic drugs also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fludrocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, acrometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fludrocortolone caproate, fludrocortolone pivalate, and fluprednidene acetate; immune selective anti-inflammatory peptide (ImSAID), for example, phenylalanine-glutamine-glycine (FEG) and its D-isomer (feG) (IMULAN BioTherapeutics, LLC); antirheumatic agents, for example, azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers, for example, etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), certolizumab pegol (CIMZIA®), golimumab (SIMPONI®), interleukin 1 (interleukin 1, IL-1) blockers, for example, anakinra (KINERET®), T-cell costimulation blockers, for example, abatacept (ORENCIA®), interleukin 6 (interleukin 6, IL-6) blockers, for example, tocilizumab (ACTEMERA®); interleukin 13 (Interleukin 13, IL-13) blockers, for example, lebrikizumab; interferon alpha (Interferon alpha, IFN) blockers, for example, lonafarnib; beta7 integrin blockers, for example, rhuMAb beta7;IgE pathway blockers, e.g., anti-M1 prime; secretory homotrimeric LTa3 and membrane-bound heterotrimeric LTa / β2 blockers, e.g., anti-lymphotoxin alpha (LTa); a variety of investigational drugs, e.g., thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols, e.g., quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanol, procyanidin, betulinic acid, and their derivatives; autophagy inhibitors, e.g., chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scoplectin, and 9-aminocamptothecin); podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); bisphosphonates, e.g., clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines, e.g., THERATOPE® vaccine; periphosin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); CCI-779, tipifamib (R11577); orafenib, ABT510; Bcl-2 inhibitors, e.g., oblimersen sodium (GENASENSE®); pixantrone; farnesyltransferase inhibitors, e.g., lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above;And combinations of two or more of the above can be cited;

[0202] 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 is dependent on PD-L1 expression), either in vitro or in vivo. A growth inhibitor can be one that significantly reduces the proportion of cells in the S phase. Non-limiting examples of growth inhibitors include agents that block cell cycle progression (at positions other than the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as the anthracycline antibiotic doxorubicin ((8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione), epirubicin, daunorubicin, etoposide, and bleomycin. Agents that arrest G1 also affect S phase arrest and are, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Taxanes (paclitaxel and docetaxel) are both anticancer agents derived from the yew tree. Docetaxel (TAXOTERE (registered trademark), Rhone-Poulenc Rorer), which is derived from the European yew, is a semi-synthetic analogue of paclitaxel (TAXOL (registered trademark), Bristol-Myers Squibb). Paclitaxel and docetaxel stabilize microtubules by promoting the assembly of microtubules from tubulin dimers and preventing depolymerization, resulting in the inhibition of mitosis in cells.

[0203] VIII. Therapeutic Uses

[0204] The target system can be introduced into various immune cells, including any cells involved in the immune response. In some embodiments, the immune cells include granulocytes such as neutrophils, eosinophils, and basophils; mast cells; monocytes that can develop into macrophages; antigen-presenting cells such as dendritic cells; and lymphocytes such as natural killer cells (NK cells), B cells, and T cells. In some embodiments, the immune cells are immune effector cells. Immune effector cells refer to immune cells that can perform specific functions in response to stimulation. In some embodiments, the immune cells are immune effector cells that can induce cell death. In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are NK cells. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are activated T cells. T cells include both naive cells and memory cells (e.g., central memory or T 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 Trl cells), natural killer T cells, tumor infiltrating lymphocytes (TIL), lymphocyte-activated killer cells (LAK), αβ T cells, γδ T cells, and similar specific classes of the T cell lineage are included. 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 target system can perform multiple functions including killing infected cells and activating or mobilizing other immune cells. CD8+ T cells are called cytotoxic T cells or cytotoxic T lymphocytes (CTL). CTL expressing the target system can be involved in recognizing and removing virus-infected cells and cancer cells. CTL have specialized compartments or granules containing cytotoxins that cause apoptosis, e.g., programmed cell death. CD4+ T cells can be subdivided into four subsets - Th1, Th2, Th17, and Treg, where "Th" refers to "T helper cells", although additional subsets may exist. Th1 cells can regulate the immune response against intracellular microorganisms, particularly bacteria. They can produce and secrete molecules that alert and activate other immune cells such as bacterium-ingesting macrophages. Th2 cells are involved in regulating the immune response against extracellular pathogens such as helminths (parasites) by alerting B cells, granulocytes, and mast cells. Th17 cells can produce interleukin 17 (IL-17), a signaling molecule that activates immune and non-immune cells. Th17 cells are important for mobilizing neutrophils.

[0205] The ligand or antigen (i.e., target antigen) of the antigen-binding portion disclosed herein can be a cell surface marker, a secreted marker, or an intracellular marker.

[0206] Non-limiting examples of antigens (i.e., target antigens) of the antigen-binding portions disclosed herein 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 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2, 3, 4, EGFIR, 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 Receptor2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ light chain, kinase insert domain receptor (KDR), kappa, Lewis A (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, cancer fetal 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, protozoa that can cause diseases) can be mentioned. In some examples, the pathogen antigen is derived from HIV, HBV, EBV, HPV, Lassa virus, influenza virus, or coronavirus.

[0207] Additional examples of antigens for the antigen-binding portions disclosed herein 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, Bacillus anthracis anthrax, BAFF, beta-amyloid, B-lymphocytes, C242 antigen, C5, CA-125, Canis lupus familiaris IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eotaxin-1), CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD25 (alpha chain of the IL-2 receptor), CD27, CD274, CD28, CD3, CD3 epsilon, 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, clamping factor A, CSF1R, CSF2, CTLA-4, C-X-C chemokine receptor type 4, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, EGFL7, EGFR, endotoxin, EpCAM, epithin, ERBB3, EscherichiaE. coli, Respiratory Syncytial Virus F protein, FAP, Fibrin II beta chain, Fibronectin Extradomain B, Folic Acid Hydrolase, Folic Acid Receptor 1, Folic Acid Receptor Alpha, Frizzled Receptor, Ganglioside GD2, GD2, GD3 Ganglioside, Glypican 3, GMCSF Receptor alpha 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 Dispersal Factor Receptor Kinase, Human TNF, Human beta Amyloid, ICAM-1 (CD54), IFN-alpha, IFN-gamma, IgE, IgE Fc region, IGF-1 Receptor, IGF-1, IGHE, IL17A, IL17F, IL20, IL-12, IL-13, IL-17, IL-1beta, 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 alpha4beta7, Integrin alpha4, Integrin alpha5beta1, Integrin alpha7beta7, Integrin alphaIIbbeta3, Integrin alphavbeta3, Interferon alpha / beta Receptor, Interferon gamma 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 Regulatory Protease 1, NGF, N-Glycolylneuraminic Acid, NOGO-A, Notch Receptor, NRP1, Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-Ralpha, Sodium Phosphate Cotransporter, Phosphatidylserine, Platelet-Derived Growth Factor Receptor beta, Prostate Cancer Cells, PseudomonasExamples include Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, baboon 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-related calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA, VEGFR1, VEGFR2, vimentin, and VWF.

[0208] Additional examples of antigens for the antigen-binding portions disclosed herein include 707-AP, biotinylated molecules, alpha-actinin-4, abl-bcr alb-b3 (b2a2), abl-bcr alb-b4 (b3a2), adipophilin, AFP, AIM-2, annexin II, ART-4, BAGE, beta-catenin, bcr-abl, bcr-abl p190 (e1a2), bcr-abl p210 (b2a2), bcr-ablp210(b3a2), BING-4, CAG-3, CAIX, CAMEL, Caspase-8, CD171, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v7 / 8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6 / AML, FBP, Fetal Acetylcholine Receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2M, 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, Cancer Fetal 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 can be mentioned.

[0209] Additional examples of antigens for the antigen-binding portions disclosed herein include antibodies, fragments thereof, or variants thereof. Such antibodies can 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 for the antigen-binding portions disclosed herein are 20-(74)-(74) (miratuzumab; bertuzumab), 20-2b-2b, 3F8, 74-(20)-(20) (miratuzumab; bertuzumab), 8H9, A33, AB-16B5, abagovomab, abciximab, abevacizumab, zolintuzumab), actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afucosylated antibodies, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG228, AMG820, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003 / SIM-BD0801 (cevastuzumab), APX005M, arcitumomab, ARX788, asclizumab, aselizumab, ASG-15ME, atezolizumab, atinumomab, ATL101, atorizumab (also called tocilizumab), atorlimomab, avelumab, B-701, bapineuzumab, basiliximab, batuximab, BAY1129980, BAY1187982, bectumomab, begelomab, belimumab, bendralizumab, beltilimumab, besilesomab, betalutin (177Lu-tetraxetan-tetomab), bevacizumab, BEVZ92 (bevacizumab biosimilar), bezlotoxumab, BGB-A317, BHQ880, BI836880, BI-505, bispecific antibodies, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, brodalumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bokokizumab, brentuximab vedotin, brevarex, briakinumab, brodalumab,Brolucizumab, Bronchiticizumab, C2-2b-2b, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, CBR96-doxorubicin immune complex, CBT124 (Bevacizumab), CC-90002, CDX-014, CDX-1401, Certolizumab pegol, Cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, Cituximab bogatox, Sixizumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, CM-24, Codrituzumab, Coltuximab ravtansine, Conatumumab, Consizumab, Cotara (Iodine I-131 deloxizumab biotin), cR6261, Crenezumab, DA-3111 (Trastuzumab biosimilar), Dacetuzumab, Daclizumab, Darolutamide, Dapirorizumab pegol, Daratumumab, Daratumumab enhertu (Daratumumab), Deruxtecan, Decitabine, Demcizumab, Denintuzumab mafodotin, Denosumab, Depatuxizumab, Depatuxizumab mafodotin, Deloxizumab biotin, Detumumab, DI-B4, Dinutuximab, Ziludimab, DKN-01, DMOT4039A, Doliromab aritox, Dorzigatox, DS-1123, DS-8895, Durigotuximab, Dupilumab, Durvalumab, Dusigitumab, Eculizumab, Eclizumab, Edobacumab, Edrecolomab, Efalizumab, Efungumab, Erdabulin, Ergemtuzumab, Ero1izumab, Ersilimomab, Emactuzumab, Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumorab situxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exvivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, FF-21101, FGFR2 antibody-drug conjugate, Fibromun, Figlatuzumab, Figitumumab,Filibuvimab, flanvotumab, fretikumab, fontolizumab, folarvab, folavilumab, FPA144, fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavrilimomab, gemtuzumab ozogamicin, gerilimuzumab, gebokizumab, gilenuximab, glenbatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hu14.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (clazukizumab), imalumab, IMC-CS4, IMC-D11, imusiromab, imigatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan), IMMU-114, ImmuTune IMP701 antagonist antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimumab, inotuzumab ozogamicin, intezumab, ipafricept, IPH4102, ipilimumab, ilatumab, isatuximab, istiratumab, itritumumab, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2 / L19TNF, labesimab, labesimab govitecan, LAG525, ramucirumab, rampalizumab, L-DOS47, remlizumab, remaresumab, rengedizumab, reldelimumab, leucozumab, rexsatumab, ribivab, rifatumumab vedotin, rigelimumab, rilotumumab tetraxetan, lintuzumab, ririlumab, LKZ145, rodessizumab, roquibetomab, rolbotuzumab mertansine, lucatumumab, rurelizumab pegol, rumiliximab, rumelesumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mabrilimomab, MB311, MCS-110, MEDI0562, MEDI-0639,MEDI0680, MEDI-3617, MEDI-551 (inebilizumab), MEDI-565, MEDI6469, mepolizumab, motavizumab, MGB453, MGD006 / S80880, MGD007, MGD009, MGD011, miratuximab, miratuximab-SN-38, minretomomab, milatuzumab soravtansine, mitsumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, molorizumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, naxitamab, namilumab, napumomab estafenatox, naratuximab, natalizumab, nebacumab, nesitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, NOV-10, obinutuzumab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimab, ofatumumab, olaratumab, orocizumab, omalizumab, OMP-131R10, OMP-305B83, ofatumumab, onartuzumab, opicinumab, oportuzumab monatox, oregovomab, orituximab, oteracizumab, otrexup, OX002 / MEN1309, ocrelizumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pancitumumab, pancitumumab-GEX, panobacumab, palsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pecilizumab, PF-05082566 (utomilumab), PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponesimod, prilixizumab, pritolaxaximab, pritumumab, PRO140, proxeninium, PSMA ADC, kirilizumab, lacotumomab, radretumab, ravulizumab, laropaxizumab, ramucirumab, ranibizumab, loxibacumab, refametinib, regorafenib, REGN1400, REGN2810 / SAR439684, reslizumab, RFM-203, RG7356,RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, Rilotumumab, Linukumab, Rituximab, RM-1929, RO7009789, Robatumumab, Loredanumab, Romosozumab, Lonartizumab, Roberizumab, Rupilizumab, Sacituzumab Govitecan, Samalizumab, SAR408701, SAR566658, Sarilumab, SAT012, Satumumab Pentide, SCT200, SCT400, SEA-CD40, Secukinumab, Seribantumab, Cetuximab, Sevirumab, SGN-CD19A, SGN-CD19B, SGN-CD33A, SGN-CD70A, SGN-LIV1A, Sibrotuzumab, Siparlimumab, Siluximab, Simtuzumab, Cipilizumab, Silkumab, Sofituzumab Vedotin, Soranizumab, Solitomab, Sonepcizumab, Sonituzumab, Stimulumab, Sulesomab, Subizumab, SYD985, SYM004 (Futuximab and Moduximab), Sym015, TAB08, Tabalumab, Tacatuzumab Tetraxetan, Tadocizumab, Talizumab, Tanezumab, Tanibirumab, Taptumomab Paptox, Tarextumab, TB-403, Tefibazumab, Telokin, Telimomab Arcitox, Tenatumomab, Teneliximab, Tepilizumab, Teplizumab, Tecidolumab, Teturomab, TG-1303, TGN1412, Thorium-227-Epratuzumab Conjugate, Tisilimumab, Tigatuzumab, Tildrakizumab, Tisotumab Vedotin, TNX-650, Tocilizumab, Tralizumab, Tosatoxumab, Tositumomab, Tobetumab, Tralokinumab, Trastuzumab, Trastuzumab Emtansine, TRBS07, TRC105, Tregalizumab, Tremelimumab, Treboglumab, TRPH011, TRX518, TSR-042, TTI-200.7, Tukotuzumab Celmoleukin, Tubilumab, U3-1565, U3-1784, Ublituximab, Urocuplumab, Urelumab, Ultuximab, Ustekinumab, Badastuximab Talirin, Vandortuzumab Vedotin, Bantictuzumab, Vanucizumab, Bapaliximab, Barilumab, Baterilizumab, VB6-845, Bedrizumab, Bertuzumab, Bepalimumab, Besencumab, Bisirilumab, Brolociximab,It can include the Fc domain of an antibody from the group including volociximab mafodotin, bortsomab, YYB-101, zalutumumab, zanolimumab, zatuximab, daclizumab, and zolimomab aritox.

[0210] Any of the systems disclosed herein can be utilized to regulate the expression or activity of an endogenous protein of a cell. Exemplary genes encoding the endogenous proteins disclosed herein are provided in Tables 4, 5, and 6. Exemplary genes related to specific diseases and disorders are provided in Tables 4 and 5. Examples of genes and polynucleotides related to signal transduction biochemical pathways are listed in Table 6. [Table 4] [Table 5-1] [Table 5-2] [Table 5-3] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8]

Table 6-9

Table 6-10

[0211] Any one of the systems and methods disclosed in this specification can be utilized to treat a target cell, target tissue, target condition, or target disease.

[0212] The target disease can be a virus, bacteria, and / or parasite infection, an inflammatory and / or autoimmune disease, or a neoplasm such as cancer and / or tumor.

[0213] The target cell can be a diseased cell. The diseased cell can 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 an affected subject. Exemplary diseases can include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart diseases, etc.

[0214] Using any one of the methods or compositions disclosed herein, various target cells can be killed. The target cells can include a wide variety of cell types. The target cells can be in vitro. The target cells can be in vivo. The target cells can be ex vivo. The target cells can be isolated cells. The target cells can be cells inside an organism. The target cells can be an organism. The target cells can be cells in cell culture. The target cells can be one of a collection of cells. The target cells can be mammalian cells or can be derived from mammalian cells. The target cells can be rodent cells or can be derived from rodent cells. The target cells can be human cells or can be derived from human cells. The target cells can be prokaryotic cells or can be derived from prokaryotic cells. The target cells can be bacterial cells or can be derived from bacterial cells. The target cells can be archaeal cells or can be derived from archaeal cells. The target cells can be eukaryotic cells or can be derived from eukaryotic cells. The target cells can be pluripotent stem cells. The target cells can be plant cells or can be derived from plant cells. The target cells can be animal cells or can be derived from animal cells. The target cells can be invertebrate cells or can be derived from invertebrate cells. The target cells can be vertebrate cells or can be derived from vertebrate cells. The target cells can be microbial cells or can be derived from microbial cells. The target cells can be fungal cells or can be derived from fungal cells. The target cells can be derived from a specific organ or tissue.

[0215] 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 (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. Clonal cells can include the progeny of the cell. The target cell can contain a target nucleic acid. The target cell can be in a living organism. The target cell can be a genetically modified cell. The target cell can be a host cell.

[0216] The target cell can be a totipotent stem cell, but in some embodiments of the present disclosure, the term "cell" may be used and may not refer to a totipotent stem cell. The target cell can be a plant cell, but in some embodiments of the present disclosure, the term "cell" may be used and 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 may not be able to differentiate into any other non-hematopoietic cells. The target cell may be able to develop into an entire organism. The target cell may or may not be able to develop into an entire organism. The target cell can be an entire organism.

[0217] The target cell can be a primary cell. For example, the primary cell culture can be passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15 times, or more. The cell can be a unicellular organism. The cell can grow in culture.

[0218] The target cells can be diseased cells. Diseased cells can have altered metabolism, gene expression, and / or morphological characteristics. Diseased cells can be cancer cells, diabetic cells, and apoptotic cells. Diseased cells can be cells from an affected subject. Exemplary diseases can include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart diseases, and the like.

[0219] When the target cells are primary cells, they can be obtained from an individual by any method. For example, white blood cells can be obtained by apheresis, leukapheresis, density gradient separation, and the like. 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 for the dispersion or suspension of the collected cells. Such a solution can generally be a balanced salt solution (e.g., physiological saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.) supplemented advantageously with fetal bovine serum or other naturally occurring factors, in combination with a low concentration of an acceptable buffer. The buffer can include HEPES, phosphate buffer, lactate buffer, and the like. The cells can be used immediately or (e.g., by freezing) stored. Frozen cells can be thawed and reused. The cells can be frozen in DMSO, serum, medium buffer (e.g., 10% DMSO, 50% serum, 40% buffered medium), and / or some other such common solutions used to store the cells at freezing temperature.

[0220] Non-limiting examples of cells that can be target cells include lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, cytokine induced killer (CIK) cells (see, for example, U.S. Patent Application Publication No. 20080241194); myeloid cells such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, dendritic cells; cells from the endocrine system including thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, oxyphil cells), adrenal (chromaffin cells), pineal (pinealocytes) cells; cells of the nervous system including glial cells (astrocytes, microglia), magnocellular neurosecretory cells, stellate cells, Betz cells, and cells of the pituitary gland (gonadotropin-producing cells, adrenocorticotropic hormone-producing cells, thyroid-stimulating hormone-producing cells, growth hormone-producing cells, mammotropin-secreting cells); cells of the respiratory system including lung cells (type I lung cells, type II lung cells), Clara cells, goblet cells, dust cells; cells of the circulatory system including cardiomyocytes, pericytes; cells of the digestive system including stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enterochromaffin cells, APUD cells, cells of the intestine including endocrine cells, liver (hepatocytes, Kupffer cells), cartilage / bone / muscle; cells of the skeletal system including osteoblasts, osteocytes, osteoclasts, cells of the teeth (cementoblasts, ameloblasts); cartilage cells including chondroblasts, chondrocytes; cells of the skin including hair follicles, keratinocytes, melanocytes (nevus cells); muscle cells including myocytes; cells of the urinary system including podocytes, juxtaglomerular cells, mesangial cells within / outside the glomerulus, renal proximal tubule brush border cells, macula densa cells; cells of the genital system including sperm, Sertoli cells, Leydig cells, oocytes; and adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of the fingernails and toenails, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, keratinized hair shaft cells, keratinized hair root sheath cells, hair root sheath cells of the Huxley layer, hair root sheath cells of the Henle layer, outer hair root sheath cells, hair matrix cells (stem cells).Wet stratified barrier epithelial cells, surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, basal cells (stem cells) of the corneal epithelium, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, urothelial cells (inner wall of the bladder and ureters), exocrine secretory epithelial cells, salivary gland mucous cells (secretion rich in polysaccharides), salivary gland serous cells (secretion rich in glycoprotein enzymes), von Ebner's gland cells of the tongue (cleaning of taste buds), mammary gland cells (lactation), lacrimal gland cells (tear secretion), ceruminous gland cells of the ear canal (earwax secretion), dark cells of eccrine sweat glands (glycoprotein secretion), clear cells of eccrine sweat glands (secretion of small molecules). Apocrine sweat gland cells (odor secretion, sex hormone sensitivity), Moll's gland cells of the eyelid (special sweat glands), sebaceous gland cells (secretion of high-lipid sebum), Bowman's gland cells of the nose (cleaning of the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secretion of semen components containing fructose for sperm motility), prostate gland cells (secretion of semen components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal fluid secretion), Littre's gland cells (mucus secretion), endometrial cells of the uterus (carbohydrate secretion), goblet cells of the respiratory and digestive tracts (mucus secretion), mucus cells of the inner wall of the stomach (mucus secretion), zymogenic cells of gastric glands (secretion of pepsinogen), acid-secreting cells of gastric glands (secretion of hydrochloric acid), acinar cells of the pancreas (secretion of bicarbonate and digestive enzymes), Paneth cells of the small intestine (secretion of lysozyme), type II pneumocytes of the lung (secretion of surfactant), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, growth hormone-producing cells, mammotropin-secreting cells, thyrotropin-producing cells, gonadotropin-producing cells, adrenocorticotropin-producing cells, intermediate pituitary cells, magnocellular neurosecretory cells, intestinal and airway cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, epithelioid cells, eosinophils, adrenal cells, chromaffin cells, Leydig cells of the testis, inner theca cells of follicles, luteal cells of ruptured follicles, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (secretion of renin), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands, and urogenital tract), kidney, type I pneumocytes (covering the lumen of the lung), pancreatic duct cells (centroacinar cells), non-linear duct cells (sweat glands, salivary glands, mammary glands, etc.), duct cells (seminal vesicles, prostate, etc.), epithelial cells covering a closed internal cavity, ciliated cells with a 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 precursor cells), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (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, stem cells and defined progenitor cells (various types) of the blood and immune system, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic nerve cells, sensory organ and peripheral neuron support cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, nurse cells, follicular cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, and interstitial kidney cells, but not limited to these.,

[0221] Particularly interesting are cancer cells. In some embodiments, the target cells are cancer cells. Non-limiting examples of cancer cells include acanthoma, acinar cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angioleiomyolipoma, angiosarcoma, appendiceal cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, bile duct cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, canceroma, carcinoma in situ, penile cancer, cancer of unknown primary site, carcinosarcoma, Castleman disease, central nervous system fetal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, fetal cancer, endodermal sinus tumor, endometrial cancer, endometrial carcinoma, endometrial tumor, enteropathy-associated T-cell lymphoma, epithelioblastoma, epithelioma, epithelioid sarcoma, erythroleukemia, esophageal cancer,esthesioneuroblastoma, Ewing family tumor, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget disease, fallopian tube cancer, encapsulated teratoma, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, glioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone,Glioblastoma multiforme, glioma, cerebral gliomatosis, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer, hemangioblastoma, perivascular cell tumor, 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 sarcoma, Kaposi sarcoma, kidney cancer, clathrin skin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, malignant acral lentiginous melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, corpus luteum tumor, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, bone malignant fibrous histiocytoma, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin, metastatic urothelial carcinoma, mixed Müllerian duct tumor, monocytic leukemia, oral cavity cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, fungating polyposis, fungating polyposis, myelodysplastic disorders, myelodysplastic syndrome, myeloid leukemia, myelosarcoma, myeloproliferative disorders, myxoma, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal cancer, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ophthalmic tumor, anaplastic astrocytoma, oligodendroglioma, tumor cell tumor, optic nerve sheath meningioma, oral cavity cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, low-grade ovarian tumor, breast Paget's disease, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epitheloid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, polyembryoma, 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, airway cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland cancer, secondary neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell cancer, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatic statinoma, soot wart, spinal cord tumor, spinal cord tumor, splenic marginal zone lymphoma, squamous cell carcinoma, stomach cancer, superficial 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, advanced lymphoma, testicular cancer, sheath tumor, pharyngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, urogenital neoplasm, uterine sarcoma, choroidal melanoma, vaginal cancer, Werner-Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenström macroglobulinemia, Warthin tumor, Wilms tumor, and cancer cells including combinations thereof are included. In some embodiments, the target cancer cells represent a subpopulation within a cancer cell population such as cancer stem cells. In some embodiments, the cancer is of the hematopoietic system such as lymphoma. The antigen can be a tumor-associated antigen.,

[0222] In some cases, the subject may have or 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, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplant rejection, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic neuropathy, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal & neuronal neuropathy, Baló disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture syndrome, granulomatosis withpolyangiitis, GPA), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing diseases, immunomodulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes (type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, Ménière's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), monoclonal gammopathy of undetermined significance (MGUS), Mouret's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (paroxysmal nocturnalhemoglobinuria, PNH), Parry-Romberg syndrome, Personnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, type II, type III autoimmune polyendocrine syndrome, rheumatoid polymyalgia, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, erythrocytapheresis, Raynaud phenomenon, reflex sympathetic dystrophy, Reiter syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren syndrome, sperm and testicular autoimmunity, stiff-man syndrome, subacute bacterial 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, vesiculobullous dermatosis, vitiligo, Waldenstrom macroglobulinemia (WM), Wegener granulomatosis (granulomatosis with polyangiitis (GPA)) can be mentioned.

[0223] In some cases, the autoimmune disease includes one or more members selected from the group consisting of rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE), myasthenia gravis, multiple sclerosis, scleroderma, Addison's disease, bullous pemphigoid, pemphigus vulgaris, Guillain - Barré syndrome, Sjögren's syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia (WM), chronic inflammatory demyelinating polyneuropathy (CIDP), Hashimoto's Encephalopathy (HE), Hashimoto's thyroiditis, Graves' disease, Wegener's granulomatosis, and antibody - mediated transplant rejection (e.g., in tissue transplantation such as kidney transplantation). In an example, the autoimmune disease can be type 1 diabetes, lupus, or rheumatoid arthritis.

[0224] In some cases, the target cells form a tumor (i.e., a solid tumor). Tumors treated by the methods of the present specification can result in stable tumor growth (e.g., one or more tumors do not increase in size by more than 1%, 5%, 10%, 15%, or 20% and / or do not metastasize). In some cases, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or longer. In some cases, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer. In some cases, the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years, or longer. 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 undetectable levels. 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 weeks, or longer 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 months, or longer 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 longer after treatment.

Example

[0225] Example 1: Modulating an endogenous target protein

[0226] Jacket cells were transduced with a lentivirus containing ef1a-dCas9-VPR-Q8 (hereinafter referred to as "Q8"), and then Q8-positive cells were sorted (for example, about 1 week later). Subsequently, the sorted Q8-positive cells were sorted again (for example, about 2 weeks later) to establish a dCas9-VPR-expressing cell line. Then, these cells (for example, about 200,000 cells per reaction) were transfected with sgRNA (for example, about 250 - 500 ng of sgRNA) using a transfection agent. The cells were seeded in a medium (for example, RPMI1640 + 10% FCS) (for example, in a 96-well plate) and incubated at 37°C for a certain period (for example, 48 - 72 hours) before gene expression analysis.

[0227] The gene expression of the target protein was measured using SYBR Green qPCR using the delta-delta Ct method with the primers provided in Table 7 (for example, forward (F) primer, reverse (R) primer).

[0228]

Table 7

[0229] a.ID3

[0230] The positions of the target polynucleotide sequences of multiple guide RNAs related to the gene encoding ID3 are shown in Figure 5A, and the sequences of multiple guide RNAs for ID3 are provided in Figure 5B (upper).

[0231] Upon activation by a system comprising Q8 and one of a plurality of guide RNAs for ID3 as disclosed herein, enhanced expression of endogenous ID3 in Jackett cells is shown in Figure 5B (bottom). In some cases, use of such a system promotes enhanced expression levels of endogenous ID3 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 control Jackett cells having a control, e.g., a control gRNA that binds to a different location in the ID3 gene or does not exhibit specific binding affinity for the ID3 gene.

[0232] b.c-Jun

[0233] The positions of the target polynucleotide sequences of a plurality of guide RNAs for the gene encoding c-Jun are shown in Figure 6A, and the sequences of a plurality of guide RNAs for c-Jun are provided in Figure 6B (top).

[0234] Upon activation by a system comprising Q8 and one of a plurality of guide RNAs for c-Jun as disclosed herein, enhanced expression of endogenous c-Jun in Jackett cells is shown in Figure 6B (bottom). In some cases, use of such a system promotes enhanced expression levels of endogenous c-Jun by about 19-fold (e.g., JUN_UP_gR94f), about 13-fold (e.g., JUN_UP_gR31f or JUN_UP_gR53f), or about 4-fold (e.g., JUN_UP_gR53f) compared to control Jackett cells having a control, e.g., a control gRNA that binds to a different location in the c-Jun gene or does not exhibit specific binding affinity for the c-Jun gene.

[0235] c.TBX21

[0236] The positions of the target polynucleotide sequences of a plurality of guide RNAs for the gene encoding TBX21 are shown in Figure 7A, and the sequences of a plurality of guide RNAs for TBX21 are provided in Figure 7B (top).

[0237] Upon activation by a system comprising Q8 and one of a plurality of guide RNAs for TBX21, as disclosed herein, enhanced expression of endogenous TBX21 in Jurkat cells is shown in FIG. 7B (bottom). In some cases, use of such a system promotes an enhanced expression level 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), compared to control Jurkat cells having a control, e.g., a control gRNA that binds to a different location of the TBX21 gene or does not exhibit specific binding affinity for the TBX21 gene.

[0238] d.IL-21

[0239] The positions of the target polynucleotide sequences of a plurality of guide RNAs for the gene encoding IL-21 are shown in FIG. 8A, and the sequences of a plurality of guide RNAs for IL-21 are provided in FIG. 8B (top).

[0240] Upon activation by a system comprising Q8 and one of a plurality of guide RNAs for IL-21, as disclosed herein, enhanced expression of endogenous IL-21 in Jurkat cells is shown in FIG. 8B (bottom). In some cases, use of such a system promotes an enhanced expression level of endogenous IL-21 by about 10-fold (e.g., IL-21_UP_gR8r), about 100-fold (e.g., IL-21_UP_gR16r), or about 1,000-fold (e.g., IL-21_UP_gR42f), compared to control Jurkat cells having a control gRNA (e.g., IL21_UP_gR92f) that binds to a different location of the IL-21 gene or does not exhibit specific binding affinity for the IL-21 gene.

[0241] TOX1

[0242] The positions of the target polynucleotide sequences of multiple guide RNAs related to the gene encoding TOX1 are shown in FIG. 9A, and the sequences of multiple guide RNAs against TOX1 are provided in FIG. 9B (top).

[0243] Upon expression by a system comprising Q8 and one of the multiple guide RNAs against TOX1 as disclosed herein, the expression of endogenous TOX1 in Jurkat cells is shown in FIG. 9B (bottom). In some cases, the use of such a system promotes a decreased expression level of endogenous TOX1, as shown by expression levels of endogenous TOX1 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) compared to control Jurkat cells having a control gRNA that binds to different positions of the TOX1 gene or does not exhibit specific binding affinity for the TOX1 gene.

[0244] f. TOX2

[0245] The positions of the target polynucleotide sequences of multiple guide RNAs related to the gene encoding TOX2 are shown in FIG. 10A, and the sequences of multiple guide RNAs against TOX2 are provided in FIG. 10B (top).

[0246] Upon expression by a system comprising Q8 and one of the multiple guide RNAs against TOX2 as disclosed herein, the expression of endogenous TOX2 in Jurkat cells is shown in FIG. 10B (bottom). In some cases, the use of such a system promotes a decreased expression level of endogenous TOX2, as shown by expression levels of endogenous TOX2 of about 0.1 (e.g., TOX2_3), about 0.2 (e.g., TOX2_1), about 0.5 (e.g., TOX2_4), or about 0.8 (e.g., TOX2_2) compared to control Jurkat cells having a control gRNA that binds to different positions of the TOX2 gene or does not exhibit specific binding affinity for the TOX2 gene.

[0247] g. SHIP1

[0248] The positions of the target polynucleotide sequences of multiple guide RNAs related to the gene encoding SHIP1 are shown in FIG. 11A, and the sequences of multiple guide RNAs for SHIP1 are provided in FIG. 11B (upper).

[0249] Upon expression by a system comprising Q8 and one of the multiple guide RNAs for SHIP1 as disclosed herein, the expression of endogenous SHIP1 in Jurkat cells is shown in FIG. 11B (lower). In some cases, the use of such a system promoted a decreased expression level of endogenous SHIP1, as shown by an expression level of endogenous SHIP1 of about 0.2 (e.g., SHIPgRr7, SHIPgR32f, or SHIPgR49r) or about 0.3 (e.g., SHIPgR60f) compared to control Jurkat cells having a control gRNA that binds to different positions of the SHIP1 gene or does not exhibit specific binding affinity for the SHIP1 gene.

[0250] h.B2M

[0251] The positions of the target polynucleotide sequences of multiple guide RNAs related to the gene encoding B2M are shown in FIG. 12A, and the sequences of multiple guide RNAs for B2M are provided in FIG. 12B (upper).

[0252] Upon expression by a system comprising Q8 and one of the multiple guide RNAs for B2M as disclosed herein, the expression of endogenous B2M in Jurkat cells is shown in FIG. 12B (lower). In some cases, the use of such a system promoted a decreased expression level of endogenous B2M, as shown by an expression level of endogenous B2M of about 0.7 (e.g., B2M_d_gR56f), about 0.6 (e.g., B2M_d_gR21r), or about 0.4 (e.g., B2M_gR21r) compared to control Jurkat cells having a control gRNA that binds to different positions of the B2M gene or does not exhibit specific binding affinity for the B2M gene.

[0253] i. BATF

[0254] The positions of the target polynucleotide sequences of multiple guide RNAs related to the gene encoding BATF are shown in Figure 13A, and the sequences of multiple guide RNAs against BATF are provided in Figure 13B (top).

[0255] Upon expression by the system, which includes Q8 and one of the multiple guide RNAs against BATF as disclosed herein, the expression of endogenous BATF in Jurkat cells is shown in Figure 13B (bottom). In some cases, the use of such a system promoted a decreased expression level of endogenous BATF, as shown by an expression level of endogenous BATF of 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) compared to control Jurkat cells having a control gRNA that binds to different positions of the BATF gene or does not exhibit specific binding affinity for the BATF gene.

[0256] j. SOCS1

[0257] The positions of the target polynucleotide sequences of multiple guide RNAs related to the gene encoding SOCS1 are shown in Figure 14A, and the sequences of multiple guide RNAs against SOCS1 are provided in Figure 14B (top).

[0258] Upon expression by a system comprising Q8 and one of a plurality of guide RNAs against SOCS1 as disclosed herein, the expression of endogenous SOCS1 in Jurkat cells is shown in Figure 14B (bottom). In some cases, use of such a system promoted a decreased expression level of endogenous SOCS1, as shown by an expression level of endogenous SOCS1 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) compared to control Jurkat cells having a control gRNA that binds to different positions of the SOCS1 gene or does not exhibit specific binding affinity for the SOCS1 gene.

[0259] k.TGFbR2

[0260] Transforming growth factor beta (TGFb) can be a multifaceted cytokine that can be secreted by tumor cells in the tumor micro-environment (TME). T cells can express a receptor for TGFb, such as TGFbR2. When TGFb binds to a TGFbR (e.g., TGFbR2), downstream signaling of the TGFbR can prevent T cells from differentiating into T cell subtypes (e.g., Th1 cells) and can reduce anti-tumor responses (e.g., IFNg secretion) and / or tumor cell cytotoxicity.

[0261] To screen for guide nucleic acid molecules that can be used to suppress the signaling or activity of TGFbR2, 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), the cells were harvested, stained with anti-TGFbR2-PE antibody, and expression was determined by flow cytometry. This screening was repeated (e.g., 3 times) to select the most effective gRNA. The top lead gRNAs were mainly located in regions 50 - 100 base pairs (bp) downstream of the TSS or 30 - 70 bp upstream of the TSS. The positions of the target polynucleotide sequences of multiple guide RNAs for the gene encoding TGFbR2 are shown in FIG. 15A.

[0262] Upon repression by a system comprising dCAS9-KRAB and one of a plurality of guide RNAs against TGFbR2, as disclosed herein, the expression of endogenous TGFbR2 in Jurkat cells is shown in FIG. 15B. In some cases, use of such a system promoted a decreased expression level of endogenous TGFbR2, as shown by an endogenous TGFbR2 expression level that was approximately 35% - approximately 20% lower than that of control Jurkat cells having a control gRNA that binds to different positions of the TGFbR2 gene or does not exhibit specific binding affinity for the TGFbR2 gene. Embodiments

[0263] The following non-limiting embodiments provide exemplary examples of the invention but do not limit the scope of the invention.

[0264] Embodiment 1. A system for regulating the expression or activity of a target protein in a cell, the system comprising

[0265] an effector portion capable of complexing with a target polynucleotide sequence in the cell, the effector portion being heterologous to the cell and the target polynucleotide sequence being endogenous to the cell, the effector portion

[0266] Complex formation results in at least about a 10% change in the expression or activity of the target protein compared to that in control cells, and the complex formation is sufficient to effect the change without editing the target polynucleotide sequence,

[0267] 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 / inhibitor of differentiation (ID), T-box transcription factor (TBX), and c-Jun,

[0268] Optionally,

[0269] (1) the target protein is TOX, and optionally, TOX comprises TOX1 or TOX2, and / or

[0270] (2) the target protein is SOCS, and optionally, SOCS comprises SOCS1, and / or

[0271] (3) the target protein is BATF, and / or

[0272] (4) the target protein is ID, and optionally, ID comprises ID3, and / or

[0273] (5) the target protein is TBX, and optionally, TBX is TBX21 (T-Bet), and / or

[0274] (6) the target protein is c-Jun, and / or

[0275] (7) an actuator moiety that can be activated for complex formation upon exposure of the cell to an external stimulus.

[0276] Embodiment 2. A system for regulating the expression or activity of a target protein in a cell, the system comprising:

[0277] An actuator portion capable of complexing with a target polynucleotide sequence in a cell, the actuator portion being heterologous to the cell and activatable for complexing upon exposure of the cell to an external stimulus, the target polynucleotide sequence being endogenous to the cell, the actuator portion comprising

[0278] Upon exposure, the actuator portion is activated for complexing to effect a change in the expression or activity of the target protein, and the complexing is sufficient to effect the change without editing the target polynucleotide sequence.

[0279] The target protein includes Src homology 2 domain-containing inositol phosphatase (SHIP) or beta-2-microglobulin (B2M), and TGF beta receptor (TGFbR).

[0280] Optionally,

[0281] (1) Upon exposure, the actuator portion is activated for complexing to effect at least about a 10% change in the expression or activity of the target protein compared to that in control cells, and / or

[0282] (2) The target protein is SHIP, and optionally, SHIP is SHIP1, and / or

[0283] (3) The target protein is B2M, and / or

[0284] (4) The target protein is TGFbR, and optionally, TGFBR is TGFbR2. A system.

[0285] Embodiment 3. Optionally further,

[0286] (1) The target protein is not a secreted protein and / or

[0287] (2) The target polynucleotide sequence (i) includes at least a part of the transcription start site (TSS) of the gene encoding the target protein, or (ii) is about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base away from the TSS of the gene encoding the target protein and / or

[0288] (3) The target polynucleotide sequence is about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base upstream of the TSS of the gene encoding the target protein and / or

[0289] (4) The target polynucleotide sequence is about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base downstream of the TSS of the gene encoding the target protein and / or

[0290] (5) The change is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more decrease in the expression or activity of the target protein compared to that in the control cells and / or

[0291] (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 compared to that in control cells, and / or

[0292] (7) The cell is an immune cell, and the change in the expression or activity of the target protein promotes the maintenance of the stem cell property of the immune cell, the survival of the immune cell, and / or the proliferation of the immune cell, and / or

[0293] (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

[0294] (9) The external stimulus is a ligand, the system includes a chimeric receptor polypeptide (receptor) that undergoes modification upon binding to the ligand, and the actuator part can be activated by receptor modification, and / or

[0295] (10) The activation of the actuator part includes (1) release of the actuator part from a substrate, or (2) modification of the actuator part, and / or

[0296] (11) The actuator part includes a nucleic acid-induced actuator part, and the system further includes a guide nucleic acid that complexes with the actuator part, and / or

[0297] (12) The guide nucleic acid includes guide ribonucleic acid (RNA), and / or

[0298] (13) The system includes two or more guide nucleic acids having complementarity to different target polynucleotide sequences, and / or

[0299] (14) The actuator part includes an effector domain configured to regulate the expression or activity of a target protein, and / or

[0300] (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 further optionally,

[0301] (a) the effector domain is a transcriptional activation domain, and / or

[0302] (b) the effector domain is a transcriptional repressor domain, and / or

[0303] (16) The actuator part includes a heterologous endonuclease or a variant thereof, and / or

[0304] (17) The modification is a conformational change or a chemical modification, and / or

[0305] (18) The cell is an immune cell, and / or

[0306] (19) The cell is a T cell or an NK cell, the system of Embodiment 1 or Embodiment 2.

[0307] Embodiment 4. A population of engineered cells, wherein each engineered cell of the population comprises the system of Embodiments 1-3.

[0308] Embodiment 5. A composition comprising the population of engineered cells of Embodiment 4, optionally further comprising a co-therapeutic agent.

[0309] Embodiment 6. 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 of a cell, wherein the target polynucleotide sequence: (i) comprises at least a part of the transcription start site (TSS) of the gene encoding the target protein, or (ii) is separated from the TSS of the gene encoding the target protein by about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base.

[0310] The target protein is selected from the group consisting of thymocyte selection-associated high mobility group box protein (TOX), suppressor of cytokine signaling (SOCS), Src homology 2 domain-containing inositol phosphatase (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 TGF-beta receptor (TGFbR).

[0311] Optionally,

[0312] (1) the guide nucleic acid molecule can recruit an effector moiety to the target polynucleotide sequence to regulate the expression or activity of the target protein, the system further comprises an effector moiety, and / or

[0313] (2) the effector moiety comprises a heterologous endonuclease or a variant thereof, and / or

[0314] (3) the gene encoding the target protein is endogenous to the cell, and / or

[0315] (4) the TSS is endogenous to the cell.

[0316] Embodiment 7. A system comprising an effector moiety 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 part of the transcription start site (TSS) of a gene encoding the target protein, or (ii) is about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base away from the TSS of the gene encoding the target protein,

[0317] 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), Src homology 2 domain-containing inositol phosphatase (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 TGF-beta receptor (TGFbR),

[0318] Optionally,

[0319] (1) the effector moiety comprises a heterologous endonuclease or a variant thereof, and / or

[0320] (2) the gene encoding the target protein is endogenous to the cell, and / or

[0321] (3) the TSS is endogenous to the cell.

[0322] Embodiment 8. A method for regulating the expression or activity of a target protein in a cell,

[0323] (a) Forming a complex in a cell that includes an actuator portion and a target polynucleotide sequence, wherein the actuator portion is heterologous to the cell and the target polynucleotide sequence is endogenous to the cell;

[0324] (b) Inducing at least a about 10% change in the expression or activity of the target protein in response to the forming, compared to that in control cells, wherein the forming of the complex is sufficient to effect the change without editing the target polynucleotide sequence;

[0325] The target protein includes 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 / inhibitor of differentiation (ID), T-box transcription factor (TBX), c-Jun, and TGF-beta receptor (TGFbR);

[0326] Optionally,

[0327] (1) the target protein is TOX, and optionally, TOX includes TOX1 or TOX2, and / or

[0328] (2) the target protein is SOCS, and optionally, SOCS includes SOCS1, and / or

[0329] (3) the target protein is BATF, and / or

[0330] (4) the target protein is ID, and optionally, ID includes ID3, and / or

[0331] (5) the target protein is TBX, and optionally, TBX is TBX21 (T-Bet), and / or

[0332] (6) The target protein is c-Jun and / or

[0333] (7) The target protein is TGFbR, and optionally, TGFbR is TGFbR2 and / or

[0334] (8) The target protein is not a cytokine, and optionally, the target protein is not a secreted protein and / or

[0335] (9) A system in which the actuator part can be activated for complex formation by exposing the cell to an external stimulus.

[0336] Embodiment 9. A method for regulating the expression or activity of a target protein in a cell, comprising:

[0337] (a) exposing the cell to an external stimulus to activate the actuator part to complex with the target polynucleotide sequence in the cell, wherein the actuator part is heterologous to the cell and the target polynucleotide sequence is endogenous to the cell;

[0338] (b) inducing a change in the expression or activity of the target protein in response to the complex formation, wherein the formation of the complex is sufficient to effect the change without editing the target polynucleotide sequence.

[0339] The target protein comprises Src homology 2 domain-containing inositol phosphatase (SHIP) or beta-2-microglobulin (B2M),

[0340] optionally,

[0341] (1) the induced change in the expression or activity of the target protein in response to the complex formation is at least about 10% compared to that in control cells and / or

[0342] (2) The target protein is SHIP, and optionally, SHIP is SHIP1, and / or

[0343] (3) The target protein is B2M, and / or

[0344] (4) The target polynucleotide sequence (i) includes at least a part of the transcription start site (TSS) of the gene encoding the target protein, or (ii) is about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base away from the TSS of the gene encoding the target protein, and / or

[0345] (5) The target polynucleotide sequence is about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base upstream of the TSS of the gene encoding the target protein, and / or

[0346] (6) The target polynucleotide sequence is about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base downstream of the TSS of the gene encoding the target protein, and / or

[0347] (7) The change is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more decrease in the expression or activity of the target protein as compared to that in the control cells, and / or

[0348] (8) The change is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more increase in the expression or activity of the target protein as compared to that in the control cells, and / or

[0349] (9) 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 proliferation of the immune cell, and / or

[0350] (10) 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 the target cell population, and / or reduced exhaustion of the immune cell, and / or

[0351] (11) The external stimulus is a ligand, the cell contains a chimeric receptor polypeptide (receptor) that undergoes modification upon binding to the ligand, and the actuator part can be activated by receptor modification, and / or

[0352] (12) The activation of the actuator part includes (1) release of the actuator part from the substrate, or (2) modification of the actuator part, and / or

[0353] (13) The actuator part includes a nucleic acid-induced actuator part, and the method further includes contacting the cell with a guide nucleic acid that complexes with the actuator part, and / or

[0354] (14) The guide nucleic acid includes guide ribonucleic acid (RNA), and / or

[0355] (15) The guide nucleic acid includes two or more guide nucleic acids having complementarity to different target polynucleotide sequences, and / or

[0356] (16) The actuator part includes an effector domain configured to regulate the expression or activity of the target protein, and / or

[0357] (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 further optionally,

[0358] (a) The effector domain is a transcriptional activation domain, and / or

[0359] (b) The effector domain is a transcriptional repressor domain, and / or

[0360] (18) The actuator part includes a heterologous endonuclease or a variant thereof, and / or

[0361] (19) The modification is a conformational change or a chemical modification, and / or

[0362] (20) The cell is an immune cell, and / or

[0363] (21) The cell is a T cell or an NK cell, and / or

[0364] (22) Further comprising administering the cell to a subject in need thereof, and / or

[0365] (23) The cell is autologous or allogeneic to the subject, and / or

[0366] (24) Further comprising administering a co-therapeutic agent to the subject, and / or

[0367] (25) The subject is a mammal and / or

[0368] (26) The subject is a human, method.

[0369] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Without departing from the present invention, numerous variations, modifications, and substitutions will occur to those skilled in the art. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative ratios shown herein, which depend on various conditions and variables. It should be understood that various alternative forms of the embodiments of the present invention described herein may be used in practicing the present disclosure. Accordingly, the present invention is also intended to embrace any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be embraced thereby.

Claims

1. 1. A system for regulating the expression or activity of a target protein in a cell, 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 the target polynucleotide sequence is endogenous to the cell; complexing results in a change in expression or activity of the target protein of at least about 10% compared to that in a control cell, and complexing is sufficient to effect the change without editing the target polynucleotide sequence; The system, 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 / inhibitor of differentiation (ID), T-box transcription factor (TBX), and c-Jun.

2. The system of 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.

5. The system of claim 4 , wherein the SOCS comprises SOCS1.

6. The system of claim 1 , wherein the target protein is BATF.

7. The system of claim 1 , wherein the target protein is the ID.

8. The system of claim 7 , wherein the ID includes ID3.

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

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

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

12. 10. The system of claim 1, wherein the actuator portion is activatable for complexation upon exposure of the cell to an external stimulus.

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

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

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

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

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

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

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

20. The system of claim 1 , wherein the target protein is not a cytokine.

21. The system of claim 1 , wherein the target protein is not a secreted protein.

22. 2. The system of claim 1, 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 separated from the TSS of a gene encoding the target protein by about 10,000 to about 5,000 bases, about 5,000 to about 4,000 bases, about 4,000 to about 3,000 bases, about 3,000 to about 2,000 bases, about 2,500 to about 2,000 bases, about 2,000 to about 1,500 bases, about 1,500 to about 1,000 bases, about 1,000 to about 500 bases, or about 500 to 1 base.

23. 2. The system of claim 1, wherein the target polynucleotide sequence is about 10,000 to about 5,000 bases, about 5,000 to about 4,000 bases, about 4,000 to about 3,000 bases, about 3,000 to about 2,000 bases, about 2,500 to about 2,000 bases, about 2,000 to about 1,500 bases, about 1,500 to about 1,000 bases, about 1,000 to about 500 bases, or about 500 to 1 base upstream of the TSS of the gene encoding the target protein.

24. 2. The system of claim 1, wherein the target polynucleotide sequence is about 10,000 to about 5,000 bases, about 5,000 to about 4,000 bases, about 4,000 to about 3,000 bases, about 3,000 to about 2,000 bases, about 2,500 to about 2,000 bases, about 2,000 to about 1,500 bases, about 1,500 to about 1,000 bases, about 1,000 to about 500 bases, or about 500 to 1 base downstream of the TSS of the gene encoding the target protein.

25. 2. The system of claim 1, wherein the alteration is at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more decrease in expression or activity of the target protein compared to that in the control cell.

26. 2. The system of claim 1, wherein the alteration is at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more increase in expression or activity of the target protein compared to that in the control cell.

27. 2. The system of claim 1, wherein the cell is an immune cell, and the change in expression or activity of the target protein promotes maintenance of stemness of the immune cell, survival of the immune cell, and / or proliferation of the immune cell.

28. 2. The system of claim 1, wherein the cell is an immune cell and the alteration 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.

29. the external stimulus is a ligand, and the system comprises:

14. The system of claim 13, comprising a chimeric receptor polypeptide (receptor) that undergoes modification upon binding to the ligand, wherein the actuator portion can be activated by receptor modification.

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

31. 10. The system of claim 1, wherein the actuator moiety comprises a nucleic acid-derivatized actuator moiety, and the system further comprises a guide nucleic acid complexed with the actuator moiety.

32. 32. The system of Claim 31, wherein the guide nucleic acid comprises a guide ribonucleic acid (RNA).

33. 10. The system of claim 1, comprising two or more guide nucleic acids having complementarity to different target polynucleotide sequences.

34. 10. The system of claim 1, wherein the actuator portion comprises an effector domain configured to modulate the expression or activity of the target protein.

35. 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, and a transcriptional repressor domain.

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

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

38. The system of claim 1 , wherein the actuator portion comprises a heterologous endonuclease or a variant thereof.

39. 30. The system of claim 29, wherein the modification is a conformational change or a chemical modification.

40. The system of claim 1 , wherein the cell is an immune cell.

41. The system of claim 1 , wherein the cell is a T cell or an NK cell.

42. A population of engineered cells, wherein each engineered cell of the population comprises a system according to any one of claims 1 to 41.

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

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

45. 1. A system comprising a guide nucleic acid molecule designed to bind to a target polynucleotide sequence to regulate 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 separated from the TSS of the gene encoding the target protein by about 10,000 to about 5,000 bases, about 5,000 to about 4,000 bases, about 4,000 to about 3,000 bases, about 3,000 to about 2,000 bases, about 2,500 to about 2,000 bases, about 2,000 to about 1,500 bases, about 1,500 to about 1,000 bases, about 1,000 to about 500 bases, or about 500 to 1 base; The system, 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), Src homology 2 domain-containing inositol phosphatase (SHIP), basic leucine zipper transcription factor ATF-like (BATF), beta-2-microglobulin (B2M), DNA-binding / inhibitor of differentiation (ID), T-box transcription factor (TBX), c-Jun, and transforming growth factor beta receptor (TGFbR).

46. 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 regulate expression or activity of the target protein, and the system further comprises the actuator moiety.

47. 1. A system comprising an actuator moiety capable of binding to a target polynucleotide sequence to regulate 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 separated from a TSS of a gene encoding the target protein by about 10,000 to about 5,000 bases, about 5,000 to about 4,000 bases, about 4,000 to about 3,000 bases, about 3,000 to about 2,000 bases, about 2,500 to about 2,000 bases, about 2,000 to about 1,500 bases, about 1,500 to about 1,000 bases, about 1,000 to about 500 bases, or about 500 to 1 base; The system, 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), Src homology 2 domain-containing inositol phosphatase (SHIP), basic leucine zipper transcription factor ATF-like (BATF), beta-2-microglobulin (B2M), DNA-binding / inhibitor of differentiation (ID), T-box transcription factor (TBX), c-Jun, and transforming growth factor beta receptor (TGFbR).

48. 47. The system of claim 46, wherein the actuator portion comprises a heterologous endonuclease or a variant thereof.

49. 46. ​​The system of claim 45, wherein the gene encoding the target protein is endogenous to the cell.

50. The system of any one of claims 45 to 49, wherein the TSS is endogenous to the cell.

51. 1. A method for modulating the expression or activity of a target protein in a cell, comprising: (a) forming in the cell a complex comprising an actuator portion and a target polynucleotide sequence, wherein the actuator portion is heterologous to the cell and the target polynucleotide sequence is endogenous to the cell; (b) inducing, in response to said forming, a change in expression or activity of the target protein of at least about 10% compared to that in a control cell, wherein forming a complex is sufficient to effect said change without editing the target polynucleotide sequence; The method, 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 / inhibitor of differentiation (ID), T-box transcription factor (TBX), c-Jun, and transforming growth factor beta receptor (TGFbR).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

66. 52. The method of claim 51 , wherein the actuator moiety is activatable for complexation upon exposure of the cell to an external stimulus.

67. 1. A method for modulating the expression or activity of a target protein in a cell, 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 the target polynucleotide sequence is endogenous to the cell; (b) inducing a change in expression or activity of the target protein in response to the complexing, wherein formation of the complex is sufficient to effect the change without editing the target polynucleotide sequence; The method, wherein the target protein comprises Src homology 2 domain-containing inositol phosphatase (SHIP) or beta-2-microglobulin (B2M).

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

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

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

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

72. 52. The method of claim 51, 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 separated from the TSS of a gene encoding the target protein by about 10,000 to about 5,000 bases, about 5,000 to about 4,000 bases, about 4,000 to about 3,000 bases, about 3,000 to about 2,000 bases, about 2,500 to about 2,000 bases, about 2,000 to about 1,500 bases, about 1,500 to about 1,000 bases, about 1,000 to about 500 bases, or about 500 to 1 base.

73. 52. The method of claim 51, wherein the target polynucleotide sequence is about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base upstream of the TSS of the gene encoding the target protein.

74. 52. The method of claim 51, wherein the target polynucleotide sequence is about 10,000 to about 5,000 bases, about 5,000 bases to about 4,000 bases, about 4,000 bases to about 3,000 bases, about 3,000 bases to about 2,000 bases, about 2,500 bases to about 2,000 bases, about 2,000 bases to about 1,500 bases, about 1,500 bases to about 1,000 bases, about 1,000 bases to about 500 bases, or about 500 bases to 1 base downstream of the TSS of the gene encoding the target protein.

75. 52. The method of claim 51 , wherein the alteration is at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more decrease in expression or activity of the target protein compared to that in the control cell.

76. 52. The method of claim 51 , wherein the alteration is at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more increase in expression or activity of the target protein compared to that in the control cell.

77. 52. The method of claim 51 , wherein the cell is an immune cell and the alteration in expression or activity of the target protein promotes maintenance of stemness of the immune cell, survival of the immune cell, and / or proliferation of the immune cell.

78. 52. The method of claim 51 , wherein the cell is an immune cell and the alteration in 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 decreased exhaustion of the immune cell.

79. The external stimulus is a ligand, and the cell 68. The method of claim 67, comprising a chimeric receptor polypeptide (receptor) that undergoes modification upon binding to the ligand, wherein the actuator portion is capable of activation upon receptor modification.

80. 52. The method of claim 51 , wherein activation of the actuator moiety comprises (1) release of the actuator moiety from a substrate, or (2) modification of the actuator moiety.

81. 52. The method of Claim 51, wherein the actuator moiety comprises a nucleic acid-derivatized actuator moiety, and the method further comprises contacting the cell with a guide nucleic acid that complexes with the actuator moiety.

82. 82. The method of Claim 81, wherein the guide nucleic acid comprises a guide ribonucleic acid (RNA).

83. 82. The method of Claim 81, wherein the guide nucleic acid comprises two or more guide nucleic acids having complementarity to different target polynucleotide sequences.

84. 52. The method of Claim 51, wherein the actuator portion comprises an effector domain configured to modulate the expression or activity of the target protein.

85. 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, and a transcriptional repressor domain.

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

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

88. 52. The method of claim 51 , wherein the actuator portion comprises a heterologous endonuclease or a variant thereof.

89. 81. The method of claim 80, wherein the modification is a conformational change or a chemical modification.

90. 52. The method of claim 51, wherein the cell is an immune cell.

91. 52. The method of claim 51, wherein the cell is a T cell or an NK cell.

92. The composition described in claim 43 for administration to a subject in need thereof.

93. 93. The composition of claim 92, wherein the cells are autologous or allogeneic to the subject.

94. 93. The composition of claim 92, wherein the subject is a mammal.

95. 93. The composition of claim 92, wherein the subject is a human.

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