Systems and methods for programming cells into the chondrogenic lineage

Abstract

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

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 482,721, filed February 1, 2023, which is incorporated herein by reference in its entirety. [Background technology]

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

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

[0004] In some embodiments, the present disclosure provides a method for converting a plurality of stem cells into a plurality of chondrogenic cells through regulating the expression levels of a plurality of distinct target genes comprising a first homeobox protein and a second homeobox protein, 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 homeobox protein operably linked to the first polynucleotide sequence; 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 homeobox protein operably linked to the second polynucleotide sequence.

[0005] In some embodiments, the present disclosure provides a method for converting a plurality of stem cells into a plurality of chondrogenic cells via regulating the expression levels of a plurality of distinct target genes, including a first distinct target gene and a second distinct target gene, the method comprising: (a) contacting a first polynucleotide sequence in the plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression levels of the first distinct target gene operably linked 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 regulate the expression levels of the first distinct target gene operably linked to the second polynucleotide sequence. The method includes regulating the expression level of a second distinct target gene selected from the first distinct target gene and the second distinct target gene, wherein the combination of the first distinct target gene and the second distinct target gene is (i) different first and second homeobox proteins, (ii) two different members selected from the group consisting of a homeobox protein, a T-box transcription factor (TBX), and a basic helix-loop-helix transcription factor (bHLH), or (iii) a first member selected from the group consisting of a homeobox protein, a TBX, and a bHLH, and a second member comprising SOX or collagen.

[0006] In some embodiments, the present disclosure provides a method for converting a plurality of stem cells to chondrogenic differentiation, the method comprising contacting a polynucleotide sequence in the plurality of stem cells with a heterologous gene regulatory moiety to regulate the expression level of a target gene operably linked to the polynucleotide sequence, wherein within less than 7 days after contacting, the conversion rate of the plurality of stem cells to chondrogenic cells is characterized as being at least about 30%.

[0007] In some embodiments, the present disclosure provides a method for treating a subject in need thereof, the method comprising administering to the subject a plurality of chondrogenic cells, wherein the plurality of chondrogenic cells are prepared by subjecting a plurality of stem cells to ex vivo differentiation, wherein within less than 7 days of ex vivo differentiation, a conversion rate from the plurality of stem cells to a plurality of chondrogenic cells is characterized as being at least about 30%.

[0008] In some embodiments, the present disclosure provides a system for converting a plurality of stem cells into a plurality of chondrogenic cells through regulating the expression levels of a plurality of distinct target genes comprising a first homeobox protein and a second homeobox protein, the system comprising: (a) a first heterologous gene regulatory portion configured to bind to a first polynucleotide sequence in the plurality of stem cells and regulate the expression level of the first homeobox protein operably linked to the first polynucleotide sequence; and (b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and regulate the expression level of the second homeobox protein operably linked to the second polynucleotide sequence.

[0009] In some embodiments, the present disclosure provides a system for converting a plurality of stem cells into a plurality of chondrogenic cells via regulating the expression levels of a plurality of distinct target genes, including a first distinct target gene and a second distinct 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 regulate the expression levels of the first distinct target gene operably linked to the first polynucleotide sequence; and (b) a second polynucleotide sequence configured to bind to a second polynucleotide sequence in the plurality of stem cells to regulate the expression levels of the second distinct target gene operably linked to the second polynucleotide sequence. and a second heterologous gene regulatory portion configured to regulate a target gene, wherein the combination of the first distinct target gene and the second distinct target gene is (i) a different first homeobox protein and a second homeobox protein, (ii) two different members selected from the group consisting of a homeobox protein, a T-box transcription factor (TBX), and a basic helix-loop-helix transcription factor (bHLH), or (iii) a first member selected from the group consisting of a homeobox protein, a TBX, and a bHLH, and a second member comprising SOX or a collagen.

[0010] In some embodiments, the present disclosure provides a system for converting a plurality of stem cells to chondrogenic differentiation, the system comprising a heterologous gene regulatory moiety configured to bind a polynucleotide sequence in the plurality of stem cells to the heterologous gene regulatory moiety to regulate the expression level of a target gene operably linked to the polynucleotide sequence, wherein the conversion rate of the plurality of stem cells to chondrogenic cells is characterized as at least about 30% within less than 7 days after contact.

[0011] In some embodiments, the present disclosure provides a composition for treating a subject in need thereof, the composition comprising a plurality of chondrogenic cells prepared by subjecting a plurality of stem cells to ex vivo differentiation, wherein the conversion rate of the plurality of stem cells to a plurality of chondrogenic cells is characterized as being at least about 30% within less than 7 days of ex vivo differentiation.

[0012] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the present disclosure contained herein, the present specification is intended to supersede and / or supersede any such conflicting material. [Brief explanation of the drawings]

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as figures or FIG. ).

[0014] [Figure 1] Schematic diagram of a heterologous gene circuit. An activating moiety can initiate the circuit and activate a gating unit. A gating unit is composed of a gating moiety and / or a gene regulatory moiety. [Figure 2A] Figure 1 shows the progression of development from iPSCs to chondrocytes, including genes known to affect chondrocyte development at different stages. [Figure 2B] An example of a heterologous gene circuit for differentiating chondroprogenitor cells from mesodermal cells is shown. [Figure 3] 1 shows an exemplary heterologous gene circuit. [Figure 4] Scatter plots (e.g., volcano plots) are shown to identify one or more heterologous gene circuits that induced stem cell to chondroprogenitor cell conversion. [Figure 5A] FIG. 5 shows an example of chondrogenic precursor cell marker analysis data utilized to generate the scatter plot of FIG. [Figure 5B]FIG. 5 shows an example of chondrogenic precursor cell marker analysis data utilized to generate the scatter plot of FIG. [Figure 5C] FIG. 5 shows an example of chondrogenic precursor cell marker analysis data utilized to generate the scatter plot of FIG. [Figure 5D] FIG. 5 shows an example of chondrogenic precursor cell marker analysis data utilized to generate the scatter plot of FIG. [Figure 6A] We show that the best-performing heterologous gene circuit converts approximately 60% of cells into chondrogenic precursor cells in 4 days. [Figure 6B] We show that the best-performing heterologous gene circuit converts approximately 60% of cells into chondrogenic precursor cells in 4 days. [Figure 6C] A summary of the data from Figures 6A-6B is shown. DETAILED DESCRIPTION OF THE INVENTION

[0015] While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed.

[0016] Whenever "at least," "greater than," or "greater than or equal to" precedes the first number in a series of two or more numbers, the term "at least," "greater than," or "greater than or equal to" applies to each of the numbers in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0017] Whenever "at most," "up to," "no more than," "less than," or "less than or equal to" precedes the first number in a series of two or more numbers, the "at most," "up to," "less than," "less than," or "less than or equal to" applies to each and every number in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0018] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "gate unit" includes a plurality of gate units.

[0019] The terms "about" or "approximately" generally mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0020] The terms "guide nucleic acid," "guide nucleic acid molecule," and "gNA," when used interchangeably herein, generally refer to 1) a guide sequence that can hybridize to a target sequence, or 2) a scaffold sequence that can interact with or form a complex with a nucleic acid-guided nuclease. A guide nucleic acid can be a single-guide nucleic acid (e.g., sgRNA) or a double-guide nucleic acid (e.g., dgRNA). An sgRNA can be a single RNA molecule that contains both a scaffolding tracrRNA and a crRNA that can be complementary to the target sequence. Alternatively, a dgRNA can be a single RNA molecule containing a crRNA annealed to a tracrRNA via a direct repeat sequence.

[0021] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives.

[0022] The terms "genetic circuit," "biological circuit," or "circuit," as used interchangeably herein, generally refer to a collection of molecular components (e.g., biological materials, e.g., polypeptides and / or polynucleotides, non-biological materials, etc.) operably linked (e.g., operating simultaneously, sequentially, etc.) according to a circuit design. The collection of molecular components may be capable of providing one or more specific outputs (e.g., regulation of one or more genes) within a cell in response to one or more inputs (e.g., a single input or multiple inputs). Such one or more inputs may be sufficient to trigger the molecular components of the genetic circuit to provide one or more specific outputs. For example, a genetic circuit may include one or more molecular switches that are activatable by one or more inputs (FIG. 1).

[0023] A genetic circuit can be a controllable gene expression system comprising a collection of biological parts that work together (e.g., simultaneously, sequentially, etc.) as a logical function. The genetic circuit can include a plurality of gating units, at least one of which is activatable by an activating moiety (e.g., a heterologous input to a cell) to activate other gating units of the plurality of gating units (e.g., simultaneously at one time, sequentially in a cascade manner, etc.) (FIG. 1). For example, at least one of the plurality of gating units can be activatable by another of the plurality of gating units (e.g., directly or indirectly) to (i) regulate the expression or activity level of one or more target genes, (ii) activate at least one other of the plurality of gating units, and / or (ii) inactivate at least one other of the plurality of gating units, thereby collectively regulating the expression and / or activity level of one or more target genes in a predetermined and desired manner predetermined by the design of the genetic circuit (FIG. 1). As used herein, the terms "heterologous gene circuit," "HGC," "gene circuit," "cellular algorithm," or "cellgorithm" may be used interchangeably.

[0024] As referred to herein, the term "gating unit" generally refers to a portion of a genetic circuit that can control gene regulation by functioning similarly to a logic gate, which can control the flow of information and allow the circuit to multiplex decision-making at different points. More specifically, the term refers to a gene switch and a nucleic acid encoding a transcriptional / translational regulatory region or series of regions on which the gene switch acts. The input for a gating unit can be an activating moiety and / or another gating unit. The output for a gating unit can be to activate another gating unit, to inactivate another gating unit, to affect a target gene, and / or any combination of the above. For example, a gating unit can be composed of multiple gating moieties and / or multiple gene regulatory moieties (Figure 1).

[0025] The term "activating moiety" referred to herein generally refers to a moiety that can activate multiple gene circuits and / or multiple gate units. Activating moieties can be heterologous inputs to cells. For example, activating moieties can include, but are not limited to, guide nucleic acid molecules (e.g., gRNAs) or other nucleic acids, polypeptides, polynucleotides, small molecules, light, or combinations thereof.

[0026] For example, an activating moiety can be a guide nucleic acid molecule that, upon forming a complex with an endonuclease (e.g., a Cas protein), binds to the polynucleotide sequence of an inactivated gating moiety (e.g., a plasmid encoding another guide nucleic acid molecule) and activates such gating moiety (e.g., induces the expression of a functional form of an additional guide nucleic acid molecule) that can target one or more gene regulatory moieties. The term "gating moiety" referred to herein generally refers to a moiety that can affect the function of a gene regulatory moiety within a gating unit. A gating moiety can activate and / or inactivate a gene regulatory moiety. For example, a gating moiety can regulate the expression of a gene regulatory moiety by editing the nucleic acid sequence, thereby activating or inactivating the gene regulatory moiety. For example, a gating moiety can be a guide nucleic acid molecule that, upon forming a complex with an endonuclease (e.g., a Cas protein), binds to the polynucleotide sequence of a gene regulatory moiety (e.g., a plasmid encoding another guide nucleic acid molecule) and activates a gene regulatory moiety (e.g., induces the expression of a functional form of another guide nucleic acid molecule) that can target one or more endogenous genes of a cell. Alternatively or additionally, a gate moiety can activate and / or inactivate another gate unit of a genetic circuit (Figure 1). For example, a gate moiety can be a guide nucleic acid molecule that, upon forming a complex with an endonuclease (e.g., a Cas protein), binds to the polynucleotide sequence of another inactivated gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) and activates the other gate moiety (e.g., induces expression of a functional form of the other guide nucleic acid molecule). In another example, a gate moiety can be a guide nucleic acid molecule that, upon forming a complex with an endonuclease (e.g., a Cas protein), binds to the polynucleotide sequence of another activated gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) and inactivates the other gate moiety (e.g., reduces expression of a functional form of the other guide nucleic acid molecule).

[0027] The term "gate moiety" referred to herein generally refers to a moiety that can affect the function of a gene regulatory moiety within a gate unit. A gate moiety can activate and / or inactivate a gene regulatory moiety. For example, a gate moiety can regulate the expression of a gene regulatory moiety by editing a nucleic acid sequence, thereby activating or inactivating the gene regulatory moiety. Alternatively or additionally, a gate moiety can activate and / or inactivate another gate unit of a genetic circuit (Figure 1).

[0028] The term "gene regulatory moiety" or "gene editing moiety," which are used interchangeably herein, generally refers to a moiety that can regulate the expression and / or activity profile of a nucleic acid sequence or protein, regardless of whether it is exogenous or endogenous to a cell (Figure 1). For example, a gene editing moiety can regulate the expression of a gene by editing a nucleic acid sequence (e.g., CRISPR-Cas, zinc finger nuclease, TALEN, or siRNA). In some cases, a gene editing moiety can regulate the expression of a gene by editing a genomic DNA sequence. In some cases, a gene editing moiety can regulate the expression of a gene by editing an mRNA template. Editing a nucleic acid sequence can sometimes change the template underlying gene expression (e.g., CRISPR-Cas-inspired RNA targeting system). Alternatively, a gene editing moiety can suppress the translation of a gene (e.g., Cas13).

[0029] Alternatively or additionally, the gene editing moiety may be capable of regulating the expression or activity of a gene by specifically binding to a target sequence (or a target sequence within a gene) operably linked to the gene and regulating the production of mRNA from DNA, such as chromosomal DNA or cDNA. For example, the gene editing moiety may recruit or contain at least one transcription factor that binds to a specific DNA sequence, thereby controlling the transcription rate of genetic information from DNA to mRNA. The gene editing moiety may itself bind to DNA and regulate transcription by physical blockage, for example, by preventing proteins such as RNA polymerase and other related proteins from assembling on the DNA template. The gene editing moiety may regulate gene expression at the translational level, for example, by regulating the production of proteins from an mRNA template. In some cases, the gene editing moiety may regulate gene expression by affecting the stability of mRNA transcripts. In some cases, the gene editing moiety may regulate genes through epigenetic editing (e.g., Cas12).

[0030] In some cases, the plasmid can encode a non-functional form of the gene editing portion. The plasmid can be activated (e.g., genetically modified) to express a functional fragment of the gene editing portion, for example, through activation of a functional gate portion. For example, the plasmid can encode a non-functional form of a guide nucleic acid molecule that can otherwise bind to a target gene in a 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., cut at one or more sites) and then repaired via endogenous mechanisms (e.g., homologous recombination, non-homologous end joining), allowing expression of a functional form of the gene editing portion (e.g., a functional form of the guide nucleic acid molecule that specifically binds to a target gene in a cell) and allowing regulation of the target gene in the cell.

[0031] In some cases, the gene regulatory portion may include a nucleic acid molecule (e.g., a guide nucleic acid molecule that forms a complex with an endonuclease, such as a Cas protein). Alternatively or additionally, the gene regulatory portion may include an endonuclease or may be operably linked to an endonuclease. An endonuclease may be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. An endonuclease may include a restriction endonuclease that cleaves DNA at a specific site without damaging the bases. Restriction endonucleases may include type I, type II, type III, and type IV endonucleases, and may further include subtypes. In some cases, the endonuclease is Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), The endonuclease may be Cas12f (Cas14 or C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas13 (C2c2), Cas13b, Cas13c, Cas13d, Cas13x.1, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx10, Csx11, Csf1, or Csn2. The endonuclease may be a dead endonuclease that exhibits reduced cleavage activity. For example, the endonuclease may be a nuclease-inactivated Cas, such as dCas (e.g., dCas9).

[0032] The above-described Cas proteins can form a complex with a guide nucleic acid (gNA) (e.g., a guide RNA (gRNA)) and utilize the gNA to specifically bind to a target polynucleotide sequence (e.g., a target DNA sequence, a target RNA sequence). Therefore, in some cases, such Cas proteins can be referred to as "NA-guided nucleases" (e.g., RNA-guided nucleases). As used herein, the term "guide nucleic acid" (gNA) can generally refer to a nucleic acid that can hybridize to another nucleic acid. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. A guide nucleic acid can be programmed to site-specifically bind to a nucleic acid sequence. A targeted nucleic acid or target nucleic acid can comprise nucleotides. A guide nucleic acid can comprise nucleotides. A portion of a target nucleic acid can be complementary to a portion of a guide nucleic acid. A strand of a double-stranded target polynucleotide that is complementary to and hybridizes with a guide nucleic acid can be referred to as a complementary strand. A strand of a double-stranded target polynucleotide that is complementary to a complementary strand and therefore may not be complementary to a guide nucleic acid can be referred to as a non-complementary strand. A guide nucleic acid may comprise a polynucleotide strand or may be referred to as a "single guide nucleic acid." A guide nucleic acid may comprise two polynucleotide strands or may be referred to as a "dual guide nucleic acid." Unless otherwise specified, the term "guide nucleic acid" may be inclusive and refer to both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid may contain a segment that may be referred to as a "nucleic acid targeting segment," "nucleic acid targeting sequence," or "spacer sequence." A nucleic acid targeting segment may contain a subsegment that may be referred to as a "protein binding segment," "protein binding sequence," "Cas protein binding segment," or "scaffold sequence."

[0033] The gene regulatory portion can be a transcription modulator system (e.g., a gene repressor complex or a gene activator complex). For example, the gene regulatory portion can be a gene repressor complex comprising a dCas protein operably linked (e.g., linked or fused) to a transcription repressor. Non-limiting examples of transcription repressors include KRAB, SID, MBD2, MBD3, DNMT1, DNMT2A, DNMT3A, DNMT3B, DNMT3L, Mecp2, FOG1, ROM2, LSD1, ERD, SRDX repression domain, Pr-SET7 / 8, SUV4-20H1, RIZ1, JMJD2A, JHDM3A, JMJD2B, JMJD2C, GASC1, JMJD2D, Examples include 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 moiety can be a gene activator complex comprising a dCas protein operably linked (e.g., fused) to a transcriptional activator. Non-limiting examples of transcriptional activators include VP16, VP64, VP48, VP160, p65 subdomain, SET1A, SET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, SYMD2, NSD1, JHDM2a, JHDM2b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRCl, ACTR, P160, CLOCK, TET1CD, TET1, DME, DML1, DML2, and ROS1.

[0034] In some cases, the gene regulatory portion has an enzymatic activity that modifies the target gene without cleaving the target gene. Modification of the target gene can, for example, cause epigenetic modifications that can alter 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., FokI nuclease), methyltransferases (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3, ZMET2, CMT1, CMT2), demethylases (e.g., Ten-Eleven Examples of the activity include demethylase activity such as that provided by TET dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1), DNA repair activity, DNA damage activity, deamination activity such as that provided by deaminases (e.g., cytosine deaminase enzymes such as APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity such as that provided by integrases and / or resolvases (e.g., Gin invertase, e.g., a hyperactive mutant of Gin invertase, GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 degrading enzyme, etc.), transposase activity, recombinase activity such as that provided by recombinases (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0035] The gene regulatory portion can include an endonuclease. The endonuclease can be an enzyme that cleaves phosphodiester bonds in a polynucleotide chain. The endonuclease can include a restriction endonuclease that cleaves DNA at a specific site without damaging the base. The restriction endonuclease can include type I, type II, type III, and type IV endonucleases, and can further include subtypes. Optionally, the endonuclease can be Cas9. Optionally, the endonuclease can be an inactivated Cas (e.g., dCAS, dCAS9).

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

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

[0038] The term "sequence identity" generally refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid, is the number of exact matches between the two aligned sequences divided by the length of the longer sequence multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, the advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), as described 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 program can be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences, for example, using the blastp program. The program further allows the use of a SEG filter to mask segments of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired degree of sequence identity ranges from about 50% to 100% and integer values ​​therebetween.In general, the present disclosure encompasses sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any of the sequences provided herein.

[0039] The term "expression" generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides can be collectively referred to as "gene products." When a polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in eukaryotic cells. With respect to expression, "upregulated" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its expression level in the wild-type state, while "downregulated" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its expression in the wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression," the transfected gene is not transferred to daughter cells during cell division. Because its expression is limited to the transfected cell, gene expression is lost over time. During transient expression, episomal DNA can be transferred to daughter cells, but because it is not replicated, it is not permanently inherited and becomes diluted over time. In contrast, stable expression of a transfected gene can occur when the gene is cotransfected with another gene that confers a selective advantage to the transfected cell. During stable expression, plasmids can contain DNA replication elements that allow them to be inherited or integrated into the genome. Such a selective advantage can be resistance to a particular toxin presented to the cell.

[0040] The terms "peptide," "polypeptide," or "protein," when used interchangeably herein, generally refer to a polymer of at least two amino acid residues linked by a peptide bond. The terms do not imply a specific length of the polymer, nor are they intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The terms apply to naturally occurring amino acid polymers and amino acid polymers containing at least one modified amino acid. Optionally, the polymer may be interrupted by non-amino acids. The terms include amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structure (e.g., domains). The terms further encompass amino acid polymers modified by any other manipulation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and conjugation with a labeling component. As used herein, the terms "amino acid" and "amino acids" generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids, which are chemically modified to include groups or chemical moieties that do not naturally occur on amino acids. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0041] The terms "derivative," "variant," or "fragment," when used interchangeably herein with respect to polypeptide, generally refer to a polypeptide that is related to a wild-type polypeptide, for example, by any of amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of polypeptides can include one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide.

[0042] As used herein with respect to polypeptide molecules (e.g., proteins), the terms "engineered," "chimeric," or "recombinant" generally refer to polypeptide molecules having heterologous or altered amino acid sequences as a result of applying genetic engineering techniques to the nucleic acid encoding the polypeptide molecule, as well as cells or organisms that express 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 having heterologous or altered nucleic acid sequences as a result of applying genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques, transfection, transformation and other gene transfer techniques, homologous recombination, site-directed mutagenesis, and gene fusion. In some cases, genetically engineered or recombinant polynucleotides (e.g., genomic DNA sequences) can be modified or changed by gene editing moieties.

[0043] Unless otherwise specified or clear from the context, the term "nucleotide," as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can include synthetic nucleotides. A nucleotide can include synthetic nucleotide analogs. A nucleotide can be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term "nucleotide" as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Labeling can also be performed using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from PerkinElmer (Foster City, California); FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham (Arlington Heights, Illinois); and Boehringer Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP available from Mannheim (Indianapolis, IN), and chromosome-labeled nucleotides BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-UTP, Cascade Blue-7-d ... Examples of suitable dUTPs include tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP.Nucleotide can also be labeled or marked by chemical modification.Chemically modified single nucleotide can be biotin-dNTP.Some non-limiting examples of biotinylated dNTP can include biotin-dATP (for example, bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (for example, biotin-11-dCTP, biotin-14-dCTP) and biotin-dUTP (for example, biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0044] The term "cell" generally refers to a biological cell. A cell may be the basic structural, functional, and / or biological unit of a living organism. A cell may be from any organism that has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, and mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum spp., and the like), and patens, C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Cells are often not derived from natural organisms (e.g., cells can be synthetically produced and are often referred to as artificial cells).

[0045] The term "differentiation" generally refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as, for example, an immune cell. A differentiated or differentiation-induced cell is one that has adopted a more specialized ("committed") position within a cell lineage. The term "uncommitted" generally refers to a cell that, under normal circumstances, would continue to differentiate into a particular cell type or subset of cell types and has progressed down the differentiation pathway to a point where, under normal circumstances, it is unable to differentiate into a different cell type or revert to a less differentiated cell type.

[0046] The term "dedifferentiation" or "de-differentiation" generally refers to a process in which a specialized, committed, or partially specialized cell loses the characteristics of the specialized cell (e.g., a chondrogenic cell). A dedifferentiated cell or dedifferentiation-induced cell has adopted a less specialized position within a cell lineage (e.g., a stem or progenitor cell). A dedifferentiated cell (e.g., a stem or progenitor cell) can then differentiate into a different cell type or revert to a less differentiated cell type.

[0047] The term "pluripotency" generally refers to the ability of a cell to form all lineages of the body or somatic cells (e.g., embryo proper). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency can be a continuum of developmental potential ranging from incompletely or partially pluripotent cells that cannot give rise to a complete organism (e.g., primitive ectodermal stem cells) to more primitive, more pluripotent cells that can give rise to a complete organism (e.g., embryonic stem cells).

[0048] The term "induced pluripotent stem cells" (iPSCs) generally refers to stem cells derived from differentiated cells (e.g., differentiated adult, neonatal, or fetal cells) that have been induced or changed (e.g., reprogrammed) into cells capable of differentiating into tissues of all three germ layers or cortex: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells found in nature. In some cases, iPSCs can be engineered to differentiate directly into committed cells (e.g., chondrogenic cells). In some cases, iPSCs can be engineered to first differentiate into tissue-specific stem cells (e.g., mesenchymal stem cells, chondroprogenitor cells), which can be further induced to differentiate into committed cells (e.g., chondrogenic cells).

[0049] The term "embryonic stem cells" (ESCs) generally refers to cells derived from naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise to all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm during development. Optionally, ESCs can be engineered to differentiate directly into committed cells (e.g., chondrogenic cells). Optionally, ESCs can be engineered to first differentiate into tissue-specific stem cells (e.g., mesodermal stem cells), which can be further induced to differentiate into committed cells (e.g., chondrogenic cells).

[0050] The term "isolated stem cells" generally refers to any type of stem cell disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells, or mesodermal stem cells (MSCs), etc.) that is isolated from a multicellular organism. For example, HSCs can be isolated from a mammalian body, such as a human body. In another example, embryonic stem cells can be isolated from an embryo.

[0051] The term "isolated" generally refers to a cell or a population of cells separated from its original environment. For example, the new environment of an isolated cell is substantially free of at least one component found in the environment in which the "non-isolated" reference cell resides. An isolated cell can be a cell that has been removed from some or all components found in its natural environment, such as a cell isolated from a tissue or biopsy sample. The term also includes cells that have been removed from at least one, some, or all components found in an environment in which they do not naturally exist, such as a cell isolated from a cell culture or cell suspension. Thus, an isolated cell is partially or completely separated from at least one component, including other substances, cells, or cell populations, when grown, stored, or persisted in a natural or non-natural environment.

[0052] The term "chondrogenic," when applied to a cell or population, generally refers to the ability of that cell or population to produce cartilage or stimulate cartilage growth under appropriate circumstances (e.g., in vivo or ex vivo conditions). The term "chondrogenic cell" generally refers to cells comprising tissues that produce cartilage or cartilage-related proteins. Non-limiting examples of chondrogenic cells are chondroblasts, chondrocytes, and cells that themselves differentiate into chondroblasts or chondrocytes, e.g., chondrogenic precursor cells. Chondrogenic cells arise from mesodermal stem cells (MSCs), including, but not limited to, paraxial mesoderm cells, sclerotome cells, and chondrogenic precursor cells.

[0053] The terms "chondroprogenitor cells," "chondrogenic precursor cells," or "chondrogenic precursor cells," when used interchangeably herein, generally refer to chondrogenic-specific precursor cells that are more committed than stem cells (e.g., ESCs, MSCs, iPSCs, etc.) but less differentiated than chondroblasts or chondrocytes. Chondrogenic precursor cells can be generated ex vivo by manipulating isolated stem cells. Chondrogenic precursor cells can also be generated in vivo by in vivo manipulated stem cells, for example, by administering such stem cells with any one of the heterologous gene circuits disclosed herein.

[0054] Overview

[0055] Biological programming, such as cellular programming, can manipulate cells to produce desired results. The results of cellular programming can include inducing or preventing a wide range of common and / or new cellular functions, and can also include enhancing or suppressing existing cellular functions. Cellular programming can be achieved through the use of genetic circuits. Cellular programming can be achieved through the manipulation of biomolecules (e.g., DNA). For example, CRISPR or CRISPR / Cas systems have been adopted for genome editing in many species due to their versatility and easy programmability. Cellular programming can affect endogenous or exogenous genes. Cellular programming can be performed to function in a time-dependent or time-independent manner.

[0056] Genetic circuits used in cellular programming can be used to control the cell fate of a cell or multiple cells by inducing differentiation or dedifferentiation and converting one cell type to another. Cell programming is controlled through the regulation of the desired expression and / or activity levels of multiple genes in the cell.

[0057] While the CRISPR / Cas system is widely used for gene editing, Cas is essentially a single-turnover nuclease, remaining bound to the double-stranded breaks it generates, leaving many regions of the genome resistant to genome editing. Increasing understanding of CRISPR / Cas-based genome editing has encouraged the development of cascade regulatory systems to further exploit this technology for use in the development of engineered cells. By implementing a series of activatable gRNAs, genome editing can be regulated from target site to target site in a relatively transient manner, performing sequential genome edits that act like a domino effect, allowing cells to be barcoded. However, this simple barcoding, which often uses exogenous fluorophores, fails to regulate endogenous genes that drive cell differentiation.

[0058] Furthermore, cell differentiation or dedifferentiation is now possible through the use of exogenous serum and growth factors, which bypass the mechanisms underlying cellular programming. The use of exogenous serum, growth factors, and other similar methods results in cells instructed to differentiate, but these cells lack the underlying biological conditions (e.g., proper chromatin state). This lack often results in cells that prematurely terminate differentiation into an undesired cell type, or cells that undergo inefficient differentiation resulting in low yields of the target cell type, or the resulting desired differentiated cells are only semi-functional. Semi-functional cells may resemble the desired cell type but may lack important biological characteristics necessary for the normal function of the desired differentiated cell type.

[0059] Thus, there remains an unmet need for an activatable CRISPR / Cas system that uses a cascade of gRNAs to form genetic circuits and use it to edit target polynucleotides (e.g., the genome of a cell, particularly a eukaryotic cell) to independently affect gene regulation and thus cell fate decisions without the use of serum and exogenous growth factors. Preprogrammed, activatable, and self-regulating gRNA cascade CRISPR / Cas systems are useful, for example, in gene therapy, genetic circuits, and / or complex cell fate decisions and / or control.

[0060] The present disclosure provides systems and methods for engineering CRISPR / Cas9 systems, which include a Cas endonuclease and an array of cognate single guide RNAs (sgRNAs or gRNAs) that have inactivation sequences in non-essential regions and are activatable, allowing for regulation and modification of the system without the need for serum, growth factors, or other additional exogenous signals. The present disclosure further provides engineered cells that can include any of the above systems or perform any of the above methods.

[0061] Systems and methods for programming cells for the chondrogenic lineage

[0062] Various embodiments of the present disclosure provide systems for inducing the desired conversion of one type of cell to another type of cell. To this end, various embodiments of the present disclosure provide methods for inducing a desired expression and / or activity level (or profile) of one or more target genes in a cell.

[0063] In one aspect, the present disclosure provides a system for converting a plurality of cells of a first type into a plurality of cells of a second cell type. The system can include a heterologous genetic circuit comprising a plurality of gating units. The plurality of gating units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gating units. The plurality of gating units can include up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 gating unit. The plurality of gating units can be different (e.g., comprise different polynucleotide sequences). Each gating unit of the plurality of gating units may affect modulation of the expression and / or activity levels of one distinct target gene or multiple distinct target genes.

[0064] The heterologous genetic circuits disclosed herein can operate with multiple gate units in series (e.g., multiple gate units connected end-to-end in series to form a single pathway), in parallel (e.g., multiple gate units connected across each other to form, e.g., two or more parallel, continuous pathways), or a combination thereof.

[0065] The gating units disclosed herein can act in concert (e.g., as predetermined by the design of the heterologous genetic circuit) to induce an outcome in a cell. The outcome in a cell can include a cell function (e.g., migration, reproduction, response to an external stimulus, nutrient production, excretion, respiration, growth) and / or a cell state (e.g., cell fate, differentiation, quiescence, programmed cell death). Such an outcome can be confirmed in vitro, ex vivo, and / or in vivo. For example, the results disclosed herein can be confirmed in vitro by (i) measuring the expression levels of genes of interest by polymerase chain reaction (PCR) or Western blotting, (ii) staining with small molecules or antibodies, (iii) cell sorting based on cell size, morphology, and / or surface protein expression, (iv) using assays to measure phenotypic differentiation and cell function (e.g., cell proliferation assays or metabolic activity assays), (v) microscopy, and / or (iv) screening for molecular and / or genetic differences using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics.

[0066] The gating units disclosed herein may be sufficient to convert a plurality of cells of a first cell type into a plurality of cells of a second cell type. For example, the gating units disclosed herein may be sufficient to convert a plurality of pluripotent stem cells (PSCs) into a plurality of tissue-specific progenitor cells. Alternatively, the gating units disclosed herein may be necessary but insufficient to convert a plurality of cells of a first cell type into a plurality of cells of a second cell type.

[0067] The result of the cell can include the regulation of a separate target gene or a set of separate target genes.Multiple gate units can induce the separate regulation of multiple target genes (for example, sequentially), so that the accumulation of the coordinated gene regulation results in the final expression and / or activity profile of the cell.The final expression and / or activity level profile of the cell can illustrate the result, such as the cell transformation from one cell type to another cell type (or its process).

[0068] In some cases, some of the multiple gate units disclosed herein may be necessary to produce the desired expression and / or activity profile of target cells, but may be individually insufficient.Therefore, the result (for example, enhanced cell function, induced cell state, etc.) in cells induced by multiple gate units may not be possible without any one of the multiple gate units.Alternatively, the degree or scale of the result in cells induced by multiple gate units may be different from the degree or scale of the result in control cells that are not induced by any of the multiple gate units, or are induced by one or more (but not all) of them, and / or by all of the multiple gate units that occur through various sequences of events (for example, greater for positive markers or smaller for negative markers).

[0069] The second gating unit can be activated (e.g., directly or indirectly) by the first gating unit. For example, the second gating unit can be activated directly by the first gating unit. Alternatively, the second gating unit can be activated by one or more additional gating units that are activated (e.g., directly or indirectly) by the first gating unit. The one or more additional gating units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gating units. The one or more additional gate units may comprise 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 can be activated via a separate moiety that participates in the activation of the first gate unit (e.g., an activation moiety, a different gate unit, etc.). In yet another alternative, the first gate unit and the second gate unit can be activated by different activation moieties (e.g., different polynucleotide molecules, such as different guide nucleic acid molecules).

[0070] In some cases, the first gate unit regulates a first target gene. Alternatively or additionally, the first gate unit can also regulate a second gate unit. The regulation of the second gate unit can be at least or at most about 1 millisecond, about 2 milliseconds, about 3 milliseconds, about 4 milliseconds, about 5 milliseconds, about 6 milliseconds, about 7 milliseconds, about 8 milliseconds, about 9 milliseconds, about 10 milliseconds, about 20 milliseconds, about 30 milliseconds, about 40 milliseconds, about 50 milliseconds, about 60 milliseconds, about 70 milliseconds, about 80 milliseconds, about 90 milliseconds, about 100 milliseconds, about 200 milliseconds, about 300 milliseconds, about 400 milliseconds, about 500 milliseconds, or about 600 milliseconds, as confirmed by rt-qPCR, Western blotting, or other methods. The time may occur after about 100 milliseconds, about 700 milliseconds, about 800 milliseconds, about 900 milliseconds, about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, or more.

[0071] Optionally, the second gate unit can modulate a second target gene, where the modulation of the second target gene is at least or at most about 1 millisecond, about 2 milliseconds, about 3 milliseconds, about 4 milliseconds, about 5 milliseconds, about 6 milliseconds, about 7 milliseconds, about 8 milliseconds, about 9 milliseconds, about 10 milliseconds, about 20 milliseconds, about 30 milliseconds, about 40 milliseconds, about 50 milliseconds, about 60 milliseconds, about 70 milliseconds, about 80 milliseconds, about 90 milliseconds, about 100 milliseconds, about 200 milliseconds, about 300 milliseconds, about 400 milliseconds, about 500 milliseconds, or about 600 milliseconds later than the modulation of the first target gene, as determined by rt-qPCR, Western blotting, or other methods. The time may occur after about 100 milliseconds, about 700 milliseconds, about 800 milliseconds, about 900 milliseconds, about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, or more.

[0072] In some cases, the term "proGuide" as generally used herein may refer to such a vector (e.g., a plasmid) encoding an activatable gNA. ProGuide may be an example of a gate portion. ProGuide may be an example of a gene regulatory portion.

[0073] The proGuides provided herein can encode, for example, an activatable guide nucleic acid molecule having an inactivating polynucleotide sequence (e.g., one or more polyX sequences, such as one or more polyT sequences). In some embodiments, the portion of the proGuide encoding the activatable guide nucleic acid molecule can include various regions linked sequentially, including a spacer sequence, an extra sequence (e.g., a linker sequence or backbone sequence), an upstream stem, a polyT unit, and a downstream stem. In some embodiments, the extra sequence can exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to GTTTTAGAGCTA (SEQ ID NO: 1935). In some embodiments, the portion of a proGuide encoding an activatable guide nucleic acid molecule can comprise various contiguously linked regions, including a spacer sequence, an extra sequence (e.g., a linker sequence or backbone sequence), an upstream stem, a polyT unit, and a downstream stem, as shown in Table 1 (SEQ ID NOS: 1-1932). Optionally, upon modification or removal of the polyT unit, the upstream stem and the downstream stem can form part of the scaffold sequence of a functional guide nucleic acid molecule. In some embodiments, the portion of a proGuide encoding an activatable guide nucleic acid molecule can comprise various contiguously linked regions, including a spacer sequence, an extra sequence (e.g., a linker sequence or backbone sequence), an upstream stem, a polyT unit, and a downstream stem, as shown in Table 1 (SEQ ID NOS: 1-1932) as a concatenated sequence with "-" to distinguish the various regions. Optionally, upon modification or removal of the polyT unit, the upstream stem and the downstream stem can form part of the scaffold sequence of a functional guide nucleic acid molecule.In some embodiments, if the spacer sequence does not begin with a G, a G is added before the spacer sequence. In some embodiments, adding a G before the spacer sequence serves to increase expression of the RNA portion from the promoter. In some embodiments, stem 1 and stem are reverse complements of each other. In some embodiments, the upstream stem and downstream stem are reverse complements of each other. In some embodiments, stem 1 may exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a member of the polynucleotide sequences set forth in Table 3 (SEQ ID NOS: 2024-2046). In some embodiments, Stem 2 may exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a member of the polynucleotide sequences set forth in Table 3 (SEQ ID NOs:2047-2069).

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

[0075] A domain of a polynucleotide sequence encoding (or corresponding to) a molecule of interest can include a polyX sequence. The polyX sequence can be sufficient to reduce expression of the molecule of interest (e.g., a guide nucleic acid molecule) from the polynucleotide sequence. For example, a polyX sequence can be placed within a domain encoding the molecule of interest (e.g., not at the 5' or 3' end of such domain) such that expression of the molecule of interest (e.g., transcription of an RNA molecule of interest) is disrupted (e.g., terminated) during expression.

[0076] Thus, a polyX sequence (e.g., within a polynucleotide sequence encoding a molecule of interest) may also be referred to as a termination sequence (e.g., a non-standard termination sequence for that sequence and / or its position), a disruption sequence (e.g., for disruption of all expression of a molecule of interest), or an inactivation sequence (e.g., for inactivating the function of a polynucleotide sequence or molecule of interest).

[0077] Optionally, the non-standard termination sequence is at least or at most about 40%, at least or at most about 45%, at least or at most about 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 86%, at least or at most about 87%, at least or at most about 88%, at least or at most about 89%, at least or at most about 90%, at least or at most about 91%, at least or at most about 92%, at least or at most about 93%, at least or at most about 94%, at least or at most about 95%, at least or at most about 96%, at least or at most about 97%, at least or at most about 98%, at least or at most about 99%, at least or at most about 100%, at least or at most about 101%, at least or at most about 102%, at least or at most about 103%, at least or at most about 104%, at least or at most about 105%, at least or at most about 106%, at least or at most about 107%, at least or at most about 108%, at least or at most about 109%, at least or at most about 110%, at least or at most about 111%, at least or at most about 112%, at least or at most about 113%, at least or at most about 114%, at least or at most about 115%, at least or at most about 116%, at least or at most about 117%, at least or at most about 118%, at least or at most about 119%, at least or at most about 120%, at least or at most about 121%, at least or at most about 122%, at least or at most about It may comprise or consist essentially of a polynucleotide sequence exhibiting up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity.

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

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

[0080] The polyX sequence (e.g., polyT or polyU sequence) can contain at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50X, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 bases. The polyX sequence can contain up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 14, up to about 13, up to about 12, up to about 11, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, or up to about 2X bases. The polyX sequence can be represented by a complementary polyX sequence in a corresponding complementary DNA strand (e.g., a polyT disclosed herein as a DNA sequence may also be referred to as a polyA in the complementary DNA strand). The disclosed polyX sequences can contain multiple X bases. The multiple X bases can be disclosed contiguously adjacent to each other (e.g., TT, TTT, TTTT, TTTTT, etc.). Alternatively or additionally, the multiple X bases may be separated by one or more additional nucleotides that are not X. The one or more additional nucleotides may comprise a single type of nucleotide or different types of nucleotides.

[0081] In some embodiments, the proGuide may comprise an inactivated polynucleotide sequence that exhibits at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to TTTTTTTTT or SEQ ID NO: 1933 or its complementary sequence.

[0082] In some embodiments, a proGuide can include a target polynucleotide domain at or adjacent to an inactivating polynucleotide sequence (e.g., at or adjacent to the 5' and / or 3' end of the inactivating polynucleotide sequence), which can be targeted (e.g., via the sequential activation mechanisms of heterologous gene circuits provided herein) to modify (e.g., edit, cleave) the inactivating polynucleotide sequence, thereby enabling the proGuide to express an activated guide nucleic acid molecule. The target polynucleotide domain of the proGuide does not exhibit sequence identity to any comparable endogenous polynucleotide sequence in the cell, thereby avoiding inadvertent targeting and regulation of endogenous target genes.

[0083] In some embodiments, the inactivation polynucleotide sequence of the proGuide can be placed between two target polynucleotide domains, which may or may not be targetable by a common guide nucleic acid sequence. In some cases, the two target polynucleotide domains can be reverse-complementary to each other so that the inactivation polynucleotide sequences can be modified or cleaved by the same mechanism (e.g., the same spacer sequence of the guide nucleic acid molecule).

[0084] In some embodiments, proGuide is selected from the group consisting of SEQ ID NOs: 1-92 (e.g., targeting ACAN), SEQ ID NOs: 93-184 (e.g., targeting COL2A1), SEQ ID NOs: 185-276 (e.g., targeting MEF2D), SEQ ID NOs: 277-460 (e.g., targeting pax9), SEQ ID NOs: 461-644 (e.g., targeting RUNX2), SEQ ID NOs: 645-920 (e.g., targeting SOX5), SEQ ID NOs: 921-1288 (e.g., targeting SOX6), SEQ ID NO: 1289 SEQ ID NOs: 1380 (e.g., SOX8 targeting), SEQ ID NOs: 1381-1472 (e.g., SOX9 targeting), SEQ ID NOs: 1473-1564 (e.g., SP1 targeting), SEQ ID NOs: 1565-1656 (e.g., TCF15 targeting), SEQ ID NOs: 1657-1748 (e.g., TCF7L2 targeting), SEQ ID NOs: 1749-1840 (e.g., TWIST1 targeting), SEQ ID NOs: 1841-1932 (e.g., UNCX targeting), or one or more members of a complementary sequence thereof. 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to

[0085] The second gate unit can be activated to induce the inactivation of the activated first gate unit.The terms "inactivation" and "destruction" can be used interchangeably herein.The inactivation disclosed herein can be induced by generating modifications (such as single-strand or double-strand breaks, indels, etc.) in at least a part of the first gate unit (such as the gate portion and / or gene regulatory portion of the first gate unit) that is involved in inducing the first separate regulation of the target gene.

[0086] Inactivation of the gate portion and / or gene regulatory portion of the first gate unit disclosed herein can be achieved through an endonuclease-based system (e.g., a CRISPR / Cas system). Alternatively or additionally, inactivation can be achieved by using a transcriptional modulator system (e.g., a transcriptional repressor). Alternatively or additionally, inactivation can be achieved by CRISPRi steric hindrance without the need for an additional transcriptional modulator. An endonuclease transcriptional modulator system (e.g., a Cas repressor) can be used to achieve polynucleotide cleavage (e.g., to inactivate the gate portion and / or gene regulatory portion). Polynucleotide cleavage can create nucleic acid modifications such as single-strand breaks, double-strand breaks, insertions, deletions, or insertion-deletions (indels). Alternatively or additionally, an endonuclease transcriptional modulator system (e.g., a Cas repressor) can be used to regulate target gene expression. Alternatively or additionally, CAS transcriptional modulator systems lacking endonuclease activity (dCAS or naked CAS with a shortened spacer insufficient to support cleavage) can target DNA regions and physically arrest transcription elongation, resulting in the repression of target genes (CRISPRi).

[0087] Alternatively, the second gate unit can be activated to amplify or enhance the activation of the activated first gate unit.The amplification or enhancement of the first gate unit can be induced by generating modifications (such as single-strand or double-strand breaks, indels, etc.) in at least a part of the first gate unit (such as the gate portion and / or gene regulatory portion of the first gate unit) that are involved in inducing the first distinct regulation of the target gene.

[0088] A heterologous gene circuit can include multiple gating units that are activated sequentially, e.g., serially, one after the other. The multiple gating units can include a functional gating unit that is preconfigured 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 gating units can further include one or more additional gating units that are preconfigured (i) to be activated before the functional gating unit and (ii) to result in the subsequent activation of the functional gating unit. Optionally, the one or more additional gating units can be preconfigured to be activated to regulate one or more additional target genes. Alternatively, the one or more additional gating units may not be preconfigured to regulate any target gene (e.g., any endogenous target gene) when activated. Such one or more additional gating units may instead serve to delay (e.g., in time) the activation of the functional gating unit during operation of the heterologous gene circuit, thereby delaying the expression and / or epigenetic profile of the functional gating unit's target gene; therefore, the one or more additional gating units may also be referred to as "blank" gating units.The heterologous gene circuit may have 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, at least or at most about 13 blank gate units, at least It can include at least or at most about 14 blank gate units, at least or at most about 15 blank gate units, at least or at most about 16 blank gate units, at least or at most about 27 blank gate units, at least or at most about 18 blank gate units, at least or at most about 19 blank gate units, at least or at most about 20 blank gate units, at least or at most about 25 blank gate units, at least or at most about 30 blank gate units, at least or at most about 35 blank gate units, at least or at most about 40 blank gate units, at least or at most about 45 blank gate units, or at least or at most about 50 blank gate units.

[0089] Optionally, the use of one or more blank gating units may inhibit activation of a functional gating unit (e.g., as confirmed by measuring the expression / epigenetic profile of the target gene or by measuring the expression of a functional variant or transcript of the functional gating unit) for 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 at most about 10 hours, , at least or at most about 11 hours, at least or at most about 12 hours, at least or at most about 13 hours, at least or at most about 14 hours, at least or at most about 15 hours, at least or at most about 16 hours, at least or at most about 17 hours, at least or at most about 18 hours, at least or at most about 19 hours, at least or at most about 20 hours, at least or at most about 21 hours, at least or at most about 22 hours, at least or at most about 23 hours, at least or at most about 24 hours, at least or at most about 2 days, at least or at most about 3 days, at least or at most about 4 days, at least or at most about 5 days, at least or at most about 6 days, or at least or at most about 7 days.

[0090] In some cases, the modification of the target gene by the gate unit can inactivate the gene. For example, the modification of the gene can stop the expression and / or activity level of the target gene. Alternatively, the modification of the gene can reduce the expression and / or activity level of the target gene. In some cases, the modification of the gene can increase the expression and / or activity level of the target gene. Alternatively, the modification of the gene can maintain the expression and / or activity level of the target gene.

[0091] In some cases, the genetic 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. The genetic modification may increase the expression and / or activity level of the target gene by up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 10%. The decrease can be as large as about 8%, by up to about 7%, by up to about 6%, by up to about 5%, by up to about 4%, by up to about 3%, by up to about 2%, by up to about 1%, by up to about 0.9%, by up to about 0.8%, by up to about 0.7%, by up to about 0.6%, by up to about 0.5%, by up to about 0.4%, by up to about 0.3%, by up to about 0.2%, by up to about 0.1%, or less.

[0092] In some cases, the genetic modification increases or decreases 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 10 ... The increase may be 0%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, at least about 1,000,000%, or more.The genetic modification may increase the expression and / or activity level of the target gene by up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, or %, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, up to about 0.1%, or less.

[0093] In some cases, the genetic modification increases the expression and / or activity level of the target gene by at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least or at most about 0.6-fold, at least or at most about 0.7-fold, at least or at most about 0.8-fold, at least or at most about 0.9-fold, at least or at most about 1-fold, at least or at most about 2-fold, at least or at most about 3-fold, at least or at most about 4-fold, at least or at most about 5-fold, at least or at most about The decrease may be 6-fold, at least or at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 60-fold, at least or at most about 70-fold, at least or at most about 80-fold, at least or at most about 90-fold, at least or at most about 100-fold, at least or at most about 500-fold, at least or at most about 1,000-fold, at least or at most about 5,000-fold, or at least or at most about 10,000-fold.The genetic modification may increase the expression and / or activity level of the target gene by up to about 10,000-fold or less than about 10,000-fold, up to about 5,000-fold or less than about 5,000-fold, up to about 1,000-fold or less than about 1,000-fold, up to about 500-fold or less than about 500-fold, up to about 100-fold or less than about 100-fold, up to about 90-fold or less than about 90-fold, up to about 80-fold or less than about 80-fold, up to about 70-fold or less than about 70-fold, up to about 60-fold or less than about 60-fold, up to about 50-fold or less than about 50-fold, up to about 40-fold or less than about 40-fold, up to about 30-fold or less than about 30-fold, up to about 20-fold or less than about 20-fold, up to about 10-fold or less than about 10-fold, or up to about 100-fold. The decrease can be at most about 9-fold or less than about 9-fold, at most about 8-fold or less than about 8-fold, at most about 7-fold or less than about 7-fold, at most about 6-fold or less than about 6-fold, at most about 5-fold or less than about 5-fold, at most about 4-fold or less than about 4-fold, at most about 3-fold or less than about 3-fold, at most about 2-fold or less than about 2-fold, at most about 1-fold or less than about 1-fold, at most about 0.9-fold or less than about 0.9-fold, at most about 0.8-fold or less than about 0.8-fold, at most about 0.7-fold or less than about 0.7-fold, at most about 0.6-fold or less than about 0.6-fold, at most about 0.5-fold or less than about 0.5-fold, at most about 0.4-fold or less than about 0.4-fold, at most about 0.3-fold or less than about 0.3-fold, at most about 0.2-fold or less than about 0.2-fold, or at most about 0.1-fold or less than about 0.1-fold.

[0094] In some cases, the genetic modification increases the expression and / or activity level of the target gene by at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least or at most about 0.6-fold, at least or at most about 0.7-fold, at least or at most about 0.8-fold, at least or at most about 0.9-fold, at least or at most about 1-fold, at least or at most about 2-fold, at least or at most about 3-fold, at least or at most about 4-fold, at least or at most about 5-fold, at least or at most about The increase can be 6-fold, at least or at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 60-fold, at least or at most about 70-fold, at least or at most about 80-fold, at least or at most about 90-fold, at least or at most about 100-fold, at least or at most about 500-fold, at least or at most about 1,000-fold, at least or at most about 5,000-fold, or at least or at most about 10,000-fold.The genetic modification may increase the expression and / or activity level of the target gene by up to about 10,000-fold or less than about 10,000-fold, up to about 5,000-fold or less than about 5,000-fold, up to about 1,000-fold or less than about 1,000-fold, up to about 500-fold or less than about 500-fold, up to about 100-fold or less than about 100-fold, up to about 90-fold or less than about 90-fold, up to about 80-fold or less than about 80-fold, up to about 70-fold or less than about 70-fold, up to about 60-fold or less than about 60-fold, up to about 50-fold or less than about 50-fold, up to about 40-fold or less than about 40-fold, up to about 30-fold or less than about 30-fold, up to about 20-fold or less than about 20-fold, up to about 10-fold or less than about 10-fold, or up to about 100-fold. The increase can be at most about 9-fold or less than about 9-fold, at most about 8-fold or less than about 8-fold, at most about 7-fold or less than about 7-fold, at most about 6-fold or less than about 6-fold, at most about 5-fold or less than about 5-fold, at most about 4-fold or less than about 4-fold, at most about 3-fold or less than about 3-fold, at most about 2-fold or less than about 2-fold, at most about 1-fold or less than about 1-fold, at most about 0.9-fold or less than about 0.9-fold, at most about 0.8-fold or less than about 0.8-fold, at most about 0.7-fold or less than about 0.7-fold, at most about 0.6-fold or less than about 0.6-fold, at most about 0.5-fold or less than about 0.5-fold, at most about 0.4-fold or less than about 0.4-fold, at most about 0.3-fold or less than about 0.3-fold, at most about 0.2-fold or less than about 0.2-fold, or at most about 0.1-fold or less than about 0.1-fold.

[0095] The expression and / or activity profile of a gene of interest (e.g., a differentiation marker) can be compared to a control gene (e.g., a housekeeping gene such as GAPDH), the relative expression levels of two or more genes of interest (e.g., the ratio of expression or activity levels between a stem cell marker and a differentiation marker), the relative average expression level of a gene of interest compared to the average expression level of the same gene of interest in a cell type of interest, etc.

[0096] In some cases, activation of the multiple gate units may be the result of a single activation of the heterologous gene circuit (e.g., by a single activation moiety at a single time point). The multiple gate units may include one of a first gate unit and a second gate preconfigured to be sequentially activated upon activation of the heterologous gene circuit by a single activation. In some cases, one of the first and second gate units may be activated by a single activation moiety (e.g., a guide nucleic acid), while the other of the first and second gate units may be activated by an additional activation moiety (e.g., a different guide nucleic acid) that is different from the activation moiety of the heterologous gene circuit. The additional activation moiety may be a part of the heterologous gene circuit that is generated (e.g., expressed) only upon activation of the heterologous gene circuit. Alternatively or additionally, the first and second gate units may each be activated by a different activation moiety that is not the same as the activation moiety of the heterologous gene circuit. Such a different activation moiety may be a part of the heterologous gene circuit that is generated (e.g., expressed) only upon activation of the heterologous gene circuit.

[0097] In some embodiments of any one of the systems disclosed herein, the gating unit can include a gating moiety (e.g., at least or about 1 gating moiety, at least or about 2 gating moieties, at least or about 3 gating moieties, at least or about 4 gating moieties, at least or about 5 gating moieties, etc.) and / or a gene regulatory moiety (e.g., at least or about 1 gene regulatory moiety, at least or about 2 gene regulatory moieties, at least or about 3 gene regulatory moieties, at least or about 4 gene regulatory moieties, at least or about 5 gene regulatory moieties, at least or about 6 gene regulatory moieties, at least or about 7 gene regulatory moieties, at least or about 8 gene regulatory moieties, at least or about 9 gene regulatory moieties, at least or about 10 gene regulatory moieties, etc.). The gating moieties disclosed herein can include guide nucleic acid molecules (gNAs) (e.g., at least or at most about 1 gNA molecule, at least or at most about 2 gNA molecules, at least or at most about 3 gNA molecules, at least or at most about 4 gNA molecules, at least or at most about 5 gNA molecules, etc.). The gene regulatory moieties disclosed herein can comprise gNAs (e.g., at least or at most about 1 gNA molecule, at least or at most about 2 gNA molecules, at least or at most about 3 gNA molecules, at least or at most about 4 gNA molecules, at least or at most about 5 gNA molecules, etc.). The guide nucleic acid molecules disclosed herein can comprise, but are not limited to, DNA, RNA, any analog thereof, or any combination thereof. In some embodiments of any one of the systems disclosed herein, the gate moiety and / or gene regulatory moiety can be activatable to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and the complex is configured to or can bind to a target polynucleotide, for example, to regulate the expression and / or activity level of the target polynucleotide or another polynucleotide sequence operably linked to the target polynucleotide.For example, the complex can modulate the expression and / or activity level of a gene that comprises the target polynucleotide.

[0098] In some cases, the guide nucleic acid molecule (gNA) (e.g., functional gNA) expressed by the second gate unit can cause a modification to at least a portion of the first gate unit when activated. For example, the activated gNA of the second gate unit can cause a modification to the polynucleotide sequence of the first gate unit encoding the gNA (e.g., activatable gNA), or to the promoter sequence of the first gate unit operably linked to such gNA of the same first gate unit. Such a modification can render the gNA of the first gate unit inoperable upon expression (e.g., reduce or inhibit specific binding to the target gene). Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0099] In some cases, the modification of polynucleotide sequence (for example, as a component of a gate unit such as a gate portion) or target gene can be caused by single-strand breaks, which are discontinuous in one nucleotide strand.The inactivation of 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, the inactivation of gene can be caused by up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 single-strand breaks.

[0100] In some cases, the gNA can have a size (e.g., including both spacer and scaffold sequences) of at least or at most about 60 nucleotides, at least or at most about 70 nucleotides, at least or at most about 80 nucleotides, at least or at most about 85 nucleotides, at least or at most about 90 nucleotides, at least or at most about 95 nucleotides, at least or at most about 100 nucleotides, at least or at most about 105 nucleotides, at least or at most about 110 nucleotides, at least or at most about 120 nucleotides, at least or at most about 130 nucleotides, at least or at most about 140 nucleotides, at least or at most about 150 nucleotides, or at least or at most about 200 nucleotides.

[0101] In some cases, the scaffold sequence of the gNA can have a size of at least or at most about 30 nucleotides, at least or at most about 35 nucleotides, at least or at most about 40 nucleotides, at least or at most about 45 nucleotides, at least or at most about 50 nucleotides, at least or at most about 55 nucleotides, at least or at most about 60 nucleotides, at least or at most about 65 nucleotides, at least or at most about 70 nucleotides, at least or at most about 75 nucleotides, at least or at most about 80 nucleotides, at least or at most about 85 nucleotides, at least or at most about 90 nucleotides, at least or at most about 95 nucleotides, at least or at most about 100 nucleotides, at least or at most about 100 nucleotides, at least or at most about 120 nucleotides, at least or at most about 130 nucleotides, at least or at most about 140 nucleotides, or at least or at most about 150 nucleotides.

[0102] In some cases, the spacer sequence of the gNA can have a size of at least or at most about 10 nucleotides, at least or at most about 11, at least or at most about 12, at least or at most about 13, at least or at most about 14, at least or at most about 15, at least or at most about 16, at least or at most about 17, at least or at most about 18, at least or at most about 19, at least or at most about 20, at least or at most about 21, at least or at most about 22, at least or at most about 23, at least or at most about 24, at least or at most about 25, at least or at most about 26, at least or at most about 27, at least or at most about 28, at least or at most about 29, or at least or at most about 30 nucleotides.

[0103] In some embodiments of any one of the systems disclosed herein, the initial (or first) gating unit of a heterologous genetic circuit disclosed herein can be activated (e.g., directly activated) by an activating moiety. The activating moiety can directly bind to at least a portion of the initial gating unit to activate the initial gating unit, thereby, for example, sequentially activating the heterologous genetic circuit. Alternatively, the activating moiety can activate an initial gating unit without directly binding to at least a portion of the initial gating unit (e.g., by using electromagnetic energy). In some cases, the initial gating unit can include at least one gating moiety and at least one gene regulatory moiety. In some cases, the initial gating unit can include at least one gating moiety, but may not, and need not, include a gene regulatory moiety. In some cases, the initial gating unit can include at least one gene regulatory moiety, but may not, and need not, include a gating moiety (e.g., the activating moiety can be configured to activate the initial gating unit and at least one additional gating unit).

[0104] In some embodiments of any one of the systems disclosed herein, the gNA of the gating moiety and / or gene regulatory moiety (e.g., the gNA encoded by the gating moiety and / or gene regulatory moiety) can be an activatable gNA. The activatable gNA can be, but is not limited to, a ribonucleotide (e.g., a gRNA), a deoxyribonucleotide, any analog thereof, or any combination thereof. In some embodiments, the vector (or expression cassette) encoding the activatable gNA can include an inactivation polynucleotide sequence to render the gNA inactive until activated (e.g., until the inactivation polynucleotide sequence is modified or removed from the vector). For example, the inactivation polynucleotide sequence can encode a self-cleaving polynucleotide molecule (e.g., a ribozyme). Alternatively, or in addition, the inactivation polynucleotide sequence can encode a non-canonical transcription termination sequence, as described below. The inactivating polynucleotide sequence may be part of or adjacent to the region of the vector encoding (i) the spacer sequence of the gNA, (ii) the scaffold sequence of the gNA, and / or (ii) any linker sequence between the spacer sequence and the scaffold sequence. The vector may comprise at least or up to about 1 inactivating polynucleotide sequence, at least or up to about 2 inactivating polynucleotide sequences, at least or up to about 3 inactivating polynucleotide sequences, at least or up to about 4 inactivating polynucleotide sequences, at least or up to about 5 inactivating polynucleotide sequences, at least or up to about 6 inactivating polynucleotide sequences, at least or up to about 7 inactivating polynucleotide sequences, at least or up to about 8 inactivating polynucleotide sequences, at least or up to about 9 inactivating polynucleotide sequences, or at least or up to about 10 inactivating polynucleotide sequences.

[0105] In some cases, the term "proGuide" as generally used herein may refer to such a vector (e.g., a plasmid) encoding an activatable gNA. ProGuide may be an example of a gate portion. ProGuide may be an example of a gene regulatory portion.

[0106] In some embodiments, the activatable gNA molecule can be a self-cleaving gNA (e.g., the gRNA contains a cis ribozyme). For example, when the activatable gNA is expressed in a cell, the activatable gNA can be self-cleaving such that it is non-functional (e.g., not configured to bind to a target gene) unless the gene encoding the activatable gNA is modified prior to expression of the activatable gNA. In some embodiments, the activatable gNA molecule contains a non-canonical transcription termination sequence (e.g., a polyX sequence, such as a polyU or polyT sequence) such that a functional gNA molecule is not expressed until the gene encoding the activatable gNA with the non-canonical transcription termination sequence is modified (e.g., to remove part or all of the transcription termination sequence). Thus, in the absence of transcription termination sequence modification, a non-functional variant (e.g., a non-functional fragment) of the gNA can be expressed. In some embodiments, the gNA can be synthetic. In some embodiments, the gNA can have an attached fluorescent label.

[0107] In some cases, the size of the polyT sequence is equal to or greater than a threshold length, and the threshold length is sufficient to reduce expression of the guide nucleic acid molecule from the polynucleotide sequence. Thus, a plasmid (e.g., a gate portion or gene regulatory portion) can encode an inactivated gNA comprising a polyT sequence equal to or greater than a threshold length, and editing such a plasmid to reduce the length of the polyT to less than the threshold length can express the gNA in its entirety without an initial termination, thereby activating the gNA. Optionally, the polyT sequence comprises at least 5 Ts. Optionally, the polyT sequence comprises at least 7 Ts. Optionally, the polyT sequence comprises at least 8 Ts. Optionally, the polyT sequence comprises at least 10 Ts. Optionally, the polyT sequence comprises between 5 Ts and 15 Ts. Optionally, the polyT sequence comprises one or more additional nucleotides that are not Ts.

[0108] In some cases, the gene regulatory portion (e.g., guide nucleic acid and / or endonuclease) can be configured to bind to a target polynucleotide sequence operably linked to a target gene in a cell. The target gene can include a coding polynucleotide sequence that encodes a target nucleic acid molecule or a target protein. The target polynucleotide sequence can be a part of the coding polynucleotide sequence. Alternatively, the target polynucleotide sequence may not be a part of the coding polynucleotide sequence. For example, the target polynucleotide sequence can be upstream of the coding polynucleotide sequence (e.g., a part of the promoter of the coding polynucleotide sequence, such as a transcription start site (TSS)).

[0109] As provided herein, when a heterologous gene circuit is activated to induce multiple distinct modulations of target genes, as provided herein, the multiple distinct modulations of the target genes can be different (e.g., different degrees of change in the expression and / or activity levels of the target genes). For example, a first regulation exerted by a first genetic unit and a second regulation exerted by a second gating unit may differ by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%. The first adjustment and the second adjustment can differ by up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, or up to about 0.1%. Alternatively or additionally, the separate modulations of the target genes may be substantially the same (e.g., the same). The separate modulations may be individually sufficient to induce a desired change in the expression and / or activity levels of the target genes. Alternatively, the separate modulations may be individually insufficient to induce a desired change in the expression and / or activity levels of the target genes.

[0110] The one or more target genes disclosed herein can include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0111] In some cases, the guide nucleic acid molecule (gNA) (e.g., functional gNA) expressed by the second gate unit can cause a modification to at least a portion of the first gate unit when activated. For example, the activated gNA of the second gate unit can cause a modification to the polynucleotide sequence of the first gate unit encoding the gNA (e.g., activatable gNA), or to the promoter sequence of the first gate unit operably linked to such gNA of the same first gate unit. Such a modification can render the gNA of the first gate unit inoperable upon expression (e.g., reduce or inhibit specific binding to the target gene). Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0112] In some embodiments of any one of the systems disclosed herein, the initial (or first) gating unit of a heterologous genetic circuit disclosed herein can be activated (e.g., directly activated) by an activating moiety. The activating moiety can directly bind to at least a portion of the initial gating unit to activate the initial gating unit, thereby, for example, sequentially activating the heterologous genetic circuit. Alternatively, the activating moiety can activate the initial gating unit without directly binding to at least a portion of the initial gating unit (e.g., by using electromagnetic energy). In some cases, the initial gating unit can include at least one gating moiety and at least one gene regulatory moiety. In some cases, the initial gating unit can include at least one gating moiety, but may not, and need not, include a gene regulatory moiety. In some cases, the initial gating unit can include at least one gene regulatory moiety, but may not, and need not, include a gating moiety (e.g., the activating moiety can be configured to activate the initial gating unit and at least one additional gating unit).

[0113] In some embodiments of any one of the systems disclosed herein, the gNA of the gating moiety and / or gene regulatory moiety (e.g., the gNA encoded by the gating moiety and / or gene regulatory moiety) can be an activatable gNA. The activatable gNA can be, but is not limited to, a ribonucleotide (e.g., a gRNA), a deoxyribonucleotide, any analog thereof, or any combination thereof. In some embodiments, the activatable gNA molecule can be a self-cleaving gNA (e.g., the gRNA contains a cis-ribozyme). For example, when the activatable gNA is expressed in a cell, the activatable gNA can be self-cleavable such that it is non-functional (e.g., not configured to bind to a target gene) unless the gene encoding the activatable gNA is modified prior to expression of the activatable gNA. In some embodiments, the gNA can be synthetic. In some embodiments, the gNA can have an attached fluorescent label.

[0114] In some cases, the guide nucleic acid molecule (gNA) (e.g., functional gNA) expressed by the second gate unit can cause a modification to at least a portion of the first gate unit when activated. For example, the activated gNA of the second gate unit can cause a modification to the polynucleotide sequence of the first gate unit encoding the gNA (e.g., activatable gNA), or to the promoter sequence of the first gate unit operably linked to such gNA of the same first gate unit. Such a modification can render the gNA of the first gate unit inoperable upon expression (e.g., reduce or inhibit specific binding to the target gene). Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0115] In some cases, the modification of polynucleotide sequence (for example, as a component of a gate unit such as a gate portion) or target gene can be caused by single-strand breaks, which are discontinuous in one nucleotide strand.The inactivation of 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, the inactivation of gene can be caused by up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 single-strand breaks.

[0116] In some cases, the gNA can have a size (e.g., including both spacer and scaffold sequences) of at least or at most about 60 nucleotides, at least or at most about 70 nucleotides, at least or at most about 80 nucleotides, at least or at most about 85 nucleotides, at least or at most about 90 nucleotides, at least or at most about 95 nucleotides, at least or at most about 100 nucleotides, at least or at most about 105 nucleotides, at least or at most about 110 nucleotides, at least or at most about 120 nucleotides, at least or at most about 130 nucleotides, at least or at most about 140 nucleotides, at least or at most about 150 nucleotides, or at least or at most about 200 nucleotides.

[0117] In some cases, the scaffold sequence of the gNA can have a size of at least or at most about 30 nucleotides, at least or at most about 35 nucleotides, at least or at most about 40 nucleotides, at least or at most about 45 nucleotides, at least or at most about 50 nucleotides, at least or at most about 55 nucleotides, at least or at most about 60 nucleotides, at least or at most about 65 nucleotides, at least or at most about 70 nucleotides, at least or at most about 75 nucleotides, at least or at most about 80 nucleotides, at least or at most about 85 nucleotides, at least or at most about 90 nucleotides, at least or at most about 95 nucleotides, at least or at most about 100 nucleotides, at least or at most about 100 nucleotides, at least or at most about 120 nucleotides, at least or at most about 130 nucleotides, at least or at most about 140 nucleotides, or at least or at most about 150 nucleotides.

[0118] In some cases, the spacer sequence of the gNA can have a size of at least or at most about 10 nucleotides, at least or at most about 11, at least or at most about 12, at least or at most about 13, at least or at most about 14, at least or at most about 15, at least or at most about 16, at least or at most about 17, at least or at most about 18, at least or at most about 19, at least or at most about 20, at least or at most about 21, at least or at most about 22, at least or at most about 23, at least or at most about 24, at least or at most about 25, at least or at most about 26, at least or at most about 27, at least or at most about 28, at least or at most about 29, or at least or at most about 30 nucleotides.

[0119] In some embodiments of any one of the systems disclosed herein, the gNA of the gate portion and / or gene regulatory portion (e.g., the gNA encoded by the gate portion and / or gene regulatory portion) can include a spacer sequence. In some cases, the spacer sequence can be specific to the target gene. Alternatively, the spacer sequence can be independent of the target gene.

[0120] As described above, the length of the spacer sequence of a gNA can affect the ability of the gNA to mediate Cas nuclease activity. In some cases, gNAs with spacer sequences of different lengths can be used in the same heterologous gene circuit to affect different types of cleavage, activation, inactivation, and / or regulation of one or more target nucleic acids. In some cases, a gNA spacer sequence shorter than a threshold length (e.g., about 16 nucleotides) can eliminate the nuclease activity of a Cas transcriptional modulator while still mediating DNA binding for transcriptional regulation of a target gene. In some cases, a gNA spacer sequence shorter than at least about 25 nucleotides, at least about 20 nucleotides, at least about 19 nucleotides, at least about 18 nucleotides, at least about 17 nucleotides, at least about 16 nucleotides, at least about 15 nucleotides, at least about 15 nucleotides, at least about 14 nucleotides, at least about 13 nucleotides, at least about 12 nucleotides, at least about 11 nucleotides, or at least about 10 nucleotides can eliminate the nuclease activity of a Cas protein while still mediating DNA binding.

[0121] For example, a gNA containing a 20-nucleotide spacer sequence (e.g., a gNA encoded by a gate portion for targeting a gene regulatory portion plasmid) may be sufficient to promote the nuclease activity of an endonuclease (e.g., a Cas or Cas transcriptional modulator fusion protein) at the target polynucleotide sequence. Alternatively or additionally, a gNA containing a 14-nucleotide spacer sequence (e.g., a gNA encoded by a gene regulatory portion) may be able to hybridize to DNA but may not be long enough to mediate nuclease activity; it may only promote endonuclease binding to the cognate DNA sequence. Thus, shorter gNAs can selectively allow transcriptional regulation of target genes through the use of an endonuclease-transcriptional modulator system (e.g., a Cas activator system, a Cas repressor system) without cleaving the target gene.

[0122] In some cases, the modification of polynucleotide sequence (for example, as a component of a gate unit such as a gate portion) or target gene can be caused by double-strand breaks, where discontinuities exist in both nucleotide strands.In some cases, the number of such double-strand breaks (for example, required for such modification) can be at least or at most about 1, at least or at most about 2, at least or at most about 3, at least or at most about 4, at least or at most about 5, at least or at most about 6, at least or at most about 7, at least or at most about 8, at least or at most about 9, or at least or at most about 10.

[0123] In some cases, modifications of polynucleotide sequences (e.g., as components of gate units such as gate segments) or target genes can be caused by indels, also known as insertion-deletion mutations. Indel mutations can include frameshift or non-frameshift mutations. Indel mutations can include point mutations, also known as base substitutions, in which only one base or base pair is modified. Indel mutations can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, or more bases or base pairs in length. Indel mutations comprise a length of up to about 2000, up to about 1000, up to about 900, up to about 800, up to about 700, up to about 600, up to about 500, up to about 400, up to about 300, up to about 200, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 base or base pair.

[0124] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can be achieved without cleavage of the polynucleotide sequence or the target gene. For example, a gene regulatory portion (e.g., a nucleic acid molecule and / or an endonuclease, e.g., a complex comprising a CRISPR / Cas protein and a guide nucleic acid molecule) can specifically bind to a polynucleotide sequence or a target gene so that the expression and / or activity of the polynucleotide sequence or the target gene is modified. The gene regulatory portion can include a transcriptional repressor or transcriptional activator provided herein. Alternatively or additionally, the gene regulatory portion can induce epigenetic modification (or epigenomic modification) provided herein.

[0125] In some cases, the modification of the polynucleotide sequence or target gene provided herein can inactivate the polynucleotide sequence or target gene.For example, the modification of the polynucleotide sequence or target gene can suppress or reduce the expression and / or activity level of the polynucleotide sequence or target gene.In some cases, the modification of the polynucleotide sequence or target gene provided herein can activate the polynucleotide sequence or target gene.For example, the modification of the polynucleotide sequence or target gene can increase the expression and / or activity level of the polynucleotide sequence or target gene.

[0126] In some cases, the modifications of the polynucleotide sequences or target genes provided herein may increase the expression and / or activity levels of the polynucleotide sequences or target genes by at least or at most about 0.1%, at least or at most about 0.2%, at least or at most about 0.3%, at least or at most about 0.4%, at least or at most about 0.5%, at least or at most about 1%, at least or at most about 2%, at least or at most about 3%, at least or at most about 4% (e.g., compared to a control lacking the modification). , by at least or at most about 5%, by at least or at most about 10%, by at least or at most about 15%, by at least or at most about 20%, by at least or at most about 30%, by at least or at most about 40%, by at least or at most about 50%, by at least or at most about 60%, by at least or at most about 70%, by at least or at most about 80%, by at least or at most about 90%, by at least or at most about 95%, by at least or at most about 99%, or by about 100%.

[0127] In some cases, modifications of a polynucleotide sequence or target gene provided herein increase the expression and / or activity level of the polynucleotide sequence or target gene by at least or at most about 0.1 fold (e.g., compared to a control lacking the modification), at least or at most about 0.2 fold, at least or at most about 0.3 fold, at least or at most about 0.4 fold, at least or at most about 0.5 fold, at least or at most about 0.6 fold, at least or at most about 0.7 fold, at least or at most about 0.8 fold, at least or at most about 0.9 fold, at least or at most about 1 fold, at least or at most about 1.5 fold, at least or at most about 2 fold, at least or at most about 3 fold, at least or at most about 4 fold, at least or at most about 5 fold, at least or at most about 6 fold, at least or at most about 7 fold, at least or at most about 8 fold, at least or at most about 9 fold, at least or at most about 10 fold, at least or at most about 11 fold, at least or at most about 12 fold, at least or at most about 13 fold, at least or at most about 14 fold, at least or at most about 15 fold, at least or at most about 16 fold, at least or at most about 17 fold, at least or at most about 18 fold, at least or at most about 19 fold, at least or at most about 20 fold, at least or at most about 21 fold, at least or at most about 22 fold, at least or at most about 23 fold, at least or at most about 24 fold, at least or at most about 25 fold, at least or at most about 26 fold, at least or at most about 28 fold, at least or at most about 29 fold, at least or at most about 3 Alternatively, it may be decreased by at most about 3-fold, at least or at most about 4-fold, at least or at most about 5-fold, at least or at most about 6-fold, at least or at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 11-fold, at least or at most about 12-fold, at least or at most about 13-fold, at least or at most about 14-fold, at least or at most about 15-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, or at least or at most about 100-fold.

[0128] In some cases, modifications of the polynucleotide sequence or target gene provided herein may increase the expression and / or activity level of the polynucleotide sequence or target gene by at least or at most about 0.1%, at least or at most about 0.2%, at least or at most about 0.3%, at least or at most about 0.4%, at least or at most about 0.5%, at least or at most about 1%, at least or at most about 2%, at least or at most about 3%, at least or at most about 4%, at least or at most about 5%, at least or at most about 10%, (e.g., compared to a control lacking the modification). This can include an increase of at least or at most about 15%, at least or at most about 20%, at least or at most about 30%, at least or at most about 40%, at least or at most about 50%, at least or at most about 60%, at least or at most about 70%, at least or at most about 80%, at least or at most about 90%, at least or at most about 100%, at least or at most about 150%, at least or at most about 200%, at least or at most about 300%, at least or at most about 400%, or at least or at most about 500%.

[0129] In some cases, modifications of a polynucleotide sequence or target gene provided herein increase the expression and / or activity level of the polynucleotide sequence or target gene by at least or at most about 0.1 fold (e.g., compared to a control lacking the modification), at least or at most about 0.2 fold, at least or at most about 0.3 fold, at least or at most about 0.4 fold, at least or at most about 0.5 fold, at least or at most about 0.6 fold, at least or at most about 0.7 fold, at least or at most about 0.8 fold, at least or at most about 0.9 fold, at least or at most about 1 fold, at least or at most about 1.5 fold, at least or at most about 2 fold, at least or at most about 3 fold, at least or at most about 4 fold, at least or at most about 5 fold, or more. The increase can be at least about 6-fold, at least or at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 11-fold, at least or at most about 12-fold, at least or at most about 13-fold, at least or at most about 14-fold, at least or at most about 15-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 100-fold, at least or at most about 200-fold, at least or at most about 300-fold, at least or at most about 400-fold, at least or at most about 500-fold, or at least or at most about 1,000-fold.

[0130] In some embodiments of any one of the systems disclosed herein, the gNA of a gating moiety and / or a gene regulatory moiety (e.g., a gNA encoded by the gating moiety and / or a gene regulatory moiety) can include a spacer sequence. In some cases, the spacer sequence can exhibit specific binding to a target gene (e.g., an endogenous target gene). Alternatively, the spacer sequence can be target gene-independent, but rather, can exhibit specific binding to a target polynucleotide sequence of another gating moiety or another gene regulatory moiety. Exemplary spacer sequences can be found in Table 2 (SEQ ID NOs: 1940-2023).

[0131] Non-limiting examples of the one or more target genes can include ACAN, COL2A1, MEF2D, pax9, RUNX2, SOX5, SOX6, SOX8, SOX9, SP1, TCF15, TCF7L2, TWIST1, and / or UNCX.In some cases, the spacer sequence of the guide nucleic acid (e.g., guide RNA) for the target gene provided herein may be any of SEQ ID NOs: 1940-1943 (e.g., for targeting ACAN), 1944-1947 (e.g., for targeting COL2A1), 1948-1951 (e.g., for targeting MEF2D), 1952-1955, 1956-1959 (e.g., for targeting pax9), 1960-1963, 1964-1967 (e.g., for targeting RUNX2), 1968-1971, 1972-1975, 1976-1977, 1978-1979, 1980-1981, 1982-1983, 1984-1985, 1986-1987, 1988-1989, 1990-1991, 1992-1993, 1994-1995, 1996-1997, 1998-2000, 1999-2001, 2002-2003, 2004-2005, 2006-2007, 2008-2009, 2010-2011, 2012-2013, 2014-2015, 2016-2017, 2018-2019, 2019-2020, 2019-2021, 2019-2022, 2019-2023, 2019-2024, 2019-2025, 20 9 (e.g., SOX5 targeting), SEQ ID NOs: 1980-1983, 1984-1987, 1988-1991, 1992-1995 (e.g., SOX6 targeting), SEQ ID NOs: 1996-1999 (e.g., SOX8 targeting), SEQ ID NOs: 2000-2003 (e.g., SOX9 targeting), SEQ ID NOs: 2004-2007 (e.g., SP1 targeting), SEQ ID NOs: 2008-2011 (e.g., TCF15 targeting), SEQ ID NOs: 2012-2015 (e.g., TCF7L2 targeting), SEQ ID NOs: 2016-2019 (e.g., TWIST1 targeting), SEQ ID NO: 2 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 86%, at least or at most about 87%, at least or at most about 88%, at least or at most about 90%, at least or at most about 91%, at least or at most about 92%, at least or at most about 93%, at least or at most about 94%, at least or at most about 95%, at least or at most about 96%, at least or at most about 97%, at least or at most about 98%, at least or at most about 99%, at least or at most about 100%, at least or at most about 101%, at least or at most about 102%, at least or at most about 103%, at least or at most about 104%, at least or at most about 105%, at least or at most about 106%, at least or at most about 107%, at least or at most about 108%, at least or at most about 109%, at least or at most about 110%, at least or at most about 111%, at least or at most about 112%, at least or at most about 113%, at least or at most about 114%, at least or at most about 115%, at least or at most about 116%, at least or at most about 117%, at least or at most about 118%, at least or at most about 119%, at least or at most about 120%, at least or at most about 121%, at least or at most about 122%, at least or at most about 123%, at least or at most about 124%, at least or at most about 125%, at least or at most about 126%, at least or at most about 127 The polynucleotide sequences may comprise polynucleotide sequences (e.g., contiguous polynucleotide sequences) exhibiting at least or at most about 89%, at least or at most about 90%, at least or at most about 91%, at least or at most about 92%, at least or at most about 93%, at least or at most about 94%, at least or at most about 95%, at least or at most about 96%, at least or at most about 97%, at least or at most about 98%, at least or at most about 99%, or substantially about 100% sequence identity.In some cases, the heterologous gene modulators provided herein are selected from the group consisting of SEQ ID NOs: 1940-1943 (e.g., targeting ACAN), SEQ ID NOs: 1944-1947 (e.g., targeting COL2A1), SEQ ID NOs: 1948-1951 (e.g., targeting MEF2D), SEQ ID NOs: 1952-1955, 1956-1959 (e.g., targeting pax9), SEQ ID NOs: 1960-1963, 1964-1967 (e.g., targeting RUNX2), SEQ ID NOs: 1968-1971, 1972-1975, 1976-1979 (e.g., targeting SOX5), SEQ ID NOs: 1980-1983, 1984-1985, 1986-1987 (e.g., targeting SOX5), SEQ ID NOs: 1987-1989, 1988-1990, 1991-1992, 1993-1994, 1995-1996 (e.g., targeting SOX5), SEQ ID NOs: 1996-2000, 2000-2001, 2001-2002, 2002-2003, 2003-2004, 2004-2005, 2005-2006, 2006-2007, 2008-2009, 2010-2011, 2012-2013, 2014-2015, 2016-2017, 2018 SEQ ID NOs: 83, 1984-1987, 1988-1991, 1992-1995 (e.g., SOX6 targeting), SEQ ID NOs: 1996-1999 (e.g., SOX8 targeting), SEQ ID NOs: 2000-2003 (e.g., SOX9 targeting), SEQ ID NOs: 2004-2007 (e.g., SP1 targeting), SEQ ID NOs: 2008-2011 (e.g., TCF15 targeting), SEQ ID NOs: 2012-2015 (e.g., TCF7L2 targeting), SEQ ID NOs: 2016-2019 (e.g., TWIST1 targeting), SEQ ID NOs: 2020-2023 (e.g., UNCX targeting), or their complementary sequences. For one or more members selected from the sequences (e.g., having a uracil to thymine conversion), at least or at most about 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 86%, at least or at most about 87%, at least or at most about 88%, at least or at most about 89%, at least or at most about 90%, at least or at most about 91%, at least or at most about 92%, at least or at most about 93%, at least or at most about 94%, at least or at most about 95%, at least or at most about 96%, at least or at most about 97%, at least or at most about 98%, at least or at most about 99%, at least or at most about 100%, at least or at most about 101%, at least or at most about 102%, at least or at most about 103%, at least or at most about 104%, at least or at most about 105%, at least or at most about 106%, at least or at most about 107%, at least or at most about 108%, at least or at most about 109%, at least or at most about 110%, at least or at most about 111%, at least or at most about 112%, at least or at most about 113%, at least or at most about 114%, at least or at most about 115%, at least or at most about 116%, at least or at most about 117%, at least or at most about 118%, at least or at most about 119%, at least or at most about 120%, at least or at most about 121%, at least or at most about 122%, at least or at most about 123%, at 9%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to a target gene (e.g., a contiguous polynucleotide sequence).

[0132] The gene regulatory moieties disclosed herein can include an endonuclease, such as a CRISPR-Cas protein, that exhibits at least some of its nuclease activity. For example, the nuclease activity can be used to activate the expression or activity of a guide nucleic acid molecule, thereby activating at least a portion of a heterologous gene circuit described herein.

[0133] The gene regulatory moieties disclosed herein can include an endonuclease operably linked to a transcriptional effector, including a transcriptional activator or transcriptional repressor, heterologous to the cell. The endonuclease can be native or engineered to exhibit reduced (or substantially no) nuclease activity, so that the endonuclease can be used to specifically bind to a target gene (e.g., an endogenous gene such as TBX, bHLH, SOX, or collagen) without cleaving the target gene. In some cases, the nuclease can be an inactivated Cas (dCas). Alternatively, once the endonuclease identifies and binds to a target gene, a transcriptional effector linked (e.g., covalently or non-covalently) to the endonuclease can interact with the target gene to increase or decrease its expression level, thereby increasing or decreasing the activity level of the target gene. For example, the endonuclease and transcriptional effector can be part of a fusion protein encoded by the same expression cassette.

[0134] Figure 10 illustrates the use of a heterologous gene circuit in combination with an endonuclease-transcription effector fusion (e.g., a CRISPR-Cas transcription activator, such as Cas9-VPR). Each gate moiety can be a modified self-inactivating (e.g., self-destructing) guide RNA that, when not inactivated, is configured to form a complex with a CRISPR-Cas transcription effector fusion. An initial activating moiety (denoted as an activating guide RNA or "aGuide") can convert the first gate moiety into an activating guide RNA (denoted as a matureGuide). Each matureGuide targets an additional gene regulatory moiety encoding an activatable guide RNA (denoted as a ramGuide) for a target gene, activating such a ramGuide. The activated ramGuide can then form a complex with a CRISPR-Cas transcription effector fusion protein to bind to the target gene and regulate the expression level of the target gene. The activated matureGuide can then target additional gate moieties downstream in the signaling cascade of the heterologous gene circuit, subsequently regulating the expression of one or more additional genes.

[0135] In some embodiments, the transcription effector can be a histone epigenetic modifier (or histone modifier). Optionally, the histone epigenetic modifier can regulate histones through methylation (e.g., a histone methylation modifier, e.g., an amino acid methyltransferase, e.g., KRAB). Optionally, the histone epigenetic modifier can regulate histones through acetylation. Optionally, the histone epigenetic modifier can regulate histones through phosphorylation. Optionally, the histone epigenetic modifier can regulate histones through ADP-ribosylation. Optionally, the histone epigenetic modifier can regulate histones through glycosylation. Optionally, the histone epigenetic modifier can regulate histones through sumoylation. Optionally, the histone epigenetic modifier can regulate histones through ubiquitination. In some cases, histone epigenetic modifiers can regulate histones by remodeling histone structure, for example, through an ATP hydrolysis-dependent process.

[0136] In some embodiments, the transcription effector can be a gene epigenetic modifier (or gene modifier). In some cases, the gene modifier can regulate genes through methylation (e.g., gene methylation modifiers such as DNA methyltransferases or DNMTs). In some cases, the gene modifier can regulate genes through acetylation.

[0137] In some embodiments, transcriptional effectors may be derived from families of related histone acetyltransferases, non-limiting examples of which include the GNAT subfamily, the MYST subfamily, the p300 / CBP subfamily, the HAT1 subfamily, GCN5, PCAF, Tip60, MOZ, MORF, MOF, HBO1, p300, CBP, HAT1, ATF-2, SRC1, and TAFII250.

[0138] In some embodiments, the transcription effector may be derived from a histone lysine methyltransferase. Non-limiting examples of histone lysine methyltransferases include EZH subfamily, non-SET subfamily, other SET subfamily, PRDM subfamily, SET1 subfamily, SET2 subfamily, SUV39 subfamily, SYMD subfamily, ASH1L, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, NSD2, NSD3, PRDM1, PRDM10, PRDM11, PRDM12, These include PRDM13, PRDM14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM7, PRDM8, PRDM9, SET1, SET1L, SET2L, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETDB1, SETDB2, SETMAR, SUV39H1, SUV39H2, SUV420H1, SUV420H2, SYMD1, SYMD2, SYMD3, SYMD4, and SYMD5.

[0139] Non-limiting examples of transcription effectors that enhance target gene expression or activity include, but are not limited to, transcription activators such as VP16, VP64, VP48, VP160, p65 subdomains (e.g., from NFkB), VP64-p65-rta fusion proteins (VPR), and the activation domain of EDLL and / or TAL activation domains (e.g., for activity in plants), histone lysine methyltransferases such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, histone lysine demethylases such as JHDM2a / b, UTX, JMJD3, histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI60, CLOCK, and the like. Examples of DNA demethylases include TET translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1.

[0140] Non-limiting examples of transcriptional effectors that reduce the expression or activity of target genes include, but are not limited to, transcriptional repressors, such as Kruppel-associated box (KRAB or SKD), KOX1 repression domain, Mad mSIN3 interaction domain (SID), ERF repressor domain (ERD), SRDX repression domain (e.g., for repression in plants), etc., histone lysine methyltransferases, e.g., Pr-SET7 / 8, SUV4-20H1, RIZ1, etc., histone lysine demethylases, e.g., JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARIDID / SMCY, etc., histone lysine deacetylases, e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc., DNA methylases, e.g., Hhal DNA Examples include m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc., as well as peripherally recruited elements such as lamin A and lamin B.

[0141] Various aspects of the present disclosure provide multiple heterologous gene circuits that are individually activatable to sequentially regulate the expression and / or activity levels of multiple distinct target genes. In some embodiments, a first heterologous gene circuit is activated to convert a plurality of cells from a first cell type to a second cell type, and then a second heterologous gene circuit is activated to convert the plurality of cells from the second cell type to the target cell type.

[0142] In some embodiments, activation of the second genetic circuit can be performed immediately after activation of the first heterologous genetic circuit. Alternatively, activation of the second genetic circuit can be performed at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 1 year, or more after activation of the first genetic circuit.

[0143] The one or more target genes disclosed herein can include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0144] The one or more target genes disclosed herein can include a cell differentiation regulator, a molecular function regulator, a binding factor, a fusogenic factor, a protein folding chaperone, a protein tag, an RNA folding chaperone, a cell signaling factor, an immune response factor, a sensory receptor, a cell structure factor, a protein binding factor, a cargo receptor, a catalytic factor, or a small molecule sensor.

[0145] The one or more target genes disclosed herein can include cell differentiation regulators including growth factors, transcription factors, myogenic regulators, immune cell regulators, neuroregulators, stem cell differentiation factors, chondrogenic regulators, osteogenic regulators, senescence factors, stemness (e.g., dedifferentiation factors), and the like.

[0146] In some cases, the one or more target genes (e.g., one or more chondrogenic regulators) can include Sox2, Sox6, Sox9, Shox2, Gli3, Trps1, Oct4, NANOG, Chondrogenicx3.2, Brachyury, Mixl1, Tbx6, Msgn1, Parxis, Pax9, Runx2, Runx3, Smad1, Smad5, and Smad8.

[0147] In some cases, one or more target genes can comprise a homeobox gene.Homeobox genes are, for example, genes that regulate large-scale anatomical features in the early stages of embryonic development.Types of homeobox genes include HOX genes, LIM genes, PAX genes, POU genes, CERS genes, HNF genes, SINE genes, CUT genes, ZF genes, paraHOX genes, DLX genes, TALE genes, PRD genes, and NKL genes. Non-limiting examples of homeobox genes include HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXB13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, and HOXC11. , HOXC12, HOXC13, HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13, CDX1, CDX2, CDX4, GSX1, GSX2 , PDX1, EVX1, EVX2, GBX1, GBX2, MEOX1, MEOX2, MNX1, DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, IRX1, IRX2, IRX3, IRX4, IRX5, IRX6, MEIS1, MEIS2, MEIS3, MKX, PBX1, PBX2, PBX3, PBX4, PKNOX1, PKNOX2, TGIF1, TGIF2, TGIF2LX, TGIF2LY, ISL1, ISL 2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, LMX1B, HDX, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F 2, POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1, POU5F1P1, POU5F1P4, POU5F2, POU6F1, POU6F2, LASS2, LASS3, LAS S4, LASS5, LASS6, HMBOX1, HNF1A, HNF1B, SIX1, SIX2, SIX3, SIX4, SIX5, SIX6, ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2,SATB1, SATB2, ADNP, ADNP2, TSHZ1, TSHZ2, TSHZ3, ZEB1, ZEB2, ZFHX2, ZFHX3, ZFHX4, ZHX1, HOME Z, ALX1(CART1), ALX3, ALX4, ARGFX, ARX, DMBX1, DPRX, DRGX, DUXA, DUXB, DUX(1, 2, 3, 4, 4c, 5), E SX1, GSC, GSC2, HESX1, HOPX, ISX, LEUTX, MIXL1, NOBOX, OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, Examples of such proteins include RHOXF1, RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, VSX2, BARHL1, BARHL2, BARX1, BARX2, BSX, DBX1, DBX2, EMX1, EMX2, EN1, EN2, HHEX, HLX1, LBX1, LBX2, MSX1, MSX2, NANOG, NOTO, TLX1, TLX2, TLX3, TSHZ1, TSHZ2, TSHZ3, VAX1, VAX2, VENTX, NKX2-1, NKX2-4, NKX2-2, NKX2-8, NKX3-1, NKX3-2, NKX2-3, NKX2-5, NKX2-6, HMX1, HMX2, HMX3, NKX6-1, NKX6-2, and NKX6-3.

[0148] Optionally, the target gene can include MIXL1, a transcription factor that preferentially binds to the DNA sequence TAAT on the MIX gene and plays a role in mesoderm patterning and tissue specification during gastrulation.

[0149] In some cases, the target gene may include UNCX, a transcription factor involved in somatogenesis and neurogenesis. UNCX is also required for the maintenance and differentiation of specific elements of the axial skeleton.

[0150] Optionally, the target gene can include PAX9, a transcription factor required for the development of the thymus, parthyroid glands, ultimobranchial body, teeth, skeleton, and limbs.

[0151] In some cases, one or more target genes may comprise a T-box transcription factor (TBX gene). TBX transcription factors are involved in development. T-box proteins have a relatively large DNA binding domain. Non-limiting examples of TBX transcription factors include TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, TBX22, and TBXT (Brachyury protein).

[0152] In some cases, the target gene can include TBXT. TBXT, also known as T-box transcription factor T or Brachyury protein, functions as a transcription factor in the T-box family of genes. TBXT plays a role in defining the midline in bilateral organisms and helps establish the anterior-posterior axis. It also helps define the mesoderm during gastrulation.

[0153] In some cases, the target gene can include TBX6. TBX6, also known as T-box transcription factor 6, is involved in the segmentation of paraxial mesoderm into somites.

[0154] In some cases, one or more target genes may comprise a basic helix-loop-helix transcription factor (bHLH gene). bHLH genes are involved in the regulation of the cell cycle and many other developmental processes. bHLH proteins have a basic helix-loop-helix protein structure. Non-limiting examples of bHLH transcription factors include AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATOH1, ATOH7, ATOH8, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, EPAS1, FERD3L, FIGLA, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2, HIF1A, ID1, ID2, ID3, ID4, KIAA2018, LYL1, MASH1, MATH2, MAX, MESP1, MESP2, MIST1, MITF, MLX, MLXIP, MLXIPL, MNT, MSC, MSGN1, MXD1, MXD3, MXD4, and MXD5. 4, MXI1, MYC, MYCL1, MYCL2, MYCN, MYF5, MYF6, MYOD1, MYOG, NCOA1, NCOA3, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, NHLH1, NHLH2, NPAS1, NPAS2, NPAS3, NPAS4, OAF1, OLIG1, OLIG2, OLIG3, PTF1A, SCL, SCXB, SIM1, SIM2, SOHLH1, SOHLH2, SREBF1, SREBF2, TAL1, TAL2, TCF12, TCF15, TCF21, TCF3, TCF4, TCFL5, TFAP4, TFE3, TFEB, TFEC, TWIST1, TWIST2, USF1, and USF2.

[0155] In some cases, target gene can include MSGN1.MSGN1, also known as mesogenin 1, is a Wnt-activated bHLH transcription factor that is involved in mesoderm formation and the regulation of RNA polymerase II transcription.MSGN1 can also be involved in somatic cell formation.

[0156] Optionally, the target gene can include TCF15, an early transcription factor that plays a role in regulating somatic cell formation, paraxial mesoderm development, and stem cell pluripotency.

[0157] In some cases, one or more target genes can comprise SRY-related box transcription factor (SOX gene).SOX transcription factor is involved in developmental regulation.Non-limiting examples of SOX transcription factor can include SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21, SOX30 and SRY.

[0158] Optionally, one or more target genes can include SOX group A, which includes SRY. Optionally, one or more target genes can include SOX group B1, which includes SOX1, SOX2, and / or SOX3. Optionally, one or more target genes can include SOX group B2, which includes SOX14 and / or SOX21. Optionally, one or more target genes can include SOX group C, which includes SOX4, SOX11, and / or SOX12. Optionally, one or more target genes can include SOX group D, which includes SOX5, SOX6, and / or SOX13. Optionally, one or more target genes can include SOX group E, which includes SOX8, SOX9, and / or SOX10. Optionally, one or more target genes can include SOX group F, which includes SOX7, SOX17, and / or SOX18. Optionally, one or more target genes can include SOX group G, which includes SOX15. Optionally, the one or more target genes can include SOX group H, which includes SOX30.

[0159] Optionally, the target gene can include SOX6, a transcription factor required for central nervous system development, chondrogenesis, and the maintenance of cardiac and skeletal muscle cells.

[0160] Optionally, the target gene can include SOX9, a transcription factor that acts during chondrocyte differentiation to regulate transcription of the anti-Müllerian hormone (AMH) gene.

[0161] In some cases, the one or more target genes can include collagen. Collagen is a fibrous protein and is a major component of skin, bone, tendon, cartilage, blood vessels, and teeth. Collagen forms insoluble fibers with high tensile strength. Non-limiting examples of collagen genes include COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, COL6A6, COL7A1, COL8A1, COL8A2, and COL9. A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, COL12A1, COL13A1, COL14A1, COL15A1, COL16A1, COL17A1, COL18A1, COL19A1, COL20A1, COL21A1, COL22A1, COL23A1, COL24A1, COL25A1, COL26A1, COL27A1, and COL28A1 can be mentioned.

[0162] In some cases, the target gene can include COL2A1. COL2A1 is a component of the pro-alpha1 chain of type II collagen. Type II collagen adds structure and strength to the connective tissue that supports the muscles, joints, organs, and skin of the body. Type II collagen is found primarily in cartilage.

[0163] In some cases, the use of the heterologous gene circuits disclosed herein can be used to differentiate mesodermal stem cells (MSCs) into chondroprogenitor cells, whereby at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the resulting cells generated by using the heterologous gene circuits disclosed herein are of the target cell type.

[0164] In some cases, the heterologous gene circuits disclosed herein can be used to differentiate mesodermal stem cells (MSCs) into chondroprogenitor cells, for example, in the absence of feeder cells, serum, and one, two, or all of exogenous growth factors. By using the heterologous gene circuits disclosed herein, up to about 1 x 10 6 , up to about 9 × 10 5 , up to about 8 × 10 5 , up to about 7 × 10 5 , up to about 6 × 10 5 , up to about 5 × 10 5 , up to about 4 × 10 5 , up to about 3 × 10 5 , up to about 2 × 10 5 , up to about 1 × 10 5 , up to about 5 × 10 4 , up to about 2 × 10 4 , up to about 1 × 10 4 , or more chondrogenic precursor cells from at least about 1 x 10 4 , at least about 2 × 10 4 , at least about 5 × 10 4 , at least about 1 x 10 5 , at least about 2 × 10 5 , at least about 5 × 10 5 , at least about 1 x 10 6 , at least about 2 × 10 6 , at least about 5 × 10 6 , at least about 1 x 10 7, at least about 2 × 10 7 , at least about 5 × 10 7 , at least about 1 x 10 8 , at least about 2 × 10 8 , at least about 5 × 10 8 , at least about 1 x 10 9 , at least about 2 × 10 9 , at least about 5 × 10 9 , at least about 1 x 10 10 , at least about 2 × 10 10 , at least about 5 × 10 10 , at least about 1 x 10 15 , at least about 2 × 10 15 , at least about 5 × 10 15 , or more chondrogenic cells can be generated.

[0165] In some cases, the use of the heterologous gene circuits disclosed herein can be used to differentiate pluripotent stem cells (PSCs, e.g., induced PSCs or iPSCs) into chondrogenic cells, for example, in the absence of feeder cells, serum, and one, two, or all of exogenous growth factors. By using the heterologous gene circuits disclosed herein, up to about 1 x 10 6 , up to about 9 × 10 5 , up to about 8 × 10 5 , up to about 7 × 10 5 , up to about 6 × 10 5 , up to about 5 × 10 5 , up to about 4 × 10 5 , up to about 3 × 10 5 , up to about 2 × 10 5 , up to about 1 × 10 5 , up to about 5 × 10 4 , up to about 2 × 10 4 , up to about 1 × 10 4 , or more chondrogenic precursor cells from at least about 1 x 10 4 , at least about 2 × 10 4 , at least about 5 × 10 4 , at least about 1 x 10 5 , at least about 2 × 10 5 , at least about 5 × 10 5, at least about 1 x 10 6 , at least about 2 × 10 6 , at least about 5 × 10 6 , at least about 1 x 10 7 , at least about 2 × 10 7 , at least about 5 × 10 7 , at least about 1 x 10 8 , at least about 2 × 10 8 , at least about 5 × 10 8 , at least about 1 x 10 9 , at least about 2 × 10 9 , at least about 5 × 10 9 , at least about 1 x 10 10 , at least about 2 × 10 10 , at least about 5 × 10 10 , at least about 1 x 10 15 , at least about 2 × 10 15 , at least about 5 × 10 15 , or more chondrogenic cells can be generated.

[0166] Such generation of chondrogenic cells by using the heterologous gene circuits disclosed herein can be achieved within a period of up to about 60 days, up to about 55 days, up to about 50 days, up to about 45 days, up to about 40 days, up to about 35 days, up to about 30 days, up to about 25 days, up to about 20 days, up to about 15 days, up to about 10 days, up to about 7 days, up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, up to about 1 day, or less.

[0167] In some cases, chondrogenic cells generated by this method produce more cartilage than chondrogenic cells obtained via directed differentiation. Chondrogenic cells generated using the provided methods 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 cartilage than chondrogenic cells obtained via directed differentiation. Alternatively, or in addition, chondrogenic cells generated by this method can have an equivalent amount of cartilage produced compared to chondrogenic cells obtained via directed differentiation.

[0168] In some cases, the chondrogenic or chondroprogenitor cells generated by this method exhibit higher expression levels of two or more positive chondroprogenitor markers compared to control chondroprogenitor cells. Non-limiting examples of positive chondroprogenitor markers can include CD146, CD73, CD112, and BMPR1.

[0169] Optionally, the chondrogenic or chondroprogenitor cells produced by this method have a cellular morphology that is at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cellular morphology, compared to control chondroprogenitor cells. or at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, or at least about 1,000,000% higher expression levels of positive chondroprogenitor markers.

[0170] Optionally, the chondrogenic or prechondrogenic cells produced by this method are at least or at most about 0.1 fold, at least or at most about 0.2 fold, at least or at most about 0.3 fold, at least or at most about 0.4 fold, at least or at most about 0.5 fold, at least or at most about 0.6 fold, at least or at most about 0.7 fold, at least or at most about 0.8 fold, at least or at most about 0.9 fold, at least or at most about 1 fold, at least or at most about 2 fold, at least or at most about 3 fold, at least or at most about 4 fold, at least or at most about 5 fold, at least or at most about 6 fold, at least or at most about 7 fold, at least or at most about 8 fold, at least or at most about 9 fold, at least or at most about 10 fold, at least or at most about 11 fold, at least or at most about 12 fold, at least or at most about 13 fold, at least or at most about 14 fold, at least or at most about 15 fold, at least or at most about 16 fold, at least or at most about 17 fold, at least or at most about 18 fold, at least or at most about 19 fold, at least or at most about 20 fold, at least or at most about 21 fold, at least or at most about 22 fold, at least or at most about 23 fold, at least or at most about 24 fold, at least or at most about 25 fold, at least or at most about 26 fold, at least or at most about 27 fold, at least or at most about 28 fold, at least or at most about 29 fold, at least or at most about 30 fold, at least or at most about 31 fold, at least or at most about 32 exhibits expression levels of positive chondroprogenitor markers that are at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 60-fold, at least or at most about 70-fold, at least or at most about 80-fold, at least or at most about 90-fold, at least or at most about 100-fold, at least or at most about 500-fold, at least or at most about 1,000-fold, at least or at most about 5,000-fold, or at least or at most about 10,000-fold higher.

[0171] In some cases, the chondrogenic or chondroprogenitor cells generated by this method exhibit higher expression levels of two or more negative chondroprogenitor markers compared to control chondroprogenitor cells. Non-limiting examples of negative chondroprogenitor markers can include CD326 and CD309.

[0172] Optionally, the chondrogenic or chondroprogenitor cells produced by this method have a cellular morphology that is at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cellular morphology, compared to control chondroprogenitor cells. or at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, or at least about 1,000,000% lower expression levels of negative chondroprogenitor markers.

[0173] Optionally, the chondrogenic or prechondrogenic cells produced by this method are at least or at most about 0.1 fold, at least or at most about 0.2 fold, at least or at most about 0.3 fold, at least or at most about 0.4 fold, at least or at most about 0.5 fold, at least or at most about 0.6 fold, at least or at most about 0.7 fold, at least or at most about 0.8 fold, at least or at most about 0.9 fold, at least or at most about 1 fold, at least or at most about 2 fold, at least or at most about 3 fold, at least or at most about 4 fold, at least or at most about 5 fold, at least or at most about 6 fold, at least or at most about 7 fold, at least or at most about 8 fold, at least or at most about 9 fold, at least or at most about 10 fold, at least or at most about 11 fold, at least or at most about 12 fold, at least or at most about 13 fold, at least or at most about 14 fold, at least or at most about 15 fold, at least or at most about 16 fold, at least or at most about 17 fold, at least or at most about 18 fold, at least or at most about 19 fold, at least or at most about 20 fold, at least or at most about 21 fold, at least or at most about 22 fold, at least or at most about 23 fold, at least or at most about 24 fold, at least or at most about 25 fold, at least or at most about 26 fold, at least or at most about 27 fold, at least or at most about 28 fold, at least or at most about 29 fold, at least or at most about 30 fold, at least or at most about 31 fold, at least or at most about 32 exhibits an expression level of the negative chondroprogenitor marker that is at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 60-fold, at least or at most about 70-fold, at least or at most about 80-fold, at least or at most about 90-fold, at least or at most about 100-fold, at least or at most about 500-fold, at least or at most about 1,000-fold, at least or at most about 5,000-fold, or at least or at most about 10,000-fold lower.

[0174] Optionally, the expression levels of chondroprogenitor cell markers can be measured using methods such as, but not limited to, RT-PCR, Western blotting, Northern blotting, protein staining, mRNA staining, and RNA sequencing.

[0175] Optionally, the expression level can be measured at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 28 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days or more after introduction of the gene circuit.

[0176] Optionally, the chondrogenic or chondroprogenitor cells generated through this method may be chondrocytes. Alternatively, the chondrogenic or chondroprogenitor cells generated by this method may be cells other than chondrocytes (e.g., chondroblasts). Optionally, the generated progenitor cells may be substantially mitotically quiescent. Alternatively, the generated progenitor cells may be substantially mitotically active.

[0177] Optionally, the first gating unit may be configured to decrease the expression and / or activity level of one or more target genes. Optionally, the first gating unit may be configured to increase the expression and / or activity level of one or more target genes. Optionally, the first gating unit may be configured to maintain the expression and / or activity level of one or more target genes.

[0178] In some cases, the regulation of the first target gene can occur before the regulation of the second target gene.In some cases, the regulation of the first target gene can occur after the regulation of the second target gene.In some cases, the regulation of the first target gene can occur almost simultaneously with the regulation of the second target gene.

[0179] In some cases, heterologous gene circuits can be used to induce cells to differentiate into a desired cell type in the absence of growth factors, serum (such as fetal bovine serum or human serum AB), or other exogenous cell differentiation regulators or media. Serum can include the liquid fraction of coagulated blood that contains trophic and macromolecular factors essential for cell growth.

[0180] Alternatively, through the use of heterologous genetic circuits, cells can be induced to differentiate into the desired cell type using reduced amounts of serum and / or growth factors (e.g., at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% reduced, or substantially serum-free). Reducing serum amounts may allow for increased consistency across experiments or batches of cells, increased growth and / or productivity of differentiated cells, better control of physiological responsiveness, and reduced risk of contamination with serum-derived factors in cell culture.

[0181] In some cases, heterologous gene circuits in stem cells (e.g., iSPCs, MSCs) can be used to induce MSCs to differentiate into chondrogenic cells in the absence of growth factors, serum (such as fetal bovine serum, human serum AB, etc.), or other exogenous cell differentiation regulators or media. In some cases, the heterologous gene circuits disclosed herein are used to differentiate stem cells into chondrogenic cells in the absence of one or both of growth factors and serum. The resulting chondrogenic cells generated by using the heterologous gene circuits disclosed herein are at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population.

[0182] In some cases, conversion of one cell type (e.g., PSCs, MSCs, or chondroprogenitor cells) to another cell type (e.g., chondrogenic cells) using a heterologous gene circuit can result in a target cell type. Alternatively, conversion of one cell type (e.g., PSCs, MSCs, or chondroprogenitor cells) to another cell type (e.g., chondrogenic cells) using a heterologous gene circuit can result in an intermediate cell type. The intermediate cell type can undergo a second conversion using a second gene circuit to result in the target cell type.

[0183] Conversion of a cell from one cell type to another can involve modulation of multiple target genes. For example, conversion can involve 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. Conversion can involve modulation of up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 target gene. Each gene disclosed herein may be subject to at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more modulations. Each gene disclosed herein may be subject to up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 modulation. The regulation of one or more target genes (e.g., endogenous genes) as induced by a heterologous gene circuit of the present disclosure can be artificial regulation (or heterologous regulation) that would not otherwise occur in the cell in the absence of (i) the heterologous gene circuit and / or (ii) the activating portion of the heterologous gene circuit.

[0184] As demonstrated in Figure 3, various heterologous gene circuits can be designed to regulate the expression or activity levels of multiple genes (e.g., multiple endogenous genes) in a cell over multiple different time points.

[0185] For example, a heterologous gene circuit can be designed to (i) regulate the expression level of a first gene and (ii) then regulate the expression level of a second gene. For example, a heterologous gene circuit can be designed to (i) activate the expression level of a first gene and (ii) then 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) then reduce the expression level of a second gene. Alternatively, a heterologous gene circuit can be designed to (i) reduce the expression level of a first gene and (ii) then activate the expression level of a second gene. Alternatively, a heterologous gene circuit can be designed to (i) reduce the expression level of a first gene and (ii) then reduce the expression level of a second gene. The first gene and the second gene can be the same gene. Alternatively, the first gene and the second gene can be different genes. A heterologous gene circuit can be designed to regulate the expression level of an additional gene. A heterologous gene circuit can be designed to include at least 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. The additional genes can be activated. Alternatively, the additional genes can be reduced. A heterologous gene circuit can be designed to regulate expression of the additional genes before regulating the expression levels of both the first gene and the second gene. Alternatively or additionally, a heterologous gene circuit can be designed to regulate expression of the additional genes after regulating the expression level of the first gene and before regulating the expression level of the second gene. Alternatively or additionally, a heterologous gene circuit can be designed to regulate expression of the additional genes after regulating the expression levels of both the first gene and the second gene.

[0186] For example, a heterologous gene circuit can be designed to (i) regulate the expression level of a first homeobox gene, and (ii) then regulate the expression level of a second homeobox gene. For example, a heterologous gene circuit can be designed to (i) activate the expression level of a first homeobox gene, and (ii) then activate the expression level of a second homeobox gene. Alternatively, a heterologous gene circuit can be designed to (i) activate the expression level of a first homeobox gene, and (ii) then reduce the expression level of a second homeobox gene. Alternatively, a heterologous gene circuit can be designed to (i) reduce the expression level of a first homeobox gene, and (ii) then activate the expression level of a second homeobox gene. Alternatively, a heterologous gene circuit can be designed to (i) reduce the expression level of a first homeobox gene, and (ii) then reduce the expression level of a second homeobox gene. The first homeobox gene and the second homeobox gene can be the same gene, or the first homeobox gene and the second homeobox gene can be different genes.

[0187] A heterologous gene circuit can be designed to include another gene in addition to the first and second homeobox genes. The additional gene can be regulated before, simultaneously with, or after the first homeobox protein. Alternatively or additionally, the additional gene can be regulated before, simultaneously with, or after the second homeobox protein. A heterologous gene circuit can be designed to activate the additional gene. Alternatively, a heterologous gene circuit can be designed to reduce the additional gene. The additional gene can be, but is not limited to, a T-box transcription factor (TBX), a basic helix-loop-helix transcription factor (bHLH), SOX, and collagen.

[0188] A heterologous gene circuit can be designed to contain two members selected from the group consisting of a homeobox protein, a T-box transcription factor (TBX), and a basic helix-loop-helix transcription factor (bHLH). The heterologous gene circuit can be designed to contain a homeobox protein and a TBX. The heterologous gene circuit can be designed to contain a homeobox protein and a bHLH. The heterologous gene circuit can be designed to contain a TBX and a bHLH.

[0189] Heterologous gene circuits can be designed to contain a first member that is a homeobox protein, TBX, or bHLH, and a second member that is SOX or collagen. Heterologous gene circuits can be designed to contain a homeobox protein and SOX. Heterologous gene circuits can be designed to contain a homeobox protein and collagen. Heterologous gene circuits can be designed to contain TBX and SOX. Heterologous gene circuits can be designed to contain TBX and collagen. Heterologous gene circuits can be designed to contain a bHLH and SOX. Heterologous gene circuits can be designed to contain a bHLH and collagen.

[0190] For example, heterologous gene circuit #10 can be designed to (i) activate the expression levels of TBXT and MIXL1 at time point 1 (denoted as step 1), (ii) subsequently activate the expression levels of MSGN1 and TBX6 at a time point after step 1 (denoted as step 2), (iii) subsequently activate the expression levels of UNCX, TCF15, and PAX9 at a time point after step 2 (denoted as step 3), and (iv) subsequently activate the expression level of SOX6 at a time point after step 3 (denoted as step 4). For comparison, a control heterologous gene circuit (denoted All-at-once 1) can be designed to simultaneously activate the same target endogenous genes from heterologous gene circuit #10.

[0191] Activation of heterologous gene circuits in cells disclosed herein can modulate the expression or activity levels of multiple genes over multiple different time points to convert the cells into different cell types (e.g., stem cells into tissue-specific progenitor cells, etc.). The rate of such cell type conversion through use of a heterologous gene circuit can be at least or at most about 1 percent (%), at least or at most about 2%, at least or at most about 5%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 40%, at least or at most about 45%, at least or at most about 50%, at least or at most about 60%, at least or at most about 70%, at least or at most about 80%, at least or at most about 90%, or at least or at most about 95% faster than the rate of cell type conversion through use of a control heterologous gene circuit (e.g., with respect to simultaneous activation of multiple target genes).

[0192] Activation of heterologous gene circuits in cells disclosed herein can modulate the expression or activity levels of multiple genes over multiple different time points to convert cells into different cell types (e.g., stem cells into tissue-specific progenitor cells). Conversion of cells into different cell types can occur in less than about 20 days, less than about 19 days, less than about 18 days, less than about 17 days, less than about 16 days, less than about 15 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, less than about 2 days, or less than about 1 day.

[0193] The cells (e.g., the initial cells that are modified into the engineered cells disclosed herein, the final cell product produced from the engineered cells disclosed herein, etc.) can include muscle cells, immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells, epithelial cells, stem cells, secretory cells, blood cells, germ cells, nurse cells, storage cells, enteroendocrine cells, pituitary cells, mesodermal cells, chondrocyte precursor cells, neurosecretory cells, duct cells, odontoblasts, cementoblasts, glial cells, or stromal cells.

[0194] Non-limiting examples of such cells include lymphoid cells, e.g., B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, cytokine-induced killer (CIK) cells (see, e.g., US20080241194), myeloid cells, e.g., granulocytes (basophilic granulocytes, eosinophilic granulocytes, neutrophilic granulocytes / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, dendritic cells, thyroid gland (thyroid epithelial cells, parafollicular cells), parathyroid gland (parathyroid chief cells, eosinophilic cells), parathyroid gland (parathyroid chief cells, eosinophilic cells), and the like. Cells from the endocrine system, including kidney (chromaffin cells) and pineal gland (pineal gland) cells; cells of the nervous system, including glial cells (astrocytes, microglia), magnocellular neurosecretory cells, astrocytes, Bechtel cells, and pituitary gland (gonadotropes, corticotropes, thyrotropes, somatotropes, and lactotropes); cells of the respiratory system, including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, and dust cells; cells of the circulatory system, including cardiac myocytes and pericytes; stomach (chief cells, parietal cells), including goblet cells, parietal cells, G cells, and D cells. Cells of the digestive system, including ECL cells, I cells, K cells, and S cells; enterochromaffin cells; APUD cells; liver (hepatocytes, Kupffer cells); enteroendocrine cells, including cartilage / bone / muscle; osteoblasts, osteocytes, osteoclasts; bone cells, including teeth (cementoblasts, ameloblasts); paraxial mesodermal cells, including sclerotocytes, chondroblasts, or chondrocytes; skin cells, including trichocytes, keratinocytes, and melanocytes (nevus cells); muscle cells, including myocytes; podocytes; juxtaglomerular cells; intraglomerular / extraglomerular mesangial cells; and kidney proximal tubule brush border cells. Cells of the urinary tract, including follicles and macula densa cells, cells of the reproductive system, including sperm, Sertoli cells, Leydig cells, and eggs, as well as other cells (adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, keratinocyte hair stem cells, keratinocyte root sheath cells, root sheath cells of Huxley's layer, root sheath cells of Henle's layer, outer root sheath cells, hair matrix cells (stem cells), moist stratified barrier epithelial cells, surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina,Basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina; urothelial cells (lining of the bladder and ureters); exocrine secretory epithelial cells; salivary gland mucous cells (secretion rich in polysaccharides); salivary gland serous cells (secretion rich in glycoprotein enzymes); von Ebner's gland cells of the tongue (cleansing the taste buds); mammary gland cells (secretion of milk); lacrimal gland cells (secretion of tears); ear canal gland cells (secretion of earwax); eccrine sweat gland dark cells (secretion of glycoproteins); eccrine sweat gland clear cells (secretion of small molecules); apocrine sweat gland cells (secretion of odorous secretions, sex hormone sensitive); M of the eyelids Oll gland cells (specialized sweat glands), sebaceous gland cells (secreting lipid-rich sebum), Bowman's gland cells in the nose (cleansing the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting seminal fluid components, including fructose for sperm swimming), prostate cells (secreting seminal fluid components), bulbourethral gland cells (secreting mucus), Bartholin's gland cells (secreting vaginal lubrication), Littré gland cells (secreting mucus), endometrial cells (secreting carbohydrates), isolated goblet cells in the respiratory and digestive tracts (secreting mucus), mucous cells lining the stomach (secreting mucus), enzyme-producing cells in the gastric glands (secreting pepsinogens) secretions), gastric oxyntic cells (secreting hydrochloric acid), pancreatic acinar cells (secreting bicarbonate and digestive enzymes), Paneth cells of the small intestine (secreting lysozyme), type II alveolar epithelial cells of the lung (secreting surfactant), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, growth hormone-producing cells, lactotrophs, thyrotrophs, gonadotropes, corticotrophs, intermediate pituitary gland, magnocellular neurosecretory cells, gastrointestinal and respiratory system cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, eosinophils, adrenal cells, Chromaffin cells, Leydig cells of the testis, theca cells of the ovarian follicle, luteinized follicles of ruptured follicles, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (renin secreting), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, digestive tract, exocrine glands, and urogenital tract), kidney, type I alveolar epithelial cells (lining of lung air spaces), pancreatic duct cells (central acinar cells), non-striated duct cells (sweat glands, salivary glands, mammary glands, etc.), duct cells (seminal vesicles, prostate, etc.), epithelial cells lining closed internal body cavities, ciliated cells with propulsive function, extracellular matrix secreting cells, contractile cells, skeletal muscle cells,Stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursor cells), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglial cells (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 committed progenitor cells of the blood and immune system (various types), Examples of such cells include pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transduction cells, autonomic nerve cells, sensory organ and peripheral nerve supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, nurse cells, ovarian follicle cells, Sertoli cells (in the testes), thymic epithelial cells, interstitial cells, and interstitial kidney cells.

[0195] In one aspect, the present disclosure provides systems and methods by which a plurality of pluripotent stem cells (PSCs) can be converted into a plurality of tissue-specific progenitor cells.

[0196] The pluripotent stem cells can include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). The tissue-specific progenitor cells can include mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), myeloid progenitor cells, muscle stem cells, chondrocyte progenitor cells, neural stem cells, epithelial stem cells, mammary stem cells, intestinal stem cells, neural crest stem cells, or testicular stem cells.

[0197] Various embodiments of the present disclosure provide engineered cells that are programmed to induce a desired expression and / or activity level (or profile) of one or more target genes in a cell.

[0198] In some embodiments, the engineered cells (e.g., engineered chondrogenic cells) of the present disclosure can be generated from isolated stem cells (e.g., isolated MSCs or iPSCs). The heterologous gene circuits and / or components thereof (e.g., gating units, gating moieties, activating moieties, etc.) disclosed herein can be introduced into any stage (or cell state) between (a) the isolated stem cell and (b) its differentiated chondrogenic cell state (e.g., a terminally differentiated chondrogenic cell state such as a chondrocyte).

[0199] The engineered cells (e.g., engineered chondrogenic cells) of the present disclosure can be used (e.g., administered) to treat a subject in need thereof. The subject 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 ex vivo and genetically modified to generate any of the subject engineered cells (e.g., chondrogenic cells) disclosed herein. The engineered immune cells can then be administered to the subject for adaptive immunotherapy. Thus, the engineered cells can be autologous to the subject in need thereof. Alternatively, the engineered cells can be allogeneic to the subject (e.g., allogeneic stem cell transplantation, allogeneic adoptive immunotherapy, etc.).

[0200] The engineered cells disclosed herein can be administered to a subject prior to, concurrently with, or after activation of the heterologous gene circuit in the engineered stem cells, for example, the engineered cells can be activated after administration to the subject, e.g., by administering to the subject an activator of the heterologous gene circuit.

[0201] A subject may be treated (e.g., administered) with a population of engineered cells (e.g., engineered muscle cells) of the present disclosure for at least or up to about 1 dose, at least or up to about 2 doses, at least or up to about 3 doses, at least or up to about 4 doses, at least or up to about 5 doses, at least or up to about 6 doses, at least or up to about 7 doses, at least or up to about 8 doses, at least or up to about 9 doses, or at least or up to about 10 doses. Alternatively, or in addition, a subject may be treated (e.g., administered) with a population of engineered cells (e.g., engineered T cells) of the present disclosure for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, at least about 30 years, at least about 40 years, at least about 50 years, at least about 60 years, at least about 70 years, at least about 80 years, at least about 90 years, or at least about 100 years.

[0202] Any one of the methods disclosed herein can be utilized to treat a target cell, target tissue, target condition, or target disease in a subject.

[0203] The target disease of subject can be the disease that affects cartilage.The disease that affects cartilage can include but is not limited to arthritis (for example, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis), gout, systemic lupus erythematosus, seronegative spondyloarthropathy, costochondritis, herniation, achondroplasia or polychondritis.The target condition of subject can be injury (for example, joint injury, such as knee injury).

[0204] The target disease of interest may be cancer or tumor. Non-limiting examples of cancers include cancer cells, and cancers include acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, mature acute myeloblastic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenomatous cystic carcinoma, adenoma, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, malignant NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, odontogranulomatous fibroma, anal cancer, undifferentiated ectomized large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal cell-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary origin, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, cancer of unknown primary origin, carcinosarcoma, Castleman disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical Cancer, bile duct carcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, embryonal neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, euthyroid Ewing's tumor family, Ewing's sarcoma family, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct carcinoma, extramammary Paget's disease, fallopian tube carcinoma, inclusion fetal tumor, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid carcinoma, gallbladder carcinoma, gallbladder carcinoma, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor,Hairy cell leukemia, Hairy cell leukemia, Head and neck cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematopoietic malignancies, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast and ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, Hypopharyngeal cancer, Hypothalamic glioma, Inflammatory breast cancer, Intraocular melanoma, Pancreatic islet cell carcinoma, Pancreatic islet cell tumor, Juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, Kidney cancer, Klatskin tumor, Krukenberg tumor, Laryngeal cancer, Laryngeal cancer, Laryngeal melanoma, Leukemia, Leukemia, Lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant Triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloepithelioma, melanoma, meningeal tumor, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell cervical cancer of unknown primary, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorder, myxoma, nasal cavity cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumor, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor (intermediately differentiated) Parenchymal Tumor of Intermediate Differentiation), pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, polyposis, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer,Primary neuroectodermal tumors, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory cancer related to the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasms, seminoma, serous tumors, Sertoli-Leydig cell tumors, sex cord-stromal tumors, Sézary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, sooty warts wart), spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, peripheral lymphatic duct carcinoma, testicular cancer, meningioma, pharyngeal cancer, thymic carcinoma, thymoma, thyroid cancer, renal pelvis and ureteral transitional cell carcinoma, transitional cell carcinoma, ureteral cancer, urethral cancer, genitourinary tumor, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morrison syndrome, verrucous carcinoma, optic pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation within a cancer cell population, such as cancer stem cells. In some embodiments, the cancer is a hematopoietic cancer, such as lymphoma. The antigen may be a tumor-associated antigen.

[0205] Non-limiting examples of target tissues include cells, such as chondrogenic cells, obtainable from a subject, including humans, dogs, cats, mice, rats, and transgenic species thereof. Examples of samples from a subject from which cells can be derived include, but are not limited to, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, feces, semen, vaginal fluid, interstitial fluid from tumor tissue, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernail, plasma, nasal swab or nasopharyngeal washing, spinal fluid, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, umbilical cord blood, emphatic fluids, cavity fluid, sputum, pus, microbiota, meconium, breast milk, and / or other excretions or bodily tissues.

[0206] The present disclosure further provides a composition comprising the engineered genetic circuit disclosed herein. The composition can further comprise an actuator of a heterologous genetic circuit. The present disclosure further provides a kit comprising the composition. The kit can further comprise an activator of the heterologous genetic circuit. The activator can be in the same composition as the engineered cells. Alternatively or additionally, the activator can be in a separate composition from the engineered cells.

[0207] In some cases, the engineered progenitor cells disclosed herein may exhibit (i) equivalent or enhanced regenerative capacity, (ii) equivalent or enhanced in vitro expression, (iii) equivalent or enhanced gene editing capabilities, (iv) equivalent or enhanced immune tolerance, (v) equivalent or shorter manufacturing timelines, (vi) equivalent or fewer growth factor or culture requirements, and / or (vii) equivalent or enhanced safety compared to control progenitor cells.

[0208] Control progenitor cells can be generated by any method, including proliferation of progenitor cells (e.g., chondroprogenitor cells) isolated from tissue, directed iPSC differentiation (e.g., using exogenous growth factors), and / or transgenic iPSC differentiation (e.g., viral transduction of heterologous genes).

[0209] Optionally, the tissue-specific progenitor cells may be stored in a container (eg, a sterile vial). Optionally, the tissue-specific progenitor cells are stored at a temperature of up to about 10°C, up to about 5°C, up to about 4°C, up to about 0°C, up to about -5°C, up to about -10°C, up to about -20°C, up to about -30°C, up to about -40°C, up to about -50°C, up to about -60°C, up to about -70°C, up to about -80°C, up to about -90°C, up to about -100°C, up to about -110°C, up to about -120°C, up to about -130°C, up to about -140°C, up to about -150°C, up to about -160°C, up to about -170°C, up to about -180°C, up to about -190°C, up to about -200°C, or colder.

[0210] Pharmaceutical Composition In some cases, the method disclosed herein comprises administering at least one tissue-specific progenitor cell to a subject in need thereof. The subject can be an animal. The subject can be a mammal (e.g., a primate, a horse, a cat, a dog, a cow, a pig, a sheep, a goat, a mouse, a rabbit, a rat, a guinea pig). The subject can be a human subject.

[0211] The pharmaceutical compositions of the present disclosure can be a combination of any of the pharmaceutical compounds described herein with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. The pharmaceutical composition can be administered in a therapeutically effective amount as a pharmaceutical composition by a variety of forms and routes, including, for example, intravenous, subcutaneous, intramuscular, inhalation, oral, parenteral, ocular, otic, subcutaneous, transdermal, intranasal, intravitreal, intratracheal, intrapulmonary, transmucosal, vaginal, and topical administration.

[0212] The formulation can vary depending on the route of administration selected. Pharmaceutical compositions containing the compounds described herein can be prepared, for example, by mixing, dissolving, emulsifying, encapsulating, entrapping, or compressing processes. [Example]

[0213] Example 1: Differentiation of chondrogenic precursor cells

[0214] Tissue-specific cells (eg, chondroprogenitor cells) can be generated from less differentiated cells (eg, stem cells such as iPSCs) by the systems and methods of the present disclosure.

[0215] A. Generation of chondroprogenitor cells.

[0216] In this example, mesodermal stem cells (MSCs) were transformed into chondroprogenitors using a heterologous gene circuit. Differentiation of stem cells (e.g., MSCs) into chondroprogenitors can be a complex process that requires turning on multiple endogenous genes at different times and turning off multiple endogenous genes at different times. See Figure 2A for an example of different endogenous genes induced for expression at different stages of stem cell differentiation into chondroprogenitors and then into chondrocytes. Thus, one or more heterologous gene circuits disclosed herein can be utilized to automatically facilitate such a cascade of different endogenous gene expression. Optionally, each heterologous gene circuit can be configured to regulate the expression levels of multiple genes at multiple different times upon a single activation of such a heterologous gene circuit.

[0217] MSCs were transiently transfected with plasmid DNA encoding one of the heterologous gene circuits described in Figure 2B or Figure 3, targeting a combination of TBXT, MIXL1, TBX6, MSGN1, UNCX, TCF15, PAX9, SOX6, SOX9, PAX3, PAX7, and COL2A1. All targeted genes were activated. Flow cytometry was used to analyze CD146 / CD73 double-positive cells, CD146 / CD112 double-positive cells, and CD326 / CD309 double-positive cells, each of which indicated the formation of chondrogenic precursor cells (Figure 6A-6B). Top-performing HCGs, such as Cellgorithm 10, were found to convert approximately 60% of cells into chondrogenic precursor cells in 4 days (Figure 6C).

[0218] B. Characterization of chondroprogenitor cells generated by heterologous gene circuits.

[0219] Transient plasmid delivery of heterologous gene circuits induced double-positive and double-negative chondrogenic progenitor cell markers that appeared after 4 days. As shown in Figure 6C, flow analysis revealed that at least one of the heterologous gene circuits provided in Figure 3, heterologous gene circuit 10 (e.g., Cellgorithm10), resulted in at least approximately 60% MSC conversion into chondrogenic progenitors in 4 days. This conversion rate was greater than a control population of iPSCs treated by simultaneously activating all of the same endogenous genes.

[0220] Separately, cells treated with the various heterogeneous gene circuits from Figure 3 were analyzed (e.g., via flow) for positive chondroprogenitor markers (e.g., CD146+ / CD73+, CD146+ / CD73+, CD326- / CD309-, etc.) and then plotted in a volcano plot, as shown in Figure 4. Volcano plots were generated to compare the efficiency of each heterogeneous gene circuit (i) in terms of statistical significance (p-value) compared to an all-at-once control circuit, which activated all target endogenous genes at once, and (ii) in terms of the magnitude of change (fold change) in the positive chondroprogenitor markers compared to the all-at-once control circuit.

[0221] Figures 5A-5D show representative data utilized in the Volcano Plots of Figure 4. In each plot in Figures 5A-5D, the y-axis represents the percentage of each cell sample expressing the indicated chondroprogenitor markers (e.g., CD146+ / CD73+, CD146+ / CD73+, BMPR1+, or CD326- / CD309-) upon treatment with one of the heterologous gene circuits from Figure 3. The x-axis indicates which heterologous gene circuit was utilized for each cell sample. The plots in Figures 5A-5D demonstrate that various heterologous gene circuits (e.g., Cellgorithm 9, Cellgorithm 10, Cellgorithm 12, etc.) are capable of generating chondroprogenitor cells, as indicated by multiple chondroprogenitor marker panels.

[0222] Example 2: Chondrogenesis assay

[0223] In this prophetic example, iPSCs and mesenchymal cells are contacted with the top-performing HGCs of Example 1 and grown in culture until chondrocytes are formed. A control chondrocyte population is collected and purified from a mouse sample. Chondrocytes from both the HGC population and the control population are tested for chondrogenesis, including the amount of cartilage formed and the mechanical properties of the cartilage tested.

[0224] Example 3: Transplantation and engraftment of chondrogenic precursor cells

[0225] The tissue-specific cells (e.g., chondroprogenitor cells) prepared by the disclosed system and method can be administered (e.g., injected into articular tissue) to a subject in need thereof to treat a joint-related or cartilage-related disorder in the subject.

[0226] A. Generation of chondroprogenitor cells.

[0227] Stem cells (e.g., MPSCs) can be transduced or transfected (e.g., transiently transfected) with one or more heterologous genes (e.g., plasmid DNA) encoding at least one heterologous gene circuit, such as one of each of the heterologous gene circuits shown in Figure 3, according to the methods described in Example 1 to generate chondroprogenitor cells.

[0228] B. In vivo administration of chondroprogenitor cells.

[0229] Once chondroprogenitor cells are generated by the disclosed system and method, they can be purified using an anti-CD146 antibody (e.g., CD146 cells). The cells can be concentrated, resuspended in a buffer (e.g., PBS), and then administered to mice by direct injection into a joint or other site of interest. After 8-10 weeks, the mice can be sacrificed and the tissue surrounding the injection site can be dissected. Sections can be immunostained for human cartilage to confirm engraftment of the ex vivo-generated chondroprogenitor cells.

[0230] Further protocols for chondroprogenitor cell transplantation are provided herein. Upon generation as described herein, chondroprogenitor cells can be suspended in chondrogenic cell medium. These cells can be transplanted into target sites within the joint, with or without further expansion. For expansion, chondroprogenitor cells can be plated as sparse cultures (e.g., 1,000–2,000 cells / well in a 24-well plate) in tissue culture wells containing hydrogel (flat or patterned) or thin gel-coated plastic (flat or patterned) and cultured with medium changes every three days. On the day of transplantation, NOD / SCID mice can be anesthetized by intraperitoneal injection of ketamine (2.4 mg / mouse) and xylazine (240 g / mouse) and irradiated in the hind limbs as previously described (A. Sacco et al. (2008) Nature 456, 502). Generated chondroprogenitor cells can be counted, resuspended, and then injected into recipient mice.

[0231] The engraftment of transplanted chondroprogenitor cells (e.g., differentiation and integration into local chondrogenic tissue) can be visualized by various methods. For example, chondroprogenitor cells can be engineered to express a heterologous marker (e.g., a fluorescent protein, e.g., green fluorescent protein) that is not present in the transplanted animal. Alternatively or additionally, the chondroprogenitor cells can be allogeneic to the animal so that any cartilage differentiated from the chondroprogenitor cells upon transplantation can be identified (e.g., immunostained) by an antigen not found in the transplanted animal.

[0232] Example 4: Generation of chondrogenic cells in vitro

[0233] For improved cell production, cells of interest can be engineered to exhibit increased expression of genes that change their phenotype toward the chondrogenic lineage. In some embodiments, improved cell production can be demonstrated by a shorter time to differentiate stem cells (e.g., pluripotent stem cells) into target cells (e.g., chondrogenic precursors or chondrogenic cells), an increased number of target cells compared to other protocols, enhanced levels of chondrogenesis as evidenced by collagen, aggrecan, and / or ECM protein production, reduced costs to produce the same number of target cells, etc. Upon nucleofection of Cellgorithm into the cells of interest, these genes are sequentially expressed in the cells of interest. The cells of interest can be induced pluripotent stem cells. Manipulated cells may exhibit increased presence of markers of the chondrogenic lineage by measuring RNA transcription, may exhibit DNA accessibility indicative of cells of the chondrogenic lineage, and / or may exhibit increased function in chondrogenic assays measuring 1) collagen, aggrecan, and desired ECM protein production, and 2) cartilage pellet formation (whereby the cartilage pellet expresses desired levels of collagen, aggrecan, and desired ECM proteins).

[0234] For example, a library of Cellgorithms, including but not limited to the Cellgorithms labeled in Table 1, constructed from sequences having core function units set forth in SEQ ID NOS: 1-1932, can be introduced into cells of interest. Table 1 shows 1932 unique chondro constructs (e.g., there are 23 unique stem combinations. There are 21 unique "4-pool" cartilage spacers. For each gene, there are 4 pools because all 4 constructs targeting that gene are used together. Thus, 21 * There are 23 = 483 different combinations. To count each individual construct, multiply by 4. 483 * 4 = 1932 unique cartilage constructs present).

[0235] Example 5: In vivo validation of Cellgorithm-derived chondrogenic cells

[0236] Chondrogenic cells generated using Cellgorithm technology can be used as a regenerative therapy for individuals with cartilage dysfunction, cartilage damage, or arthritic cartilage. Delivery of these cells to such individuals can result in pain relief in the associated joint, improved joint function, and improved longevity of function in each treated joint. Once produced by Cellgorithm-driven instructions, chondrogenic cells can be used in the same manner as chondrogenic cells produced by other methods, such as isolation from cadaveric donors or differentiation of pluripotent stem cells using protocols for varying growth factors and other small molecules over a period of time (so-called directed differentiation). Regardless of their origin, chondrogenic cells can be evaluated and used in vivo by injecting them into the joints of animal models that have suffered acute cartilage injury or have arthritis through genetic or induced models. Chondrogenic cells can be characterized phenotypically based on gene expression and cell surface marker properties and functionally for their ability to make cartilage. Both in vitro and in vivo characterization provide significant insight into the quality of chondrogenic cells.

[0237] To evaluate the functionality of the produced chondrogenic cells, they can be implanted into osteochondral defects (e.g., full thickness, 1 mm diameter) in the knee joints of athymic rats (e.g., n=6) or 6-month-old Yucatan pigs. After chondrogenic cell implantation, histological examination can be performed to measure the expression of genes of interest (e.g., COL2A1, COL2A2, COL10, AGGN) and extracellular matrix proteins of interest. Chondrogenic cells must be implanted to produce cartilage that expresses these proteins above vehicle controls, resulting in healing of the defective cartilage leading to functional joint outcomes.

[0238] Table 1-1

[0239] Table 1-2

[0240] Table 1-3

[0241] Table 1-4

[0242] Table 1-5

[0243] Table 1-6

[0244] Table 1-7

[0245] Table 1-8

[0246] Table 1-9

[0247] Table 1-10

[0248] Table 1-11

[0249] Table 1-12

[0250] Table 1-13

[0251] Table 1-14

[0252] Table 1-15

[0253] Table 1-16

[0254] Table 1-17

[0255] Table 1-18

[0256] Table 1-19

[0257] Table 1-20

[0258] Table 1-21

[0259] Table 1-22

[0260] Table 1-23

[0261] Table 1-24

[0262] Table 1-25

[0263] Table 1-26

[0264] Table 1-27

[0265] Table 1-28

[0266] Table 1-29

[0267] Table 1-30

[0268] Table 1-31

[0269] Table 1-32

[0270] Table 1-33

[0271] Table 1-34

[0272] Table 1-35

[0273] Table 1-36

[0274] Table 1-37

[0275] Table 1-38

[0276] Table 1-39

[0277] Table 1-40

[0278] Table 1-41

[0279] Table 1-42

[0280] Table 1-43

[0281] Table 1-44

[0282] Table 1-45

[0283] Table 1-46

[0284] Table 1-47

[0285] Table 1-48

[0286] Table 1-49

[0287] Table 1-50

[0288] Table 1-51

[0289] Table 1-52

[0290] Table 1-53

[0291] Table 1-54

[0292] Table 1-55

[0293] Table 1-56

[0294] Table 1-57

[0295] Table 1-58

[0296] Table 1-59

[0297] Table 1-60

[0298] Table 1-61

[0299] Table 1-62

[0300] Table 1-63

[0301] Table 1-64

[0302] Table 1-65

[0303] Table 1-66

[0304] Table 1-67

[0305] Table 1-68

[0306] Table 1-69

[0307] Table 1-70

[0308] Table 1-71

[0309] Table 1-72

[0310] Table 1-73

[0311] Table 1-74

[0312] Table 1-75

[0313] Table 1-76

[0314] Table 1-77

[0315] Table 1-78

[0316] Table 1-79

[0317] Table 1-80

[0318] Table 1-81

[0319] Table 1-82

[0320] Table 1-83

[0321] Table 1-84

[0322] Table 1-85

[0323] Table 1-86

[0324] Table 1-87

[0325] Table 1-88

[0326] Table 1-89

[0327]

Table 1-90

[0328] Table 1-91

[0329] Table 1-92

[0330] Table 1-93

[0331] Table 1-94

[0332] Table 1-95

[0333] Table 1-96

[0334] Table 1-97

[0335] Table 1-98

[0336] Table 1-99

[0337]

Table 1-100

[0338] Table 1-101

[0339] Table 1-102

[0340] Table 1-103

[0341] Table 1-104

[0342] Table 1-105

[0343] Table 1-106

[0344] Table 1-107

[0345] Table 1-108

[0346] Table 1-109

[0347]

Table 1-110

[0348] Table 1-111

[0349] Table 1-112

[0350] Table 1-113

[0351] Table 1-114

[0352] Table 1-115

[0353] Table 1-116

[0354] Table 1-117

[0355] Table 1-118

[0356] Table 1-119

[0357]

Table 1-120

[0358] Table 1-121

[0359] Table 1-122

[0360] Table 1-123

[0361] Table 1-124

[0362] Table 1-125

[0363] Table 1-126

[0364] Table 1-127

[0365] Table 1-128

[0366] Table 1-129

[0367] Table 1-130

[0368] Table 1-131

[0369] Table 1-132

[0370] Table 1-133

[0371] Table 1-134

[0372] Table 1-135

[0373] Table 1-136

[0374] Table 1-137

[0375] Table 1-138

[0376] Table 1-139

[0377] Table 1-140

[0378] Table 1-141

[0379] Table 1-142

[0380] Table 1-143

[0381] Table 1-144

[0382] Table 1-145

[0383] Table 1-146

[0384] Table 1-147

[0385] Table 1-148

[0386] Table 1-149

[0387] Table 1-150

[0388] Table 1-151

[0389] Table 1-152

[0390] Table 1-153

[0391] Table 1-154

[0392] Table 1-155

[0393] Table 1-156

[0394] Table 1-157

[0395] Table 1-158

[0396] Table 1-159

[0397] Table 1-160

[0398] Table 1-161

[0399] Table 1-162

[0400] Table 2-1

[0401] [Table 2-2]

[0402] [Table 2-3]

[0403] [Table 3-1]

[0404] [Table 3-2]

[0405] Embodiment The following non-limiting embodiments provide illustrative examples of the present invention, but do not limit the scope of the invention. 1. A method for converting a plurality of stem cells into a plurality of chondrogenic cells through modulation of the expression levels of a plurality of distinct target genes comprising a first homeobox protein and a second homeobox protein, the method comprising: (a) contacting a first polynucleotide sequence in a plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of a first homeobox protein operably linked to the first polynucleotide sequence; (b) contacting the second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory portion to regulate the expression level of a second homeobox protein operably linked to the second polynucleotide sequence. 2. The method of embodiment 1, wherein (b) occurs after (a), and steps (a) and (b) sequentially result in modulation of the first homeobox protein and the second homeobox protein. 3. The method of embodiment 1, wherein (i) the first polynucleotide sequence is upstream of or encodes a first homeobox protein, or (ii) the second polynucleotide sequence is upstream of or encodes a second homeobox protein. 4. The method of embodiment 1, wherein (iii) the expression level of the first homeobox protein is enhanced upon contact with the first heterologous gene regulatory portion, or (iv) the expression level of the second homeobox protein is enhanced upon contact with the second heterologous gene regulatory portion. 5. The method of embodiment 1, further comprising modulating expression of additional target genes among the plurality of target genes, wherein the additional target genes comprise one or more members selected from the group consisting of T-box transcription factors (TBXs), basic helix-loop-helix transcription factors (bHLHs), Sox, and collagens. 6. The method of embodiment 5, wherein the expression level of the additional target gene is regulated (i) before the second homeobox protein, (ii) simultaneously with the second homeobox protein, or (iii) after the second homeobox protein. 7. The method of embodiment 5, wherein the expression level of an additional target gene is enhanced. 8. The method includes contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to effect transformation, the plurality of gating units: (i) a first gating unit preconfigured to provide a first heterologous gene regulatory portion to regulate the expression level of a first homeobox protein; and (ii) a second gating unit preconfigured to provide a second heterologous gene regulatory moiety to regulate the expression level of a second homeobox protein; 2. The method of embodiment 1, wherein, upon activation of the heterologous gene circuit, the plurality of gating units operates to effect the transformation. 9. The method of embodiment 8, wherein (i) the first gating unit is activatable to express a first heterologous gene regulatory portion, or (ii) the second gating unit is activatable to express a second heterologous gene regulatory portion. 10. The method of embodiment 9, wherein the plurality of gating units comprises an additional gating unit preconfigured to modulate the expression level of an additional target gene from the plurality of distinct target genes, wherein the additional target gene encodes one or more members selected from the group consisting of T-box transcription factors (TBXs), basic helix-loop-helix transcription factors (bHLHs), Soxs, and collagens. 11. The method of embodiment 1, wherein the conversion occurs in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 5 days. 12. A method for converting a plurality of stem cells into a plurality of chondrogenic cells through modulation of the expression levels of a plurality of distinct target genes, including a first distinct target gene and a second distinct target gene, the method comprising: (a) contacting a first polynucleotide sequence in a plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene operably linked to the first polynucleotide sequence; (b) contacting the second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to regulate the expression level of a second distinct target gene operably linked to the second polynucleotide sequence; The combination of the first distinct target gene and the second distinct target gene is (i) a first homeobox protein and a second homeobox protein that are different; (ii) two distinct members selected from the group consisting of homeobox proteins, T-box transcription factors (TBXs), and basic helix-loop-helix transcription factors (bHLHs); or (iii) The method, wherein the first member is selected from the group consisting of a homeobox protein, TBX, and bHLH, and the second member comprises SOX or collagen. 13. The method of embodiment 12, wherein the combination is (i) a first homeobox protein and a second homeobox protein. 14. The method of embodiment 12, wherein the combination is (ii) two different members selected from the group consisting of homeobox proteins, TBXs, and bHLHs. 15. The method of embodiment 12, wherein the combination is (iii) a first member selected from the group consisting of a homeobox protein, TBX, and a bHLH, and a second member comprising SOX or a collagen. 16. The method of embodiment 12, wherein (b) is performed after (a), thereby sequentially resulting in regulation of a first distinct target gene and a second distinct target gene. 17. The method of embodiment 12, wherein (i) the first polynucleotide sequence is upstream of or encodes a first distinct target gene, or (ii) the second polynucleotide sequence is upstream of or encodes a second distinct target gene. 18. The method of embodiment 12, wherein (ii) the expression level of a first distinct target gene is enhanced upon contact with a first heterologous gene regulatory portion, or (ii) the expression level of a second distinct target gene is enhanced upon contact with a second heterologous gene regulatory portion. 19. The method includes contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to effect transformation, the plurality of gating units comprising: (i) a first gating unit preconfigured to provide a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene; and (ii) a second gating unit preconfigured to provide a second heterologous gene regulatory moiety to regulate the expression level of a second distinct target gene; 13. The method of embodiment 12, wherein, upon activation of the heterologous gene circuit, the plurality of gating units operates to effect the transformation. 20. The method of embodiment 12, wherein the conversion occurs in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 5 days. 21. A method for converting a plurality of stem cells into chondrogenic differentiation, said method comprising: contacting a polynucleotide sequence in the plurality of stem cells with a heterologous gene regulatory moiety to regulate the expression level of a target gene operably linked to the polynucleotide sequence; The method, wherein the conversion rate of the plurality of stem cells to chondrogenic cells is characterized as at least about 30% within less than seven days after contacting. 22. The method of embodiment 21, wherein the conversion rate is at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. 23. The method of embodiment 21, wherein the conversion rate is observed within less than about 6 days after contacting or within less than about 5 days after contacting. 24. The method of embodiment 21, wherein the expression level of the target gene is enhanced upon contact with a heterologous gene regulatory moiety. 25. The method of embodiment 21, wherein the target gene comprises one or more members selected from the group consisting of homeobox proteins, T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), SOX, and collagen. 26. The target gene comprises a plurality of distinct target genes, including a first distinct target and a second distinct target gene, and the contacting comprises: (a) contacting a first polynucleotide sequence in a plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene operably linked to the first polynucleotide sequence; (b) contacting the second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to regulate the expression level of a second distinct target gene operably linked to the second polynucleotide sequence. 27. The combination of the first distinct target gene and the second distinct target gene is (i) a first homeobox protein and a second homeobox protein that are different; (ii) two distinct members selected from the group consisting of homeobox proteins, T-box transcription factors (TBXs), and basic helix-loop-helix transcription factors (bHLHs); or (iii) The method of embodiment 26, wherein the first member is selected from the group consisting of a homeobox protein, TBX, and a bHLH, and the second member comprises SOX or a collagen. 28. The method of embodiment 26, wherein steps (a) and (b) sequentially result in the regulation of a first distinct target gene and a second distinct target gene. 29. Contact contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to effect the transformation, the plurality of gating units comprising: (i) a first gating unit preconfigured to provide a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene; and (ii) a second gating unit preconfigured to provide a second heterologous gene regulatory moiety to regulate the expression level of a second distinct target gene; 27. The method of embodiment 26, wherein, upon activation of the heterologous gene circuit, the plurality of gating units operates to effect the transformation. 30. The method of embodiment 21, wherein the target gene is an endogenous target gene. 31. A method of treating a subject in need thereof, the method comprising: administering to a subject a plurality of chondrogenic cells, wherein the plurality of chondrogenic cells are prepared by subjecting a plurality of stem cells to ex vivo differentiation; The method, wherein the conversion rate of the plurality of stem cells to the plurality of chondrogenic cells is characterized as at least about 30% within less than 7 days of ex vivo differentiation. 32. The method of embodiment 31, wherein the conversion rate is at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. 33. The method of embodiment 31, wherein the conversion rate is observed within less than about 6 days after contacting or within less than about 5 days after contacting. 34. The method of embodiment 31, wherein the plurality of chondrogenic cells are subjected to ex vivo culture for less than about 2 weeks, less than about 10 days, less than about 8 days, less than about 7 days, less than about 6 days, or less than about 5 days. 35. The method of embodiment 31, wherein the ex vivo differentiation comprises modulating the expression levels of target genes comprising one or more members selected from the group consisting of homeobox proteins, T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), SOX, and collagen. 36. The method of embodiment 35, wherein the target gene is an endogenous target gene. 37. The target gene is (i) a first homeobox protein and a second homeobox protein that are different; (ii) two distinct members selected from the group consisting of homeobox proteins, T-box transcription factors (TBXs), and basic helix-loop-helix transcription factors (bHLHs); or (iii) The method of embodiment 35, comprising a plurality of distinct target genes, wherein the plurality of distinct target genes comprises a first member selected from the group consisting of a homeobox protein, TBX, and bHLH, and a second member comprising SOX or a collagen. 38. The method of any one of embodiments 1-37, wherein the plurality of chondrogenic cells comprises chondroprogenitor cells or chondrocytes. 39. The method of any one of embodiments 1-38, wherein the plurality of chondrogenic cells are characterized as being CD146+ / CD73+, CD146+ / CD112+, or CD326+ / CD309+. 40. The method of any one of embodiments 1-39, wherein the plurality of stem cells comprises pluripotent stem cells (PSCs) or mesodermal cells. 41. The method of any one of embodiments 1 to 40, wherein the plurality of distinct target genes is a plurality of endogenous genes of stem cells. 42. The method of any one of embodiments 1 to 41, wherein the first heterologous gene regulatory portion or the second heterologous gene regulatory portion comprises (i) an endonuclease or (ii) a guide nucleic acid (gNA) molecule. 43. The method of embodiment 42, wherein the endonuclease and gNA form a complex capable of binding to their respective target polynucleotide sequences. 44. The method of embodiment 42, wherein the endonuclease is a Cas protein. 45. The method of any one of embodiments 1 to 44, wherein the conversion occurs under conditions that are substantially free of (i) serum and / or (ii) exogenous cell differentiation regulators. 46. ​​The method of embodiment 45, wherein the exogenous cell differentiation regulatory factor comprises one or more members selected from the group consisting of TGF beta 1, TGF beta 2, TGF beta 3, BMP2, BMP4, BMP6, MP7, and IGF1. 47. The method of embodiment 45, wherein the exogenous cell differentiation regulating factor is a chondrogenic factor comprising one or more members selected from the group consisting of dexamethasone, ascorbic acid, insulin, transferrin, and selenate. 48. The method of any one of embodiments 1-47, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is a paired-box (PRD) class homeobox protein. 49. The method of embodiment 48, wherein the PRD class homeobox protein is MIXL1 or UNCX. 50. The method of any one of embodiments 1-49, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is not a member of Pax3 and Pax7. 51. The method of any one of embodiments 1 to 50, wherein TBX comprises one or more members selected from the group consisting of TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, and TBX22. 52. The method of embodiment 51, wherein TBX is TBXT or TBX6. 53. The method of any one of embodiments 1-52, 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. 54. The method of embodiment 53, wherein the bHLH is a group A bHLH. 55. The method of embodiment 53, wherein the bHLH is MSGN1 or TCF15. 56. The method of any one of embodiments 1-55, wherein SOX comprises one or more members selected from the group consisting of SOXA, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH. 57. The method of embodiment 56, wherein SOX is SOXD. 58. The method of embodiment 57, wherein SOXD is SOX6. 59. The method of embodiment 56, wherein SOX is SOXE. 60. The method of embodiment 59, wherein SOXE is SOX9. 61. The method of any one of embodiments 1-60, wherein the collagen comprises one or more members selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen. 62. The method of embodiment 61, wherein the collagen is type II collagen. 63. The method of embodiment 62, wherein the type II collagen is COL2A1. 64. The method of any one of embodiments 1-63, wherein the conversion occurs in less than about 10 days, less than about 7 days, or less than about 5 days. 65. The method of any one of embodiments 1-64, further comprising storing the plurality of tissue-specific progenitor cells or the plurality of chondrogenic cells in a sterile vial. 66. The method of any of embodiments 1-65, further comprising administering a plurality of tissue-specific progenitor cells or a plurality of chondrogenic cells to a subject in need thereof. 67. The method of any one of embodiments 1-66, wherein administering comprises administering to one or more knees. 68. A system for converting a plurality of stem cells into a plurality of chondrogenic cells through regulating the expression levels of a plurality of distinct target genes comprising a first homeobox protein and a second homeobox protein, the system comprising: a) a first heterologous gene regulatory portion configured to bind to a first polynucleotide sequence in a plurality of stem cells and regulate the expression level of a first homeobox protein operably linked to the first polynucleotide sequence; and b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and regulate the expression level of a second homeobox protein operably linked to the second polynucleotide sequence. 69. The system of embodiment 68, wherein the first heterologous gene regulatory portion and the second heterologous gene regulatory portion are configured to sequentially effect regulation of the first homeobox protein and the second homeobox protein. 70. The system of embodiment 68, wherein (i) the first polynucleotide sequence is upstream of or encodes a first homeobox protein, or (ii) the second polynucleotide sequence is upstream of or encodes a second homeobox protein. 71. The system of embodiment 68, wherein (ii) the expression level of a first homeobox protein is enhanced upon contact with a first heterologous gene regulatory portion, or (ii) the expression level of a second homeobox protein is enhanced upon contact with a second heterologous gene regulatory portion. 72. The system of embodiment 68, further comprising an additional heterologous gene regulatory portion configured to regulate expression of an additional target gene among the plurality of target genes, wherein the additional target gene comprises one or more members selected from the group consisting of T-box transcription factors (TBXs), basic helix-loop-helix transcription factors (bHLHs), Sox, and collagens. 73. The system of embodiment 72, wherein the expression level of the additional target gene is regulated (i) before the second homeobox protein, (ii) simultaneously with the second homeobox protein, or (iii) after the second homeobox protein. 74. The system of embodiment 72, wherein the expression level of the additional target gene is enhanced. 75. A heterologous gene circuit comprising a plurality of gating units, wherein the heterologous gene circuit is activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to effect transformation, wherein the plurality of gating units: (i) a first gating unit preconfigured to provide a first heterologous gene regulatory portion to regulate the expression level of a first homeobox protein; and (ii) a second gating unit preconfigured to provide a second heterologous gene regulatory moiety to regulate the expression level of a second homeobox protein; 69. The system of embodiment 68, wherein the plurality of gating units operates to effect the transformation upon activation of the heterologous gene circuit. 76. The system of embodiment 75, wherein (i) the first gating unit is activatable to express a first heterologous gene regulatory portion, or (ii) the second gating unit is activatable to express a second heterologous gene regulatory portion. 77. The system of embodiment 75, wherein the plurality of gating units comprises an additional gating unit preconfigured to regulate the expression level of an additional target gene from the plurality of distinct target genes, wherein the additional target gene encodes one or more members selected from the group consisting of T-box transcription factors (TBXs), basic helix-loop-helix transcription factors (bHLHs), Sox, and collagens. 78. The system of embodiment 68, wherein the conversion occurs in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 5 days. 79. A system for converting a plurality of stem cells into a plurality of chondrogenic cells through modulation of the expression levels of a plurality of distinct target genes, including a first distinct target gene and a second distinct target gene, the system comprising: a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in a plurality of stem cells and regulate the expression level of a first distinct target gene operably linked to the first polynucleotide sequence; and b) a second heterologous gene regulatory moiety configured to bind to a second polynucleotide sequence in the plurality of stem cells to regulate the expression level of a second distinct target gene operably linked to the second polynucleotide sequence; The combination of the first distinct target gene and the second distinct target gene is (i) a first homeobox protein and a second homeobox protein that are different; (ii) two different members selected from the group consisting of homeobox proteins, T-box transcription factors (TBXs), and basic helix-loop-helix transcription factors (bHLHs); or (iii) The system of embodiment 26, wherein the first member is selected from the group consisting of a homeobox protein, TBX, and a bHLH, and the second member comprises SOX or a collagen. 80. The system of embodiment 79, wherein the combination is (i) a first homeobox protein and a second homeobox protein. 81. The system of embodiment 79, wherein the combination is (ii) two different members selected from the group consisting of homeobox proteins, TBXs, and bHLHs. 82. The system of embodiment 79, wherein the combination is (iii) a first member selected from the group consisting of a homeobox protein, TBX, and a bHLH, and a second member comprising SOX or a collagen. 83. The system of embodiment 79, wherein the first heterologous gene regulatory portion and the second heterologous gene regulatory portion are configured to sequentially effect regulation of the first distinct target gene prior to regulation of the second distinct target gene. 84. The system of embodiment 79, wherein (i) the first polynucleotide sequence is upstream of or encodes a first distinct target gene, or (ii) the second polynucleotide sequence is upstream of or encodes a second distinct target gene. 85. The system of embodiment 79, wherein (iii) the expression level of a first distinct target gene is enhanced upon contact with a first heterologous gene regulatory portion, or (iv) the expression level of a second distinct target gene is enhanced upon contact with a second heterologous gene regulatory portion. 86. A heterologous gene circuit comprising a plurality of gating units, wherein the heterologous gene circuit is activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to effect transformation, wherein the plurality of gating units: (i) a first gating unit preconfigured to provide a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene; and (ii) a second gating unit preconfigured to provide a second heterologous gene regulatory moiety to regulate the expression level of a second distinct target gene; 80. The system of embodiment 79, wherein the plurality of gating units operates to effect the transformation upon activation of the heterologous gene circuit. 87. The system of embodiment 79, wherein the conversion occurs in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 5 days. 88. A system for converting a plurality of stem cells into chondrogenic differentiation, said system comprising: a heterologous gene regulatory moiety configured to bind to a polynucleotide sequence in the plurality of stem cells and regulate the expression level of a target gene operably linked to the polynucleotide sequence; The system is characterized by a conversion rate of the plurality of stem cells to chondrogenic cells of at least about 30% within less than seven days after contact. 89. The system of embodiment 88, wherein the conversion rate is at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. 90. The system of embodiment 88, wherein the conversion rate is observed within less than about 6 days after contacting or within less than about 5 days after contacting. 91. The system of embodiment 88, wherein the expression level of the target gene is enhanced upon contact with a heterologous gene regulatory moiety. 92. The system of embodiment 88, wherein the target gene comprises one or more members selected from the group consisting of homeobox proteins, T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), SOX, and collagen. 93. The target gene comprises a plurality of distinct target genes, including a first distinct target and a second distinct target gene, and the system comprises: a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in a plurality of stem cells and regulate the expression level of a first distinct target gene operably linked to the first polynucleotide sequence; and b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and regulate the expression level of a second, distinct target gene operably linked to the second polynucleotide sequence. 94. The combination of the first distinct target gene and the second distinct target gene is (i) a first homeobox protein and a second homeobox protein that are different; (ii) two different members selected from the group consisting of homeobox proteins, T-box transcription factors (TBXs), and basic helix-loop-helix transcription factors (bHLHs); or (iii) The composition of embodiment 93, wherein the first member is selected from the group consisting of a homeobox protein, TBX, and a bHLH, and the second member comprises SOX or a collagen. 95. The system of embodiment 93, wherein the first heterologous gene regulatory portion and the second heterologous gene regulatory portion are configured to sequentially effect regulation of a first distinct target gene and a second distinct target gene. 96. A heterologous gene circuit comprising a plurality of gating units, wherein the heterologous gene circuit is activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to effect transformation, wherein the plurality of gating units: (i) a first gating unit preconfigured to provide a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene; and (ii) a second gating unit preconfigured to provide a second heterologous gene regulatory moiety to regulate the expression level of a second distinct target gene; 94. The system of embodiment 93, wherein the plurality of gating units operates to effect the transformation upon activation of the heterologous gene circuit. 97. The system of embodiment 88, wherein the target gene is an endogenous target gene. 98. A composition comprising any one of embodiments 1-97. 99. A composition for treating a subject, the composition comprising: a plurality of chondrogenic cells prepared by subjecting a plurality of stem cells to ex vivo differentiation; The composition, wherein the conversion rate of the plurality of stem cells to the plurality of chondrogenic cells is characterized by at least about 30% within less than 7 days of ex vivo differentiation. 100. The composition of embodiment 99, wherein the conversion rate is at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. 101. The composition of embodiment 99, wherein the conversion rate is observed within less than about 6 days after contact or within less than about 5 days after contact. 102. The composition of embodiment 99, wherein the plurality of chondrogenic cells are subjected to ex vivo culture for less than about 2 weeks, less than about 10 days, less than about 8 days, less than about 7 days, less than about 6 days, or less than about 5 days. 103. The composition of embodiment 99, wherein the ex vivo differentiation is characterized by modulation of the expression levels of target genes comprising one or more members selected from the group consisting of homeobox proteins, T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), SOX, and collagen. 104. The composition of embodiment 99, wherein the target gene is an endogenous target gene. 105. The target gene is (i) a first homeobox protein and a second homeobox protein that are different; (ii) two different members selected from the group consisting of homeobox proteins, T-box transcription factors (TBXs), and basic helix-loop-helix transcription factors (bHLHs); or (iii) a first member selected from the group consisting of a homeobox protein, TBX, and bHLH, and a second member comprising SOX or a collagen; 94. The composition of embodiment 93, comprising a plurality of distinct target genes comprising: 106. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the plurality of chondrogenic cells comprises chondroprogenitor cells or chondrocytes. 107. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the plurality of chondrogenic cells are CD146+ / CD73+, CD146+ / CD112+, or CD326+ / CD309+. 108. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the plurality of stem cells comprises pluripotent stem cells (PSCs) or mesodermal cells. 109. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the plurality of distinct target genes are endogenous genes of a plurality of stem cells. 110. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the first heterologous gene regulatory portion or the second heterologous gene regulatory portion comprises (i) an endonuclease or (ii) a guide nucleic acid (gNA) molecule. 111. A system according to any one of the preceding embodiments, or a composition according to any one of the preceding embodiments, wherein the endonuclease and gNA form a complex capable of binding to their respective target polynucleotide sequences. 112. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the endonuclease is a Cas protein. 113. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the conversion occurs under conditions that are substantially free of (i) serum and / or (ii) exogenous cell differentiation regulators. 114. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the exogenous cell differentiation regulator comprises one or more members selected from the group consisting of TGF beta 1, TGF beta 2, TGF beta 3, BMP2, BMP4, BMP6, MP7, and IGF1. 115. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the exogenous cell differentiation regulator is a chondrogenic factor comprising one or more members selected from the group consisting of dexamethasone, ascorbic acid, insulin, transferrin, and selenate. 116. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is a paired-box (PRD) class homeobox protein. 117. The system according to any one of the preceding embodiments, or the composition according to any one of the preceding embodiments, wherein the PRD class homeobox protein is MIXL1 or UNCX. 118. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is not a member of Pax3 and Pax7. 119. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein TBX comprises one or more members selected from the group consisting of TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, and TBX22. 120. A system according to any one of the preceding embodiments, or a composition according to any one of the preceding embodiments, wherein TBX is TBXT or TBX6. 121. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, 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. 122. The system according to any one of the preceding embodiments, or the composition according to any one of the preceding embodiments, wherein the bHLH is a Group A bHLH. 123. A system according to any one of the preceding embodiments, or a composition according to any one of the preceding embodiments, wherein the bHLH is MSGN1 or TCF15. 124. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein SOX comprises one or more members selected from the group consisting of SOXA, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH. 125. The system according to any one of the preceding embodiments, or the composition according to any one of the preceding embodiments, wherein SOX is SOXD. 126. The system according to any one of the preceding embodiments, or the composition according to any one of the preceding embodiments, wherein SOXD is SOX6. 127. A system according to any one of the preceding embodiments, or a composition according to any one of the preceding embodiments, wherein SOX is SOXE. 128. The system according to any one of the preceding embodiments, or the composition according to any one of the preceding embodiments, wherein SOXE is SOX9. 129. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the collagen comprises one or more members selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen. 130. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the collagen is type II collagen. 131. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the type II collagen is COL2A1. 132. The system of any one of the preceding embodiments, or the composition of any one of the preceding embodiments, wherein the conversion occurs in less than about 10 days, less than about 7 days, or less than about 5 days.

[0406] The systems and methods of the present disclosure may be combined with or modified by other systems and methods for cellular programming, such as those described in, for example, International Patent Application No. PCT / US2018 / 052211, International Patent Application No. PCT / US2018 / 052211, International Patent Application No. PCT / US2023 / 028169, International Patent Application No. PCT / US2023 / 028255, and International Patent Application No. PCT / US2023 / 028033, each of which is incorporated herein by reference in its entirety.

[0407] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, depending upon a variety of conditions and variables. It is to be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the invention. Therefore, it is contemplated that the present invention shall encompass any and all such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for converting a plurality of stem cells into a plurality of chondrogenic cells via regulating the expression levels of a plurality of distinct target genes comprising a first homeobox protein and a second homeobox protein, the method comprising: (a) contacting a first polynucleotide sequence in a plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of a first homeobox protein operably linked to the first polynucleotide sequence; (b) contacting the second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory portion to regulate the expression level of a second homeobox protein operably linked to the second polynucleotide sequence.

2. 2. The method of claim 1, wherein (b) is performed after (a), and steps (a) and (b) sequentially result in modulation of the first homeobox protein and the second homeobox protein.

3. 2. The method of claim 1, wherein (i) the first polynucleotide sequence is upstream of or encodes a first homeobox protein, or (ii) the second polynucleotide sequence is upstream of or encodes a second homeobox protein.

4. The method of claim 1, wherein (iii) the expression level of a first homeobox protein is enhanced upon contact with a first heterologous gene regulatory portion, or (iv) the expression level of a second homeobox protein is enhanced upon contact with a second heterologous gene regulatory portion.

5. 10. The method of claim 1, further comprising modulating expression of additional target genes among the plurality of target genes, wherein the additional target genes comprise one or more members selected from the group consisting of T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), Sox, and collagen.

6. 6. The method of claim 5, wherein the expression level of the additional target gene is regulated (i) before the second homeobox protein, (ii) simultaneously with the second homeobox protein, or (iii) after the second homeobox protein.

7. The method of claim 5, wherein the expression level of the additional target gene is enhanced.

8. The method includes contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to effect the transformation, the plurality of gating units comprising: (i) a first gating unit preconfigured to provide a first heterologous gene regulatory moiety to regulate the expression level of a first homeobox protein; and (ii) a second gating unit preconfigured to effect a second heterologous gene regulatory moiety to regulate the expression level of a second homeobox protein, wherein upon activation of the heterologous gene circuit, the plurality of gating units operates to effect conversion.

9. 9. The method of claim 8, wherein (i) the first gating unit is activatable to express a first heterologous gene regulatory portion, or (ii) the second gating unit is activatable to express a second heterologous gene regulatory portion.

10. 10. The method of claim 9, wherein the plurality of gating units comprises an additional gating unit preconfigured to modulate the expression level of an additional target gene of the plurality of distinct target genes, wherein the additional target gene encodes one or more members selected from the group consisting of T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), Sox, and collagen.

11. 10. The method of claim 1, wherein the conversion occurs in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 5 days.

12. 1. A method for converting a plurality of stem cells into a plurality of chondrogenic cells through modulation of expression levels of a plurality of distinct target genes, including a first distinct target gene and a second distinct target gene, the method comprising: (a) contacting a first polynucleotide sequence in a plurality of stem cells with a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene operably linked to the first polynucleotide sequence; (b) contacting the second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to regulate the expression level of a second distinct target gene operably linked to the second polynucleotide sequence, wherein the combination of the first distinct target gene and the second distinct target gene comprises: (i) a first homeobox protein and a second homeobox protein that are different; (ii) two different members selected from the group consisting of homeobox proteins, T-box transcription factors (TBX), and basic helix-loop-helix transcription factors (bHLH), or (iii) the first member is selected from the group consisting of homeobox protein, TBX, and bHLH, and the second member comprises SOX or collagen.

13. 13. The method of claim 12, wherein the combination is (i) a first homeobox protein and a second homeobox protein.

14. 13. The method of claim 12, wherein the combination is (ii) two different members selected from the group consisting of homeobox proteins, TBX, and bHLH.

15. 13. The method of claim 12, wherein the combination is (iii) a first member selected from the group consisting of homeobox proteins, TBX, and bHLH, and a second member comprising SOX or collagen.

16. 13. The method of claim 12, wherein (b) is performed after (a), thereby sequentially resulting in modulation of the first distinct target gene and the second distinct target gene.

17. 13. The method of claim 12, wherein (i) the first polynucleotide sequence is upstream of or encodes a first distinct target gene, or (ii) the second polynucleotide sequence is upstream of or encodes a second distinct target gene.

18. 13. The method of claim 12, wherein (ii) the expression level of a first distinct target gene is enhanced upon contact with a first heterologous gene regulatory portion, or (ii) the expression level of a second distinct target gene is enhanced upon contact with a second heterologous gene regulatory portion.

19. The method includes contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to effect the transformation, the plurality of gating units comprising: (i) a first gating unit preconfigured to provide a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene; and 13. The method of claim 12, comprising (ii) a second gating unit preconfigured to effect a second heterologous gene regulatory moiety to modulate the expression level of a second distinct target gene, wherein upon activation of the heterologous gene circuit, the plurality of gating units operates to effect the conversion.

20. 13. The method of claim 12, wherein the conversion occurs in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 5 days.

21. 1. A method for converting a plurality of stem cells into chondrogenic differentiation, said method comprising: contacting a polynucleotide sequence in the plurality of stem cells with a heterologous gene regulatory moiety to regulate the expression level of a target gene operably linked to the polynucleotide sequence; The method, wherein the conversion rate of the plurality of stem cells to chondrogenic cells is characterized as at least about 30% within less than seven days after contacting.

22. 22. The method of claim 21, wherein the conversion rate is at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%.

23. 22. The method of claim 21, wherein the conversion is observed within less than about 6 days after contacting or within less than about 5 days after contacting.

24. 22. The method of claim 21, wherein the expression level of the target gene is enhanced upon contact with the heterologous gene regulatory moiety.

25. 22. The method of claim 21, wherein the target gene comprises one or more members selected from the group consisting of homeobox proteins, T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), SOX, and collagen.

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

27. The combination of the first distinct target gene and the second distinct target gene is (i) a first homeobox protein and a second homeobox protein that are different; (ii) two different members selected from the group consisting of homeobox proteins, T-box transcription factors (TBXs), and basic helix-loop-helix transcription factors (bHLHs); or 27. The method of claim 26, wherein (iii) a first member is selected from the group consisting of a homeobox protein, TBX, and bHLH, and a second member comprises SOX or collagen.

28. 27. The method of claim 26, wherein steps (a) and (b) sequentially result in the regulation of a first distinct target gene and a second distinct target gene.

29. The contacting includes contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, the heterologous genetic circuit being activatable to induce the plurality of gating units to sequentially regulate the expression levels of a plurality of distinct target genes to effect the transformation, the plurality of gating units comprising: (i) a first gating unit preconfigured to provide a first heterologous gene regulatory moiety to regulate the expression level of a first distinct target gene; and (ii) a second gating unit preconfigured to provide a second heterologous gene regulatory moiety to regulate the expression level of a second distinct target gene; 27. The method of claim 26, wherein the plurality of gating units operates to effect the transformation upon activation of the heterologous gene circuit.

30. 22. The method of claim 21, wherein the target gene is an endogenous target gene.

31. 1. A method of treating a subject in need thereof, said method comprising: administering to a subject a plurality of chondrogenic cells, wherein the plurality of chondrogenic cells are prepared by subjecting a plurality of stem cells to ex vivo differentiation; The method, wherein the conversion rate of the plurality of stem cells to the plurality of chondrogenic cells is characterized as at least about 30% within less than 7 days of ex vivo differentiation.

32. 32. The method of claim 31, wherein the conversion rate is at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%.

33. 32. The method of claim 31 , wherein the conversion is observed within less than about 6 days after contacting or within less than about 5 days after contacting.

34. 32. The method of claim 31, wherein the plurality of chondrogenic cells are subjected to ex vivo culture for less than about 2 weeks, less than about 10 days, less than about 8 days, less than about 7 days, less than about 6 days, or less than about 5 days.

35. 32. The method of claim 31, wherein the ex vivo differentiation comprises modulating the expression levels of target genes comprising one or more members selected from the group consisting of homeobox proteins, T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), SOX, and collagen.

36. 36. The method of claim 35, wherein the target gene is an endogenous target gene.

37. The target gene is (i) a first homeobox protein and a second homeobox protein that are different; (ii) two different members selected from the group consisting of homeobox proteins, T-box transcription factors (TBX), and basic helix-loop-helix transcription factors (bHLH), or 36. The method of claim 35, comprising: (iii) a plurality of distinct target genes comprising a first member selected from the group consisting of homeobox proteins, TBX, and bHLH, and a second member comprising SOX or collagen.

38. 38. The method of any one of claims 1 to 37, wherein the plurality of chondrogenic cells comprises chondroprogenitor cells or chondrocytes.

39. 39. The method of any one of claims 1 to 38, wherein the plurality of chondrogenic cells are characterized as CD146+ / CD73+, CD146+ / CD112+, or CD326+ / CD309+.

40. 40. The method of any one of claims 1 to 39, wherein the plurality of stem cells comprises pluripotent stem cells (PSCs) or mesodermal cells.

41. 41. The method of any one of claims 1 to 40, wherein the plurality of distinct target genes are a plurality of endogenous genes of stem cells.

42. 42. The method of any one of claims 1 to 41, wherein the first heterologous gene regulatory portion or the second heterologous gene regulatory portion comprises (i) an endonuclease or (ii) a guide nucleic acid (gNA) molecule.

43. 43. The method of claim 42, wherein the endonuclease and gNA form a complex capable of binding to their respective target polynucleotide sequences.

44. 43. The method of claim 42, wherein the endonuclease is a Cas protein.

45. 45. The method of any one of claims 1 to 44, wherein the conversion occurs under conditions that are substantially free of (i) serum and / or (ii) exogenous cell differentiation regulators.

46. 46. ​​The method of claim 45, wherein the exogenous cell differentiation regulatory factor comprises one or more members selected from the group consisting of TGF beta 1, TGF beta 2, TGF beta 3, BMP2, BMP4, BMP6, MP7, and IGF1.

47. 46. ​​The method of claim 45, wherein the exogenous cell differentiation regulator is a chondrogenic factor comprising one or more members selected from the group consisting of dexamethasone, ascorbic acid, insulin, transferrin, and selenate.

48. 48. The method of any one of claims 1 to 47, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is a paired box (PRD) class homeobox protein.

49. 49. The method of claim 48, wherein the PRD class homeobox protein is MIXL1 or UNCX.

50. 50. The method of any one of claims 1 to 49, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is not a member of Pax3 and Pax7.

51. 51. The method of any one of claims 1-50, wherein TBX comprises one or more members selected from the group consisting of TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, and TBX22.

52. 52. The method of claim 51, wherein the TBX is TBXT or TBX6.

53. 53. The method of any one of claims 1 to 52, 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.

54. 54. The method of claim 53, wherein the bHLH is a Group A bHLH.

55. 54. The method of claim 53, wherein the bHLH is MSGN1 or TCF15.

56. 56. The method of any one of claims 1 to 55, wherein SOX comprises one or more members selected from the group consisting of SOXA, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH.

57. 57. The method of claim 56, wherein the SOX is SOXD.

58. 58. The method of claim 57, wherein the SOXD is SOX6.

59. 57. The method of claim 56, wherein the SOX is SOXE.

60. 60. The method of claim 59, wherein the SOXE is SOX9.

61. 61. The method of any one of claims 1 to 60, wherein the collagen comprises one or more members selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen.

62. 62. The method of claim 61, wherein the collagen is type II collagen.

63. 63. The method of claim 62, wherein the type II collagen is COL2A1.

64. 64. The method of any one of claims 1-63, wherein the conversion occurs in less than about 10 days, less than about 7 days, or less than about 5 days.

65. 65. The method of any one of claims 1 to 64, further comprising storing the plurality of tissue-specific progenitor cells or the plurality of chondrogenic cells in a sterile vial.

66. 66. The method of any of claims 1 to 65, further comprising administering the plurality of tissue-specific progenitor cells or the plurality of chondrogenic cells to a subject in need thereof.

67. 67. The method of any one of claims 1-66, wherein the administering step comprises one or more administrations to the knee.

68. 1. A system for converting a plurality of stem cells into a plurality of chondrogenic cells through modulation of the expression levels of a plurality of distinct target genes comprising a first homeobox protein and a second homeobox protein, the system comprising: a) a first heterologous gene regulatory portion configured to bind to a first polynucleotide sequence in a plurality of stem cells to regulate the expression level of a first homeobox protein operably linked to the first polynucleotide sequence; and b) a second heterologous gene regulatory portion configured to bind to a second polynucleotide sequence in the plurality of stem cells and regulate the expression level of a second homeobox protein operably linked to the second polynucleotide sequence.

69. 1. A system for converting a plurality of stem cells into a plurality of chondrogenic cells via regulating the expression levels of a plurality of distinct target genes, including a first distinct target gene and a second distinct target gene, the system comprising: a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in a plurality of stem cells to regulate the expression level of a first distinct target gene operably linked to the first polynucleotide sequence; and b) a second heterologous gene regulatory moiety configured to bind to a second polynucleotide sequence in the plurality of stem cells to regulate the expression level of a second distinct target gene operably linked to the second polynucleotide sequence; The combination of the first distinct target gene and the second distinct target gene is (i) a first homeobox protein and a second homeobox protein that are different; (ii) two different members selected from the group consisting of homeobox proteins, T-box transcription factors (TBX), and basic helix-loop-helix transcription factors (bHLH), or (iii) A system, wherein the first member is selected from the group consisting of homeobox proteins, TBX, and bHLH, and the second member comprises SOX or collagen.

70. 1. A system for converting a plurality of stem cells into chondrogenic differentiation, said system comprising: a heterologous gene regulatory moiety configured to bind to a polynucleotide sequence in the plurality of stem cells and regulate the expression level of a target gene operably linked to the polynucleotide sequence; The system is characterized by a conversion rate of the plurality of stem cells to chondrogenic cells of at least about 30% within less than seven days after contact.

71. 1. A composition for treating a subject, said composition comprising: a plurality of chondrogenic cells prepared by subjecting a plurality of stem cells to ex vivo differentiation; The composition is characterized by a conversion rate of at least about 30% of the stem cells to a plurality of chondrogenic cells within less than 7 days of ex vivo differentiation.

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