Systems and methods for cellular programming - Patents.com

Abstract

Provided herein are systems for regulating gene expression to differentiate cells, such as immune cells, methods of use, and engineered cells.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 311,122, filed February 17, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] Heterologous proteins and / or nucleic acid molecules can be utilized to induce desired responses in cells. Heterologous proteins and / or nucleic acid molecules can regulate genes of interest (e.g., transgenes and / or endogenous genes) to program cells (e.g., differentiate, dedifferentiate). In some cases, endonuclease-based techniques (e.g., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins or "CRISPR / Cas") are employed to manipulate polynucleotide sequences, their genetic modifications, and / or their expression levels. For example, CRISPR / Cas techniques can be characterized by their versatility and easy programmability, and can be used to facilitate genome editing across a variety of species. Summary of the Invention

[0003] The present disclosure provides a method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising contacting the cell with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in concert to sequentially induce a plurality of distinct modulations of the target gene, each of the plurality of distinct modulations being required but individually insufficient to achieve the desired expression and / or activity profile of the target gene, and wherein the plurality of gating units (i) act to induce a first distinct modulation of the plurality of distinct modulations upon activation of the heterologous genetic circuit. and (ii) a second gating unit activatable to induce a second individual regulation of the plurality of individual regulations upon activation of the heterologous genetic circuit, the second individual regulation being induced after the first individual regulation such that the first individual regulation and the second individual regulation both increase or both decrease the expression and / or activity level of the target gene in the cell, wherein upon contacting, the plurality of gating units cooperate to achieve a desired expression and / or activity profile of the target gene in the cell.

[0004] The present disclosure also provides a system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in concert to sequentially induce a plurality of distinct modulations of the target gene, each of the plurality of distinct modulations being required but individually insufficient to achieve the desired expression and / or activity profile of the target gene, and wherein the plurality of gating units are (i) activatable to induce a first distinct modulation of the plurality of distinct modulations upon activation of the heterologous genetic circuit. The heterologous genetic circuit comprises a first gating unit and (ii) a second gating unit that is activatable to induce a second individual regulation of the plurality of individual regulations upon activation of the heterologous genetic circuit, the second individual regulation being induced after the first individual regulation such that the first individual regulation and the second individual regulation both increase or both decrease expression and / or activity levels of the target gene in the cell, and upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to achieve a desired expression and / or activity profile of the target gene in the cell.

[0005] The present disclosure also provides a method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising: contacting the cell with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units cooperate to sequentially induce a plurality of individual regulation of the target gene, each of the plurality of individual regulation being required but not individually sufficient to achieve a desired expression and / or activity profile of the target gene, the plurality of gating units comprising: (i) a first gating unit that is activatable upon activation of the heterologous genetic circuit to induce a first individual regulation of the plurality of individual regulation; and (ii) a second gating unit that is activatable to induce disruption of the first gating unit that is activated, the second individual regulation being induced after the first individual regulation to attenuate the first individual regulation, wherein upon contacting, the plurality of gating units cooperate to achieve a desired expression and / or activity profile of the target gene in the cell.

[0006] The present disclosure also provides a system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in concert to sequentially induce a plurality of distinct modulations of the target gene, each of the plurality of distinct modulations being required but individually insufficient to achieve the desired expression and / or activity profile of the target gene, the plurality of gating units (i) inducing a first distinct modulation of the plurality of distinct modulations upon activation of the heterologous genetic circuit, and (ii) a second gating unit that is activatable to induce disruption of the activated first gating unit, the second gating unit inactivating to induce a second individual regulation of the plurality of individual regulations, the second individual regulation being induced after the first individual regulation to attenuate the first individual regulation, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to achieve a desired expression and / or activity profile of target genes in the cell.

[0007] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material. [Brief description of the drawings]

[0008] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG.") in which:

[0009] [Figure 1] FIG. 1 illustrates the autologous T cell development pathway within a circuit model. [Diagram 2] FIG. 1 shows genes known to affect T cell differentiation at various stages during T cell development. [Diagram 3] FIG. 1 illustrates how heterologous genetic circuits (HGCs) function at various stages for further T cell development, compared to the theorized function of autologous T cell developmental pathways. [Figure 4] FIG. 1 illustrates a guide nucleic acid molecule (e.g., gRNA) in accordance with the disclosed systems and methods. [Figure 5A]

[0023] Figure 5 illustrates examples of homologous genetic circuits (HGCs) according to the systems and methods of the present disclosure. Figure 5A represents T cell HGC1. [Figure 5B] 5A-5C are diagrams illustrating examples of homologous genetic circuits (HGCs) in accordance with the systems and methods of the present disclosure. FIG. 5B depicts T cell HGC2. [Figure 5C] 5A-5C are diagrams illustrating examples of homologous genetic circuits (HGCs) in accordance with the systems and methods of the present disclosure. [Figure 5D] 5A-5D are diagrams illustrating examples of homologous genetic circuits (HGCs) according to the systems and methods of the present disclosure. FIG. 5D represents T cell HGC4. [Figure 5E]

[0023] Figure 5A illustrates an example of a homologous genetic circuit (HGC) in accordance with the systems and methods of the present disclosure. Figure 5E depicts T cell HGC5. [Figure 5F]

[0023] Figure 5A illustrates an example of a homologous genetic circuit (HGC) in accordance with the systems and methods of the present disclosure. Figure 5F represents T cell HGC6. [Figure 5G]

[0023] Figure 5G illustrates an example of a homologous genetic circuit (HGC) in accordance with the systems and methods of the present disclosure. Figure 5G represents T cell HGC7. [Figure 5H]

[0023] Figure 5 illustrates an example of a homologous genetic circuit (HGC) in accordance with the systems and methods of the present disclosure. Figure 5H represents T cell HGC8. [Figure 5I]

[0023] Figure 5I illustrates an example of a homologous genetic circuit (HGC) in accordance with the systems and methods of the present disclosure. Figure 5I represents T cell HGC9. [Figure 6] FIG. 13 is a diagram depicting intermediate T cell stages induced by HGCs overlaid on the conventional T cell developmental pathway, used for reference. [Figure 7] Schematic diagram of a homologous genetic circuit. An activating moiety can initiate the circuit and activate a gating unit. A gating unit is composed of a gate moiety and / or a gene regulatory moiety. [Figure 8] FIG. 1 depicts an exemplary heterologous genetic circuit. [Figure 9] FIG. 1 shows scatter plots (i.e., volcano plots) at days 3, 5, and 9 to identify one or more heterologous genetic circuits in induced pluripotent stem cells (iPSCs) differentiated into hematopoietic progenitor cells. [Figure 10A] Figure 10 shows an example of the analytical data of hematopoietic progenitor cell markers used to generate the scatter plots of Figure 9. Figure 10A shows the results for the cell marker CD34+. The points on the left and right represent replicate samples for each condition. The center point represents the average value. [Figure 10B] FIG. 10B shows an example of the hematopoietic progenitor cell marker analysis data used to generate the scatter plots in FIG. 9. FIG. 10B shows the results for the cell marker CD43+. The dots on the left and right represent replicate samples for each condition. The center dot represents the average value. [Figure 10C] FIG. 10C shows an example of the hematopoietic progenitor cell marker analysis data used to generate the scatter plots in FIG. 9. FIG. 10C shows the results for the cell marker CD45+. The dots on the left and right represent replicate samples for each condition. The center dot represents the average value. [Figure 10D]FIG. 10C shows an example of the hematopoietic progenitor cell marker analysis data used to generate the scatter plots of FIG. 9. FIG. 10D shows the results for the combination of cell markers CD34+ / CD43- / CD45+. The points on the left and right represent replicate samples for each condition. The center point represents the average value. The points on the left and right represent replicate samples for each condition. The center point represents the average value. [Figure 11A] Figure 11 shows that heterologous genetic circuits generate substantially more hematopoietic progenitor cells after 5 days in culture compared to directed differentiation.Figure 11A shows cell marker results for CD34+ and CD45+ markers. [Figure 11B] (B) shows that heterologous genetic circuits generate substantially more hematopoietic progenitor cells after 5 days in culture compared to directed differentiation. (C) shows additional cell marker results for CD34+, CD43+, and CD45+ markers. [Figure 11C] Figure 11C shows that heterologous genetic circuits generate substantially more hematopoietic progenitors after 5 days in culture compared to directed differentiation. [Figure 11D] (D) shows that heterologous genetic circuits generate substantially more hematopoietic progenitors after 5 days in culture compared to directed differentiation. (E) shows a summary of results from replicate experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0011] As used in this specification and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. For example, the term "a gate unit" includes a plurality of gate units.

[0012] The term "about" or "approximately" generally means within an acceptable error range in 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., on the limitations of the measurement system. For example, "about" may mean within one or more standard deviations, as is customary in the art. Alternatively, "about" may mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0013] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives.

[0014] definition The terms "genetic circuit," "biological circuit," or "circuit," as used interchangeably herein, generally refer to a collection of molecular components (e.g., biological materials such as polypeptides and / or polynucleotides, non-biological materials, etc.) operably linked (e.g., operating simultaneously, operating sequentially, etc.) according to a circuit design. The collection of molecular components may be capable of providing one or more specific outputs (e.g., regulation of one or more genes) within a cell in response to one or more inputs (e.g., a single input or multiple inputs). Such one or more inputs may be sufficient to induce the molecular components of the genetic circuit to provide one or more specific outputs. For example, a genetic circuit may include one or more molecular switches that are activatable by one or more inputs (Figure 7).

[0015] A genetic circuit may be a controllable gene expression system that includes an assembly of biological parts that function together (e.g., simultaneously, sequentially, etc.) as a logical function. A genetic circuit may include a plurality of gating units, where at least one gating unit of the plurality of gating units is activatable by an activating moiety (e.g., a heterologous input to a cell) that activates other gating units of the plurality of gating units (e.g., simultaneously at once, sequentially in a cascade manner, etc.) (FIG. 7). For example, at least one gating unit of the plurality of gating units may be activatable (e.g., directly or indirectly) by another gating unit of the plurality of gating units to (i) regulate the expression or activity level of one or more target genes, (ii) activate at least one other gating unit of the plurality of gating units, and / or (ii) inactivate at least one other gating unit of the plurality of gating units, thereby collectively regulating the expression and / or activity level of one or more target genes in a desired manner, as predetermined by the design of the genetic circuit (FIG. 7). As used herein, the terms "heterologous genetic circuit," "HGC," "cellular algorithm," or "cellgorithm" may be used interchangeably.

[0016] The term "gate unit" referred to herein generally refers to a portion of a genetic circuit that can control gene regulation by functioning similarly to a logic gate, which can control the flow of information and allow the circuit to multiplex decisions at different points. More specifically, the term refers to a nucleic acid that encodes a genetic switch and a transcription / translation regulatory region or set of regions on which the genetic switch acts. The input to the gate unit can be an activation portion and / or another gate unit. The output to the gate unit can be used to activate another gate unit, to inactivate another gate unit, to affect a target gene, and / or any combination of the above. For example, a gate unit can be composed of multiple gate portions and / or multiple gene regulatory portions (Figure 7).

[0017] The term "activating moiety" referred to herein generally refers to a moiety that can activate multiple genetic circuits and / or multiple gate units. The activating moiety can be a heterologous input to the cell. In some cases, the activating moiety can include, but is not limited to, a guide nucleic acid molecule (e.g., gRNA) or other nucleic acid, polypeptide, polynucleotide, small molecule, light, or a combination thereof. For example, the activating moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., Cas protein) to bind to a polynucleotide sequence of a gate moiety to be inactivated (e.g., a plasmid encoding another guide nucleic acid molecule) and activate such a gate moiety that can target one or more gene regulatory moieties (e.g., induce expression of a functional form of an additional guide nucleic acid molecule).

[0018] The term "gate moiety" referred to herein generally refers to a moiety that can affect the function of a gene regulatory moiety in a gate unit. A gate moiety can activate and / or deactivate a gene regulatory moiety. For example, a gate moiety can regulate the expression of a gene regulatory moiety by editing a nucleic acid sequence, thereby activating or deactivating the gene regulatory moiety. For example, a gate moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of a gene regulatory moiety (e.g., a plasmid encoding another guide nucleic acid molecule) and activates a gene regulatory moiety that can target one or more endogenous genes of a cell (e.g., induces expression of a functional form of another guide nucleic acid molecule). Alternatively or additionally, a gate moiety can activate and / or deactivate another gate unit of a genetic circuit (Figure 7). For example, a gate moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of another gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) that is to be inactivated and activates another gate moiety (e.g., induces expression of a functional form of another guide nucleic acid molecule). In another example, a gate moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of another gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) that is activated and inactivates the other gate moiety (e.g., reduces expression of a functional form of the other guide nucleic acid molecule).

[0019] The term "gene regulation moiety" or "gene editing moiety", which are used interchangeably herein, generally refers to a moiety that can regulate the expression and / or activity profile of a nucleic acid sequence or protein, whether exogenous or endogenous to a cell (Figure 7). For example, a gene editing moiety can regulate the expression of a gene by editing a nucleic acid sequence (e.g., CRISPR-Cas, zinc finger nuclease, TALEN, or siRNA). In some cases, a gene editing moiety can regulate the expression of a gene by editing a genomic DNA sequence. In some cases, a gene editing moiety can regulate the expression of a gene by editing an mRNA template. Editing a nucleic acid sequence can in some cases change the template underlying gene expression (e.g., CRISPR-Cas inspired RNA targeting system). Alternatively, a gene editing moiety can suppress the translation of a gene (e.g., Cas13).

[0020] Alternatively or additionally, the gene editing moiety may be capable of regulating the expression or activity of a gene by specifically binding to a target sequence (or a target sequence within a gene) operably linked to the gene and regulating the production of mRNA from DNA, such as chromosomal DNA or cDNA. For example, the gene editing moiety may recruit or include 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 gene editing moiety may itself bind to DNA and regulate transcription by physical occlusion, for example, preventing proteins such as RNA polymerase and other associated proteins from assembling on the DNA template. The gene editing moiety may regulate the expression of a gene at the translational level, for example, by regulating the production of a protein from an mRNA template. In some cases, the gene editing moiety may regulate gene expression by affecting the stability of the mRNA transcript. In some cases, the gene editing moiety may regulate a gene by epigenetic editing (e.g., Cas12).

[0021] In some cases, the plasmid can code for a non-functional form of the gene editing portion. The plasmid can be activated to express a functional form of the gene editing portion, for example, by activating a functional gate portion. For example, the gene editing portion can code for a non-functional form of a guide nucleic acid molecule that can otherwise bind to a target gene of a cell. When a functional gate portion (e.g., another guide nucleic acid molecule complexed with a Cas protein) is bound to the plasmid, the plasmid can be edited to allow expression of a functional form of the gene editing portion (e.g., a functional form of a guide nucleic acid molecule that specifically binds to a target gene of a cell) to allow regulation of the target gene in the cell.

[0022] Thus, the gene regulatory portion can include a nucleic acid molecule (e.g., a guide nucleic acid molecule that forms a complex with an endonuclease, such as a Cas protein). Alternatively or additionally, the gene regulatory portion can include an endonuclease or be operably linked to an endonuclease. The endonuclease can be an enzyme that cleaves phosphodiester bonds in a polynucleotide chain. The endonuclease can include a restriction endonuclease that cleaves DNA at a specific site without damaging the bases. The restriction endonuclease can include type I, type II, type III, and type IV endonucleases, and further include subtypes thereof. In some cases, the endonuclease is Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12, Cas12a(Cpf1), Cas12b(C2c1), Cas12c(C2c3), Cas12d(CasY), Cas12e(CasX), The endonuclease may be Cas12f (Cas14 or C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas13 (C2c2), Cas13b, Cas13c, Cas13d, Cas13x.1, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx1O, Csx11, Csf1, Csn2. The endonuclease may be a dead endonuclease that exhibits reduced cleavage activity. For example, the endonuclease may be a nuclease-inactivated Cas, such as dCas (e.g., dCas9).

[0023] The gene regulatory portion can be a transcriptional modulator system (e.g., a gene repressor complex or a gene activator complex). For example, the gene regulatory portion can be a gene repressor complex that includes a dCas protein operably linked (e.g., fused) to a transcriptional repressor. Non-limiting examples of transcriptional repressors include KRAB, SID, MBD2, MBD3, DNMT1, DNMT2A, DNMT3A, DNMT3B, DNMT3L, Mecp2, FOG1, ROM2, LSD1, ERD, SRDX repression domain, Pr-SET7 / 8, SUV4-20H1, RIZ1, JMJD2A, JHDM3A, JMJD2B, JMJD2C, GASC1, JMJD2D, JHDM3A, JHDM3B, JHDM3L, JHDM3B ... Examples of such proteins include ARID1A, RBP2, JARID1B / PLU-1, JARIDIC / SMCX, JARIDID / SMCY, HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, M.Hhal, METI, DRM3, ZMET2, CMT1, CMT2, lamin A, and lamin B. Alternatively, the gene regulatory moiety can be a gene activator complex comprising a dCas protein operably linked (e.g., fused) to a transcriptional activator. Non-limiting examples of transcriptional activators include VP16, VP64, VP48, VP160, p65 subdomain, SET1A, SET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, SYMD2, NSD1, JHDM2a, JHDM2b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, p1 60, CLOCK, TET1CD, TET1, DME, DML1, DML2, and ROS1.

[0024] In some cases, the gene regulatory portion has an enzymatic activity that modifies the target gene without cleaving the target gene. Modification of the target gene can result in, for example, an epigenetic modification that can modify gene expression and / or activity levels. Examples of enzymatic activities that can be provided by the gene regulatory portion include nuclease activity, such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity, such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3, ZMET2, CMT1, CMT2), demethylase (e.g., Ten-Eleven Examples of the activity of the enzyme include, but are not limited to, demethylase activity such as that provided by TET dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1), DNA repair activity, DNA damage activity, deamination activity such as that provided by deaminases (e.g., cytosine deaminase enzymes such as APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity such as that provided by integrases and / or resolvases (e.g., Gin invertases such as GinH106Y, a hyperactive mutant of Gin invertase, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase), transposase activity, recombinase activity such as that provided by recombinases (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0025] Unless otherwise specified or clear from the context, the terms "polynucleotide", "oligonucleotide", or "nucleic acid", as used interchangeably herein, generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof, in either single-stranded, double-stranded, or multiple-stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may exist in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may assume any three-dimensional structure and perform any function, known or unknown. A polynucleotide may contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xenonucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine.Non-limiting examples of polynucleotide include coding or non-coding regions of gene or gene fragment, locus determined by linkage analysis, exon, intron, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotide including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probe, and primer.Nucleotide sequence can be interfered with by non-nucleotide components.

[0026] The term "gene" generally refers to a nucleic acid (e.g., DNA, such as genomic DNA or cDNA) involved in coding an RNA transcript and its corresponding nucleotide sequence. As used herein with respect to genomic DNA, the term includes regulatory regions as well as intervening non-coding regions and may include 5' and 3' ends. In some applications, the term encompasses the transcribed sequence, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region contains an "open reading frame" that encodes a polypeptide. In some applications of the term, a "gene" includes only the coding sequence (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, a gene does not encode a polypeptide, e.g., a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only the transcribed sequence, but also includes non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene", i.e., a native gene in its natural location in the genome of an organism. A gene can refer to an "exogenous gene" or a non-native gene. A non-native gene can refer to a gene that is not normally found in a 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 location 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.

[0027] The term "expression" generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. "Upregulated" in the context of expression generally refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to the expression level in the wild-type state, while "downregulated" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to the 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 during cell division. Expression of the gene decreases over time because its expression is restricted to the transfected cell. In contrast, stable expression of a transfected gene can occur when a gene is co-transfected with another gene that confers a selective advantage to the transfected cell, such a selective advantage could be resistance to a particular toxin presented to the cell.

[0028] The terms "peptide," "polypeptide," or "protein," as used interchangeably herein, generally refer to a polymer of at least two amino acid residues linked by peptide bonds. The terms do not imply a particular length of the polymer, and are not intended to suggest or distinguish whether the peptide is produced using recombinant techniques, chemical synthesis, or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers that contain at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The terms include amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structure (e.g., domains). The terms also encompass amino acid polymers 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. As used herein, the terms "amino acid" and "amino acids" generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural and unnatural amino acids, which are chemically modified to include groups or chemical moieties that do not naturally occur on the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0029] The terms "derivative," "variant," or "fragment," as used interchangeably herein with respect to a polypeptide, generally refer to a polypeptide that is related to a wild-type polypeptide, for example, by any of amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Polypeptide derivatives, variants, and fragments can include one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, as compared to the wild-type polypeptide.

[0030] The terms "engineered", "chimeric", or "recombinant" as used herein with respect to a polypeptide molecule (e.g., a protein) generally refer to a polypeptide molecule having a non-homologous or modified amino acid sequence as a result of the application of genetic engineering techniques to the nucleic acid encoding the polypeptide molecule, as well as to a cell or organism expressing the polypeptide molecule. The terms "engineered" or "recombinant" as used herein with respect to a polynucleotide molecule (e.g., a DNA molecule or an RNA molecule) generally refer to a polynucleotide molecule having a non-homologous or modified nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques, transfection, transformation, and other gene transfer techniques, homologous recombination, site-directed mutagenesis, and gene fusion. In some cases, an engineered or recombinant polynucleotide (e.g., a genomic DNA sequence) may be modified or altered by a gene editing moiety.

[0031] Unless otherwise specified or clear from the context, the term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination. A nucleotide can include synthetic nucleotides. A nucleotide can include synthetic nucleotide analogs. A nucleotide can be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, 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 triphosphate (ddNTP) and its derivatives. Examples of dideoxyribonucleoside triphosphate 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.Fluorescent labels for nucleotides can 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-dUTP available from Boehringer Mannheim, Indianapolis, Indiana; ATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, as well as chromosome-labeled nucleotides available from Molecular Probes, Eugene, Oregon, 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 Examples of suitable nucleotides include tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides can also be labeled or marked by chemical modification.Chemically modified single nucleotide can be biotin-dNTP.Some non-limiting examples of biotinylated dNTP can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP) and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0032] The term "cell" generally refers to a biological cell. A cell may be the basic structural, functional, and / or biological unit of a living organism. A cell may originate 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., cells from plant crops, fruits, vegetables, cereals, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, forage, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, liverworts, mosses), algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardlii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, etc.), and / or microbial cells (e.g., microbial cells from plants such as microbial cells from ... patens, C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Sometimes the cells do not originate from a natural organism (e.g., cells can be synthetically produced and are sometimes called artificial cells).

[0033] The terms "reprogramming", "dedifferentiation", "increasing cell potential" or "increasing developmental potential" used interchangeably herein generally refer to a method of increasing the potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potential has more developmental flexibility (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell in a less differentiated state than the same cell in a non-reprogrammed state.

[0034] The term "differentiation" generally refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as, for example, an immune cell. A differentiated or differentiation-induced cell is a cell that is acquired at a more specialized ("committed") position within a cell lineage. The term "specialized" generally refers to a cell that has progressed in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a particular cell type or subset of cell types, but cannot differentiate into a different cell type or revert to a less differentiated cell type under normal circumstances.

[0035] The term "pluripotency" generally refers to the ability of a cell to form all lineages of body or somatic cells (i.e., embryo proper). For example, an embryonic stem cell is a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency can be a continuum of developmental potential ranging from incompletely or partially pluripotent cells that cannot give rise to a complete organism (e.g., epiblast stem cells) to more primitive, more pluripotent cells that can give rise to a complete organism (e.g., embryonic stem cells).

[0036] The term "induced pluripotent stem cells" (iPSCs) generally refers to stem cells derived from differentiated cells (e.g., differentiated adult, neonatal, or fetal cells) that have been induced or changed (i.e., reprogrammed) into cells that can differentiate into tissues of all three germ layers, mesoderm, endoderm, and ectoderm, or the dermal layer. The iPSCs produced do not refer to cells found in nature. In some cases, iPSCs can be engineered to directly differentiate into specialized cells (e.g., natural killer (NK) cells). In some cases, iPSCs can be engineered to first differentiate into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) or hematopoietic progenitor cells), which can be further induced to differentiate into specialized cells (e.g., NK cells).

[0037] The term "embryonic stem cells" (ESCs) generally refers to cells derived from naturally occurring pluripotent stem cells of the inner cell mass of embryonic blastocysts. Embryonic stem cells are pluripotent and give rise to all derivatives of the three primary germ layers, ectoderm, endoderm, and mesoderm, during development. In some cases, ESCs can be engineered to differentiate directly into specialized cells (e.g., NK cells). In some cases, ESCs can be engineered to first differentiate into tissue-specific stem cells (e.g., HSCs), which can be further induced to differentiate into specialized cells (e.g., NK cells).

[0038] The term "isolated stem cells" refers to any type of stem cell disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells (MSCs), etc.) that is generally isolated from a multicellular organism. For example, HSCs can be isolated from a mammalian body, such as a human body. In another example, embryonic stem cells can be isolated from an embryo.

[0039] The term "isolated" generally refers to a cell or cell population that is separated from its environment of origin. For example, the new environment of the isolated cell is substantially free of at least one component that is found in the environment in which the "non-isolated" reference cell is present. An isolated cell can be a cell that is removed from some or all components when found in its natural environment, such as a cell isolated from a tissue sample or biopsy sample. The term also includes a cell that is removed from at least one, some, or all components when found in an environment in which the cell does not naturally occur, such as a cell isolated from a cell culture or cell suspension. Thus, an isolated cell is partially or completely separated from at least one component, including other substances, cells, or cell populations, when found in nature or when grown, stored, or sustained in an environment in which it does not naturally occur.

[0040] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursor cells", as used interchangeably herein, generally refer to cells that are specified into the hematopoietic lineage but capable of further hematopoietic differentiation (e.g., into NK cells), including multipotent hematopoietic stem cells (hematopoietic blasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells). In some cases, HSCs can be CD34+ hematopoietic cells that can give rise to both mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells.

[0041] The term "immune cells" generally refers to differentiated hematopoietic cells. Non-limiting examples of immune cells include NK cells, T cells, monocytes, innate lymphocytes, tumor-infiltrating lymphocytes, macrophages, granulocytes, and the like.

[0042] overview

[0043] Biological programming, such as cellular programming, allows cells to be engineered to produce desired results. The results of cellular programming can include inducing or preventing a wide range of common and / or new cellular functions. The results can also include enhancing or suppressing cellular functions that have already occurred. Cellular programming can be achieved through the use of genetic circuits. Cellular programming can be achieved through the manipulation of biomolecules (e.g., DNA). For example, CRISPR or CRISPR / Cas systems have been adopted for genome editing across many species due to their versatility and easy programmability. Cellular programming can affect endogenous or exogenous genes. Cellular programming can be performed to function in a time-dependent or non-time-dependent manner.

[0044] The genetic circuits used for cellular programming can be used to control a cascade of multiple desired expression and / or activity profiles of multiple genes in a cell. Genetic circuits can be multiplexed to create positive and / or negative feedback systems to allow for better control of specific cellular outcomes.

[0045] Although the CRISPR / Cas system has been widely used for gene editing, Cas is essentially a single-turnover nuclease as it remains bound to the double-stranded breaks it generates, making many regions of the genome resistant to genome editing. Increasing understanding of CRISPR / Cas-based genome editing has encouraged the development of cascade regulatory systems to further exploit this technology for use in the generation of engineered cells. By implementing a series of activatable gRNAs, genome editing can be regulated more temporally from target site to target site, sequential genome editing can be performed to function like a domino effect, and cells can be barcoded. However, this simple barcoding, which often uses exogenous fluorophores, does not allow for multiplexed regulation of endogenous genes to achieve cell differentiation.

[0046] Thus, there remains an unmet need for an activatable multiplexed CRISPR / Cas system and its use to edit target polynucleotides (e.g., the genome of a cell, specifically a eukaryotic cell) using a cascade of gRNAs to form a genetic circuit that includes a feedback loop to affect gene regulation and thus cell fate decision by itself.Given the improved multiplexing capacity due to the use of internal positive and / or negative feedback loops, the pre-programmed, activatable, self-regulating gRNA cascade CRISPR / Cas system finds utility in, for example, gene therapy, genetic circuitry, and / or complex cell fate decision and / or control.

[0047] The present disclosure provides systems and methods for engineering a CRISPR / Cas9 system that includes a Cas endonuclease and an array of cognate single guide RNAs (sgRNAs or gRNAs) that are activatable and have inactivating sequences in non-essential regions to allow for regulation and modification of the system through the use of positive and negative feedback loops. The present disclosure also provides engineered cells that can include any of the above systems or perform any of the above methods.

[0048] Systems and methods for engineering genetic circuits, cells containing same, and methods of use thereof

[0049] Various embodiments of the present disclosure provide systems for inducing a desired expression and / or activity level (or profile) of one or more target genes in a cell. Various embodiments of the present disclosure provide methods for inducing a desired expression and / or activity level (or profile) of one or more target genes in a cell.

[0050] In one embodiment, the present disclosure provides a system for inducing a desired expression and / or activity profile of a target gene in a cell. The system can include a heterologous genetic circuit comprising a plurality of gate units. The plurality of gate units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gate units. The plurality of gate units can include up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 gate unit. The plurality of gate units can be different (e.g., include different polynucleotide sequences).

[0051] The heterologous genetic circuits disclosed herein can operate with multiple gate units in series (e.g., multiple gate units connected serially in an end-to-end manner to form a single pathway), in parallel (e.g., multiple gate units connected across each other to form, e.g., two or more parallel pathways), or a combination thereof.

[0052] The gating units disclosed herein can operate in concert (e.g., as predetermined by the design of the heterologous genetic circuit) to induce an outcome in a cell. The outcome in a cell can include cell function (e.g., migration, reproduction, response to external stimuli, nutrient production, excretion, respiration, growth) and / or cell state (e.g., cell fate, differentiation, dormancy, programmed cell death). Such outcomes can be confirmed in vitro, ex vivo, and / or in vivo. For example, the outcomes disclosed herein can be confirmed in vitro by (i) measuring expression levels of genes of interest by polymerase chain reaction (PCR) or western blotting, (ii) staining with small molecules or antibodies, (iii) cell sorting based on cell size, morphology, and / or surface protein expression, (iv) using assays to measure phenotypic differentiation and cell function (e.g., cell proliferation assays, metabolic activity assays, cell death assays), (v) microscopy, and / or (iv) screening for molecular and / or genetic differences using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics.

[0053] The intracellular outcome can include modulation of the target gene. The modulation of the target gene can include multiple individual modulations of the target gene. The multiple gating units can each induce one of the multiple individual modulations of the target gene, such that the collection of individual modulations cooperate to result in a final expression and / or activity profile of the target gene. At least two individual modulations of the multiple individual modulations can increase both the expression and / or activity level of the target gene. At least two individual modulations of the multiple individual modulations can decrease both the expression and / or activity level of the target gene. Alternatively, a first individual modulation of the multiple individual modulations can increase the expression and / or activity level of the target gene, and a second individual modulation of the multiple individual modulations can decrease the expression and / or activity level of the target gene. In such a case, the first individual modulation can occur before the second individual modulation, or vice versa. Alternatively, one individual modulation of the multiple individual modulations (e.g., the first and / or second modulation) can maintain the expression and / or activity level of the target gene at the expression and / or activity level before modulation.

[0054] In some cases, each of the individual regulation of the multiple individual regulation of target genes disclosed herein may be necessary to produce the desired expression and / or activity profile of target genes, but may not be sufficient individually.Therefore, the outcome in a cell induced by the multiple individual regulation of target genes (e.g., improving cell function, inducing cell state, etc.) may not be possible in the absence of any one of the multiple individual regulation of target genes.Alternatively, the degree or scale of the outcome in a cell induced by the multiple individual regulation of target genes may be higher than the degree or scale of the outcome in a control cell induced by any one or more, but not all, of the multiple individual regulation of target genes, and / or by all of the multiple individual regulation of target genes that occurs through different sequential events.

[0055] The second gating unit can be activated (e.g., directly or indirectly) by the first gating unit. For example, the second gating unit can be directly activated by the first gating unit. Alternatively, the second gating unit can be activated by one or more additional gating units that are activated (e.g., directly or indirectly) by the first gating unit. The one or more additional gating units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gating units. The one or more additional gating units can include up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 gating unit. In yet another alternative, the second gating unit may be activated via another moiety (eg, an activation moiety, a different gating unit, etc.) that is involved in the activation of the first gating unit.

[0056] The second gate unit can be activatable to induce the inactivation of the activated first gate unit. The terms "inactivation" or "disruption" can be used interchangeably herein. The inactivation disclosed herein can be induced by generating a modification (e.g., a break, such as a single-strand or double-strand break, and an indel, etc.) in at least a portion of the first gate unit (e.g., the gate portion and / or gene regulatory portion of the first gate unit) that is involved in inducing the first individual regulation of the target gene.

[0057] Inactivation of the gate portion and / or gene regulatory portion of the first gate unit disclosed herein can be achieved by an endonuclease-based system (e.g., CRISPR / Cas system). Alternatively or additionally, inactivation can be achieved by the use of a transcription regulator system (e.g., a transcription repressor). An endonuclease transcription regulator system (e.g., a Cas repressor) can be used to achieve polynucleotide cleavage (e.g., to inactivate the gate portion and / or gene regulatory portion). Polynucleotide cleavage can create nucleic acid modifications such as single-strand breaks, double-strand breaks, insertions, deletions, or insertion-deletions (indels). Alternatively or additionally, an endonuclease transcription regulator system (e.g., a Cas repressor) can be used to regulate target gene expression.

[0058] Alternatively, the second gate unit can be activatable to amplify or enhance the activation of the activated first gate unit.The amplification or enhancement of the first gate unit can be induced by generating a modification (e.g., a break such as a single-strand or double-strand break, and an indel, etc.) in at least a portion of the first gate unit (e.g., the gate portion and / or gene regulatory portion of the first gate unit) that is involved in inducing the first individual regulation of the target gene.

[0059] In some cases, the guide nucleic acid molecule (gNA) expressed by the second gate unit (e.g., functional gNA) can create a modification in at least a portion of the first gate unit upon activation. For example, the activated gNA of the second gate unit can create a modification in the polynucleotide sequence of the first gate unit that encodes the gNA (e.g., activatable gNA), or in the promoter sequence of the first gate unit that is operably linked to such gNA of the same first gate unit. Such a modification can render the gNA of the first gate unit inoperable (e.g., reduce or inhibit specific binding to a target gene) when expressed. Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0060] In some cases, the modification of polynucleotide sequence (e.g., as a component of gate unit such as gate portion) or target gene can occur by single-strand breaks, which are discontinuous in one nucleotide strand.The inactivation of polynucleotide sequence or target gene can occur by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more single-strand breaks.In some cases, the inactivation of gene can occur by up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 single-strand breaks.

[0061] Optionally, the gNA is at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, or more in length.

[0062] In some cases, the disclosed system and method can utilize at least two different endonucleases. A first endonuclease (e.g., a Cas protein that is not bound to a transcriptional modulator) can be used in conjunction with a guide nucleic acid to induce the cleavage of the target polynucleotide sequence of a gate portion or gene regulatory portion plasmid, activating and / or deactivating the gate portion or gene regulatory portion, respectively. In addition, a second endonuclease (e.g., a Cas protein that is bound to a transcriptional modulator) can be used in conjunction with another guide nucleic acid to bind to a target gene (e.g., a target endogenous gene) of a cell, and regulate the expression and / or activity of the target gene without causing cleavage in the target gene.

[0063] In some cases, the disclosed systems and methods can utilize a single endonuclease system (e.g., a Cas repressor) to achieve both (i) polynucleotide cleavage (e.g., to activate / inactivate a gate portion and / or a gene regulatory portion) and (ii) regulation of target gene expression. When using a single endonuclease transcription regulator system, unique guide nucleic acid molecules (gNAs) with different spacer sequence lengths can be used to determine whether the single endonuclease transcription regulator system can (i) hybridize to a polynucleotide sequence and induce the Cas-mediated nuclease activity of the polynucleotide sequence, or (ii) hybridize to a target gene (e.g., genomic DNA) and regulate the expression and / or activity level of the target gene by the action of a transcription activator without mediating Cas nuclease activity as desired by individual heterologous genetic circuits. For example, the use of gNAs with different spacer sequence lengths that bind to different targets can allow the second gating unit provided herein to induce inactivation of an activated first gating unit and / or induce individual regulation of a second target gene.

[0064] As described above, the length of the spacer sequence of the gNA can affect the ability of the gNA to mediate Cas nuclease activity. Optionally, gNAs with spacer sequences of different lengths can be used in the same heterologous genetic circuit to affect different types of cleavage, activation, inactivation, and / or regulation of one or more target nucleic acids. Optionally, a gNA spacer sequence shorter than a threshold length (e.g., about 16 nucleotides) can eliminate the nuclease activity of the Cas transcription regulator while still mediating DNA binding for transcriptional regulation of the target gene. Optionally, a gNA spacer sequence shorter than at least about 25 nucleotides, at least about 20 nucleotides, at least about 19 nucleotides, at least about 18 nucleotides, at least about 17 nucleotides, at least about 16 nucleotides, at least about 15 nucleotides, at least about 15 nucleotides, at least about 14 nucleotides, at least about 13 nucleotides, at least about 12 nucleotides, at least about 11 nucleotides, or at least about 10 nucleotides can eliminate the nuclease activity of the Cas protein while still mediating DNA binding.

[0065] For example, a gNA containing a 20 nucleotide spacer sequence (e.g., a gNA encoded by a gate portion to target a gene regulatory portion plasmid) may be sufficient to promote the nuclease activity of an endonuclease (e.g., a Cas or Cas transcription regulator fusion protein) in a target polynucleotide sequence. Alternatively or additionally, a gNA containing a 14 nucleotide spacer sequence (e.g., a gNA encoded by a gene regulatory portion) may be able to hybridize to DNA but may not be long enough to mediate nuclease activity and may only promote endonuclease binding to the cognate DNA sequence. Thus, a shorter gNA may selectively allow transcriptional regulation of a target gene by the use of an endonuclease transcription regulator system (e.g., a Cas activator system, a Cas repressor system) without cleaving the target gene.

[0066] In some cases, the modification of polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or target gene can occur by double-strand breaks, where there is a discontinuity in both nucleotide strands.In some cases, the inactivation of gene can occur by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more double-strand breaks.In some cases, the inactivation of polynucleotide sequence or target gene can occur by up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 double-strand breaks.

[0067] In some cases, the modification of polynucleotide sequence (e.g., as a component of gate unit such as gate portion) or target gene can be caused by indel, also known as insertion-deletion mutation.Indel mutation can include frameshift mutation or non-frameshift mutation.Indel mutation can include point mutation, also known as base substitution, in which only one base or base pair is modified. Indel mutations can comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, or more bases or base pairs in length. Indel mutations can comprise up to about 2000, up to about 1000, up to about 900, up to about 800, up to about 700, up to about 600, up to about 500, up to about 400, up to about 300, up to about 200, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 base or base pair in length.

[0068] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be achieved without cleaving the polynucleotide sequence or the target gene. For example, a gene regulatory portion (e.g., a nucleic acid molecule and / or an endonuclease, such as a complex comprising a CRISPR / Cas protein and a guide nucleic acid molecule) can specifically bind to a polynucleotide sequence or a target gene such that the expression and / or activity of the polynucleotide sequence or the target gene is modified. The gene regulatory portion can include a transcriptional repressor or a transcriptional activator as provided herein.

[0069] In some cases, the modification of the polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or the target gene can inactivate the gene. For example, the modification of the polynucleotide sequence or the target gene can stop the expression and / or activity level of the polynucleotide sequence or the target gene. Alternatively, the modification of the polynucleotide sequence or the target gene can reduce the expression and / or activity level of the polynucleotide sequence of the target gene. In some cases, the modification of the polynucleotide sequence or the target gene can increase the expression and / or activity level of the polynucleotide sequence or the target gene. Alternatively, the modification of the polynucleotide sequence or the target gene can maintain the expression and / or activity level of the polynucleotide sequence or the target gene.

[0070] In some cases, modification of the polynucleotide sequence (e.g., as a component of a gating unit, such as a gating portion) or the target gene reduces the expression and / or activity level of the polynucleotide sequence or the target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, or at least about 2%. , at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. Modification of the polynucleotide sequence or target gene may include reducing the expression and / or activity levels of the polynucleotide sequence or target gene, respectively, by up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, up to about 0.1%, or less.

[0071] In some cases, modification of the polynucleotide sequence (e.g., as a component of a gating unit, such as a gating portion) or the target gene may increase the expression and / or activity level of the polynucleotide sequence or the target gene, respectively, by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 42%, at least about 44%, at least about 46%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, at least about 79%, at least about 80%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 9 This may include increasing by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, at least about 1,000,000%, or more. Modification of the polynucleotide sequence or the target gene may increase the expression and / or activity levels of the polynucleotide sequence or the target gene by up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 500%, up to about 500%, up to about 600%, up to about 700%, up to about 800%, up to about 900%, up to about 900%, up to about 100%, up to about 100%, up to about 15 ... In some embodiments, the increase may include increasing the concentration of the amine by 0%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, up to about 0.1%, or less.

[0072] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gating unit, such as a gate portion) or a target gene increases the expression and / or activity level of the polynucleotide sequence or target gene by at least or at most about 0.1 fold, at least or at most about 0.2 fold, at least or at most about 0.3 fold, at least or at most about 0.4 fold, at least or at most about 0.5 fold, at least or at most about 0.6 fold, at least or at most about 0.7 fold, at least or at most about 0.8 fold, at least or at most about 0.9 fold, at least or at most about 1 fold, at least or at most about 2 fold, at least or at most about 3 fold, at least or at most about 4 fold, at least or at most about 5 fold, at least or at most about 6 fold, at least or at most about 7 fold, at least or at most about 8 fold, at least or at most about 9 fold, at least or at most about 10 fold, at least or at most about 15 ...5 fold, at least or at most about 15 fold, at least or at most about 15 fold, at least or at most about 15 fold, at least or at most about 15 fold, at least or at most about 15 fold, at least or at most about 15 fold, at least or at most about 15 fold, at least or This may include reducing by at least or at most about 4-fold, at least or at most about 5-fold, at least or at most about 6-fold, at least or at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 60-fold, at least or at most about 70-fold, at least or at most about 80-fold, at least or at most about 90-fold, at least or at most about 100-fold, at least or at most about 500-fold, at least or at most about 1,000-fold, at least or at most about 5,000-fold, or at least or at most about 10,000-fold. Modification of the polynucleotide sequence or the target gene can increase the expression and / or activity level of the polynucleotide sequence or the target gene by up to about 10,000-fold or less, up to about 5,000-fold or less, up to about 1,000-fold or less, up to about 500-fold or less, up to about 100-fold or less, up to about 90-fold or less, up to about 80-fold or less, up to about 70-fold or less, up to about 60-fold or less, up to about 50-fold or less, up to about 40-fold or less, up to about This may include reducing by 30-fold or less, up to about 20-fold or less, up to about 10-fold or less, up to about 9-fold or less, up to about 8-fold or less, up to about 7-fold or less, up to about 6-fold or less, up to about 5-fold or less, up to about 4-fold or less, up to about 3-fold or less, up to about 2-fold or less, up to about 1-fold or less, up to about 0.9-fold or less, up to about 0.8-fold or less, up to about 0.7-fold or less, up to about 0.6-fold or less, up to about 0.5-fold or less, up to about 0.4-fold or less, up to about 0.3-fold or less, up to about 0.2-fold or less, or up to about 0.1-fold or less.

[0073] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gating unit, such as a gating portion) or a target gene increases the expression and / or activity level of the polynucleotide sequence or target gene, respectively, by at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least or at most about 0.6-fold, at least or at most about 0.7-fold, at least or at most about 0.8-fold, at least or at most about 0.9-fold, at least or at most about 1-fold, at least or at most about 2-fold, at least or at most about 3-fold, compared to the expression and / or activity level of a control. , at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold. Modification of the polynucleotide sequence or the target gene can increase the expression and / or activity level of the target gene by up to about 10,000 fold or less, up to about 5,000 fold or less, up to about 1,000 fold or less, up to about 500 fold or less, up to about 100 fold or less, up to about 90 fold or less, up to about 80 fold or less, up to about 70 fold or less, up to about 60 fold or less, up to about 50 fold or less, up to about 40 fold or less, up to about 30 fold or less, This may include increasing by up to about 20-fold or less, up to about 10-fold or less, up to about 9-fold or less, up to about 8-fold or less, up to about 7-fold or less, up to about 6-fold or less, up to about 5-fold or less, up to about 4-fold or less, up to about 3-fold or less, up to about 2-fold or less, up to about 1-fold or less, up to about 0.9-fold or less, up to about 0.8-fold or less, up to about 0.7-fold or less, up to about 0.6-fold or less, up to about 0.5-fold or less, up to about 0.4-fold or less, up to about 0.3-fold or less, up to about 0.2-fold or less, or up to about 0.1-fold or less.

[0074] In some cases, (i) the sequential change (e.g., improvement or decrease) in expression and / or activity level of the target gene upon a plurality of individual modulations of the target gene is at least or at most about 1%, at least or at most about 2%, at least or at most about 3%, at least or at most about 4%, at least or at most about 5%, at least or at most about 6%, at least or at most about 7%, at least or at most about 8%, at least or at most about 9%, at least or at most about 10%, at least or at most about 11%, at least or at most about 12%, at least or at most about 13%, at least or at most about 14%, at least or at most about 15%, at least or at most about 16%, at least or at most about 17%, at least or at most about 19%, at least or at most about 20%, at least or at most about 21%, at least or at most about 22%, at least or at most about 23%, at least or at most about 24%, at least or at most about 25%, at least or at most about 26%, at least or at most about 27%, at least or at most about 28%, at least or at most about 29%, at least or at most about 30%, at least or at most about 31%, at least or at most about 32%, at least or at most about 33%, at least or at most about 34%, at least or at most about 35%, at least or at most about 36%, at least or at most about 37%, at least or at most about 38%, at least or at most about 39%, at least or at most about 40%, at least or at most about 41%, at least or at most about 42%, at least or at most about 43%, at least or at most about 44%, at least or at most about 45%, at least or at most about 46%, at least or at most about 47%, at least or at most about 48%, at least or at most about 8%, at least or up to about 19%, 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%, at least or up to about 95%, at least or up to about 100%, at least or up to about 125%, at least or up to about 150%, at least or up to about 175%, at least or up to about 200%, at least or up to about 225%, at least or up to about 250%, at least or up to about 275%, or at least or up to about 300% more.

[0075] In some cases, (i) the sequential change (e.g., improvement or decrease) in expression and / or activity level of the target gene upon a plurality of individual modulations of the target genes is (ii) at least or at most about 1%, at least or at most about 2%, at least or at most about 3%, at least or at most about 4%, at least or at most about 5%, at least or at most about 6%, at least or at most about 7%, at least or at most about 8%, at least or at most about 9%, at least or at most about 10%, at least or at most about 11%, at least or at most about 12%, at least or at most about 13%, at least or at most about 14%, at least or at most about 15%, at least or at most about 16%, at least or at most about 17%, at least or at most about 18%, at least or up to about 19%, 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%, at least or up to about 95%, at least or up to about 100%, at least or up to about 125%, at least or up to about 150%, at least or up to about 175%, at least or up to about 200%, at least or up to about 225%, at least or up to about 250%, at least or up to about 275%, or at least or up to about 300% longer.

[0076] In some cases, (i) the expression and / or activity level of a target gene upon multiple individual modulation of consecutive target genes (e.g., including sequential disruption) is at least or at most about 1%, at least or at most about 2%, at least or at most about 3%, at least or at most about 4%, at least or at most about 5%, at least or at most about 6%, at least or at most about 7%, at least or at most about 8%, at least or at most about 9%, at least or at most about 10%, at least or at most about 11%, at least or at most about 12%, at least or at most about 13%, at least or at most about 14%, at least or at most about 15%, at least or at most about 16%, at least or at most about 17%, at least or at most about 18%, at most about 19%, at least or at most about 20%, at least or at most about 21%, at least or at most about 22%, at least or at most about 23%, at least or at most about 24%, at least or at most about 25%, at least or at most about 26%, at least or at most about 27%, at least or at most about 28%, at least or at most about 29%, at least or at most about 30%, at least or at most about 31%, at least or at most about 32%, at least or at most about 33%, at least or at most about 34%, at least or at most about 35%, at least or at most about 36%, at least or at most about 37%, at least or at most about 38%, at least or at most about 39%, at least or at most about 40%, at least or at most about 41%, at least or at most about 42%, at least or at most about 43%, at least or at most about 44%, at least or at most about 45%, at least or at most about 46%, at least or at most about 47%, at least or at most about 48%, at least or at most may be as low as or up to about 19%, 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%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or at least or up to about 100% less.

[0077] In some cases, (i) the expression and / or activity level of a target gene upon multiple individual modulation of consecutive target genes (e.g., including sequential disruption) is at least or at most about 1%, at least or at most about 2%, at least or at most about 3%, at least or at most about 4%, at least or at most about 5%, at least or at most about 6%, at least or at most about 7%, at least or at most about 8%, at least or at most about 9%, at least or at most about 10%, at least or at most about 11%, at least or at most about 12%, at least or at most about 13%, at least or at most about 14%, at least or at most about 15%, at least or at most about 16%, at least or at most about 17%, at least or at most about 18%, It may last at least or up to about 19%, 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%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or at least or up to about 100% shorter.

[0078] In some cases, the first gate unit and the second gate unit provided herein can be activated to induce a first individual regulation and then a second individual regulation of a common gene (e.g., expression and / or activity profile of a common gene).The first gate unit and the second gate unit can be activated sequentially (e.g., at different times) to achieve the first and second individual regulation of the common gene in succession.Alternatively, the first gate unit and the second gate unit can be activated simultaneously thereafter, and these gates can be pre-configured to always achieve the first and second individual regulation of the common gene in this way in succession.

[0079] Optionally, the activation of the multiple gate units can be the result of a single activation of the heterologous genetic circuit (e.g., by a single activation moiety at a single time). The multiple gate units can include one of a first gate unit and a second gate unit preconfigured to be activated sequentially upon activation of the heterologous genetic circuit by a single activation. Optionally, one of the first and second gate units can be activated by a single activation moiety (e.g., a guide nucleic acid), while the other of the first and second gate units can be activated by an additional activation moiety (e.g., a different guide nucleic acid) that is different from the activation moiety of the heterologous genetic circuit. The additional activation moiety can be a portion of the heterologous genetic circuit that is generated (e.g., expressed) only upon activation of the heterologous genetic circuit. Alternatively or additionally, the first and second gate units can each be activated by a different activation moiety that is not the same as the activation moiety of the heterologous genetic circuit. Such a different activation moiety can be a portion of the heterologous genetic circuit that is generated (e.g., expressed) only upon activation of the heterologous genetic circuit.

[0080] In some embodiments of any one of the systems disclosed herein, the gating unit can include a gating portion (e.g., at least 1, 2, 3, 4, 5, or more different gating portions) and / or a gene regulatory portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different gene regulatory portions). The gating portion disclosed herein can include a guide nucleic acid molecule (gNA) (e.g., at least 1, 2, 3, 4, 5, or more gNA). The gene regulatory portion disclosed herein can include a gNA (e.g., at least 1, 2, 3, 4, 5, or more gNA). The guide nucleic acid molecule disclosed herein can include, but is not limited to, DNA, RNA, any analog thereof, or any combination thereof. In some embodiments of any one of the systems disclosed herein, the gating moiety and / or gene regulatory moiety can be activatable to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and the complex can be configured to or capable of binding to a target polynucleotide, e.g., to regulate the expression and / or activity level of the target polynucleotide or another polynucleotide sequence operably linked to the target polynucleotide. For example, the complex can regulate the expression and / or activity level of a gene that comprises the target polynucleotide.

[0081] In some embodiments of any one of the systems disclosed herein, the initial (or first) gating unit of the heterologous genetic circuit disclosed herein may be activated (e.g., directly activated) by an activating moiety. The activating moiety may directly bind to at least a portion of the initial gating unit to activate the initial gating unit, thereby, for example, sequentially activating the heterologous genetic circuit. Alternatively, the activating moiety (e.g., electromagnetic energy) may activate the initial gating unit without directly binding to at least a portion of the initial gating unit. In some cases, the initial gating unit may include at least one gating moiety and at least one gene regulatory moiety. In some cases, the initial gating unit may include at least one gating moiety, but does not or does not need to include a gene regulatory moiety. In some cases, the initial gating unit may include at least one gene regulatory moiety, but does not or does not need to include a gating moiety (e.g., the activating moiety may be configured to activate the initial gating unit and at least one additional gating unit).

[0082] In some embodiments of any one of the systems disclosed herein, the gNA of the gate moiety and / or gene regulatory moiety (e.g., the gNA encoded by the gate moiety and / or gene regulatory moiety) can be an activatable gNA. The activatable gNA can be, but is not limited to, any one of ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogs thereof, or any combination thereof. In some embodiments, the activatable gNA molecule can be a self-cleaving gNA (e.g., the gRNA contains a cis ribozyme). For example, when the activatable gNA is expressed in a cell, the activatable gNA can be self-cleavable such that it is non-functional (e.g., not configured to bind to a target gene) unless the gene encoding the activatable gNA is modified prior to expression of the activatable gNA. In some aspects, the activatable gNA molecule comprises a non-standard transcription termination sequence such that a functional gNA molecule is not expressed until a gene encoding an activatable gNA having a non-standard transcription termination sequence (e.g., a polyX sequence, such as a polyU sequence or a polyT sequence) can be modified (e.g., to remove part or all of the transcription termination sequence). Thus, in the absence of modification of the transcription termination sequence, a non-functional variant (e.g., a non-functional fragment) of the gNA can be expressed. In some embodiments, the gNA can be synthetic. In some embodiments, the gNA can have a fluorescent label attached thereto.

[0083] Optionally, the size of the polyT sequence is equal to or greater than a threshold length, and the threshold length is sufficient to reduce expression of the guide nucleic acid molecule from the polynucleotide sequence. Thus, a plasmid (e.g., a gate portion or a gene regulatory portion) can encode an inactivated gNA that includes a polyT sequence equal to or greater than a threshold length, and editing such a plasmid to reduce the length of the polyT to less than the threshold length can allow expression of the entire gNA without premature termination, thereby activating the gNA. Optionally, the polyT sequence includes at least 5 Ts. Optionally, the polyT sequence includes at least 7 Ts. Optionally, the polyT sequence includes at least 8 Ts. Optionally, the polyT sequence includes at least 10 Ts. Optionally, the polyT sequence includes between 5 Ts and 15 Ts. Optionally, the polyT sequence includes one or more additional nucleotides that are not Ts.

[0084] In some cases, the gene regulatory portion (e.g., guide nucleic acid and / or endonuclease) can be configured to bind to a target polynucleotide sequence operably linked to a target gene in a cell. The target gene can include a coding polynucleotide sequence that encodes a target nucleic acid molecule or a target protein. The target polynucleotide sequence can be a part of the coding polynucleotide sequence. Alternatively, the target polynucleotide sequence may not be a part of the coding polynucleotide sequence. For example, the target polynucleotide sequence can be upstream of the coding polynucleotide sequence (e.g., a part of the promoter of the coding polynucleotide sequence, such as a transcription start site (TSS)).

[0085] The individual modulations of the target genes may differ (eg, differing degrees of change in the expression and / or activity levels of the target genes). For example, the first adjustment exerted by the first gating unit and the second adjustment exerted by the second gating unit can differ by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%. The first and second modulations may differ by up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, or up to about 0.1%. Alternatively or additionally, the individual modulations of the target genes may be substantially the same (e.g., the same).

[0086] Multiple individual modulations may be sufficient to individually induce a desired change in the expression and / or activity levels of a target gene. Alternatively, individual modulations may be insufficient to individually induce a desired change in the expression and / or activity levels of a target gene.

[0087] The one or more target genes disclosed herein can include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0088] The one or more target genes disclosed herein can include a cell differentiation regulator, a molecular function regulator, a binding factor, a fusion factor, a protein folding chaperone, a protein tag, an RNA folding chaperone, a cell signaling factor, an immune response factor, a sensory receptor, a cell structure factor, a protein binding factor, a cargo receptor, a catalytic factor, or a small molecule sensor.

[0089] The one or more target genes disclosed herein can include cell differentiation regulators, including growth factors, transcription factors, myogenic regulators, immune cell regulators, neuronal regulators, stem cell differentiation factors, chondrogenic regulators, osteogenic regulators, senescence factors, stemness factors (e.g., dedifferentiation factors), and the like.

[0090] In some cases, the one or more target genes (e.g., one or more myogenic regulatory factors) can include Msgn1, Pou5f1, Pax7, Myog, Myf5, Myf6, Myof, Srf, Ccnd2, Fgf2, Sox2, Tbx6, Tbxt, Ctnnb1, Mycl, Bcl2l2, Bcl2l1, Bcl2a1, Myod1, Bcl2, Tdgf1, Pax3, Chrd, Ccnd3, Hgf, Ccnd1, Foxm1, Myc, Tgfb1, Mdm2, Mcl1, Igf1, Mef2c, Mrf4, MyHC, MCK, Six1, Six4, Hdac4, Notch1, Ezh2, p21, Myh1, Myh2, Myh4, and / or Smad4.

[0091] In some cases, the one or more target genes (e.g., one or more immune cell regulators) are selected from the group consisting of Mycn, Ptcra, Bcl11b, Hhex, Notch1, Tcf3, Rag2, Dtx1, Runx1, Hoxa9, Rag1, Spi1, Ets1, Id2, Bcl11a, Id3, Tcf7, Ikzf1, Tcf12, Runx3, Lmo2, Lef1, Gfi1, Lyl1, Aqp3, Meis1, Gata3, Hes1, St18, Nr4a1, C20orf100, Ikaros, Spib, Rorc, Tcf1, Myc, Ahr, Foxo1, Notch1, Notch3, IL-2, IL4, IL7, IL15, The target genes may include Ebf1, Pax5, Tal1, Myb, Erg, Hhex, E4pb4, Gfi1b, pTα, Tbx21 Etsl, Ets2, or Mef2c (FIGS. 1-2). For example, the one or more target genes for inducing T cell differentiation may be selected from the group consisting of Hes1, Gata3, Tcf1, Tcf3, Tcf7, Spi1, Lmo2, Ikzf1, Notch1, IL7, IL2, Runx1, and Bcl11b (FIG. 3). In another example, the one or more target genes for inducing B cell differentiation may be selected from the group consisting of Ebf1, IL4, Tcf3, Foxo1, and Pax5. In another example, the one or more target genes for inducing NK cell differentiation may be selected from the group consisting of E4bp4, Spi1, ZBTB16, Tbx21, IL7, IL15, and Notch1.

[0092] In some cases, the one or more target genes (e.g., one or more stem cell differentiation factors) may include HoxB4, Notch1, Sox11, Sox17, Runx1, Gata2, Fli1, Erg, Wnt, Hnf1, Hnf3, Hnf4, Cdx2, and Lin28A.

[0093] In some cases, the one or more target genes (e.g., one or more neuronal regulatory factors) can include Ezh2, Sox1, Sox3, Sox4, Sox9, Sox10, Sox11, Sox21, Hes1, Dlk1, Ascl1, Ngn1, Ngn2, Lhx6, Zeb1, Zeb2, Smad7, Myt1l, Pax6, Wnt7a, Nptx1, Nab2, Lhx6, Olig2, Nfia, Nfib, Dlx5, Nurr1, Gpx1, Otx2, Pbx1, Foxa2, Isl1, Phox2a, Phox2b, Pitx3, Bm3a, and Brn4.

[0094] Optionally, the one or more target genes (e.g., one or more chondrogenic regulators) can include Sox9, Gli3, Trps1, Nkx3.2, Runx2, Runx3, Smad1, Smad5, and Smad8.

[0095] In some cases, the one or more target genes (e.g., one or more bone formation regulators) may include Fos, Crebbp, Hnf4a, Irf1, Irf9, Foxo4, Meis1, Egr1, Pou2f1, Anf281, Sp1, Esr1, Sox9, Znf6, Znf162, and Znf281.

[0096] Optionally, the one or more target genes (eg, one or more senescence factors) can include p21, p53, Bax, Apaf1, Mdm2, E2f1, and Foxo3.

[0097] In some cases, the one or more target genes (e.g., one or more stemness factors) can include Hes1, Hes5, Cbf1, Soc2, Hmga2, Olig2, Id2, Id4, Hesr1, Hesr2, Gli1, Gli2, Gli3, SoxB, and Bmi1.

[0098] In some cases, one or more target genes can include T-box transcription factors (TBX genes). TBX transcription factors are involved in development. T-box proteins have relatively large DNA binding domains. Non-limiting examples of TBX transcription factors can include TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, TBX22, and TBXT (Brachyury protein).

[0099] In some cases, one or more target genes are basic helix-loop-helix transcription factors (bHLH genes). bHLH transcription factors are involved in the cell cycle and many other developmental processes. bHLH proteins have a basic helix-loop-helix protein structure. Non-limiting examples of bHLH transcription factors include AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATOH1, ATOH7, ATOH8, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, EPAS1, FERD3L, FIGLA, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2, HIF1A, ID1, ID2, ID3, ID4, KIAA2018, LYL1, MASH1, MATH2, MAX, MESP1, MESP2, MIST1, MITF, MLX, MLXIP, MLXIPL, MNT, MSC, MSGN1, MXD1, MXD3, MXD 4, MXI1, MYC, MYCL1, MYCL2, MYCN, MYF5, MYF6, MYODI, MYOG, NCOA1, NCOA3, NEUROD1, NEUR0D2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, NHLH1, NHLH2, NPAS1, NPAS2, NPAS3, NPAS4, OAF1, OLIG1, OLIG2, OLIG3, PTF1A, SCL, SCXB, SIM1, SIM2, SOHLH1, SOHLH2, SREBF1, SREBF2, TAL1, TAL2, TCF12, TCF15, TCF21, TCF3, TCF4, TCFL5, TFAP4, TFE3, TFEB, TFEC, TWIST1, TWIST2, USF1, and USF2.

[0100] Optionally, one or more target genes can comprise SRY-associated box transcription factor (SOX gene).SOX transcription factor is involved in regulating development.Non-limiting examples of SOX transcription factor can include SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21, SOX30 and SRY.

[0101] Optionally, one or more target genes can include SOX group A, which includes SRY. Optionally, one or more target genes can include SOX group B1, which includes SOX1, SOX2, and / or SOX3. Optionally, one or more target genes can include SOX group B2, which includes SOX14 and / or SOX21. Optionally, one or more target genes can include SOX group C, which includes SOX4, SOX11, and / or SOX12. Optionally, one or more target genes can include SOX group D, which includes SOX5, SOX6, and / or SOX13. Optionally, one or more target genes can include SOX group E, which includes SOX8, SOX9, and / or SOX10. Optionally, one or more target genes can include SOX group F, which includes SOX7, SOX17, and / or SOX18. Optionally, one or more target genes can include SOX group G, which includes SOX15. Optionally, the one or more target genes can include a SOX group H, including SOX30.

[0102] In some cases, one or more target genes can include Forkhead Box (FOX). FOX is a transcription factor that plays a role in regulating the expression of genes involved in cell growth, proliferation, differentiation and life span. Some FOX genes can bind to chromatin during cell differentiation process. Non-limiting examples of FOX genes can include FOXA, FOXB, FOXC, FOXD, FOXE, FOXF, FOXG, FOXH, FOXI, FOXJ, FOXK, FOXL, FOXM, FOXN, FOXO, FOXP, FOXQ, FOXR and FOXS.

[0103] In some cases, the one or more target genes can include erythroblast transformation specific (ETS) genes. ETS is a transcription factor specific to animals and is involved in tissue development. Non-limiting examples of ETS genes can include ELF1, ELF2 (NERF), ELF4 (MEF), GABPα, ERG, FLl1, FEV, ERF (PE2), ETV3 (PE1), ELF3 (ESE1 / ESX), ELF5 (ESE2), ESE3 (EHF), ETS1, ETS2, SPDEF (PDEF / PSE), ETV4 (PEA3 / E1AF), ETV5 (ERM), ETV1 (ER81), ETV2 (ER71), SPI1 (PU.1), SPIB, SPIC, ELK1, ELK4 (SAP1), ELK3 (NET / SAP2), ETV6 (TEL), and ETV7 (TEL2).

[0104] In some cases, the one or more target genes may include collagen. Collagen is a fibrous protein and is a major component of skin, bone, tendon, cartilage, blood vessels, and teeth. Collagen forms insoluble fibers with high tensile strength. Non-limiting examples of collagen genes include COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, COL6A6, COL7A1, COL8A1, COL8A2, COL9A, COL10A, COL11A, COL12A, COL13A, COL14A, COL15A, COL16A, COL17A, COL18A, COL19A, COL20A, COL21A, COL22A, COL23A, COL24A, COL25A, COL26A, COL27A, COL28A, COL29A, COL30A, COL31A, COL32A, COL33A, COL34A, COL35A, COL36A, COL37A, COL38A, COL39A, COL39B, COL39C, COL39D, COL39D, COL39E, COL39F ... 1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, COL12A1, COL13A1, COL14A1, COL15A1, COL16A1, COL17A1, COL18A1, COL19A1, COL20A1, COL21A1, COL22A1, COL23A1, COL24A1, COL25A1, COL26A1, COL27A1, and COL28A1 can be mentioned.

[0105] Optionally, one or more target genes can comprise homeobox gene.Homeobox gene is, for example, the gene that regulates large-scale anatomical features in early embryonic development.Types of homeobox gene include HOX gene, LIM gene, PAX gene, POU gene, CERS gene, HNF gene, SINE gene, CUT gene, ZF gene, paraHOX gene, DLX gene, TALE gene, PRD gene and NKL gene. Non-limiting examples of homeobox genes include HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXB13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13, HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13, CDX1, CDX2, CDX4, GSX1, GSX2, PDX1, EVX1, EVX2, GBX1, GBX2, MEOX1, MEOX2, MNX1, DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, IRX1, IRX2, IRX3, IRX4, IRX5, I RX6, MEIS1, MEIS2, MEIS3, MKX, PBX1, PBX2, PBX3, PBX4, PKNOX1, PKNOX2, TGIF1, TGIF2, TGIF2LX, TGIF2LY, ISL1, ISL2 , LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, LMX1B, HDX, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2 , POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, P0U5F1, POU5F1P1, POU5F1P4, POU5F2, POU6F1, POU6F2, LASS2, LASS3, LAS S4, LASS5, LASS6, HMBOX1, HNF1A, HNF1B, SIX1, SIX2, SIX3, SIX4, SIX5, SIX6, ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2,SATB1, SATB2, ADNP, ADNP2, TSHZ1, TSHZ2, TSHZ3, ZEB1, ZEB2, ZFHX2, ZFHX3, ZFHX4, ZHX1, HOME Z, ALX1(CART1), ALX3, ALX4, ARGFX, ARX, DMBX1, DPRX, DRGX, DUXA, DUXB, DUX(1, 2, 3, 4, 4c, 5), E SX1, GSC, GSC2, HESX1, HOPX, ISX, LEUTX, MIXL1, NOBOX, OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, Examples of such nucleotides include RHOXF1, RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, VSX2, BARHL1, BARHL2, BARX1, BARX2, BSX, DBX1, DBX2, EMX1, EMX2, EN1, EN2, HHEX, HLX1, LBX1, LBX2, MSX1, MSX2, NANOG, NOTO, TLX1, TLX2, TLX3, TSHZ1, TSHZ2, TSHZ3, VAX1, VAX2, VENTX, NKX2-1, NKX2-4, NKX2-2, NKX2-8, NKX3-1, NKX3-2, NKX2-3, NKX2-5, NKX2-6, HMX1, HMX2, HMX3, NKX6-1, NKX6-2, and NKX6-3.

[0106] Optionally, one or more target genes can include GATA gene. GATA gene is a transcription factor characterized by its ability to bind to the DNA sequence "GATA". Non-limiting examples of GATA gene can include GATA1, GATA2, GATA3, GATA4, GATA5 and GATA6.

[0107] In some cases, the use of heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) into hematopoietic cells, whereby at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the resulting cells generated by using the heterologous genetic circuits disclosed herein are of the target cell type. The target cell type can include T cells, natural killer cells, B cells, dendritic cells, macrophages, or other hematopoietic cells.

[0108] In some cases, the use of heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) into immune cells (T cells, NK cells, etc.) in the absence of, for example, one, two, or all of feeder cells, serum, and exogenous Notch. Using the heterologous genetic circuits disclosed herein, it is possible to differentiate at least about 1×10 4 , at least about 2×10 4 , at least about 5×10 4 , at least about 1×10 5 , at least about 2×10 5 , at least about 5×10 5 , at least about 1×10 6 , at least about 2×10 6 , at least about 5×10 6 , at least about 1×10 7 , at least about 2×10 7 , at least about 5×10 7 , at least about 1×10 8 , at least about 2×10 8 , at least about 5×10 8 , at least about 1×10 9 , at least about 2×10 9 , at least about 5×10 9 , at least about 1×10 10 , at least about 2×10 10 , at least about 5×10 10 , at least about 1×10 15, at least about 2×10 15 , at least about 5×10 15 , or more T cells, up to approximately 1 x 10 6 , up to about 9×10 5 , up to about 8×10 5 , up to about 7×10 5 , up to about 6×10 5 , up to about 5×10 5 , up to about 4×10 5 , up to about 3×10 5 , up to about 2×10 5 , up to about 1×10 5 , up to about 5×10 4 , up to about 2×10 4 , up to about 1×10 4 , or a smaller number of EMP cells (e.g., human EMP cells).

[0109] Generation of such T cells by using the heterologous genetic circuits disclosed herein can be accomplished in a span of up to about 60 days, up to about 55 days, up to about 50 days, up to about 45 days, up to about 40 days, up to about 35 days, up to about 30 days, up to about 25 days, up to about 20 days, up to about 15 days, up to about 10 days, up to about 5 days, or less. The resultant T cells generated by using the heterologous genetic circuits disclosed herein have the ability to produce T cells that are effective when confirmed (e.g., in vitro, in vivo) at an effector to target (E:T) ratio of at least about 1:3, at least about 1:4, at least about 1:5, at least about 1:6, at least about 1:7, at least about 1:8, at least about 1:9, at least about 1:10, at least about 1:12, at least about 1:15, at least about 1:20, at least about 1:30, at least about 1:40, at least about 1:50, or less (e.g., an E:T ratio of 1:100 is less than an E:T ratio of 1:50). In vivo), the resulting T cells generated by using the heterologous genetic circuits disclosed herein can induce at least about 30%, at least about 40%, 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 more of target cells (e.g., cancer cells). The resulting T cells generated by using the heterologous genetic circuits disclosed herein, when injected into a mouse, can extend the mouse lifespan by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more.

[0110] In some cases, T cells generated by this method have a greater killing function than T cells obtained by directed differentiation. T cells generated using the provided methods can have a killing function that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% greater than the killing function of T cells obtained by directed differentiation. Alternatively or additionally, T cells generated by this method can have comparable killing function compared to primary peripheral blood-derived T cells.

[0111] In some cases, the generated T cells other than primary peripherally derived T cells have the same killing ability as primary peripherally derived T cells measured through CAR activity.T cell types can include, but are not limited to, gamma-delta T cells, NKT cells, and MAIT cells.In some cases, the use of heterologous genetic circuits disclosed herein can be used to obtain cells with non-native phenotypes (e.g., non-T cells with killing ability).

[0112] In some cases, the use of heterologous genetic circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) can induce differentiation of stem cells into immune cells in the absence of feeder cells. In some cases, the use of heterologous genetic circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) can induce differentiation of stem cells into immune cells in the absence of serum. In some cases, the use of heterologous genetic circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) can induce differentiation of stem cells into immune cells in the absence of exogenous Notch. In some cases, the use of heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) into T cells, e.g., in the absence of one or both of embryoid bodies and serum. The resulting T cells generated by using the heterologous genetic circuits disclosed herein are at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population. In some cases, the use of the heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) into natural killer cells, for example, in the absence of one or both of embryoid bodies and serum. The resulting natural killer cells generated by using the heterologous genetic circuits disclosed herein are at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population.

[0113] In some cases, the use of heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) into B cells. The resulting B cells generated by using the heterologous genetic circuits disclosed herein are at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population after differentiation.

[0114] In some cases, the use of heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) into dendritic cells. The resulting dendritic cells generated by using the heterologous genetic circuits disclosed herein are at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population after differentiation.

[0115] In some cases, the use of the heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), subpathogenic progenitor cells, hematopoietic progenitor cells) into macrophages. The resulting macrophages generated by using the heterologous genetic circuits disclosed herein are similar to the naive cell state in their ability to detect, phagocytose, and destroy harmful cells, as measured by their ability to act as a surrogate for a CAR or by their ability to kill cancerous cells.

[0116] In some cases, the target gene may be subjected to at least two separate regulation, including a first regulation and a second regulation. The timing of the first regulation and the second regulation can be controlled (e.g., as predetermined by the design of the heterologous genetic circuit). For example, the initiation of the second regulation (e.g., by at least a portion of the second gate unit, such as the second gene regulatory portion) can be at least about 1 second, at least about 2 seconds, at least about 3 seconds, at least about 4 seconds, at least about 5 seconds, at least about 6 seconds, at least about 7 seconds, at least about 8 seconds, at least about 9 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes ... minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least about 10 minutes, at least This can occur in at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days. The initiation of this second regulation (e.g., by at least a portion of a second gating unit, such as a second gene regulatory portion) may occur up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, up to about 1 day, up to about 20 hours, up to about 10 hours, up to about 9 hours, up to about 8 hours, up to about 7 hours, up to about 6 hours, up to about 10 ... About 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1 minute, about 50 seconds, about 40 seconds, about 30 seconds, about 20 seconds, about 10 seconds, about 9 seconds, about 8 seconds, about 7 seconds, about 6 seconds, about 5 seconds, about 4 seconds, about 3 seconds, about 2 seconds. Or it can occur in about 1 second.

[0117] In some cases, the number of gate units that need to be activated (e.g., sequentially activated) between the activation of the first regulation by the first gate unit and the subsequent activation of the second regulation by the second gate unit can at least partially determine (e.g., substantially determine) the timing between the first regulation and the second regulation. Upon activation of the first regulation of the target gene by the first gate unit, it may be necessary to activate (e.g., sequentially activate) at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more additional gate units to activate the second gate unit to induce the second regulation. Upon activation of a first regulation of a target gene by a first gating unit, it may be necessary to activate (e.g., sequentially activate) up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 additional gating units to induce a second regulation.

[0118] The cellular outcome can include modulation of multiple target genes. For example, the outcome can include modulation of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more target genes. The outcome can include modulation of up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 target gene. Each of the genes disclosed herein can be subject to at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more modulations. Each of the genes disclosed herein can be subject to up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 modulation. One or more modulations of a target gene (e.g., an endogenous gene) induced by a heterologous genetic circuit of the present disclosure can be artificial modulation (or heterologous modulation) that would not otherwise occur in a cell in the absence of (i) the heterologous genetic circuit and / or (ii) the active portion of the heterologous genetic circuit.

[0119] The multiple gate units can operate sequentially (e.g., each of the multiple gate units is activated sequentially). For example, multiple gate units are activated to activate subsequent multiple gate units. The sequential operation of the gate units can be linear. Alternatively, the sequential operation of the gate units can be routed back to each other as inputs forming a loop. For example, the multiple gate units can induce a feedback loop, such as a positive feedback loop or a negative feedback loop.

[0120] In some embodiments of any one of the systems disclosed herein, the first gating unit can include a first gene regulatory portion that exhibits specific binding to the target gene and can be activatable to induce a first individual regulation. Alternatively or additionally, the first gating unit can include a first gene regulatory portion that exhibits non-specific binding to the target gene and can be activatable to induce a first individual regulation.

[0121] The first individual modulation can induce a change (e.g., an increase or decrease) in the expression and / or activity level of the target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. The first individual modulation can induce a change (e.g., an increase or decrease) in the expression and / or activity level of the target gene by up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, up to about 0.1%, or less.

[0122] The first individual modulation disclosed herein (e.g., induced by a first gating unit) can be at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least or at most about 0.6-fold, at least or at most about 0.7-fold, at least or at most about 0.8-fold, at least or at most about 0.9-fold, at least or at most about 1-fold, at least or at most about 2-fold, at least or at most about 3-fold, at least or at most about 4-fold, at least or at most about A change (e.g., an increase or decrease) of at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold can be induced. The first individual modulation can be up to about 10,000-fold or less, up to about 5,000-fold or less, up to about 1,000-fold or less, up to about 500-fold or less, up to about 100-fold or less, up to about 90-fold or less, up to about 80-fold or less, up to about 70-fold or less, up to about 60-fold or less, up to about 50-fold or less, up to about 40-fold or less, up to about 30-fold or less, up to about 20-fold or less, up to about 10-fold or less, with respect to the expression and / or activity level of the target gene compared to the expression and / or activity level of a control. A change (e.g., increase or decrease) of up to about 9-fold or less, up to about 8-fold or less, up to about 7-fold or less, up to about 6-fold or less, up to about 5-fold or less, up to about 4-fold or less, up to about 3-fold or less, up to about 2-fold or less, up to about 1-fold or less, up to about 0.9-fold or less, up to about 0.8-fold or less, up to about 0.7-fold or less, up to about 0.6-fold or less, up to about 0.5-fold or less, up to about 0.4-fold or less, up to about 0.3-fold or less, up to about 0.2-fold or less, or up to about 0.1-fold or less can be induced.

[0123] Subsequently, the second individual modulation disclosed herein (e.g., induced by a second gating unit) can have a magnitude of at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about Further alterations (e.g., increases, decreases, or selective attenuations) of 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, or at least about 1,000,000% can be induced. The second individual modulation can be up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, Further changes (e.g., increases or decreases) of up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, or up to about 0.1% can be induced.

[0124] The further change due to the second individual modulation may be at least or at most about 0.1 fold, at least or at most about 0.2 fold, at least or at most about 0.3 fold, at least or at most about 0.4 fold, at least or at most about 0.5 fold, at least or at most about 0.6 fold, at least or at most about 0.7 fold, at least or at most about 0.8 fold, at least or at most about 0.9 fold, at least or at most about 1 fold, at least or at most about 2 fold, at least or at most about 3 fold, at least or at most about 4 fold, at least or at most about 5 fold, at least or at most about 6 fold, Further changes (e.g., increases or decreases) of at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold can be induced. The second individual modulation is, relative to the expression and / or activity level of the target gene, up to about 10,000 fold or less, up to about 5,000 fold or less, up to about 1,000 fold or less, up to about 500 fold or less, up to about 100 fold or less, up to about 90 fold or less, up to about 80 fold or less, up to about 70 fold or less, up to about 60 fold or less, up to about 50 fold or less, up to about 40 fold or less, up to about 30 fold or less, up to about 20 fold or less, up to about 10 fold or less, relative to the expression and / or activity level of a control. , up to about 9-fold or less, up to about 8-fold or less, up to about 7-fold or less, up to about 6-fold or less, up to about 5-fold or less, up to about 4-fold or less, up to about 3-fold or less, up to about 2-fold or less, up to about 1-fold or less, up to about 0.9-fold or less, up to about 0.8-fold or less, up to about 0.7-fold or less, up to about 0.6-fold or less, up to about 0.5-fold or less, up to about 0.4-fold or less, up to about 0.3-fold or less, up to about 0.2-fold or less, up to about 0.1-fold or less can be induced.

[0125] Further changes due to the second individual regulation can occur when the expression and / or activity level of the target gene reaches a target level by the action of the first individual regulation, for example by engineering a heterologous genetic circuit.

[0126] The further change due to the second individual modulation may be such that the expression and / or activity level of the target gene is at least or at most about 0.1 fold, at least or at most about 0.2 fold, at least or at most about 0.3 fold, at least or at most about 0.4 fold, at least or at most about 0.5 fold, at least or at most about 0.6 fold, at least or at most about 0.7 fold, at least or at most about 0.8 fold, at least or at most about 0.9 fold, at least or at most about 1 fold, at least or at most about 2 fold, at least or at most about 3 fold, at least or at most about 4 fold, at least or at most about 5 fold, at least or at most about 6 fold, at least or at most about 7 fold, at least or at most about 8 fold, at least or at most about 9 fold, at least or at most about 10 fold, at least or at most about 11 fold, at least or at most about 12 fold, at least or at most about 13 fold, at least or at most about 14 fold, at least or at most about 15 fold, at least or at most about 16 fold, at least or at most about 17 fold, at least or at most about 18 fold, at least or at most about 19 fold, at least or at most about 20 fold, at least or at most about 21 fold, at least or at most about 22 fold, at least or at most about 23 fold, at least or at most about 24 fold, at least or at most about 25 fold, at least or at most about 26 fold, at least or at most about 27 fold, at least or at most about 28 fold, at least or at most about 29 fold, at least or at most about 30 fold, at least or at most about 31 fold, at least or at most about or may occur when the change (e.g., increase or decrease) is at least about 6-fold, at least or at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 60-fold, at least or at most about 70-fold, at least or at most about 80-fold, at least or at most about 90-fold, at least or at most about 100-fold, at least or at most about 500-fold, at least or at most about 1,000-fold, at least or at most about 5,000-fold, or at least or at most about 10,000-fold. The further change due to the second individual modulation may be that the expression and / or activity level of the target gene is increased by the effect of the first individual modulation by up to about 10,000 fold or less, up to about 5,000 fold or less, up to about 1,000 fold or less, up to about 500 fold or less, up to about 100 fold or less, up to about 90 fold or less, up to about 80 fold or less, up to about 70 fold or less, up to about 60 fold or less, up to about 50 fold or less, up to about 40 fold or less, up to about 30 fold or less, up to about This may occur when the concentration changes (e.g., increases or decreases) by 20-fold or less, a maximum of about 10-fold or less, a maximum of about 9-fold or less, a maximum of about 8-fold or less, a maximum of about 7-fold or less, a maximum of about 6-fold or less, a maximum of about 5-fold or less, a maximum of about 4-fold or less, a maximum of about 3-fold or less, a maximum of about 2-fold or less, a maximum of about 1-fold or less, a maximum of about 0.9-fold or less, a maximum of about 0.8-fold or less, a maximum of about 0.7-fold or less, a maximum of about 0.6-fold or less, a maximum of about 0.5-fold or less, a maximum of about 0.4-fold or less, a maximum of about 0.3-fold or less, a maximum of about 0.2-fold or less, or a maximum of about 0.1-fold or less.

[0127] Alternatively or additionally, the second individual regulation disclosed herein (e.g., induced by a second gating unit) may be at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 15 ... %, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, or at least about 1,000,000% change (e.g., increase or decrease). The second individual modulation can be up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 150%, up to about 200%, up to about 300%, up to about 350%, up to about 400%, up to about 500%, up to about 500%, up to about 600%, up to about 700%, up to about 800%, up to about 9 ... The method can induce a change (e.g., increase or decrease) of up to about 0.00%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, or up to about 0.1%. The cell can include a prokaryotic cell, a eukaryotic cell, or an engineered cell.

[0128] The cells (e.g., initial cells that are modified into engineered cells disclosed herein, final cell products generated from engineered cells disclosed herein, etc.) can include muscle cells, immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells, epithelial cells, stem cells, secretory cells, blood cells, germ cells, nurse cells, storage cells, enteroendocrine cells, pituitary cells, neurosecretory cells, duct cells, odontoblasts, cementoblasts, glial cells, or stromal cells.

[0129] Non-limiting examples of such cells include lymphoid cells, such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, cytokine-induced killer (CIK) cells (see, e.g., U.S. Patent Application Publication No. 20080241194); myeloid cells, such as granulocytes (basophilic granulocytes, eosinophilic granulocytes, neutrophilic granulocytes / hypersegmented neutrophils), monocytes / macrophages, red blood cells (reticulocytes), mast cells, monocytes / macrophages, dendritic cells; thyroid (thyroid epithelial cells, follicular cells), Cells from the endocrine system, including parathyroid (chief parathyroid cells, eosinophil cells), adrenal (chromaffin cells), and pineal (pinealocyte) cells; cells of the nervous system, including glial cells (astrocytes, microglia), giant cell neurosecretory cells, stellate cells, Bechtel cells, and pituitary (gonadotropes, corticotropes, thyrotropes, somatotropes, and lactotropes); cells of the respiratory system, including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, and dust cells; myocardial cells and pericytes. cells of the circulatory system including; 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; enteroendocrine cells including enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage / bone / muscle; bone cells including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts); chondrocytes including chondroblasts and chondrocytes; skin cells (nevus cells) including hair follicle cells, keratinocytes, and melanocytes; muscle cells including myocytes; podocytes, juxtamembrane cells Urinary system cells including glomerular cells, intraglomerular / extraglomerular mesangial cells, renal proximal tubule brush border cells, macula densa cells; germ line cells including sperm, Sertoli cells, Leydig cells, and eggs; as well as adipocytes, fibroblasts, tendon cells, epithelial keratinocytes (differentiated epithelial cells), epithelial basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, reticular hair stem cells, reticular root sheath cells, root sheath cells of the Hussuri layer, root sheath cells of the Henle layer, outer root sheath cells, hair matrix cells (stem cells), wet stratified barrier cells,Other cells include epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, urothelial cells (lining the bladder and ureters), exocrine epithelial cells, salivary gland mucous cells (secretes rich in polysaccharides), salivary gland serous cells (secretes rich in glycoprotein enzymes), von Ebner's gland cells of the tongue (wash the taste buds), mammary gland cells (milk secretions), lacrimal gland cells (tear secretions), skin gland cells of the ear (wax secretions), eccrine sweat gland dark cells (secretes glycoproteins), eccrine sweat gland clear cells (secretes small molecules). Apocrine sweat gland cells (odor secretion, sex hormone sensitive), Moll's gland cells of the eyelid (specialized sweat gland), sebaceous gland cells (secreting lipid-rich sebum), Bowman's gland cells of the nose (cleansing the olfactory epithelium), Brunner's gland cells of the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting seminal fluid, including fructose necessary for sperm swimming), prostate cells (secreting seminal fluid), bulbourethral gland cells (secreting mucus), Bartholin's gland cells (secreting vaginal lubrication), Littré gland cells (secreting mucus), endometrial cells (secreting carbohydrates), isolated goblet cells of the respiratory and digestive tracts (secreting mucus), mucous cells of the stomach lining (secreting mucus), gastric gland enzyme-producing cells (secreting pepsinogen), gastric gland acid-secreting cells (secreting hydrochloric acid), pancreatic acinar cells (secreting bicarbonate and digestive enzymes), Paneth cells of the small intestine (secreting lysozyme), type II alveolar epithelium of the lungs cells (surfactant secretion), Clara cells of the lung, hormone secreting cells, anterior pituitary cells, growth hormone secreting cells, lactotrophic cells, thyrotrophic cells, gonadotrophic cells, corticotrophic cells, intermediate pituitary cells, giant cell neurosecretory cells, cells of the digestive tract and respiratory system, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, acid secreting cells, adrenal cells, chromaffin cells, Leydig cells of the testis, luteal membranous cells of the follicle, luteal cells of ruptured follicles, granulosa luteal cells, membranous luteal cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lungs, digestive tract, exocrine glands and urogenital tract), kidney, type I alveolar epithelial cells (lining of air spaces in the lungs), pancreatic duct cells (central acinar cells), nonstriated duct cellscell) (sweat glands, salivary glands, mammary glands, etc.), duct cells (seminal vesicles, prostate, etc.), epithelial cells lining closed internal body cavities, ciliated cells with propulsive functions, extracellular matrix secreting cells, contractile cells. Skeletal muscle cells, stem cells, cardiac muscle cells, blood and immune system cells, red blood cells, megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglial cells (in the central nervous system), neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and differentiated precursors of the blood and immune system (committed progenitors (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transduction cells, autonomic nerve cells, sensory and peripheral nerve supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, sperm cells, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, nurse cells, ovarian follicle cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, and interstitial kidney cells.

[0130] The stem cells can include induced pluripotent stem cells (iPSCs), pluripotent stem cells (PSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), erythromyeloid progenitor cells (EMPs), sub-pathogenic progenitor cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells, muscle stem cells, neural stem cells, epithelial stem cells, epidermal stem cells, mammary stem cells, intestinal stem cells, neural crest stem cells, or testicular stem cells.

[0131] Various embodiments of the present disclosure provide engineered cells that are programmed to induce a desired expression and / or activity level (or profile) of one or more target genes in the cell.

[0132] In some embodiments, the engineered cells (e.g., engineered muscle cells, engineered immune cells) of the present disclosure can be generated from isolated stem cells (e.g., isolated ESCs, iPSCs, EMPs, etc.). The heterologous genetic circuits and / or components thereof (e.g., gating units, gating moieties, activation moieties, etc.) disclosed herein can be introduced during any stage (or cell state) between and including (a) the isolated stem cell and (b) its differentiated immune cell state (e.g., a terminally differentiated immune cell state such as a terminally differentiated T cell).

[0133] In some cases, the engineered cell, a T cell, can be derived from an EMP, and the heterologous genetic circuit and / or components thereof (e.g., heterologous gating units, heterologous activation moieties, heterologous gating moieties, etc.) can be introduced into the cell at (A) an EMP state, (B) a hematopoietic stem cell state, (C) a T cell state, or (D) any other intermediate cell state. The heterologous genetic circuit and / or components thereof (e.g., heterologous gating units, heterologous activation moieties, heterologous gating moieties, etc.) can be introduced into the cell once during one of (A), (B), (C), or (D). Alternatively, the heterologous genetic circuit and / or components thereof (e.g., heterologous gating units, heterologous activation moieties, heterologous gating moieties, etc.) can be introduced into the cell multiple times during two, three, or all of (A), (B), (C), or (D).

[0134] The engineered cells (e.g., engineered T cells) of the present disclosure can be used (e.g., administered) to treat a subject in need of treatment. The subject can have or be suspected of having a condition, such as a disease (e.g., cancer). Cells (e.g., stem cells or differentiated cells) can be obtained from the subject, and such cells can be cultured ex vivo and genetically modified to generate engineered cells (e.g., any immune cell) of any of the subject matters disclosed herein. The engineered immune cells can then be administered to the subject for adaptive immunotherapy. Thus, the engineered cells can be autologous to the subject in need thereof. Alternatively, the engineered cells can be allogeneic to the subject (e.g., allogeneic stem cell transplantation, allogeneic adaptive immunotherapy, etc.).

[0135] The cells or constructs of the present disclosure can be used (e.g., administered) in a pharmaceutical preparation. The pharmaceutical preparation can further include an additional therapeutic agent. The additional therapeutic agent can include a chemotherapeutic agent, an immunosuppressant, and / or an antibiotic.

[0136] Chemotherapeutic agents, also known as antitumor agents, are a type of cancer treatment that is used to directly or indirectly inhibit the growth and proliferation of cancer cells.Chemotherapeutic agents can be alkylating agents (e.g., oxazaphosphorines, nitrogen mustards, imidazotetrazines, nitrosourea, alkylsulfonates, hydrazines, or platinum-based agents), antimetabolites (e.g., antifolates, pyrimidine antagonists, purine antagonists, or ribonuclease reductase inhibitors), topoisomerase inhibitors (e.g., topoisomerase I inhibitors or topoisomerase II inhibitors), antibiotics (e.g., bleomycin, actinomycin D, anthracyclines, or mitomycin), mitotic inhibitors (e.g., vinca alkaloids, taxanes, or nonataxanes microtubule inhibitors), protein kinase inhibitors (e.g., tyrosine kinase inhibitors, MEK inhibitors, CDK inhibitors), proteasome inhibitors, or PARP inhibitors.

[0137] Immunosuppressants are drugs that reduce immune response.Non-limiting examples of immunosuppressants can include steroids (e.g., prednisone, methylprednisolone, dexamethasone), colchicine, hydroxychloroquine, sulfasalazine, dapsone, methotrexate, mycophenolate mofetil, azathioprine, anti-IL-1 biologics, anti-TNF biologics, anti-IL-6 biologics, B cell growth factor targeting biologics, T cell, cytokine, or JAK inhibitors.

[0138] Antibiotics are drugs that destroy or inhibit the growth of microorganisms. Non-limiting examples of antibiotic agents include tetracycline, oxytetracycline, methacycline, doxycycline, minocycline, erythromycin, lincomycin, penicillin G, clindamycin, kanamycin, chloramphenicol, fradiomycin, streptomycin, norfloxacin, ciprofloxacin, ofloxacin, grepafloxacin, levofloxacin, sparfloxacin, ampicillin, fusidic acid, ciprofloxacin, and other quinolones, erythromycin, gentamicin, sulfonamides, trimethoprim, dapsone, and the like. In some embodiments, the medicaments may include, but are not limited to, isoniazid, teicoplanin, avoparcin, cinarcid, virginiamycin, piperacillin, ticarcillin, cefepime, cefpirome, rifampicin, pyrazinamide, enrofloxacin, amikacin, netilmicin, imipenem, meropenem, inezolid, cefuroxime, ceftriaxone, cefadroxil, cefazolin, ceftazidime, cefotaxime, roxithromycin, cefaclor, cephalexin, cefoxitin, amoxicillin, co-amoxiclav, mupirocin, cloxacillin, and cotrimoxazole.

[0139] The pharmaceutical formulation may further comprise an excipient, which may be a buffer, carrier, stabilizer, solubilizer, filler, preservative, diluent, vehicle, detergent, salt, peptide, surfactant, oligosaccharide, amino acid, adjuvant, carbohydrate, and / or bulking agent.

[0140] The engineered cells disclosed herein can be administered to a subject prior to, concurrently with, or after activation of a heterologous genetic circuit in the engineered stem cells. For example, the engineered cells can be activated after administration to a subject, for example, by administering an activator of a heterologous genetic circuit to the subject.

[0141] A subject can be treated with (e.g., administered) a collection of engineered cells (e.g., engineered T cells) of the present disclosure for at least or at most about 1 dose, at least or at most about 2 doses, at least or at most about 3 doses, at least or at most about 4 doses, at least or at most about 5 doses, at least or at most about 6 doses, at least or at most about 7 doses, at least or at most about 8 doses, at least or at most about 9 doses, or at least or at most about 10 doses. Alternatively or additionally, a subject can be treated with (e.g., administered with) a collection of engineered cells (e.g., engineered T cells) of the present disclosure for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, or 100 years.

[0142] Any one of the methods disclosed herein can be utilized to treat a target cell, target tissue, target condition, or target disease in a subject.

[0143] Non-limiting examples of target tissues include cells that can be obtained from a subject, such as immune cells (e.g., lymphocytes, including T cells and NK cells). Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Examples of samples from a subject from which cells may be derived include, but are not limited to, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, feces, semen, vaginal fluid, interstitial fluid from tumor tissue, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, plasma, nasal swabs or nasopharyngeal washings, spinal fluid, cerebrospinal fluid, tissue, throat swabs, biopsies, placental fluid, amniotic fluid, umbilical cord blood, lung fluid, cavity fluid, sputum, pus, microbiota, meconium, breast milk, and / or other excretions or bodily tissues.

[0144] The target disease of interest can be cancer or tumor.Non-limiting examples of cancer can include cancer cells, including acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrohidroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, mature acute myeloblastic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, malignant NK cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastic fibrosis, and leukemia. tumor, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, carcinoma of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, carcinoma of unknown primary site, carcinosarcoma, Castleman disease, central nervous system embryonal tumor, small Brain astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid Sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing family tumors, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, inclusion fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, 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, cardiac cancer, hemangioblastoma, hemangiopericytoma, hemangiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast cancer-ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, pancreatic islet cell carcinoma, pancreatic islet cell tumor, juvenile myelomonocytic leukemia, kapo Ziz sarcoma, Kaposi's sarcoma, kidney cancer, Kratoskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, lentigo maligna melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve tumor ... sheath tumor, malignant rhabdoid tumor, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, recurrent metastatic cervical squamous cell carcinoma of unknown primary, metastatic urothelial carcinoma, mixed mullerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine tumor Tumor syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, tumor, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular oncology oncology), oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, multiple embryos, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primary neuroectodermal tumor, prostate cancer,Pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory cancer associated with the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannoma disease, sebaceous gland carcinoma, secondary tumors, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord stromal tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, sooty mold, spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading malignant melanoma, supratentorial primitive These include neuroectodermal tumors, surface epithelial stromal tumors, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoid cancer, testicular cancer, meningioma, pharyngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, ureteral cancer, urethral cancer, genitourinary tumors, uterine sarcoma, uveal melanoma, vaginal cancer, Werner-Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subset within a cancer cell population, such as cancer stem cells. In some embodiments, the cancer is a cancer of the hematopoietic system, such as lymphoma. The antigen can be a tumor-associated antigen.

[0145] The present disclosure also provides a composition comprising the engineered genetic circuit disclosed herein. The composition may further comprise an activator of a heterologous genetic circuit. The present disclosure also provides a kit comprising the composition. The kit may further comprise an activator of a heterologous genetic circuit. The activator may be in the same composition as the engineered cells. Alternatively or additionally, the activator may be in a separate composition different from the engineered cells. EXAMPLES

[0146] Example 1: T cell differentiation

[0147] In this prospective example, we test the CRISPR all-at-once approach against a variety of heterologous genetic circuits (HGCs) and show that the performance of HGCs is superior to that of the CRISPR all-once approach.

[0148] Figure 4 shows the nine gRNAs (E2A, Ikaros, Runx1, Lmo2, PU.1, Hes1, Tcf7, Gata3, and Bcl11b) used in the CRISPR all-at-once method. In the CRISPR all-in-one method, gRNAs are added simultaneously to cells by nucleofection. Cells are incubated until T cell differentiation is complete.

[0149] Figure 5A-Figure 5I show the T cell HGCs (T cell HGCs 1-9) used in these experiments. In the HGC method, the activation portion of the HGC is added to cells by nucleofection. The cells are incubated until T cell differentiation is complete.

[0150] FIG. 5A shows an example of an engineered cell in contact with T cell HGC1. The engineered cell can be activated to show a profile of expression and activity of multiple genes associated with T cell differentiation. T cell HGC1 contains four gate units that contain four different steps. Gate unit 1 of HGC1 activates genes E2A, Ikaros, Runx1, Lmo2, and PU1 to activate gate unit 2. Gate unit 2 of HGC1 activates only gate unit 3, which acts as a hold phase used as a filler to aid in development. Gate unit 3 of HGC1 activates genes Hes1, Tcf7, Gata3, and Bcl11b, which in turn activates gate unit 4 to negatively regulate the early activated genes Lmo2 and PU1. FIG. 6 shows how the four Cellgorithm steps work together to achieve differentiation from starter cells to T cells. Gating unit 1 of T cell HGC1 can be activated by injection containing a CRISPR sequence aligned to activated gating unit 1. Once the first gating unit is activated, the celgorhythm proceeds without any external activation until the heterologous genetic circuit is completed (e.g., gating unit 4 / step 4). The result is an engineered T cell. Other celgorhythms not shown here function by having a third gating unit reinforce the expression and activity profile of genes regulated by the first gating unit.

[0151] One control cell population is generated using standard T cell culture methods. iPSCs are formed into embryoid bodies and grown for 12 days in medium containing BMP-4, VEGF, and bFGF. On day 12, CD34+ cells are purified using magnetic beads and seeded onto DLL4-coated plates. Cells are grown for 21 days in medium containing TPO, IL-7, SDF, FLT3L, and SCF. On day 21, cells are stimulated with CD3 antibody in medium containing H-7, IL-2, DXM to allow for the proliferation of mature T cells. Another control T cell population is collected and purified from mouse blood samples.

[0152] T cells generated by HGC can grow in larger quantities than T cells generated using CRISPR all-at-once methods. T cells generated by HGC show normal signaling, gene regulation, and growth control compared to natural T cells collected from blood samples. Furthermore, unlike cells cultured in medium, T cells generated by HGC do not lose cytotoxic activity over time and may have high killing efficiency (e.g., lower effector-to-target ratio), longer persistence, and better control and interaction with APCs. Through the generation of CD4 cells, the ratio of CD4+ cells to CD8+ cells can also be controlled, affecting the persistence of cells and their ability to interact with other immune cells that kill similar NK cells.

[0153] It should be understood that the various aspects of the present invention can be realized individually, collectively, or in combination with each other. The various aspects of the present invention described herein may be applied to any of the specific applications disclosed herein. The compositions comprising any of the compounds of the formulas disclosed herein in the composition section of the present disclosure may be utilized in the method section including the methods of use and production disclosed herein, or vice versa.

[0154] Example 2: Differentiation of hematopoietic progenitor cells

[0155] In this example, induced pluripotent stem cells (iPSCs) are differentiated into hematopoietic progenitor cells using various heterologous genetic circuits (HGCs).

[0156] Maintenance of iPSCs

[0157] Episomal iPSCs (Gibco, Cat#A18945) were maintained in complete Essential 8™ Flex medium (Gibco, Cat#A2858501) or StemFlex™ medium (Gibco, Cat#A3349401) on flasks coated with Vitronectin (Gibco, Cat#A31804) and passaged to maintain a confluency of 50-90% using Versene (Gibco, Cat#15040066) to lift cells while maintaining cell clumps.

[0158] Nucleofaction

[0159] On experimental day 0, iPSC cells were harvested as a single cell suspension using ACCUTASE™ (Stemcell Technologies, Cat#07920). Cells were resuspended at 1.2e9 cells / ml in P3Primary Cell Nucleofector™ solution and 8e5 cells were nucleofected per well in a 96-well nucleocuvette (Lonza, Cat#V4SP-3096) on a Lonza's 4D Nucleofector with 96-well shuttle (Cat#AAF-1003B / S) using an optimized pulse code.

[0160] Cells were nucleofected with plasmids encoding Cas9-VPR, core cascade, and gene targeting spacer. Cas9-VPR is a Cas transcriptional modulator system that can be used for both cleavage gene regulation (e.g., of the gate portion plasmid, of the gene regulation portion plasmid) and non-cleavage gene regulation (e.g., CRISPR activation of target endogenous genes, CRISPR inhibition of target endogenous genes) depending on the particular gNA used. The activatable gNA used is only a polyT tract, with a constant core cascade that executes four consecutive steps. Nucleotide gNA spacers were utilized to target the genome according to the sequences in Table 1. Figure 8 describes the gene targets used for each heterologous genetic circuit cascade. These steps refer to the order in which genes are expected to be expressed as a result of Cas9-VPR-mediated targeting and activation.

[0161] [Table 1-1]

[0162] [Table 1-2]

[0163] [Table 1-3]

[0164] cell culture

[0165] 1.8e5 nucleofected cells were plated per well at 500 μl / well in 24-well tissue culture plates treated with anti-adhesion rinse solution (Stemcell Technologies, Cat#07010) plates rotated at 100 RPM under standard normoxic conditions in a humidified 5% CO2 tissue culture incubator. Day 0 medium contained: StemPro-34 (Gibco, Cat#10639011), 1X penicillin-streptomycin (Gibco, Cat#15140122), 2mL Glutamax (Gibco, Cat#35050061), 50ug / mL ascorbic acid (Santa Cruz Biotechnology, Cat#sc-39430), 4mM 1-thioglycerol (Sigma-Aldrich, Cat#M1753), 1X insulin-transferrin-selenium (ITS-G) (Gibco, Cat#41400045), and 10mM Y-27632 (Bio-Techne, Cat#1254), 50ng / ml rhBMP4 (Peprotec, Cat#120-05ET), 50ng / ml FGF-2 (Peprotech, Cat#3718-FB), and 10uM CHIR99021 (Bio-Techne, Cat#4423).

[0166] On day 1, cultures were supplemented with StemPro-34 (Gibco, Cat#10639011), 1X penicillin-streptomycin (Gibco, Cat#15140122), 2mL Glutamax (Gibco, Cat#35050061), 50ug / mL ascorbic acid (Santa Cruz Biotechnology, Cat#sc-39430), 4mM 1-thioglycerol (Sigma-Aldrich, Cat#M1753), 1X insulin-transferrin-selenium (ITS-G) (Gibco, Cat#41400045), 50ng / ml rhBMP4 (Peprotec, Cat#120-05ET), 50ng / ml FGF-2 (Peprotech, Cat#3718-FB), and 50ng / ml The medium was replaced with complete medium containing VEGF-165 (Peprotech, Cat#100-20). On day 2, 6 uM SB431542 (Selleckchem, Cat#101762-616) is added to the cultures. On day 4, cultures were supplemented with StemPro-34 (Gibco, Cat#10639011), 1X penicillin-streptomycin (Gibco, Cat#15140122), 2mL Glutamax (Gibco, Cat#35050061), 50ug / mL ascorbic acid (Santa Cruz Biotechnology, Cat#sc-39430), 4mM 1-thioglycerol (Sigma-Aldrich, Cat#M1753), 1X insulin-transferrin-selenium (ITS-G) (Gibco, Cat#41400045), 50ng / ml FGF-2 (Peprotech, Cat#3718-FB), 50ng / ml VEGF-165 (Peprotech, Cat#100-20) and 50ng / ml The medium was replaced with one containing SCF (Peprotech, Cat#300-07).

[0167] Flow cytometry

[0168] Cultures were analyzed by flow cytometry on a Penteon (Agilent, Cat#2010284AA) on days 3 and 5. Embryoid bodies were harvested and placed into 96 deep-well plates (Nest, Cat#503162) and disaggregated in 500ml TrypLE™ Express Enzyme (Gibco, Cat#12605028) for 20-30 minutes at 37°C before being mechanically disrupted by pipetting. TrypLE™ Express Enzyme was quenched with DMEM-F12 (Stemcell Technologies, Cat#36254). Cells were stained with Zombie Aqua™ Fixable (Biolegend, Cat#423102, 1:2000) for 20 minutes at room temperature, washed with 1XDPBS, 2% FBS, 0.02% NaN3, and blocked with human IgG (Lee Biosolutions, Cat#340-21) for 10 minutes on ice. Cells were stained for 30 min on ice with PDGFR-BV421 (BD Biosciences, Cat#562799, 1:400), CD73-BV785 (Biolegend, Cat#344028, 1:400), CD144 / VE-cadherin-PE (Biolegend, Cat#348505, 1:100), CD34-PE-Dazz (Biolegend, Cat#343534, 1:400), CD309-PE-Cy7 (Biolegend, Cat#359912, 1:100), CD43-APC (Biolegend, Cat#343206, 1:800), CD45-AF700 (Biolegend, Cat#304024, 1:200). Cells were washed with 1XDPBS, 2% FBS, 0.02% NaN3. For analysis, cells were resuspended in 1XDPBS, 2% FBS, 0.02% NaN3. Data was analyzed using FlowJo™ v10.8 software (BD Life Sciences). Dead cells and doublets were excluded from analysis with Zombie Aqua and doublet discrimination gates.

[0169] Flow cytometry data were compiled to form Figures 10A-D, which depict the relative frequency of cells in appropriately sized live singlets at day 5 of culture. Scatter plots were generated at various time points to visualize the magnitude of the increase in hematopoietic gene expression induced by HGCs over controls (Figure 9).

[0170] Exemplary results of flow cytometry analysis showing the frequency of cells expressing the indicated surface markers in live singlet cells on day 5 of culture for HSC(Cergorhythm) #7 and #12 are shown in Figures 11A-11B compared to no DNA controls (nucleofected in the absence of exogenous DNA). Summary graphs are shown in Figures 11C-11D.

[0171] Embodiment The following non-limiting embodiments provide illustrations of the invention, but do not limit the scope of the invention.

[0172] Embodiment 1. A method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising: contacting a cell with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in concert to sequentially induce a plurality of distinct modulations of a target gene, each of the plurality of distinct modulations being required but individually insufficient to achieve a desired expression and / or activity profile of the target gene, and wherein the plurality of gating units: (i) a first gating unit that is activatable to induce a first distinct regulation of the plurality of distinct regulations upon activation of the heterologous genetic circuit; (ii) a second gating unit that is activatable to induce a second individual regulation of the plurality of individual regulations upon activation of the heterologous genetic circuit, the second individual regulation being induced after the first individual regulation such that the first individual regulation and the second individual regulation together increase or both decrease the expression and / or activity level of the target gene in the cell. During the contacting step, the gating units operate in concert to achieve a desired expression and / or activity profile of the target genes in the cell; Optionally, (1) the method further comprises contacting the cell with an activating moiety to activate the heterologous genetic circuit; and / or (2)(i) the first gating unit comprises a first gene regulatory portion that is activated upon activation of the first gating unit to induce a first individual regulation via specific binding of the first gene regulatory portion to a target gene; (ii) the second gating unit comprises a second gene regulatory portion that is activated upon activation of the second gating unit to induce a second individual regulation via specific binding of the first gene regulatory portion to a target gene; and / or (3) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to substantially the same polynucleotide sequence of the target gene; and / or (4) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to different polynucleotide sequences of the target gene; and / or (5) the second gating unit further comprises a second gating portion that is activated upon activation of the second gating unit to induce activation of the second gene regulatory portion via specific binding of the second gating portion to the second gene regulatory portion; Optionally, (i) the first gating unit further comprises a first gating moiety that is activated upon activation of the first gating unit to induce (a) activation of the first gene regulatory moiety via specific binding of the first gating moiety to the first gene regulatory moiety, and (b) activation of the second gating moiety via specific binding of the first gating moiety to the second gating moiety; and / or (ii) the activating moiety is capable of inducing (a) activation of the first gene regulatory moiety through specific binding of the activating moiety to the first gene regulatory moiety, and (b) activation of the second gating moiety; and / or (6) the first individual regulation and the second individual regulation together increase the expression and / or activity level of the target gene; and / or (7) the first individual regulation and the second individual regulation together reduce the expression and / or activity level of the target gene; and / or (8) the first gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and the second gating unit is activatable to selectively induce a second individual regulation of the target gene without modulating the expression and / or activity profile of the additional target gene; and / or (9) The method, wherein the second gating unit is activatable to modulate the expression and / or activity profile of the additional target gene, and the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene.

[0173] Embodiment 2. A method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising: contacting a cell with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in concert to sequentially induce a plurality of distinct modulations of a target gene, each of the plurality of distinct modulations being required but individually insufficient to achieve a desired expression and / or activity profile of the target gene, and wherein the plurality of gating units: (i) a first gating unit that is activatable to induce a first distinct regulation of the plurality of distinct regulations upon activation of the heterologous genetic circuit; (ii) a second gating unit that is activatable to induce disruption of the activated first gating unit, the second gating unit being inactivated to induce a second individual regulation of the plurality of individual regulations, the second individual regulation being induced after the first individual regulation to attenuate the first individual regulation; During the contacting step, the gating units operate in concert to achieve a desired expression and / or activity profile of the target genes in the cell; Optionally, (1) the method further comprises contacting the cell with an activating moiety to activate the heterologous genetic circuit; and / or (2)(i) the first gating unit comprises a first gene regulatory portion that is activated upon activation of the first gating unit to induce a first individual regulation via specific binding of the first gene regulatory portion to a target gene; (ii) the second gating unit comprises a second gene regulatory portion that is activated upon activation of the second gating unit to induce a second individual regulation via specific binding of the first gene regulatory portion to a target gene; and / or (3) the second gating unit further comprises a second gating portion that is activated upon activation of the second gating unit and induces activation of the second gene regulatory portion via specific binding of the second gating portion to the second gene regulatory portion; Optionally, (i) the first gating unit further comprises a first gating moiety that is activated upon activation of the first gating unit to induce (a) activation of the first gene regulatory portion via specific binding of the first gating portion to the first gene regulatory portion, and (b) activation of the second gating portion via specific binding of the first gating portion to the second gating portion; Additionally, optionally, (A) upon activation of the second gene regulatory portion, the second gene regulatory portion induces the destruction of the first gating unit through the destruction of the first gating portion and / or the first gene regulatory portion; and / or (B) the disruption comprises modifying a polynucleotide sequence encoding the first gating portion and / or the first gene regulatory portion; and / or (ii) the activating portion is capable of inducing (a) activation of the first gene regulatory portion via specific binding of the activating portion to the first gene regulatory portion, and (b) activation of the second gating portion; Additionally, optionally, (A) upon activation of the second gene regulatory portion, the second gene regulatory portion induces the destruction of the first gating unit through the destruction of the first gene regulatory portion; and / or (B) the disruption comprises modifying a polynucleotide sequence encoding the first gene regulatory portion; and / or (4) the first gating unit is activatable to modulate the expression and / or activity profile of the additional target gene, and the second gating unit is activatable to selectively induce a second individual regulation of the target gene without modulating the expression and / or activity profile of the additional target gene; and / or (5) the second gating unit is activatable to modulate the expression and / or activity profile of the additional target gene, and the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene; and / or (6) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to substantially the same polynucleotide sequence of the target gene; and / or (7) A method, wherein the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to different polynucleotide sequences of the target gene.

[0174] Embodiment 3. Optionally, (1) the target gene comprises multiple target genes; and / or (2) a second individual regulation induces selective regulation of one of the plurality of target genes but not the remainder of the plurality of target genes; and / or (3) the activating moiety comprises a guide nucleic acid (gNA) capable of forming a complex with an endonuclease, the gNA being capable of binding to at least a portion of the heterologous genetic circuit, thereby activating the heterologous genetic circuit; and / or (4) each of the first gating unit, the first gating portion, the first gene regulatory portion, the second gating unit, the second gating portion, and / or the second gene regulatory portion comprises an activatable gNA; Upon activation of gNA, gNA forms a complex with an endonuclease, and / or (5) (i) the activatable gNA comprises a self-cleaving gNA, and / or (ii) the activatable gNA comprises a non-canonical termination sequence, and / or (6) the endonuclease contains a CRISPR / Cas protein; Optionally, the CRISPR / Cas protein is operably linked to a gene activator or a gene repressor, and / or (7) the induction of the second separate adjustment is configured to occur at least about 5 minutes, at least about 1 hour, at least about 6 hours, or at least about 12 hours after the induction of the first separate adjustment; and / or (8) the activation moiety consists of a single activation moiety; and / or (9) upon activation of the second gating unit, the second gating unit is further capable of regulating the expression and / or activity of an additional target gene in the cell; and / or (10) the second gating unit comprises an additional gene regulatory portion that is activated upon activation of the second gating unit to induce regulation of the expression and / or activity of the additional target gene through specific binding of the additional gene regulatory portion to the additional target gene; and / or (11) the target gene is endogenous to the cell; and / or (12) the desired expression and / or activity profile of the target gene induces differentiation of cells towards a target cell type; and / or (13) the target cell type exhibits a target phenotype; and / or (14) The target cell type is a tissue-specific cell, and optionally the tissue-specific cell is selected from the group consisting of immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells, and epithelial cells; and / or (15) the target phenotype comprises (i) the expression or activity level of a target protein, (ii) the average cell size, and / or (iii) a desired function; and / or (16) The target gene encodes a cell differentiation regulator, and / or (17) The cell differentiation regulator comprises a growth factor, and / or (18) The cell differentiation regulator comprises a transcription factor, and / or (19) The cell differentiation regulator comprises an immune cell regulator including E2A, Ikaros, Runx1, Lmo2, PU.1, Hes1, Tcf7, Gata3, and Bcl11b; and / or (20) The cell differentiation regulator comprises a T-box transcription factor (TBX), a basic helix-loop-helix transcription factor (bHLH), a homeobox protein, a forkhead box (FOX), an SRY-related HMG box (SOX), and / or a GATA protein; and / or (20) The method of embodiment 1 or embodiment 2, wherein the stem cells are selected from the group consisting of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), neural stem cells, and epithelial stem cells.

[0175] Embodiment 4. A system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising: A heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in concert to sequentially induce a plurality of distinct modulations of a target gene, each of the plurality of distinct modulations being required but individually insufficient to achieve a desired expression and / or activity profile of the target gene, and wherein the plurality of gating units: (i) a first gating unit that is activatable to induce a first distinct regulation of the plurality of distinct regulations upon activation of the heterologous genetic circuit; (ii) a second gating unit that is activatable to induce a second individual regulation of the plurality of individual regulations upon activation of the heterologous genetic circuit, the second individual regulation being induced after the first individual regulation such that the first individual regulation and the second individual regulation together increase or both decrease expression and / or activity levels of a target gene in the cell; Upon activation of the heterologous genetic circuit, the gating units operate in a coordinated manner to achieve a desired expression and / or activity profile of target genes in the cell; Optionally, (1) the system further comprises an activating moiety for activating a heterologous genetic circuit; and / or (2)(i) the first gating unit comprises a first gene regulatory portion that is activated upon activation of the first gating unit to induce a first individual regulation via specific binding of the first gene regulatory portion to a target gene; (ii) the second gating unit comprises a second gene regulatory portion that is activated upon activation of the second gating unit to induce a second individual regulation via specific binding of the first gene regulatory portion to a target gene; and / or (3) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to substantially the same polynucleotide sequence of the target gene; and / or (4) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to different polynucleotide sequences of the target gene; and / or (5) the second gating unit further comprises a second gating portion that is activated upon activation of the second gating unit to induce activation of the second gene regulatory portion via specific binding of the second gating portion to the second gene regulatory portion; Optionally, (i) the first gating unit further comprises a first gating moiety that is activated upon activation of the first gating unit to induce (a) activation of the first gene regulatory moiety via specific binding of the first gating moiety to the first gene regulatory moiety, and (b) activation of the second gating moiety via specific binding of the first gating moiety to the second gating moiety; and / or (ii) the activating moiety is capable of inducing (a) activation of the first gene regulatory moiety through specific binding of the activating moiety to the first gene regulatory moiety, and (b) activation of the second gating moiety; and / or (6) the first individual regulation and the second individual regulation together increase the expression and / or activity level of the target gene; and / or (7) the first individual regulation and the second individual regulation together reduce the expression and / or activity level of the target gene; and / or (8) the first gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and the second gating unit is activatable to selectively induce a second individual regulation of the target gene without modulating the expression and / or activity profile of the additional target gene; and / or (9) The system, wherein the second gating unit is activatable to modulate the expression and / or activity profile of the additional target gene, and the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene.

[0176] Embodiment 5. A system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising: A heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in concert to sequentially induce a plurality of distinct modulations of a target gene, each of the plurality of distinct modulations being required but individually insufficient to achieve a desired expression and / or activity profile of the target gene, and wherein the plurality of gating units: (i) a first gating unit that is activatable to induce a first distinct regulation of the plurality of distinct regulations upon activation of the heterologous genetic circuit; (ii) a second gating unit that is activatable to induce disruption of the activated first gating unit, whereby a second individual regulation of the plurality of individual regulations is induced upon inactivation, the second individual regulation being induced after the first individual regulation to attenuate the first individual regulation; Upon activation of the heterologous genetic circuit, the gating units operate in a coordinated manner to achieve a desired expression and / or activity profile of target genes in the cell; Optionally, (1) the system further comprises an activating moiety for activating a heterologous genetic circuit; and / or (2)(i) the first gating unit comprises a first gene regulatory portion that is activated upon activation of the first gating unit to induce a first individual regulation via specific binding of the first gene regulatory portion to a target gene; (ii) the second gating unit comprises a second gene regulatory portion that is activated upon activation of the second gating unit to induce a second individual regulation via specific binding of the first gene regulatory portion to a target gene; and / or (3) the second gating unit further comprises a second gating portion that is activated upon activation of the second gating unit and induces activation of the second gene regulatory portion via specific binding of the second gating portion to the second gene regulatory portion; Optionally, (i) the first gating unit further comprises a first gating moiety that is activated upon activation of the first gating unit to induce (a) activation of the first gene regulatory portion via specific binding of the first gating portion to the first gene regulatory portion, and (b) activation of the second gating portion via specific binding of the first gating portion to the second gating portion; Additionally, optionally, (A) upon activation of the second gene regulatory portion, the second gene regulatory portion induces the destruction of the first gating unit through the destruction of the first gating portion and / or the first gene regulatory portion; and / or (B) the disruption comprises modifying a polynucleotide sequence encoding the first gating portion and / or the first gene regulatory portion; and / or (ii) the activating portion is capable of inducing (a) activation of the first gene regulatory portion via specific binding of the activating portion to the first gene regulatory portion, and (b) activation of the second gating portion; Additionally, optionally, (A) upon activation of the second gene regulatory portion, the second gene regulatory portion induces the destruction of the first gating unit through the destruction of the first gene regulatory portion; and / or (B) the disruption comprises modifying a polynucleotide sequence encoding the first gene regulatory portion; and / or (4) the first gating unit is activatable to modulate the expression and / or activity profile of the additional target gene, and the second gating unit is activatable to selectively induce a second individual regulation of the target gene without modulating the expression and / or activity profile of the additional target gene; and / or (5) the second gating unit is activatable to modulate the expression and / or activity profile of the additional target gene, and the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene; and / or (6) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to substantially the same polynucleotide sequence of the target gene; and / or (7) A system in which the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to different polynucleotide sequences of the target gene.

[0177] Embodiment 6. Optionally, (1) the target gene comprises multiple target genes; and / or (2) a second individual regulation induces selective regulation of one of the plurality of target genes but not the remainder of the plurality of target genes; and / or (3) the activating moiety comprises a guide nucleic acid (gNA) capable of forming a complex with an endonuclease, the gNA being capable of binding to at least a portion of the heterologous genetic circuit, thereby activating the heterologous genetic circuit; and / or (4) each of the first gating unit, the first gating portion, the first gene regulatory portion, the second gating unit, the second gating portion, and / or the second gene regulatory portion comprises an activatable gNA; Upon activation of gNA, gNA forms a complex with an endonuclease, (5) the activatable gNA comprises a self-cleaving gNA, and / or (ii) the activatable gNA comprises a non-canonical termination sequence, and / or (6) the endonuclease contains a CRISPR / Cas protein; Optionally, the CRISPR / Cas protein is operably linked to a gene activator or a gene repressor, and / or (7) the induction of the second separate adjustment is configured to occur at least about 5 minutes, at least about 1 hour, at least about 6 hours, or at least about 12 hours after the induction of the first separate adjustment; and / or (8) the activation moiety consists of a single activation moiety; and / or (9) upon activation of the second gating unit, the second gating unit is further capable of regulating the expression and / or activity of an additional target gene in the cell; and / or (10) the second gating unit comprises an additional gene regulatory portion that is activated upon activation of the second gating unit to induce regulation of the expression and / or activity of the additional target gene through specific binding of the additional gene regulatory portion to the additional target gene; and / or (11) the target gene is endogenous to the cell; and / or (12) the desired expression and / or activity profile of the target gene induces differentiation of cells towards a target cell type; and / or (13) the target cell type exhibits a target phenotype; and / or (14) The target cell type is a tissue-specific cell, and optionally the tissue-specific cell is selected from the group consisting of an immune cell, a neuron, an osteoblast, an endothelial cell, a mesenchymal cell, and an epithelial cell; and / or (15) the target phenotype comprises (i) the expression or activity level of a target protein, (ii) the average cell size, and / or (iii) a desired function; and / or (16) The target gene encodes a cell differentiation regulator, and / or (17) The cell differentiation regulator comprises a growth factor, and / or (18) The cell differentiation regulator comprises a transcription factor, and / or (19) The cell differentiation regulator comprises an immune cell regulator including E2A, Ikaros, Runx1, Lmo2, PU.1, Hes1, Tcf7, Gata3, and Bcl11b; and / or (20) The cell differentiation regulator comprises a T-box transcription factor (TBX), a basic helix-loop-helix transcription factor (bHLH), a homeobox protein, a forkhead box (FOX), an SRY-related HMG box (SOX), and / or a GATA protein; and / or (20) The system of embodiment 4 or embodiment 5, wherein the stem cells are selected from the group consisting of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), neural stem cells, and epithelial stem cells.

[0178] Embodiment 7. An engineered cell comprising the system of any one of embodiments 4 to 6.

[0179] Embodiment 8. A composition comprising the system of any one of embodiments 4 to 6 or the engineered cells of embodiment 7, Optionally, (1) the composition further comprises a separate therapeutic agent; Further optionally, the separate therapeutic agent comprises a chemotherapeutic agent, an immunosuppressant agent, and / or an antibiotic agent; and / or (2) the composition further comprises an activating moiety; and / or (3) A composition, wherein the composition comprises an engineered cell and is substantially free of an activating moiety.

[0180] The systems and methods of the present disclosure may be combined with or modified by other systems and methods, such as those described in International Patent Application PCT / US2018 / 052211, the entirety of which is incorporated herein by reference.

[0181] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. 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 intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, depending upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the practice of the present invention. Therefore, it is contemplated that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

1. 1. A method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising: contacting the cell with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in concert to sequentially induce a plurality of distinct modulations of the target gene, each of the plurality of distinct modulations being required but individually insufficient to achieve the desired expression and / or activity profile of the target gene, and wherein the plurality of gating units: (i) a first gating unit that is activatable to induce a first distinct regulation of the plurality of distinct regulations upon said activation of said heterologous genetic circuit; (ii) a second gating unit that is activatable upon the activation of the heterologous genetic circuit to induce a second individual regulation among the plurality of individual regulations, wherein the second individual regulation is induced after the first individual regulation such that the first individual regulation and the second individual regulation together increase or decrease the expression and / or activity level of the target gene in the cell; During the contacting step, the plurality of gating units operate cooperatively to achieve the desired expression and / or activity profile of the target gene in the cell.

2. 10. The method of claim 1, further comprising contacting the cell with an activating moiety to activate the heterologous genetic circuit.

3. 2. The method of claim 1, wherein the activation moiety consists of a single activation moiety, the activation moiety comprises a guide nucleic acid (gNA) capable of forming a complex with an endonuclease, the gNA capable of binding to at least a portion of the heterologous genetic circuit, thereby activating the heterologous genetic circuit, and the first individual regulation and the second individual regulation together increase the expression and / or activity level of the target gene.

4. The method described in claim 1, wherein the first gating unit is activatable to regulate the expression and / or activity profile of an additional target gene, and the second gating unit is activatable to selectively induce the second individual regulation of the target gene without regulating the expression and / or activity profile of the additional target gene.

5. The method described in claim 1, wherein the target gene comprises multiple target genes.

6. The method described in claim 1, wherein the induction of the second individual adjustment is configured to occur at least about 5 minutes, at least about 1 hour, at least about 6 hours, or at least about 12 hours after the induction of the first individual adjustment.

7. The method described in claim 1, wherein the target gene is endogenous to the cell.

8. The method described in claim 1, wherein the target gene encodes a cell differentiation regulatory factor.

9. The method described in claim 1, wherein the desired expression and / or activity profile of the target gene induces differentiation of the cells toward a target cell type.

10. The method of claim 1, wherein the cells are stem cells. (i) the first gating unit comprises a first gene regulatory portion that is activated upon activation of the first gating unit to induce the first individual regulation through specific binding of the first gene regulatory portion to the target gene; (ii) the second gating unit comprises a second gene regulatory moiety that is activated upon activation of the second gating unit to induce the second individual regulation through specific binding of the first gene regulatory moiety to the target gene; The method of claim 1.

12. The method of claim 11, wherein the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to substantially the same polynucleotide sequence of the target gene, or the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to different polynucleotide sequences of the target gene, and the second gating unit further comprises a second gating portion that is activated upon the activation of the second gating unit and induces activation of the second gene regulatory portion through specific binding of the second gating portion to the second gene regulatory portion.

13. The first gate unit is a first gate portion that is activated upon the activation of the first gate unit, (a) activation of the first gene regulatory moiety via specific binding of the first gating moiety to the first gene regulatory moiety; and (b) activating the second gating moiety via specific binding of the first gating moiety to the second gating moiety.

13. The method of claim 12, further comprising a first gate portion that induces 14. The activating moiety is (a) activation of the first gene regulatory moiety via specific binding of the activation moiety to the first gene regulatory moiety; and (b) activating the second gate portion. The method of claim 12, wherein the 15. The method of claim 1, wherein each of the first gate unit, the first gate portion, the first gene regulatory portion, the second gate, the second gate portion, and / or the second gene regulatory portion comprises an activatable gNA; 15. The method of any one of claims 1 to 14, wherein upon activation of the gNA, the gNA forms a complex with an endonuclease, and the activatable gNA comprises a non-canonical termination sequence.

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