System for cell programming of pancreatic lineage and method thereof

Abstract

Provided herein are systems that modulate gene expression, methods of use thereof, and engineered cells (e.g., beta cells) for cell differentiation.

Description

[0001] Cross-referencing

[0002] 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

[0003] Heterogeneous proteins and / or nucleic acid molecules can be used to elicit desired responses in cells. They can also modulate genes of interest (e.g., transgenic and / or endogenous genes) to program cells (e.g., differentiation, dedifferentiation). In some cases, endonuclease-based techniques (e.g., clustered regularly spaced short palindromic repeats (CRISPR)-associated proteins, or “CRISPR / Cas”) have been used to manipulate polynucleotide sequences, their epigenetic modifications, and / or their expression levels. For example, CRISPR / Cas technology can be characterized by its versatility and programmability, and can be used to facilitate genome editing across different species and cell types. Summary of the Invention

[0004] This disclosure provides methods and systems for programming cells, for example, for evoking desired responses in cells. The systems and methods of this disclosure can facilitate the transformation of cells from one type to another. The systems and methods of this disclosure can utilize genetic circuits to control the cascade of multiple desired expression and / or activity profiles of multiple genes in a cell to influence such transformation. The systems and methods of this disclosure can utilize heterologous proteins and / or nucleic acid molecules as building blocks for such genetic circuits.

[0005] In some embodiments, this disclosure provides a method for converting multiple stem cells into multiple pancreatic lineage cells, the method comprising: contacting the multiple stem cells with a heterologous gene circuit comprising multiple gate units, wherein the heterologous gene circuit is activatable to induce the multiple gate units to sequentially regulate the expression levels of multiple different target genes to achieve the conversion, wherein the multiple gate units comprise: (a) a first gate unit pre-configured to regulate the expression level of a first target gene among the multiple different target genes, wherein the first target gene includes one or more members selected from forkhead box (FOX), SRY-associated HMG box (SOX), and GATA; and (b) a second gate unit pre-configured to regulate the expression level of a second target gene among the multiple different target genes such that the expression levels of the first target gene and the second target gene are regulated sequentially, wherein, upon activation of the heterologous gene circuit, the multiple gate units operate to achieve the conversion.

[0006] In some embodiments, this disclosure provides a method for converting a plurality of stem cells into a plurality of pancreatic lineage cells by regulating the expression levels of a plurality of different 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 portion to regulate the expression level of the first target gene operatively coupled to the first polynucleotide sequence, wherein the first target gene includes one or more members selected from forkhead boxes (FOX), SRY-associated HMG boxes (SOX), and GATA; and (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 the second target gene operatively coupled to the second polynucleotide sequence, wherein the second target gene includes one or more members selected from basic helical-loop-helical transcription factors (bHLH), homeoboxes, and Maf transcription factors.

[0007] In some embodiments, this disclosure provides a method for converting multiple stem cells into multiple pancreatic lineage cells by regulating the expression levels of multiple different target genes, including a first target gene and a second target gene, the method comprising: (a) contacting a first polynucleotide sequence in the multiple stem cells with a first heterologous gene regulatory portion to regulate the expression level of the first target gene operatively coupled to the first polynucleotide sequence, wherein the first target gene includes one or more members selected from forkhead box (FOX), SRY-associated HMG box (SOX), GATA, basic helical-loop-helical transcription factor (bHLH), homeobox, and Maf transcription factor; and (b) contacting a second polynucleotide sequence in the multiple stem cells with a second heterologous gene regulatory portion to reduce the expression level of the second target gene operatively coupled to the second polynucleotide sequence, wherein the second target gene includes one or more members selected from additional SOX, additional homeobox, and ETS transcription factor.

[0008] In some embodiments, this disclosure provides a method for converting multiple stem cells into pancreatic lineage cells, the method comprising: contacting a polynucleotide sequence in the multiple stem cells with a heterologous gene regulatory portion to regulate the expression level of a target gene operatively coupled to the polynucleotide sequence, wherein, within less than or equal to about 2 weeks after the contact, the conversion rate from the multiple stem cells to multiple pancreatic lineage cells is characterized as at least about 5%.

[0009] In some embodiments, this disclosure provides a method for treating a subject in need, the method comprising: administering a plurality of pancreatic lineage cells to the subject, wherein the plurality of pancreatic lineage cells are prepared by causing a plurality of stem cells to undergo in vitro differentiation, wherein the conversion rate from the plurality of stem cells to the plurality of pancreatic lineage cells is characterized as at least about 5% within a period of less than or equal to about 2 weeks of the in vitro differentiation.

[0010] In some embodiments, this disclosure provides a system for converting multiple stem cells into multiple pancreatic lineage cells, the system comprising: a heterogeneous gene circuit including multiple gate units, wherein the heterogeneous gene circuit is activatable to induce the multiple gate units to sequentially regulate the expression levels of multiple different target genes to achieve the conversion, wherein the multiple gate units include: (i) a first gate unit pre-configured to regulate the expression level of a first target gene among the multiple different target genes, wherein the first target gene includes one or more members selected from forkhead boxes (FOX), SRY-associated HMG boxes (SOX), and GATA; and (ii) a second gate unit pre-configured to regulate the expression level of a second target gene among the multiple different target genes such that the expression levels of the first target gene and the second target gene are regulated in the sequential manner, wherein, upon activation of the heterogeneous gene circuit, the multiple gate units operate to achieve the conversion.

[0011] In some embodiments, this disclosure provides a system for converting a plurality of stem cells into a plurality of pancreatic lineage cells by regulating the expression levels of a plurality of different target genes, including a first target gene and a second target gene, the system comprising: (a) a first heterologous gene regulation portion configured to bind a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of the first target gene operably coupled to the first polynucleotide sequence, wherein the first target gene includes one or more members selected from forkhead boxes (FOX), SRY-associated HMG boxes (SOX), and GATA; and (b) a second heterologous gene regulation portion configured to bind a second polynucleotide sequence in the plurality of stem cells to regulate the expression level of the second target gene operably coupled to the second polynucleotide sequence, wherein the second target gene includes one or more members selected from basic helical-loop-helical transcription factors (bHLH), homeoboxes, and Maf transcription factors.

[0012] In some embodiments, this disclosure provides a system for converting multiple stem cells into multiple pancreatic lineage cells by regulating the expression levels of multiple different target genes, including a first target gene and a second target gene, the system comprising: (a) a first heterologous gene regulation portion configured to bind a first polynucleotide sequence in the multiple stem cells to regulate the expression level of the first target gene operably coupled to the first polynucleotide sequence, wherein the first target gene includes one or more members selected from forkhead boxes (FOX), SRY-associated HMG boxes (SOX), GATA, basic helical-loop-helical transcription factor (bHLH), homeoboxes, and Maf transcription factors; and (b) a second heterologous gene regulation portion configured to bind a second polynucleotide sequence in the multiple stem cells to reduce the expression level of the second target gene operably coupled to the second polynucleotide sequence, wherein the second target gene includes one or more members selected from additional SOX, additional homeoboxes, and EST transcription factors.

[0013] In some embodiments, this disclosure provides a system for converting multiple stem cells into pancreatic lineage cells, the system comprising: a heterologous gene regulatory portion configured to bind a polynucleotide sequence in the multiple stem cells to regulate the expression level of a target gene operably coupled to the polynucleotide sequence, wherein, within less than or equal to about 2 weeks after the contact, the conversion rate from the multiple stem cells to multiple pancreatic lineage cells is characterized as at least about 5%.

[0014] In some embodiments, this disclosure provides a composition for treating a subject in need, the composition comprising: a plurality of pancreatic lineage cells prepared by in vitro differentiation of a plurality of stem cells, wherein the conversion rate from the plurality of stem cells to the plurality of pancreatic lineage cells is characterized as at least about 5% within a period of less than or equal to about 2 weeks of said in vitro differentiation.

[0015] Incorporation

[0016] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is expressly and individually indicated to be incorporated by reference. Where a publication, patent, or patent application incorporated by reference contradicts the disclosure contained in this specification, this specification is intended to supersede and / or give precedence to any such contradictory material. Attached Figure Description

[0017] The novel features of the invention are set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as “Figure” and “FIG.”), in which:

[0018] Figure 1 This is a schematic diagram of a heterologous gene circuit. Activation of the initiation circuit can also activate the gate unit. The gate unit consists of a gate portion and / or a gene regulatory portion.

[0019] Figure 2 The developmental process from iPSCs to mature β cells was depicted, including genes known to affect β cell development at different stages and biomarkers for screening associated expression patterns.

[0020] Figure 3 An exemplary heterologous gene circuit is depicted.

[0021] Figures 4A-4D Scatter plots (e.g., volcano plots) are shown to identify one or more heterologous gene circuits that induce stem cell transformation into β cells under conditions such as: serum-free + B27 + T3 + β-cytokinin + ALK5i ( Figure 4A Serum + B27 + T3 + β-cytokinin + ALK5i Figure 4B ); serum only Figure 4C ); and serum +ALK5i ( Figure 4D The x-axis represents the log2 change factor enriched on the core cascade, and the y-axis represents the p-value, with the dashed line indicating a p-value of 0.05.

[0022] Figure 5 A scatter plot is shown to identify one or more heterologous gene circuits that induce stem cell transformation into β cells after five days.

[0023] Figure 6 Key surface markers for cross-conditional upregulation of high-performing heterologous gene circuits were revealed.

[0024] Figure 7 The image shows that heterologous gene circuits #9 and #16 (Cell Algorithm 9 and Cell Algorithm 16, respectively) produced cultures with islet-like morphology after eight days of culture. The arrows point to islet cells.

[0025] Figure 8A The study showed that the top-performing heterologous gene circuits produced insulin-positive cells after eight days of culture. Figure 8B The percentage of insulin-positive cells for each heterologous gene circuit tested is shown.

[0026] Figure 9The comparison between directed differentiation and heterologous gene circuits during β-cell production is shown. Detailed Implementation

[0027] While various embodiments of the 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. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0028] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least," "greater than," or "greater than or equal to" applies to each value in that series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0029] Whenever the terms “at most,” “up to,” “not exceeding,” “less than,” or “less than or equal to” precede the first value in a series of two or more values, the terms “at most,” “up to,” “not exceeding,” “less than,” or “less than or equal to” apply to each value in that series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0030] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used in this specification and claims include plural references. For example, the term “a gate cell” includes multiple gate cells.

[0031] The terms "about" or "approximately" generally mean within an acceptable range of error for a particular value, as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within or greater than one standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude of a value, preferably within 5 times, and more preferably within 2 times. Where a particular value is described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable range of error for that particular value.

[0032] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. The term "and / or" should be understood to mean any one or both of the alternatives.

[0033] As used interchangeably throughout this document, the terms “guide nucleic acid,” “guide nucleic acid molecule,” and “gNA” generally refer to either a guide sequence that can hybridize with a target sequence, or a scaffold sequence that can interact with or complex with a nucleic acid-guided nuclease. A guide nucleic acid can be a single guide nucleic acid (e.g., sgRNA) or a dual guide nucleic acid (e.g., dgRNA). An sgRNA can be a single RNA molecule containing both a scaffold tracrRNA and a crRNA that can complement the target sequence. Alternatively, a dgRNA can be a single RNA molecule containing a crRNA that has been annealed to tracrRNA via a direct repeat sequence.

[0034] 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 peptides and / or polynucleotides, non-biological materials, etc.) that are operatively coupled (e.g., simultaneously, sequentially, etc.) according to circuit design. The collection of molecular components may be able to provide one or more specific outputs in the cell (e.g., regulation of one or more genes) 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 contain one or more molecular switches that can be activated by one or more inputs (…). Figure 1 ).

[0035] A gene circuit can be a controllable gene expression system comprising the assembly of biological parts that work together as logical functions (e.g., simultaneously, sequentially, etc.). A gene circuit can contain multiple gate units, wherein at least one of the gate units is activated by an activation component (e.g., heterologous input to the cell) to activate other gate units (e.g., simultaneously at once, sequentially in a cascaded manner, etc.). Figure 1 For example, at least one of a plurality of phylum ... Figure 1The terms “heterogeneous gene circuit”, “HGC”, “gene circuit”, “cellular algorithm”, or “cellgorithm” used in this article are interchangeable.

[0036] As mentioned herein, the term "gate unit" generally refers to a portion of a gene circuit that controls gene regulation by functioning similarly to a logic gate, where it controls the flow of information and allows the circuit to make multiplexed decisions at different points. More specifically, the term refers to nucleic acids encoding genetic switches and transcriptional / translational regulatory regions, or a series of regions on which genetic switches act. The input to a gate unit can be an activating part and / or another gate unit. The output of a gate unit can activate another gate unit, deactivate another gate unit, affect a target gene, and / or any combination thereof. For example, a gate unit can consist of multiple gate parts and / or multiple gene regulatory parts (…). Figure 1 )composition.

[0037] As mentioned herein, the term "activation moiety" generally refers to a portion that can activate multiple gene circuits and / or multiple phylogenetic units. The activation moiety can be a heterologous input to the cell. In some cases, the activation moiety may include, but is not limited to, guiding nucleic acid molecules (e.g., gRNA) or other nucleic acids, peptides, polynucleotides, small molecules, light, or combinations thereof.

[0038] For example, the activating part can be a guide nucleic acid molecule that forms a complex with a nuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of an inactivated phylogenetic part (e.g., a plasmid encoding another guide nucleic acid molecule) to activate such a phylogenetic part that can target one or more gene regulatory parts (e.g., induce the expression of a functional form of another guide nucleic acid molecule). As mentioned herein, the term "phylogenetic part" generally refers to a part that can affect the function of a gene regulatory part within a phylogenetic unit. A phylogenetic part can activate and / or deactivate a gene regulatory part. For example, a phylogenetic part can regulate the expression of a gene regulatory part by editing a nucleic acid sequence and thereby activating or deactivating the gene regulatory part. For example, a phylogenetic part can be a guide nucleic acid molecule that forms a complex with a nuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of a gene regulatory part (e.g., a plasmid encoding another guide nucleic acid molecule) to activate a gene regulatory part that can target one or more endogenous genes in the cell (e.g., induce the expression of a functional form of another guide nucleic acid molecule). Alternatively or additionally, a phylogenetic part can activate and / or deactivate another phylogenetic unit of a gene circuit ( Figure 1For example, the phylum moiety can be a guide nucleic acid molecule that forms a complex with a nuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of an inactivated phylum moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to activate that other phylum moiety (e.g., to induce the expression of a functional form of the other guide nucleic acid molecule). In another example, the phylum moiety can be a guide nucleic acid molecule that forms a complex with a nuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of an activated phylum moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to inactivate that other phylum moiety (e.g., to reduce the expression of a functional form of the other guide nucleic acid molecule).

[0039] As mentioned herein, the term "gate portion" generally refers to a portion that can influence the function of the gene regulatory portion within a gate unit. A gate portion can activate and / or deactivate the gene regulatory portion. For example, a gate portion can regulate the expression of the gene regulatory portion by editing the nucleic acid sequence and thereby activating or deactivating it. Alternatively or additionally, a gate portion can activate and / or deactivate another gate unit of the gene circuit ( Figure 1 ).

[0040] As used interchangeably in this article, the terms "gene regulation portion" or "gene editing portion" generally refer to the portion that regulates the expression and / or activity profile of a nucleic acid sequence or protein (whether exogenous or endogenous to the cell). Figure 1 For example, gene editing can regulate gene expression by editing nucleic acid sequences (e.g., CRISPR-Cas, zinc finger nucleases, TALENs, or siRNA). In some cases, gene editing can regulate gene expression by editing genomic DNA sequences. In others, it can regulate gene expression by editing mRNA templates. In still others, editing nucleic acid sequences can alter the underlying template used for gene expression (e.g., CRISPR-Cas-inspired RNA targeting systems). Alternatively, gene editing can suppress gene translation (e.g., Cas13).

[0041] Alternatively or additionally, gene-editing portions can regulate gene expression or activity by specifically binding to a target sequence (or a target sequence within a gene) operatively coupled to the gene, and regulate the production of mRNA from DNA (such as chromosomal DNA or cDNA). For example, a gene-editing portion 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 portion itself can bind to DNA and regulate transcription by physically hindering it, for example, by preventing the assembly of proteins (such as RNA polymerases and other associated proteins) on a DNA template. Gene-editing portions can regulate gene expression at the translational level, for example, by regulating the production of proteins from an mRNA template. In some cases, gene-editing portions can regulate gene expression by affecting the stability of mRNA transcripts. In some cases, gene-editing portions can regulate genes (e.g., Cas12) through epigenetic editing.

[0042] In some cases, plasmids can encode non-functional forms of gene-edited portions. Plasmids can be activated (e.g., through gene modification) to express functional forms of the gene-edited portions, for example, via activation of functional gate portions. For instance, plasmids can encode non-functional forms of guide nucleic acid molecules that would otherwise be able to bind to target genes in the cell. When a functional gate portion (e.g., another guide nucleic acid molecule complexed with a Cas protein) binds to the plasmid, the plasmid can be edited (e.g., cleaved at one or more sites and then repaired via endogenous mechanisms (e.g., homologous recombination, non-homologous end joining) to allow expression of functional forms of the gene-edited portions (e.g., functional forms of guide nucleic acid molecules that specifically bind to target genes in the cell), thereby permitting the regulation of target genes in the cell.

[0043] In some cases, the gene regulatory portion may contain a nucleic acid molecule (e.g., a guide nucleic acid molecule that forms a complex with an endonuclease such as a Cas protein). Alternatively or additionally, the gene regulatory portion may contain or be operatively coupled to an endonuclease. An endonuclease may be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. Endonucleases may be restriction endonucleases that cleave DNA at specific sites without damaging bases. Restriction endonucleases may include endonucleases type I, II, III, and IV, which may further include subtypes. In some cases, the endonuclease can be Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14 or C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas13 (C2c2), Cas13b, Cas13c, Cas13d, Cas13x.1, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx10, Csx11, Csf1, or Csn2. Endonucleases can be dead endonucleases that exhibit reduced cleavage activity. For example, endonucleases can be nuclease-inactivated Cas, such as dCas (e.g., dCas9).

[0044] The aforementioned Cas proteins can form complexes with guide nucleic acids (gNAs, e.g., guide RNA (gRNA)) and utilize gNA to specifically bind to target polynucleotide sequences (e.g., target DNA sequences, target RNA sequences). Therefore, in some cases, such Cas proteins may 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 with another nucleic acid. The guide nucleic acid can be RNA. The guide nucleic acid can be DNA. The guide nucleic acid can be programmed to bind to a nucleic acid sequence specifically at a specific site. The target nucleic acid or the nucleic acid to be targeted may contain nucleotides. The guide nucleic acid may contain nucleotides. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. Double strand. The strand of the target polynucleotide that is complementary to and hybridizes with the guide nucleic acid can be called 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 can be called the non-complementary strand. A guide nucleic acid may contain a polynucleotide chain and can be called a "single guide nucleic acid." A guide nucleic acid may include two polynucleotide chains and can be called a "double guide nucleic acid." Unless otherwise specified, the term "guide nucleic acid" may be inclusive, referring to both single and double guide nucleic acids. A guide nucleic acid may contain a segment that can be called a "nucleic acid target region," "nucleic acid target sequence," or "spacer region sequence." A nucleic acid target region may contain sub-segments that can be called a "protein-binding region," "protein-binding sequence," "Cas protein-binding region," or "scaffold sequence."

[0045] The gene regulatory portion can be a transcriptional regulator 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 operatively coupled to a transcriptional repressor (e.g., coupled to or fused to a transcriptional repressor). Non-limiting examples of transcriptional repressors may include KRAB, SID, MBD2, MBD3, DNMT1, DNMT2A, DNMT3A, DNMT3B, DNMT3L, Mecp2, FOG1, ROM2, LSD1, ERD, SRDX repressor domain, Pr-SET7 / 8, SUV4-20H1, RIZ1, JMJD2A, JHDM3A, JMJD2B, JMJD2C, GASC1, JMJD2 D, JARID1A, RBP2, JARID1B / PLU-1, JARIDIC / SMCX, JARIDID / SMCY, HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, M.Hhal, METI, DRM3, ZMET2, CMT1, CMT2, lamin A, and lamin B. Alternatively, the gene regulatory portion may be a gene activator complex comprising a dCas protein operatively coupled to (e.g., fused to) a transcription activator. Non-restricted examples of transcriptional activators may include VP16, VP64, VP48, VP160, p65 subdomain, SET1A, SET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, SYMD2, NSD1, JHDM2a, JHDM2b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK, TET1CD, TET1, DME, DML1, DML2, and ROS1.

[0046] In some cases, the gene regulatory portion possesses enzymatic activity that modifies the target gene without cleaving it. Modification of the target gene can induce epigenetic modifications, such as those that modify gene expression and / or activity levels. Examples of enzymatic activities that can be provided by the gene regulatory portion include, but are not limited to: nuclease activity (such as that provided by restriction enzymes (e.g., Fokl nucleases)); methyltransferase activity (such as that provided by methyltransferases (e.g., HhalDNA)). Methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3, ZMET2, CMT1, CMT2) provide methyltransferase activity; demethylase activity (such as demethylase activity provided by demethylases (e.g., deca-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1) provide demethylase activity); DNA repair activity; DNA damage activity; deamination activity (such as deamination activity provided by demethylases (e.g., cytosine dioxygenase 1, TET1CD, TET1, DME, DML1, DML2, ROS1) provide demethylase activity); DNA repair activity; DNA damage activity; deamination activity (such as deamination activity provided by deamination ...CD, TET1CD, DME, DML1, DML2, ROS1) provide demethylase activity); DNA repair activity; DNA damage activity; deamination activity (such as deamination activity provided by deaminations (e.g., cytosine dioxygenase 1, TET1CD, TET2CD, TET1CD, TET The activities of pyrimidine deaminases (such as APOBEC1) include deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity (such as integrase activity provided by integrase and / or dissociation enzymes (e.g., Gin convertase, such as the hyperactive mutant of Gin convertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 dissociation enzyme, etc.), transposase activity, recombinase activity (such as recombinase activity provided by recombinase (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylation enzyme activity).

[0047] Gene regulatory components may include endonucleases. Endonucleases can be enzymes that cleave phosphodiester bonds within a polynucleotide chain. Endonucleases can be restriction endonucleases that cleave DNA at specific sites without damaging bases. Restriction endonucleases can include type I, II, III, and IV endonucleases, which may further include subtypes. In some cases, the endonuclease may be Cas9. In some cases, the endonuclease may be a deactivated Cas (e.g., dCas, dCas9).

[0048] Unless otherwise specified or apparent from the context, as used interchangeably herein, the terms “polynucleotide,” “oligonucleotide,” or “nucleic acid” generally refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or their analogues, and whether single-stranded, double-stranded, or multi-stranded. Polynucleotides can be exogenous or endogenous to the cell. Polynucleotides can exist in cell-free environments. Polynucleotides can be genes or fragments thereof. Polynucleotides can be DNA. Polynucleotides can be RNA. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Polynucleotides can include one or more analogues (e.g., modified backbones, sugars, or nucleobases). Where modifications are present, the nucleotide structure can be modified before or after polymer assembly. Some non-limiting examples of analogues include: 5-bromouracil, peptide nucleic acids, heteronucleic acids, morpholino derivatives, locked nucleic acids, glycol nucleic acids, threonine nucleic acids, dideoxynucleotides, cordycepin, 7-denitro-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogues, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine nucleoside, brassinoside, and wyoside. Non-restrictive examples of polynucleotides include coding or non-coding regions of genes or gene segments, loci (locus) as 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-chain 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.

[0049] The term "gene" generally refers to nucleic acids (e.g., DNA, such as genomic DNA and cDNA) that encode RNA transcripts and their corresponding nucleotide sequences. As used herein with respect to genomic DNA, the term includes intercalated non-coding regions as well as regulatory regions and may include 5′ and 3′ ends. In some uses, the term encompasses transcribed sequences, including 5′ and 3′ untranslated regions (5′-UTR and 3′-UTR), exons, and introns. In some genes, the transcribed region will contain an "open reading frame" encoding a polypeptide. In some uses of the term, "gene" contains only the coding sequence necessary to encode a polypeptide (e.g., "open reading frame" or "coding region"). In some cases, genes do not encode polypeptides, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only transcribed sequences but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene" or a natural gene located in its native position within an organism's genome. A gene can also refer to an "exogenous gene" or a non-natural gene. Non-natural genes can refer to genes that are not normally found in a host organism but are introduced into the host organism through gene transfer. Non-natural genes can also refer to genes in an organism's genome that are not in their natural location. Furthermore, non-natural genes can refer to naturally occurring nucleic acid or polypeptide sequences that contain mutations, insertions, and / or deletions (e.g., non-natural sequences).

[0050] The term "sequence identity" typically refers to the exact nucleotide-nucleotide or amino acid-amino acid correspondence between two polynucleotide or polypeptide sequences, respectively. Typically, techniques used to determine sequence identity involve determining the nucleotide sequence of the polynucleotide and / or the amino acid sequence it encodes, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percentage of identity." Whether it's a nucleic acid or amino acid sequence, the percentage of identity between two sequences is the number of exact matches between the two aligned sequences divided by the length of the longer sequence and multiplied by 100. The percentage of identity can also be determined, for example, by comparing sequence information using the advanced BLAST computer program (including version 2.2.9) available from the National Institutes of Health. The BLAST procedure is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990) and discussed in 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 procedure can be used to determine the percentage of identity across the entire length of the compared proteins. Default parameters are provided to optimize retrieval with short query sequences in procedures such as blastp. The procedure also allows the use of SEG filters to mask segments of the query sequence, as determined by the SEG procedure in 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 ​​in between. Generally, this disclosure includes 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 with any sequence provided herein.

[0051] The term "expression" generally refers to one or more processes of transcription (such as transcription into mRNA or other RNA transcripts) of polynucleotides from a DNA template and / or the subsequent translation of transcribed mRNA into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include the splicing of mRNA. In terms of expression, "upregulation" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression level in the wild-type state, while "downregulation" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression level in the wild-type state. Expression of transfected genes can occur transiently or stably in cells. During "transient expression," the transfected gene does not transfer to daughter cells during cell division. Because its expression is limited to the transfected cell, gene expression disappears over time. During transient expression, free DNA can be transferred to daughter cells, but because free DNA is not replicated, it is not permanently inherited and will dilute over time. Conversely, stable expression of a transfected gene can occur when it is co-transfected with another gene that confers a selective advantage to the transfected cells. During stable expression, plasmids can possess DNA replication elements that allow them to be inherited or integrated into the genome. Such a selective advantage can be resistance to a certain toxin presented to the cell.

[0052] As used interchangeably herein, the terms “peptide,” “polypeptide,” or “protein” generally refer to a polymer of at least two amino acid residues linked by peptide bonds. This term does not imply a specific length of polymer, nor is it intended to suggest or distinguish whether a peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is 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 acid chains. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structures (e.g., domains). The term also covers modified amino acid polymers, for example, through disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeled component. As used herein, the terms “amino acid” and “amino acids” generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include both natural and non-natural amino acids that have been chemically modified to include groups or chemical moieties not naturally present on the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0053] As used interchangeably herein, with respect to polypeptides, the terms “derivative,” “variant,” or “fragment” generally refer to a polypeptide that is associated with a wild-type polypeptide, for example, by its amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of polypeptides may contain one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to wild-type polypeptides.

[0054] As used herein, with respect to polypeptide molecules (e.g., proteins), the terms "engineered," "chimeric," or "recombinant" generally refer to polypeptide molecules that have heterologous or altered amino acid sequences due to the application of genetic engineering techniques to the nucleic acids encoding the polypeptide molecule and to the cells or organisms expressing the polypeptide molecule. As used herein, with respect to polynucleotide molecules (e.g., DNA or RNA molecules), the terms "engineered" or "recombinant" generally refer to polynucleotide molecules that have heterologous or altered nucleic acid sequences due to 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, engineered or recombinant polynucleotides (e.g., genomic DNA sequences) can be modified or altered by gene editing.

[0055] Unless otherwise specified or apparent from the context, as used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphate adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS]dATP, 7-denitro-dGTP, and 7-denitro-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled using known techniques. Labeling may also be performed using quantum dots. Detectable labeling may include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labeling of nucleotides may include, but is not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-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 may 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, all available from Perkin Elmer, Foster City, Calif.; and FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink, all available from Amersham, Arlington Heights, Ill. Cy5-dUTP; luciferin-15-dATP, luciferin-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, luciferin-12-ddUTP, luciferin-12-UTP, and luciferin-15-2′-dATP, available from Boehringer Mannheim, Indianapolis, Ind.; and luciferin-15-2′-dATP, available from Molecular Nucleotides with chromosome markers obtained by Probes, Eugene, and 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 can also be labeled or marked by chemical modifications. Chemically modified mononucleotides can be biotin-dNTPs. 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).

[0056] The term "cell" generally refers to a biological cell. A cell can be the basic structural, functional, and / or biological unit of a living organism. Cells can originate from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaea cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycophytes, hornwort, liverwort, mosses), algal cells (e.g., *Botryococcus braunii*, *Chlamydomonas reinhardtii*, *Nannochloropsis gaditana*, *Chlorella pyrenoidosa*, *Sargassum*). Cells can be derived from various organisms, including: patens, C. Agardh, algae (e.g., kelp), fungal cells (e.g., yeast cells, mushroom cells), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Sometimes, cells are not derived from natural organisms (e.g., cells can be synthetically manufactured, sometimes called artificial cells).

[0057] The term "differentiation" generally refers to the process by which undifferentiated ("uncommitted") or less specialized cells acquire the characteristics of specialized cells (such as, for example, immune cells). Differentiated or differentiation-induced cells are cells that have reached a more specialized ("committed") position within a cell lineage. The term "committed" generally refers to cells that, under normal circumstances, will continue to differentiate into a specific cell type or a subset of cell types, and under normal circumstances, cannot differentiate into different cell types or revert to a less differentiated cell type.

[0058] The term "dedifferentiation" or "de-differentiation" generally refers to the process by which specialized, fixed, or partially specialized cells lose the characteristics of specialized cells (e.g., β cells). Dedifferentiated cells, or dedifferentiation-induced cells, are cells that have acquired fewer specialized positions within a cell lineage (e.g., stem cells or progenitor cells). Dedifferentiated cells (e.g., stem cells or progenitor cells) may subsequently differentiate into different cell types or may revert to less differentiated cell types.

[0059] The term "pluripotent" generally refers to the ability of a cell to form all lineages of somatic cells (e.g., the embryonic body). For example, embryonic stem cells are a type of pluripotent stem cell that is capable of forming cells from each of the three germ layers (ectoderm, mesoderm, and endoderm). Pluripotency can be a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., ectoderm stem cells) (which cannot produce a complete organism) to more primitive, more pluripotent cells (which can produce a complete organism) (e.g., embryonic stem cells).

[0060] 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 altered (e.g., reprogrammed) to differentiate into tissues capable of differentiating from all three germ layers or cortical layers (mesoderm, endoderm, and ectoderm). The resulting iPSCs do not refer to cells found in nature. In some cases, iPSCs can be engineered to differentiate directly into morphological cells (e.g., β cells). In other cases, iPSCs can be engineered to first differentiate into tissue-specific stem cells (e.g., endoderm stem cells, endocrine progenitor cells), which can then be further induced to differentiate into morphological cells (e.g., β cells).

[0061] The term "embryonic stem cell" (ESC) generally refers to naturally occurring pluripotent stem cells derived from the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and, during development, produce all derivatives of the three proteroderms (ectoderm, endoderm, and mesoderm). In some cases, ESCs can be engineered to differentiate directly into morphological cells (e.g., β cells). In other cases, ESCs can be engineered to first differentiate into tissue-specific stem cells (e.g., endoderm stem cells), which can then be further induced to differentiate into morphological cells (e.g., pancreatic lineage cells or β cells).

[0062] The term "isolated stem cells" generally refers to any type of stem cells disclosed herein (e.g., ESCs, HSCs, endoderm stem cells, etc.) isolated from a multicellular organism. For example, HSCs can be isolated from the body of a mammal such as a human. In another example, embryonic stem cells can be isolated from an embryo.

[0063] The term "isolated" generally refers to cells or groups of cells that have been separated from their original environment. For example, the new environment of isolated cells is substantially free of at least one component found in the environment where "unisolated" reference cells were present. Isolated cells can be cells from which some or all of the components found in their natural environment have been removed, such as cells isolated from tissue or biopsy samples. The term also includes cells from which at least one, some, or all of the components have been removed when cells are found in a non-natural environment, such as cells isolated from cell cultures or cell suspensions. Thus, isolated cells are cells that have been partially or completely separated from at least one component, including other substances, cells, or groups of cells when they are found in nature or when they are grown, stored, or survive in a non-natural environment.

[0064] As used interchangeably in this document, the terms “β cell,” “β-cell,” or “pancreatic β cell” generally refer to any cell in the pancreas that produces insulin. Non-limiting examples of β cells are immature β cells, mature β cells, and cells that differentiate themselves into immature or mature β cells (such as pancreatic lineage cells).

[0065] When applied to cells or populations, the term "pancreatic lineage" generally refers to the ability of that cell or population to produce glucagon, insulin, somatostatin, and / or pancreatic polypeptide (PP) (e.g., in vivo or in vitro conditions). Pancreatic lineage cells can be at least partially differentiated alpha (α) cells (e.g., glucagon-producing), beta (β) cells (e.g., insulin-producing), delta (δ) cells (e.g., somatostatin-producing), or PP cells (e.g., pancreatic polypeptide (PP)-producing). Alternatively, pancreatic lineage cells can be cells that differentiate themselves into pancreatic cells, such as pancreatic progenitor cells.

[0066] The terms “pancreatic progenitor cell,” “pancreatic endocrine progenitor cell,” “pancreatic precursor,” and “pancreatic endocrine precursor” are used interchangeably in this document and generally refer to tissue-specific stem cells capable of becoming pancreatic hormone-expressing cells that can form pancreatic endocrine cells, pancreatic exocrine cells, or pancreatic duct cells. Pancreatic progenitor cells can dedicate themselves to differentiating into at least one type of pancreatic cell (such as alpha (α) cells, beta (β) cells, delta (δ) cells, or PP cells).

[0067] The term "pancreatic lineage cells" generally refers to cells that produce or are capable of producing glucagon, insulin, somatostatin, and / or pancreatic polypeptides. Pancreatic lineage cells can be generated in vitro from engineered stem cells. Pancreatic lineage cells can develop from endoderm stem cells and may include, but are not limited to, well-defined endoderm cells, primitive intestinal cells, foregut cells, pancreatic progenitor cells, endocrine progenitor cells, pancreatic endocrine cells, pancreatic exocrine cells, pancreatic duct cells, α cells, δ cells, PP cells, immature β cells, and mature β cells. Pancreatic lineage cells can be generated in vivo from engineered stem cells, for example, by administering any of the heterologous gene circuits disclosed herein to such stem cells.

[0068] Overview

[0069] Biological programming (such as cell programming) allows cells to be engineered to produce desired results. The results of cell programming can include inducing or preventing a wide range of common and / or novel cellular functions; results can also include enhancing or suppressing cellular functions that have already occurred. Cell programming can be accomplished by using genetic circuits. Cell programming can be accomplished by manipulating biomolecules (e.g., DNA). For example, CRISPR or CRISPR / Cas systems have been used for genome editing across many species due to their versatility and ease of programming. Cell programming can affect endogenous or exogenous genes. Cell programming can be implemented to operate in a time-dependent or time-independent manner. Cell programming can be implemented to directly affect endogenous or exogenous genes. Alternatively, cell programming can be implemented to indirectly affect endogenous or exogenous genes (e.g., by regulating portions of or using heterologous gene circuits for gene regulation).

[0070] The gene circuits used in cell programming can be used to control the cell fate of one or more cells by inducing differentiation or dedifferentiation and by converting from one cell type to another. Cell programming controls this fate by regulating the desired expression and / or activity levels of multiple genes within the cell.

[0071] While the CRISPR / Cas system is widely used for gene editing, Cas is essentially a single-turnover nuclease because it remains bound to the double-strand breaks it produces, and many regions of the genome are resistant to genome editing. Increased understanding of CRISPR / Cas-based genome editing has encouraged the development of cascade regulatory systems to further utilize this technology for engineered cell development. By implementing a series of activatable gRNAs, genome editing can be regulated more temporally from target site to target site, sequential genome editing can be performed to act like a domino effect, and cells can be barcoded. However, this simple barcoding, which typically uses exogenous fluorophores, does not allow for the regulation of endogenous genes to achieve cell differentiation.

[0072] Furthermore, cell differentiation or dedifferentiation is currently achieved using exogenous serum and growth factors, which bypass the underlying mechanisms of cell programming. Using exogenous serum, growth factors, and other similar methods results in cells being instructed to differentiate, but lacking the accompanying underlying biology (e.g., chromatin in the correct state). This lack often leads to premature termination of cell differentiation into a non-desired cell type or inefficient differentiation, resulting in low yields of the target cell type or only semi-functional cells. Semi-functional cells may resemble the cell type of interest but may lack the key biological characteristics necessary for the normal function of the desired cell type.

[0073] Therefore, there remains an unmet need for activatable CRISPR / Cas systems and their use in editing target polynucleotides (e.g., the genomes of cells, particularly eukaryotic cells) that utilize gRNA cascades to form gene circuits to independently influence gene regulation and consequently cell fate determination without the use of serum and exogenous growth factors. Preprogrammed, activatable, and self-regulating gRNA cascade CRISPR / Cas systems are being used, for example, in gene therapy, gene circuitry, and / or complex cell fate determination and / or control.

[0074] This disclosure provides systems and methods for engineering a CRISPR / Cas9 system comprising a Cas endonuclease and an array of homologous single guide RNAs (sgRNAs or gRNAs) having inactivating sequences in non-essential regions and being activatable to allow for regulation and modification of the system without the need for serum, growth factors, or other additional exogenous signals. This disclosure also provides engineered cells that may contain any of the above-described systems or be capable of performing any of the above-described methods.

[0075] Systems and methods for cell programming of pancreatic lineages

[0076] Various aspects of this disclosure provide systems for inducing a desired transformation from one type of cell to another. For this purpose, various aspects of this disclosure provide methods for inducing a desired expression and / or activity level (or spectrum) of one or more target genes in a cell.

[0077] In one aspect, this disclosure provides a system for converting multiple cells of a first type into multiple cells of a second cell type. The system may include a heterologous gene circuit comprising multiple gate units. The multiple gate 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 gate units. The multiple gate units may include 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. The multiple gate units may be different (e.g., containing different polynucleotide sequences). Each of the multiple phylogenetic units can influence the regulation of the expression and / or activity levels of different target genes or multiple different target genes.

[0078] The heterologous gene circuits disclosed herein can be operated using multiple gate units in series (e.g., multiple gate units connected sequentially in an end-to-end manner to form a single path), multiple gate units in parallel (e.g., multiple gate units cross-connected to each other to form, for example, two or more parallel sequential paths), or combinations thereof.

[0079] As disclosed herein, multiple phylogenetic units can operate synergistically (e.g., as predetermined by the design of heterologous gene circuits) to induce outcomes in cells. These outcomes can include cell function (e.g., motility, reproduction; response to external stimuli, nutrient output, excretion, respiration, growth) and / or cell state (e.g., cell fate, differentiation, quiescence, programmed cell death). Such outcomes can be determined in vitro, ex vivo, and / or in vivo. For example, outcomes as disclosed herein can be determined 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) measuring phenotypic differentiation and cell function using assays (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.

[0080] The multiple gate units disclosed herein are sufficient to achieve the conversion of multiple cells from a first cell type to multiple cells from a second cell type. For example, the multiple gate units disclosed herein are sufficient to achieve the conversion of multiple pluripotent stem cells (PSCs) to multiple tissue-specific progenitor cells. Alternatively, the multiple gate units disclosed herein may be necessary but insufficient to achieve the conversion of multiple cells from a first cell type to multiple cells from a second cell type.

[0081] Cellular outcomes can include regulation of different target genes or different sets of target genes. Multiple phylogenetic units can induce different regulation of multiple target genes (e.g., sequentially), such that the regulatory set of genes works synergistically to produce the final expression and / or activity profile of the cell. The final expression and / or activity level profile of the cell can illustrate outcomes such as the transformation of cells from one cell type to another (or its process).

[0082] In some cases, multiple phylogenetic units, as disclosed herein, may be necessary, but individually may be insufficient to achieve the desired expression and / or activity profile of the target cells. Therefore, in the absence of any of the multiple phylogenetic units, outcomes in cells induced by multiple phylogenetic units (e.g., enhanced cell function, induced cell state, etc.) may not be possible. Alternatively, the extent or measurement of outcomes in cells induced by multiple phylogenetic units may differ (e.g., greater for positive markers or smaller for negative markers) from the extent or measurement of outcomes in control cells induced by the absence of, one or more but not all of, and / or by all of the multiple phylogenetic units that occur in different sequences of events.

[0083] The second gate unit can be activated by the first gate unit (e.g., directly or indirectly). 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 other gate units that are activated by the first gate unit (e.g., directly or indirectly). The one or more other gate 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 gate units. One or more additional gate units may be up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 gate unit. In yet another alternative, the second gate unit may be activated via another portion responsible for activating the first gate unit (e.g., an activation portion, a different gate unit, etc.). In yet another alternative, the first and second gate units may be activated by different activation portions (e.g., different polynucleotide molecules, such as different guide nucleic acid molecules).

[0084] In some cases, such as the term "proGuide" as commonly used herein, it can refer to a vector (e.g., a plasmid) that encodes an activating gNA. A proGuide can be an example of a phylogenetic part. A proGuide can be an example of a gene regulatory part.

[0085] In some embodiments, the proGuide, as provided herein, may encode an activatable guide nucleic acid molecule, for example, 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 the proGuide encoding the activatable guide nucleic acid molecule may comprise multiple regions sequentially linked (e.g., from 5' to 3'), including an upstream stem (e.g., an upstream cleavage site), a polyT unit (or "proUnit" or "proGuide" as used interchangeably herein), and a downstream stem (e.g., a downstream cleavage site). The upstream and downstream stems may correspond to "stem region" polynucleotide sequences that are at least partially complementary to each other.

[0086] The proGuide provided herein can encode an activatable guide nucleic acid molecule, for example, having an inactivating polynucleotide sequence (e.g., one or more polyX sequences, such as one or more polyT sequences). In some embodiments, the portion of the proGuide encoding the activatable guide nucleic acid molecule may comprise a plurality of sequentially linked regions, including a spacer sequence, an additional sequence (e.g., an adapter sequence or backbone sequence), an upstream stem, a polyT unit, and a downstream stem. In some embodiments, the additional sequence 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 with GTTTTAGAGCTA (SEQ ID NO: 2027). In some embodiments, the portion of the proGuide encoding the activatable guide nucleic acid molecule may comprise a plurality of sequentially linked regions, including a spacer sequence, an additional sequence (e.g., an adapter sequence or backbone sequence), an upstream stem, a polyT unit, and a downstream stem, as shown in Table 4 (SEQ ID No: 1-2024). In some embodiments, the portion encoding the proGuide that activates the guide nucleic acid molecule may comprise multiple sequentially linked regions, including a spacer sequence, additional sequences (e.g., adapter or backbone sequences), an upstream stem, a polyT unit, and a downstream stem, as shown in Table 4 (SEQ ID No: 1-2024), presented as sequences linked by "-" to distinguish the respective regions. In some cases, after modification or removal of the polyT unit, the upstream and downstream stems may form part of a scaffold sequence for the functional guide nucleic acid molecule. In some embodiments, when the spacer sequence does not begin with G, a G is added before the spacer sequence. In some embodiments, adding a G before the spacer sequence helps increase the expression of the RNA portion from the promoter. In some embodiments, stem 1 and stem 2 are anticomplements of each other. In some embodiments, the upstream and downstream stems are anticomplements 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 with members of the polynucleotide sequences shown in Table 6 (SEQ ID No: 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 with members of the polynucleotide sequences shown in Table 6 (SEQ ID No: 2143-2165).

[0087] The domains encoding (or corresponding to) the polynucleotide sequence of interest may contain polyX sequences. The polyX sequence may be sufficient to reduce the expression of the molecule of interest (e.g., a guide nucleic acid molecule) from the polynucleotide sequence. For example, the polyX sequence may be positioned within the domain encoding the molecule of interest (e.g., not at the 5' or 3' end of such a domain) such that the expression of the molecule of interest (e.g., transcription of the RNA molecule of interest) will be disrupted (e.g., terminated) midway through expression.

[0088] Therefore, a polyX sequence (e.g., in a polynucleotide sequence encoding a molecule of interest) can be referred to as a termination sequence (e.g., a non-canonical termination sequence for its sequence and / or its position), a disruption sequence (e.g., for disrupting the full expression of the molecule of interest), or an inactivation sequence (e.g., for inactivating the function of the polynucleotide sequence or the molecule of interest).

[0089] In some cases, the polyX sequence may be located within a polynucleotide sequence (such as a DNA sequence or an RNA sequence) (e.g., not at the end). In some cases, the polyX sequence may 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 from the 3' end of the polynucleotide sequence. In some cases, the polyX sequence can be located at least about 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, or 100 bases from the 5' end of the polynucleotide sequence. In some cases, the polyX sequence can be located at the end of the nucleic acid sequence.

[0090] In some cases, the polyT or polyU sequence may be located within a polynucleotide sequence (such as a DNA sequence or an RNA sequence) (e.g., not at the end). In some cases, the polyT or polyU sequence may be located at a distance of 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 from the 3' end of the polynucleotide sequence. In some cases, the polyT or polyU sequence can be located at at least about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 bases from the 5' end of the polynucleotide sequence. In some cases, the polyT or polyU sequence can be located at the end of the nucleic acid sequence. In some cases, RNA containing a polyU sequence can also be represented by DNA containing a polyT sequence.

[0091] The polyX sequence (e.g., the polyT sequence or the polyU sequence) may 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 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 X bases. The polyX sequence may 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 2 X bases. The polyX sequence may be represented by a complementary polyX sequence in the corresponding complementary DNA strand (e.g., polyT, as disclosed herein as a DNA sequence, may also be referred to as polyA in the complementary DNA strand). The disclosed polyX sequence may contain multiple X bases. Multiple X bases that are sequentially adjacent to each other may be disclosed (e.g., TT, TTT, TTTT, TTTTT, etc.). Alternatively or additionally, multiple X bases may be separated by one or more other nucleotides that are not X. These one or more other nucleotides may include a single type of nucleotide or different types of nucleotides.

[0092] In some embodiments, proGuide may comprise an inactivated polynucleotide sequence or its complementary sequence that exhibits at least or more than about 50%, at least or more than about 55%, at least or more than about 60%, at least or more than about 65%, at least or more than about 70%, at least or more than about 75%, at least or more than about 80%, at least or more than about 85%, at least or more than about 86%, at least or more than about 87%, at least or more than about 88%, at least or more than about 89%, at least or more than about 90%, at least or more than about 91%, at least or more than about 92%, at least or more than about 93%, at least or more than about 94%, at least or more than about 95%, at least or more than about 96%, at least or more than about 97%, at least or more than about 98%, at least or more than about 99%, or substantially about 100% sequence identity with TTTTTTTTT (SEQ ID NO:2025).

[0093] In some cases, a non-canonical termination sequence may contain or consist essentially of the following: [the sequence is missing from the original text]. NO:2026) is a polynucleotide sequence or its complementary sequence that exhibits at least or more than about 40%, at least or more than about 45%, at least or more than about 50%, at least or more than about 55%, at least or more than about 60%, at least or more than about 65%, at least or more than about 70%, at least or more than about 75%, at least or more than about 80%, at least or more than about 85%, at least or more than about 86%, at least or more than about 87%, at least or more than about 88%, at least or more than about 89%, at least or more than about 90%, at least or more than about 91%, at least or more than about 92%, at least or more than about 93%, at least or more than about 94%, at least or more than about 95%, at least or more than about 96%, at least or more than about 97%, at least or more than about 98%, at least or more than about 99%, or substantially about 100% sequence identity.

[0094] In some embodiments, proGuide may include a target polynucleotide domain located at or near an inactivating polynucleotide sequence (e.g., at or near the 5' and / or 3' ends of the inactivating polynucleotide sequence), which can be targeted (e.g., via a sequential activation mechanism of a heterologous gene circuit as described herein) to modify (e.g., edit, cleave) the inactivating polynucleotide sequence, thereby causing proGuide to express an activated guide nucleic acid molecule. The target polynucleotide domain of proGuide may not exhibit sequence identity with any comparable endogenous polynucleotide sequence in the cell, thereby avoiding unintentional targeting and regulation of endogenous target genes.

[0095] In some implementations, the inactivating polynucleotide sequence of proGuide can be positioned between two target polynucleotide domains, which may or may not be targeted by a common guide nucleic acid sequence. In some cases, the two target polynucleotide domains can be inversely related and complementary to each other, allowing the inactivating polynucleotide sequence to be modified or cleaved via the same mechanism (e.g., the same spacer sequence of the guide nucleic acid molecule).

[0096] In some implementations, proGuide may comprise the following: SEQ ID NO:1-92 (e.g., EGF targeting), SEQ ID NO:93-184 (e.g., FEV targeting), SEQ ID NO:185-276 (e.g., FOXA2 targeting), SEQ ID NO:277-368 (e.g., GATA4 targeting), SEQ ID NO:369-460 (e.g., gata6 targeting), SEQ ID NO:461-552 (e.g., GCG targeting), SEQ ID NO:553-644 (e.g., INS targeting), SEQ ID NO:645-736 (e.g., isl1 targeting), SEQ ID NO:737-828 (e.g., LMX1A targeting), SEQ ID NO:829-920 (e.g., MAFA targeting), SEQ ID NO:921-1012 (e.g., NEUROD1), SEQ ID NO:1013-1104 (e.g., NEUROG3 targeting), SEQ ID NO:1-92 (e.g., EGF targeting), SEQ ID NO:93-184 (e.g., FEV targeting), SEQ ID NO:185-276 (e.g., FOXA2 targeting), SEQ ID NO:277-368 (e.g., GATA4 targeting), SEQ ID NO:369-460 (e.g., gata6 targeting), SEQ ID NO:461-552 (e.g., GCG targeting), SEQ ID NO:553-644 (e.g., INS targeting), SEQ ID NO:645-736 (e.g., isl1 targeting), SEQ ID NO:737-828 (e.g., LMX1A targeting), SEQ ID NO:829-920 (e.g., MA SEQ ID NO:1105-1196 (e.g., NKX2-2 targeting), SEQ ID NO:1197-1288 (e.g., NKX6-1 targeting), SEQ ID NO:1289-1380 (e.g., ONECUT1 targeting), SEQ ID NO:1381-1472 (e.g., PDX1 targeting), SEQ ID NO:1473-1564 (e.g., PTF1a targeting), SEQ ID NO:1565-1656 (e.g., SHH targeting), SEQ ID NO:1657-1748 (e.g., sox17 targeting), SEQ ID NO:1749-1840 (e.g., SOX9 targeting), SEQ ID NO:1841-1932 (e.g., SST targeting), SEQ ID One or more members of NO:1933-2024 (e.g., TBXT targeting) exhibit at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 86%, at least or more to about 87%, at least or more to about 88%, at least or more to about 89%, at least or more to about 90%, at least or more to about 91%, at least or more to about 92%, at least or more to about 93%, at least or more to about 94%, at least or more to about 95%, at least or more to about 96%, at least or more to about 97%, at least or more to about 98%, at least or more to about 99%, or substantially about 100% sequence identity of a polynucleotide sequence or its complementary sequence.

[0097] The second gate unit may be activatable to induce the inactivation of an already activated first gate unit. The terms “inactivation” or “disruption” are used interchangeably herein. Inactivation and, as disclosed herein, can be induced by modifying (e.g., cleavage, such as single- or double-strand breaks, and insertion-deletion, etc.) at least a portion of the first gate unit (e.g., the gate portion and / or gene regulatory portion of the first gate unit) responsible for inducing a first distinct regulation of the target gene.

[0098] Inactivation of the phylum portion and / or gene regulatory portion of the first phylum unit as disclosed herein can be achieved using a nuclease-based system (e.g., a CRISPR / Cas system). Alternatively or additionally, inactivation can be achieved using a transcription regulator system (e.g., a transcriptional repressor). Alternatively or additionally, inactivation can be achieved using CRISPRi steric hindrance without requiring an additional transcription regulator. Nuclease transcription regulator systems (e.g., Cas-repressors) can be used to achieve polynucleotide cleavage (e.g., for inactivating the phylum portion and / or gene regulatory portion). Polynucleotide cleavage can create nucleic acid modifications such as single-strand breaks, double-strand breaks, insertions, deletions, or insertion-deletion. Alternatively or additionally, nuclease transcription regulator systems (e.g., Cas-repressors) can be used to regulate target gene expression. Alternatively or additionally, CAS transcription regulator systems lacking nuclease activity (dCAS or naked CAS with a shortened spacer insufficient to support cleavage) target DNA regions and physically stop transcriptional elongation, resulting in repression of the target gene (CRISPRi).

[0099] Alternatively, the second gate unit may be activatable to amplify or enhance the activation of the already activated first gate unit. Amplification or enhancement of the first gate unit can be induced by modifying (e.g., cleavage, such as single- or double-strand breaks, and insertion-deletion) at least a portion of the first gate unit (e.g., the gate portion and / or gene regulatory portion of the first gate unit), which is responsible for inducing a first distinct regulation of the target gene.

[0100] In some cases, the first phylacting unit regulates the first target gene. Alternatively or additionally, the first phylacting unit may also regulate the second phylacting unit. Regulation of the second phylacting unit may occur at least or at most about 1 millisecond, at least or at most about 2 milliseconds, at least or at most about 3 milliseconds, at least or at most about 4 milliseconds, at least or at most about 5 milliseconds, at least or at most about 6 milliseconds, at least or at most about 7 milliseconds, at least or at most about 8 milliseconds, at least or at most about 9 milliseconds, at least or at most about 10 milliseconds, at least or at most about 20 milliseconds, at least or at most about 30 milliseconds, at least or at most about 40 milliseconds, at least or at most about 50 milliseconds, at least or at most about 60 milliseconds, at least or at most about 70 milliseconds, at least or at most about 1 millisecond. Approximately 80 milliseconds, at least or more than approximately 90 milliseconds, at least or more than approximately 100 milliseconds, at least or more than approximately 200 milliseconds, at least or more than approximately 300 milliseconds, at least or more than approximately 400 milliseconds, at least or more than approximately 500 milliseconds, at least or more than approximately 600 milliseconds, at least or more than approximately 700 milliseconds, at least or more than approximately 800 milliseconds, at least or more than approximately 900 milliseconds, at least or more than approximately 1 second, at least or more than approximately 2 seconds, at least or more than approximately 3 seconds, at least or more than approximately 4 seconds, at least or more than approximately 5 seconds, at least or more than approximately 6 seconds, at least or more than approximately 7 seconds, at least or more than approximately 8 seconds Seconds, at least or at most about 9 seconds, at least or at most about 10 seconds, at least or at most about 15 seconds, at least or at most about 20 seconds, at least or at most about 30 seconds, at least or at most about 40 seconds, at least or at most about 50 seconds, at least or at most about 1 minute, at least or at most about 2 minutes, at least or at most about 3 minutes, at least or at most about 4 minutes, at least or at most about 5 minutes, at least or at most about 6 minutes, at least or at most about 7 minutes, at least or at most about 8 minutes, at least or at most about 9 minutes, at least or at most about 10 minutes, at least or at most about 20 minutes, at least or at most about 30 minutes, to The time may be as short as or as long as about 40 minutes, at least or as long as about 50 minutes, at least or as long as about 1 hour, at least or as long as about 2 hours, at least or as long as about 3 hours, at least or as long as about 4 hours, at least or as long as about 5 hours, at least or as long as about 6 hours, at least or as long as about 7 hours, at least or as long as about 8 hours, at least or as long as about 9 hours, at least or as long as about 10 hours, at least or as long as about 12 hours, at least or as long as about 16 hours, at least or as long as about 20 hours, or at least or as long as about 24 hours or longer, as determined by rt-qPCR, Western blotting or other methods.

[0101] In some cases, the second gate unit can regulate a second target gene. Regulation of the second target gene can occur at least or at most approximately 1 millisecond, approximately 2 milliseconds, approximately 3 milliseconds, approximately 4 milliseconds, approximately 5 milliseconds, approximately 6 milliseconds, approximately 7 milliseconds, approximately 8 milliseconds, approximately 9 milliseconds, approximately 10 milliseconds, approximately 20 milliseconds, approximately 30 milliseconds, approximately 40 milliseconds, approximately 50 milliseconds, approximately 60 milliseconds, approximately 70 milliseconds, approximately 80 milliseconds, approximately 90 milliseconds, approximately 1 second, approximately 2 seconds, approximately 3 seconds, approximately 4 seconds, approximately 5 seconds, approximately 6 seconds, or approximately 7 seconds after regulation of the first target gene. Approximately 8 seconds, 9 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours or longer, as determined by rt-qPCR, Western blotting or other methods.

[0102] A heterogeneous gene circuit may include multiple gate units that are activated sequentially (e.g., tandemly activated one after another). These gate units may include functional gate units pre-configured to be 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 pre-configured to (i) be activated prior to the functional gate unit and (ii) to enable subsequent activation of the functional gate unit. In some cases, one or more additional gate units may be pre-configured to be activated to regulate one or more additional target genes. Alternatively, one or more additional gate units may not be pre-configured to regulate any target gene (e.g., any endogenous target gene) upon activation. Such one or more additional gate units may instead be used to delay (e.g., temporally) the activation of the functional gate unit during operation of the heterogeneous gene circuit, thereby delaying the expression and / or epigenetic profile of the target gene of the functional gate unit; therefore, one or more additional gate units may be referred to as “blank” gate units. The heterologous gene circuit may include at least or at most about 1 blank gate unit, at least or at most about 2 blank gate units, at least or at most about 3 blank gate units, at least or at most about 4 blank gate units, at least or at most about 5 blank gate units, at least or at most about 6 blank gate units, at least or at most about 7 blank gate units, at least or at most about 8 blank gate units, at least or at most about 9 blank gate units, at least or at most about 10 blank gate units, at least or at most about 11 blank gate units, at least or at most about 12 blank gate units, or at least or at most about 13 blank gate units. At least or at most about 14 blank door units, at least or at most about 15 blank door units, at least or at most about 16 blank door units, at least or at most about 27 blank door units, at least or at most about 18 blank door units, at least or at most about 19 blank door units, at least or at most about 20 blank door units, at least or at most about 25 blank door units, at least or at most about 30 blank door units, at least or at most about 35 blank door units, at least or at most about 40 blank door units, at least or at most about 45 blank door units, at least or at most about 50 blank door units.

[0103] In some cases, using one or more blank gate units can delay the activation of functional gate units (e.g., as determined by measuring the expression / epigenetic profile of target genes, or as determined by measuring the expression of functional variants or transcripts of functional gate units) by at least or at most about 1 minute, at least or at most about 5 minutes, at least or at most about 10 minutes, at least or at most about 30 minutes, at least or at most about 1 hour, at least or at most about 2 hours, at least or at most about 3 hours, at least or at most about 4 hours, at least or at most about 5 hours, at least or at most about 6 hours, at least or at most about 7 hours, at least or at most about 8 hours, at least or at most about 9 hours. At least or more than about 10 hours, at least or more than about 11 hours, at least or more than about 12 hours, at least or more than about 13 hours, at least or more than about 14 hours, at least or more than about 15 hours, at least or more than about 16 hours, at least or more than about 17 hours, at least or more than about 18 hours, at least or more than about 19 hours, at least or more than about 20 hours, at least or more than about 21 hours, at least or more than about 22 hours, at least or more than about 23 hours, at least or more than about 24 hours, at least or more than about 2 days, at least or more than about 3 days, at least or more than about 4 days, at least or more than about 5 days, at least or more than about 6 days, or at least or more than about 7 days.

[0104] In some cases, phylogenetic modifications to a target gene can inactivate it. For example, gene modification can prevent the expression and / or activity level of the target gene. Alternatively, gene modification can reduce or decrease the expression and / or activity level of the target gene. In some cases, gene modification can increase the expression and / or activity level of the target gene. Alternatively, gene modification can maintain the expression and / or activity level of the target gene.

[0105] In some cases, gene modification can reduce 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. Gene modification can reduce the expression and / or activity levels of target genes by up to approximately 500%, up to approximately 400%, up to approximately 300%, up to approximately 200%, up to approximately 100%, up to approximately 90%, up to approximately 80%, up to approximately 70%, up to approximately 60%, up to approximately 50%, up to approximately 40%, up to approximately 30%, up to approximately 20%, up to approximately 10%, up to approximately 9%, up to approximately 8%, up to approximately 7%, up to approximately 6%, up to approximately 5%, up to approximately 4%, up to approximately 3%, up to approximately 2%, up to approximately 1%, up to approximately 0.9%, up to approximately 0.8%, up to approximately 0.7%, up to approximately 0.6%, up to approximately 0.5%, up to approximately 0.4%, up to approximately 0.3%, up to approximately 0.2%, up to approximately 0.1%, or less.

[0106] In some cases, gene modification can increase 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%, or at least about 80%. At least approximately 90%, at least approximately 100%, at least approximately 200%, at least approximately 300%, at least approximately 400%, at least approximately 500%, at least approximately 600%, at least approximately 700%, at least approximately 800%, at least approximately 900%, at least approximately 1,000%, at least approximately 2,000%, at least approximately 3,000%, at least approximately 4,000%, at least approximately 5,000%, at least approximately 6,000%, at least approximately 7,000%, at least approximately 8,000%, at least approximately 9,000%, at least approximately 10,000%, at least approximately 100,000%, at least approximately 1,000,000%, or more. Gene modification can increase the expression and / or activity levels of target genes by up to approximately 1,000,000%, up to approximately 100,000%, up to approximately 9,000%, up to approximately 8,000%, up to approximately 7,000%, up to approximately 6,000%, up to approximately 5,000%, up to approximately 4,000%, up to approximately 3,000%, up to approximately 2,000%, up to approximately 1,000%, up to approximately 900%, up to approximately 800%, up to approximately 700%, up to approximately 600%, up to approximately 500%, up to approximately 400%, up to approximately 300%, and up to approximately 200%. Up to approximately 100%, up to approximately 90%, up to approximately 80%, up to approximately 70%, up to approximately 60%, up to approximately 50%, up to approximately 40%, up to approximately 30%, up to approximately 20%, up to approximately 10%, up to approximately 9%, up to approximately 8%, up to approximately 7%, up to approximately 6%, up to approximately 5%, up to approximately 4%, up to approximately 3%, up to approximately 2%, up to approximately 1%, up to approximately 0.9%, up to approximately 0.8%, up to approximately 0.7%, up to approximately 0.6%, up to approximately 0.5%, up to approximately 0.4%, up to approximately 0.3%, up to approximately 0.2%, up to approximately 0.1% or less.

[0107] In some cases, gene modification can reduce the expression and / or activity level of the target gene by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 2-fold, at least or more than about 3-fold, at least or more than about 4-fold, at least or more than about 5-fold, compared to the control expression and / or activity level. At least or more than about 6 times, at least or more than about 7 times, at least or more than about 8 times, at least or more than about 9 times, at least or more than about 10 times, at least or more than about 20 times, at least or more than about 30 times, at least or more than about 40 times, at least or more than about 50 times, at least or more than about 60 times, at least or more than about 70 times, at least or more than about 80 times, at least or more than about 90 times, at least or more than about 100 times, at least or more than about 500 times, at least or more than about 1,000 times, at least or more than about 5,000 times, or at least or more than about 10,000 times. Compared to control expression and / or activity levels, gene modification can reduce the expression and / or activity levels of the target gene by at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than Approximately 10 times, up to or less than approximately 9 times, up to or less than approximately 8 times, up to or less than approximately 7 times, up to or less than approximately 6 times, up to or less than approximately 5 times, up to or less than approximately 4 times, up to or less than approximately 3 times, up to or less than approximately 2 times, up to or less than approximately 1 time, up to or less than approximately 0.9 times, up to or less than approximately 0.8 times, up to or less than approximately 0.7 times, up to or less than approximately 0.6 times, up to or less than approximately 0.5 times, up to or less than approximately 0.4 times, up to or less than approximately 0.3 times, up to or less than approximately 0.2 times, up to or less than approximately 0.1 times.

[0108] In some cases, gene modification can increase the expression and / or activity level of the target gene by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 2-fold, at least or more than about 3-fold, at least or more than about 4-fold, at least or more than about 5-fold, compared to the control expression and / or activity level. At least or more than about 6 times, at least or more than about 7 times, at least or more than about 8 times, at least or more than about 9 times, at least or more than about 10 times, at least or more than about 20 times, at least or more than about 30 times, at least or more than about 40 times, at least or more than about 50 times, at least or more than about 60 times, at least or more than about 70 times, at least or more than about 80 times, at least or more than about 90 times, at least or more than about 100 times, at least or more than about 500 times, at least or more than about 1,000 times, at least or more than about 5,000 times, or at least or more than about 10,000 times. Compared to control expression and / or activity levels, gene modification can increase the expression and / or activity levels of the target gene by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than Approximately 10 times, up to or less than approximately 9 times, up to or less than approximately 8 times, up to or less than approximately 7 times, up to or less than approximately 6 times, up to or less than approximately 5 times, up to or less than approximately 4 times, up to or less than approximately 3 times, up to or less than approximately 2 times, up to or less than approximately 1 time, up to or less than approximately 0.9 times, up to or less than approximately 0.8 times, up to or less than approximately 0.7 times, up to or less than approximately 0.6 times, up to or less than approximately 0.5 times, up to or less than approximately 0.4 times, up to or less than approximately 0.3 times, up to or less than approximately 0.2 times, up to or less than approximately 0.1 times.

[0109] The expression and / or activity profile of a gene of interest (e.g., a differentiation marker) can be compared with control genes (e.g., housekeeping genes, such as GAPDH), the relative expression levels of two or more genes of interest (e.g., the ratio of expression or activity levels between stem cell markers and differentiation markers), the relative average expression level of a gene of interest compared with the average expression level of the same gene of interest in the cell type of interest, etc.

[0110] In some cases, the activation of multiple gate units may be the result of a single activation of the heterogeneous gene circuit (e.g., through a single activation portion at a single time point). The multiple gate units may include one of a first gate unit and a second gate unit that is pre-configured to be sequentially activated after a single activation of the heterogeneous gene circuit. In some cases, one of the first and second gate units may be activated by a single activation portion (e.g., a guide nucleic acid), while the other of the first and second gate units may be activated by a different activation portion (e.g., a different guide nucleic acid) that is different from the activation portion of the heterogeneous gene circuit. This additional activation portion may be a portion of the heterogeneous gene circuit that is generated (e.g., expressed) only after activation of the heterogeneous gene circuit. Alternatively or additionally, the first and second gate units may each be activated by a different activation portion that is different from the activation portion of the heterogeneous gene circuit. This different activation portion may be a portion of the heterogeneous gene circuit that is generated (e.g., expressed) only after activation of the heterogeneous gene circuit.

[0111] In some embodiments of any of the systems disclosed herein, a gate unit may include a gate (e.g., at least or more to about one gate portion, at least or more to about two gate portions, at least or more to about three gate portions, at least or more to about four gate portions, at least or more to about five gate portions, etc.) and / or a gene regulatory portion (e.g., at least or more to about one gene regulatory portion, at least or more to about two gene regulatory portions, at least or more to about three gene regulatory portions, at least or at most about four gene regulatory portions, at least or more to about five gene regulatory portions, at least or more to about six gene regulatory portions, at least or more to about seven gene regulatory portions, at least or more to about eight gene regulatory portions, at least or more to about nine gene regulatory portions, at least or more to about ten gene regulatory portions, etc.). A gate portion as disclosed herein may include a guide nucleic acid molecule (gNA) (e.g., at least or more to about one gNA molecule, at least or more to about two gNA molecules, at least or more to about three gNA molecules, at least or more to about four gNA molecules, at least or more to about five gNA molecules, etc.). The gene regulatory portion disclosed herein may contain gNA (e.g., at least or more than about one gNA molecule, at least or more than about two gNA molecules, at least or more than three gNA molecules, at least or more than about four gNA molecules, at least or more than about five gNA molecules, etc.). The guide nucleic acid molecule disclosed herein may include, but is not limited to, DNA, RNA, any analogues of such, or any combination thereof. In some embodiments of any of the systems disclosed herein, the gate portion and / or gene regulatory portion may be activated to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and this complex may be configured or capable of binding to a target polynucleotide, for example, to regulate the expression and / or activity level of the target polynucleotide or another polynucleotide sequence operatively coupled to the target polynucleotide. For example, the complex may regulate the expression and / or activity level of a gene containing the target polynucleotide.

[0112] In some cases, the guide nucleic acid molecule (gNA) (e.g., a functional gNA) expressed by the second gate unit upon activation can modify at least a portion of the first gate unit. For example, the activated gNA of the second gate unit can modify 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 operatively coupled to the same first gate unit. This modification can render the gNA of the first gate unit inoperable when expressed (e.g., reduce or inhibit specific binding to a target gene). Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0113] In some cases, modifications to a polynucleotide sequence (e.g., as a component of a phylum unit, such as a phylum portion) or a target gene can be caused by a single-strand break that is discontinuous in one of the nucleotide chains. Inactivation of a polynucleotide sequence or 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, gene inactivation 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.

[0114] In some cases, gNA may have a size of at least or more than about 60 nucleotides, at least or more than about 70 nucleotides, at least or more than about 80 nucleotides, at least or more than about 85 nucleotides, at least or more than about 90 nucleotides, at least or more than about 95 nucleotides, at least or more than about 100 nucleotides, at least or more than about 105 nucleotides, at least or more than about 110 nucleotides, at least or more than about 120 nucleotides, at least or more than about 130 nucleotides, at least or more than about 140 nucleotides, at least or more than about 150 nucleotides, or at least or more than about 200 nucleotides (e.g., including both spacer and scaffold sequences).

[0115] In some cases, the scaffold sequence of gNA may have a size of at least or more than about 30 nucleotides, at least or more than about 35 nucleotides, at least or more than about 40 nucleotides, at least or more than about 45 nucleotides, at least or more than about 50 nucleotides, at least or more than about 55 nucleotides, at least or more than about 60 nucleotides, at least or more than about 65 nucleotides, at least or more than about 70 nucleotides, at least or more than about 75 nucleotides, at least or more than about 80 nucleotides, at least or more than about 85 nucleotides, at least or more than about 90 nucleotides, at least or more than about 95 nucleotides, at least or more than about 100 nucleotides, at least or more than about 100 nucleotides, at least or more than about 120 nucleotides, at least or more than about 130 nucleotides, at least or more than about 140 nucleotides, or at least or more than about 150 nucleotides.

[0116] In some cases, the spacer region sequence of gNA may have a size of at least or more than about 10 nucleotides, at least or more than about 11, at least or more than about 12, at least or more than about 13, at least or more than about 14, at least or more than about 15, at least or more than about 16, at least or more than about 17, at least or more than about 18, at least or more than about 19, at least or more than about 20, at least or more than about 21, at least or more than about 22, at least or more than about 23, at least or more than about 24, at least or more than about 25, at least or more than about 26, at least or more than about 27, at least or more than about 28, at least or more than about 29, or at least or more than about 30 nucleotides.

[0117] In some embodiments of any of the systems disclosed herein, the initial (or first) gate unit of the heterologous gene circuit disclosed herein may be activated by an activation portion (e.g., direct activation). The activation portion may directly bind to at least a portion of the initial gate unit to activate the initial gate unit, e.g., thereby sequentially activating the heterologous gene circuit. Alternatively, the activation portion may activate the initial gate unit without directly binding to at least a portion of the initial gate unit (e.g., by using electromagnetic energy). In some cases, the initial gate unit may comprise at least one gate portion and at least one gene regulatory portion. In some cases, the initial gate unit may comprise at least one gate portion, but may not include, and need not include, a gene regulatory portion. In some cases, the initial gate unit may comprise at least one gene regulatory portion, but may not include, and need not include, a gate portion (e.g., the activation portion may be configured to activate the initial gate unit and at least one additional gate unit).

[0118] In some embodiments of any of the systems disclosed herein, the gNA of the phylum and / or gene regulatory portion (e.g., the gNA encoded by the phylum and / or gene regulatory portion) may be an activatable gNA. An activatable gNA may be one of, but not limited to, ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogues of such, or any combination thereof. In some embodiments, the vector (or expression cassette) encoding the activatable gNA may contain an inactivating polynucleotide sequence to inactivate the gNA until it is activated (e.g., until the inactivating polynucleotide sequence is modified or removed from the vector). For example, the inactivating polynucleotide sequence may encode a self-cleaving polynucleotide molecule (e.g., a ribozyme). Alternatively or additionally, the inactivating polynucleotide sequence may encode a non-canonical transcription termination sequence, as described below. The inactivating polynucleotide sequence may be part of or adjacent to a region of the vector that encodes (i) a spacer sequence of the gNA, (ii) a scaffold sequence of the gNA, and / or (ii) any linker sequence between the spacer sequence and the scaffold sequence. The vector may contain at least or at most about 1 inactivated polynucleotide sequence, at least or at most about 2 inactivated polynucleotide sequences, at least or at most about 3 inactivated polynucleotide sequences, at least or at most about 4 inactivated polynucleotide sequences, at least or at most about 5 inactivated polynucleotide sequences, at least or at most about 6 inactivated polynucleotide sequences, at least or at most about 7 inactivated polynucleotide sequences, at least or at most about 8 inactivated polynucleotide sequences, at least or at most about 9 inactivated polynucleotide sequences, or at least or at most about 10 inactivated polynucleotide sequences.

[0119] In some cases, such as the term "proGuide" as commonly used herein, it can refer to a vector (e.g., a plasmid) that encodes an activating gNA. A proGuide can be an example of a phylogenetic part. A proGuide can be an example of a gene regulatory part.

[0120] In some embodiments, the activatable gNA molecule may be a self-cleaving gNA (e.g., gRNA containing a cis-ribozyme). For example, when the activatable gNA is expressed in a cell, the activatable gNA may be self-cleavable to become non-functional (e.g., not configured to bind a target gene) unless the gene encoding the activatable gNA is modified prior to its expression. In some embodiments, the activatable gNA molecule contains a non-canonical transcription termination sequence (e.g., a polyX sequence, such as a polyU sequence or a polyT sequence) such that the functional gNA molecule is not expressed until the gene encoding the activatable gNA with the non-canonical transcription termination sequence can be modified (e.g., to remove some or all of the transcription termination sequence). Thus, in the absence of modification with the transcription termination sequence, a non-functional variant of the gNA (e.g., a non-functional fragment) can be expressed. In some embodiments, the gNA may be synthetic. In some embodiments, the gNA may have an attached fluorescent label.

[0121] In some cases, the size of the polyT sequence is greater than or equal to a threshold length, where the threshold length is sufficient to reduce the expression of the guiding nucleic acid molecule from the polynucleotide sequence. Therefore, plasmids (e.g., phylogenetic or gene regulatory portions) can encode inactive gNAs containing polyT sequences greater than or equal to the threshold length, and editing such plasmids to reduce the length of the polyT to below the threshold length can allow gNAs to be expressed in their entirety without premature termination, thereby activating gNAs. 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 5 to 15 Ts. In some cases, the polyT sequence contains one or more additional nucleotides that are not Ts.

[0122] In some cases, gene regulatory portions (e.g., guide nucleic acids and / or endonucleases) can be configured to bind to a target polynucleotide sequence operatively coupled to a target gene in the cell. The target gene may contain a coding polynucleotide sequence encoding a target nucleic acid molecule or a target protein. The target polynucleotide sequence may be part of a coding polynucleotide sequence. Alternatively, the target polynucleotide sequence may not be part of a coding polynucleotide sequence. For example, the target polynucleotide sequence may be upstream of a coding polynucleotide sequence (e.g., part of a promoter encoding a polynucleotide sequence, such as a transcription start site (TSS)).

[0123] As presented herein, when a heterologous gene circuit is activated to induce multiple different regulation of a target gene, these multiple different regulation of the target gene can be different (e.g., different degrees of alteration in the expression and / or activity levels of the target gene). For example, the different degrees of the first regulation exerted by the first gene unit and the second regulation exerted by the second phylum unit can be at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%. The different degrees of the first and second adjustments can be up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, or up to about 0.1%. Alternatively or additionally, the different regulation of the target gene can be substantially the same (e.g., identical). Multiple different regulation may individually be sufficient to induce the desired change in the expression and / or activity level of the target gene. Alternatively, different regulation may individually be insufficient to induce the desired change in the expression and / or activity level of the target gene.

[0124] As disclosed herein, one or more target genes may include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0125] In some cases, the guide nucleic acid molecule (gNA) (e.g., a functional gNA) expressed by the second gate unit upon activation can modify at least a portion of the first gate unit. For example, the activated gNA of the second gate unit can modify 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 operatively coupled to the same first gate unit. This modification can render the gNA of the first gate unit inoperable when expressed (e.g., reduce or inhibit specific binding to a target gene). Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0126] In some embodiments of any of the systems disclosed herein, the initial (or first) gate unit of the heterologous gene circuit disclosed herein may be activated by an activation portion (e.g., direct activation). The activation portion may directly bind to at least a portion of the initial gate unit to activate the initial gate unit, e.g., thereby sequentially activating the heterologous gene circuit. Alternatively, the activation portion may activate the initial gate unit without directly binding to at least a portion of the initial gate unit (e.g., by using electromagnetic energy). In some cases, the initial gate unit may comprise at least one gate portion and at least one gene regulatory portion. In some cases, the initial gate unit may comprise at least one gate portion but may not include, and is not required to include, a gene regulatory portion. In some cases, the initial gate unit may comprise at least one gene regulatory portion but may not include, and is not required to include, a gate portion (e.g., the activation portion may be configured to activate the initial gate unit and at least one additional gate unit).

[0127] In some embodiments of any of the systems disclosed herein, the gNA of the phylum and / or gene regulatory portion (e.g., the gNA encoded by the phylum and / or gene regulatory portion) may be an activatable gNA. The activatable gNA may be, but is not limited to, any of the following: ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogues of such, or any combination thereof. In some embodiments, the activatable gNA molecule may be a self-cleaving gNA (e.g., gRNA containing a cis-ribozyme). For example, when the activatable gNA is expressed in a cell, the activatable gNA may be self-cleavable to become non-functional (e.g., not configured to bind a target gene) unless the gene encoding the activatable gNA is modified prior to the expression of the activatable gNA. In some embodiments, the gNA may be synthetic. In some embodiments, the gNA may have an attached fluorescent label.

[0128] In some embodiments of any of the systems disclosed herein, the gNA of the phylum and / or gene regulatory portion (e.g., the gNA encoded by the phylum and / or gene regulatory portion) may include a spacer sequence. In some cases, the spacer sequence may be specific to the target gene. Alternatively, the spacer sequence may be independent of the target gene.

[0129] As described above, the length of the spacer region sequence of gNA can affect the ability of gNA to mediate Cas nuclease activity. In some cases, gNAs with spacer region sequences of different lengths can be used in the same heterologous gene circuit to influence different types of cleavage, activation, inactivation, and / or regulation of one or more target nucleic acids. In some cases, gNA spacer region sequences shorter than a threshold length (e.g., about 16 nucleotides) can impede the nuclease activity of Cas-transcriptional regulators while still mediating DNA binding for transcriptional regulation of target genes. In some cases, gNA spacer region sequences 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 impede the nuclease activity of Cas proteins while still mediating DNA binding.

[0130] For example, a gNA containing a 20-nucleotide spacer sequence (e.g., a gNA encoded by the phylogenetic part of a plasmid used for targeting gene regulation) may be sufficient to promote the nuclease activity of endonucleases (e.g., Cas or Cas-transcriptional regulator fusion proteins). Alternatively or additionally, a gNA containing a 14-nucleotide spacer sequence (e.g., a gNA encoded by a gene regulatory part) may hybridize with DNA, but may not be long enough to mediate nuclease activity—it may only promote endonuclease binding to homologous DNA sequences. Thus, shorter gNAs can selectively allow transcriptional regulation of target genes, even with the use of endonuclease-transcriptional regulator systems (e.g., Cas-activator systems, Cas-repressor systems), without cleaving the target gene.

[0131] In some cases, modifications to polynucleotide sequences (e.g., as components of phylum units, such as phylum portions) or target genes can be caused by double-strand breaks in which discontinuities exist in both nucleotide chains. In some cases, the number of such double-strand breaks (e.g., those necessary for the modification) can be at least or more than about 1, at least or more than about 2, at least or more than about 3, at least or more than about 4, at least or more than about 5, at least or more than about 6, at least or more than about 7, at least or more than about 8, at least or more than about 9, or at least or more than about 10.

[0132] In some cases, modifications to polynucleotide sequences (e.g., as components of phylum units, such as phylum portions) or target genes can be caused by insertion-deletion (also known as insertion-deletion mutations). Insertion-deletion mutations can include frameshift or non-frameshift mutations. Insertion-deletion mutations can also include point mutations (also known as base substitutions), where only one base or base pair is modified. The length of an insertion-deletion mutation 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. The length of an insertion-deletion mutation can include up to approximately 2000, up to approximately 1000, up to approximately 900, up to approximately 800, up to approximately 700, up to approximately 600, up to approximately 500, up to approximately 400, up to approximately 300, up to approximately 200, up to approximately 100, up to approximately 90, up to approximately 80, up to approximately 70, up to approximately 60, up to approximately 50, up to approximately 40, up to approximately 30, up to approximately 20, up to approximately 15, up to approximately 10, up to approximately 9, up to approximately 8, up to approximately 7, up to approximately 6, up to approximately 5, up to approximately 4, up to approximately 3, up to approximately 2, or up to approximately 1 base or base pair.

[0133] In some cases, it is possible to modify polynucleotide sequences (e.g., as components of phylogenetic units, such as phylogenetic parts) or target genes without cleaving the polynucleotide sequence or the target gene. For example, gene regulatory parts (e.g., nucleic acid molecules and / or endonucleases, such as complexes containing CRISPR / Cas proteins and guide nucleic acid molecules) can specifically bind to polynucleotide sequences or target genes, thereby modifying the expression and / or activity of those polynucleotide sequences or target genes. Gene regulatory parts may contain transcriptional repressors or transcriptional activators as described herein. Alternatively or additionally, gene regulatory parts may induce epigenetic modifications (or epigenomic modifications) as described herein.

[0134] In some cases, as described herein, modifications to polynucleotide sequences or target genes can inactivate them. For example, modifications can inhibit or reduce the expression and / or activity levels of polynucleotide sequences or target genes. In other cases, as described herein, modifications can activate polynucleotide sequences or target genes. For example, modifications can increase the expression and / or activity levels of polynucleotide sequences or target genes.

[0135] In some cases, as provided herein, modification of a polynucleotide sequence or target gene may include reducing the expression and / or activity level of the polynucleotide sequence or target gene by at least or more than about 0.1%, at least or more than about 0.2%, at least or more than about 0.3%, at least or more than about 0.4%, at least or more than about 0.5%, at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 10%, at least or more than about 15%, at least or more than about 20%, at least or more than about 30%, at least or more than about 40%, at least or more than about 50%, at least or more than about 60%, at least or more than about 70%, at least or more than about 80%, at least or more than about 90%, at least or more than about 95%, at least or more than about 99%, or about 100% (e.g., compared to a control, for example, lacking the modification).

[0136] In some cases, as provided herein, modification of a polynucleotide sequence or target gene may include reducing the expression and / or activity level of the polynucleotide sequence or target gene by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 1.5-fold, at least or more than about 2-fold, at least or more than About 3 times, at least or more than about 4 times, at least or more than about 5 times, at least or more than about 6 times, at least or more than about 7 times, at least or more than about 8 times, at least or more than about 9 times, at least or more than about 10 times, at least or more than about 11 times, at least or more than about 12 times, at least or more than about 13 times, at least or more than about 14 times, at least or more than about 15 times, at least or more than about 20 times, at least or more than about 30 times, at least or more than about 40 times, at least or more than about 50 times, or at least or more than about 100 times (e.g., compared to a control, for example, lacking the modification).

[0137] In some cases, as provided herein, modification of a polynucleotide sequence or target gene may include increasing the expression and / or activity level of the polynucleotide sequence or target gene by at least or more than about 0.1%, at least or more than about 0.2%, at least or more than about 0.3%, at least or more than about 0.4%, at least or more than about 0.5%, at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 10%, at least or more than About 15%, at least or more to about 20%, at least or more to about 30%, at least or more to about 40%, at least or more to about 50%, at least or more to about 60%, at least or more to about 70%, at least or more to about 80%, at least or more to about 90%, at least or more to about 100%, at least or more to about 150%, at least or more to about 200%, at least or more to about 300%, at least or more to about 400%, or at least or more to about 500% (e.g., compared to a control, for example, lacking the modification).

[0138] In some cases, as provided herein, modification of a polynucleotide sequence or target gene may include increasing the expression and / or activity level of the polynucleotide sequence or target gene by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 1.5-fold, at least or more than about 2-fold, at least or more than about 3-fold, at least or more than about 4-fold, at least or more than about 5 ... 6 times, at least or more than about 7 times, at least or more than about 8 times, at least or more than about 9 times, at least or more than about 10 times, at least or more than about 11 times, at least or more than about 12 times, at least or more than about 13 times, at least or more than about 14 times, at least or more than about 15 times, at least or more than about 20 times, at least or more than about 30 times, at least or more than about 40 times, at least or more than about 50 times, at least or more than about 100 times, at least or more than about 200 times, at least or more than about 300 times, at least or more than about 400 times, at least or more than about 500 times, or at least or more than about 1,000 times (e.g., compared to a control, for example, lacking the modification).

[0139] In some embodiments of any of the systems disclosed herein, the gNA of the phylum and / or gene regulatory portion (e.g., gNA encoded by the phylum and / or gene regulatory portion) 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 unrelated to a target gene but may exhibit specific binding to a target polynucleotide sequence of another phylum or another gene regulatory portion. Example spacer sequences can be found in Table 5.

[0140] Non-limiting examples of 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, the spacer region sequence of the guide nucleic acid (e.g., guide RNA) for a target gene as provided herein may contain sequence identity with one or more members selected from SEQ ID NO:2030-2117 that exhibits at least to about 50%, at least to about 55%, at least to about 60%, at least to about 65%, at least to about 70%, at least to about 75%, at least to about 80%, at least to about 85%, at least to about 86%, at least to about 87%, at least to about 88%, at least to about 89%, at least to about 90%, at least to about 91%, at least to about 92%, at least to about 93%, at least to about 94%, at least to about 95%, at least to about 96%, at least to about 97%, at least to about 98%, at least to about 99%, or substantially about 100% sequence identity (e.g.,The following are selected from SEQ ID NO:2030-2033 for EGF targeting, SEQ ID NO:2034-2037 for FEV targeting, SEQ ID NO:2038-2041 for FOXA2 targeting, SEQ ID NO:2041-2045 for GATA4 targeting, SEQ ID NO:2046-2049 for gata6 targeting, SEQ ID NO:2050-2053 for GCG targeting, SEQ ID NO:2054-2057 for INS targeting, SEQ ID NO:2058-2061 for isl1 targeting, SEQ ID NO:2062-2065 for LMX1A targeting, SEQ ID NO:2066-2069 for MAFA targeting, SEQ ID NO:2070-2073 for NEUROD1 targeting, and SEQ ID NO:2070-2073 for NEUROG3 targeting. The polynucleotide sequence (e.g., a continuous polynucleotide sequence) or its complementary sequence thereof, representing the sequence identity of one or more members of the following: SEQ ID NO:2074-2077 for NKX2-2 targeting, SEQ ID NO:2078-2081 for NKX6-1 targeting, SEQ ID NO:2082-2085 for ONECUT1 targeting, SEQ ID NO:2086-2089 for PDX1 targeting, SEQ ID NO:2090-2093 for PTF1a targeting, SEQ ID NO:2094-2097 for SHH targeting, SEQ ID NO:2098-2101 for sox17 targeting, SEQ ID NO:2102-2105 for SOX17 targeting, SEQ ID NO:2106-2109 for SOX9 targeting, SEQ ID NO:2110-2113 for SST targeting, and SEQ ID NO:2114-2117 for TBXT targeting. In some cases, such as the heterologous gene regulators presented in this article, they can exhibit specific binding to target genes.The target gene contains sequence identity with one or more members selected from SEQ ID NO:2030-2117 that exhibits at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 86%, at least or more to about 87%, at least or more to about 88%, at least or more to about 89%, at least or more to about 90%, at least or more to about 91%, at least or more to about 92%, at least or more to about 93%, at least or more to about 94%, at least or more to about 95%, at least or more to about 96%, at least or more to about 97%, at least or more to about 98%, at least or more to about 99%, or substantially about 100% (e.g., with SEQ ID NO:2030-2033 for EGF targeting, SEQ ID NO:2030-2033 for FEV targeting). SEQ ID NO:2034-2037, for FOXA2 targeting, SEQ ID NO:2038-2041, for GATA4 targeting, SEQ ID NO:2041-2045, for gata6 targeting, SEQ ID NO:2046-2049, for GCG targeting, SEQ ID NO:2050-2053, for INS targeting, SEQ ID NO:2054-2057, for isl1 targeting, SEQ ID NO:2058-2061, for LMX1A targeting, SEQ ID NO:2062-2065, for MAFA targeting, SEQ ID NO:2066-2069, for NEUROD1 targeting, SEQ ID NO:2070-2073, for NEUROG3 targeting, SEQ ID NO:2074-2077, for NKX2-2 targeting, SEQ ID NO:2078-2081, for NKX6-1 targeting, SEQ ID NO:2034-2045, for GATA4 targeting, SEQ ID NO:2046-2049, for GATA6 targeting, SEQ ID NO:2050-2053, for INS targeting, SEQ ID NO:2054-2057, for GATA4 targeting, SEQ ID NO:2058-2045, for GATA6 targeting, SEQ ID NO:2046-2049, for LMX1A targeting, SEQ ID NO:2062-2065, for MAFA targeting, SEQ ID NO:2066-2069, for NEUROD1 targeting, SEQ ID NO:2070-2073, for NEUROG3 targeting, SEQ ID NO:2074-2077, for NKX2-2 targeting, SEQ ID NO:2078-2081, for NKX6-1 targeting, SEQ ID NO:2 The polynucleotide sequence (e.g., a continuous polynucleotide sequence) or its complementary sequence (e.g., with uracil-to-thymine conversion) of one or more members of SEQ ID NO:2082-2085 for ONECUT1 targeting, SEQ ID NO:2086-2089 for PDX1 targeting, SEQ ID NO:2090-2093 for PTF1a targeting, SEQ ID NO:2094-2097 for SHH targeting, SEQ ID NO:2098-2101 for sox17 targeting, SEQ ID NO:2102-2105 for SOX9 targeting, SEQ ID NO:2106-2109 for SST targeting, SEQ ID NO:2110-2113 for SST targeting, and SEQ ID NO:2114-2117 for TBXT targeting.

[0141] The gene regulatory components disclosed herein may include endonucleases, such as CRISPR-Cas proteins exhibiting at least a portion of their nuclease activity. For example, nuclease activity can be used to activate the expression or activity of a guide nucleic acid molecule, thereby activating at least a portion of the heterologous gene circuit as described herein.

[0142] The gene regulation portion disclosed herein may include a nuclease operatively coupled to a transcriptional effector (including a transcriptional activator or repressor) that is heterologous to the cell. The nuclease may be native or engineered to exhibit reduced (or substantially no) nuclease activity, allowing it to specifically bind to a target gene without cleaving it (e.g., endogenous genes such as FOX, SOX, GATA, MAF, bHLH, homeoboxes, TBX, EST, etc.). In some cases, the nuclease may be a deactivated Cas (dCas). Conversely, once the nuclease identifies and binds to the target gene, the transcriptional effector coupled (e.g., covalently or non-covalently coupled) to the nuclease can interact with the target gene to increase or decrease its expression level, thereby increasing or decreasing its activity level. For example, the nuclease and the transcriptional effector may be part of a fusion protein encoded by the same expression cassette.

[0143] Figure 10 schematically illustrates the use of heterologous gene circuits in conjunction with endonuclease-transcriptional effector fusions (e.g., CRISPR Cas-transcriptional activators, such as Cas9-VPR). Each gate portion can be a modified self-deactivating (e.g., self-destructing) guide RNA, which, if not deactivated, will be configured to form a complex with the CRISPR Cas-transcriptional effector fusion. An initial activation portion (denoted as activated guide RNA or "aGuide") can convert the first gate portion into activated guide RNA (denoted as mature Guide). Each mature Guide can target an additional gene regulatory portion (denoted as ramGuide) encoding an activating guide RNA against a target gene to activate such a ramGuide. Subsequently, the activated ramGuide can form a complex with the CRISPR Cas-transcriptional effector fusion protein to bind to the target gene and regulate its expression level. The activated mature Guide can also target additional gate portions downstream within the heterologous gene circuit signaling cascade to subsequently regulate the expression of one or more additional genes.

[0144] In some implementations, transcriptional effectors can be histone epigenetic modifiers (or histone modifiers). In some cases, histone epigenetic modifiers can regulate histones via methylation (e.g., histone methylation modifiers, such as amino acid methyltransferases, e.g., KRAB). In some cases, histone epigenetic modifiers can regulate histones via acetylation. In some cases, histone epigenetic modifiers can regulate histones via phosphorylation. In some cases, histone epigenetic modifiers can regulate histones via ADP-ribosylation. In some cases, histone epigenetic modifiers can regulate histones via glycosylation. In some cases, histone epigenetic modifiers can regulate histones via SUMOylation. In some cases, histone epigenetic modifiers can regulate histones via ubiquitination. In some cases, histone epigenetic modifiers can regulate histones by remodeling histone structure, for example, via an ATP-dependent process.

[0145] In some implementations, transcriptional effectors can be gene epigenetic modifiers (or gene modifiers). In some cases, gene modifiers can regulate genes via methylation (e.g., gene methylation modifiers such as DNA methyltransferases or DNMT). In some cases, gene modifiers can regulate genes via acetylation.

[0146] In some implementations, transcriptional effectors may be derived from the relevant histone acetyltransferase family. Non-limiting examples of histone acetyltransferases include the GNAT subfamily, MYST subfamily, p300 / CBP subfamily, HAT1 subfamily, GCN5, PCAF, Tip60, MOZ, MORF, MOF, HBO1, p300, CBP, HAT1, ATF-2, SRC1, and TAFII250.

[0147] In some implementations, transcriptional effectors may be derived from histone lysine methyltransferases. Non-limiting examples of histone lysine methyltransferases include the 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, PRDM13, PRDM14, and PRD. M15, 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.

[0148] Non-limiting examples of transcriptional effectors that enhance the expression or activity of target genes may include, but are not limited to, transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomains (e.g., from NFkB), VP64-p65-rta fusion proteins (VPR), and activation domains and / or TAL activation domains of EDLL (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3; and histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI 60. CLOCK; and DNA demethylases, such as deca-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1.

[0149] Non-restricted examples of transcriptional effectors that reduce the expression or activity of target genes may include, but are not limited to, transcriptional repressors such as Kruppel-associated boxes (KRAB or SKD), KOX1 repressor domains, Mad mSIN3 interaction domains (SID), ERF repressor domains (ERD), SRDX repressor domains (e.g., for repression in plants); histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1; and histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD1A / RBP2, JARID1B / PLU-1, JARID... 1C / SMCX, JARIDID / SMCY, etc.; histone lysine deacetylases, such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.; DNA methyltransferases, such as Hhal DNAm5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant), etc.; and peripheral recruitment elements, such as lamin A, lamin B, etc.

[0150] Various aspects of this disclosure provide multiple heterologous gene circuits that are individually activatable to sequentially regulate the expression and / or activity levels of multiple different target genes. In some embodiments, a first heterologous gene circuit is activated to convert multiple cells from a first cell type to a second cell type, and subsequently a second heterologous gene circuit is activated to convert multiple cells from the second cell type to the target cell type.

[0151] In some implementations, 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 longer after activation of the first genetic circuit.

[0152] As disclosed herein, one or more target genes may include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0153] One or more target genes disclosed herein may include cell differentiation regulators, molecular function regulators, binding factors, membrane fusion (fusogenic) factors, protein folding chaperones, protein tags, RNA folding chaperones, cell signaling factors, immune response factors, sensory receptors, cell structural factors, protein binding factors, cargo receptors, catalytic factors, or small molecule sensors.

[0154] One or more target genes disclosed herein may include cell differentiation regulators, including growth factors, transcription factors, myogenic regulators, immune cell regulators, neuronal regulators, stem cell differentiation factors, endocrine regulators, β-cell regulators, pancreatic lineage regulators, chondrogenic regulators, osteogenic regulators, aging factors, stemness factors (e.g., dedifferentiation factors), etc.

[0155] In some cases, 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.

[0156] In some cases, one or more target genes may include homeobox genes. Homeobox genes are genes that regulate, for example, a wide range of anatomical features during 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-restrictive examples of homeobox genes may include HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXB13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13, HOXD1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA ...A9, HOXA10, HOXA11, HOXC12, HOXC13, HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA12, HOXC13, HOXA1, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA12, HOXC13, HOXA1, HOXA3, HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA OXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13, CDX1, CDX2, CDX4, GSX1, GSX2, PDX1, EVX1, EVX2, GBX1, GBX2, MEOX 1. MEOX2, MNX1, DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, IRX1, IRX2, IRX3, IRX4, IRX5, IRX6, MEIS1, MEIS2, MEIS3, MKX, PBX1, PBX2, P BX3, PBX4, PKNOX1, PKNOX2, TGIF1, TGIF2, TGIF2LX, TGIF2LY, ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1 A. LMX1B, HDX, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2, POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1, POU5F1P1, P OU5F1P4, POU5F2, POU6F1, POU6F2, LASS2, LASS3, LASS4, LASS5, LASS6, HMBOX1, HNF1A, HNF1B, SIX1, SIX2, SIX3, SIX4, SIX5, SI X6,ONECUT1,ONECUT2,ONECUT3,CUX1,CUX2,SATB1,SATB2,ADNP,ADNP2,TSHZ1,TSHZ2,TSHZ3,ZEB1,ZEB2,ZFHX2,ZFHX3,ZFHX4,ZHX1, HOMEZ, ALX1(CART1), 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, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, 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, NANOG, NOTO, TLX1, TLX2, T LX3, 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. ,

[0157] In some cases, target genes may include CDX1. CDX1 is a pahahox class homeobox. CDX1 is a protein expressed in the developing endoderm, and its expression persists in the gut throughout adulthood.

[0158] In some cases, target genes may include CDX2. CDX2 is a pahahox class homeobox. CDX2 is a transcription factor expressed in the nucleus of intestinal epithelial cells and plays a crucial role in the development and function of the digestive system.

[0159] In some cases, target genes may include CDX4. CDX4 is a pahahox homeobox. CDX4 is a transcription factor that participates in embryonic tissue formation, anterior-posterior patterning, and hematopoiesis during embryogenesis.

[0160] In some cases, the target gene may include LMX1A. LMX1A is a protein that binds to an A / T-rich sequence in the insulin promoter and stimulates insulin transcription.

[0161] In some cases, target genes may include LMX1B. LMX1B is a transcription factor that plays a central role in dorsoventral patterning of vertebrate limbs.

[0162] In some cases, target genes may include GSX1. GSX1 is a pahahox homeobox. GSX1 is a transcription factor that plays a role in pituitary development.

[0163] In some cases, target genes may include GSX2. GSX2 is a pahahox homeobox. GSX2 is a transcription factor that plays a role in brain development.

[0164] In some cases, target genes may include PDX1. PDX1 is a pahahox class homeobox. PDX1 is essential for pancreatic development, including β-cell maturation and duodenal differentiation.

[0165] In some cases, target genes may include NKX6.1 (also known as NKX6-1). NKX6.1 is an NKX homeobox. NKX6.1 is a bifunctional transcriptional regulator required for β-cell development.

[0166] In some cases, target genes may include NKX2.2 (also known as NKX2-2). NKX2.2 is an NKX homeobox. NKX2.2 is a transcription factor involved in morphogenesis in the central nervous system.

[0167] In some cases, target genes may include PAX6. PAX6 is a PRD homeobox. PAX6 is a transcription factor and is important in the development of the eye and other sensory organs, certain neural and epidermal tissues, and other structures derived from ectoderm tissues.

[0168] In some cases, target genes may include ONECUT1. ONECUT1 is a CUT-like homeobox. ONECUT1 is a transcription factor abundant in the liver, where it stimulates the transcription of hepatic genes.

[0169] In some cases, target genes may include ISL1. ISL1 is a LIM class homeobox. ISL1 is a transcription factor that plays an important role in the formation of pancreatic islets.

[0170] 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-restrictive examples of TBX transcription factors may include TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, TBX22, and TBXT (Brachyury protein).

[0171] In some cases, target genes may include TBXT. TBXT, also known as T-box transcription factor T or brachyyury protein, functions as a transcription factor within the T-box gene family. TBXT plays a role in determining the midline of bilateral organisms, contributing to the establishment of the anterior-posterior axis. It can also assist in determining the mesoderm during gastrulation.

[0172] In some cases, one or more target genes may include basic helical-loop-helical transcription factors (bHLH genes). bHLH transcription factors are involved in the regulation of the cell cycle and many other developmental processes. bHLH proteins have a basic helical-loop-helical protein structure. Non-restrictive examples of bHLH transcription factors may 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, MX D3, MXD4, MXI1, MYC, MYCL1, MYCL2, MYCN, MYF5, MYF6, MYOD1, MYOG, NCOA1, NCOA3, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, NHLH1, NHLH2, NPAS1, NPAS2, NPAS3, NPAS4, OAF 1. OLIG1, OLIG2, OLIG3, PTF1A, SCL, SCXB, SIM1, SIM2, SOHLH1, SOHLH2, SREBF1, SREBF2, TAL1, TAL 2. TCF12, TCF15, TCF21, TCF3, TCF4, TCFL5, TFAP4, TFE3, TFEB, TFEC, TWIST1, TWIST2, USF1 and USF2.

[0173] In some cases, target genes may include NEUROG3. NEUROG3, or neuro-element 3, is an endocrine transcription factor that activates gene transcription in endocrine progenitor cells. NEUROG3 is important for pancreatic islet differentiation and regeneration, and its function is to directly enhance the expression of lineage-defining transcription factors, which are required for endocrine progenitor cells to differentiate into each endocrine cell subtype.

[0174] In some cases, target genes may include NEUROD1. NEUROD1 is a transcription factor that regulates insulin gene expression.

[0175] In some cases, target genes may include PTF1a. PTF1a is a transcription factor that plays a role in pancreatic development.

[0176] In some cases, one or more target genes may include SRY-associated box transcription factors (SOX genes). SOX transcription factors are involved in developmental regulation. Non-restrictive examples of SOX transcription factors may include SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21, SOX30, and SRY.

[0177] In some cases, one or more target genes may include SOX A group, which includes SRY. In some cases, one or more target genes may include SOX B1 group, which includes SOX1, SOX2, and / or SOX3. In some cases, one or more target genes may include SOX B2 group, which includes SOX14 and / or SOX21. In some cases, one or more target genes may include SOX C group, which includes SOX4, SOX11, and / or SOX12. In some cases, one or more target genes may include SOX D group, which includes SOX5, SOX6, and / or SOX13. In some cases, one or more target genes may include SOX E group, which includes SOX8, SOX9, and / or SOX10. In some cases, one or more target genes may include SOX F group, which includes SOX7, SOX17, and / or SOX18. In some cases, one or more target genes may include SOX G group, which includes SOX15. In some cases, one or more target genes may include SOX H group, which includes SOX30.

[0178] In some cases, target genes may include SOX17. SOX17 is a transcription factor that is involved in the regulation of vertebrate embryonic development and the determination of endoderm cell fate.

[0179] In some cases, target genes may include SOX9. SOX9 is a transcription factor that plays a role in cell differentiation and regulates the transcription of the anti-Müllerian hormone (AMH) gene.

[0180] In some cases, one or more target genes may include forkhead boxes (FOX). FOX is a transcription factor that regulates the expression of genes involved in cell growth, proliferation, differentiation, and lifespan. Some FOX genes can bind to chromatin during cell differentiation. Non-restrictive 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.

[0181] In some cases, target genes may include FOXA1. FOXA1 is a transcriptional activator of liver-specific transcripts.

[0182] In some cases, target genes may include FOXA2. FOXA2 is a transcription factor that plays an important role in development, in mature tissues, and in cancer when dysregulated or mutated.

[0183] In some cases, target genes may include FOXA3. FOXA3 is a transcriptional activator of liver-specific transcripts.

[0184] In some cases, one or more target genes may include erythroblast transformation-specific (ETS) genes. ETS are animal-specific transcription factors and are involved in tissue development. Non-restrictive examples of ETS genes may 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).

[0185] In some cases, target genes may include ERG. ERG is a transcriptional regulator that also functions as an oncogene.

[0186] In some cases, target genes may include FLI1. FLI1 is a transcription factor, a proto-oncogene, and is involved in proliferation and terminal differentiation.

[0187] In some cases, target genes may include FEVs. FEVs are transcriptional repressors.

[0188] 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.

[0189] In some cases, target genes may include GATA4. GATA4 is a zinc finger transcription factor that regulates genes involved in embryogenesis and cardiac muscle differentiation and function.

[0190] In some cases, one or more target genes may include the MAF transcription factor. MAF is an oncogene involved in immune cell differentiation.

[0191] In some cases, the use of heterologous gene circuits as disclosed herein can be used to differentiate endoderm 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%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the resulting cells produced by using heterologous gene circuits as disclosed herein are of the target cell type.

[0192] In some cases, the heterologous gene circuits disclosed herein can be used, for example, to differentiate endoderm stem cells into pancreatic lineage cells in the absence of one, two, or all of feeder cells, serum, and exogenous growth factors. Using heterologous gene circuits disclosed herein can reduce the number of cells from up to approximately 1 x 10⁻⁶. 6 One, at most about 9x10 5 One, at most about 8x10 5 One, at most about 7x10 5 One, at most about 6x10 5 One, at most about 5x10 5 One, at most about 4x10 5 One, at most about 3x10 5 One, at most about 2x10 5 One, at most about 1x10 5 One, at most about 5x10 4 One, at most about 2x10 4 One, at most about 1x10 4 One or more pancreatic lineage cells produce at least approximately 1 x 102 4 One, at least about 2 x 10 4 One, at least about 5 x 10 4 One, at least about 1x10 5 One, at least about 2 x 10 5One, at least about 5 x 10 5 One, at least about 1x10 6 One, at least about 2 x 10 6 One, at least about 5 x 10 6 One, at least about 1x10 7 One, at least about 2 x 10 7 One, at least about 5 x 10 7 One, at least about 1x10 8 One, at least about 2 x 10 8 One, at least about 5 x 10 8 One, at least about 1x10 9 One, at least about 2 x 10 9 One, at least about 5 x 10 9 One, at least about 1x10 10 One, at least about 2 x 10 10 One, at least about 5 x 10 10 One, at least about 1x10 15 One, at least about 2 x 10 15 One, at least about 5 x 10 15 One or more β cells.

[0193] In some cases, the heterologous gene circuits disclosed herein can be used, for example, to differentiate pluripotent stem cells (PSCs, such as induced PSCs or iPSCs) into β cells in the absence of one, two, or all of feeder cells, serum, and exogenous growth factors. Using heterologous gene circuits disclosed herein can differentiate up to approximately 1 x 102 6 One, at most about 9x10 5 One, at most about 8x10 5 One, at most about 7x10 5 One, at most about 6x10 5 One, at most about 5x10 5 One, at most about 4x10 5 One, at most about 3x10 5 One, at most about 2x10 5 One, at most about 1x10 5 One, at most about 5x10 4 One, at most about 2x10 4 One, at most about 1x10 4 One or more pancreatic lineage cells produce at least approximately 1 x 102 4 One, at least about 2 x 10 4 One, at least about 5 x 10 4 One, at least about 1x10 5 One, at least about 2 x 10 5 One, at least about 5 x 10 5 One, at least about 1x106 One, at least about 2 x 10 6 One, at least about 5 x 10 6 One, at least about 1x10 7 One, at least about 2 x 10 7 One, at least about 5 x 10 7 One, at least about 1x10 8 One, at least about 2 x 10 8 One, at least about 5 x 10 8 One, at least about 1x10 9 One, at least about 2 x 10 9 One, at least about 5 x 10 9 One, at least about 1x10 10 One, at least about 2 x 10 10 One, at least about 5 x 10 10 One, at least about 1x10 15 One, at least about 2 x 10 15 One, at least about 5 x 10 15 One or more β cells.

[0194] Such β-cell production, achieved through the use of heterologous gene circuits as disclosed herein, can be achieved within a time span of up to approximately 60 days, up to approximately 55 days, up to approximately 50 days, up to approximately 45 days, up to approximately 40 days, up to approximately 35 days, up to approximately 30 days, up to approximately 25 days, up to approximately 20 days, up to approximately 15 days, up to approximately 10 days, up to approximately 7 days, up to approximately 6 days, up to approximately 5 days, up to approximately 4 days, up to approximately 3 days, up to approximately 2 days, up to approximately 1 day, or less.

[0195] In some cases, β cells produced by this method produce more insulin than β cells obtained through directed differentiation. β cells produced using the provided method can 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 β cells obtained through directed differentiation. Alternatively or additionally, β cells produced by this method can produce an equivalent amount of insulin compared to β cells obtained through directed differentiation.

[0196] In some cases, β 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.

[0197] In some cases, β cells or pancreatic lineage cells produced by this method exhibit 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%, and so on, compared to control pancreatic lineage cells. At least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 2,000%, at least 3,000%, at least 4,000%, at least 5,000%, at least 6,000%, at least 7,000%, at least 8,000%, at least 9,000%, at least 10,000%, at least 100,000%, or at least 1,000,000% of the expression level of positive pancreatic lineage cell markers.

[0198] In some cases, β cells or pancreatic lineage cells produced by this method showed at least or more than about 0.1 times, at least or more than about 0.2 times, at least or more than about 0.3 times, at least or more than about 0.4 times, at least or more than about 0.5 times, at least or more than about 0.6 times, at least or more than about 0.7 times, at least or more than about 0.8 times, at least or more than about 0.9 times, at least or more than about 1 time, at least or more than about 2 times, at least or more than about 3 times, at least or more than about 4 times, at least or more than about 5 times, at least or more than about 6 times, at least The expression levels of positive pancreatic lineage cell markers were at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 500 times, at least 1,000 times, at least 5,000 times, or at least 10,000 times.

[0199] In some cases, β 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 may include CD49.

[0200] In some cases, β cells or pancreatic lineage cells produced by this method showed a reduction of at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, and so on, compared to control pancreatic lineage cells. At least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 2,000%, at least 3,000%, at least 4,000%, at least 5,000%, at least 6,000%, at least 7,000%, at least 8,000%, at least 9,000%, at least 10,000%, at least 100,000%, or at least 1,000,000% of the expression level of negative pancreatic lineage cell markers.

[0201] In some cases, β cells or pancreatic lineage cells produced by this method showed a level at least or more than about 0.1, at least or more than about 0.2, at least or more than about 0.3, at least or more than about 0.4, at least or more than about 0.5, at least or more than about 0.6, at least or more than about 0.7, at least or more than about 0.8, at least or more than about 0.9, at least or more than about 1, at least or more than about 2, at least or more than about 3, at least or more than about 4, at least or more than about 5, at least or more than about 6, at least The expression levels of negative pancreatic lineage cell markers were at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 500 times, at least 1,000 times, at least 5,000 times, or at least 10,000 times.

[0202] 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, RNA blotting, protein staining, mRNA staining, and RNA sequencing.

[0203] In some cases, expression levels can be measured at least approximately 12 hours, at least approximately 13 hours, at least approximately 14 hours, at least approximately 15 hours, at least approximately 16 hours, at least approximately 17 hours, at least approximately 18 hours, at least approximately 19 hours, at least approximately 20 hours, at least approximately 21 hours, at least approximately 22 hours, at least approximately 23 hours, at least approximately 24 hours, at least approximately 28 hours, at least approximately 32 hours, at least approximately 36 hours, at least approximately 40 hours, at least approximately 44 hours, at least approximately 48 hours, at least approximately 3 days, at least approximately 4 days, at least approximately 5 days, at least approximately 6 days, at least approximately 7 days, at least approximately 8 days, at least approximately 9 days, at least approximately 10 days, at least approximately 11 days, at least approximately 12 days, at least approximately 13 days, at least approximately 14 days, or more days after the introduction of the gene circuit.

[0204] In some cases, the β cells or pancreatic lineage cells produced by this method may be immature β cells. Alternatively, the β cells or pancreatic lineage cells produced by this method may be mature β cells. In some cases, the progenitor cells produced may be substantially mitotically dormant. Alternatively, the progenitor cells produced may be substantially mitotically active.

[0205] In some cases, the first gate unit can be configured to reduce the expression and / or activity levels of one or more target genes. In some cases, the first gate unit can be configured to enhance the expression and / or activity levels of one or more target genes. In some cases, the first gate unit can be configured to maintain the expression and / or activity levels of one or more target genes.

[0206] In some cases, the regulation of the first target gene can occur before the regulation of the second target gene. In other cases, the regulation of the first target gene can occur after the regulation of the second target gene. In still other cases, the regulation of the first target gene can occur approximately simultaneously with the regulation of the second target gene.

[0207] In some cases, regulation of a target gene may include increasing the expression level of a second target gene. Alternatively, regulation of a target gene may include decreasing the expression level of a second target gene. Alternatively, regulation of a target gene may include maintaining the expression level of a second target gene.

[0208] In some cases, the use of heterologous gene circuits can induce cell differentiation into the cell type of interest in the absence of growth factors, serum (fetal bovine serum, human serum AB, etc.), or other exogenous cell differentiation regulators or culture media. Serum may contain the liquid portion of blood clots, including essential nutrients and macromolecules for cell growth.

[0209] Alternatively, the use of heterologous gene circuits can induce cell differentiation into the cell type of interest using reduced amounts of serum and / or growth factors (e.g., reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially no serum). Reduced serum amounts can allow for more consistent cell distribution across experiments or batches, increase the growth and / or productivity of differentiated cells, better control over physiological responsiveness, and reduce the risk of contamination by serum-born agents in cell culture.

[0210] In some cases, the use of heterologous gene circuits in stem cells (e.g., iSPCs, endoderm stem cells) can induce the differentiation of endoderm stem cells into pancreatic lineage cells (e.g., β cells) in the absence of growth factors, serum (fetal bovine serum, human serum AB, etc.) or other exogenous cell differentiation regulators or culture media. In some cases, the use of heterologous gene circuits as 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 produced by using heterologous gene circuits as disclosed herein constitute at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population.

[0211] In some cases, using a heterologous gene circuit to convert one cell type (e.g., PSC, endoderm stem cells, or pancreatic lineage cells) into another cell type (e.g., β cells) can produce a target cell type. Alternatively, using a heterologous gene circuit to convert one cell type (e.g., PSC, endoderm stem cells, or pancreatic lineage cells) into another cell type (e.g., β cells) can produce an intermediate cell type. The intermediate cell type can then undergo a second conversion using a second gene circuit to produce the target cell type.

[0212] The transformation of cells from one cell type to another can involve the regulation of multiple target genes. For example, transformation can involve 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. Transformation can involve 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 regulation. 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 regulation. One or more regulation of a target gene (e.g., an endogenous gene) induced by the heterologous gene circuit of this disclosure may be artificial regulation (or heterologous regulation), which may additionally not occur in the cell in the absence of (i) the heterologous gene circuit and / or (ii) the activation portion of the heterologous gene circuit.

[0213] like Figure 3 As demonstrated in the study, various heterologous gene circuits can be designed to regulate the expression or activity levels of multiple genes (e.g., multiple endogenous genes) in cells at multiple different time points.

[0214] 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. Alternatively, 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 depress the expression level of a second gene. Alternatively, a heterologous gene circuit can be designed to (i) depress 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) depress the expression level of a first gene and (ii) subsequently depress 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 another gene. The heterologous gene circuit can be designed to include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more additional genes. These additional genes can be activated. Alternatively, the number of additional genes can be reduced. The heterologous gene circuit can be designed to regulate the expression of the additional genes before regulating the expression levels of both the first and second genes. Alternatively or additionally, the heterologous gene circuit can be designed to regulate the 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 additionally, the heterologous gene circuit can be designed to regulate the expression of the additional genes after regulating the expression levels of both the first and second genes.

[0215] Heterogeneous gene circuits can be designed to include FOX, SOX, and / or GATA as the first member and bHLH, a homeobox, and / or MAF as the second member. Heterogeneous gene circuits can be designed to include FOX and bHLH. Heterogeneous gene circuits can be designed to include FOX and a homeobox. Heterogeneous gene circuits can be designed to include FOX and MAF. Heterogeneous gene circuits can be designed to include SOX and bHLH. Heterogeneous gene circuits can be designed to include SOX and a homeobox. Heterogeneous gene circuits can be designed to include SOX and MAF. Heterogeneous gene circuits can be designed to include GATA and bHLH. Heterogeneous gene circuits can be designed to include GATA and a homeobox. Heterogeneous gene circuits can be designed to include GATA and MAF.

[0216] The heterologous gene circuits in activated cells disclosed herein can regulate the expression or activity levels of multiple genes at multiple different time points to achieve cell transformation into different cell types (e.g., transformation of stem cells into tissue-specific progenitor cells). The transformation rate of such cell types using heterologous gene circuits can be at least or more than about 1%, at least or more than about 2%, at least or more than about 5%, at least or more than about 10%, at least or more than about 15%, at least or more than about 20%, at least or more than about 25%, at least or more than about 30%, at least or more than about 35%, at least or more than about 40%, at least or more than about 45%, at least or more than about 50%, at least or more than about 60%, at least or more than about 70%, at least or more than about 80%, at least or more than about 90%, or at least or more than about 95%.

[0217] As disclosed in this paper, activating heterologous gene circuits in cells can regulate the expression or activity levels of multiple genes at multiple different time points to achieve cell transformation into different cell types (e.g., the transformation of stem cells into tissue-specific progenitor cells). This transformation can occur in less than approximately 20 days, less than approximately 19 days, less than approximately 18 days, less than approximately 17 days, less than approximately 16 days, less than approximately 15 days, less than approximately 14 days, less than approximately 13 days, less than approximately 12 days, less than approximately 11 days, less than approximately 10 days, less than approximately 9 days, less than approximately 8 days, less than approximately 7 days, less than approximately 6 days, less than approximately 5 days, less than approximately 4 days, less than approximately 3 days, less than approximately 2 days, or less than approximately 1 day.

[0218] Cells (e.g., initial cells to be modified into engineered cells as disclosed herein, final cell products derived from engineered cells as disclosed herein) may include muscle cells, immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells, epithelial cells, stem cells, secretory cells, blood cells, germ cells, nurturing cells, storage cells, enteroendocrine cells, pituitary cells, mesodermal cells, endoderm cells, pancreatic lineage cells, neurosecretory cells, ductal cells, odontoblasts, cementoblasts, glial cells, or mesenchymal cells.

[0219] Non-limiting examples of such cells may include lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, cytokine-induced killer (CIK) cells (see, for example, US20080241194); myeloid cells such as granulocytes (basophils, eosinophils, neutrophils / segmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, coagulating cells / megakaryocytes, dendritic cells; cells from the endocrine system, including thyroid cells (thyroid epithelial cells, parafollicular cells), parathyroid cells (parathyroid chief cells, eosinophils), adrenal cells (chromaffin cells), pineal cells (pinealocytes); and cells from the nervous system, including glial cells (astrocytes, microglia), large neurosecreting cells, astrocytes, and Boettcher cells.Cells of the respiratory system, including lung cells (type I and type II lung cells), Clara cells, goblet cells, and dust cells; cells of the circulatory system, including cardiomyocytes and pericytes; cells of the digestive system, including gastric cells (chief cells and peripheral cells), goblet cells, Panette cells, G cells, D cells, ECL cells, I cells, K cells, and S cells; and intestinal cells. Endocrine cells, including enterochromaffin cells, APUD cells, hepatocytes (hepatocytes, Kupffer cells), cartilage / bone / muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, and dental cells (cementoblasts, ameloblasts); adaxial mesodermal cells and chondrocytes, including osteoblasts, chondrocytes, and chondrocytes; skin cells, including hair cells, keratinocytes, and melanocytes (nevus cells); muscle cells, including myocytes; and urinary system cells, including podocytes, juxtaglobinocytes, and intraglobinic mesangial cells. Cells / extraglomerular mesangial cells, brush border cells of the proximal renal tubules, dense maculocytes; reproductive system cells, including sperm, Sartori cells, testicular interstitial cells, oocytes; and other cells, including adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of the nails and toenails, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, keratinized hair stem cells, keratinized root sheath cells, root sheath cells of Huxley's stratum, Henle's stratum The text appears to be a list of cells and related terms, possibly related to epithelial cells, basal cells, and urethral epithelial cells. A direct translation wouldn't be meaningful without further context or clarification.Ebner's cells include: Nabothian gland cells (for washing taste buds), mammary gland cells (for milk secretion), lacrimal gland cells (for tear secretion), ceruminous gland cells in the ear (for wax secretion), dark cells of eccrine sweat glands (for glycoprotein secretion), and light cells of eccrine sweat glands (for small molecule secretion). Apocrine sweat gland cells (odor secretion, sex hormone sensitivity), eyelash gland cells in the eyelids (dedicated sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Baumann's gland cells in the nose (washing olfactory epithelium), Brenner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting semen components, including fructose from motile sperm), prostate cells (secreting semen components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (secreting vaginal lubricant), Litrex's gland cells (mucus secretion), endometrial cells (carbohydrate secretion), separate goblet cells of the respiratory and digestive tracts (mucus secretion), gastric lining mucus cells (mucus secretion), gastric gland zymogen cells (pepsinogen secretion), gastric gland acid-secreting cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), Panthen's cells of the small intestine (lysozyme secretion), type II lung cells of the lungs (surface... (Active agent secretion), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, somatic cells, prolactin cells, thyroid-stimulating hormone cells, gonadotropin cells, adrenocorticotropic hormone cells, intermediate pituitary cells, large neurosecreting cells, intestinal and respiratory tract cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophilic cells, adrenal cells, chromaffin cells, testicular interstitial cells, follicular endometrial cells, luteal cells of ruptured follicles, granulosa luteal cells, membranous luteal cells, juxtaglomerular cells (renin secretion), dendritic cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands and urogenital tract), kidney cells, immature β cells, mature β cells, type I lung cells (lung lining air space) air space), pancreatic duct cells (vacuole cells), streaked duct cells (of sweat glands, salivary glands, mammary glands, etc.), duct cells (of seminal vesicles, prostate glands, etc.), epithelial cells lining closed internal body cavities, propulsive ciliated cells, extracellular matrix secretory cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells)Cells, megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in the central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and progenitor cells of the blood and immune system (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells. Cells), autonomic neurons, sensory organ and peripheral neurons supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oocytes (Oogonium / Oocyte), sperm cells, spermatocytes, spermatogonia (sperm stem cells of spermatocytes), sperm, nurse cells, ovarian follicle cells, Setolly cells (in the testes), thymic epithelial cells, interstitial cells and interstitial kidney cells.

[0220] In one aspect, this disclosure provides systems and methods for converting multiple pluripotent stem cells (PSCs) into multiple tissue-specific progenitor cells.

[0221] Pluripotent stem cells can include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Tissue-specific progenitor cells can include endoderm stem cells, hematopoietic stem cells (HSCs), myeloid progenitor cells, muscle stem cells, pancreatic lineage cells, neural stem cells, epithelial stem cells, epidermal stem cells, breast stem cells, intestinal stem cells, neural crest stem cells, or testicular stem cells.

[0222] 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., from the bone marrow of a subject). Alternatively, in some cases, the tissue-specific progenitor cells or tissue-specific cells generated as disclosed herein may not be derived from isolated MSCs.

[0223] In some cases, the conversion of stem cells into tissue-specific progenitor cells has been characterized by the production of fewer non-endodermal cells or fewer enterochromaffin cells compared to control conversions in a culture medium containing (i) serum and / or (ii) exogenous cell differentiation regulators. 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, bone, dermis, connective tissue, or mesothelial cells. Ectodermal cells can differentiate into ectodermal organs such as the brain, spinal cord, adrenal medulla, epidermis, hair / nail / skin glands, sensory organs, peripheral nerves, or lens.

[0224] In some cases, the transformation of stem cells into tissue-specific progenitors is characterized by the production of up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, or less of non-endoderm cells. In some cases, the transformation of stem cells into tissue-specific progenitors is characterized by the production of up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, or less of enterochromaffin cells. In some cases, regulation of genes such as SOX2, CDX2, LMX1a, and FEV can be used to repress the fate of non-endoderm and / or enterochromaffin cells.

[0225] Various aspects of this disclosure provide engineered cells programmed to induce desired levels (or profiles) of expression of one or more target genes in cells.

[0226] In some embodiments, the engineered cells of this disclosure (e.g., engineered β cells) can be generated from isolated stem cells (e.g., isolated endoderm stem cells or iPSCs). The heterologous gene circuits and / or components thereof (e.g., phylogenetic units, phylogenetic portions, activation portions, etc.) disclosed herein can be introduced during any stage (or cell state) between and including: (a) isolated stem cells, and (b) their differentiated β cell state (e.g., terminally differentiated β cell state).

[0227] The engineered cells disclosed herein (e.g., engineered β cells) can be used (e.g., administered) to treat subjects in need. Subjects may 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 in vitro and genetically modified to produce engineered cells (e.g., β cells) of any subject matter disclosed herein. Subsequently, engineered immune cells can be administered to the subject for adaptive immunotherapy. Thus, the engineered cells can be autologous for the subject in need. Alternatively, the engineered cells can be allogeneic for the subject (e.g., allogeneic stem cell transplantation, allogeneic adoptive immunotherapy, etc.).

[0228] As disclosed herein, engineered cells can be administered to a subject before, simultaneously with, or after the activation of heterologous genetic circuits in engineered stem cells. For example, engineered cells can be activated after administration to a subject, for instance, by administering an activator of a heterologous genetic circuit to the subject.

[0229] Subjects may be treated (e.g., administered) with the engineered cell (e.g., engineered myocyte) population disclosed herein for at least or more than about 1 dose, at least or more than about 2 doses, at least or more than about 3 doses, at least or more than about 4 doses, at least or more than about 5 doses, at least or more than about 6 doses, at least or more than about 7 doses, at least or more than about 8 doses, at least or more than about 9 doses, or at least or more than about 10 doses. Alternatively or additionally, the subject may be treated (e.g., administered) with the engineered cell (e.g., engineered T cell) population disclosed herein 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.

[0230] The target cells, target tissues, target conditions, or target diseases of the subject can be treated using any of the methods disclosed herein.

[0231] The target disease of the subject can be a disease that affects the pancreas. Diseases affecting the pancreas can include, but are not limited to, pancreatitis, cholangitis, cholecystitis, diabetes, gallstones, and pancreatic cancer.

[0232] The target disease of the subject can be a disease that affects insulin production. Diseases that affect insulin production can include, but are not limited to, diabetes, hypertension, dyslipidemia, and cardiovascular disease.

[0233] The target disease of the subject can be cancer or tumor. Non-limiting examples of cancer may include cancerous cells, including acanthoma, acinar carcinoma, acoustic neuroma, acral lentigines-like melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloid leukemia with maturation, acute myeloid dendritic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, amelomas, adenocarcinomas, adenoid cystic carcinomas, adenomas, odontogenic adenomatoid tumors, and kidneys. Adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, undifferentiated thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdomyosarcoma, basal cell carcinoma, basoid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini ductal carcinoma. Carcinoma, biliary tract cancer, bladder cancer, germ cell tumor, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown origin, carcinoid tumor, carcinoma, carcinoma in situ, penile carcinoma, carcinoma of unknown origin, carcinosarcoma, Castleman's diseaseDiseases, embryonic tumors of the central nervous system, cerebellar astrocytoma, brain astrocytoma, cervical cancer, chondroma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease. Disease), dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, olfactory neuroblastoma, Ewing family tumor, Ewing family sarcoma, Ewing's sarcoma, extracranial Germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct carcinoma, extramammary Paget's disease, fallopian tube cancer, fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid carcinoma, gallbladder cancer, ganglioglioma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma Tumors, gestational trophoblastic tumors, giant cell tumors of bone, glioblastoma multiforme, gliomas, gliomatosis, glomus tumors, glucagonomas, gonadoblastomas, granulosa cell tumors, hairy cell leukemia, head and neck cancer, heart cancer, hemangioblastomas, hemangiopericytomas, angiosarcomas, hematologic malignancies, hepatocellular carcinomas, hepatocellular T-cell lymphomas, hereditary breast cancer and ovarian cancer syndromes, Hodgkin's lymphoma. Lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, renal cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, malignant lentigines melanoma, leukemia, lip and oral cancer, liposarcoma, lung cancer, corpus luteum tumor, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoid leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, bone malignant fibrous histiocytoma, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdomyosarcoma, malignant salamander tumorTumor), MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, occult primary metastatic squamous neck cancer, metastatic urothelial carcinoma, mixed Müllerian tumor. Tumor), monocytic leukemia, oral cancer, myxoma, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorders, myxoma, nasal cavity carcinoma, nasopharyngeal carcinoma, nasopharyngeal carcinoma, vegetation, neurofibroma, neuroblastoma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular tumors. Oncology, oligodendroastrocytoma, oligodendroglioma, eosinophilic cytoma, optic nerve sheath meningioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, low-grade ovarian potential tumor, Paget's disease of the breast, superior sulcus tumor of the lung, pancreatic cancer, papillary thyroid carcinoma, papilloma, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor. Differentiation), pineal blastoma, pituitary adenoma, pituitary tumor, plasma cell tumor, pleural pulmonary blastoma, polyembryoma, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, peritoneal pseudomyxoma, rectal cancer, renal cell carcinoma, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland carcinoma, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary vegetations, seminoma, serous tumor, Sertoli-Leydig cell tumor.Cell tumors, sex cord-stromal tumors, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestinal cancer, soft tissue sarcoma, somatostatinoma, sootoma. Spinal cord tumors, spinal tumors, marginal zone lymphoma of the spleen, squamous cell carcinoma, gastric cancer, superficial diffuse melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphoblastic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, advanced lymphoma, testicular cancer, theca cell tumor, laryngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, urogenital neoplasm, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia. Macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation within a cancer cell population, such as cancer stem cells. In some embodiments, the cancer is a hematopoietic lineage cancer, such as lymphoma. The antigen may be a tumor-associated antigen.

[0234] Non-limiting examples of target tissues may include cells that can be obtained from the object, such as β cells. Non-limiting examples of objects include humans, dogs, cats, mice, rats, and their transgenic species. Examples of samples from objects from which cells may originate include, but are not limited to, skin, heart, lungs, kidneys, bone marrow, mammary glands, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestines, brain, prostate, esophagus, thyroid gland, serum, saliva, urine, gastric juice and digestive juices, tears, feces, semen, vaginal fluid, interstitial fluid derived from tumor tissue, eye discharge, sweat, mucus, earwax, oil, glandular secretions, cerebrospinal fluid, hair, nails, plasma, nasal swabs or nasopharyngeal washes, cerebrospinal fluid, cerebrospinal fluid, tissue, pharyngeal swabs, biopsy, amniotic fluid, amniotic fluid, umbilical cord blood, emphatic fluids, cavity fluid, sputum, pus, microbiota, meconium, breast milk, and / or other excretions or body tissues.

[0235] This disclosure also provides compositions comprising engineered gene circuits as disclosed herein. The composition may further comprise an actuator for a heterologous gene circuit. This disclosure also provides a kit comprising the composition. The kit may further comprise an activator of the heterologous gene circuit. The activator may be in the same composition as the engineered cells. Alternatively or additionally, the activator may be in a different and separate composition from the engineered cells.

[0236] In some cases, compared with control progenitor cells, the engineered progenitor cells disclosed herein may exhibit (i) comparable or enhanced regenerative capacity; (ii) comparable or enhanced in vitro expression; (iii) comparable or enhanced gene editing capacity; (iv) comparable or enhanced immune tolerance; (v) comparable or shorter manufacturing timeline; (vi) comparable or fewer growth factors or culture requirements; and / or (vii) comparable or enhanced safety.

[0237] Control progenitor cells can be generated by any method, including expanding progenitor cells isolated from tissues (e.g., pancreatic lineage cells), directing iPSC differentiation (e.g., using exogenous growth factors), and / or transgenic iPSC differentiation (e.g., viral transduction of heterologous genes).

[0238] In some cases, tissue-specific progenitor cells can be stored in containers (e.g., sterile vials). In some cases, tissue-specific progenitor cells are stored at temperatures up to approximately 10°C, up to approximately 5°C, up to approximately 4°C, up to approximately 0°C, up to approximately -5°C, up to approximately -10°C, up to approximately -20°C, up to approximately -30°C, up to approximately -40°C, up to approximately -50°C, up to approximately -60°C, up to approximately -70°C, up to approximately -80°C, up to approximately -90°C, up to approximately -100°C, up to approximately -110°C, up to approximately -120°C, up to approximately -130°C, up to approximately -140°C, up to approximately -150°C, up to approximately -160°C, up to approximately -170°C, up to approximately -180°C, up to approximately -190°C, up to approximately -200°C, or lower.

[0239] Pharmaceutical Composition

[0240] In some cases, the methods disclosed herein involve administering at least one tissue-specific progenitor cell to a subject in need. The subject may be an animal. The subject may be a mammal (e.g., primates, horses, cats, dogs, cattle, pigs, sheep, goats, mice, rabbits, rats, guinea pigs). The subject may be a human subject.

[0241] The pharmaceutical compositions disclosed herein can be any pharmaceutical compound described herein combined with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical compositions facilitate the administration of the compound to a living organism. The pharmaceutical compositions can be administered as a therapeutically effective amount in various forms and routes, including, for example, intravenous, subcutaneous, intramuscular, inhalation, oral, parenteral, ocular, ocular, subcutaneous, transdermal, nasal, intravitreal, intratracheal, intrapulmonary, transmucosal, vaginal, and topical administration.

[0242] The formulation can be modified according to the chosen route of administration. Pharmaceutical compositions containing the compounds described herein can be manufactured, for example, by mixing, dissolving, emulsifying, encapsulating, embedding, or compressing processes.

[0243] Example

[0244] Example 1: Differentiation of pancreatic lineage cells

[0245] Tissue-specific cells (e.g., pancreatic lineage cells) can be generated from less differentiated cells (e.g., stem cells, such as iPSCs) using the systems and methods disclosed herein.

[0246] A. Production of pancreatic lineage cells

[0247] In this embodiment, a heterologous gene circuit is used to induce endoderm stem cells to differentiate into pancreatic lineage cells. The differentiation of stem cells (e.g., endoderm stem cells) into pancreatic lineage cells can be a complex process, requiring the activation of multiple endogenous genes at different time points and the deactivation of multiple endogenous genes at different time points. See also Figure 2 Examples of different endogenous genes induced for expression at different stages of stem cell differentiation into pancreatic lineage cells and then into β cells. Therefore, one or more heterologous gene circuits as disclosed herein can be used to automatically promote the regulation of such cascades 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 time points following single activation of such a heterologous gene circuit.

[0248] Endoderm stem cells were encoded as follows Figure 3Transient transfection with plasmid DNA of one of the heterologous gene circuits described herein, for example, these heterologous gene circuits target combinations of TBXT, FOXA2, SOX17, PDX1, GATA4, p53shRNA, 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 (endoderm progenitor cell markers), ECAD / CD142 double-positive cells (pancreatic progenitor cell markers), and CD49a-positive cells (immature β-cell markers), each of which indicates the formation of pancreatic lineage cells. Figure 6 In step 1, poorly performing HCGs, such as cell algorithms 1, 2, 3, 4, 5, 6, 7, 13, 14, 23, and 24, were found to share the same TBXT gene. Highly performing HCGs, such as cell algorithms 9 and 16, produced cultures with islet-like morphology within eight days. Figure 7 It was also found that top-performing HCGs, such as Cell Algorithm 11, converted at least 15% or more of the cells into insulin-positive cells within eight days. Figures 8A-8B ).

[0249] B. Characterization of pancreatic lineage cells generated by heterologous gene circuits

[0250] Transient plasmid delivery of heterologous gene circuits induced the appearance of pancreatic lineage cell markers four days later. Analysis revealed that... Figure 3 Several heterologous gene circuits provided in the study, such as heterologous gene circuits 9, 11, and 16 (i.e., cell algorithms 9, 11, and 16), generated at least approximately 15% conversion of endoderm stem cells into pancreatic lineage cells within four days. This conversion occurred much faster than conversion of pancreatic lineage cells through directed differentiation. Figure 9 ).

[0251] Figures 4A-4D and Figure 5 A volcano diagram depicting the results of cells grown under different conditions is shown. Figures 4A-4D small pictures and Figure 5 Various heterogeneous gene circuits (e.g., Cell Algorithm 15, Cell Algorithm 16, Cell Algorithm 18, etc.) have been shown to generate pancreatic lineage cells.

[0252] Example 2: β-cell function assay

[0253] In this predictive embodiment, iPSCs and endoderm stem cells were contacted with high-performing HGCs from Example 1 and allowed to grow in culture medium until β cells formed. A control β cell population was collected and purified from mouse samples. Insulin production from β cells of both the HGC and control populations was tested.

[0254] Example 3: Implantation and transplantation of pancreatic lineage cells

[0255] Tissue-specific cells (e.g., pancreatic lineage cells) prepared using the systems and methods of this disclosure can be administered to subjects in need (e.g., injected into pancreatic tissue) to treat pancreatic-related conditions (e.g., diabetes).

[0256] A. Production of pancreatic lineage cells

[0257] According to the method described in Example 1, stem cells (e.g., endoderm stem cells) can be used to encode at least one heterologous gene circuit (such as, for example, Figure 3 One or more heterologous genes (e.g., plasmid DNA) are transduced or transfected (e.g., transiently transfected) in order to generate pancreatic lineage cells.

[0258] B. In vivo administration of pancreatic lineage cells

[0259] After pancreatic lineage cells are generated using the systems and methods of this disclosure, the cells can be purified. The cells can be concentrated and resuspended in a buffer (e.g., PBS) and then administered to mice via direct injection into the pancreas or other sites of interest. After 8 to 10 weeks, the mice can be sacrificed, and tissue sections from the injection site can be prepared. Immunostaining of the sections against islet cells or insulin production can be performed to confirm the transplantation of ex vivo generated pancreatic lineage cells.

[0260] This article provides an alternative protocol for pancreatic lineage cell transplantation. Following 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 into tissue culture wells containing hydrogel (flat or patterned) or thin gel-coated plastic (flat or patterned) as a sparse culture (e.g., 24-well plates of 1000–2000 cells / well) and cultured with the medium changed every 3 days. On the day of transplantation, NOD / SCID mice can be anesthetized by intraperitoneal injection of ketamine (2.4 mg / mouse) and toluidine (240 g / mouse) and irradiated with their hind limbs as previously described (A. Sacco et al. (2008) Nature 456, 502). The generated pancreatic lineage cells can be counted and resuspended, and then injected into recipient mice.

[0261] Transplantation of pancreatic lineage cells (e.g., differentiation and integration into local pancreatic tissue) can be visualized using various methods. For example, pancreatic lineage cells can be engineered to express heterologous markers not present in the transplant animal (e.g., fluorescent proteins, such as green fluorescent protein). Alternatively or additionally, pancreatic lineage cells can be allogeneic to the animal, such that any insulin produced by the pancreatic lineage cells after transplantation can be identified by antigens not found in the transplant animal (e.g., immunostaining).

[0262] Example 4: In vitro generation of β cells

[0263] To improve the cell manufacturing process, cells of interest can be engineered to exhibit increased gene expression, thereby altering their phenotype to that of the pancreatic β-cell lineage. In some embodiments, improved cell manufacturing can be demonstrated by: shorter time to differentiate stem cells (e.g., pluripotent stem cells) into target cells (e.g., pancreatic progenitor cells or β-cells); increased number of target cells compared to other approaches; improved levels of insulin and / or C-peptide produced and / or secreted; lower cost of producing a similar number of target cells; and so on. Figure 9 This demonstrates some advantages of the system and method disclosed in this paper compared to directed differentiation. After transfecting the cellular algorithm nucleus into cells of interest, these genes can be expressed sequentially within the cells of interest.

[0264] The cells of interest could be induced pluripotent stem cells. Engineered cells could exhibit an increased presence of pancreatic β-cell lineage markers obtained by measuring RNA transcription, and could exhibit increased DNA accessibility and / or function in a glucose responsiveness assay that measures 1) insulin production, 2) insulin release, 3) C-peptide production, and / or 4) C-peptide release.

[0265] Example 5: Generation of cells in the pancreatic β-cell lineage

[0266] For example, a library of cell algorithms (including, but not limited to, the cell algorithms labeled in Table 1) constructed from sequences described in SEQ ID NO:1-2024 with core functional units can be introduced into cells of interest. Table 4 (SEQ ID NO:1-2024) shows 2024 unique βproGuide constructs (e.g., there are 23 unique stem combinations. There are 22 unique “4 pools” for β-spacer regions. There are 4 pools because for each gene, all 4 constructs targeting that gene are used together. So this means there are 22*23=506 different combinations. If you count each individual construct, then it will be multiplied 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 using live-cell imaging, phenotypic assays for insulin production and release, and C-peptide production and release on days 1-14.Measurements of pancreatic cell identity include RNA-seq markers such as the following 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, and COL21A1. SOX9, SOX17, ASCL1, HOXB2, PYY, DNAJB1, ALDH1A1, MAFA, MAFB, NEUROD1, DLK1, LDHB, ITGB1, SUSD2, F3, GP2, CLU, NTS, NKX2.2, ACSL1, PPY, POU5F1, NANOG, OTX2, SOX2, CDX2, TBXT, GATA4, GATA6, EOMES, CER1, GSC, HLXB9, MNX1, HNF1B, HNF4A, HNF6, ONECUT 1. TBX3, PTF1A, NR5A2, PROX1, HES1, NGN3, PAX6, SIX2, RFX6, GLIS3, BRN4, LRX2, PAX4, CDH1, EPCAM, ITGA1, CFTR, KRT19, SPP1, NKX6-1, NKX2-2, TFAP2A, TFAP2B, NES, VIM, ACVR1C, DHRS2, HOPX, NKX2.1, G6PC2, NPTX2, CXCR4, FOXA2, FOXA1, RNA28SN4, RNA2 8SN2, RNA28SN1, NEUROG1, 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, ID3.

[0267] Table 1: Example of a multi-step cascaded cell algorithm. Condition 1 is a negative empty control.

[0268]

[0269]

[0270]

[0271] Table 2: Examples of M3 cell culture media used for producing β cells

[0272]

[0273] Table 3: Examples of activin A cell culture media used for β-cell production

[0274]

[0275]

[0276] Example 6: In vivo validation of cells obtained by the cell algorithm

[0277] background

[0278] Pancreatic β-cells and other islet cells generated using cellular algorithm technology can be used as regenerative therapies for individuals with pancreatic dysfunction, particularly those related to insulin production. Delivering these cells to such individuals can provide relief from exogenous insulin requirements. Once the cellular algorithm-driven instructions are generated, islets can be used in the same manner as islets generated through other methods, such as islet isolation from cadaveric donors or differentiation of pluripotent stem cells using protocols that alter growth factors and other small molecules over a period of time (e.g., directed differentiation). Regardless of their origin, islets can be evaluated and used in vivo by injecting them subcapsularly, into the portal vein, intramuscularly, or in a subcutaneously encapsulated device. Islets can be phenotypically characterized based on gene expression and cell surface markers, and functionally characterized for their ability to regulate blood glucose and appetite. In vitro and in vivo characterization of glucose responsiveness and subsequent insulin secretion can provide important insights into islet quality.

[0279] To assess the function of the generated islets, an induced diabetes animal model was used. These mice were injected with streptozotocin (STZ) into their pancreas to induce organ destruction, thereby inducing diabetes. Subsequently, islets were injected into the tissue of interest and fed to the mice. Blood / serum glucose and insulin levels were measured to characterize islet performance.

[0280] method

[0281] Immunodeficient mice aged 8–10 weeks (e.g., SCID-Beige) can be procured from Taconic or The Jackson Laboratory, and 8–12-week-old NOD.Cg-Rag1tm1MomIL2rgtm1WjlIns2Akita (NRG-Akita) can also be procured for validation studies. Human islets (e.g., 500–1000 IEQ per animal for SCID-Beige, or 4,000 IEQ per animal for NRG-Akita mice) or iPS-derived cell clusters (e.g., 5e6 cells per animal) can be loaded into catheters for cell delivery beneath the renal sac of the mouse. The mice can then be analyzed at selected time points by performing glucose challenge, measuring blood glucose, and collecting serum to measure human insulin. After fasting the mice overnight (e.g., 16 hours), D-(+)-glucose (e.g., 2 g glucose / 1 kg body weight) can be injected into the mice (e.g., intraperitoneally (IP)). Blood glucose levels can be measured and serum collected at specified time points (e.g., 0 min and 30 min) after glucose injection. The blood glucose levels observed in mice containing transplanted islets should be significantly reduced. Serum human insulin levels can be quantified as a measure of transplanted islet cell function (e.g., using a human high-sensitivity insulin ELISA). Human insulin levels in mouse serum should increase significantly within a short period after feeding (e.g., 30 min to 60 min). The kidney containing the transplant can then be dissected from the mouse, fixed overnight (e.g., in 4% PFA), embedded in paraffin, and sectioned for histological analysis to determine the composition of the transplanted islets (e.g., the ratio of α to β to γ ​​cells, etc.). The presence of β cells should be observed and identified (e.g., by immunofluorescence staining for insulin protein expression).

[0282] Table 4: Concatenation sequence of proGuide

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453] Table 5: Sequences of spacer regions and target genes

[0454]

[0455]

[0456]

[0457] Table 6: Stem Sequence

[0458]

[0459]

[0460] Implementation Plan

[0461] The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

[0462] 1. A method for converting multiple stem cells into multiple pancreatic lineage cells, the method comprising:

[0463] The plurality of stem cells are contacted with a heterologous gene circuit comprising multiple gate units, wherein the heterologous gene circuit is activatable to induce the multiple gate units to sequentially regulate the expression levels of multiple different target genes, thereby achieving the transformation, wherein the multiple gate units include:

[0464] a) A first gate unit, pre-configured to regulate the expression level of a first target gene among the plurality of different target genes, wherein the first target gene includes one or more members selected from the forkhead box (FOX), the SRY-associated HMG box (SOX), and GATA; and

[0465] b) A second gate unit, pre-configured to regulate the expression level of a second target gene among the plurality of different target genes, such that the expression levels of the first target gene and the second target gene are regulated in the stated order.

[0466] In this process, after the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

[0467] 2. The method as described in embodiment 1, wherein the first target gene comprises two or more members selected from the FOX, the SOX, and the GATA.

[0468] 3. The method as described in embodiment 1, wherein the first target gene includes the FOX, the SOX, and the GATA.

[0469] 4. The method of embodiment 1, wherein the first target gene further includes a T-box transcription factor (TBX).

[0470] 5. The method of embodiment 1, wherein the second target gene comprises one or more members selected from basic helical-loop-helical transcription factors (bHLH), homeoboxes, and Maf transcription factors.

[0471] 6. The method of embodiment 1, wherein 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.

[0472] 7. A method for converting multiple stem cells into multiple pancreatic lineage cells by regulating the expression levels of multiple different target genes, including a first target gene and a second target gene, said method comprising:

[0473] a) A first heterologous gene regulatory portion contacts a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of a first target gene operatively coupled to the first polynucleotide sequence, wherein the first target gene includes one or more members selected from forkhead boxes (FOX), SRY-associated HMG boxes (SOX), and GATA; and

[0474] b) The second heterologous gene regulatory portion contacts a second polynucleotide sequence in the plurality of stem cells to regulate the expression level of a second target gene operatively coupled to the second polynucleotide sequence, wherein the second target gene includes one or more members selected from basic helical-loop-helical transcription factors (bHLH), homeoboxes, and Maf transcription factors.

[0475] 8. The method of embodiment 7, wherein (b) is performed after (a) to achieve the regulation of the first target gene and the second target gene in a sequential manner, respectively.

[0476] 9. The method of embodiment 7, wherein the first target gene comprises two or more members selected from the FOX, the SOX, and the GATA.

[0477] 10. The method of embodiment 7, wherein the first target gene includes the FOX, the SOX, and the GATA.

[0478] 11. The method of embodiment 7, wherein the second target gene comprises two or more members selected from the bHLH, the homeo frame, and the Maf transcription factor.

[0479] 12. The method of embodiment 7, wherein the second target gene includes the bHLH, the homeo frame, and the Maf transcription factor.

[0480] 13. The method of embodiment 7, wherein (i) the first polynucleotide sequence is located upstream of the first target gene or encodes the first target gene, or (ii) the second polynucleotide sequence is located upstream of the second target gene or encodes the second target gene.

[0481] 14. The method of embodiment 7, wherein (i) the expression level of the first target gene is enhanced after contact with the first heterologous gene regulatory portion, or (ii) the expression level of the second target gene is enhanced after contact with the second heterologous gene regulatory portion.

[0482] 15. The method of embodiment 7, the method comprising contacting the plurality of stem cells with a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to sequentially regulate the expression levels of the plurality of different target genes to achieve the transformation, and wherein the plurality of gate units comprises:

[0483] (i) a first gate unit, pre-configured to implement the first heterologous gene regulatory portion to regulate the expression level of the first target gene; and

[0484] (ii) A second gate unit, pre-configured to enable the second heterologous gene regulatory portion to regulate the expression level of the second target gene.

[0485] In this process, after the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

[0486] 16. The method of embodiment 15, wherein (i) the first gate unit is activated to express the first heterogeneous regulatory portion, or (ii) the second gate unit is activated to express the second heterogeneous regulatory portion.

[0487] 17. A method for converting multiple stem cells into multiple pancreatic lineage cells by regulating the expression levels of multiple different target genes, including a first target gene and a second target gene, said method comprising:

[0488] a) A first heterologous gene regulatory portion contacts a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of a first target gene operatively coupled to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from forkhead box (FOX), SRY-associated HMG box (SOX), GATA, basic helical-loop-helical transcription factor (bHLH), homeobox, and Maf transcription factor; and

[0489] b) The second heterologous gene regulatory portion contacts the second polynucleotide sequence in the plurality of stem cells to reduce the expression level of the second target gene operatively coupled to the second polynucleotide sequence, wherein the second target gene includes one or more members selected from additional SOX, additional homeoboxes, and ETS transcription factors.

[0490] 18. The method of embodiment 17, wherein (b) is performed before (a) to achieve, respectively, the regulation of the first target gene and the reduction of the expression level of the second target gene in a sequential manner.

[0491] 19. The method of embodiment 17, wherein (b) is performed after (a) to achieve, respectively, the reduction of expression of the second target gene and the regulation of the first target gene in a sequential manner.

[0492] 20. The method as described in implementation scheme 17, wherein (a) and (b) are performed simultaneously.

[0493] 21. The method as described in embodiment 17, wherein the additional SOX is not a member of SOX9 and SOX17.

[0494] 22. The method as described in embodiment 17, wherein the additional SOX comprises SOX2.

[0495] 23. The method as described in embodiment 17, wherein the additional homologous box is not a member of PDX1, NKX6-1, NKX2-2, PAX6, ONECUT1, and ISL1.

[0496] 24. The method of embodiment 17, wherein the additional source block includes CDX1, CDX2 or CDX4.

[0497] 25. The method of embodiment 24, wherein the additional source box includes CDX2.

[0498] 26. The method of embodiment 17, wherein the additional source block includes LMX1A or LMX1B.

[0499] 27. The method of embodiment 26, wherein the additional source block includes LMX1A.

[0500] 28. The method of embodiment 17, wherein the ETS transcription factor includes ERG, FLI1, or FEV.

[0501] 29. The method of embodiment 28, wherein the ETS transcription factor includes FEV.

[0502] 30. The method of embodiment 17, wherein the first target gene comprises two or more members selected from the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

[0503] 31. The method of embodiment 17, wherein the first target gene comprises one or more members selected from the FOX, the SOX, and the GATA.

[0504] 32. The method of embodiment 17, wherein the first target gene includes one or more members selected from the bHLH, the homeobox, and the Maf transcription factor.

[0505] 33. The method of embodiment 17, wherein (i) the first polynucleotide sequence is located upstream of the first target gene or encodes the first target gene, or (ii) the second polynucleotide sequence is located upstream of the second target gene or encodes the second target gene.

[0506] 34. The method of embodiment 17, wherein the expression level of the first target gene is enhanced upon contact with the portion regulated by the first heterologous gene.

[0507] 35. The method of embodiment 17, wherein the method includes contacting the plurality of stem cells with a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to sequentially regulate the expression levels of the plurality of different target genes to achieve the transformation, and wherein the plurality of gate units comprises:

[0508] (i) a first gate unit, pre-configured to implement the first heterologous gene regulatory portion to regulate the expression level of the first target gene; and

[0509] (ii) A second gate unit, pre-configured to reduce the expression level of the second target gene by the second heterologous gene regulatory portion.

[0510] In this process, after the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

[0511] 36. The method of embodiment 35, wherein (i) the first gate unit is activated to express the first heterogeneous regulatory portion, or (ii) the second gate unit is activated to express the second heterogeneous regulatory portion.

[0512] 37. A method for converting multiple stem cells into pancreatic lineages, the method comprising:

[0513] The heterologous gene regulatory portion contacts polynucleotide sequences in the plurality of stem cells to regulate the expression levels of target genes operatively coupled to the polynucleotide sequences.

[0514] Specifically, within approximately 2 weeks after the contact, the conversion rate from the plurality of stem cells to the plurality of pancreatic lineage cells was characterized as at least approximately 5%.

[0515] 38. The method of embodiment 37, wherein the plurality of pancreatic lineage cells include pancreatic progenitor cells.

[0516] 39. The method of embodiment 38, 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%.

[0517] 40. The method of embodiment 37, wherein the plurality of pancreatic lineage cells include β cells.

[0518] 41. The method as described in embodiment 40, wherein the conversion rate from the plurality of stem cells to the β cells is at least about 10%.

[0519] 42. The method of embodiment 37, wherein the conversion rate is observed in less than or equal to about 12 days, less than or equal to about 10 days, or less than or equal to about 8 days.

[0520] 43. The method of embodiment 37, wherein the expression level of the target gene is enhanced upon contact with the heterologous gene-regulated portion.

[0521] 44. The method of embodiment 37, wherein the target gene comprises one or more members selected from the forkhead box (FOX), SRY-associated HMG box (SOX), GATA, basic helical-loop-helical transcription factor (bHLH), homeobox, and Maf transcription factor.

[0522] 45. The method of embodiment 44, wherein the target gene comprises two or more members selected from the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

[0523] 46. ​​The method of embodiment 37, wherein the target gene comprises a plurality of different target genes, the plurality of different target genes comprising a first different target gene and a second different target gene, and wherein the contact comprises:

[0524] (a) The first heterologous gene regulatory portion contacts a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of the first distinct target gene operatively coupled to the first polynucleotide sequence; and

[0525] (b) The second heterologous gene regulatory portion contacts the second polynucleotide sequence in the plurality of stem cells to regulate the expression level of the second different target gene operatively coupled to the second polynucleotide sequence.

[0526] 47. The method of embodiment 46, wherein steps (a) and (b) are performed sequentially to regulate the first different target gene and the second different target gene.

[0527] 48. The method of embodiment 46, wherein the contact comprises:

[0528] The plurality of stem cells are contacted with a heterologous gene circuit comprising multiple gate units, wherein the heterologous gene circuit is activatable to induce the multiple gate units to sequentially regulate the expression levels of multiple different target genes to achieve the transformation, and wherein the multiple gate units comprise:

[0529] (i) a first gate unit, pre-configured to enable the first heterologous gene regulatory portion to regulate the expression level of the first different target gene; and

[0530] (ii) A second gate unit, pre-configured to enable the second heterologous gene regulatory portion to regulate the expression level of the second different target gene.

[0531] In this process, after the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

[0532] 49. The method of embodiment 46, wherein the plurality of different target genes include two or more members selected from the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

[0533] 50. The method of embodiment 37, wherein the target gene is an endogenous gene.

[0534] 51. A method for treating a person in need, the method comprising:

[0535] Multiple pancreatic lineage cells are administered to the subject, wherein the multiple pancreatic lineage cells are prepared by in vitro differentiation of multiple stem cells.

[0536] Specifically, within approximately 2 weeks of in vitro differentiation, the conversion rate from the plurality of stem cells to the plurality of pancreatic lineage cells was characterized as at least approximately 5%.

[0537] 52. The method of embodiment 51, wherein the plurality of pancreatic lineage cells include pancreatic progenitor cells.

[0538] 53. The method of embodiment 52, 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%.

[0539] 54. The method of embodiment 51, wherein the plurality of pancreatic lineage cells include β cells.

[0540] 55. The method as described in embodiment 53, wherein the conversion rate from the plurality of stem cells to the β cells is at least about 10%.

[0541] 56. The method of embodiment 51, wherein the conversion rate is observed in less than or equal to about 12 days, less than or equal to about 10 days, or less than or equal to about 8 days.

[0542] 57. The method of embodiment 51, wherein the plurality of pancreatic lineage cells are cultured in vitro for less than or equal to about 4 weeks, less than or equal to about 3 weeks, less than or equal to about 2 weeks, less than or equal to about 12 days, less than or equal to about 10 days, or less than or equal to about 8 days.

[0543] 58. The method of embodiment 51, wherein the in vitro differentiation includes regulating the expression level of a target gene, said target gene including one or more members selected from the forkhead box (FOX), SRY-associated HMG box (SOX), GATA, basic helical-loop-helical transcription factor (bHLH), homeobox, and Maf transcription factor.

[0544] 59. The method of embodiment 58, wherein the target gene comprises two or more members selected from the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

[0545] 60. The method of embodiment 58, wherein the target gene is an endogenous target gene.

[0546] 61. The method as described in any of the preceding embodiments, wherein the plurality of pancreatic lineage cells include pancreatic progenitor cells or pancreatic β cells.

[0547] 62. The method as described in any of the preceding embodiments, wherein the plurality of pancreatic lineage cells are characterized as ECAD+ / CD142+ or CD49a+.

[0548] 63. The method of any one of the foregoing embodiments, wherein the plurality of pancreatic lineage cells are characterized as producing insulin.

[0549] 64. The method of any one of the foregoing embodiments, wherein the plurality of stem cells comprises pluripotent stem cells (PSCs) or endoderm cells.

[0550] 65. The method as described in any of the preceding embodiments, wherein the plurality of different target genes are endogenous genes of the plurality of stem cells.

[0551] 66. The method as described in any of the preceding embodiments, wherein the first heterologous gene regulatory portion or the second heterologous gene regulatory portion comprises (i) a nuclease, or (ii) a guide nucleic acid (gNA) molecule.

[0552] 67. The method of embodiment 66, wherein the endonuclease and the gNA form a complex capable of binding to their respective target polynucleotide sequences.

[0553] 68. The method of embodiment 66, wherein the endonuclease is a Cas protein.

[0554] 69. The method of any of the preceding embodiments, wherein the transformation occurs in conditions substantially free of (i) serum and / or (ii) exogenous cell differentiation regulators.

[0555] 70. The method of embodiment 69, wherein the exogenous cell differentiation regulator comprises one or more members selected from insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sound hedgehog factor (SHH), vascular endothelial growth factor (VEGF), the transforming growth factor-β (TGFβ) superfamily, bone morphogenetic protein-2 (BMP2), and bone morphogenetic protein-7 (BMP7).

[0556] 71. The method of embodiment 69, wherein the exogenous cell differentiation regulator comprises one or more members selected from GSK3β inhibitors, ALK inhibitors, BMP type 1 receptor inhibitors, and retinoic acid.

[0557] 72. The method as described in any of the foregoing embodiments, wherein the FOX includes one or more members selected from FOXA, FOXB, FOXC, FOXD, FOXE, FOXF, FOXG, FOXH, FOXXI, FOXJ, FOXK, FOXL, FOXM, FOXN, FOXO, FOXP, FOXQ, FOXR, and FOXS.

[0558] 73. The method of embodiment 72, wherein the FOX is FOXA1, FOXA2 or FOXA3.

[0559] 74. The method as described in embodiment 73, wherein the FOX is FOXA2.

[0560] 75. The method as described in any of the foregoing embodiments, wherein the SOX comprises one or more members selected from SOXA, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH.

[0561] 76. The method of embodiment 75, wherein the SOX is SOXE.

[0562] 77. The method of embodiment 76, wherein the SOXE is SOX9.

[0563] 78. The method of embodiment 75, wherein the SOX is SOXF.

[0564] 79. The method of embodiment 78, wherein the SOXF is SOX17.

[0565] 80. The method as described in any of the foregoing embodiments, wherein the SOX is not SOX2.

[0566] 81. The method as described in any of the foregoing embodiments, wherein the SOX is not SOXB1 or SOXB2.

[0567] 82. The method as described in any of the foregoing embodiments, wherein the GATA includes one or more members selected from GATA1, GATA2, GATA3, GATA4, GATA5, and GATA6.

[0568] 83. The method as described in embodiment 82, wherein the GATA is GATA4.

[0569] 84. The method as described in any of the foregoing embodiments, wherein the TBX includes one or more members selected from TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21 and TBX22.

[0570] 85. The method of embodiment 84, wherein the TBX is TBXT.

[0571] 86. The method of any of the foregoing embodiments, wherein the plurality of different target genes does not include TBXT.

[0572] 87. The method as described in any of the preceding embodiments, wherein the bHLH comprises one or more members selected from group A bHLH, group B bHLH, group C bHLH, group D bHLH, group E bHLH and group F bHLH.

[0573] 88. The method as described in embodiment 87, wherein the bHLH is group A bHLH.

[0574] 89. The method of embodiment 87, wherein the bHLH is NEUROG3, NEUROD1, or PTF1a.

[0575] 90. The method of any of the foregoing embodiments, wherein the homology box includes the ParaHox gene, the ParaHox gene including one or more members selected from CDX1, CDX2, CDX4, GSX1, GSX2 and PDX1.

[0576] 91. The method of embodiment 90, wherein the ParaHox gene is PDX1.

[0577] 92. The method as described in any of the foregoing embodiments, wherein the homologous frame includes an NKX-class homologous frame, the NKX-class homologous frame including one or more members selected from 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.

[0578] 93. The method as described in embodiment 92, wherein the NKX class homologous frame is NKX6-1 or NKX2-2.

[0579] 94. The method as described in any of the foregoing embodiments, wherein the homologous frames include paired-frame (PRD) homologous frames, and the paired-frame (PRD) homologous frames include those selected from 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, and NOBOX. One or more members of OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, RHOXF1, RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, and VSX2.

[0580] 95. The method as described in embodiment 94, wherein the PRD class is PAX6.

[0581] 96. The method as described in any of the foregoing embodiments, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is not a member of Pax3 and Pax7.

[0582] 97. The method of any of the foregoing embodiments, wherein the same source frame includes a CUT-class same source frame, the CUT-class same source frame including one or more members selected from ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2, SATB1, and SATB2.

[0583] 98. The method of embodiment 97, wherein the CUT class homologous frame is ONECUT1.

[0584] 99. The method as described in any of the foregoing embodiments, wherein the same source frame includes a LIM-class same source frame, the LIM-class same source frame including one or more members selected from ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, and LMX1B.

[0585] 100. The method as described in embodiment 99, wherein the LIM class source box is ISL1.

[0586] 101. The method as described in any of the foregoing embodiments, wherein the conversion occurs in less than about 14 days or less than about 10 days.

[0587] 102. The method as described in any of the foregoing embodiments, the method further comprising storing the plurality of pancreatic lineage cells in sterile vials.

[0588] 103. The method as described in any of the foregoing embodiments, wherein the transformation achieves the formation of islets comprising the plurality of pancreatic lineage cells.

[0589] 104. The method as described in any of the foregoing embodiments, the method further comprising generating islets containing the plurality of pancreatic lineage cells.

[0590] 105. The method of embodiment 104, wherein the islets are used for application to a subject in need.

[0591] 106. The method as described in any of the foregoing embodiments, the method further comprising loading the plurality of pancreatic lineage cells into or onto a scaffold.

[0592] 107. The method of embodiment 106, wherein the scaffold and the plurality of pancreatic lineage cells are combined for transplantation to a recipient.

[0593] 108. The method as described in any of the foregoing embodiments, the method further comprising administering the plurality of pancreatic lineage cells to a subject in need.

[0594] 109. The method as described in any of the foregoing embodiments, wherein the administration comprises one or more portal vein injections.

[0595] 110. The method as described in any of the foregoing embodiments, wherein the application comprises one or more subcutaneous implantations.

[0596] 111. The method as described in any of the preceding embodiments, wherein the subject has or is suspected of having diabetes.

[0597] 112. The method as described in any of the preceding embodiments, wherein the transformation is characterized as producing fewer non-endodermal cells or fewer enterochromaffin cells compared to a control transformation of multiple stem cells in a culture medium containing (i) serum and / or (ii) exogenous cell differentiation regulators.

[0598] 113. A system for converting multiple stem cells into multiple pancreatic lineage cells, the system comprising:

[0599] A heterologous gene circuit comprising multiple gate units, wherein the heterologous gene circuit is activatable to induce the multiple gate units to sequentially regulate the expression levels of multiple different target genes to achieve the transformation, wherein the multiple gate units include:

[0600] (i) A first gate unit pre-configured to regulate the expression level of a first target gene among the plurality of different target genes, wherein the first target gene includes one or more members selected from the forkhead box (FOX), the SRY-associated HMG box (SOX), and GATA; and

[0601] (ii) A second gate unit, pre-configured to regulate the expression level of a second target gene among the plurality of different target genes, such that the expression levels of the first target gene and the second target gene are regulated in the aforementioned order.

[0602] In this process, after the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

[0603] 114. The system of embodiment 113, wherein the first target gene comprises two or more members selected from the FOX, the SOX and the GATA.

[0604] 115. The system of embodiment 113, wherein the first target gene includes the FOX, the SOX, and the GATA.

[0605] 116. The system of embodiment 113, wherein the first target gene further comprises a T-box transcription factor (TBX).

[0606] 117. The system of embodiment 113, wherein the second target gene comprises one or more members selected from basic helical-loop-helical transcription factors (bHLH), homeoboxes, and Maf transcription factors.

[0607] 118. The system of 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.

[0608] 119. A system for converting multiple stem cells into multiple pancreatic lineage cells by regulating the expression levels of multiple different target genes, including a first target gene and a second target gene, said system comprising:

[0609] a) A first heterologous gene regulatory portion configured to bind a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of a first target gene operatively coupled to the first polynucleotide sequence, wherein the first target gene includes one or more members selected from forkhead boxes (FOX), SRY-associated HMG boxes (SOX), and GATA; and

[0610] b) A second heterologous gene regulatory portion configured to bind a second polynucleotide sequence in the plurality of stem cells to regulate the expression level of a second target gene operatively coupled to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from basic helical-loop-helical transcription factors (bHLH), homeoboxes, and Maf transcription factors.

[0611] 120. The system of embodiment 119, wherein the first heterologous gene regulation portion and the second heterologous gene regulation portion are configured to regulate the first target gene and the second target gene in a sequential manner, respectively.

[0612] 121. The system of embodiment 119, wherein the first target gene comprises two or more members selected from the FOX, the SOX and the GATA.

[0613] 122. The system of embodiment 119, wherein the first target gene includes the FOX, the SOX, and the GATA.

[0614] 123. The system of embodiment 119, wherein the second target gene comprises two or more members selected from the bHLH, the homologous frame, and the Maf.

[0615] 124. The system as described in embodiment 119, wherein the second target gene includes the bHLH, the homology frame, and the Maf.

[0616] 125. The system of embodiment 119, wherein (i) the first polynucleotide sequence is located upstream of the first target gene or encodes the first target gene, or (ii) the second polynucleotide sequence is located upstream of the second target gene or encodes the second target gene.

[0617] 126. The system of embodiment 119, wherein (i) the expression level of the first target gene is enhanced after contact with the first heterologous gene regulatory portion, or (ii) the expression level of the second target gene is enhanced after contact with the second heterologous gene regulatory portion.

[0618] 127. The system of embodiment 119, the system comprising a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to sequentially regulate the expression levels of the plurality of different target genes to achieve the transformation, and wherein the plurality of gate units comprises:

[0619] (i) a first gate unit, pre-configured to implement the first heterologous gene regulatory portion to regulate the expression level of the first target gene; and

[0620] (ii) A second gate unit, pre-configured to enable the second heterologous gene regulatory portion to regulate the expression level of the second target gene.

[0621] In this process, after the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

[0622] 128. The system of embodiment 127, wherein (i) the first gate unit is activated to express the first heterogeneous regulatory portion, or (ii) the second gate unit is activated to express the second heterogeneous regulatory portion.

[0623] 129. A system for converting multiple stem cells into multiple pancreatic lineage cells by regulating the expression levels of multiple different target genes, including a first target gene and a second target gene, said system comprising:

[0624] a) A first heterologous gene regulatory portion configured to bind a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of a first target gene operatively coupled to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from forkhead boxes (FOX), SRY-associated HMG boxes (SOX), GATA, basic helical-loop-helical transcription factor (bHLH), homeoboxes, and Maf transcription factors; and

[0625] b) A second heterologous gene regulatory portion configured to bind a second polynucleotide sequence in the plurality of stem cells to reduce the expression level of a second target gene operatively coupled to the second polynucleotide sequence, wherein the second target gene includes one or more members selected from additional SOX, additional homeoboxes, and EST transcription factors.

[0626] 130. The system of embodiment 129, wherein the first heterologous gene regulation portion and the second heterologous gene regulation portion are configured to sequentially regulate the expression level of the first target gene and reduce the expression level of the second target gene, respectively.

[0627] 131. The system of embodiment 129, wherein the first heterologous gene regulation portion and the second heterologous gene regulation portion are configured to sequentially reduce the expression of the second target gene and regulate the first target gene, respectively.

[0628] 132. The system of embodiment 129, wherein the first heterologous gene regulation portion and the second heterologous gene regulation portion are configured to simultaneously regulate the first and second target genes.

[0629] 133. The system as described in embodiment 129, wherein the additional SOX is not a member of SOX9 and SOX17.

[0630] 134. The system as described in embodiment 129, wherein the additional SOX comprises SOX2.

[0631] 135. The system as described in embodiment 129, wherein the additional homologous box is not a member of PDX1, NKX6-1, NKX2-2, PAX6, ONECUT1, and ISL1.

[0632] 136. The system as described in embodiment 129, wherein the additional homologous frame includes CDX1, CDX2, or CDX4.

[0633] 137. The system of embodiment 136, wherein the additional homologous block includes CDX2. 138. The system of embodiment 129, wherein the additional homologous block includes LMX1A or LMX1B.

[0634] 139. The system of embodiment 138, wherein the additional homology box includes LMX1A. 140. The system of embodiment 129, wherein the EST transcription factor includes ERG, FLI1, or FEV.

[0635] 141. The system of embodiment 140, wherein the EST transcription factor includes FEV.

[0636] 142. The system of embodiment 129, wherein the first target gene comprises two or more members selected from the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

[0637] 143. The system of embodiment 129, wherein the first target gene includes one or more members selected from the FOX, the SOX and the GATA.

[0638] 144. The system of embodiment 129, wherein the first target gene includes one or more members selected from the bHLH, the homeobox, and the Maf transcription factor.

[0639] 145. The system of embodiment 129, wherein (i) the first polynucleotide sequence is located upstream of the first target gene or encodes the first target gene, or (ii) the second polynucleotide sequence is located upstream of the second target gene or encodes the second target gene.

[0640] 146. The system of embodiment 129, wherein the expression level of the first target gene is enhanced upon contact with the portion regulated by the first heterologous gene.

[0641] 147. The system of embodiment 129, the system comprising a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to sequentially regulate the expression levels of the plurality of different target genes to achieve the transformation, and wherein the plurality of gate units comprises:

[0642] (i) a first gate unit, pre-configured to implement the first heterologous gene regulatory portion to regulate the expression level of the first target gene; and

[0643] (ii) A second gate unit, pre-configured to enable the second heterologous gene regulatory portion to regulate the expression level of the second target gene.

[0644] In this process, after the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

[0645] 148. The system of embodiment 147, wherein (i) the first gate unit is activated to express the first heterogeneous regulatory portion, or (ii) the second gate unit is activated to express the second heterogeneous regulatory portion.

[0646] 149. A system for converting multiple stem cells into pancreatic lineages, the system comprising:

[0647] The heterologous gene regulation component is configured to bind to polynucleotide sequences in the plurality of stem cells to regulate the expression levels of target genes operatively coupled to the polynucleotide sequences.

[0648] Specifically, within approximately 2 weeks after the contact, the conversion rate from the plurality of stem cells to the plurality of pancreatic lineage cells was characterized as at least approximately 5%.

[0649] 150. The system of embodiment 149, wherein the plurality of pancreatic lineage cells include pancreatic progenitor cells.

[0650] 151. The system of 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%.

[0651] 152. The system as described in embodiment 149, wherein the plurality of pancreatic lineage cells include β cells.

[0652] 153. The system as described in embodiment 152, wherein the conversion rate from the plurality of stem cells to the β cells is at least about 10%.

[0653] 154. The system as described in embodiment 149, wherein the conversion rate is observed in less than or equal to about 12 days, less than or equal to about 10 days, or less than or equal to about 8 days.

[0654] 155. The system of embodiment 149, wherein the expression level of the target gene is enhanced upon contact with the heterologous gene-regulated portion.

[0655] 156. The system of embodiment 149, wherein the target gene comprises one or more members selected from the forkhead box (FOX), SRY-associated HMG box (SOX), GATA, basic helical-loop-helical transcription factor (bHLH), homeobox, and Maf transcription factor.

[0656] 157. The system of embodiment 156, wherein the target gene comprises two or more members selected from the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

[0657] 158. The system of embodiment 149, wherein the target gene comprises a plurality of different target genes, the plurality of different target genes comprising a first different target gene and a second different target gene, and wherein the system comprises:

[0658] a) A first heterologous gene regulatory portion configured to bind a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of the first distinct target gene operably coupled to the first polynucleotide sequence; and

[0659] b) A second heterologous gene regulatory portion configured to bind a second polynucleotide sequence in the plurality of stem cells to regulate the expression level of a second different target gene operatively coupled to the second polynucleotide sequence.

[0660] 159. The system of embodiment 158, wherein the first and second heterogeneous gene regulation portions are configured to sequentially regulate the first different target gene and the second different target gene.

[0661] 160. The system of embodiment 158, the system comprising a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to sequentially regulate the expression levels of a plurality of different target genes to achieve the transformation, and wherein the plurality of gate units comprises:

[0662] (i) a first gate unit, pre-configured to enable the first heterologous gene regulatory portion to regulate the expression level of the first different target gene; and

[0663] (ii) A second gate unit, pre-configured to enable the second heterologous gene regulatory portion to regulate the expression level of the second different target gene.

[0664] In this process, after the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

[0665] 161. The system of embodiment 158, wherein the plurality of different target genes include two or more members selected from the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

[0666] 162. The system as described in embodiment 149, wherein the target gene is an endogenous gene.

[0667] 163. A composition comprising the system as described in any of the foregoing embodiments.

[0668] 164. A composition for treating a subject in need, said composition comprising:

[0669] Multiple pancreatic lineage cells were prepared by in vitro differentiation of multiple stem cells.

[0670] Specifically, within approximately 2 weeks of in vitro differentiation, the conversion rate from the plurality of stem cells to the plurality of pancreatic lineage cells was characterized as at least approximately 5%.

[0671] 165. The composition of embodiment 164, wherein the plurality of pancreatic lineage cells include pancreatic progenitor cells.

[0672] 166. The composition of embodiment 165, 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%.

[0673] 167. The composition of embodiment 164, wherein the plurality of pancreatic lineage cells include β cells.

[0674] 168. The composition as described in embodiment 167, wherein the conversion rate from the plurality of stem cells to the β cells is at least about 10%.

[0675] 169. The composition of embodiment 164, wherein the conversion rate is observed in less than or equal to about 12 days, less than or equal to about 10 days, or less than or equal to about 8 days.

[0676] 170. The composition of embodiment 164, wherein the plurality of pancreatic lineage cells are cultured in vitro for less than or equal to about 4 weeks, less than or equal to about 3 weeks, less than or equal to about 2 weeks, less than or equal to about 12 days, less than or equal to about 10 days, or less than or equal to about 8 days.

[0677] 171. The composition of embodiment 164, wherein the in vitro differentiation comprises regulating the expression level of a target gene, said target gene comprising one or more members selected from the forkhead box (FOX), SRY-associated HMG box (SOX), GATA, basic helical-loop-helical transcription factor (bHLH), homeobox, and Maf transcription factor.

[0678] 172. The composition of embodiment 171, wherein the target gene comprises two or more members selected from the FOX, the SOX, the GATA, the bHLH, the homeobox, and the Maf transcription factor.

[0679] 173. The composition of embodiment 171, wherein the target gene is an endogenous target gene. 174. The system or composition of any of the preceding embodiments, wherein the plurality of pancreatic lineage cells comprises pancreatic progenitor cells or pancreatic β cells.

[0680] 175. The system or composition as described in any of the foregoing embodiments, wherein the plurality of pancreatic lineage cells are characterized as ECAD+ / CD142+ or CD49a+.

[0681] 176. The system or composition as described in any of the foregoing embodiments, wherein the plurality of pancreatic lineage cells are characterized to produce insulin.

[0682] 177. The system or composition as described in any of the foregoing embodiments, wherein the plurality of stem cells comprises pluripotent stem cells (PSCs) or mesodermal cells.

[0683] 178. The system or composition as described in any of the foregoing embodiments, wherein the plurality of different target genes are endogenous genes of the plurality of stem cells.

[0684] 179. The system or composition as described in any of the preceding embodiments, wherein the first heterogeneous regulatory portion or the second heterogeneous regulatory portion comprises (i) a nuclease, or (ii) a guide nucleic acid (gNA) molecule.

[0685] 180. The system or composition as described in any of the foregoing embodiments, wherein the endonuclease and the gNA form a complex capable of binding to their respective target polynucleotide sequences.

[0686] 181. The system or composition as described in any of the foregoing embodiments, wherein the endonuclease is a Cas protein.

[0687] 182. The system or composition as described in any of the preceding embodiments, wherein the transformation occurs in conditions substantially free of (i) serum and / or (ii) exogenous cell differentiation regulators. 183. The system or composition as described in any of the preceding embodiments, wherein the exogenous cell differentiation regulator comprises one or more members selected from insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sound hedgehog factor (SHH), and vascular endothelial growth factor (VEGF), the transforming growth factor-β (TGFβ) superfamily, bone morphogenetic protein-2 (BMP2), and bone morphogenetic protein-7 (BMP7).

[0688] 184. The system or composition as described in any of the foregoing embodiments, wherein the exogenous cell differentiation regulator comprises one or more members selected from GSK3β inhibitors, ALK inhibitors, BMP type 1 receptor inhibitors, and retinoic acid.

[0689] 185. The system or composition as described in any of the foregoing embodiments, wherein the FOX comprises one or more members selected from FOXA, FOXB, FOXC, FOXD, FOXE, FOXF, FOXG, FOXH, FOXXI, FOXJ, FOXK, FOXL, FOXM, FOXN, FOXO, FOXP, FOXQ, FOXR, and FOXS.

[0690] 186. The system or composition as described in any of the foregoing embodiments, wherein the FOX is FOXA1, FOXA2, or FOXA3.

[0691] 187. The system or composition as described in any of the foregoing embodiments, wherein the FOX is FOXA2.

[0692] 188. The system or composition as described in any of the foregoing embodiments, wherein the SOX comprises one or more members selected from SOXA, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH.

[0693] 189. The system or composition as described in any of the foregoing embodiments, wherein the SOX is SOXE.

[0694] 190. The system or composition as described in any of the foregoing embodiments, wherein the SOXE is SOX9.

[0695] 191. The system or composition as described in any of the foregoing embodiments, wherein the SOX is SOXF.

[0696] 192. The system or composition as described in any of the foregoing embodiments, wherein the SOXF is SOX17.

[0697] 193. The system or composition as described in any of the foregoing embodiments, wherein the SOX is not SOX2.

[0698] 194. The system or composition as described in any of the foregoing embodiments, wherein the SOX is not SOXB1 or SOXB2.

[0699] 195. The system or composition as described in any of the foregoing embodiments, wherein the GAGA includes one or more members selected from GATB1, GATA2, GATA3, GATA4, GATA5, and GATA6.

[0700] 196. The system or composition as described in any of the foregoing embodiments, wherein the GATA is GATA4.

[0701] 197. The system or composition as described in any of the foregoing embodiments, wherein the TBX comprises one or more members selected from TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21 and TBX22.

[0702] 198. The system or composition as described in any of the foregoing embodiments, wherein the TBX is TBXT.

[0703] 199. The system or composition as described in any of the foregoing embodiments, wherein the plurality of different target genes does not include TBXT.

[0704] 200. The system or composition as described in any of the foregoing embodiments, wherein the bHLH comprises one or more members selected from group A bHLH, group B bHLH, group C bHLH, group D bHLH, group E bHLH and group F bHLH.

[0705] 201. The system or composition as described in any of the foregoing embodiments, wherein the bHLH is a group A bHLH.

[0706] 202. The system or composition as described in any of the foregoing embodiments, wherein the bHLH is NEUROG3, NEUROD1, or PTF1a.

[0707] 203. The system or composition as described in any of the foregoing embodiments, wherein the homology box includes the ParaHox gene, the ParaHox gene including one or more members selected from CDX1, CDX2, CDX4, GSX1, GSX2 and PDX1.

[0708] 204. The system or composition as described in any of the foregoing embodiments, wherein the ParaHox gene is PDX1.

[0709] 205. The system or composition as described in any of the foregoing embodiments, wherein the homologous frame includes an NKX-class homologous frame, the NKX-class homologous frame including one or more members selected from 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.

[0710] 206. The system or composition as described in any of the foregoing embodiments, wherein the NKX class homologous box is NKX6-1 or NKX2-2.

[0711] 207. The system or composition as described in any of the foregoing embodiments, wherein the homologous frame includes a paired-frame (PRD) class homologous frame, the paired-frame (PRD) class homologous frame including those selected from 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, NOB One or more members of OX, OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, RHOXF1, RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, and VSX2.

[0712] 208. The system or composition as described in any of the foregoing embodiments, wherein the PRD class is PAX6.

[0713] 209. The system or composition as described in any of the foregoing embodiments, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is not a member of Pax3 and Pax7.

[0714] 210. The system or composition as described in any of the foregoing embodiments, wherein the homologous frame includes a CUT-class homologous frame, the CUT-class homologous frame including one or more members selected from ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2, SATB1, and SATB2.

[0715] 211. The system or composition as described in any of the foregoing embodiments, wherein the CUT class-related box is ONECUT1.

[0716] 212. The system or composition as described in any of the foregoing embodiments, wherein the homologous frame includes a LIM-class homologous frame, the LIM-class homologous frame including one or more members selected from ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, and LMX1B.

[0717] 213. The system or composition as described in any of the foregoing embodiments, wherein the LIM class homologous box is ISL1.

[0718] 214. The system or composition as described in any of the foregoing embodiments, wherein the conversion occurs in less than about 14 days or less than about 10 days.

[0719] 215. The system or composition as described in any of the foregoing embodiments, wherein the conversion achieves the formation of islets comprising the plurality of pancreatic lineage cells.

[0720] 216. The system or composition as described in any of the preceding embodiments, wherein the transformation is characterized as producing fewer non-endodermal cells or fewer enterochromaffin cells compared to a control transformation of multiple stem cells in a culture medium containing (i) serum and / or (ii) exogenous cell differentiation regulators.

[0721] The systems and methods disclosed herein can be combined with or modified by other systems and methods for cell programming, such as those described, for example, in International Patent Application Nos. PCT / US2018 / 052211, PCT / US2018 / 052211, PCT / US2023 / 028169, PCT / US2023 / 028255 and PCT / US2023 / 028033, each of which is incorporated herein by reference in its entirety.

[0722] Although preferred embodiments of the 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 invention is not intended to be limited to the specific embodiments provided herein. While the invention has been described with reference to the foregoing description, the description and illustration of embodiments herein are not intended to be construed as limiting. Various modifications, alterations, and substitutions will now be conceived by those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, and depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. Therefore, it is contemplated that the invention should also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

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 gate modules, wherein the heterologous genetic circuit is activatable to induce the plurality of gate modules to modulate expression levels of a plurality of different target genes in a sequential manner to effect the conversion, wherein the plurality of gate modules comprises: a) a first gate module preconfigured to modulate an expression level of a first target gene of the plurality of different target genes, wherein the first target gene comprises one or more members selected from a forkhead box (FOX), a SRY-related HMG box (SOX), and a GATA; and b) a second gate module preconfigured to modulate an expression level of a second target gene of the plurality of different target genes, such that the expression levels of the first target gene and the second target gene are modulated in the sequential manner, wherein upon activation of the heterologous genetic circuit, the plurality of gate modules operate to effect the conversion.

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

3. The method of claim 1, wherein the first target gene comprises the FOX, the SOX, and the GATA.

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

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

6. The method of claim 1, the expression level of the first target gene is enhanced by the first gate module, or (ii) the expression level of the second target gene is enhanced by the second gate module.

7. A method for converting a plurality of stem cells into a plurality of pancreatic lineage cells via modulation of expression levels of a plurality of different target genes comprising 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 genetic regulatory moiety to modulate an expression level of the first target gene operably coupled to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from a forkhead box (FOX), a SRY-related HMG box (SOX), and a GATA; and b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous genetic regulatory moiety to modulate an expression level of the second target gene operably coupled to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from a basic helix-loop-helix transcription factor (bHLH), a homeobox, and a Maf transcription factor.

8. The method of claim 7, wherein (b) is performed after (a) to effect modulation of the first target gene and the second target gene, respectively, in a sequential manner.

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

10. The method of claim 7, wherein the first target gene comprises the FOX, the SOX, and the GATA.

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

12. The method of claim 7, wherein the second target gene comprises the bHLH, the homeobox, and the Maf transcription factor.

13. 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. The method of claim 7, wherein (i) the expression level of the first target gene is enhanced upon contact by the first heterologous gene regulatory moiety, or (ii) the expression level of the second target gene is enhanced upon contact by the second heterologous gene regulatory moiety.

15. The method of claim 7, comprising contacting the plurality of stem cells with a heterologous gene circuit comprising a plurality of gate modules, wherein the heterologous gene circuit is activatable to induce the plurality of gate modules to modulate expression levels of the plurality of different target genes in a sequential manner to achieve the conversion, and wherein the plurality of gate modules comprises: (i) a first gate module preconfigured to achieve the first heterologous gene regulatory moiety modulating the expression level of the first target gene; and (ii) a second gate module preconfigured to achieve the second heterologous gene regulatory moiety modulating the expression level of the second target gene, wherein, upon activation of the heterologous gene circuit, the plurality of gate modules operate to achieve the conversion.

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

17. A method for converting a plurality of stem cells into a plurality of pancreatic lineage cells via modulating expression levels of a plurality of different target genes comprising a first target gene and a second target gene, the method comprising: a) contacting a first polynucleotide sequence in the plurality of stem cells by a first heterologous gene regulatory moiety to modulate an expression level of the first target gene operably coupled to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from a forkhead box (FOX), a SRY-related HMG box (SOX), a GATA, 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 regulatory moiety to decrease the expression level of a second target gene operably coupled 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. The method of claim 17, wherein (b) is performed prior to (a) to achieve modulation of the first target gene and decreased expression level of the second target gene, respectively, in a sequential manner.

19. The method of claim 17, wherein (b) is performed after (a) to achieve decreased expression of the second target gene and modulation of the first target gene, respectively, in a sequential manner.

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

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

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

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

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

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

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

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

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

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

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

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

35. The method of claim 17, wherein the method comprises contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gate units, wherein the heterologous genetic circuit is activatable to induce the plurality of gate units to modulate expression levels of the plurality of different target genes in a sequential manner to achieve the conversion, and wherein the plurality of gate units comprises: (i) a first gate unit preconfigured to achieve the first heterologous genetic regulatory moiety modulating the expression level of the first target gene; and (ii) a second gate unit preconfigured to achieve the second heterologous genetic regulatory moiety decreasing the expression level of the second target gene, wherein, upon activation of the heterologous genetic circuit, the plurality of gate units operate to achieve the conversion.

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

37. A method for converting a plurality of stem cells to a pancreatic lineage, the method comprising: contacting a polynucleotide sequence in the plurality of stem cells with a heterologous genetic regulatory moiety to modulate an expression level of a target gene operably coupled to the polynucleotide sequence, wherein, within less than or equal to about 2 weeks after the contacting, a conversion rate from the plurality of stem cells to a plurality of pancreatic lineage cells is characterized as at least about 5%.

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

39. The method of claim 38, 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%.

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

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

42. The method of claim 37, wherein the conversion rate is observed within less than or equal to about 12 days, less than or equal to about 10 days, or less than or equal to about 8 days.

43. The method of claim 37, wherein the expression level of the target gene is enhanced upon contacting by the heterologous genetic regulatory moiety.

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

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

46. The method of claim 37, wherein the target gene comprises a plurality of different target genes, the plurality of different target genes comprising a first different target gene and a second different target gene, and wherein the contacting comprises: ​ (a) contacting a first polynucleotide sequence in the plurality of stem cells with a first heterologous gene regulatory moiety to modulate an expression level of the first different target gene operably coupled to the first polynucleotide sequence; and (b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to modulate an expression level of the second different target gene operably coupled to the second polynucleotide sequence.

47. The method of claim 46, wherein the steps (a) and (b) effect modulation of the first different target gene and the second different target gene in a sequential manner.

48. The method of claim 46, wherein the contacting comprises: contacting the plurality of stem cells with a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to modulate expression levels of the plurality of different target genes in a sequential manner to effect the transformation, and wherein the plurality of gate units comprises: (i) a first gate unit preconfigured to effect the first heterologous gene regulatory moiety modulating the expression level of the first different target gene; and (ii) a second gate unit preconfigured to effect the second heterologous gene regulatory moiety modulating the expression level of the second different target gene, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the transformation.

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

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

51. 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 an ex vivo differentiation, wherein a transformation rate from the plurality of stem cells to the plurality of pancreatic lineage cells is characterized as at least about 5% within less than or equal to about 2 weeks of the ex vivo differentiation.

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

53. The method of claim 52, wherein the transformation 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%.

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

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

56. The method of claim 51, wherein the transformation rate is observed within less than or equal to about 12 days, less than or equal to about 10 days, or less than or equal to about 8 days.

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

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

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

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

61. The method of any one of the preceding claims, wherein the plurality of pancreatic lineage cells comprise pancreatic progenitor cells or pancreatic beta cells.

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

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

64. The method of any one of the preceding claims, wherein the plurality of stem cells comprise pluripotent stem cells (PSCs) or endoderm cells.

65. The method of any one of the preceding claims, wherein the plurality of different target genes are endogenous genes of the plurality of stem cells.

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

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

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

69. The method of any one of the preceding claims, wherein the transformation occurs in conditions that are substantially free of (i) serum and / or (ii) exogenous cell differentiation regulatory factors.

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

71. The method of claim 69, wherein the exogenous cell differentiation regulatory factor comprises one or more members selected from the group consisting of GSK3B inhibitors, ALK inhibitors, BMP type 1 receptor inhibitors, and retinoids.

72. The method of any of the preceding claims, wherein the 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. The method of claim 72, wherein the FOX is FOXA1, FOXA2, or FOXA3.

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

75. The method of any of the preceding claims, 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. The method of claim 75, wherein the SOX is SOXE.

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

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

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

80. The method of any of the preceding claims, wherein the SOX is not SOX2.

81. The method of any of the preceding claims, wherein the SOX is not SOXB1 or SOXB2.

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

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

84. The method of any of the preceding claims, 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. The method of claim 84, wherein the TBX is TBXT.

86. The method of any of the preceding claims, wherein the plurality of different target genes does not comprise TBXT.

87. The method of any of the preceding claims, 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. The method of claim 87, wherein the bHLH is a group A bHLH.

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

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

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

92. The method of any of the preceding claims, wherein the homeobox comprises an NKX class homeobox comprising one or more members selected from 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 method of any of the preceding claims, wherein the homeobox comprises a paired box (PRD) class homeobox comprising one or more members selected from 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, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, RHOXF1, RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, and VSX2.

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

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

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

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

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

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

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

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

103. The method of any one of the preceding claims, wherein the conversion effects formation of an islet comprising the plurality of pancreatic lineage cells.

104. The method of any one of the preceding claims, further comprising generating an islet comprising the plurality of pancreatic lineage cells.

105. The method of claim 104, wherein the islet is for administration to a subject in need thereof.

106. The method of any one of the preceding claims, further comprising loading the plurality of pancreatic lineage cells into or onto a scaffold.

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

108. The method of any one of the preceding claims, further comprising administering the plurality of pancreatic lineage cells to a subject in need thereof.

109. The method of any one of the preceding claims, wherein the administering comprises one or more portal vein injections.

110. The method of any one of the preceding claims, wherein the administering comprises one or more subcutaneous implants.

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

112. The method of any one of the preceding claims, 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 culture medium comprising (i) serum and / or (ii) an exogenous cellular differentiation regulatory factor.

113. A system for converting a plurality of stem cells to a plurality of pancreatic lineage cells, the system comprising: a heterologous genetic circuit comprising a plurality of gate elements, wherein the heterologous genetic circuit is activatable to induce the plurality of gate elements to modulate expression levels of a plurality of different target genes in a sequential manner to effect the conversion, wherein the plurality of gate elements comprises: (i) a first gate element preconfigured to modulate an expression level of a first target gene of the plurality of different target genes, wherein the first target gene comprises one or more members selected from a forkhead box (FOX), a SRY-related HMG box (SOX), and a GATA; and 114. The system of claim 113, wherein the first target gene is a FOX gene. (ii) a second gate unit preconfigured to modulate an expression level of a second target gene among the plurality of different target genes such that the expression levels of the first target gene and the second target gene are modulated in the sequential manner, wherein upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion.

114. A system for converting a plurality of stem cells into a plurality of pancreatic lineage cells via modulation of expression levels of a plurality of different target genes comprising 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 to modulate an expression level of the first target gene operably coupled to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from a forkhead box (FOX), a SRY-related HMG box (SOX), and a GATA; and b) a second heterologous gene regulatory moiety configured to bind to a second polynucleotide sequence in the plurality of stem cells to modulate an expression level of the second target gene operably coupled to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from a basic helix-loop-helix transcription factor (bHLH), a homeobox, and a Maf transcription factor.

115. A system for converting a plurality of stem cells into a plurality of pancreatic lineage cells via modulation of expression levels of a plurality of different target genes comprising 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 to modulate an expression level of the first target gene operably coupled to the first polynucleotide sequence, wherein the first target gene comprises one or more members selected from a forkhead box (FOX), a SRY-related HMG box (SOX), a GATA, a basic helix-loop-helix transcription factor (bHLH), a homeobox, and a Maf transcription factor; and b) a second heterologous gene regulatory moiety configured to bind to a second polynucleotide sequence in the plurality of stem cells to reduce an expression level of the second target gene operably coupled to the second polynucleotide sequence, wherein the second target gene comprises one or more members selected from a further SOX, a further homeobox, and an EST transcription factor.

116. A system for converting a plurality of stem cells into a pancreatic lineage, the system comprising: a heterologous gene regulatory moiety configured to bind to a polynucleotide sequence in the plurality of stem cells to modulate an expression level of a target gene operably coupled to the polynucleotide sequence, wherein a conversion rate from the plurality of stem cells to a plurality of pancreatic lineage cells is characterized as at least about 5% within less than or equal to about 2 weeks following the contacting.

117. A composition for use in 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, a plurality of pancreatic lineage cells prepared by subjecting a plurality of stem cells to ex vivo differentiation, wherein The conversion rate from the plurality of stem cells to the plurality of pancreatic lineage cells is characterized as at least about 5% within less than or equal to about 2 weeks of the ex vivo differentiation. The conversion rate from the plurality of stem cells to the plurality of pancreatic lineage cells is characterized as at least about 5% within less than or equal to about 2 weeks of the ex vivo differentiation.

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