Systems and methods for programming cells to the pancreatic lineage

Abstract

Provided herein are systems for regulating gene expression, methods for their use, and cells genetically engineered for the purpose of differentiating cells, such as beta cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 442,612, filed February 1, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Heterologous proteins and / or nucleic acid molecules can be utilized to induce desired responses in cells. The 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 technologies (e.g., clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, or "CRISPR / Cas") have been employed to manipulate polynucleotide sequences, their epigenetic modifications, and / or their expression levels. For example, CRISPR / Cas technology can be characterized by its versatility and easy programmability and can be used to facilitate genome editing across different species and cell types. Summary of the Invention

[0003] The present disclosure provides methods and systems for programming cells, for example, to induce a desired response in the cell. The disclosed systems and methods can promote the conversion of cells from one type to another. The disclosed systems and methods can utilize genetic circuits to control a cascade of multiple desired expression and / or activity profiles of multiple genes in a cell to affect this conversion. The disclosed systems and methods can utilize heterologous proteins and / or nucleic acid molecules as building blocks of such genetic circuits.

[0004] In some embodiments, the present disclosure provides a method for converting a plurality of stem cells into a plurality of pancreatic lineage cells, the method comprising contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce a plurality of gating units to sequentially modulate expression levels of a plurality of distinct target genes to achieve the conversion, the plurality of gating units including: (a) a first gating unit preconfigured to modulate the expression level of a first target gene among the plurality of distinct target genes, the first target gene being selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SRY-related HMG box), and the like; (b) a first gating unit comprising one or more members selected from the group consisting of HMG-box (SOX), and GATA; and (b) a second gating unit preconfigured to regulate the expression level of a second target gene among a plurality of distinct target genes such that the expression levels of the first target gene and the second target gene are sequentially regulated, wherein upon activation of the heterologous gene circuit, the plurality of gating units operate to effect conversion.

[0005] In some embodiments, the present disclosure provides a method for converting a plurality of stem cells into a plurality of pancreatic lineage cells via regulating the expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the method comprising: (a) contacting a first polynucleotide sequence in the plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression levels of the first target gene operably linked to the first polynucleotide sequence; The method includes the steps of (a) contacting a second polynucleotide sequence in a plurality of stem cells with a second heterologous gene regulatory portion, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), and GATA; and (b) regulating the expression level of a second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor.

[0006] In some embodiments, the present disclosure provides a method for converting a plurality of stem cells into a plurality of pancreatic lineage cells via regulating the expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the method comprising: (a) contacting a first polynucleotide sequence in the plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression levels of the first target gene operably linked to the first polynucleotide sequence, wherein the first target gene is selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GAT A, a basic helix-loop-helix transcription factor (bHLH), a homeobox, and a Maf transcription factor; and (b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to reduce the expression level of a second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of an additional SOX, an additional homeobox, and an ETS transcription factor.

[0007] In some embodiments, the present disclosure provides a method for converting a plurality of stem cells into a pancreatic lineage, the method comprising contacting a polynucleotide sequence in the plurality of stem cells with a heterologous gene regulatory moiety to regulate the expression level of a target gene operably linked to the polynucleotide sequence, wherein within about two weeks after contacting, the conversion rate of the plurality of stem cells into a plurality of pancreatic lineage cells is characterized as being at least about 5%.

[0008] In some embodiments, the present disclosure provides a method for treating a subject in need thereof, the method comprising administering to the subject a plurality of pancreatic lineage cells, wherein the plurality of pancreatic lineage cells is prepared by subjecting a plurality of stem cells to ex vivo differentiation; The method includes a step in which the conversion rate of the plurality of stem cells to the plurality of pancreatic lineage cells is characterized as being at least about 5% within about two weeks of ex vivo differentiation.

[0009] In some embodiments, the present disclosure provides a system for converting a plurality of stem cells into a plurality of pancreatic lineage cells, the system comprising a heterologous genetic circuit including a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to achieve the conversion by sequentially regulating expression levels of a plurality of different target genes, the plurality of gating units comprising: (i) a first gating unit preconfigured to regulate the expression level of a first target gene among the plurality of distinct target genes, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), and GATA; and (ii) a second gating unit preconfigured to regulate the expression level of a second target gene among the plurality of distinct target genes, wherein the expression levels of the first target gene and the second target gene are sequentially regulated; Upon activation of the heterologous gene circuit, multiple gating units operate to effect the transformation.

[0010] In some embodiments, the present disclosure provides a system for converting a plurality of stem cells into a plurality of pancreatic lineage cells via regulating the expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the system comprising: (a) a first heterologous gene regulatory portion configured to bind to a first polynucleotide sequence in the plurality of stem cells and regulate the expression level of the first target gene operably linked to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), and GATA; and (b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and regulate the expression level of a second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of basic helix-loop-helix transcription factors (bHLH), homeobox, and Maf transcription factors.

[0011] In some embodiments, the present disclosure provides a system for converting a plurality of stem cells into a plurality of pancreatic lineage cells via regulating the expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the system comprising: (a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in the plurality of stem cells and regulate the expression levels of the first target gene operably linked to the first polynucleotide sequence, wherein the first target gene is selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GATA, basic helix, and the like. (b) a first heterologous gene regulatory portion comprising one or more members selected from the group consisting of a bHLH transcription factor, a homeobox, and a Maf transcription factor; and (b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in a plurality of stem cells and reduce the expression level of a second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of an additional SOX, an additional homeobox, and an EST transcription factor.

[0012] In some embodiments, the present disclosure provides a system for converting a plurality of stem cells to a pancreatic lineage, the system comprising a heterologous gene regulatory portion configured to bind to a polynucleotide sequence in the plurality of stem cells and regulate the expression level of a target gene operably linked to the polynucleotide sequence, wherein the conversion rate of the plurality of stem cells to a plurality of pancreatic lineage cells within about two weeks after contact is characterized as being at least about 5%.

[0013] In some embodiments, the present disclosure provides a composition for treating a subject in need thereof, the composition comprising a plurality of pancreatic lineage cells prepared by subjecting a plurality of stem cells to ex vivo differentiation, wherein the conversion rate of the plurality of stem cells to a plurality of pancreatic lineage cells is characterized as being at least about 5% within about two weeks of ex vivo differentiation.

[0014] Incorporation by Reference All publications, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent 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 explanation of the drawings]

[0015] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG."). [Figure 1] Schematic of a heterologous gene circuit. An activating moiety can initiate the circuit and activate a gating unit. A gating unit is composed of a gating moiety and / or a gene regulatory moiety. [Figure 2] FIG. 1 shows the developmental progression from iPSCs to mature beta cells, including genes known to affect beta cell development at different stages and markers for screening associated expression patterns. [Figure 3] 1 shows an exemplary heterologous gene circuit. [Figure 4A] Scatter plots (e.g., volcano plots) are shown to identify one or more heterogeneous gene circuits that induced stem cell to beta cell conversion under conditions such as no serum + B27 + T3 + betacellulin + ALK5i (e.g., Figure 4A), serum + B27 + T3 + betacellulin + ALK5i (Figure 4B), serum only (Figure 4C), and serum + ALK5i (Figure 4D). The x-axis is the log2 fold change in enrichment across the core cascade, and the y-axis is the p-value, with the dashed line indicating a p-value of 0.05. [Figure 4B] Scatter plots (e.g., volcano plots) are shown to identify one or more heterogeneous gene circuits that induced stem cell to beta cell conversion under conditions such as no serum + B27 + T3 + betacellulin + ALK5i (e.g., Figure 4A), serum + B27 + T3 + betacellulin + ALK5i (Figure 4B), serum only (Figure 4C), and serum + ALK5i (Figure 4D). The x-axis is the log2 fold change in enrichment across the core cascade, and the y-axis is the p-value, with dashed lines indicating p=0.05. [Figure 4C] Scatter plots (e.g., volcano plots) are shown to identify one or more heterogeneous gene circuits that induced stem cell to beta cell conversion under conditions such as no serum + B27 + T3 + betacellulin + ALK5i (e.g., Figure 4A), serum + B27 + T3 + betacellulin + ALK5i (Figure 4B), serum only (Figure 4C), and serum + ALK5i (Figure 4D). The x-axis is the log2 fold change in enrichment across the core cascade, and the y-axis is the p-value, with dashed lines indicating p=0.05. [Figure 4D] Scatter plots (e.g., volcano plots) to identify one or more heterogeneous gene circuits that induced stem cell to beta cell conversion under conditions such as no serum + B27 + T3 + betacellulin + ALK5i (e.g., Figure 4A), serum + B27 + T3 + betacellulin + ALK5i (Figure 4B), serum only (Figure 4C), and serum + ALK5i (Figure 4D). The x-axis is the log2 fold change in enrichment across the core cascade, and the y-axis is the p-value, with dashed lines indicating p=0.05. [Figure 5] Scatter plots are shown to identify one or more heterologous gene circuits that induced stem cell to beta cell conversion after 5 days. [Figure 6] We show that top-performing heterologous gene circuits upregulate key surface markers across conditions. [Figure 7]Heterologous gene circuits #9 and #16 (Cell Algorithm 9 and Cell Algorithm 16, respectively) produce cultures with islet-like morphology after 8 days in culture. Arrows indicate islet cells. [Figure 8A] We show that a high-performance heterologous gene circuit generates insulin-positive cells after 8 days in culture. [Figure 8B] The percentage of insulin positive cells is shown for each heterologous gene circuit tested. [Figure 9] A comparison of directed differentiation and heterologous gene circuits in the generation of β cells is shown. Detailed Description of the Invention

[0016] 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 invention described herein may be employed.

[0017] Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" apply to each and every number in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0018] Whenever the terms "at most," "up to," "no more than," "less than," or "less than or equal to" precede the first number in a series of two or more numbers, the terms "at most," "up to," "no more than," "less than," or "less than or equal to" apply to each and every number in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0019] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "gate unit" includes a plurality of gate units.

[0020] The term "about" or "approximately" generally means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0021] 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.

[0022] As used interchangeably herein, the terms "guide nucleic acid," "guide nucleic acid molecule," and "gNA" generally refer to 1) a guide sequence capable of hybridizing to a target sequence, or 2) a scaffold sequence capable of interacting with or forming a complex with a nucleic acid-guided nuclease. A guide nucleic acid can be a single-guide nucleic acid (e.g., sgRNA) or a double-guide nucleic acid (e.g., dgRNA). An sgRNA can be a single RNA molecule containing both a scaffold tracrRNA and a crRNA, which can be complementary to a target sequence. Alternatively, a dgRNA can be a single RNA molecule containing a crRNA annealed to a tracrRNA via a direct repeat sequence.

[0023] As used interchangeably herein, the terms "genetic circuit," "biological circuit," or "circuit" generally refer to a collection of molecular components (e.g., biological materials such as polypeptides and / or polynucleotides, non-biological materials, etc.) that are operably linked (e.g., operating simultaneously, 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) in 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 trigger 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 (FIG. 1).

[0024] A genetic circuit can be a controllable gene expression system comprising an assembly of biological parts that work together (e.g., simultaneously, sequentially, etc.) as a logical function. The genetic circuit can include a plurality of gating units, at least one of which can be activated by an activating moiety (e.g., a heterologous input to a cell) and activates other gating units of the plurality of gating units (e.g., simultaneously, sequentially in a cascading manner, etc.) (FIG. 1). For example, at least one of the gating units can be activated by another of the plurality of gating units (e.g., directly or indirectly) to (i) regulate the expression or activity level of one or more target genes, (ii) activate at least one other of the plurality of gating units, and / or (ii) deactivate at least one other of the plurality of gating units, thereby collectively regulating the expression and / or activity level of one or more target genes in a predetermined and desired manner according to the design of the genetic circuit (FIG. 1). As used herein, the terms "heterologous genetic circuit," "HGC," "genetic circuit," "cellular algorithm," or "cellgorithm" may be used interchangeably.

[0025] As referred to herein, the term "gate unit" generally refers to a portion of a genetic circuit that can control gene regulation by functioning similarly to a logic gate, where it controls the flow of information and allows the circuit to make multiple decisions at different points. More specifically, the term refers to nucleic acids that encode gene switches and transcription / translation regulatory regions, or the set of regions on which a gene switch acts. The input to a gate unit can be an activating moiety and / or another gate unit. The output to a gate unit can be to activate another gate unit, to deactivate 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 moieties and / or multiple gene regulatory moieties (Figure 1).

[0026] As referred to herein, the term "activating moiety" generally refers to a moiety that can activate multiple gene 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, a polypeptide, a polynucleotide, a small molecule, light, or a combination thereof.

[0027] For example, an activation moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) and binds to the polynucleotide sequence of an inactivated gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule), thereby activating such gate moiety (e.g., inducing the expression of a functional form of an additional guide nucleic acid molecule), and thereby targeting one or more gene regulatory moieties. The term "gate moiety," as referred to herein, generally refers to a moiety that can affect the function of a gene regulatory moiety within a gate unit. A gate moiety can activate and / or inactivate 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 inactivating 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), binds to the polynucleotide sequence of a gene regulatory moiety (e.g., a plasmid encoding another guide nucleic acid molecule), and activates the gene regulatory moiety (e.g., inducing the expression of a functional form of another guide nucleic acid molecule), and can target one or more endogenous genes of a cell. Alternatively, or in addition, a gate moiety can activate and / or deactivate another gate unit of a genetic circuit (Figure 1). For example, a gate moiety can be a guide nucleic acid molecule that can form a complex with an endonuclease (e.g., a Cas protein), bind to a polynucleotide sequence of another gate moiety to be inactivated (e.g., a plasmid encoding another guide nucleic acid molecule), and activate the other gate moiety (e.g., induce expression of a functional form of the other guide nucleic acid molecule). In another example, a gate moiety can be a guide nucleic acid molecule that can form a complex with an endonuclease (e.g., a Cas protein), bind to a polynucleotide sequence of another gate moiety to be activated (e.g., a plasmid encoding another guide nucleic acid molecule), and inactivate the other gate moiety (e.g., reduce expression of a functional form of the other guide nucleic acid molecule).

[0028] As referred to herein, the term "gate moiety" generally refers to a moiety that can affect the function of a gene regulatory moiety within a gate unit. A gate moiety can activate and / or inactivate 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 inactivating the gene regulatory moiety. Alternatively, or in addition, a gate moiety can activate and / or inactivate another gate unit of a genetic circuit (Figure 1).

[0029] As used interchangeably herein, the terms "gene regulating moiety" or "gene editing moiety" generally refer 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 1). 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 nucleases, 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, alter the template underlying gene expression (e.g., CRISPR-Cas-inspired RNA targeting systems). Alternatively, a gene editing moiety can suppress the translation of a gene (e.g., Cas13).

[0030] Alternatively, or in addition, the gene editing moiety may be capable of regulating gene expression or activity 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 can recruit or contain 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 can itself bind to DNA and regulate transcription by physical obstruction, for example, preventing proteins such as RNA polymerase and other related proteins from assembling on the DNA template. The gene editing moiety can regulate gene expression at the translational level, for example, by regulating the production of proteins from an mRNA template. In some cases, the gene editing moiety can regulate gene expression by affecting the stability of mRNA transcripts. In some cases, the gene editing moiety can regulate genes through epigenetic editing (e.g., Cas12).

[0031] In some cases, the plasmid can encode a non-functional form of the gene editing moiety. The plasmid can be activated (e.g., genetically modified) to express a functional form of the gene editing moiety, for example, through activation of a functional gate moiety. For example, the plasmid can encode a non-functional form of a guide nucleic acid molecule that can otherwise bind to a target gene of a cell. Upon binding of a functional gate moiety (e.g., another guide nucleic acid molecule complexed with a Cas protein) to the plasmid, the plasmid is edited (e.g., cut at one or more sites and then repaired via endogenous mechanisms (e.g., homologous recombination, nonhomologous end joining)), allowing expression of a functional form of the gene editing moiety (e.g., a functional form of the guide nucleic acid molecule that specifically binds to a target gene of a cell) and allowing regulation of the target gene in the cell.

[0032] In some cases, the gene regulatory portion may 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 in addition, the gene regulatory portion may include an endonuclease or be operably linked to an endonuclease. An endonuclease may be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. An endonuclease may include a restriction endonuclease that cleaves DNA at specific sites without damaging the bases. Restriction endonucleases may include type I, type II, type III, and type IV endonucleases, and may further include subtypes. 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 (Cas The endonuclease may be any of Cas12, Cas13c, Cas13d, Cas13x.1, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx10, Csx11, Csf1, and Csn2. The endonuclease may be a dead endonuclease that exhibits reduced cleavage activity. For example, the endonuclease may be a nuclease-inactivating Cas, such as dCas (e.g., dCas9).

[0033] The above-described Cas proteins can form a complex with a guide nucleic acid (gNA) (e.g., a guide RNA (gRNA)) and utilize the gNA to specifically bind to a target polynucleotide sequence (e.g., a target DNA sequence, a target RNA sequence). Thus, in some cases, such Cas proteins can be referred to as "NA-guided nucleases" (e.g., RNA-guided nucleases). As used herein, the term "guide nucleic acid" (gNA) can generally refer to a nucleic acid that can hybridize to another nucleic acid. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. A guide nucleic acid can be programmed to site-specifically bind to a sequence of a nucleic acid. A targeted nucleic acid or target nucleic acid can comprise nucleotides. A guide nucleic acid can comprise nucleotides. A portion of a target nucleic acid can be complementary to a portion of a guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with a guide nucleic acid may be referred to as the complementary strand. The strand of a double-stranded target polynucleotide that is complementary to the complementary strand and therefore may not be complementary to the guide nucleic acid may be referred to as the noncomplementary strand. A guide nucleic acid may comprise a polynucleotide strand and may be referred to as a "single guide nucleic acid." A guide nucleic acid may comprise two polynucleotide strands and may be referred to as a "double guide nucleic acid." Unless otherwise specified, the term "guide nucleic acid" may be inclusive and refer to both single and dual guide nucleic acids. A guide nucleic acid may comprise a segment that may be referred to as a "nucleic acid-targeting segment," "nucleic acid-targeting sequence," or "spacer sequence."The nucleic acid targeting segment may include a subsegment that may be referred to as a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment" or "scaffold sequence."

[0034] The gene regulatory portion can be a transcription 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 comprising a dCas protein operably linked (e.g., linked or fused) to a transcription 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, JARID1A, RBP2, JARID1B / PLU-1, JARID1B / SMCX, JARID1B / SMCY, HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, M.Hal, METI, DRM3, ZMET2, CMT1, CMT2, Lamin A, and Lamin B. Alternatively, the gene regulatory portion 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 can 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, SRCl, ACTR, P160, CLOCK, TET1CD, TET1, DME, DML1, DML2, and ROS1.

[0035] In some cases, the gene regulatory moiety has an enzymatic activity that modifies the target gene without cleaving the target gene. Modification of the target gene can result in, for example, epigenetic modifications that can modify gene expression and / or activity levels. Examples of enzymatic activities that can be provided by the gene regulatory moiety include nuclease activity, such as that provided by restriction enzymes (e.g., Fokl nuclease), methyltransferases (e.g., Hhal DNA cleavage enzymes), and the like. Methyltransferase activity, such as that provided by m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3, ZMET2, CMT1, and CMT2; demethylase activity, such as that provided by demethylases (e.g., Ten-Eleven Translocation (TET) Dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1); DNA repair activity, DNA damage activity, and deaminases (e.g., cytosine deaminase enzymes such as APOBEC1). These include, but are not limited to, deaminating activity such as that provided by a Gin recombinase, dismutase activity, alkylating activity, depurinating activity, oxidizing activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and / or resolvase (e.g., Gin invertase, e.g., a hyperactive mutant of Gin invertase, GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 degrading enzyme, etc.), transposase activity, recombinase activity such as that provided by a recombinase (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0036] The gene regulatory portion may comprise an endonuclease. The endonuclease may be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. The endonuclease may comprise a restriction endonuclease that cleaves DNA at a specific site without damaging the base. The restriction endonuclease may comprise type I, type II, type III, and type IV endonuclease, and may further comprise subtypes. In some instances, the endonuclease may be Cas9. In some instances, the endonuclease may be an inactivated Cas (e.g., dCas, dCas9).

[0037] Unless otherwise specified or apparent from context, the terms "polynucleotide," "oligonucleotide," or "nucleic acid," when used interchangeably herein, generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either single-stranded, double-stranded, or multi-stranded. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide can contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached 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 polynucleotides include coding or non-coding regions of genes or gene fragments, loci (locuses) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0038] The term "gene" generally refers to nucleic acids (e.g., DNA such as genomic DNA and cDNA) and their corresponding nucleotide sequences involved in encoding an RNA transcript. When used herein with reference to genomic DNA, this term includes intervening non-coding regions and regulatory regions, and may include the 5' and 3' ends. In some uses, this 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 includes "open reading frames" that encode a polypeptide. In some uses of this 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, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or a native gene in its natural location in the genome of an organism. A gene may refer to an "exogenous gene" or a non-native gene. A non-native gene may 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 may also refer to a gene that is not in its natural location in the genome of an organism. A non-native gene may also refer to a naturally occurring nucleic acid or polypeptide sequence that contains mutations, insertions, and / or deletions (e.g., a non-native sequence).

[0039] The term "sequence identity" generally refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid, is the number of exact matches between the two aligned sequences divided by the length of the longer sequence multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, the advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method discussed in Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), and Altschul, et al., J. Mol. Biol., 215:403-410 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). This program can be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences, for example, using the blastp program.The program also allows for the use of SEG filters to mask segments of query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired degree of sequence identity ranges from about 50% to 100% and integer values ​​therebetween. Generally, the present disclosure encompasses sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any of the sequences provided herein.

[0040] The term "expression" generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. A 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. With respect to expression, "up-regulated" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its expression level in the wild-type state, and "down-regulated" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its expression in the wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression," the transfected gene is not transferred to daughter cells during cell division. Because its expression is limited to the transfected cell, gene expression is lost over time. During transient expression, episomal DNA can be transferred to daughter cells, but because episomal DNA is not replicated, it is not permanently heritable and is diluted over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selective advantage to the transfected cell. During stable expression, plasmids can contain DNA replication elements that allow them to be inherited or integrated into the genome. Such a selective advantage can be resistance to a specific toxin presented to the cell.

[0041] The terms "peptide," "polypeptide," or "protein," when used interchangeably herein, generally refer to a polymer of at least two amino acid residues linked by a peptide bond. The term does not imply a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structure (e.g., domains). The term also encompasses amino acid polymers modified by any other manipulation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and conjugation with a labeling component. The terms "amino acid" and "amino acids," as used herein, 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 have been 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.

[0042] The terms "derivative," "variant," or "fragment," when 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.

[0043] As used herein with respect to polypeptide molecules (e.g., proteins), the terms "engineered," "chimeric," or "recombinant" generally refer to polypeptide molecules having heterologous or altered amino acid sequences as a result of the application of genetic engineering techniques to nucleic acids encoding the polypeptide molecules, as well as cells or organisms that express the polypeptide molecules. As used herein with respect to polynucleotide molecules (e.g., DNA or RNA molecules), the terms "engineered" or "recombinant" generally refer to polynucleotide molecules having heterologous or altered nucleic acid sequences 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, genetically engineered or recombinant polynucleotides (e.g., genomic DNA sequences) can be modified or altered by gene editing moieties.

[0044] Unless otherwise specified or clear from the context, as used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. A nucleotide may include synthetic nucleotides. A nucleotide may include synthetic nucleotide analogs. A nucleotide may be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may 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 may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide may refer to dideoxyribonucleoside triphosphate (ddNTP) and its derivatives. Examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled by well-known techniques. Labeling may be performed using quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.Fluorescent labels for nucleotides may 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, IL; and Fluorolink Cy5-dUTP, available from Boehringer Ingelheim. Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP available from Mannheim, Indianapolis, Ind., and Molecular Chromosomal labeled nucleotides available from Probes, Eugene, Oreg., include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides may also be labeled or unlabeled by chemical modification. The chemically modified single nucleotide may be a biotin-dNTP.Some non-limiting examples of biotinylated dNTPs can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0045] 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 be derived from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, cells from mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidaria, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes the cells are not derived from a natural organism (e.g., the cells can be synthetically produced, sometimes referred to as artificial cells).

[0046] The term "differentiation" generally refers to the process by which an uncommitted or less specialized cell acquires the characteristics of a specialized cell, such as an immune cell. A differentiated or differentiation-induced cell is one that has adopted a more specialized ("uncommitted") position within a cell lineage. The term "uncommitted" generally refers to a cell that, under normal circumstances, would continue to differentiate into a particular cell type or subset of cell types and has progressed down the differentiation pathway to a point where, under normal circumstances, it is unable to differentiate into a different cell type or revert to a less differentiated cell type.

[0047] The terms "dedifferentiation" or "de-differentiation" generally refer to a process in which a specialized, uncommitted, or partially specialized cell loses the characteristics of a specialized cell (e.g., a beta cell). A dedifferentiated cell or dedifferentiation-induced cell is one that has committed to a less specialized position within the lineage of a cell (e.g., a stem or progenitor cell). A dedifferentiated cell (e.g., a stem or progenitor cell) can then differentiate into a different cell type or revert to a less differentiated cell type.

[0048] The term "pluripotent" generally refers to the ability of a cell to form all lineages of the body or soma (e.g., 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 potencies ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells) that cannot give rise to a complete organism to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0049] 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 (e.g., reprogrammed) into cells that can differentiate into tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. Produced iPSCs do not refer to cells found in nature. In some cases, iPSCs can be engineered to differentiate directly into committed cells (e.g., beta cells). In some cases, iPSCs can be engineered to first differentiate into tissue-specific stem cells (e.g., endodermal stem cells, endocrine progenitor cells), which can be further induced to differentiate into committed cells (e.g., beta cells).

[0050] The term "embryonic stem cell" (ESC) generally refers to cells derived from naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and, during development, give rise to derivatives of all three primary germ layers: ectoderm, endoderm, and mesoderm. In some cases, ESCs can be engineered to differentiate directly into committed cells (e.g., beta cells). In some cases, ESCs can be engineered to first differentiate into tissue-specific stem cells (e.g., endodermal stem cells), which can be further induced to differentiate into committed cells (e.g., pancreatic lineage cells or beta cells).

[0051] The term "isolated stem cells" generally refers to any type of stem cell disclosed herein (e.g., ESCs, HSCs, endodermal stem cells, etc.) that is 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.

[0052] The term "isolated" generally refers to a cell or population of cells that has been separated from its original environment. For example, the new environment of an isolated cell is substantially free of at least one component that would be found in the environment in which the "un-isolated" reference cell resides. An isolated cell can be a cell that has been removed from some or all components as it would be found in its natural environment, e.g., a cell isolated from a tissue or biopsy sample. The term also includes cells that have been removed from at least one, some, or all components as they would be found in a non-naturally occurring environment, e.g., 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, as it is found in nature or grown, stored, or sustained in a non-naturally occurring environment.

[0053] The terms "beta cell," "β-cell," or "pancreatic beta cell," when used interchangeably herein, generally refer to any insulin-producing cell in the pancreas. Non-limiting examples of beta cells are immature beta cells, mature beta cells, and cells that themselves differentiate into immature or mature beta cells, e.g., pancreatic lineage cells.

[0054] The term "pancreatic lineage," when applied to a cell or population, generally refers to the ability of the cell or population to produce glucagon, insulin, somatostatin, and / or pancreatic polypeptide (PP) (e.g., under in vivo or ex vivo conditions). Pancreatic lineage cells can be at least partially differentiated alpha (α) cells (e.g., producing glucagon), beta (β) cells (e.g., producing insulin), delta (δ) cells (e.g., producing somatostatin), or PP cells (e.g., producing pancreatic polypeptide (PP)). Alternatively, pancreatic lineage cells can be cells that themselves differentiate into pancreatic cells, such as pancreatic progenitor cells.

[0055] The terms "pancreatic progenitor," "pancreatic endocrine progenitor," "pancreatic precursor," and "pancreatic endocrine precursor" are used interchangeably herein and generally refer to tissue-specific stem cells that can become pancreatic hormone-expressing cells that can form pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. Pancreatic progenitor cells can be committed to differentiate into at least one type of pancreatic cell, such as alpha (α) cells, beta (β) cells, delta (δ) cells, or PP cells.

[0056] The term "pancreatic lineage cells" generally refers to cells that produce or have the ability to produce glucagon, insulin, somatostatin, and / or pancreatic polypeptide. Pancreatic lineage cells can be generated ex vivo by manipulating isolated stem cells. Pancreatic lineage cells can arise from endodermal stem cells and can include, but are not limited to, definitive endoderm cells, primitive gut cells, posterior foregut cells, pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, pancreatic exocrine cells, pancreatic duct cells, alpha cells, delta cells, PP cells, immature beta cells, and mature beta cells. Pancreatic lineage cells can be generated in vivo by in vivo manipulated stem cells, for example, by administering such stem cells with any one of the heterologous gene circuits disclosed herein.

[0057] Overview

[0058] Biological programming, such as cellular programming, allows for the manipulation of cells to produce desired results. The results of cellular programming can include inducing or preventing a wide range of common and / or new cellular functions, and outcomes can also include enhancing or suppressing cellular functions that are already occurring. Cellular programming can be achieved through the use of genetic circuits. Cellular programming can be achieved through the manipulation of biological molecules (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 implemented to function in a time-dependent or time-independent manner. Cellular programming can be performed to directly affect endogenous or exogenous genes. Alternatively, cellular programming can be performed to indirectly affect endogenous or exogenous genes (e.g., via heterologous gene regulatory moieties or heterologous gene circuits for gene regulation).

[0059] Genetic circuits used in cellular programming can be used to control the cell fate of a cell or multiple cells by inducing differentiation or dedifferentiation and converting one cell type to another. Cell programming is controlled through the regulation of the desired expression and / or activity levels of multiple genes in the cell.

[0060] While the CRISPR / Cas system is widely used for gene editing, Cas is essentially a single-turnover nuclease, remaining bound to the double-strand breaks it generates, leaving many regions of the genome resistant to genome editing. Increased understanding of CRISPR / Cas-based genome editing has facilitated the development of cascade regulatory systems to further exploit this technology for use in the development of genetically engineered cells. By implementing a series of activatable gRNAs, genome editing can be regulated from target site to target site in a more temporal manner, and sequential genome editing can be performed in a domino effect-like fashion, allowing cells to be barcoded. However, this simple barcoding, which often uses exogenous fluorophores, does not allow for the regulation of endogenous genes to achieve cell differentiation.

[0061] Furthermore, cell differentiation or dedifferentiation can now be enabled by the use of exogenous serum and growth factors, which bypass the mechanisms underlying cellular programming. The use of exogenous serum, growth factors, and other similar methods results in cells that are instructed to differentiate but lack the accompanying underlying biology (e.g., correct chromatin state). This lack often results in cells that either undergo premature termination of differentiation into an undesired cell type, or undergo inefficient differentiation with a low yield of the target cell type, or the resulting desired differentiated cells are only semi-functional. Semi-functional cells may resemble the desired cell type but may lack important biological characteristics necessary for the normal function of the desired differentiated cell type.

[0062] Thus, there is an unmet need for activatable CRISPR / Cas systems and their use to edit target polynucleotides (e.g., the genome of a cell, particularly a eukaryotic cell) to form genetic circuits using a cascade of gRNAs to solely affect gene regulation and cell fate decisions, without the use of serum and exogenous growth factors. Preprogrammed, activatable, and self-regulating gRNA cascade CRISPR / Cas systems are useful, for example, in gene therapy, genetic circuits, and / or complex cell fate decisions and / or control.

[0063] The present disclosure provides systems and methods for designing CRISPR / Cas9 systems that contain an array of activatable cognate single guide RNAs (sgRNAs or gRNAs) with inactivating sequences in cas endonucleases and nonessential regions, allowing for regulation and modification of the system without the need for serum, growth factors, or other additional exogenous signals. The present disclosure also provides engineered cells that may contain any of the above systems or perform any of the above methods.

[0064] Systems and methods for cell programming to the pancreatic lineage

[0065] Various embodiments of the present disclosure provide systems for inducing the desired conversion of one type of cell to another type of cell. To this end, 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.

[0066] In one aspect, the present disclosure provides a system for converting a plurality of cells of a first type into a plurality of cells of a second type. The system may include a heterologous genetic circuit comprising a plurality of gating units. The plurality of gating units may 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 plurality of gating units may 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. The plurality of gating units may be different (e.g., comprise different polynucleotide sequences). Each gating unit of the plurality of gating units may affect modulation of the expression and / or activity level of a distinct target gene or multiple distinct target genes.

[0067] The heterologous genetic circuits disclosed herein may operate multiple gate units in series (e.g., multiple gate units connected end-to-end sequentially to form a single pathway), in parallel (e.g., multiple gate units connected across each other, e.g., to form two or more parallel sequential pathways), or a combination thereof.

[0068] The multiple gating units disclosed herein can operate together to induce a cellular outcome (e.g., as predetermined by the design of the heterologous gene circuit). The cellular outcome can include cellular function (e.g., migration, reproduction, response to external stimuli, nutrient production, excretion, respiration, growth) and / or cellular state (e.g., cell fate, differentiation, quiescence, 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 the 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 or metabolic activity assays), (v) microscopy, and / or (iv) screening for molecular and / or genetic differences using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics.

[0069] The multiple gate units disclosed herein may be sufficient to achieve the conversion of a plurality of cells of a first cell type into a plurality of cells of a second cell type. For example, the multiple gate units disclosed herein may be sufficient to achieve the conversion of a plurality of pluripotent stem cells (PSCs) into a plurality of tissue-specific progenitor cells. Alternatively, the multiple gate units disclosed herein may be necessary but insufficient to achieve the conversion of a plurality of cells of a first cell type into a plurality of cells of a second cell type.

[0070] The outcome of a cell can include the regulation of a separate target gene or a set of separate target genes.Multiple gate units can induce the separate regulation of multiple target genes (for example, sequentially), so that the collection of gene regulation coordinates to achieve the final expression and / or activity profile of the cell.The final expression and / or activity level profile of the cell can exemplify an outcome, such as the transformation of a cell from one cell type to another cell type (or the process thereof).

[0071] In some cases, some of the multiple gating units as disclosed herein may be necessary to achieve the desired expression and / or activity profile of a target cell, but may be individually insufficient. Thus, the outcome in a cell induced by the multiple gating units (e.g., enhanced cell function, induced cell state, etc.) may not be possible in the absence of any one of the multiple gating units. Alternatively, the degree or scale of the outcome in a cell induced by the multiple gating units may be different (e.g., greater for a positive marker or less for a negative marker) than the degree or scale of the outcome in a control cell induced by one or more, but not all, of the multiple gating units and / or by all of the multiple gating units occurring through a different sequential order of events.

[0072] The second gate unit can be activated (e.g., directly or indirectly) by the first gate unit. For example, the second gate unit can be directly activated by the first gate unit. Alternatively, the second gate unit can be activated by one or more additional gate units that are activated (e.g., directly or indirectly) by the first gate unit. The one or more additional 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 one or more additional gate units are at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 gate unit. In yet another alternative, the second gate unit can be activated via a separate moiety (e.g., an activation moiety, a different gate unit, etc.) involved in the activation of the first gate unit. In yet another alternative, the first gate unit and the second gate unit can be activated by different activation moieties (e.g., different polynucleotide molecules, such as different guide nucleic acid molecules).

[0073] In some cases, the term "proGuide" as used generally herein may refer to such a vector (e.g., a plasmid) encoding an activatable gNA. A proGuide may be an example of a gate portion. A proGuide may be an example of a gene regulatory portion.

[0074] In some embodiments, a proGuide provided herein can encode, for example, an activatable guide nucleic acid molecule having an inactivating polynucleotide sequence (e.g., one or more polyX sequences, such as one or more polyT sequences). In some cases, the portion of a proGuide encoding an activatable guide nucleic acid molecule can include various regions linked sequentially (e.g., 5' to 3'), including an upstream stem (e.g., an upstream cleavage site), a polyT unit (or, as used interchangeably herein, a "proUnit" or "proGuide Unit"), and a downstream stem (e.g., a downstream cleavage site). The upstream stem and downstream stem can correspond to "stem region" polynucleotide sequences that are at least partially complementary to each other.

[0075] The proGuides provided herein can encode, for example, an activatable guide nucleic acid molecule having an inactivating polynucleotide sequence (e.g., one or more poly-X sequences, such as one or more poly-T sequences). In some embodiments, the portion of the proGuide encoding the activatable guide nucleic acid molecule can include various regions linked sequentially, including a spacer sequence, an extra sequence (e.g., a linker sequence or backbone sequence), an upstream stem, a poly-T unit, and a downstream stem. In some embodiments, the extra sequence can exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to GTTTAGAGCTA (SEQ ID NO: 2027). In some embodiments, the portion of a proGuide encoding an activatable guide nucleic acid molecule may include various regions sequentially linked together, including a spacer sequence, extra sequence (e.g., a linker sequence or backbone sequence), upstream stem, polyT unit, and downstream stem, as shown in Table 4 (SEQ ID NOS: 1-2024). In some embodiments, the portion of a proGuide encoding an activatable guide nucleic acid molecule may include various regions sequentially linked together, including a spacer sequence, extra sequence (e.g., a linker sequence or backbone sequence), upstream stem, polyT unit, and downstream stem, as shown in Table 4 (SEQ ID NOS: 1-2024) as a concatenated sequence with a "-" to distinguish the various regions. In some cases, upon modification or removal of the polyT unit, the upstream stem and downstream stem may form part of the scaffold sequence of a functional guide nucleic acid molecule. In some embodiments, if the spacer sequence does not begin with a G, a G is added before the spacer sequence. In some embodiments, adding a G before the spacer sequence helps increase expression of the RNA portion from the promoter. In some embodiments, stem 1 and stem 2 are reverse complements of each other. In some embodiments, the upstream stem and downstream stem are reverse complements of each other.In some embodiments, stem 1 may exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a member of the polynucleotide sequences set forth in Table 6 (SEQ ID NOS:2120-2142). In some embodiments, stem 2 may exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a member of the polynucleotide sequences set forth in Table 6 (SEQ ID NOS:2143-2165).

[0076] A domain of a polynucleotide sequence encoding (or corresponding to) a molecule of interest can contain a polyX sequence. The PolyX sequence can be sufficient to reduce expression of the molecule of interest (e.g., a guide nucleic acid molecule) from the polynucleotide sequence. For example, a polyX sequence can be placed within a domain encoding a molecule of interest (e.g., not at either the 5' or 3' end of such a domain) such that expression of the molecule of interest (e.g., transcription of an RNA molecule of interest) is disrupted (e.g., terminated) midway through expression.

[0077] Thus, a polyX sequence (e.g., in a polynucleotide sequence encoding a molecule of interest) may be referred to as a termination sequence (e.g., a non-standard termination sequence for that sequence and / or its position), a disruption sequence (e.g., to disrupt all expression of the molecule of interest), or an inactivation sequence (e.g., to inactivate the function of the polynucleotide sequence or molecule of interest).

[0078] In some cases, the poly X sequence can be located within (e.g., not at) the end of a polynucleotide sequence, such as a DNA or RNA sequence. In some cases, the poly X sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 3' end of the polynucleotide sequence. In some cases, the poly X sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 5' end of the polynucleotide sequence. In some cases, the poly X sequence can be located at the end of the nucleic acid sequence.

[0079] In some cases, a poly-T or poly-U sequence can be located within (e.g., not at) the end of a polynucleotide sequence, such as a DNA or RNA sequence. In some cases, the poly-T or poly-U sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 3' end of the polynucleotide sequence. In some cases, the poly-T or poly-U sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 5' end of the polynucleotide sequence. In some cases, the poly-T or poly-U sequence can be located at the end of the nucleic acid sequence. In some cases, RNA containing a poly-U sequence can also be represented by DNA containing a poly-T sequence.

[0080] The poly-X sequence (e.g., poly-T or poly-U sequence) can contain 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 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50x, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 bases. The poly X sequence can contain 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 14, up to about 13, up to about 12, up to about 11, 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, or up to about 2X bases. The poly X sequence can be represented by a complementary poly X sequence in a corresponding complementary DNA strand (e.g., a poly T disclosed herein as a DNA sequence can also be referred to as a poly A in the complementary DNA strand). The disclosed poly X sequences can contain multiple X bases. The multiple X bases can be disclosed contiguously adjacent to each other (e.g., TT, TTT, TTTT, TTTTT, etc.). Alternatively, or in addition, multiple X bases can be separated by one or more additional nucleotides that are not X. The one or more additional nucleotides can comprise a single type of nucleotide or different types of nucleotides.

[0081] In some embodiments, the proGuide may comprise an inactivating polynucleotide sequence that exhibits 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 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, 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 substantially about 100% sequence identity to TTTTTTTTT (SEQ ID NO: 2025), or its complementary sequence.

[0082] In some cases, the non-canonical termination sequence is 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 86%, at least or up to about 90%, 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%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111%, at least or up to about 112%, at least or up to about 113%, at least or up to about 114%, at least or up to about 115%, at least or up to about 116%, at least or up to about 117%, at least or up to about 118%, at least or up to about 119%, at least or up to about 120%, at least or up to about 121%, at least or up to about 122%, at least or up to about 123%, at least or up to about 124%, at least or up to about 125%, at least or up to about 126%, at least or up to about 128%, at least or up to about 129%, at least or up to about 13 The polynucleotide sequence may comprise or consist essentially of a polynucleotide sequence exhibiting at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, 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 substantially about 100% sequence identity.

[0083] In some embodiments, a proGuide may include a target polynucleotide domain at or adjacent to an inactivating polynucleotide sequence (e.g., at or adjacent to the 5' and / or 3' end of the inactivating polynucleotide sequence), which can be targeted (e.g., via the sequential activation mechanism of a heterologous gene circuit provided herein) to modify (e.g., edit, cleave) the inactivating polynucleotide sequence, thereby causing the proGuide to express an activated guide nucleic acid molecule. The target polynucleotide domain of the proGuide does not exhibit sequence identity to any comparable endogenous polynucleotide sequence in the cell, thereby avoiding unintended targeting and regulation of endogenous target genes.

[0084] In some embodiments, the inactivation polynucleotide sequence of the proGuide can be placed between two target polynucleotide domains, which may or may not be targetable by a common guide nucleic acid sequence. In some cases, the two target polynucleotide domains can be reverse-complementary to each other so that the inactivation polynucleotide sequences can be modified or cleaved by the same mechanism (e.g., the same spacer sequence of the guide nucleic acid molecule).

[0085] In some embodiments, the proGuide is selected from the group consisting of SEQ ID NOS: 1-92 (e.g., EGF targeting), SEQ ID NOS: 93-184 (e.g., FEV targeting), SEQ ID NOS: 185-276 (e.g., FOXA2 targeting), SEQ ID NOS: 277-368 (e.g., GATA4 targeting), SEQ ID NOS: 369-460 (e.g., GATA6 targeting), SEQ ID NOS: 461-552 (e.g., GCG targeting), SEQ ID NOS: 553-644 (e.g., INS targeting), SEQ ID NOS: 645-736 (e.g., isl1 targeting), SEQ ID NOS: 737-828 (e.g., LMX1A targeting), and SEQ ID NOS: 829-920 (e.g., MAFA targeting). SEQ ID NOs: 921-1012 (e.g., NEUROD1), SEQ ID NOs: 1013-1104 (e.g., NEUROG3 targeting), SEQ ID NOs: 1105-1196 (e.g., NKX2-2 targeting), SEQ ID NOs: 1197-1288 (e.g., NKX6-1 targeting).SEQ ID NOs: 1289-1380 (e.g., ONECUT1 targeting), SEQ ID NOs: 1381-1472 (e.g., PDX1 targeting), SEQ ID NOs: 1473-1564 (e.g., PTF1a targeting), SEQ ID NOs: 1565-1656 (e.g., SHH targeting), SEQ ID NOs: 1657-1748 (e.g., sox17 targeting), SEQ ID NOs: 1749-1840 (e.g., SOX9 targeting), SEQ ID NOs: 1841-1932 (e.g., SST targeting), SEQ ID NOs: 1933-2024 (e.g., TBXT targeting), or a complementary sequence thereof, 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 or may include polynucleotide sequences exhibiting up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, 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 substantially about 100% sequence identity to one or more members from SEQ ID NOs.

[0086] The second gate unit can be activated to induce the inactivation of the activated first gate unit.The terms " inactivation " and " disruption " can be used interchangeably herein.As disclosed herein, inactivation can be induced by generating modifications (such as single-strand or double-strand breaks, and breaks such as indels) to at least a portion of the first gate unit (for example, the gate portion and / or gene regulatory portion of the first gate unit) that are involved in inducing the first distinct regulation of target gene.

[0087] As disclosed herein, inactivation of the first gate unit by the gate portion and / or gene regulatory portion can be achieved through an endonuclease-based system (e.g., a CRISPR / Cas system). Alternatively, or in addition, inactivation can be achieved by using a transcriptional modulator system (e.g., a transcriptional repressor). Alternatively, or in addition, inactivation can be achieved by CRISPRi steric hindrance without the need for an additional transcriptional modulator. An endonuclease transcriptional modulator 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 indels. Alternatively, or in addition, an endonuclease transcriptional modulator system (e.g., a Cas repressor) can be used to regulate target gene expression. Alternatively, or in addition, CAS transcriptional modulator systems lacking endonuclease activity (dCAS or naked CAS with a shortened spacer insufficient to support cleavage) can target DNA regions and physically arrest transcription elongation, achieving repression of target genes (CRISPRi).

[0088] Alternatively, the second gate unit can be activated 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 modifications (e.g., breaks such as single-strand or double-strand breaks, indels, 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 are involved in inducing specific regulation of the target gene.

[0089] In some cases, the first gate unit regulates the first target gene.Alternatively or in addition, the first gate unit can also modulate the second gate unit.The modulation of the second gate unit, as confirmed by rt-qPCR, Western blotting, or other methods, can be at least or up to about 1 millisecond, at least or up to about 2 milliseconds, at least or up to about 3 milliseconds, at least or up to about 4 milliseconds, at least or up to about 5 milliseconds, at least or up to about 6 milliseconds, at least or up to about 7 milliseconds, at least or up to about 8 milliseconds, at least or up to about 9 milliseconds, at least or up to about 10 milliseconds, at least or up to about 20 milliseconds, at least or up to about 30 milliseconds, at least or up to about 40 milliseconds, at least or up to about 50 milliseconds, at least or up to about 60 milliseconds, at least or up to about 70 milliseconds, at least or up to about 80 milliseconds, at least or up to about 90 milliseconds, at least or up to about 100 milliseconds, at least or up to about 200 milliseconds, at least or up to about 300 milliseconds, at least or up to about 400 milliseconds, at least or up to about 50 ...0 milliseconds, at least or up to about 700 milliseconds, at least or about 400 milliseconds, at least or up to about 500 milliseconds, at least or up to about 600 milliseconds, at least or up to about 700 milliseconds, at least or up to about 800 milliseconds, at least or up to about 900 milliseconds, at least or up to about 1 second, at least or up to about 2 seconds, at least or up to about 3 seconds, at least or up to about 4 seconds, at least or up to about 5 seconds, at least or up to about 6 seconds, at least or up to about 7 seconds, at least or up to about 8 seconds, at least or up to about 9 seconds, at least or up to about 10 seconds, at least or up to about 15 seconds, at least or up to about 20 seconds, at least or up to about 30 seconds, at least or up to about 40 seconds, at least or up to about 50 seconds, at least or up to about 1 minute, at least or up to about 2 minutes, at least or up to about 3 minutes, at least or up to about 4 minutes, at least or up to about 5 minutes, at least or up to about 6 minutes, at least or up to about 7 minutes, at least up to about 8 minutes,This may occur after at least or up to about 9 minutes, at least or up to about 10 minutes, at least or up to about 20 minutes, at least or up to about 30 minutes, at least or up to about 40 minutes, at least or up to about 50 minutes, at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 12 hours, at least or up to about 16 hours, at least or up to about 20 hours, or at least or up to about 24 hours, or more.

[0090] In some cases, the second gating unit can modulate a second target gene, where the modulation of the second target gene is at least or up to about 1 millisecond, about 2 milliseconds, about 3 milliseconds, about 4 milliseconds, about 5 milliseconds, about 6 milliseconds, about 7 milliseconds, about 8 milliseconds, about 9 milliseconds, about 10 milliseconds, about 20 milliseconds, about 30 milliseconds, about 40 milliseconds, about 50 milliseconds, about 60 milliseconds, about 70 milliseconds, about 80 milliseconds, about 90 milliseconds, about 100 milliseconds, about 200 milliseconds, about 300 milliseconds, about 400 milliseconds, about 500 milliseconds, about 600 milliseconds, or about 700 milliseconds later than the modulation of the first target gene, as confirmed by rt-qPCR, Western blotting, or other methods. seconds, about 800 milliseconds, about 900 milliseconds, about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, or more.

[0091] A heterologous gene circuit may include multiple gate units that are activated sequentially, e.g., serially. The multiple gate units may include preconfigured functional gate units that are activated to regulate (e.g., directly regulate) the expression and / or epigenetic profile of a target gene (e.g., an endogenous target gene). The multiple gate units may further include one or more additional gate units that are preconfigured (i) to be activated before the functional gate unit and (ii) to achieve subsequent activation of the functional gate unit. In some cases, the one or more additional gate units may be preconfigured to be activated to regulate one or more additional target genes. Alternatively, the one or more additional gate units may not be preconfigured to regulate any target gene (e.g., any endogenous target gene) upon activation. Such one or more additional gate units may instead serve to delay (e.g., in terms of time) the activation of the functional gate unit during operation of the heterologous gene circuit, thereby delaying the expression and / or epigenetic profile of the target gene of the functional gate unit; therefore, the one or more additional gate units may be referred to as "blank" gate units.The heterologous genetic circuit may comprise at least or up to about 1 blank gate unit, at least or up to about 2 blank gate units, at least or up to about 3 blank gate units, at least or up to about 4 blank gate units, at least or up to about 5 blank gate units, at least or up to about 6 blank gate units, at least or up to about 7 blank gate units, at least or up to about 8 blank gate units, at least or up to about 9 blank gate units, at least or up to about 10 blank gate units, at least or up to about 11 blank gate units, at least or up to about 12 blank gate units, at least or up to about 13 blank gate units, at least The blank gate units may include at least or up to about 14 blank gate units, at least or up to about 15 blank gate units, at least or up to about 16 blank gate units, at least or up to about 27 blank gate units, at least or up to about 18 blank gate units, at least or up to about 19 blank gate units, at least or up to about 20 blank gate units, at least or up to about 25 blank gate units, at least or up to about 30 blank gate units, at least or up to about 35 blank gate units, at least or up to about 40 blank gate units, at least or up to about 45 blank gate units, or at least or up to about 50 blank gate units.

[0092] In some cases, the use of one or more blank gating units may inhibit activation of a functional gating unit for at least or up to about 1 minute, at least or up to about 5 minutes, at least or up to about 10 minutes, at least or up to about 30 minutes, at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 11 hours, at least or up to about 12 hours, at least or up to about 13 hours, at least or up to about 14 hours, at least or up to about The delay may be by about 15 hours, at least or up to about 16 hours, at least or up to about 17 hours, at least or up to about 18 hours, at least or up to about 19 hours, at least or up to about 20 hours, at least or up to about 21 hours, at least or up to about 22 hours, at least or up to about 23 hours, at least or up to about 24 hours, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, or at least or up to about 7 days (e.g., as determined by measuring the expression / epigenetic profile of the target gene or by measuring the expression of a functional variant or transcript of the functional gating unit).

[0093] In some cases, the modification of the target gene by the gate unit can inactivate the gene. For example, the modification of the gene can stop the expression and / or activity level of the target gene. Alternatively, the modification of the gene can decrease or reduce the expression and / or activity level of the target gene. In some cases, the modification of the gene can increase the expression and / or activity level of the target gene. Alternatively, the modification of the gene can maintain the expression and / or activity level of the target gene. In some cases, genetic modification may decrease 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 gene modification may increase 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% , by up to about 8%, by up to about 7%, by up to about 6%, by up to about 5%, by up to about 4%, by up to about 3%, by up to about 2%, by up to about 1%, by up to about 0.9%, by up to about 0.8%, by up to about 0.7%, by up to about 0.6%, by up to about 0.5%, by up to about 0.4%, by up to about 0.3%, by up to about 0.2%, by up to about 0.1%, or less.

[0094] In some cases, the genetic modification increases or decreases the expression and / or activity levels 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%, The increase may be 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.Genetic modification can increase the expression and / or activity levels of a 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 200%, up to about 1 The increase may be 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%, up to about 0.1%, or less.

[0095] In some cases, genetic modification increases the expression and / or activity level of the 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 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 32 fold, The amount of ATP may be decreased by at least 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.Genetic modification can increase the expression and / or activity level of the target gene by more or less about 10,000-fold, more or less about 5,000-fold, more or less about 1,000-fold, more or less about 500-fold, more or less about 100-fold, more or less about 90-fold, more or less about 80-fold, more or less about 70-fold, more or less about 60-fold, more or less about 50-fold, more or less about 40-fold, more or less about 30-fold, more or less about 20-fold, more or less It may be reduced by more or less about 10 times, more or less about 9 times, more or less about 8 times, more or less about 7 times, more or less about 6 times, more or less about 5 times, more or less about 4 times, more or less about 3 times, more or less about 2 times, more or less about 1 time, more or less 0.9 times, more or less about 0.8 times, more or less about 0.7 times, more or less 0.6 times, more or less 0.5 times, more or less 0.4 times, more or less about 0.3 times, more or less 0.2 times, or more or less about 0.1 times.

[0096] In some cases, genetic modification increases the expression and / or activity level of the 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 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 32 fold, The increase may be at least 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.Genetic modification can increase the expression and / or activity level of the target gene by more or less about 10,000-fold, more or less about 5,000-fold, more or less about 1,000-fold, more or less about 500-fold, more or less about 100-fold, more or less about 90-fold, more or less about 80-fold, more or less about 70-fold, more or less about 60-fold, more or less about 50-fold, more or less about 40-fold, more or less about 30-fold, more or less about 20-fold, more or less about For example, the increase may be about 10-fold, more or less about 9-fold, more or less about 8-fold, more or less about 7-fold, more or less about 6-fold, more or less about 5-fold, more or less about 4-fold, more or less about 3-fold, more or less about 2-fold, more or less about 1-fold, more or less 0.9-fold, more or less about 0.8-fold, more or less about 0.7-fold, more or less 0.6-fold, more or less 0.5-fold, more or less 0.4-fold, more or less about 0.3-fold, more or less 0.2-fold, or more or less about 0.1-fold.

[0097] The expression and / or activity profile of a gene of interest (e.g., a differentiation marker) can be compared to a control gene (e.g., a housekeeping gene such as GAPDH), the relative expression levels of two or more genes of interest (e.g., the ratio of expression or activity levels between a stem cell marker and a differentiation marker), the relative average expression level of a gene of interest compared to the average expression level of the same gene of interest in a cell type of interest, etc.

[0098] In some cases, activation of the multiple gate units may be the result of a single activation of the heterologous gene circuit (e.g., by a single activation moiety at a single time point). The multiple gate units may include one of a first gate unit and a second gate unit preconfigured to be sequentially activated upon activation of the heterologous gene circuit by a single activation. In some cases, one of the first and second gate units may be activated by a single activation moiety (e.g., a guide nucleic acid), while the other of the first and second gate units may be activated by an additional activation moiety (e.g., a different guide nucleic acid) that is different from the activation moiety of the heterologous gene circuit. The additional activation moiety may be a portion of the heterologous gene circuit that is generated (e.g., expressed) only upon activation of the heterologous gene circuit. Alternatively, or in addition, the first and second gate units may each be activated by a different activation moiety that is not the same as the activation moiety of the heterologous gene circuit. Such a different activation moiety may be a portion of the heterologous gene circuit that is generated (e.g., expressed) only upon activation of the heterologous gene circuit.

[0099] In some embodiments of any of the systems disclosed herein, the gating unit may include gates (e.g., at least or up to about 1 gating moiety, at least or up to about 2 gating moieties, at least or up to about 3 gating moieties, at least or up to about 4 gating moieties, at least or up to about 5 gating moieties, etc.), and / or gene regulatory moieties (e.g., at least or up to about 1 gene regulatory moiety, at least or up to about 2 gene regulatory moieties, at least or up to about 3 gene regulatory moieties, at least or up to about 4 gene regulatory moieties, at least or up to about 5 gene regulatory moieties, at least or up to about 6 gene regulatory moieties, at least or up to about 7 gene regulatory moieties, at least or up to about 8 gene regulatory moieties, at least or up to about 9 gene regulatory moieties, at least or up to about 10 gene regulatory moieties, etc.). The gating moieties disclosed herein may include guide nucleic acid molecules (gNAs) (e.g., at least or up to about 1 gNA molecule, at least or up to about 2 gNA molecules, at least or up to about 3 gNA molecules, at least or up to about 4 gNA molecules, at least or up to about 5 gNA molecules, etc.). The gene regulatory portion disclosed herein may comprise a gNA (e.g., at least or up to about 1 gNA molecule, at least or up to about 2 gNA molecules, at least or up to about 3 gNA molecules, at least or up to about 4 gNA molecules, at least or up to about 5 gNA molecules, etc.). The guide nucleic acid molecule disclosed herein may comprise, but is not limited to, DNA, RNA, any analog thereof, or any combination thereof. In some embodiments of any of the systems disclosed herein, the gate portion and / or gene regulatory portion may be activatable to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and the complex is configured to or may bind to a target polynucleotide, for example, 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 may modulate the expression and / or activity level of a gene comprising the target polynucleotide.

[0100] In some cases, the guide nucleic acid molecule (gNA) expressed by the second gate unit (e.g., a functional gNA) can cause a modification to at least a portion of the first gate unit when activated. For example, the activated gNA of the second gate unit can generate a modification to the polynucleotide sequence of the first gate unit encoding the gNA (e.g., an activatable gNA) or the promoter sequence of the first gate unit operably linked to such a 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.

[0101] 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 caused by a single-strand break where there is a discontinuity in one nucleotide strand. Inactivation of a polynucleotide sequence or a target gene can be caused by 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, or more single-strand breaks. In some cases, inactivation of a gene can be caused by 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 single-strand break.

[0102] In some cases, a gNA may have a size (e.g., including both spacer and scaffold sequences) of at least or up to about 60 nucleotides, at least or up to about 70 nucleotides, at least or up to about 80 nucleotides, at least or up to about 85 nucleotides, at least or up to about 90 nucleotides, at least or up to about 95 nucleotides, at least or up to about 100 nucleotides, at least or up to about 105 nucleotides, at least or up to about 110 nucleotides, at least or up to about 120 nucleotides, at least or up to about 130 nucleotides, at least or up to about 140 nucleotides, at least or up to about 150 nucleotides, or at least or up to about 200 nucleotides.

[0103] In some cases, the scaffold sequence of the gNA may have a size of at least or up to about 30 nucleotides, at least or up to about 35 nucleotides, at least or up to about 40 nucleotides, at least or up to about 45 nucleotides, at least or up to about 50 nucleotides, at least or up to about 55 nucleotides, at least or up to about 60 nucleotides, at least or up to about 65 nucleotides, at least or up to about 70 nucleotides, at least or up to about 75 nucleotides, at least or up to about 80 nucleotides, at least or up to about 85 nucleotides, at least or up to about 90 nucleotides, at least or up to about 95 nucleotides, at least or up to about 100 nucleotides, at least or up to about 100 nucleotides, at least or up to about 120 nucleotides, at least or up to about 130 nucleotides, at least or up to about 140 nucleotides, or at least or up to about 150 nucleotides.

[0104] In some cases, the spacer sequence of the gNA may have a size of at least or up to about 10 nucleotides, at least or up to about 11, at least or up to about 12, at least or up to about 13, at least or up to about 14, at least or up to about 15, at least or up to about 16, at least or up to about 17, at least or up to about 18, at least or up to about 19, at least or up to about 20, at least or up to about 21, at least or up to about 22, at least or up to about 23, at least or up to about 24, at least or up to about 25, at least or up to about 26, at least or up to about 27, at least or up to about 28 nucleotides, at least or up to about 29 nucleotides, or at least or up to about 30 nucleotides.

[0105] In some embodiments of any of the systems disclosed herein, the initial (or first) gating unit of a heterologous genetic circuit disclosed herein may be activated (e.g., directly activated) by an activation moiety. The activation moiety may directly bind to at least a portion of the initial gating unit to activate the initial gating unit, e.g., thereby sequentially activating the heterologous genetic circuit. Alternatively, the activation moiety may activate the initial gating unit (e.g., through the use of electromagnetic energy) without directly binding 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 may not and need not include a gene regulatory moiety. In some cases, the initial gating unit may include at least one gene regulatory moiety, but may not and need not include a gating moiety (e.g., the activation moiety may be configured to activate the initial gating unit and at least one additional gating unit).

[0106] In some embodiments of any of the systems disclosed herein, the gNA of the gating moiety and / or gene regulatory moiety (e.g., the gNA encoded by the gating moiety and / or gene regulatory moiety) can be an activatable gNA. The activatable gNA can be, but is not limited to, any one of the following: ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogs thereof, or any combination thereof. In some embodiments, the vector (or expression cassette) encoding the activatable gNA can include an inactivation polynucleotide sequence to render the gNA inactive until activated (e.g., until the inactivation polynucleotide sequence is modified or removed from the vector). For example, the inactivation polynucleotide sequence can encode a self-cleaving polynucleotide molecule (e.g., a ribozyme). Alternatively, or in addition, the inactivation polynucleotide sequence can encode a non-canonical transcription termination sequence, as described below. The inactivating polynucleotide sequence may be part of or adjacent to the region of the vector encoding (i) the spacer sequence of the gNA, (ii) the scaffold sequence of the gNA, and / or (ii) any linker sequence between the spacer sequence and the scaffold sequence. The vector may comprise at least or up to about 1 inactivating polynucleotide sequence, at least or up to about 2 inactivating polynucleotide sequences, at least or up to about 3 inactivating polynucleotide sequences, at least or up to about 4 inactivating polynucleotide sequences, at least or up to about 5 inactivating polynucleotide sequences, at least or up to about 6 inactivating polynucleotide sequences, at least or up to about 7 inactivating polynucleotide sequences, at least or up to about 8 inactivating polynucleotide sequences, at least or up to about 9 inactivating polynucleotide sequences, or at least or up to about 10 inactivating polynucleotide sequences.

[0107] In some cases, the term "proGuide" as used generally herein may refer to such a vector (e.g., a plasmid) encoding an activatable gNA. A proGuide may be an example of a gate portion. A proGuide may be an example of a gene regulatory portion.

[0108] 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-cleaving and 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 embodiments, the activatable gNA molecule contains a non-canonical transcription termination sequence (e.g., a poly-X sequence, such as a poly-U or poly-T sequence), such that a functional gNA molecule is not expressed until the gene encoding the activatable gNA with the non-canonical transcription termination sequence is modified (e.g., to remove some 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 an attached fluorescent label.

[0109] In some cases, the size of the polyT sequence is equal to or greater than a threshold length, which is sufficient to reduce expression of the guide nucleic acid molecule from the polynucleotide sequence. Thus, a plasmid (e.g., a gate portion or gene regulatory portion) can encode an inactivated gNA containing a polyT sequence equal to or greater than the threshold length, and editing such a plasmid to reduce the length of the polyT to less than the threshold length allows expression of the entire gNA without premature termination, thereby activating the gNA. In some cases, the polyT sequence contains at least 5 Ts. In some cases, the polyT sequence contains at least 7 Ts. In some cases, the polyT sequence contains at least 8 Ts. In some cases, the polyT sequence contains at least 10 Ts. In some cases, the polyT sequence contains 5Ts to 15Ts. In some cases, the polyT sequence contains one or more additional nucleotides that are not Ts.

[0110] 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 portion of the coding polynucleotide sequence. Alternatively, the target polynucleotide sequence may not be a portion of the coding polynucleotide sequence. For example, the target polynucleotide sequence can be upstream of the coding polynucleotide sequence (e.g., a portion of the promoter of the coding polynucleotide sequence, such as a transcription start site (TSS)).

[0111] As provided herein, when a heterologous gene circuit is activated to induce multiple distinct modulations of target genes, as provided herein, the multiple distinct modulations of the target genes can be different (e.g., different degrees of change in the expression and / or activity levels of the target genes). For example, the first modulation provided by the first genetic unit and the second modulation provided 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 modulation and the second modulation 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 in addition, distinct modulations of target genes can be substantially the same (e.g., identical). Multiple distinct modulations can be individually sufficient to induce a desired change in expression and / or activity levels of the target gene. Alternatively, the distinct modulations can be individually insufficient to induce a desired change in expression and / or activity levels of the target gene.

[0112] 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.

[0113] In some cases, the guide nucleic acid molecule (gNA) (e.g., functional gNA) expressed by the second gate unit can cause a modification to at least a portion of the first gate unit upon activation. For example, the activated gNA of the second gate unit can generate a modification to the polynucleotide sequence of the first gate unit encoding the gNA (e.g., activatable gNA) or the promoter sequence of the first gate unit 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.

[0114] In some embodiments of any of the systems disclosed herein, the initial (or first) gating unit of a heterologous genetic circuit disclosed herein can be activated (e.g., directly activated) by an activation moiety. The activation moiety can 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 activation moiety can activate the initial gating unit without directly binding at least a portion of the initial gating unit (e.g., through the use of electromagnetic energy). In some cases, the initial gating unit can include at least one gating moiety and at least one gene regulatory moiety. In some cases, the initial gating unit can include at least one gating moiety, but may not and need not include a gene regulatory moiety. In some cases, the initial gating unit can include at least one gene regulatory moiety, but may not and need not include a gating moiety (e.g., the activation moiety can be configured to activate the initial gating unit and at least one additional gating unit).

[0115] In some embodiments of any 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 a ribonucleotide (e.g., a gRNA), a deoxyribonucleotide, any analog 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 and 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 embodiments, the gNA can be synthetic. In some embodiments, the gNA can have an attached fluorescent label.

[0116] In some embodiments of any of the systems disclosed herein, the gNA of the gate portion and / or gene regulatory portion (e.g., the gNA encoded by the gate portion and / or gene regulatory portion) can include a spacer sequence. In some cases, the spacer sequence can be specific to the target gene. Alternatively, the spacer sequence can be agnostic to the target gene.

[0117] As described above, the length of the spacer sequence of a gNA can affect the ability of the gNA to mediate Cas nuclease activity. In some cases, gNAs with spacer sequences of different lengths can be used in the same heterologous gene circuit to affect different types of cleavage, activation, inactivation, and / or regulation of one or more target nucleic acids. In some cases, a gNA spacer sequence shorter than a threshold length (e.g., about 16 nucleotides) can eliminate the nuclease activity of a Cas transcriptional regulator while still mediating DNA binding for transcriptional regulation of a target gene. In some cases, 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 a Cas protein while still mediating DNA binding.

[0118] For example, a gNA containing a 20-nucleotide spacer sequence (e.g., a gNA encoded by a gate moiety for targeting a gene regulatory portion plasmid) may be sufficient to promote the nuclease activity of an endonuclease (e.g., a Cas or Cas transcriptional modulator fusion protein) at the target polynucleotide sequence. Alternatively, or in addition, a gNA containing a 14-nucleotide spacer sequence (e.g., a gNA encoded by a gene regulatory portion) may hybridize to DNA but may not be long enough to mediate nuclease activity; it may only promote endonuclease binding to the cognate DNA sequence. Thus, shorter gNAs can selectively allow transcriptional regulation of target genes through the use of an endonuclease transcriptional modulator system (e.g., a Cas activator system, a Cas repressor system) without cleaving the target gene.

[0119] 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 caused by a double-strand break in which there is a discontinuity in both nucleotide strands. In some cases, the number of such double-strand breaks (e.g., required for such modification) can be 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, or at least or at most about 10.

[0120] In some cases, modifications of polynucleotide sequences (e.g., as components of gate units, such as gate segments) or target genes can be caused by indels, also known as insertion-deletion mutations. Indel mutations can include frameshift or non-frameshift mutations. Indel mutations can include point mutations, also known as base substitutions, in which only one base or base pair is modified. Indel mutations 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, 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.

[0121] 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 cleavage of 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 so 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 transcriptional activator provided herein. Alternatively or additionally, the gene regulatory portion can induce epigenetic modifications (or epigenomic modifications) provided herein.

[0122] In some cases, the modification of the polynucleotide sequence or target gene provided herein can inactivate the polynucleotide sequence or target gene. For example, the modification of the polynucleotide sequence or target gene can suppress or reduce the expression and / or activity level of the polynucleotide sequence or target gene. In some cases, the modification of the polynucleotide sequence or target gene provided herein can activate the polynucleotide sequence or target gene. For example, the modification of the polynucleotide sequence or target gene can increase the expression and / or activity level of the polynucleotide sequence or target gene.

[0123] In some cases, as provided herein, modification of a polynucleotide sequence or target gene may comprise decreasing the expression and / or activity level of the polynucleotide sequence or target gene by at least or at most about 0.1%, at least or at most about 0.2%, at least or at most about 0.3%, at least or at most about 0.4%, at least or at most about 0.5%, 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 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 30%, at least or at most about 40%, at least or at most about 50%, at least or at most about 60%, at least or at most about 70%, at least or at most about 80%, at least or at most about 90%, at least or at most about 95%, at least or at most about 99%, or about 100% (e.g., compared to a control lacking the modification, for example).

[0124] In some cases, modifications of a polynucleotide sequence or target gene provided herein increase 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 1.5 fold, at least or at most about 2 fold, at least or at most about 3 fold, or at least It may include a decrease of 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 11-fold, at least or up to about 12-fold, at least or up to about 13-fold, at least or up to about 14-fold, at least or up to about 15-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, or at least or up to about 100-fold (e.g., compared to a control lacking the modification).In some cases, modifications of the polynucleotide sequence or target gene provided herein may increase the expression and / or activity level of the polynucleotide sequence or target gene by at least or up to about 0.1%, at least or up to about 0.2%, at least or up to about 0.3%, at least or up to about 0.4%, at least or up to about 0.5%, at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about It may include an increase of 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500% (e.g., compared to a control lacking the modification).

[0125] In some cases, modifications of a polynucleotide sequence or target gene provided herein increase 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 1.5 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, or at least The modification may include increasing the expression level by 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 11-fold, at least or up to about 12-fold, at least or up to about 13-fold, at least or up to about 14-fold, at least or up to about 15-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 100-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, at least or up to about 500-fold, or at least or up to about 1,000-fold (e.g., as compared to a control lacking the modification).

[0126] In some embodiments of any of the systems disclosed herein, the gNA of a gate moiety and / or gene regulatory moiety (e.g., a gNA encoded by the gate moiety and / or gene regulatory moiety) may include a spacer sequence. In some cases, the spacer sequence may exhibit specific binding to a target gene (e.g., an endogenous target gene). Alternatively, the spacer sequence may be target gene-independent, but rather may exhibit specific binding to a target polynucleotide sequence of another gate moiety or another gene regulatory moiety. Exemplary spacer sequences can be found in Table 5.

[0127] Non-limiting examples of the one or more target genes may include EGF, FEV, FOXA2, GATA4, gata6, GCG, INS, isl1, LMX1A, MAFA, NEUROD1, NEUROG3, NKX2-2, NKX6-1, ONECUT1, PDX1, PTF1a, SHH, SOX17, SOX9, SST, and / or TBXT. In some cases, as provided herein, the spacer sequence of a guide nucleic acid (e.g., guide RNA) for a target gene is 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 86%, at least or up to about 90%, 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%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 110%, at least or up to about 111%, at least or up to about 112%, at least or up to about 113%, at least or up to about 114%, at least or up to about 115%, at least or up to about 116%, at least or up to about 117%, at least or up to about 118%, at least or up to about 119%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, 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 substantially about 100% sequence identity (e.g., SEQ ID NOS: 2030-2033, SEQ ID NOS: 2034-2037 for EGF targeting). FEV targeting, SEQ ID NOs: 2038-2041 FOXA2 targeting, SEQ ID NOs: 2041-2045 GATA4 targeting, SEQ ID NOs: 2046-2049 GATA6 targeting, SEQ ID NOs: 2050-2053 GCG targeting, SEQ ID NOs: 2054-2057 INS targeting, SEQ ID NOs: 2058-2061 isl1 targeting, SEQ ID NOs: 2062-2065 LMX1A targeting, SEQ ID NOs: 2066-2069 MAFA targeting, SEQ ID NOs: 2070-2073 NEUROD1 targeting, SEQ ID NOs: 2074-2077 NEUROG3 targeting, SEQ ID NOs: 2078-2081 NKX2-2 targeting, SEQ ID NOs: 2082-2085 NKX6-1 targeting, SEQ ID NOs: 2086-2079 ONECUT1 targeting, SEQ ID NOs: 2090-20932110-2117, or its complementary sequence. In some cases, the heterologous gene modulators provided herein may comprise a polynucleotide sequence (e.g., a contiguous polynucleotide sequence) that exhibits sequence identity to one or more members selected from PDX1 targeting, SEQ ID NOS:2094-2097 PTF1a targeting, SEQ ID NOS:2098-2101 SHH targeting, SEQ ID NOS:2102-2105 SOX17, SEQ ID NOS:2106-2109 SOX9 targeting, SEQ ID NOS:2110-2113 SST targeting, and SEQ ID NOS:2114-2117 TBXT targeting. In some cases, the heterologous gene modulators provided herein may exhibit sequence identity to one or more members selected from SEQ ID NOS:2030-2117, or a complementary sequence thereof. In some cases, the heterologous gene modulators provided herein exhibit sequence identity to one or more members selected from SEQ ID NOS:2030-2117, or a complementary sequence thereof. At least about 88%, at least or up to about 89%, 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 substantially about 100% sequence identity (e.g., SEQ ID NOS: 2030-2033, SEQ ID NOS: 2034-2037 for EGF targeting). FEV targeting, SEQ ID NOs: 2038-2041 FOXA2 targeting, SEQ ID NOs: 2041-2045 GATA4 targeting, SEQ ID NOs: 2046-2049 gata6 targeting, SEQ ID NOs: 2050-2053 GCG targeting, SEQ ID NOs: 2054-2057 INS targeting, SEQ ID NOs: 2058-2061 isl1 targeting, SEQ ID NOs: 2062-2065 LMX1A targeting, SEQ ID NOs: 2066-2069 MAFA targeting, SEQ ID NOs: 2070-2073 NEUROD1 targeting, SEQ ID NOs: 2074-2077 NEUROG3 targeting, SEQ ID NOs: 2078-2081 NKX2-2 targeting, SEQ ID NOs: 2082-2085 NKX6-1 targeting, SEQ ID NOs: 2086-2079The polynucleotide sequence may exhibit specific binding to a target gene comprising a polynucleotide sequence (e.g., a contiguous polynucleotide sequence) exhibiting sequence identity to one or more members selected from: ONECUT1 targeting, PDX1 targeting of SEQ ID NOs: 2090-2093, PTF1a targeting of SEQ ID NOs: 2094-2097, SHH targeting of SEQ ID NOs: 2098-2101, sox17 of SEQ ID NOs: 2102-2105, SOX9 targeting of SEQ ID NOs: 2106-2109, SST targeting of SEQ ID NOs: 2110-2113, and TBXT targeting of SEQ ID NOs: 2114-2117, or a complementary sequence thereof (e.g., using uracil to thymine conversion).

[0128] As disclosed herein, the gene regulatory portion can include an endonuclease, such as a CRISPR-Cas protein, that exhibits at least some of its nuclease activity. For example, the nuclease activity can be utilized to activate the expression or activity of a guide nucleic acid molecule, thereby activating at least a portion of the heterologous gene circuit described herein.

[0129] The gene regulatory portion disclosed herein may include an endonuclease operably linked to a transcriptional effector, including a transcriptional activator or repressor heterologous to the cell. The endonuclease may be native or engineered to exhibit reduced (or substantially no) nuclease activity, so that the endonuclease can be used to specifically bind to a target gene (e.g., an endogenous gene such as FOX, SOX, GATA, MAF, bHLH, homeobox, TBX, or EST) without cleaving the target gene. In some cases, the nuclease may be an inactivated Cas (dCas). Alternatively, once the endonuclease identifies and binds to the target gene, a transcriptional effector bound (e.g., covalently or non-covalently) to the endonuclease can interact with the target gene to increase or decrease the expression level of the target gene, thereby increasing or decreasing the activity level of the target gene. For example, the endonuclease and the transcription effector can be part of a fusion protein encoded by the same expression cassette.

[0130] Figure 10 illustrates the use of a heterologous gene circuit in combination with an endonuclease-transcription effector fusion (e.g., a CRISPR Cas transcription activator, such as Cas9-VPR). Each gate moiety can be a modified self-inactivating (e.g., self-destructing) guide RNA that, when not inactivated, forms a complex with the CRISPR Cas transcription effector fusion. A first activating moiety (denoted as an activating guide RNA or "aGuide") can convert the first gate moiety into an activating guide RNA (denoted as a matureGuide). Each matureGuide can target an additional gene regulatory moiety encoding an activatable guide RNA for a target gene (denoted as a ramGuide) to activate the aGuide. The activated ramGuide can then form a complex with a CRISPR Cas transcription effector fusion protein to bind to the target gene and regulate the expression level of the target gene. Activated matureGuide may also target additional gates downstream in the signaling cascade of the heterologous gene circuit, and subsequently regulate the expression of one or more additional genes.

[0131] In some embodiments, the transcriptional effector can be a histone epigenetic modifier (or histone modifier). In some cases, the histone epigenetic modifier can regulate histones through methylation (e.g., a histone methylation modifier such as an amino acid methyltransferase, e.g., KRAB). In some cases, the histone epigenetic modifier can regulate histones through acetylation. In some cases, the histone epigenetic modifier can regulate histones through phosphorylation. In some cases, the histone epigenetic modifier can regulate histones through ADP-ribosylation. In some cases, the histone epigenetic modifier can regulate histones through glycosylation. In some cases, the histone epigenetic modifier can regulate histones through SUMOylation. In some cases, histone epigenetic modifiers can regulate histones through ubiquitination. In some cases, histone epigenetic modifiers can regulate histones by remodeling histone structure, for example, through an ATP hydrolysis-dependent process.

[0132] In some embodiments, the transcription effector can be a gene epigenetic modifier (or gene modifier). In some cases, the gene modifier can regulate genes through methylation (e.g., a gene methylation modifier such as DNA methyltransferase or DNMT). In some cases, the gene modifier can regulate genes through acetylation.

[0133] In some embodiments, the transcriptional effector may be derived from a family of related histone acetyltransferases, non-limiting examples of which include the GNAT subfamily, the MYST subfamily, the p300 / CBP subfamily, the HAT1 subfamily, GCN5, PCAF, Tip60, MOZ, MORF, MOF, HBO1, p300, CBP, HAT1, ATF-2, SRC1, and TAFII250.

[0134] In some embodiments, the transcription effector may be derived from a histone lysine methyltransferase. Non-limiting examples of histone lysine methyltransferases include EZH subfamily, non-SET subfamily, other SET subfamily, PRDM subfamily, SET1 subfamily, SET2 subfamily, SUV39 subfamily, SYMD subfamily, ASH1L, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, NSD2, NSD3, PRDM1, PRDM10, PRDM11, PRDM12, P These include RDM13, PRDM14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM7, PRDM8, PRDM9, SET1, SET1L, SET2L, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETDB1, SETDB2, SETMAR, SUV39H1, SUV39H2, SUV420H1, SUV420H2, SYMD1, SYMD2, SYMD3, SYMD4, and SYMD5.

[0135] Non-limiting examples of transcription effectors that enhance target gene expression or activity include transcription activators such as VP16, VP64, VP48, VP160, p65 subdomains (e.g., from NFkB), VP64-p65-rta fusion proteins (VPR), and the activation domain of EDLL and / or TAL activation domains (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI60, CLOCK; and Ten-Eleven These include, but are not limited to, DNA demethylases such as Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1.

[0136] Non-limiting examples of transcriptional effectors that reduce the expression or activity of target genes include transcriptional repressors such as Kruppel-associated box (KRAB or SKD), KOX1 repression domain, Mad histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, and RIZ1; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, and JARIDID / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, and HDAC11; and Hal DNA repressors. These may include, but are not limited to, DNA methylases such as m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, and CMT2 (plants), as well as periphery recruitment elements such as lamin A and lamin B.

[0137] Various aspects of the present disclosure provide multiple heterologous gene circuits that can be individually activated to sequentially modulate the expression and / or activity levels of multiple different target genes. In some embodiments, a first heterologous gene circuit is activated to convert a plurality of cells from a first cell type to a second cell type, and then a second heterologous gene circuit is activated to convert the plurality of cells from the second cell type to the target cell type.

[0138] In some embodiments, activation of the second genetic circuit can be performed immediately after activation of the first heterologous genetic circuit. Alternatively, activation of the second genetic circuit can be performed at least about 30 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 11 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, 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 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 1 year, or more after activation of the first genetic circuit.

[0139] 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.

[0140] The one or more target genes disclosed herein may 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.

[0141] The one or more target genes disclosed herein may comprise cell differentiation regulators including growth factors, transcription factors, myogenic regulators, immune cell regulators, neuroregulators, stem cell differentiation factors, endocrine regulators, beta cell regulators, pancreatic lineage regulators, chondrogenic regulators, osteogenic regulators, senescence factors, stemness factors (e.g., dedifferentiation factors), and the like.

[0142] In some cases, the one or more target genes (e.g., one or more pancreatic lineage regulators) may include TBXT, FOXA2, SOX17, PDX1, GATA4, p53shRNA, HNF6, PTF1A, NGN3, NEUROG3, NKX6.1, SOX9, NKX2.2, MAFA, ONECUT1, NEUROD3, OCT4, NANOG, SOX2, CXCR4, HNF1β, INS, GCG, SST, and ISL1.

[0143] In some cases, the one or more target genes may include a homeobox gene. Homeobox genes are genes that regulate large-scale anatomical features, for example, in the early stages of embryonic development. Types of homeobox genes include HOX genes, LIM genes, PAX genes, POU genes, CERS genes, HNF genes, SINE genes, CUT genes, ZF genes, paraHOX genes, DLX genes, TALE genes, PRD genes, and NKL genes. 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, HOXC14, HOXC15, HOXC16, HOXC17, HOXC18, HOXC19, HOXC20, HOXC21, HOXC22, HOXC23, HOXC24, HOXC25, HOXC26, HOXC28, HOXC29, HOXC26, HOXC29, HOXC28, HOXC29, HOXC29, HOXC20, HOXC21, HOXC22, HOXC23, HOXC24, HOXC25, HOXC26, HOXC27, HOXC28, HOXC29 ...0, HOXC21, HOXC22, HOXC23, HOXC24, HOXC25, HOXC26, HOXC28, HOXC29, HOXC29, HOXC20, HOXC21, HOXC22, HOXC23, C10, 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, IRX6, MEIS1, MEIS2, MEIS3, MKX, PBX1, PBX2, PBX3, PBX4, PKNOX1, PKNOX2, TGIF1TGIF2 , TGIF2LX, TGIF2LY, ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, LMX1B, HDX, POU1F1, PO U2F1, POU2F2, POU2F3, POU3F1, POU3F2, POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1, POU5F1P1, POU5F2, P OU6F1, POU6F2, LASS2, LASS3, LASS4, LASS5, LASS6, HMBOX1, HNF1A, HNF1B, SIX1, SIX2, SIX3, SIX4, SIX5, SIX6,ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2, SATB1, SATB2, ADNP, ADNP2, TSHZ1, TSHZ2, TSHZ3, ZEB1, ZE B2, ZFHX2, ZFHX3, ZFHX4, ZHX1, HOMEZ, ALX1(CART1), ALX3, ALX4, ARGFX, ARX, DMBX1, DPRX, DRGX, DUX A, DUXB, DUX(1, 2, 3, 4, 4c, 5), ESX1, GSC, GSC2, HESX1, HOPX, ISX, LEUTX, MIXL1, NOBOX, OTP, OTX1, O TX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PR RRX1, PRRX2, RAX, RAX2, 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, NA These may include NOG, 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.

[0144] In some cases, the target gene may include CDX1. CDX1 is a parahox-class homeobox protein that is expressed in the developing endoderm and whose expression persists in the intestine throughout adulthood.

[0145] In some cases, the target gene may include CDX2, a parahox-class homeobox transcription factor expressed in the nuclei of intestinal epithelial cells and playing an essential role in the development and function of the digestive system.

[0146] In some cases, the target gene may include CDX4. CDX4 is a parahox class homeobox transcription factor involved in embryonic tissue formation, anterior-posterior patterning, and blood formation during embryogenesis.

[0147] In some cases, the target gene can include LMX1A, a protein that binds to A / T-rich sequences in the insulin promoter and stimulates insulin transcription.

[0148] In some cases, the target gene may include LMX1B, a transcription factor that plays a central role in dorsoventral patterning of the vertebrate limb.

[0149] In some cases, the target gene can include GSX1, a parahox class homeobox transcription factor that plays a role in pituitary development.

[0150] In some cases, the target gene can include GSX2, which is a parahox-class homeobox transcription factor that plays a role in brain development.

[0151] In some cases, the target gene can include PDX1, a parahox class homeobox gene required for pancreatic development, including beta cell maturation and duodenal differentiation.

[0152] In some cases, the target gene may include NKX6.1 (also called NKX6-1). NKX6.1 is an NKX-class homeobox. NKX6.1 is a bifunctional transcription regulator required for β-cell development.

[0153] In some cases, the target gene may include NKX2.2 (also called NKX2-2). NKX2.2 is a homeobox of the NKX class. NKX2.2 is a transcription factor involved in the morphogenesis of the central nervous system.

[0154] In some cases, the target gene may include PAX6. PAX6 is a PRD-class homeobox. PAX6 is a transcription factor that is important in the development of eyes and other sensory organs, certain neural and epidermal tissues, and other structures derived from ectodermal tissues.

[0155] In some cases, the target gene may include ONECUT1. ONECUT1 is a CUT-class homeobox. ONCECUT1 is a transcription factor enriched in the liver, which stimulates the transcription of liver-expressed genes.

[0156] In some cases, the target gene may include ISL1, which is a LIM-class homeobox transcription factor that plays an important role in the formation of pancreatic islets of Langerhans.

[0157] In some cases, one or more target genes may 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 may include TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, TBX22, and TBXT (Brachyury protein).

[0158] In some cases, the target gene may include TBXT. TBXT, also known as T-box transcription factor T or Brachyury protein, functions as a transcription factor in the T-box family of genes. TBXT plays a role in defining the midline in bilateral organisms and helps establish the anterior-posterior axis. It also plays a role in helping define the mesoderm during gastrulation.

[0159] In some cases, one or more target genes may include basic helix-loop-helix transcription factors (bHLH genes). bHLH transcription factors are involved in regulating 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 , MXD4, MXI1, MYC, MYCL1, MYCN, MYF5, MYF6, MYOD1, MYOG, NCOA1, NCOA3, NEUROD1, NEUROD2, 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.

[0160] In some cases, the target gene may include NEUROG3. NEUROG3, or neurogenin 3, is an endocrine transcription factor that activates gene transcription in endocrine precursor cells. NEUROG3 is important for pancreatic islet differentiation and regeneration and functions to directly enhance the expression of lineage-committed transcription factors required for the differentiation of endocrine precursor cells into each of the endocrine cell subtypes.

[0161] In some cases, the target gene can include NEUROD1, a transcription factor that regulates the expression of the insulin gene.

[0162] In some cases, the target gene can include PTF1a, a transcription factor that plays a role in pancreatic development.

[0163] In some cases, one or more target genes may comprise SRY-related box transcription factor (SOX gene).SOX transcription factor is involved in developmental regulation.Non-limiting examples of SOX transcription factor may include SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21, SOX30 and SRY.

[0164] In some cases, one or more target genes may include SOX group A, which includes SRY. In some cases, one or more target genes may include SOX group B1, which includes SOX1, SOX2, and / or SOX3. In some cases, one or more target genes may include SOX group B2, which includes SOX14 and / or SOX21. In some cases, one or more target genes may include SOX group C, which includes SOX4, SOX11, and / or SOX12. In some cases, one or more target genes may include SOX group D, which includes SOX5, SOX6, and / or SOX13. In some cases, one or more target genes may include SOX group E, which includes SOX8, SOX9, and / or SOX10. In some cases, one or more target genes may include SOX group F, which includes SOX7, SOX17, and / or SOX18. In some cases, one or more target genes may include SOX group G, which includes SOX15. In some cases, the one or more target genes may include SOX group H, which includes SOX30.

[0165] In some cases, the target gene can include SOX17, a transcription factor involved in regulating vertebrate embryonic development and determining endoderm cell fate.

[0166] In some cases, the target gene can include SOX9, a transcription factor that acts during cell differentiation and regulates transcription of the anti-Mullerian hormone (AMH) gene.

[0167] In some cases, one or more target genes may comprise 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 may include FOXA, FOXB, FOXC, FOXD, FOXE, FOXF, FOXG, FOXH, FOXI, FOXJ, FOXK, FOXL, FOXM, FOXN, FOXO, FOXP, FOXQ, FOXR and FOXS.

[0168] In some cases, the target gene can include FOXA1, which is a transcriptional activator for liver-specific transcripts.

[0169] In some cases, the target gene may include FOXA2, a transcription factor that plays an important role in mature tissues during development and, when dysregulated or mutated, in cancer.

[0170] In some cases, the target gene can include FOXA3, which is a transcriptional activator of liver-specific transcripts.

[0171] In some cases, the one or more target genes may include erythroblast transformation specific (ETS) genes, which are transcription factors unique to animals and involved in tissue development. Non-limiting examples of ETS genes can include ELF1, ELF2 (NERF), ELF4 (MEF), GABPα, ERG, FLI1, 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).

[0172] In some examples, the target gene may include ERG, a transcriptional regulator that also functions as an oncogene.

[0173] In some cases, the target gene can include FLI1, a proto-oncogene and transcription factor involved in proliferation and terminal differentiation.

[0174] In some cases, the target gene can include FEV, which is a transcriptional repressor.

[0175] In some cases, one or more target genes may include GATA genes. GATA genes are transcription factors characterized by their ability to bind to the DNA sequence "GATA". Non-limiting examples of GATA genes may include GATA1, GATA2, GATA3, GATA4, GATA5, and GATA6.

[0176] In some cases, the target gene can include GATA4, a zinc finger transcription factor that regulates genes involved in embryogenesis and cardiac muscle differentiation and function.

[0177] In some cases, the one or more target genes may include a MAF transcription factor, which is an oncogene involved in immune cell differentiation.

[0178] In some cases, use of the heterologous gene circuits disclosed herein can be used to differentiate endodermal stem cells into pancreatic lineage 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%, 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 produced by using the heterologous gene circuits disclosed herein are of the target cell type.

[0179] In some cases, the use of the heterologous gene circuits disclosed herein can be used to differentiate endoderm stem cells into pancreatic lineage cells, for example, in the absence of feeder cells, serum, and one, two, or all of exogenous growth factors. The use of the heterologous gene circuits disclosed herein can be used to differentiate endoderm stem cells into pancreatic lineage cells, for example, in the absence of feeder cells, serum, and exogenous growth factors. 6 , up to about 9 × 10 5 , up to about 8 × 105 , 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 pieces, up to about 2 x 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 more pancreatic lineage cells from at least about 1 x 10 4 , at least about 2 × 10 4 , at least about 5 × 10 4 , at least about 1 x 10 5 , at least about 2 × 10 5 , at least about 5 × 10 5 , at least about 1 x 10 6 , at least about 2 × 10 6 , at least about 5 × 10 6 , at least about 1 x 10 7 , at least about 2 × 10 7 , at least about 5 × 10 7 , at least about 1 x 10 8 , at least about 2 × 10 8 , at least about 5 × 10 8 , at least about 1 x 10 9 , at least about 2 × 10 9 , at least about 5 × 10 9 , at least about 1 x 10 10 , at least about 2 × 10 10 , at least about 5 × 10 10 , at least about 1 x 10 15 , at least about 2 × 10 15 , at least about 5 × 10 15 , or more beta cells.

[0180] In some cases, the use of the heterologous gene circuits disclosed herein can be used to differentiate pluripotent stem cells (PSCs, e.g., induced PSCs or iPSCs) into beta cells, e.g., in the absence of one, two, or all of feeder cells, serum, and exogenous growth factors. The use of the heterologous gene circuits disclosed herein can be used to differentiate up to about 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 pieces, up to about 4 x 10 5 pieces, up to about 3 x 10 5 pieces, up to about 2 x 10 5 pieces, up to about 1 x 10 5 pieces, up to about 5 x 10 4 pieces, up to about 2 x 10 4 pieces, up to about 1 x 10 4 or more pancreatic lineage cells, at least about 1 x 10 4 , at least about 2 × 10 4 , at least about 5 × 10 4 , at least about 1 x 10 5 , at least about 2 × 10 5 , at least about 5 × 10 5 , at least about 1 x 10 6 , at least about 2 × 10 6 , at least about 5 × 10 6 , at least about 1 x 10 7 , at least about 2 × 10 7 , at least about 5 × 10 7 , at least about 1 x 10 8 , at least about 2 × 10 8 , at least about 5 × 10 8 , at least about 1 x 10 9 , at least about 2 × 10 9 , at least about 5 × 10 9 , at least about 1 x 10 10 , at least about 2 × 10 10 , at least about 5 × 10 10 , at least about 1 x 10 15 , at least about 2 × 1015 , at least about 5 × 10 15 , or more beta cells.

[0181] Such generation of beta cells by using the heterologous gene circuits disclosed herein can be achieved within a period 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 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, or less.

[0182] In some cases, beta cells generated by this method produce more insulin than beta cells obtained by directed differentiation. Beta cells generated using the provided methods may produce at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 120%, at least about 150%, or at least about 200% more insulin than beta cells obtained via directed differentiation. Alternatively, or in addition, beta cells generated by this method may produce equivalent amounts of insulin compared to beta cells obtained by directed differentiation.

[0183] In some cases, beta cells or pancreatic lineage cells generated by this method exhibit higher expression levels of two or more positive pancreatic lineage cell markers compared to control pancreatic lineage cells. Non-limiting examples of positive pancreatic lineage cell markers may include EpCam, ECAD, CD142, and CD49a.

[0184] In some cases, the beta cells or pancreatic lineage cells generated by this method have 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 120%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 510%, at least about 520%, at least about 53 0%, 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% higher expression levels.

[0185] In some cases, beta cells or pancreatic lineage cells generated by this method have at least or up to about 0.1 fold, at least or up to about 0.2 fold, at least or up to about 0.3 fold, at least or up to about 0.4 fold, at least or up to about 0.5 fold, at least or up to about 0.6 fold, at least or up to about 0.7 fold, at least or up to about 0.8 fold, at least or up to about 0.9 fold, at least or up to about 1 fold, at least or up to about 2 fold, at least or up to about 3 fold, 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 11 fold, at least or up to about 12 fold, at least or up to about 13 fold, at least or up to about 14 fold, at least or up to about 15 fold, at least or up to about 16 fold, at least or up to about 17 fold, at least or up to about 18 fold, at least or up to about 19 fold, at least or up to about 20 fold, at least or up to about 21 fold, at least or up to about 22 fold, at least or up to about 23 fold, at least or up to about 24 fold, at least or up to about 25 fold, at least or up to about 26 fold, at least or up to about 27 fold, at least or up to about 28 fold, at least or up to about 29 fold, at least or up to about 30 fold, at least or up to about 31 fold, at least or up to about 32 fold, exhibits an expression level that is 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 higher.

[0186] In some cases, beta cells or pancreatic lineage cells generated by this method exhibit higher expression levels of two or more negative pancreatic lineage cell markers compared to control pancreatic lineage cells. Non-limiting examples of negative pancreatic lineage cell markers can include CD49.

[0187] In some cases, the beta cells or pancreatic lineage cells generated by this method have a phenotype that is 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%, or at least about 80% higher than control pancreatic lineage cells. or 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% lower expression levels of negative pancreatic lineage cell markers.

[0188] In some cases, beta cells or pancreatic lineage cells generated by this method have a phenotype that 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 or at least 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 lower expression levels of negative pancreatic lineage cell markers.

[0189] In some cases, the expression levels of pancreatic lineage cell markers can be measured using methods such as, but not limited to, RT-PCR, Western blotting, Northern blotting, protein staining, mRNA staining, and RNA sequencing.

[0190] In some cases, the expression level can be measured at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 28 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours, at least about 48 hours, 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, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or more days after introduction of the genetic circuit.

[0191] In some cases, the beta cells or pancreatic lineage cells generated by this method can be immature beta cells. Alternatively, the beta cells or pancreatic lineage cells generated by this method can be mature beta cells. In some cases, the progenitor cells generated can be substantially mitotically dormant. Alternatively, the progenitor cells generated can be substantially mitotically active.

[0192] In some cases, the first gating unit may be configured to reduce the expression and / or activity level of one or more target genes. In some cases, the first gating unit may be configured to enhance the expression and / or activity level of one or more target genes. In some cases, the first gating unit may be configured to maintain the expression and / or activity level of one or more target genes.

[0193] In some cases, the regulation of the first target gene may occur before the regulation of the second target gene. In some cases, the regulation of the first target gene may occur after the regulation of the second target gene. In some cases, the regulation of the first target gene may occur approximately simultaneously with the regulation of the second target gene.

[0194] In some examples, regulating the target gene comprises increasing the expression level of the second target gene. Alternatively, regulating the target gene may comprise decreasing the expression level of the second target gene. Alternatively, regulating the target gene may comprise maintaining the expression level of the second target gene.

[0195] In some cases, the use of heterologous gene circuits can induce cells to differentiate into a desired cell type in the absence of growth factors, serum (such as fetal bovine serum, human serum AB, etc.), or other exogenous cell differentiation regulators or culture media. Serum can include the liquid fractions of coagulated blood, which contains trophic and macromolecular factors essential for cell growth.

[0196] Alternatively, the use of heterologous gene circuits can induce differentiation of cells into a desired cell type using reduced amounts of serum and / or growth factors (e.g., 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even to substantially serum-free conditions). A reduction in serum amount may allow for more consistency across experiments or batches of cells, increased proliferation and / or productivity of differentiated cells, better control of physiological responsiveness, and reduced risk of contamination with serum-derived factors in cell culture.

[0197] In some cases, the use of heterologous gene circuits in stem cells (e.g., iSPCs, endodermal stem cells) can induce the differentiation of endodermal stem cells into pancreatic lineage cells (e.g., β cells) in the absence of growth factors, serum (such as fetal bovine serum, human serum AB), or other exogenous cell differentiation regulators or media. In some cases, the use of heterologous gene circuits disclosed herein can be used to differentiate stem cells into β cells in the absence of one or both of growth factors and serum. The resulting β cells generated by using the heterologous gene 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 cell population obtained.

[0198] In some cases, conversion of one cell type (e.g., PSC, endodermal stem cell, or pancreatic lineage cell) to another cell type (e.g., β cell) using a heterologous gene circuit can result in a target cell type. Alternatively, conversion of one cell type (e.g., PSC, endodermal stem cell, or pancreatic lineage cell) to another cell type (e.g., β cell) using a heterologous gene circuit can result in an intermediate cell type. The intermediate cell type can undergo a second conversion using a second gene circuit to achieve the target cell type.

[0199] The conversion of a cell from one cell type to another cell type can include the regulation of multiple target genes. For example, the conversion can include the regulation 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 conversion can include the regulation 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 gene disclosed herein may 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 gene disclosed herein may 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 modulations. The regulation of one or more target genes (e.g., endogenous genes) as induced by a heterologous gene circuit of the present disclosure can be artificial regulation (or heterologous regulation) that would not otherwise occur in the cell in the absence of (i) the heterologous gene circuit and / or (ii) the activating portion of the heterologous gene circuit.

[0200] As shown in Figure 3, various heterologous gene circuits can be designed to regulate the expression or activity levels of multiple genes (e.g., multiple endogenous genes) in a cell over multiple different time points.

[0201] For example, a heterologous gene circuit can be designed to (i) regulate the expression level of a first gene and (ii) subsequently regulate the expression level of a second gene. For example, a heterologous gene circuit can be designed to (i) activate the expression level of a first gene and (ii) subsequently activate the expression level of a second gene. Alternatively, a heterologous gene circuit can be designed to (i) activate the expression level of a first gene and (ii) subsequently reduce the expression level of a second gene. Alternatively, a heterologous gene circuit can be designed to (i) reduce the expression level of a first gene and (ii) subsequently activate the expression level of a second gene. Alternatively, a heterologous gene circuit can be designed to (i) reduce the expression level of a first gene and (ii) subsequently reduce the expression level of a second gene. The first gene and the second gene can be the same gene. Alternatively, the first gene and the second gene can be different genes. A heterologous gene circuit can be designed to regulate the expression level of an additional gene. A heterologous gene circuit can be designed to include 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 additional genes. The additional genes can be activated. Alternatively, the additional genes can be decreased. A heterologous gene circuit can be designed to regulate expression of the additional genes before regulating the expression levels of both the first gene and the second gene. Alternatively, or in addition, a heterologous gene circuit can be designed to regulate expression of the additional genes after regulating the expression level of the first gene and before regulating the expression level of the second gene. Alternatively, or in addition, a heterologous gene circuit can be designed to regulate expression of the additional genes after regulating the expression levels of both the first gene and the second gene.

[0202] Heterologous gene circuits can be designed to include a first member that is a FOX, SOX, and / or GATA, and a second member that is a bHLH, homeobox, and / or MAF. Heterologous gene circuits can be designed to include a FOX and a bHLH. Heterologous gene circuits can be designed to include a FOX and a homeobox. Heterologous gene circuits can be designed to include a FOX and a MAF. Heterologous gene circuits can be designed to include a SOX and a bHLH. Heterologous gene circuits can be designed to include a SOX and a homeobox. Heterologous gene circuits can be designed to include a SOX and a MAF. Heterologous gene circuits can be designed to include a GATA and a bHLH. Heterologous gene circuits can be designed to include a GATA and a homeobox. Heterologous gene circuits can be designed to include a GATA and a MAF.

[0203] Activation of a heterologous gene circuit in a cell disclosed herein can modulate the expression or activity levels of multiple genes over multiple different time points to convert the cell into a different cell type (e.g., from a stem cell to a tissue-specific progenitor cell, etc.). The rate of such cell type conversion through use of a heterologous gene circuit can be at least or up to about 1 percent (%), at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, or at least or up to about 95% greater than the rate of cell type conversion through use of a control heterologous gene circuit (e.g., for simultaneous activation of multiple target genes).

[0204] Activation of heterologous gene circuits in cells disclosed herein can modulate the expression or activity levels of multiple genes over multiple different time points to convert the cell into a different cell type (e.g., from a stem cell to a tissue-specific progenitor cell, etc.) Conversion of a cell into a different cell type can occur in less than about 20 days, less than about 19 days, less than about 18 days, less than about 17 days, less than about 16 days, less than about 15 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, less than about 2 days, or less than about 1 day.

[0205] The cells (e.g., initial cells that are modified into the engineered cells disclosed herein, final cell products produced from the 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, mesodermal cells, endodermal cells, pancreatic lineage cells, neurosecretory cells, duct cells, odontoblasts, cementoblasts, glial cells, or stromal cells.

[0206] 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, T helper cells), natural killer cells, and cytokine-induced killer (CIK) cells (see, e.g., US20080241194), myeloid cells such as granulocytes (basophils, eosinophil granulocytes, neutrophils / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, and dendritic cells, thyroid gland (thyroid epithelial cells, parafollicular cells), parathyroid gland (parathyroid chief cells, oxyphil cells), and the like. Cells from the endocrine system, including cells of the adrenal gland (chromaffin cells), pineal gland (pineal cells), glial cells (astrocytes, microglia), magnocellular neurosecretory cells, stellate cells, Boettcher cells, and pituitary gland (gonadotropes, corticotropes, thyrotropes, somatotropes, lactotropes), cells of the nervous system, including pneumocytes (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells, and Cells of the respiratory system, including myocardiocytes, cells of the circulatory system, including pericytes, cells of the digestive system, including stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, and S cells, enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells),Enteroendocrine cells, including osteoblasts, osteocytes, osteoclasts, and teeth (cementoblasts, ameloblasts), paraxial mesoderm cells, including chondrocytes, cartilage cells, or skin cells, including trichocytes, keratinocytes, and melanocytes (nevus cells), muscle cells, including myocytes, podocytes, juxtaglomerular cells, and intraglomerular mesangial cells. Urinary tract cells including extraglomerular mesangial cells, kidney proximal tubule brush border cells, and macula densa cells; germ line cells including spermatozoon, Sertoli cells, Leydig cells, and ovum; as well as adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiating epidermal cells), epidermal basal cells (stem cells), keratinocytes of the fingernails and toenails, nail bed basal cells (stem cells), medullary hair shaft cells, and cortical hair stem cells. hair shaft cell), epidermal hair stem cell (Cuticular hair shaft cell)Hair root sheath cells, cuticular hair root sheath cells, hair root sheath cells of Huxley's layer, hair root sheath cells of Henle's layer, external hair root sheath cells, hair matrix cells (stem cells), wet stratified barrier epithelial cells, surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, basal cells of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina (stem cells), urinary epithelium cells (urinary bladder and ureter) ducts), exocrine secretory epithelial cells, salivary gland mucous cells (polysaccharide-rich secretion), salivary gland serous cells (glycoprotein enzyme-rich secretion), Von Ebner's gland cells in the tongue (cleansing the taste buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), ceruminous gland cells in the ear (wax secretion), and eccrine sweat gland dark cells.Other cells may be included, including Eccrine sweat gland clear cells (glycoprotein secretion), Eccrine sweat gland clear cells (small molecule secretion). Apocrine sweat gland cells (odoriferous secretion, sex-hormone sensitive), Gland of Moll cells in the eyelids (specialized sweat glands), Sebaceous gland cells (lipid-rich sebum secretion), Bowman's gland cells in the nose (cleansing the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), Seminal vesicle cells (secreting seminal fluid components, including fructose for sperm swimming), Prostate gland cells Bulbourethral gland cell (mucus secretion), Bartholin's gland cell (vaginal lubricant secretion), gland of Littre cell (mucus secretion), Uterus endometrium gastric gland zymogenic cell (pepsinogen secretion), gastric gland zymogenic cell (pepsinogen secretion), gastric gland zymogenic cell (pepsinogen secretion), gastric gland zymogenic cell (pepsinogen secretion)Cells (secreting hydrochloric acid), pancreatic acinar cells (secreting bicarbonate and digestive enzymes), Paneth cells of the small intestine (secreting lysozyme), type II pneumocytes of the lungs (secreting surfactant), Clara cells of the lungs, hormone-secreting cells, anterior pituitary cells, somatotropes, lactotrophs, thyrotropes, gonadotrophs, corticotrophs, intermediate pituitary cells, magnocellular neurosecretory cells, intestinal and respiratory system cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, oxyphil cells, adrenal cells, chromaffin cells cells, Leydig cells in the testis, Theca interna cells in the ovarian follicle, Corpus luteum cells in the ruptured follicle, Granulosa lutein cells, Theca lutein cells, Juxtaglomerular cells (renin secretion), Macula densa cells in the kidney, metabolic and storage cells, barrier function cells (lung, gastrointestinal tract, exocrine glands, and urogenital tract), kidney, immature beta cells, mature beta cells, type I pneumocytes (lining the air spaces in the lungs), pancreatic duct cells (centroacinar cells in the breast), non-muscularis duct cells cells (such as sweat glands, salivary glands, and mammary glands), bile duct cells, duct cells (such as seminal vesicles and prostate glands), epithelial cells lining internal body cavities, propulsive cellsCiliated cells with propulsive function, extracellular matrix secreting cells, contractile cells, skeletal muscle cells, stem cells, cardiac muscle cells, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells angerhans 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 committed progenitors for the blood and immune system (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic neuron cells, sense organ and peripheral neuron supporting cells cells), central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonium / oocyte, spermatid, spermatid, spermatogonium cell (spermatid stem cell), spermatozoon, nurse cells, ovarian follicular cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, and interstitial kidney cells.

[0207] In one aspect, the present disclosure provides systems and methods capable of converting a plurality of pluripotent stem cells (PSCs) into a plurality of tissue-specific progenitor cells.

[0208] Pluripotent stem cells may include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Tissue-specific progenitor cells may include endodermal stem cells, hematopoietic stem cells (HSCs), myeloid progenitor cells, muscle stem cells, pancreatic lineage cells, neural stem cells, epithelial stem cells, epidermal stem cells, mammary stem cells, intestinal stem cells, neural crest stem cells, or testicular stem cells.

[0209] In some cases, the tissue-specific progenitor cells or tissue-specific cells generated as disclosed herein may be derived from isolated mesenchymal stem cells (MSCs) (e.g., isolated from the subject's bone marrow). Alternatively, in some cases, the tissue-specific progenitor cells or tissue-specific cells generated as disclosed herein may not be derived from isolated MSCs.

[0210] In some cases, the conversion of stem cells into tissue-specific progenitor cells is characterized by the production of fewer non-endodermal cells or fewer enterochromaffin cells compared to control conversion of multiple stem cells in a medium containing (i) serum and / or (ii) an exogenous cell differentiation regulator. Non-endodermal cells can be mesodermal or ectodermal cells. Mesodermal cells can differentiate into mesodermal organs such as the kidney, ureter, heart, blood, gonads, adrenal cortex, muscle, skeleton, dermis, connective tissue, or mesothelium. Ectoderm cells can differentiate into ectodermal organs such as the brain, spinal cord, adrenal medulla, epidermis, hair / nails / dermal glands, sensory organs, peripheral nerves, or lens.

[0211] In some cases, the conversion of stem cells into tissue-specific progenitor cells is characterized by producing up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 5%, or fewer non-endodermal cells. In some cases, the conversion of stem cells into tissue-specific progenitor cells is characterized by producing up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 5%, or fewer enterochromaffin cells. In some cases, modulation of genes such as SOX2, CDX2, LMX1a, and FEV is used to suppress non-endodermal and / or enterochromaffin cell fates.

[0212] 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 a cell.

[0213] In some embodiments, the engineered cells (e.g., engineered beta cells) of the present disclosure can be generated from isolated stem cells (e.g., isolated endodermal stem cells or iPSCs). The heterologous gene circuits and / or components thereof (e.g., gating units, gating portions, activating portions, etc.) disclosed herein can be introduced into any stage (or cell state) between (a) the isolated stem cell and (b) its differentiated beta cell state (e.g., a terminally differentiated beta cell state).

[0214] Genetically engineered cells (e.g., engineered beta cells of the present disclosure) can be used (e.g., administered) to treat a subject in need thereof. 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 any of the subject engineered cells (e.g., beta cells) 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 adoptive immunotherapy, etc.).

[0215] The engineered cells disclosed herein can be administered to a subject prior to, concurrently with, or after activation of the heterologous gene circuit in the engineered stem cells, e.g., the engineered cells can be activated after administration to the subject, e.g., by administering to the subject an activator of the heterologous gene circuit.

[0216] A subject may be treated (e.g., administered) with a population of engineered cells (e.g., engineered muscle cells) of the present disclosure for at least or up to about 1 dose, at least or up to about 2 doses, at least or up to about 3 doses, at least or up to about 4 doses, at least or up to about 5 doses, at least or up to about 6 doses, at least or up to about 7 doses, at least or up to about 8 doses, at least or up to about 9 doses, or at least or up to about 10 doses. Alternatively, or in addition, a subject may be treated (e.g., administered) with a population of engineered cells (e.g., engineered T cells) of the present disclosure for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, at least about 30 years, at least about 40 years, at least about 50 years, at least about 60 years, at least about 70 years, at least about 80 years, at least about 90 years, or at least about 100 years.

[0217] 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.

[0218] The target disease of interest may be a disease affecting the pancreas, including, but not limited to, pancreatitis, cholangitis, cholecystitis, diabetes, gallstones, and pancreatic cancer.

[0219] The target disease of interest can be a disease that affects insulin production, including, but not limited to, diabetes, hypertension, dyslipidemia, and cardiovascular disease.

[0220] The target disease of interest may be cancer or tumor. Non-limiting examples of cancer include acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, and acute promyelocytic leukemia. leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor tumor), basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinomacarcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, Brown's tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, cancer of unknown primary site, carcinosarcoma, Castleman's disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer cholangiocarcinoma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia leukemia), chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear-cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Dego's disease, dermatofibrosarcoma protuberans. protuberans), dermoid cysts (dermoid cysts) cyst, desmoplastic small round cell tumor, diffuse large B cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinomacarcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial uterine cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumors, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Fallopian tube cancer, Fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri cerebri), glomus tumor (Glomustumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma Melanoma, pancreatic islet cell carcinoma, pancreatic islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, lentigo maligna melanoma, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, maculoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone, malignant fibrous histiocytoma of bone Bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant Triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, spinal germ cell tumor, spinal tumor, medullary thyroidcancer, medulloblastoma, medulloepithelioma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, metastatic urothelial carcinoma, mixed mullerian tumor, monocytic leukemia, mouth cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic disease Disease, Myelodysplastic Syndromes, Myeloid Leukemia, Myeloid Sarcoma, Myeloproliferative Disorders, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Neoplasm, Neuroinoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular Melanoma, Non-Hodgkin's Lymphoma, Non-Melanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular Oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic Nerve Sheath Meningioma, Oral Cancer, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor Malignant Potential Tumor, Paget's disease of the breastbreast, Pancos tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, blood Plasma cell neoplasm, neuropulmonary blastoma, polyembryoma, 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, cancer of the respiratory tract related to the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary tumors neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, Signetling cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, soot wart, spinal cord tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor tumor), surface epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyteThe cancer may include, but is not limited to, cells of a cancer, including, but not limited to, leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, terminal lymphatic cancer, testicular cancer, thecoma, throat cancer, thymic cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional cell carcinoma, brachial cancer, urethral cancer, genitourinary neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the target cancer cells represent a subpopulation within a cancer cell population, such as cancer stem cells. In some embodiments, the cancer is a cancer of the hematopoietic lineage, such as lymphoma. The antigen can be a tumor-associated antigen.

[0221] Non-limiting examples of target tissues can include cells, such as beta cells, obtainable from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Examples of samples from a subject from which cells can 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, fingernail, plasma, nasal swab or nasopharyngeal washing, spinal fluid, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, umbilical cord blood, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and / or other excretions or bodily tissues.

[0222] The present disclosure also provides a composition comprising the engineered genetic circuit disclosed herein. The composition may further comprise an actuator of a heterologous genetic circuit. The present disclosure also provides a kit comprising the composition. The kit may further comprise an activator of the heterologous genetic circuit. The activator may be in the same composition as the engineered cells. Alternatively, or in addition, the activator may be in a separate composition different from the engineered cells.

[0223] In some cases, the engineered progenitor cells disclosed herein may exhibit (i) equivalent or enhanced regenerative capacity, (ii) equivalent or enhanced in vitro expression, (iii) equivalent or enhanced gene editing capabilities, (iv) equivalent or enhanced immunotolerance, (v) equivalent or shorter manufacturing timelines, (vi) equivalent or reduced growth factor or culture requirements, and / or (vii) equivalent or enhanced safety compared to control progenitor cells.

[0224] Control progenitor cells can be generated by any method, including proliferation of progenitor cells isolated from tissue (e.g., pancreatic lineage cells), induced iPSC differentiation (e.g., using exogenous growth factors), and / or transgenic iPSC differentiation (e.g., viral transduction of heterologous genes).

[0225] In some cases, the tissue-specific progenitor cells can be stored in a container (e.g., a sterile vial). In some cases, the tissue-specific progenitor cells are stored at a temperature of up to about 10°C, up to about 5°C, up to about 4°C, up to about 0°C, up to about -5°C, up to about -10°C, up to about -20°C, up to about -30°C, up to about -40°C, up to about -50°C, up to about -60°C, up to about -70°C, up to about -80°C, up to about -90°C, up to about -100°C, up to about -110°C, up to about -120°C, up to about -130°C, up to about -140°C, up to about -150°C, up to about -160°C, up to about -170°C, up to about -180°C, up to about -190°C, up to about -0°C, or lower.

[0226] Pharmaceutical Composition In some cases, the method disclosed herein includes administering at least one tissue-specific progenitor cell to a subject in need thereof. The subject may be an animal. The subject may be a mammal (e.g., a primate, a horse, a cat, a dog, a cow, a pig, a sheep, a goat, a mouse, a rabbit, a rat, a guinea pig). The subject may be a human subject.

[0227] The pharmaceutical compositions of the present disclosure can be a combination of any of the pharmaceutical compounds described herein with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. The pharmaceutical composition can be administered in a therapeutically effective amount as a pharmaceutical composition by a variety of forms and routes, including, for example, intravenous, subcutaneous, intramuscular, inhalation, oral, parenteral, ocular, otic, subcutaneous, transdermal, intranasal, intravitreal, intratracheal, intrapulmonary, transmucosal, vaginal, and topical administration.

[0228] The formulation can vary depending on the route of administration selected. Pharmaceutical compositions containing the compounds described herein can be prepared, for example, by mixing, dissolving, emulsifying, encapsulating, entrapping, or compressing processes. [Example]

[0229] Example 1: Differentiation of pancreatic lineage cells

[0230] Tissue-specific cells (eg, pancreatic lineage cells) can be generated from less-differentiated cells (eg, stem cells such as iPSCs) by the systems and methods of the present disclosure.

[0231] Generation of pancreatic lineage cells.

[0232] In this example, endodermal stem cells were transformed into pancreatic lineage cells using a heterologous gene circuit. Differentiation of stem cells (e.g., endodermal stem cells) into pancreatic lineage cells can be a complex process that requires turning on multiple endogenous genes at different times and turning off multiple endogenous genes at different times. See Figure 2 for an example of different endogenous genes induced for expression at different stages of stem cell differentiation into pancreatic lineage cells and then beta cells. Thus, one or more heterologous gene circuits disclosed herein can be utilized to automatically facilitate such cascade regulation of different endogenous gene expression. In some cases, each heterologous gene circuit can be configured to regulate the expression levels of multiple genes at multiple different times upon a single activation of such a heterologous gene circuit.

[0233] Endodermal stem cells were transiently transfected with plasmid DNA encoding one of the heterologous gene circuits described in Figure 3, targeting a combination of TBXT, FOXA2, SOX17, PDX1, GATA4, p53 shRNA, HNF6, PTF1A, NGN3, NEUROG3, NKX6.1, SOX9, NKX2.2, MAFA, ONECUT1, NEUROD3, and ISL1. All targeted genes were activated. Flow cytometry was used to analyze CD45- / EpCam+ cells (endodermal progenitor marker), ECAD / CD142 double-positive cells (pancreatic progenitor marker), and CD49a-positive cells (immature beta cell marker), each of which indicated the formation of pancreatic lineage cells (Figure 6). Low-performing HCGs, such as Cell Algorithms 1, 2, 3, 4, 5, 6, 7, 13, 14, 23, and 24, were all found to share the same genes in step 1:TBXT. High-performing HCGs, such as Cell Algorithms 9 and 16, were found to produce cultures with islet-like morphology in 8 days (Figure 7). High-performing HCGs, such as Cell Algorithm 11, were also found to convert at least 15% of cells to insulin-positive cells in 8 days (Figures 8A-8B).

[0234] B. Characterization of pancreatic lineage cells generated by heterologous gene circuits.

[0235] Transient plasmid delivery of heterologous gene circuits resulted in the appearance of pancreatic lineage cell markers after 4 days. Analysis revealed that some of the heterologous gene circuits, such as heterologous gene circuits 9, 11, and 16 (i.e., cell algorithms 9, 11, and 16) provided in Figure 3, resulted in at least approximately 15% conversion of endoderm stem cells to pancreatic lineage cells in 4 days. This conversion occurred more rapidly than the conversion of pancreatic lineage cells by direct differentiation (Figure 9).

[0236] Figures 4A-4D and 5 show volcano plots illustrating the results of cells grown under different conditions. The plots in Figures 4A-4D and 5 demonstrate that various heterologous gene circuits (e.g., Cell Algorithm 15, Cell Algorithm 16, Cell Algorithm 18, etc.) can generate pancreatic lineage cells.

[0237] Example 2: Beta cell function assay

[0238] In this anticipated example, iPSCs and endodermal stem cells are contacted with the high-performance HGCs of Example 1 and grown in culture until β cells are formed. A control β cell population is collected and purified from a mouse sample. β cells from both the HGC population and the control population are tested for insulin production.

[0239] Example 3: Transplantation and engraftment of pancreatic lineage cells

[0240] Tissue-specific cells (e.g., pancreatic lineage cells) prepared by the systems and methods of the present disclosure can be administered (e.g., injected into pancreatic tissue) to a subject in need thereof to treat a pancreas-related disorder (e.g., diabetes) in the subject.

[0241] Generation of pancreatic lineage cells.

[0242] Stem cells (e.g., endodermal stem cells) can be transduced or transfected (e.g., transiently transfected) with one or more heterologous genes (e.g., plasmid DNA) encoding at least one heterologous gene circuit, such as one of each heterologous gene circuit, as shown in Figure 3, according to the methods described in Example 1 to generate pancreatic lineage cells.

[0243] B. In vivo administration of pancreatic lineage cells.

[0244] Upon generation of pancreatic lineage cells using the disclosed systems and methods, the pancreatic lineage cells can be purified. The cells can be concentrated, resuspended in a buffer (e.g., PBS), and then administered to mice by direct injection into the pancreas or other site of interest. After 8-10 weeks, the mice can be sacrificed and the tissue surrounding the injection area can be sectioned. Sections can be immunostained for islet cells or insulin production to confirm engraftment of the ex vivo generated pancreatic lineage cells.

[0245] Further protocols for pancreatic lineage cell transplantation are provided herein. Upon generation as described herein, pancreatic lineage cells can be suspended in cell culture medium. These cells can be transplanted into target sites in the pancreas with or without further expansion. For expansion, pancreatic lineage cells can be plated as sparse cultures (e.g., 1,000–2,000 cells / well in 24-well plates) in tissue culture wells containing hydrogel (flat or patterned) or thin gel-coated plastic (flat or patterned) and cultured with medium changes every three days. On the day of transplantation, NOD / SCID mice can be anesthetized by intraperitoneal injection of ketamine (2.4 mg / mouse) and xylazine (240 g / mouse) and irradiated in the hind limbs as previously described (A. Sacco et al. (2008) Nature 456, 502). The generated pancreatic lineage cells can be counted, resuspended, and subsequently injected into recipient mice.

[0246] The engraftment of transplanted pancreatic lineage cells (e.g., differentiation and integration into local pancreatic tissue) can be visualized by various methods. For example, pancreatic lineage cells can be engineered to express a xenogeneic marker (e.g., a fluorescent protein such as green fluorescent protein) that is not present in the transplanted animal. Alternatively, or in addition, pancreatic lineage cells can be allogeneic to the animal so that any insulin produced by the pancreatic lineage cells upon transplantation can be identified (e.g., immunostained) by an antigen not found in the transplanted animal.

[0247] Example 4: Generation of β-cells in vitro

[0248] For improved cell production processes, cells of interest can be engineered to exhibit increased expression of genes capable of shifting their phenotype toward the pancreatic beta cell lineage. In some embodiments, improved cell production can be evidenced by shorter times for differentiating stem cells (e.g., pluripotent stem cells) into target cells (e.g., pancreatic progenitor cells or beta cells), increased numbers of target cells compared to other protocols, enhanced levels of insulin and / or c-peptide produced and / or secreted, lower costs for producing the same number of target cells, and higher costs. Figure 9 illustrates some advantages of the systems and methods disclosed herein compared to induced differentiation. Upon nucleofection of cell alginates into cells of interest, these genes can be sequentially expressed in the cells of interest. The cells of interest can be induced pluripotent stem cells. The engineered cells may exhibit increased presence of markers of pancreatic beta cell lineage by measuring RNA transcription, may exhibit DNA accessibility indicative of cells of the pancreatic beta lineage, and / or may exhibit increased function in glucose-responsiveness assays measuring 1) insulin production, 2) insulin release, 3) c-peptide production, and / or 4) c-peptide release.

[0249] Example 5: Generation of cells in the pancreatic β-cell lineage

[0250] For example, a library of cell algorithms, including but not limited to those labeled in Table 1, constructed from sequences composed of core functional units set forth in SEQ ID NOS: 1-2024, can be introduced into cells of interest. Table 4 (SEQ ID NOS: 1-2024) shows 2024 unique β proGuide constructs (e.g., there are 23 unique stem combinations). There are 22 unique "4-pools" of β spacers. For each gene, there are four pools because all four constructs targeting that gene are used together. This means that there are 22 * ​​23 = 506 different combinations. To count each individual construct, multiply it by 4. Cells can be cultured on Geltrex using M3 (alk alt) medium (Table 2) or Activin A medium (Table 3). Cells can be measured on days 1-14 using live cell imaging, phenotyping assays for insulin production and release, and c-peptide production and release.Measures of pancreatic cell identity include the genes: CHGA, INS, GCG, PDX1, NKX6.1, SST, HHEX, ISL1, ARX, IRX2, TPH1, DDC, SLC18A1, LMX1A, ADRA2A, FEV, TAC1, NEUROG3, FOXJ1, TOP2A, DISP2, TUBA1B, MYL7, AFP, TTR, LGALS3, HSPA1A, HSPA6, HSPA1B, FN1, COL5A2, COL21A1, SOX9, SOX17, A SCL1, HOXB2, PYY, DNAJB1, ALDH1A1, MAFA, MAFB, NEUROD1, DLK1, LDHB, ITGB1, SUSD2, F3, GP2, CLU, NTS, NKX2.2, ACSL1, PPY, POU 5F1, NANOG, OTX2, SOX2, CDX2, TBXT, GATA4, GATA6, EOMES, CER1, GSC, HLXB9, MNX1, HNF1B, HNF4A, HNF6, ONECUT1, TBX3, PTF1A, N R5A2, PROX1, HES1, NGN3, PAX6, SIX2, RFX6, GLIS3, BRN4, LRX2, PAX4, CDH1, EPCAM, ITGA1, CFTR, KRT19, SPP1, NKX6-1, NKX2-2, T FAP2A, TFAP2B, NES, VIM, ACVR1C, DHRS2, HOPX, NKX2.1, G6PC2, NPTX2, CXCR4, FOXA2, FOXA1, RNA28SN4, RNA28SN2, RNA28SN1, NE RNA seq markers include UROG1, HNF1A, INSM1, RFX3, ATF3, ATF4, ATF5, BMAL1, CREB, CRTC2, EGR1, ESRRG, FOS, FOXO1, FOXO3, FOXO4, MYC, P53, NFATC1, NFATC2, NR4A1, PPARA, PPARG, PPARD, NR1D1, SIX3, SMAD2, SMAD3, SREBP1, SRF, THRA, THRB, BEX1, CD82, and ID3.

[0251] [Table 1-1]

[0252] [Table 1-2]

[0253] [Table 2]

[0254] [Table 3]

[0255] Example 6: In vivo validation of cells derived from the Cell Algorithm

[0256] background

[0257] Pancreatic beta cells and other cells of pancreatic islets generated using cell algorithm technology can be used as a regenerative therapy, particularly for individuals with pancreatic insufficiency related to insulin production. Delivery of these cells to such individuals can provide a reduction in the need for exogenous insulin. Once produced by cell algorithm-driven instructions, pancreatic islets can be used in the same manner as islets produced by other methods, such as isolation from cadaveric donors or differentiation of pluripotent stem cells (e.g., induced differentiation) using protocols for varying growth factors and other small molecules over a period of time. Regardless of their origin, pancreatic islets can be evaluated and used in vivo by injecting the islets into an encapsulated device that resides under the kidney capsule, in the portal vein, intramuscularly, or subcutaneously. Pancreatic islets can be characterized phenotypically based on gene expression and cell surface marker properties, and functionally for their ability to regulate blood glucose and appetite. In vitro and in vivo characterization of glucose responsiveness and subsequent insulin secretion can provide significant insight into islet quality.

[0258] Induced diabetes animal models are used to assess the function of the produced islets. The pancreases of these mice are injected with streptozotocin (STZ) to induce destruction of the organ and make the mice diabetic. After this, the islets are injected into the tissue of interest and fed to the mice. Blood / serum glucose and insulin levels are measured to characterize islet performance.

[0259] method

[0260] Eight- to ten-week-old immunodeficient mice (e.g., SCID-Beige) can be obtained from Taconic or The Jackson Laboratory, and eight- to twelve-week-old NOD.Cg-Rag1tm1Mom IL2rgtm1Wjl Ins2Akita (NRG-Akita) can also be obtained for validation studies. To deliver cells under the renal capsule of mice, the catheter can be loaded with human pancreatic islets (e.g., 500–1,000 IEQ per mouse for SCID-Beige mice, or 4,000 IEQ per mouse for NRG-Akita mice) or iPS-derived cell clusters (e.g., 5e6 cells per mouse). Mice can then be analyzed at selected times by administering a glucose challenge, measuring blood glucose, and collecting serum to measure human insulin. After fasting the mice overnight (eg, 16 hours), they can be injected (eg, intraperitoneally (IP)) with D-(+)-glucose (eg, 2 g glucose / kg body weight). Blood glucose can be measured and serum collected at specific time points (eg, 0 and 30 minutes) after glucose injection. The observed blood glucose levels should be significantly reduced in mice containing transplanted islets. As a readout of transplanted islet cell function, serum human insulin levels can be quantified (e.g., using a human ultrasensitive insulin ELISA). Human insulin levels in mouse serum should rise significantly within a short period (e.g., 30 to 60 minutes) after feeding. The kidney containing the graft can then be dissected from the mouse, fixed overnight (e.g., in 4% PFA), embedded in paraffin, and sectioned for histological analysis to confirm the composition of the transplanted islets (e.g., ratio of alpha cells to beta cells to gamma cells, etc.). The presence of beta cells should be observed and identified (eg, by insulin protein expression after immunofluorescence staining).

[0261] [Table 4-1]

[0262] [Table 4-2]

[0263] [Table 4-3]

[0264] [Table 4-4]

[0265] [Table 4-5]

[0266] [Table 4-6]

[0267] [Table 4-7]

[0268] Table 4-8

[0269] Table 4-9

[0270] Table 4-10

[0271] Table 4-11

[0272] Table 4-12

[0273] Table 4-13

[0274] Table 4-14

[0275] Table 4-15

[0276] Table 4-16

[0277] Table 4-17

[0278] Table 4-18

[0279] Table 4-19

[0280] Table 4-20

[0281] Table 4-21

[0282] Table 4-22

[0283] Table 4-23

[0284] Table 4-24

[0285] Table 4-25

[0286] Table 4-26

[0287] Table 4-27

[0288] Table 4-28

[0289] Table 4-29

[0290] Table 4-30

[0291] Table 4-31

[0292] Table 4-32

[0293] Table 4-33

[0294] Table 4-34

[0295] Table 4-35

[0296] Table 4-36

[0297] Table 4-37

[0298] Table 4-38

[0299] Table 4-39

[0300] Table 4-40

[0301] Table 4-41

[0302] Table 4-42

[0303] Table 4-43

[0304] Table 4-44

[0305] Table 4-45

[0306] Table 4-46

[0307] Table 4-47

[0308] Table 4-48

[0309] Table 4-49

[0310] Table 4-50

[0311] Table 4-51

[0312] Table 4-52

[0313] Table 4-53

[0314] Table 4-54

[0315] Table 4-55

[0316] Table 4-56

[0317] Table 4-57

[0318] Table 4-58

[0319] Table 4-59

[0320] Table 4-60

[0321] Table 4-61

[0322] Table 4-62

[0323] Table 4-63

[0324] Table 4-64

[0325] Table 4-65

[0326] Table 4-66

[0327] Table 4-67

[0328] Table 4-68

[0329] Table 4-69

[0330] Table 4-70

[0331] Table 4-71

[0332] Table 4-72

[0333] Table 4-73

[0334] Table 4-74

[0335] Table 4-75

[0336] Table 4-76

[0337] Table 4-77

[0338] Table 4-78

[0339] Table 4-79

[0340] Table 4-80

[0341] Table 4-81

[0342] Table 4-82

[0343] Table 4-83

[0344] Table 4-84

[0345] Table 4-85

[0346] Table 4-86

[0347] Table 4-87

[0348] Table 4-88

[0349] Table 4-89

[0350] Table 4-90

[0351] Table 4-91

[0352] Table 4-92

[0353] Table 4-93

[0354] Table 4-94

[0355] Table 4-95

[0356] Table 4-96

[0357] Table 4-97

[0358] Table 4-98

[0359] Table 4-99

[0360] Table 4-100

[0361] Table 4-101

[0362] Table 4-102

[0363] Table 4-103

[0364] Table 4-104

[0365] Table 4-105

[0366] Table 4-106

[0367] Table 4-107

[0368] Table 4-108

[0369] Table 4-109

[0370] Table 4-110

[0371] Table 4-111

[0372] Table 4-112

[0373] Table 4-113

[0374] Table 4-114

[0375] Table 4-115

[0376] Table 4-116

[0377] Table 4-117

[0378] Table 4-118

[0379] Table 4-119

[0380] Table 4-120

[0381] Table 4-121

[0382] Table 4-122

[0383] Table 4-123

[0384] Table 4-124

[0385] Table 4-125

[0386] Table 4-126

[0387] Table 4-127

[0388] Table 4-128

[0389] Table 4-129

[0390] Table 4-130

[0391] Table 4-131

[0392] Table 4-132

[0393] Table 4-133

[0394] Table 4-134

[0395] Table 4-135

[0396] Table 4-136

[0397] Table 4-137

[0398] Table 4-138

[0399] Table 4-139

[0400] Table 4-140

[0401] Table 4-141

[0402] Table 4-142

[0403] Table 4-143

[0404] Table 4-144

[0405] Table 4-145

[0406] Table 4-146

[0407] Table 4-147

[0408] Table 4-148

[0409] Table 4-149

[0410] Table 4-150

[0411] Table 4-151

[0412] Table 4-152

[0413] Table 4-153

[0414] Table 4-154

[0415] Table 4-155

[0416] Table 4-156

[0417] Table 4-157

[0418] Table 4-158

[0419] Table 4-159

[0420] Table 4-160

[0421] Table 4-161

[0422] Table 4-162

[0423] Table 4-163

[0424] Table 4-164

[0425] Table 4-165

[0426] Table 4-166

[0427] Table 4-167

[0428] Table 4-168

[0429] Table 4-169

[0430] [Table 5-1]

[0431] [Table 5-2]

[0432] [Table 5-3]

[0433] [Table 6-1]

[0434] [Table 6-2]

[0435] Embodiment The following non-limiting embodiments provide illustrative examples of the present invention, but do not limit the scope of the invention. Embodiment 1. A method for converting a plurality of stem cells into a plurality of pancreatic lineage cells, the method comprising: contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce a plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to achieve conversion, the plurality of gating units comprising: a) a first gate unit pre-configured to regulate the expression level of a first target gene among a plurality of distinct target genes, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), and GATA; b) a second gating unit pre-configured to regulate the expression level of a second target gene among the plurality of distinct target genes, such that the expression levels of the first target gene and the second target gene are sequentially regulated; Upon activation of the heterologous gene circuit, the plurality of gating units operate to effect the transformation. Embodiment 2. The method of embodiment 1, wherein the first target gene comprises two or more members selected from the group consisting of FOX, SOX, and GATA. Embodiment 3. The method of embodiment 1, wherein the first target genes include FOX, SOX, and GATA. Embodiment 4. The method of embodiment 1, wherein the first target gene further comprises a T-box transcription factor (TBX). Embodiment 5. The method of embodiment 1, wherein the second target gene comprises one or more members selected from the group consisting of basic helix-loop-helix transcription factors (bHLH), homeobox, and Maf transcription factors. Embodiment 6. The method of embodiment 1, wherein (i) the expression level of the first target gene is enhanced by the first gate unit, or (ii) the expression level of the second target gene is enhanced by the second gate unit. Embodiment 7. A method for converting a plurality of stem cells into a plurality of pancreatic lineage cells through modulation of the expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the method comprising: a) contacting a first polynucleotide sequence in a plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of a first target gene operably linked to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), and GATA; b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory portion to regulate the expression level of a second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of basic helix-loop-helix transcription factors (bHLH), homeobox, and Maf transcription factors. Embodiment 8. The method of embodiment 7, wherein (b) is performed subsequent to (a) to sequentially regulate the first target gene and the second target gene, respectively. Embodiment 9. The method of embodiment 7, wherein the first target gene comprises two or more members selected from the group consisting of FOX, SOX, and GATA. Embodiment 10. The method of embodiment 7, wherein the first target genes include FOX, SOX, and GATA. Embodiment 11. The method of embodiment 7, wherein said second target gene comprises two or more members selected from the group consisting of bHLH, homeobox, and Maf transcription factors. Embodiment 12 The method of embodiment 7, wherein the second target genes include bHLH, homeobox, and Maf transcription factors. Embodiment 13. The method of embodiment 7, wherein (i) the first polynucleotide sequence is upstream of or encodes the first target gene, or (ii) the second polynucleotide sequence is upstream of or encodes the second target gene. Embodiment 14. The method of embodiment 7, wherein (i) the expression level of the first target gene is enhanced upon contact with the first heterologous gene regulatory portion, or (ii) the expression level of the second target gene is enhanced upon contact with the second heterologous gene regulatory portion. Embodiment 15. The method comprises contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce a plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to achieve transformation, the plurality of gating units comprising: (i) a first gating unit preconfigured to act on a first heterologous gene regulatory portion to regulate the expression level of a first target gene; (ii) a second gating unit preconfigured to act on a second heterologous gene regulatory portion to regulate the expression level of a second target gene; 8. The method of embodiment 7, wherein upon activation of the heterologous gene circuit, the plurality of gating units operates to effect the conversion. Embodiment 16. The method of embodiment 15, wherein (i) the first gate unit is activatable to express the first heterologous gene regulatory portion, or (ii) the second gate unit is activatable to express the second heterologous gene regulatory portion. Embodiment 17. A method for converting a plurality of stem cells into a plurality of pancreatic lineage cells through modulation of the expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the method comprising: a) contacting a first polynucleotide sequence in a plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of a first target gene operably linked to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor; b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to reduce the expression level of a second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of additional SOX, additional homeobox, and ETS transcription factors. Embodiment 18. The method of embodiment 17, wherein (b) is performed before (a) to sequentially regulate the first target gene and reduce the expression level of the second target gene, respectively. Embodiment 19. The method of embodiment 17, wherein (b) is performed subsequent to (a) to sequentially reduce the expression of the second target gene and regulate the first target gene, respectively. Embodiment 20. The method of embodiment 17, wherein (a) and (b) are performed simultaneously. Embodiment 21. The method of embodiment 17, wherein the additional SOX is not a member of SOX9 and SOX17. Embodiment 22. The method of embodiment 17, wherein the additional SOX comprises SOX2. Embodiment 23 The method of embodiment 17, wherein the additional homeobox is not a member from PDX1, NKX6-1, NKX2-2, PAX6, ONECUT1, and ISL1. Embodiment 24 The method of embodiment 17, wherein the additional homeobox comprises CDX1, CDX2, or CDX4. Embodiment 25 The method of embodiment 24, wherein the additional homeobox comprises CDX2. Embodiment 26 The method of embodiment 17, wherein the additional homeobox comprises LMX1A or LMX1B. Embodiment 27 The method of embodiment 26, wherein the additional homeobox comprises LMX1A. Embodiment 28 The method of embodiment 17, wherein the ETS transcription factor comprises ERG, FLI1, or FEV. Embodiment 29 The method of embodiment 28, wherein the ETS transcription factor comprises FEV. Embodiment 30. The method of embodiment 17, wherein the first target gene comprises two or more members selected from the group consisting of FOX, SOX, GATA, bHLH, homeobox, and Maf transcription factors. Embodiment 31 The method of embodiment 17, wherein the first target gene comprises one or more members selected from the group consisting of FOX, SOX, and GATA. Embodiment 32 The method of embodiment 17, wherein the first target gene comprises one or more members selected from the group consisting of bHLH, homeobox, and Maf transcription factors. Embodiment 33. The method of embodiment 17, wherein (i) the first polynucleotide sequence is upstream of or encodes the first target gene, or (ii) the second polynucleotide sequence is upstream of or encodes the second target gene. Embodiment 34 The method of embodiment 17, wherein the expression level of said first target gene is enhanced upon contact by said first heterologous gene regulatory moiety. Embodiment 35. The method comprises contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce a plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to achieve transformation, the plurality of gating units comprising: (i) a first gating unit preconfigured to act on a first heterologous gene regulatory portion to regulate the expression level of a first target gene; (ii) a second gating unit preconfigured to act on the second heterologous gene regulatory portion to reduce the expression level of the second target gene; 18. The method of embodiment 17, wherein upon activation of the heterologous gene circuit, the plurality of gating units operates to effect the conversion. Embodiment 36. The method of embodiment 35, wherein (i) the first gate unit is activatable to express the first heterologous gene regulatory portion, or (ii) the second gate unit is activatable to express the second heterologous gene regulatory portion. Embodiment 37. A method for converting a plurality of stem cells into pancreatic lineage cells, the method comprising: A method comprising contacting a polynucleotide sequence in a plurality of stem cells with a heterologous gene regulatory moiety to regulate the expression level of a target gene operably linked to the polynucleotide sequence, wherein within about two weeks after contacting, the conversion rate of the plurality of stem cells to a plurality of pancreatic lineage cells is characterized as being at least about 5%. Embodiment 38 The method of embodiment 37, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells. Embodiment 39. The method of embodiment 38, wherein the conversion rate of the plurality of stem cells to pancreatic progenitor cells is at least about 10%, at least about 15%, at least about 20%, or at least about 25%. Embodiment 40 The method of embodiment 37, wherein the plurality of pancreatic lineage cells comprises beta cells. Embodiment 41 The method of embodiment 40, wherein the conversion rate of the plurality of stem cells to beta cells is at least about 10%. Embodiment 42 The method of embodiment 37, wherein the conversion rate is observed within about 12 days, within about 10 days, or within about 8 days. Embodiment 43 The method of embodiment 37, wherein the expression level of said target gene is enhanced upon contact with said heterologous gene regulatory moiety. Embodiment 44. The method of embodiment 37, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor. Embodiment 45. The method of embodiment 44, wherein said target genes comprise two or more members selected from the group consisting of FOX, SOX, GATA, bHLH, homeobox, and Maf transcription factors. Embodiment 46. The target gene comprises a plurality of distinct target genes, including a first distinct target and a second distinct target gene, and the contacting step comprises: (a) contacting a first polynucleotide sequence in a plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene operably linked to the first polynucleotide sequence; (b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to regulate the expression level of a second distinct target gene operably linked to the second polynucleotide sequence. Embodiment 47. The method of embodiment 46, wherein steps (a) and (b) sequentially regulate the first distinct target gene and the second distinct target gene. Embodiment 48. The contacting step comprises contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, wherein the heterologous genetic circuit is activatable to induce a plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to achieve transformation, wherein the plurality of gating units: (i) a first gating unit preconfigured to act on a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene; (ii) a second gating unit preconfigured to act on a second heterologous gene regulatory portion to regulate the expression level of a second distinct target gene; 47. The method of embodiment 46, wherein, upon activation of the heterologous gene circuit, the plurality of gating units operates to effect the conversion. Embodiment 49. The method of embodiment 46, wherein the plurality of distinct target genes comprises two or more members selected from the group consisting of FOX, SOX, GATA, bHLH, homeobox, and Maf transcription factors. Embodiment 50. The method of embodiment 37, wherein the target gene is an endogenous gene. Embodiment 51. A method of treating a subject in need thereof, the method comprising: administering to a subject a plurality of pancreatic lineage cells, wherein the plurality of pancreatic lineage cells are prepared by subjecting a plurality of stem cells to ex vivo differentiation; The method, wherein the conversion rate of said plurality of stem cells to said plurality of pancreatic lineage cells within about two weeks of ex vivo differentiation is characterized as being at least about 5%. Embodiment 52 The method of embodiment 51, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells. Embodiment 53 The method of embodiment 51, wherein the conversion rate of the plurality of stem cells to the pancreatic progenitor cells is at least about 10%, at least about 15%, at least about 20%, or at least about 25%. Embodiment 54 The method of embodiment 51, wherein the plurality of pancreatic lineage cells comprises beta cells. Embodiment 55 The method of embodiment 53, wherein the conversion rate of the plurality of stem cells to beta cells is at least about 10%. Embodiment 56 The method of embodiment 51, wherein the conversion rate is observed within about 12 days, within about 10 days, or within about 8 days. Embodiment 57. The method of embodiment 51, wherein the plurality of pancreatic lineage cells are subjected to ex vivo culture for about 4 weeks or less, about 3 weeks or less, about 2 weeks or less, about 12 days or less, about 10 days or less, or about 8 days or less. Embodiment 58. The method of embodiment 51, wherein the ex vivo differentiation comprises modulating the expression levels of target genes comprising one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor. Embodiment 59. The method of embodiment 58, wherein said target genes comprise two or more members selected from the group consisting of FOX, SOX, GATA, bHLH, homeobox and Maf transcription factors. Embodiment 60 The method of embodiment 58, wherein the target gene is an endogenous target gene. Embodiment 61. The method of any one of embodiments 1 to 60, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells or pancreatic beta cells. Embodiment 62. The method of any one of embodiments 1 to 61, wherein the plurality of pancreatic lineage cells are characterized as ECAD+ / CD142+ or CD49a+. Embodiment 63 The method of any one of embodiments 1 to 62, wherein the plurality of pancreatic lineage cells is characterized as producing insulin. Embodiment 64. The method of any one of embodiments 1 to 63, wherein the plurality of stem cells comprises pluripotent stem cells (PSCs) or endodermal cells. Embodiment 65. The method of any one of embodiments 1 to 64, wherein the plurality of distinct target genes is a plurality of endogenous genes of stem cells. Embodiment 66. The method of any one of embodiments 1 to 65, wherein the first heterologous gene regulatory portion or the second heterologous gene regulatory portion comprises (i) an endonuclease or (ii) a guide nucleic acid (gNA) molecule. Embodiment 67. The method of embodiment 66, wherein the endonuclease and gNA form a complex capable of binding to their respective target polynucleotide sequences. Embodiment 68 The method of embodiment 66, wherein the endonuclease is a Cas protein. Embodiment 69. The method of any one of embodiments 1 to 68, wherein the conversion occurs under conditions that are substantially free of (i) serum and / or (ii) exogenous cell differentiation regulators. Embodiment 70. The method of embodiment 69, wherein the exogenous cell differentiation regulatory factor comprises one or more members selected from the group consisting of insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH), and vascular endothelial growth factor (VEGF), transforming growth factor-β (TGFβ) superfamily, bone morphogenetic protein-2 (BMP2), and bone morphogenetic protein-7 (BMP7). Embodiment 71. The method of embodiment 69, wherein the exogenous cell differentiation regulator comprises one or more members selected from the group consisting of a GSK3β inhibitor, an ALK inhibitor, a BMP1 receptor inhibitor, and retinoic acid. Embodiment 72. The method of any one of embodiments 1 to 71, wherein FOX comprises one or more members selected from the group consisting of FOXA, FOXB, FOXC, FOXD, FOXE, FOXF, FOXG, FOXH, FOXI, FOXJ, FOXK, FOXL, FOXM, FOXN, FOXO, FOXP, FOXQ, FOXR, and FOXS. Embodiment 73 The method of embodiment 72, wherein FOX is FOXA1, FOXA2, or FOXA3. Embodiment 74 The method of embodiment 73, wherein FOX is FOXA2. Embodiment 75. The method of any one of embodiments 1 to 74, wherein SOX comprises one or more members selected from the group consisting of SOXA, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH. Embodiment 76 The method of embodiment 75, wherein SOX is SOXE. Embodiment 77. The method of embodiment 76, wherein SOXE is SOX9. Embodiment 78. The method of embodiment 75, wherein SOX is SOXF. Embodiment 79. The method of embodiment 78, wherein SOXF is SOX17. Embodiment 80. The method of any one of embodiments 1 to 79, wherein SOX is not SOX2. Embodiment 81. The method of any one of embodiments 1 to 80, wherein SOX is not SOXB1 or SOXB2. Embodiment 82. The method of any one of embodiments 1 to 81, wherein GATA comprises one or more members selected from the group consisting of GATA1, GATA2, GATA3, GATA4, GATA5, and GATA6. Embodiment 83 The method of embodiment 82, wherein the GATA is GATA4. Embodiment 84. The method of any one of embodiments 1 to 83, wherein the TBX comprises one or more members selected from the group consisting of TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, and TBX22. Embodiment 85. The method of embodiment 84, wherein TBX is TBXT. Embodiment 86 The method of any one of embodiments 1 to 85, wherein the plurality of distinct target genes does not include TBXT. Embodiment 87. The method of any one of embodiments 1 to 86, wherein the bHLH comprises one or more members selected from the group consisting of Group A bHLH, Group B bHLH, Group C bHLH, Group D bHLH, Group E bHLH, and Group F bHLH. Embodiment 88. The method of embodiment 87, wherein the bHLH is a Group A bHLH. Embodiment 89. The method of embodiment 87, wherein the bHLH is NEUROG3, NEUROD1, or PTF1a. Embodiment 90. The method of any one of embodiments 1 to 89, wherein the homeobox comprises a ParaHox gene comprising one or more members selected from the group consisting of CDX1, CDX2, CDX4, GSX1, GSX2, and PDX1. Embodiment 91 The method of embodiment 90, wherein the ParaHox gene is PDX1. Embodiment 92. The method of any one of embodiments 1 to 91, wherein the homeobox comprises a homeobox of the NKX class, including one or more members selected from the group consisting of 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. Embodiment 93. The method of embodiment 92, wherein the NKX class homeobox is NKX6-1 or NKX2-2. Embodiment 94. The homeobox is selected from the group consisting of ALX1, ALX3, ALX4, ARGFX, ARX, DMBX1, DPRX, DRGX, DUXA, DUXB, DUX(1, 2, 3, 4, 4c, 5), ESX1, GSC, GSC2, HESX1, HOPX, ISX, LEUTX, MIXL1, NOBOX, OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, P 94. The method of any one of embodiments 1-93, comprising a homeobox of the paired box (PRD) class comprising one or more members selected from the group consisting of HOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, RHOXF1, RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, and VSX2. Embodiment 95. The method of embodiment 94, wherein the PRD class is PAX6. Embodiment 96 The method of any one of embodiments 1 to 95, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is not a member of Pax3 and Pax7. Embodiment 97. The method of any one of embodiments 1 to 96, wherein the homeobox comprises a homeobox of the CUT class comprising one or more members selected from the group consisting of ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2, SATB1, and SATB2. Embodiment 98. The method of embodiment 97, wherein the homeobox of the CUT class is ONECUT1. Embodiment 99. The method of any one of embodiments 1 to 98, wherein the homeobox comprises a LIM class homeobox comprising one or more members selected from the group consisting of ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, and LMX1B. Embodiment 100. The method of embodiment 99, wherein the LIM class homeobox is ISL1. Embodiment 101. The method of any one of embodiments 1 to 100, wherein the conversion occurs in less than about 14 days or less than about 10 days. Embodiment 102 The method of any one of embodiments 1 to 101, further comprising storing the plurality of pancreatic lineage cells in a sterile vial. Embodiment 103. The method of any one of embodiments 1 to 102, wherein said conversion achieves the formation of pancreatic islets comprising a plurality of pancreatic lineage cells. Embodiment 104 The method of any one of embodiments 1 to 103, further comprising generating pancreatic islets comprising said plurality of pancreatic lineage cells. Embodiment 105. The method of embodiment 104, wherein the pancreatic islets are for administration to a subject. Embodiment 106 The method of any one of embodiments 1 to 105, further comprising loading a plurality of pancreatic lineage cells into or onto the scaffold. Embodiment 107. The method of embodiment 106, wherein the scaffold and the plurality of pancreatic lineage cells are combined and intended for transplantation into a subject. Embodiment 108 The method of any one of embodiments 1 to 107, further comprising administering the plurality of pancreatic lineage cells to a subject in need thereof. Embodiment 109. The method of any one of embodiments 1 to 108, wherein the administration comprises one or more hepatic portal vein injections. Embodiment 110. The method of any one of embodiments 1 to 109, wherein the administration comprises one or more subcutaneous implantations. Embodiment 111. The method of any one of embodiments 1 to 110, wherein the subject has or is suspected of having diabetes. Embodiment 112. The conversion is characterized by producing fewer non-endodermal cells or fewer enterochromaffin cells compared to control conversion of a plurality of stem cells in a medium containing (i) serum and / or (ii) an exogenous cell differentiation regulator. Embodiment 113. A system for converting a plurality of stem cells into a plurality of pancreatic lineage cells, the system comprising: A heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce a plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to achieve conversion, the plurality of gating units comprising: (i) a first gating unit pre-configured to regulate the expression level of a first target gene among a plurality of distinct target genes, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), and GATA; (ii) a second gating unit pre-configured to regulate the expression level of a second target gene among the plurality of distinct target genes, wherein the expression levels of the first target gene and the second target gene are sequentially regulated; Upon activation of a heterologous gene circuit, multiple gating units operate to achieve the transformation of the system. Embodiment 114. The system of embodiment 113, wherein the first target gene comprises two or more members selected from the group consisting of FOX, SOX, and GATA. Embodiment 115. The system of embodiment 113, wherein the first target genes include FOX, SOX, and GATA. Embodiment 116. The system of embodiment 113, wherein the first target gene further comprises a T-box transcription factor (TBX). Embodiment 117. The system of embodiment 113, wherein the second target gene comprises one or more members selected from the group consisting of basic helix-loop-helix transcription factors (bHLH), homeobox, and Maf transcription factors. Embodiment 118. The system described in embodiment 113, wherein (i) the expression level of the first target gene is enhanced by the first gate unit, or (ii) the expression level of the second target gene is enhanced by the second gate unit. Embodiment 119. A system for converting a plurality of stem cells into a plurality of pancreatic lineage cells through modulation of the expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the system comprising: (a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in a plurality of stem cells and regulate the expression level of a first target gene operably linked to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), and GATA; (b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and regulate the expression level of a second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of basic helix-loop-helix transcription factors (bHLH), homeobox, and Maf transcription factors. Embodiment 120. The system of embodiment 119, wherein the first heterologous gene regulatory portion and the second heterologous gene regulatory portion are configured to sequentially achieve regulation of the first target gene and the second target gene, respectively. Embodiment 121. The system of embodiment 119, wherein the first target gene comprises two or more members selected from the group consisting of FOX, SOX, and GATA. Embodiment 122. The system of embodiment 119, wherein the first target genes include FOX, SOX, and GATA. Embodiment 123. The system of embodiment 119, wherein the second target gene comprises two or more members selected from the group consisting of bHLH, homeobox, and Maf. Embodiment 124. The system of embodiment 119, wherein the second target genes include bHLH, homeobox, and Maf transcription factors. Embodiment 125. The system described in embodiment 119, wherein (i) the first polynucleotide sequence is upstream of or encodes the first target gene, or (ii) the second polynucleotide sequence is upstream of or encodes the second target gene. Embodiment 126. The system described in embodiment 119, wherein (i) the expression level of the first target gene is enhanced upon contact with the first heterologous gene regulatory portion, or (ii) the expression level of the second target gene is enhanced upon contact with the second heterologous gene regulatory portion. Embodiment 127. The system comprises a heterologous genetic circuit comprising a plurality of gating units, wherein the heterologous genetic circuit is activatable to induce a plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes, thereby achieving transformation, wherein the plurality of gating units: a first gating unit preconfigured to act on a first heterologous gene regulatory portion to regulate the expression level of a first target gene; a second gating unit preconfigured to act on the second heterologous gene regulatory portion to regulate the expression level of the second target gene; 120. The system of embodiment 119, wherein upon activation of the heterologous gene circuit, the plurality of gating units operate to effect the conversion. Embodiment 128. The method of embodiment 127, wherein (i) the first gate unit is activatable to express the first heterologous gene regulatory portion, or (ii) the second gate unit is activatable to express the second heterologous gene regulatory portion. Embodiment 129. A system for converting a plurality of stem cells into a plurality of pancreatic lineage cells through modulation of the expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the system comprising: a) a first heterologous gene regulatory portion configured to bind to a first polynucleotide sequence in a plurality of stem cells and regulate the expression level of a first target gene operably linked to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor; b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and reduce the expression level of a second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of an additional SOX, an additional homeobox, and an EST transcription factor. Embodiment 130. The system of embodiment 129, wherein the first heterologous gene regulatory portion and the second heterologous gene regulatory portion are configured to sequentially regulate the first target gene and reduce the expression level of the second target gene, respectively. Embodiment 131. The system of embodiment 129, wherein the first heterologous gene regulatory portion and the second heterologous gene regulatory portion are configured to sequentially achieve reduced expression of the second target gene and regulation of the first target gene, respectively. Embodiment 132 The system of embodiment 129, wherein the first heterologous gene regulatory portion and the second heterologous gene regulatory portion are configured to simultaneously regulate the first and second target genes. Embodiment 133. The system of embodiment 129, wherein the additional SOX is not a member from SOX9 and SOX17. Embodiment 134. The system described in embodiment 129, wherein the additional SOX includes SOX2. Embodiment 135. The system of embodiment 129, wherein the additional homeobox is not a member from PDX1, NKX6-1, NKX2-2, PAX6, ONECUT1, and ISL1. Embodiment 136. The system of embodiment 129, wherein the additional homeobox comprises CDX1, CDX2, or CDX4. Embodiment 137. The system of embodiment 136, wherein the additional homeobox comprises CDX2. Embodiment 138. The system described in embodiment 129, wherein the additional homeobox comprises LMX1A or LMX1B. Embodiment 139. The system of embodiment 138, wherein the additional homeobox includes LMX1A. Embodiment 140. The system of embodiment 129, wherein the ETS transcription factor comprises ERG, FLI1, or FEV. Embodiment 141. The system of embodiment 140, wherein the ETS transcription factor comprises FEV. Embodiment 142. The system of embodiment 129, wherein the first target gene comprises two or more members selected from the group consisting of FOX, SOX, GATA, bHLH, homeobox, and Maf transcription factors. Embodiment 143. The system of embodiment 129, wherein the first target gene comprises one or more members selected from the group consisting of FOX, SOX, and GATA. Embodiment 144. The system of embodiment 129, wherein the first target gene comprises one or more members selected from the group consisting of bHLH, homeobox, and Maf transcription factors. Embodiment 145. The system of embodiment 129, wherein (i) the first polynucleotide sequence is upstream of or encodes the first target gene, or (ii) the second polynucleotide sequence is upstream of or encodes the second target gene. Embodiment 146 The system of embodiment 129, wherein the expression level of the first target gene is enhanced upon contact by the first heterologous gene regulatory portion. Embodiment 147. The system comprises a heterologous genetic circuit comprising a plurality of gating units, wherein the heterologous genetic circuit is activatable to induce a plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes, thereby achieving transformation, wherein the plurality of gating units: (i) a first gating unit preconfigured to act on a first heterologous gene regulatory portion to regulate the expression level of a first target gene; (ii) a second gating unit preconfigured to act on the second heterologous gene regulatory portion to reduce the expression level of the second target gene; 130. The system of embodiment 129, wherein upon activation of the heterologous gene circuit, the plurality of gating units operate to effect the conversion. Embodiment 148. The system described in embodiment 147, wherein (i) the first gate unit is activatable to express the first heterologous gene regulatory portion, or (ii) the second gate unit is activatable to express the second heterologous gene regulatory portion. Embodiment 149. A system for converting a plurality of stem cells into pancreatic lineage cells, the system comprising: a heterologous gene regulatory moiety configured to bind to a polynucleotide sequence in the plurality of stem cells and regulate the expression level of a target gene operably linked to the polynucleotide sequence; The system is characterized in that the conversion rate of the plurality of stem cells to the plurality of pancreatic lineage cells is at least about 5% within about two weeks after contact. Embodiment 150. The system described in embodiment 149, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells. Embodiment 151. The system described in embodiment 150, wherein the conversion rate from the plurality of stem cells to the pancreatic progenitor cells is at least about 10%, at least about 15%, at least about 20%, or at least about 25%. Embodiment 152. The system described in embodiment 149, wherein the plurality of pancreatic lineage cells comprises beta cells. Embodiment 153. The system described in embodiment 152, wherein the conversion rate of the plurality of stem cells to beta cells is at least about 10%. Embodiment 154. The system of embodiment 149, wherein the conversion rate is observed within about 12 days, within about 10 days, or within about 8 days. Embodiment 155 The system of embodiment 149, wherein the expression level of the target gene is enhanced upon contact with the heterologous gene regulatory moiety. Embodiment 156. The system of embodiment 149, wherein the target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor. Embodiment 157. The system of embodiment 156, wherein the target genes comprise two or more members selected from the group consisting of FOX, SOX, GATA, bHLH, homeobox, and Maf transcription factors. Embodiment 158. The target gene comprises a plurality of distinct target genes, including a first distinct target and a second distinct target gene, and the system further comprises: (a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in a plurality of stem cells and regulate the expression level of a first distinct target gene operably linked to the first polynucleotide sequence; (b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and regulate the expression level of a second, distinct target gene operably linked to the second polynucleotide sequence. Embodiment 159. The system of embodiment 158, wherein the first and second heterologous gene regulatory portions are configured to sequentially achieve regulation of the first distinct target gene and the second distinct target gene. Embodiment 160. The system comprises a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce a plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes, thereby achieving transformation, the plurality of gating units comprising: (i) a first gating unit preconfigured to act on a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene; (ii) a second gating unit preconfigured to act on a second heterologous gene regulatory portion to regulate the expression level of a second distinct target gene; 159. The system of embodiment 158, wherein upon activation of the heterologous gene circuit, the plurality of gating units operates to effect the conversion. Embodiment 161. The system of embodiment 158, wherein the plurality of distinct target genes comprises two or more members selected from the group consisting of FOX, SOX, GATA, bHLH, homeobox and Maf transcription factors. Embodiment 162. The system described in embodiment 149, wherein the target gene is an endogenous gene. Embodiment 163. A composition comprising a system according to any one of embodiments 1 to 162. Embodiment 164. A composition for treating a subject in need thereof, comprising: a plurality of pancreatic lineage cells prepared by subjecting a plurality of stem cells to ex vivo differentiation; The composition is characterized by a conversion rate of at least about 5% of the plurality of stem cells to a plurality of pancreatic lineage cells within about two weeks of ex vivo differentiation. Embodiment 165. The composition of embodiment 164, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells. Embodiment 166. The composition of embodiment 165, wherein the conversion rate of the plurality of stem cells to the pancreatic progenitor cells is at least about 10%, at least about 15%, at least about 20%, or at least about 25%. Embodiment 167. The composition of embodiment 164, wherein the plurality of pancreatic lineage cells comprises beta cells. Embodiment 168. The composition of embodiment 167, wherein the conversion rate of the plurality of stem cells to beta cells is at least about 10%. Embodiment 169. The composition of embodiment 164, wherein the conversion rate is observed within about 12 days, within about 10 days, or within about 8 days. Embodiment 170. The composition of embodiment 164, wherein the plurality of pancreatic lineage cells are subjected to ex vivo culture for about 4 weeks or less, about 3 weeks or less, about 2 weeks or less, about 12 days or less, about 10 days or less, or about 8 days or less. Embodiment 171. The method of embodiment 164, wherein the ex vivo differentiation comprises modulating the expression levels of target genes comprising one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor. Embodiment 172. The composition of embodiment 171, wherein the target gene comprises two or more members selected from the group consisting of FOX, SOX, GATA, bHLH, homeobox, and Maf transcription factors. Embodiment 173. The composition of embodiment 171, wherein the target gene is an endogenous target gene. Embodiment 174. A system or composition described in any one of embodiments 1 to 173, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells or pancreatic beta cells. Embodiment 175. A system or composition described in any one of embodiments 1 to 174, wherein the plurality of pancreatic lineage cells are characterized as ECAD+ / CD142+ or CD49a+. Embodiment 176. A system or composition described in any one of embodiments 1 to 175, wherein the plurality of pancreatic lineage cells are characterized as producing insulin. Embodiment 177. A system or composition described in any one of embodiments 1 to 176, wherein the plurality of stem cells comprises pluripotent stem cells (PSCs) or mesodermal cells. Embodiment 178. A system or composition described in any one of embodiments 1 to 177, wherein the plurality of distinct target genes are endogenous genes of a plurality of stem cells. Embodiment 179. The system or composition of any one of embodiments 1 to 178, wherein the first heterologous gene regulatory portion or the second heterologous gene regulatory portion comprises (i) an endonuclease or (ii) a guide nucleic acid (gNA) molecule. Embodiment 180. A system or composition described in any one of embodiments 1 to 179, wherein the endonuclease and gNA form a complex capable of binding to their respective target polynucleotide sequences. Embodiment 181. A system or composition described in any one of embodiments 1 to 180, wherein the endonuclease is a Cas protein. Embodiment 182. A system or composition described in any one of embodiments 1 to 181, wherein the conversion occurs under conditions that are substantially free of (i) serum and / or (ii) exogenous cell differentiation regulators. Embodiment 183. The system or composition described in any one of embodiments 1 to 182, wherein the exogenous cell differentiation regulatory factor comprises one or more members selected from the group consisting of insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH), and vascular endothelial growth factor (VEGF), transforming growth factor-β (TGFβ) superfamily, bone morphogenetic protein-2 (BMP2), and bone morphogenetic protein-7 (BMP7). Embodiment 184. A system or composition described in any one of embodiments 1 to 183, wherein the exogenous cell differentiation regulatory factor comprises one or more members selected from the group consisting of a GSK3β inhibitor, an ALK inhibitor, a BMP1 receptor inhibitor, and retinoic acid. Embodiment 185. A system or composition described in any one of embodiments 1 to 184, wherein FOX comprises one or more members selected from the group consisting of FOXA, FOXB, FOXC, FOXD, FOXE, FOXF, FOXG, FOXH, FOXI, FOXJ, FOXK, FOXL, FOXM, FOXN, FOXO, FOXP, FOXQ, FOXR, and FOXS. Embodiment 186. A system or composition described in any one of embodiments 1 to 185, wherein FOX is FOXB1, FOXA2, or FOXA3. Embodiment 187. A system or composition described in any one of embodiments 1 to 186, wherein FOX is FOXA2. Embodiment 188. A system or composition described in any one of embodiments 1 to 187, wherein SOX comprises one or more members selected from the group consisting of SOXA, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH. Embodiment 189. A system or composition described in any one of embodiments 1 to 188, wherein SOX is SOXE. Embodiment 190. A system or composition described in any one of embodiments 1 to 189, wherein SOXE is SOX9. Embodiment 191. A system or composition described in any one of embodiments 1 to 190, wherein SOX is SOXF. Embodiment 192. A system or composition described in any one of embodiments 1 to 191, wherein SOXF is SOX17. Embodiment 193. A system or composition described in any one of embodiments 1 to 192, wherein SOX is not SOX2. Embodiment 194. A system or composition according to any one of embodiments 1 to 193, wherein SOX is not SOXB1 or SOXB2. Embodiment 195. A system or composition described in any one of embodiments 1 to 194, wherein GAGA comprises one or more members selected from the group consisting of GATB1, GATA2, GATA3, GATA4, GATA5, and GATA6. Embodiment 196. A system or composition described in any one of embodiments 1 to 195, wherein GATA is GATA4. Embodiment 197. A system or composition described in any one of embodiments 1 to 196, wherein TBX comprises one or more members selected from the group consisting of TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, and TBX22. Embodiment 198. A system or composition described in any one of embodiments 1 to 197, wherein TBX is TBXT. Embodiment 199. A system or composition described in any one of embodiments 1 to 198, wherein the plurality of distinct target genes does not include TBXT. Embodiment 200. The system or composition of any one of embodiments 1 to 199, wherein the bHLH comprises one or more members selected from the group consisting of Group A bHLH, Group B bHLH, Group C bHLH, Group D bHLH, Group E bHLH, and Group F bHLH. Embodiment 201. A system or composition according to any one of embodiments 1 to 200, wherein the bHLH is a Group A bHLH. Embodiment 202. A system or composition described in any one of embodiments 1 to 201, wherein the bHLH is NEUROG3, NEUROD1, or PTF1a. Embodiment 203. A system or composition described in any one of embodiments 1 to 202, wherein the homeobox comprises a ParaHox gene comprising one or more members selected from the group consisting of CDX1, CDX2, CDX4, GSX1, GSX2, and PDX1. Embodiment 204. A system or composition described in any one of embodiments 1 to 203, wherein the ParaHox gene is PDX1. Embodiment 205. A system or composition described in any one of embodiments 1 to 204, wherein the homeobox comprises a homeobox of the NKX class comprising one or more members selected from the group consisting of 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. Embodiment 206. A system or composition according to any one of embodiments 1 to 205, wherein the NKX class homeobox is NKX6-1 or NKX2-2. Embodiment 207. The homeobox is ALX1, ALX3, ALX4, ARGFX, ARX, DMBX1, DPRX, DRGX, DUXA, DUXB, DUX(1, 2, 3, 4, 4c, 5), ESX1, GSC, GSC2, HESX1, HOPX, ISX, LEUTX, MIXL1, NOBOX, OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHOX2 207. The system or composition of any one of embodiments 1-206, comprising a homeobox of the paired box (PRD) class comprising one or more members selected from the group consisting of A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, RHOXF1, RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, and VSX2. Embodiment 208. A system or composition described in any one of embodiments 1 to 206, wherein the PRD class is PAX6. Embodiment 209. A system or composition described in any one of embodiments 1 to 208, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is not a member of Pax3 and Pax7. Embodiment 210. A system or composition described in any one of embodiments 1 to 209, wherein the homeobox comprises a homeobox of the CUT class comprising one or more members selected from the group consisting of ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2, SATB1, and SATB2. Embodiment 211. A system or composition described in any one of embodiments 1 to 210, wherein the CUT-class homeobox is ONECUT1. Embodiment 212. A system or composition described in any one of embodiments 1 to 211, wherein the homeobox comprises a homeobox of the LIM class comprising one or more members selected from the group consisting of ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, and LMX1B. Embodiment 213. A system or composition described in any one of embodiments 1 to 212, wherein the LIM class homeobox is ISL1. Embodiment 214. A system or composition according to any one of embodiments 1 to 213, wherein the conversion occurs in less than about 14 days or less than about 10 days. Embodiment 215. A system or composition described in any one of embodiments 1 to 214, wherein the conversion achieves the formation of pancreatic islets comprising a plurality of pancreatic lineage cells. Embodiment 216. A system or composition described in any one of embodiments 1 to 215, wherein the conversion is characterized by producing fewer non-endodermal cells or fewer enterochromaffin cells compared to a control conversion of a plurality of stem cells in a medium comprising (i) serum and / or (ii) an exogenous cell differentiation regulator.

[0436] The systems and methods of the present disclosure may be combined with or modified by other systems and methods for cellular programming, such as those described in International Patent Application No. PCT / US2018 / 052211, International Patent Application No. PCT / US2018 / 052211, International Patent Application No. PCT / US2023 / 028169, International Patent Application No. PCT / US2023 / 028255, and International Patent Application No. PCT / US2023 / 028033, each of which is incorporated herein by reference in its entirety.

[0437] 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. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for converting a plurality of stem cells into a plurality of pancreatic lineage cells, the method comprising: contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to achieve conversion, the plurality of gating units comprising: a) a first gating unit pre-configured to regulate the expression level of a first target gene among the plurality of distinct target genes, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-associated HMG box (SOX), and GATA; b) a second gating unit preconfigured to regulate the expression level of a second target gene among the plurality of distinct target genes such that the expression levels of the first target gene and the second target gene are sequentially regulated; Upon activation of the heterologous gene circuit, the plurality of gating units operate to effect the conversion.

2. 2. The method of claim 1, wherein the first target gene comprises two or more members selected from the group consisting of FOX, SOX, and GATA.

3. 2. The method of claim 1, wherein the first target genes include the FOX, the SOX, and the GATA.

4. 2. The method of claim 1, wherein the first target gene further comprises a T-box transcription factor (TBX).

5. 2. The method of claim 1, wherein the second target gene comprises one or more members selected from the group consisting of basic helix-loop-helix transcription factors (bHLH), homeobox, and Maf transcription factors.

6. 2. The method of claim 1, wherein (i) the expression level of the first target gene is enhanced by the first gating unit, or (ii) the expression level of the second target gene is enhanced by the second gating unit.

7. 1. A method for converting a plurality of stem cells into a plurality of pancreatic lineage cells through modulation of expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the method comprising: a) contacting a first polynucleotide sequence in the plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of the first target gene operably linked to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-associated HMG box (SOX), and GATA; b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to regulate the expression level of a second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of basic helix-loop-helix transcription factors (bHLH), homeobox, and Maf transcription factors.

8. 8. The method of claim 7, wherein (b) is performed subsequent to (a) to sequentially achieve regulation of the first target gene and the second target gene, respectively.

9. 8. The method of claim 7, wherein the first target gene comprises two or more members selected from the group consisting of the FOX, the SOX, and the GATA.

10. 8. The method of claim 7, wherein the first target genes include the FOX, the SOX, and the GATA.

11. 8. The method of claim 7, wherein the second target gene comprises two or more members selected from the group consisting of the bHLH, the homeobox, and the Maf transcription factor.

12. The method of claim 7 , wherein the second target genes include the bHLH, the homeobox, and the Maf transcription factors.

13. 8. The method of claim 7, wherein (i) the first polynucleotide sequence is upstream of or encodes the first target gene, or (ii) the second polynucleotide sequence is upstream of or encodes the second target gene.

14. 8. The method of claim 7, wherein (i) the expression level of the first target gene is enhanced upon contact with the first heterologous gene regulatory portion, or (ii) the expression level of the second target gene is enhanced upon contact with the second heterologous gene regulatory portion.

15. The method comprises contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of the plurality of distinct target genes, thereby achieving conversion, the plurality of gating units comprising: (i) a first gating unit preconfigured to act on the first heterologous gene regulatory portion to regulate the expression level of the first target gene; (ii) a second gating unit preconfigured to act on the second heterologous gene regulatory portion to regulate the expression level of the second target gene; 8. The method of claim 7, wherein upon activation of the heterologous gene circuit, the plurality of gating units operate to effect the conversion.

16. 16. The method of claim 15, wherein (i) the first gating unit is activatable to express the first heterologous gene regulatory portion, or (ii) the second gating unit is activatable to express the second heterologous gene regulatory portion.

17. 1. A method for converting a plurality of stem cells into a plurality of pancreatic lineage cells through modulation of expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the method comprising: a) contacting a first polynucleotide sequence in the plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of the first target gene operably linked to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-associated HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor; b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to reduce the expression level of the second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of an additional SOX, an additional homeobox, and an ETS transcription factor.

18. 18. The method of claim 17, wherein (b) is performed before (a) to sequentially achieve modulation of the first target gene and reduction of the expression level of the second target gene, respectively.

19. 18. The method of claim 17, wherein (b) is performed subsequent to (a) to sequentially achieve reduced expression of the second target gene and modulation of the first target gene, respectively.

20. 18. The method of claim 17, wherein (a) and (b) are performed simultaneously.

21. 18. The method of claim 17, wherein the additional SOX is not a member from SOX9 and SOX17.

22. The method of claim 17 , wherein the additional SOX comprises SOX2.

23. 18. The method of claim 17, wherein the additional homeobox is not a member from PDX1, NKX6-1, NKX2-2, PAX6, ONECUT1, and ISL1.

24. 18. The method of claim 17, wherein the additional homeobox comprises CDX1, CDX2, or CDX4.

25. 25. The method of claim 24, wherein the additional homeobox comprises CDX2.

26. 18. The method of claim 17, wherein the additional homeobox comprises LMX1A or LMX1B.

27. 27. The method of claim 26, wherein the additional homeobox comprises LMX1A.

28. 18. The method of claim 17, wherein the ETS transcription factor comprises ERG, FLI1, or FEV.

29. 29. The method of claim 28, wherein the ETS transcription factor comprises FEV.

30. 18. The method of claim 17, wherein the first target gene comprises two or more members selected from the group consisting of the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

31. 18. The method of claim 17, wherein the first target gene comprises one or more members selected from the group consisting of the FOX, the SOX, and the GATA.

32. 18. The method of claim 17, wherein the first target gene comprises one or more members selected from the group consisting of the bHLH, the homeobox, and the Maf transcription factor.

33. 18. The method of Claim 17, wherein (i) the first polynucleotide sequence is upstream of or encodes the first target gene, or (ii) the second polynucleotide sequence is upstream of or encodes the second target gene.

34. 18. The method of claim 17, wherein the expression level of the first target gene is enhanced upon contact with the first heterologous gene regulatory moiety.

35. The method comprises contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of the plurality of distinct target genes to achieve the conversion, the plurality of gating units comprising: (i) a first gating unit preconfigured to act on the first heterologous gene regulatory portion to regulate the expression level of the first target gene; (ii) a second gating unit preconfigured to act on the second heterologous gene regulatory portion to reduce the expression level of the second target gene; 18. The method of claim 17, wherein upon activation of the heterologous gene circuit, the plurality of gating units operate to effect the conversion.

36. 36. The method of claim 35, wherein (i) the first gating unit is activatable to express the first heterologous gene regulatory portion, or (ii) the second gating unit is activatable to express the second heterologous gene regulatory portion.

37. 1. A method for converting a plurality of stem cells into pancreatic lineage cells, the method comprising: contacting a polynucleotide sequence in said plurality of stem cells with a heterologous gene regulatory moiety to regulate the expression level of a target gene operably linked to said polynucleotide sequence. wherein within about two weeks after contacting, a conversion rate of said plurality of stem cells to a plurality of pancreatic lineage cells is characterized as being at least about 5%.

38. 38. The method of claim 37, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells.

39. 39. The method of claim 38, wherein the conversion rate of the plurality of stem cells to the pancreatic progenitor cells is at least about 10%, at least about 15%, at least about 20%, or at least about 25%.

40. 38. The method of claim 37, wherein the plurality of pancreatic lineage cells comprises beta cells.

41. 41. The method of claim 40, wherein the conversion rate of the plurality of stem cells to the beta cells is at least about 10%.

42. 38. The method of claim 37, wherein the conversion is observed within about 12 days, within about 10 days, or within about 8 days.

43. 38. The method of claim 37, wherein the expression level of the target gene is enhanced upon contact with the heterologous gene regulatory moiety.

44. 38. The method of claim 37, wherein the target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor.

45. 45. The method of claim 44, wherein the target gene comprises two or more members selected from the group consisting of the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

46. wherein the target gene comprises a plurality of distinct target genes, including a first distinct target gene and a second distinct target gene, and wherein the contacting step comprises: (a) contacting a first polynucleotide sequence in said plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of said first distinct target gene operably linked to said first polynucleotide sequence; (b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to regulate the expression level of the second target gene operably linked to the second polynucleotide sequence.

47. 47. The method of Claim 46, wherein steps (a) and (b) sequentially achieve regulation of the first distinct target gene and the second distinct target gene.

48. The contacting step comprises: contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of the plurality of distinct target genes to achieve conversion, the plurality of gating units comprising: (i) a first gating unit preconfigured to act on the first heterologous gene regulatory moiety to regulate the expression level of the first distinct target gene; (ii) a second gating unit preconfigured to act on the second heterologous gene regulatory portion to regulate the expression level of the second distinct target gene; 47. The method of claim 46, wherein upon activation of the heterologous gene circuit, the plurality of gating units operate to effect the transformation.

49. 47. The method of claim 46, wherein the plurality of distinct target genes comprises two or more members selected from the group consisting of the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

50. 38. The method of claim 37, wherein the target gene is an endogenous gene.

51. 1. A method of treating a subject in need thereof, said method comprising: administering to the subject a plurality of pancreatic lineage cells, wherein the plurality of pancreatic lineage cells is prepared by subjecting a plurality of stem cells to ex vivo differentiation; The method, wherein within about two weeks of ex vivo differentiation, the conversion rate of said plurality of stem cells to said plurality of pancreatic lineage cells is characterized as being at least about 5%.

52. 52. The method of claim 51, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells.

53. 53. The method of claim 52, wherein the conversion rate of the plurality of stem cells to the pancreatic progenitor cells is at least about 10%, at least about 15%, at least about 20%, or at least about 25%.

54. 52. The method of claim 51, wherein the plurality of pancreatic lineage cells comprises beta cells.

55. 54. The method of claim 53, wherein the conversion rate of the plurality of stem cells to the beta cells is at least about 10%.

56. 52. The method of claim 51, wherein the conversion is observed within about 12 days, within about 10 days, or within about 8 days.

57. 52. The method of claim 51, wherein the plurality of pancreatic lineage cells are subjected to ex vivo culture for about 4 weeks or less, about 3 weeks or less, about 2 weeks or less, about 12 days or less, about 10 days or less, or about 8 days or less.

58. 52. The method of claim 51, wherein the ex vivo differentiation comprises modulating the expression levels of target genes comprising one or more members selected from the group consisting of forkhead box (FOX), SRY-related HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor.

59. 59. The method of claim 58, wherein the target gene comprises two or more members selected from the group consisting of the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

60. 59. The method of claim 58, wherein the target gene is an endogenous gene.

61. 61. The method of any one of claims 1-60, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells or pancreatic beta cells.

62. 62. The method of any one of claims 1-61, wherein the plurality of pancreatic lineage cells are characterized as ECAD+ / CD142+ or CD49a+.

63. 63. The method of any one of claims 1-62, wherein the plurality of pancreatic lineage cells are characterized as producing insulin.

64. 64. The method of any one of claims 1 to 63, wherein the plurality of stem cells comprises pluripotent stem cells (PSCs) or endodermal cells.

65. 65. The method of any one of claims 1 to 64, wherein the plurality of distinct target genes are endogenous genes of said plurality of stem cells.

66. 66. The method of any one of claims 1 to 65, wherein the first heterologous gene regulatory portion or the second heterologous gene regulatory portion comprises (i) an endonuclease or (ii) a guide nucleic acid (gNA) molecule.

67. 67. The method of claim 66, wherein the endonuclease and the gNA form a complex capable of binding to their respective target polynucleotide sequences.

68. 67. The method of claim 66, wherein the endonuclease is a Cas protein.

69. 69. The method of any one of claims 1 to 68, wherein the conversion occurs under conditions that are substantially free of (i) serum and / or (ii) exogenous cell differentiation regulators.

70. 70. The method of claim 69, wherein the exogenous cell differentiation regulator comprises one or more members selected from the group consisting of insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH), and vascular endothelial growth factor (VEGF), transforming growth factor-β (TGFβ) superfamily, bone morphogenetic protein-2 (BMP2), and bone morphogenetic protein-7 (BMP7).

71. 70. The method of claim 69, wherein the exogenous cell differentiation regulator comprises one or more members selected from the group consisting of a GSK3β inhibitor, an ALK inhibitor, a BMP type 1 receptor inhibitor, and retinoic acid.

72. 72. The method of any one of claims 1-71, wherein FOX comprises one or more members selected from the group consisting of FOXA, FOXB, FOXC, FOXD, FOXE, FOXF, FOXG, FOXH, FOXI, FOXJ, FOXK, FOXL, FOXM, FOXN, FOXO, FOXP, FOXQ, FOXR, and FOXS.

73. 73. The method of claim 72, wherein the FOX is FOXA1, FOXA2, or FOXA3.

74. 74. The method of claim 73, wherein the FOX is FOXA2.

75. 75. The method of any one of claims 1 to 74, wherein the SOX comprises one or more members selected from the group consisting of SOXA, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH.

76. 76. The method of claim 75, wherein the SOX is SOXE.

77. 77. The method of claim 76, wherein the SOXE is SOX9.

78. 76. The method of claim 75, wherein the SOX is SOXF.

79. 79. The method of claim 78, wherein the SOXF is SOX17.

80. 80. The method of any one of claims 1 to 79, wherein SOX is not SOX2.

81. 81. The method of any one of claims 1 to 80, wherein the SOX is not SOXB1 or SOXB2.

82. 82. The method of any one of claims 1 to 81, wherein the GATA comprises one or more members selected from the group consisting of GATA1, GATA2, GATA3, GATA4, GATA5, and GATA6.

83. 83. The method of claim 82, wherein the GATA is GATA4.

84. 84. The method of any one of claims 1-83, wherein the TBX comprises one or more members selected from the group consisting of TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, and TBX22.

85. 85. The method of claim 84, wherein the TBX is TBXT.

86. 86. The method of any one of claims 1 to 85, wherein the plurality of distinct target genes does not include TBXT.

87. 87. The method of any one of claims 1 to 86, wherein the bHLH comprises one or more members selected from the group consisting of Group A bHLH, Group B bHLH, Group C bHLH, Group D bHLH, Group E bHLH, and Group F bHLH.

88. 88. The method of claim 87, wherein the bHLH is a group A bHLH.

89. 88. The method of claim 87, wherein the bHLH is NEUROG3, NEUROD1, or PTF1a.

90. 90. The method of any one of claims 1-89, wherein the homeobox comprises a ParaHox gene comprising one or more members selected from the group consisting of CDX1, CDX2, CDX4, GSX1, GSX2, and PDX1.

91. 91. The method of claim 90, wherein the ParaHox gene is PDX1.

92. 92. The method of any one of claims 1 to 91, wherein the homeobox comprises an NKX class homeobox comprising one or more members selected from the group consisting of 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.

93. The method of claim 92, wherein the NKX class homeobox is NKX6-1 or NKX2-2.

94. The homeobox is ALX1, ALX3, ALX4, ARGFX, ARX, DMBX1, DPRX, DRGX, DUXA, DUXB, DUX (1, ​​2, 3, 4, 4c, 5), ESX1, GSC, GSC2, HESX1, HOPX, ISX, LEUTX, MIXL1, NOBOX, OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHO 94. The method of any one of claims 1 to 93, wherein the homeobox comprises a paired box (PRD) class homeobox comprising one or more members selected from the group consisting of X2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, RHOXF1, RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, and VSX2.

95. 95. The method of claim 94, wherein the PRD class is PAX6.

96. 96. The method of any one of claims 1 to 95, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is not a member of Pax3 and Pax7.

97. 97. The method of any one of claims 1 to 96, wherein the homeobox comprises a CUT class homeobox comprising one or more members selected from the group consisting of ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2, SATB1, and SATB2.

98. 98. The method of claim 97, wherein the CUT class homeobox is ONECUT1.

99. 99. The method of any one of claims 1 to 98, wherein the homeobox comprises a LIM class homeobox comprising one or more members selected from the group consisting of ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, and LMX1B.

100. 100. The method of claim 99, wherein the LIM class homeobox is ISL1.

101. 101. The method of any one of claims 1 to 100, wherein the conversion occurs in less than about 14 days or less than about 10 days.

102. 102. The method of any one of claims 1-101, further comprising storing the plurality of pancreatic lineage cells in a sterile vial.

103. 103. The method of any one of claims 1-102, wherein said conversion achieves the formation of pancreatic islets comprising said plurality of pancreatic lineage cells.

104. 104. The method of any one of claims 1 to 103, further comprising generating pancreatic islets comprising said plurality of pancreatic lineage cells.

105. 105. The method of claim 104, wherein the pancreatic islets are for administration to a subject.

106. 106. The method of any one of claims 1-105, further comprising loading said plurality of pancreatic lineage cells into or onto a scaffold.

107. 107. The method of claim 106, wherein the scaffold and the plurality of pancreatic lineage cells, in combination, are for transplantation into a subject.

108. The method of any one of claims 1 to 107, further comprising administering the plurality of pancreatic lineage cells to a subject.

109. 109. The method of any one of claims 1-108, wherein the administering step comprises one or more hepatic portal vein injections.

110. 110. The method of any one of claims 1 to 109, wherein the administering step comprises one or more subcutaneous implantations.

111. 111. The method of any one of claims 1 to 110, wherein the subject has or is suspected of having diabetes.

112. 112. The method of any one of claims 1-111, wherein the conversion is characterized by producing fewer non-endodermal cells or fewer enterochromaffin cells compared to a control conversion of a plurality of stem cells in a medium comprising (i) serum and / or (ii) an exogenous cell differentiation regulator.

113. 1. A system for converting a plurality of stem cells into a plurality of pancreatic lineage cells, the system comprising: a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to achieve conversion, the plurality of gating units comprising: (i) a first gating unit pre-configured to regulate the expression level of a first target gene among the plurality of distinct target genes, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-associated HMG box (SOX), and GATA; (ii) a second gating unit pre-configured to regulate an expression level of a second target gene among the plurality of distinct target genes, wherein the expression levels of the first target gene and the second target gene are sequentially regulated; Upon activation of the heterologous gene circuit, the plurality of gating units operate to effect the conversion.

114. 1. A system for converting a plurality of stem cells into a plurality of pancreatic lineage cells through modulation of expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the system comprising: a) a first heterologous gene regulatory portion configured to bind to a first polynucleotide sequence in the plurality of stem cells and regulate the expression level of the first target gene operably linked to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-associated HMG box (SOX), and GATA; b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and regulate the expression level of the second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of basic helix-loop-helix transcription factors (bHLH), homeobox, and Maf transcription factors.

115. 1. A system for converting a plurality of stem cells into a plurality of pancreatic lineage cells through modulation of expression levels of a plurality of distinct target genes, including a first target gene and a second target gene, the system comprising: a) a first heterologous gene regulatory portion configured to bind to a first polynucleotide sequence in the plurality of stem cells and regulate the expression level of the first target gene operably linked to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from the group consisting of forkhead box (FOX), SRY-associated HMG box (SOX), GATA, basic helix-loop-helix transcription factor (bHLH), homeobox, and Maf transcription factor; b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and reduce the expression level of the second target gene operably linked to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from the group consisting of an additional SOX, an additional homeobox, and an EST transcription factor.

116. 1. A system for converting a plurality of stem cells into pancreatic lineage cells, the system comprising: a heterologous gene regulatory moiety configured to bind to a polynucleotide sequence in said plurality of stem cells and regulate the expression level of a target gene operably linked to said polynucleotide sequence, A system comprising a heterologous gene regulatory moiety, wherein the conversion rate of said plurality of stem cells to a plurality of pancreatic lineage cells is characterized as at least about 5% within about two weeks after contact.

117. 1. A composition for treating a subject in need thereof, said composition comprising: A plurality of pancreatic lineage cells prepared by subjecting a plurality of stem cells to ex vivo differentiation, A system comprising pancreatic lineage cells, wherein the conversion rate of the plurality of stem cells to the plurality of pancreatic lineage cells within about two weeks of the ex vivo differentiation is characterized as being at least about 5%.

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