System for cell programming of chondrogenesis lineage and method thereof

Abstract

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

Description

[0001] CROSS-REFERENCE

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 482,721, filed February 1, 2023, which is incorporated by reference herein in its entirety. BACKGROUND

[0003] Heterologous proteins and / or nucleic acid molecules can be used to elicit a desired response in a cell. Heterologous proteins and / or nucleic acid molecules can modulate genes of interest (e.g., transgenes and / or endogenous genes) to program a cell (e.g., differentiate, de-differentiate). In some cases, nucleases-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 technologies can be characterized by their versatility and ease of programming, and can be used to facilitate genome editing across different species and cell types. SUMMARY

[0004] The present disclosure provides methods and systems for programming cells, e.g., for eliciting a desired response in a cell. The systems and methods of the present disclosure can facilitate the transformation of a cell from one type to another. The systems and methods of the present disclosure 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 transformation. The systems and methods of the present disclosure can utilize heterologous proteins and / or nucleic acid molecules as building blocks for such genetic circuits.

[0005] In some embodiments, the present disclosure provides a method for transforming a plurality of stem cells into a plurality of chondrogenic cells via modulating expression levels of a plurality of different 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 moiety to modulate an expression level of a first homeobox protein operably coupled to the first polynucleotide sequence; and (b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to modulate an expression level of a second homeobox protein operably coupled to the second polynucleotide sequence.

[0006] In some embodiments, the present disclosure provides a method for converting a plurality of stem cells to a plurality of chondrogenic cells via modulating expression levels of a plurality of different target genes comprising a first different target gene and a second different target gene, the method comprising: (a) contacting, by a first heterologous gene regulatory moiety, a first polynucleotide sequence in the plurality of stem cells to modulate an expression level of a first different target gene operably coupled to the first polynucleotide sequence; and (b) contacting, by a second heterologous gene regulatory moiety, a second polynucleotide sequence in the plurality of stem cells to modulate an expression level of a second different target gene operably coupled to the second polynucleotide sequence, wherein the combination of the first and second different target genes is: (i) a first homeodomain protein and a second homeodomain protein that are different; (ii) two different members selected from a homeodomain protein, a T-box transcription factor (TBX), and a basic helix-loop-helix transcription factor (bHLH); or (iii) a first member selected from a homeodomain protein, a TBX, and a bHLH, and a second member comprising a SOX or a collagen.

[0007] In some embodiments, the present disclosure provides a method for converting a plurality of stem cells to chondrogenic differentiation, the method comprising: contacting, by a heterologous gene regulatory moiety, a polynucleotide sequence in the plurality of stem cells to modulate an expression level of a target gene operably coupled to the polynucleotide sequence, wherein a conversion rate from the plurality of stem cells to chondrogenic cells is characterized as at least about 30% within less than 7 days after the contacting.

[0008] In some embodiments, the present disclosure provides a method for treating a subject in need thereof, the method comprising: administering a plurality of chondrogenic cells to the subject, wherein the plurality of chondrogenic cells is prepared by subjecting a plurality of stem cells to ex vivo differentiation, wherein a conversion rate from the plurality of stem cells to the plurality of chondrogenic cells is characterized as at least about 30% within less than 7 days of the ex vivo differentiation.

[0009] In some embodiments, the present disclosure provides a system for converting a plurality of stem cells to a plurality of chondrogenic cells via modulating expression levels of a plurality of different target genes comprising a first homeodomain protein and a second homeodomain protein, the system comprising: (a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in the plurality of stem cells to modulate an expression level of the first homeodomain protein operably coupled 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 modulate an expression level of the second homeodomain protein operably coupled to the second polynucleotide sequence.

[0010] In some embodiments, the present disclosure provides a system for converting a plurality of stem cells to a plurality of chondrogenic cells via modulating expression levels of a plurality of different target genes comprising a first different target gene and a second different target gene, the system comprising: (a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in the plurality of stem cells to modulate an expression level of a first different target gene operably coupled 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 modulate an expression level of a second different target gene operably coupled to the second polynucleotide sequence, wherein the combination of the first and second different target genes is: (i) a first homeodomain protein and a second homeodomain protein that are different; (ii) two different members selected from the group consisting of a homeodomain 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 homeodomain protein, a TBX, and a bHLH, and a second member comprising a SOX or a collagen.

[0011] 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 to a polynucleotide sequence in the plurality of stem cells to modulate an expression level of a target gene operably coupled to the polynucleotide sequence, wherein a conversion rate from the plurality of stem cells to chondrogenic cells is characterized as at least about 30% within less than 7 days after the contacting.

[0012] 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 a conversion rate from the plurality of stem cells to the plurality of chondrogenic cells is characterized as at least about 30% within less than 7 days of the ex vivo differentiation.

[0013] INCORPORATION BY REFERENCE

[0014] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent there is a contradiction between the disclosure herein and the disclosure in any publication or patent or patent application incorporated by reference, the present specification is intended to take precedence. BRIEF DESCRIPTION OF DRAWINGS

[0015] The novel features of the application are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present application will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the application are utilized, and the accompanying drawings of which:

[0016] FIG. 1 is a schematic of a heterologous gene circuit. The activation portion initiates the circuit and can activate the gate unit. The gate unit is comprised of a gate portion and / or a gene regulatory portion.

[0017] FIG. 2A depicts the developmental progression from iPSCs to chondrocytes, including genes known to influence chondrocyte development at different stages. FIG. 2B depicts an example of a heterologous gene circuit that distinguishes chondroprogenitor cells from mesodermal cells.

[0018] FIG. 3 depicts an example heterologous gene circuit.

[0019] FIG. 4 shows a scatter plot (e.g., a volcano plot) for identifying one or more heterologous gene circuits that induce stem cells to transform into chondroprogenitor cells.

[0020] FIG. 5A-5D shows an example of chondrogenic progenitor cell marker analysis data used to generate the scatter plot in FIG. 4

[0021] FIG. 6A-6B shows that the top performing heterologous gene circuits transformed approximately 60% of cells into chondrogenic progenitor cells within four days. FIG. 6C depicts a summary of data from FIG. 6A-6B DETAILED DESCRIPTION

[0022] While various embodiments of the application are shown and described herein, it will be apparent to those skilled in the art that many variations, modifications, and alternatives can be made to the embodiments described herein without departing from the application. It will be understood that various alternatives to the embodiments of the application described herein can be employed.

[0023] Whenever the term“at least,”“greater than,” or“greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term“at least,”“greater than,” or“greater than or equal to” applies to each of the numerical values in this series of numbers. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0024] ​​Whenever the term“at most,”“up to,”“no more than,”“less than,” or“less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term“at most,”“up to,”“no more than,”“less than,” or“less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0025] As used in the specification and claims, the singular form“a,”“an,” and“the” include plural references unless the context clearly dictates otherwise. For example, the term“a gate unit” includes a plurality of gate units.

[0026] The term“about” or“approximately” generally means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 standard deviation, or up to 1% of a given value. Alternatively, “about” can mean ranges approximately 20%, approximately 10%, approximately 5%, or approximately 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean in the same order of magnitude as the value, preferably within a factor of 5, and more preferably within a factor of 2. Where particular values described in the application and claims are described as being approximate, unless otherwise indicated, it should be assumed that the term“about” has been

[0027] The terms“guide nucleic acid,”“guide nucleic acid molecule,” and“gNA,” as 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 or complex with a nucleic acid-guided nuclease. A guide nucleic acid can be a single guide nucleic acid (e.g., sgRNA) or a dual guide nucleic acid (e.g., dgRNA). An sgRNA can be a single RNA molecule that includes both a scaffold tracrRNA and a crRNA that can be complementary to a target sequence. Alternatively, a dgRNA can be a single RNA molecule that contains a crRNA annealed to a tracrRNA by a direct repeat sequence.

[0028] The use of alternatives (e.g.,“or”) should be understood to mean either one, but not both, of the alternatives. The term“and / or” should be understood to mean either one or both of the alternatives.

[0029] As used interchangeably herein, the terms "gene circuit," "biological circuit," or "circuit" generally refer to a collection of molecular components (e.g., biological materials such as polypeptides and / or polynucleotides, non-biological materials, etc.) operably coupled (e.g., simultaneously, sequentially, etc.) according to a circuit design. The collection of molecular components can be capable of providing one or more specific outputs (e.g., regulation of one or more genes) in a cell in response to one or more inputs (e.g., a single input or multiple inputs). Such one or more inputs can be sufficient to trigger the molecular components of the gene circuit to provide the one or more specific outputs. For example, a gene circuit can include one or more molecular switches activatable by one or more inputs FIG. 1

[0030] A gene circuit can be a controllable gene expression system that includes an assembly of biological parts that work together (e.g., simultaneously, sequentially, etc.) as a logical function. A gene circuit can include a plurality of gate units, wherein at least one gate unit of the plurality of gate units is activated by an activation part (e.g., a heterologous input to the cell) to activate other gate units of the plurality of gate units (e.g., simultaneously at one time, sequentially in a cascade, etc.) FIG. 1 For example, at least one gate unit of the plurality of gate units can be activated (e.g., directly or indirectly) by another gate unit of the plurality of gate units to (i) regulate the expression or activity level of one or more target genes, (ii) activate at least one other gate unit of the plurality of gate units, and / or (iii) deactivate at least one other gate unit of the plurality of gate units, thereby collectively regulating the expression and / or activity level of one or more target genes in a desired manner as predetermined by the design of the gene circuit FIG. 1 As used herein, the terms "heterologous gene circuit," "HGC," "gene circuit," "cellular algorithm," or "cellgorithm" can be used interchangeably.

[0031] As referred to herein, the term "gate unit" generally refers to a part of a gene circuit that can control gene regulation by acting like a logic gate, where it can control the flow of information and allow the circuit to make multiple decisions at different points. More specifically, the term refers to a nucleic acid that encodes a genetic switch and a series of regions on which the genetic switch acts or a transcriptional / translational regulatory region. The input of a gate unit can be an activation part and / or another gate unit. The output of a gate unit can activate another gate unit, deactivate another gate unit, affect a target gene, and / or any combination of the above. For example, a gate unit can be composed of a plurality of gate parts and / or a plurality of gene regulation parts FIG. 1

[0032] ​​As referred to herein, the term“activation moiety” generally refers to a moiety that can activate a plurality of gene circuits and / or a plurality of gate units. The activation moiety can be a heterologous input to the cell. For example, the activation moiety can include, but is not limited to, a guide nucleic acid molecule (e.g., gRNA) or other nucleic acid, polypeptide, polynucleotide, small molecule, light, or combinations thereof.

[0033] For example, the activation moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., Cas protein) to bind to a polynucleotide sequence of an inactivated gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to activate such gate moiety that can target one or more gene regulatory moieties (e.g., induce expression of a functional form of another guide nucleic acid molecule). As referred to herein, the term“gate moiety” generally refers to a moiety that can affect the function of a gene regulatory moiety within a gate unit. The gate moiety can activate and / or deactivate a gene regulatory moiety. For example, the gate moiety can regulate expression of a gene regulatory moiety by editing a nucleic acid sequence and thereby activating or deactivating the gene regulatory moiety. For example, the gate moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., Cas protein) to bind to a polynucleotide sequence of a gene regulatory moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to activate the gene regulatory moiety that can target one or more endogenous genes of the cell (e.g., induce expression of a functional form of another guide nucleic acid molecule). Alternatively or additionally, the gate moiety can activate and / or deactivate another gate unit of a gene circuit. FIG. 1 For example, the gate moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., Cas protein) to bind to a polynucleotide sequence of another inactivated gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to activate the other gate moiety (e.g., induce expression of a functional form of another guide nucleic acid molecule). In another example, the gate moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., Cas protein) to bind to a polynucleotide sequence of another activated gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to inactivate the other gate moiety (e.g., reduce expression of a functional form of another guide nucleic acid molecule).

[0034] As referred to herein, the term“gate moiety” generally refers to a moiety that can affect the function of a gene regulatory moiety within a gate unit. The gate moiety can activate and / or deactivate a gene regulatory moiety. For example, the gate moiety can regulate expression of a gene regulatory moiety by editing a nucleic acid sequence and thereby activating or deactivating the gene regulatory moiety. Alternatively or additionally, the gate moiety can activate and / or deactivate another gate unit of a gene circuit. FIG. 1

[0035] ​As interchangeably used herein, the term“gene regulatory moiety” or“gene editing moiety” generally refers to a moiety that can regulate the expression and or activity profile of a nucleic acid sequence or protein, whether exogenous or endogenous to a cell FIG. 1 For example, a gene editing moiety can regulate 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 expression of a gene by editing a genomic DNA sequence. In some cases, a gene editing moiety can regulate expression of a gene by editing an mRNA template. In some cases, editing a nucleic acid sequence can change the underlying template for gene expression (e.g., CRISPR-Cas inspired RNA targeting system). Alternatively, a gene editing moiety can inhibit translation of a gene (e.g., Cas13).

[0036] Alternatively or additionally, a gene editing moiety can be capable of regulating expression or activity of a gene by specifically binding to a target sequence operably coupled to the gene (or a target sequence within the gene), and regulating the production of mRNA from DNA, such as chromosomal DNA or cDNA. For example, a gene editing moiety can recruit or comprise at least one transcription factor that binds to a particular DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. The gene editing moiety can bind to DNA and regulate transcription by physically impeding, for example, preventing proteins such as RNA polymerase and other associated proteins from assembling on the DNA template. A gene editing moiety can regulate expression of a gene at the level of translation, for example, by regulating the production of a protein from an mRNA template. In some cases, a gene editing moiety can regulate gene expression by affecting the stability of an mRNA transcript. In some cases, a gene editing moiety can regulate a gene through epigenetic editing (e.g., Cas12).

[0037] In some cases, a plasmid can encode a non-functional form of a gene editing moiety. The plasmid can be activated (e.g., genetically modified) to express a functional form of the gene editing moiety, for example, via activation of a functional gate moiety. For example, a plasmid can encode a non-functional form of a guide nucleic acid molecule that would otherwise be capable of binding to a target gene of a cell. Upon binding of a functional gate moiety (e.g., another guide nucleic acid molecule in complex with a Cas protein) to the plasmid, the plasmid can be edited (e.g., cleaved at one or more sites, then repaired via endogenous mechanisms (e.g., homologous recombination, non-homologous end joining) to allow expression of a functional form of the gene editing moiety (e.g., a functional form of a guide nucleic acid molecule that specifically binds to a target gene of a cell) to permit regulation of the target gene in a cell.

[0038] In some cases, a gene-regulatory moiety can comprise 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, a gene-regulatory moiety can comprise or be operably coupled to an endonuclease. An endonuclease can be an enzyme that cleaves a phosphodiester bond within a polynucleotide strand. An endonuclease can comprise a restriction endonuclease that cleaves DNA without damaging the base at a specific site. Restriction endonucleases can include endonucleases Type I, Type II, Type III, and Type IV, which can further include subtypes. In some cases, an endonuclease can be Casl, Cas2, Cas 3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, CaslOd, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (Casl4 or C2c10), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Cas 13 (C2c2), Casl3b, Casl3c, Casl3d, Casl3x.1, Csel, Cse2, Csyl, Csy2, Csy3, Csm2, Cmr5, CsxlO, Csxll, Csf1, Csn2. An endonuclease can be a dead endonuclease that exhibits reduced cleavage activity. For example, an endonuclease can be a nuclease-inactivated Cas, such as dCas (e.g., dCas9).

[0039] The Cas proteins described above 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). Accordingly, 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 nucleic acid to be targeted or a 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 to a guide nucleic acid can be referred to as the complementary strand. A strand of a double-stranded target polynucleotide that is complementary to the complementary strand and thus can not be complementary to a guide nucleic acid can be referred to as the non-complementary strand. A guide nucleic acid can comprise a polynucleotide strand and can be referred to as a “single guide nucleic acid.” A guide nucleic acid can comprise two polynucleotide strands and can be referred to as a “double guide nucleic acid.” If not otherwise specified, the term “guide nucleic acid” can be inclusive of both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid can comprise a segment that can be referred to as a “nucleic acid targeting segment” or “nucleic acid targeting sequence” or “spacer sequence.” A nucleic acid targeting segment can comprise a sub-segment that can be referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment” or “scaffold sequence.”

[0040] The gene regulatory moiety can be a transcriptional regulator system (e.g., a gene repressor complex or a gene activator complex). For example, the gene regulatory moiety can be a gene repressor complex comprising a dCas protein operably coupled to a transcriptional repressor (e.g., coupled to or fused with a transcriptional repressor). Non-limiting examples of transcriptional repressors can include KRAB, SID, MBD2, MBD3, DNMT1, DNMT2A, DNMT3A, DNMT3B, DNMT3L, Mecp2, FOG1, ROM2, LSD1, ERD, SRDX repressor domain, Pr-SET7 / 8, SUV4-20H1, RIZ1, JMJD2A, JHDM3A, JMJD2B, JMJD2C, GASC1, JMJD2D, JARID1A, RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / 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 coupled to (e.g., fused with) a transcriptional activator. Non-limiting examples of transcriptional activators can include VP16, VP64, VP48, VP160, p65 subdomain, SET1A, SET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, SYMD2, NSD1, JHDM2a, JHDM2b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK, TET1CD, TET1, DME, DML1, DML2, and ROS1.

[0041] In some cases, the gene-regulating moiety has an enzymatic activity that modifies the target gene without cleaving the target gene. The modification of the target gene can cause, for example, an epigenetic modification that can modify the level of gene expression and / or activity. Examples of enzymatic activities that can be provided by the gene-regulating moiety can include, but are not limited to, nuclease activity (such as nuclease activity provided by restriction enzymes (e.g., Fokl nuclease)); methyltransferase activity (such as methyltransferase activity provided by 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)); demethylase activity (such as demethylase activity provided by demethylases (e.g., ten-eleven translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1)), DNA repair activity, DNA damage activity, deamination activity (such as deamination activity provided by deaminases (e.g., cytosine deaminases such as APOBEC1)), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity (such as integrase activity provided by integrases and / or resolvases (e.g., Gin transposase, such as hyperactive mutants of Gin transposase, Gin H106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase, etc.)), transposase activity, recombinase activity (such as recombinase activity provided by recombinases (e.g., catalytic domain of Gin recombinase)), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0042] The gene-regulating moiety can comprise an endonuclease. An endonuclease can be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. An endonuclease can comprise a restriction endonuclease that cleaves DNA without damaging the bases at specific sites. Restriction endonucleases can include endonucleases Type I, Type II, Type III, and Type IV, which can further include subtypes. In some cases, the endonuclease can be Cas9. In some cases, the endonuclease can be a deactivated Cas (e.g., dCas, dCas9).

[0043] Unless specifically indicated otherwise, or as is apparent from the context, the terms "polynucleotide," "oligonucleotide," or "nucleic acid" as used interchangeably herein refer generally to polymeric forms of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs, in either single- or double-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 include one or more analogs of a natural nucleotide (e.g., altered backbone, sugar or nucleobase). Modifications can be made to the nucleotide structure after polymer assembly or before polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine nucleosides, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides (including cell-free DNA (cfDNA) and cell-free RNA (cfRNA)), nucleic acid probes, and primers. A sequence of nucleotides can be interrupted by non-nucleotide components.

[0044] The term "gene" generally refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) that is involved in the coding of an RNA transcript and its corresponding nucleotide sequence. As used herein, this term in reference to genomic DNA includes intervening non-coding regions as well as regulatory regions and can include 5' and 3' ends. In some uses, the term encompasses the transcribed sequence, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region will contain an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" contains only the coding sequences (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, a gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only the transcribed sequence, but also, in addition, non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene" or native gene in its natural location in the genome of an organism. A gene can refer to an "exogenous gene" or non-native gene. A non-native gene can refer to a gene that is not normally found in the host organism, but that has been introduced 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 includes a mutation, insertion, and / or deletion (e.g., a non-native sequence).

[0045] Generally, the term "sequence identity" refers to the precise nucleotide-nucleotide or amino acid-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining 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." Whether a nucleic acid or an amino acid sequence, the percent identity of two sequences is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity can also be determined, for example, by using the advanced BLAST computer program available from the National Institutes of Health, including version 2.2.9, which compares sequence information. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990) and as discussed in Altschul et al., J. Mol. Biol., 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). The 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, in the blastp program. The program also allows the use of a SEG filter to mask sections of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The degree of sequence identity desired ranges from about 50% to 100%, and integral values therebetween. Generally, the present disclosure includes sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any of the sequences provided herein.

[0046] The term“expression” generally refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides can be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell can include splicing of the mRNA. In reference to expression,“up-regulation” generally refers to an increase in the level of expression of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression level in a wild-type state, while“down-regulation” generally refers to a decrease in the level of expression of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression in a wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During“transient expression,” the transfected gene does not transfer to daughter cells during cell division. Because its expression is limited to the transfected cell, the expression of the gene disappears over time. During transient expression, episomal DNA can transfer to daughter cells, but because episomal DNA is not replicated, it is not permanently inherited and will dilute over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selective advantage to the transfected cell. During stable expression, plasmids can have DNA replication elements that allow them to be inherited or integrated into the genome. Such a selective advantage can be resistance to some toxin presented to the cell.

[0047] As used interchangeably herein, the terms“peptide,”“polypeptide,” or“protein” generally refer to a polymer of at least two amino acid residues linked by peptide bonds. This term does not imply a particular length of the polymer, nor is it intended to distinguish between peptides produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. The term applies to naturally occurring amino acid polymers as well as to amino acid polymers that contain at least one modified amino acid. In some instances, the polymer can be interrupted by non-amino acids. The term embraces chains of amino acid residues including full-length proteins, as well as proteins having no secondary and / or tertiary structure (e.g., a domain). The term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation and any other manipulation such as conjugation with a labeling component. As used herein, the terms“amino acid” and“amino acids” generally refer to natural and non-natural amino acids, which include, but are not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural and non-natural amino acids that have been chemically modified to include a group or chemical moiety that does not naturally occur on an amino acid. An amino acid analog can refer to an amino acid derivative. The term“amino acid” includes both D-amino acids and L-amino acids.

[0048] As used interchangeably herein with respect to polypeptides, the terms “derivative,” “variant,” or “fragment” generally refer to a polypeptide that is related to a wild-type polypeptide, e.g., by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, as compared to the wild-type polypeptide.

[0049] As used herein with respect to polypeptide molecules (e.g., proteins), the terms “engineered,” “chimeric,” or “recombinant” generally refer to polypeptide molecules that have a heterologous amino acid sequence or an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids encoding the polypeptide molecules and cells or organisms expressing the polypeptide molecules. As used herein with respect to polynucleotide molecules (e.g., DNA or RNA molecules), the terms “engineered” or “recombinant” generally refer to polynucleotide molecules that have a heterologous nucleic acid sequence or an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; transfection, transformation, and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, an engineered or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or altered by a gene editing moiety.

[0050] The term "nucleotide," as used herein, generally refers to a base-sugar-phosphate combination, unless otherwise specified or made clear from the context. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleotide triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleotide triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [aS]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 dideoxyribonucleotide triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of dideoxyribonucleotide triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Labeling can also be done with quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels. Fluorescent labels of nucleotides can include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from Perkin Elmer, Foster City, Calif.FluoroLink dideoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, III.; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and chromosome labeling nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or tagged by chemical modification. A chemically modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0051] The term“cell” generally refers to a biological cell. A cell can be a basic structural, functional, and / or biological unit of a living organism. A cell can be derived from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, a eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-celled eukaryotic organism, a protozoan cell, a cell from a plant (e.g., from a plant crop, a fruit, a vegetable, a grain, a soybean, a maize, a corn, a wheat, a seed, a tomato, a rice, a cassava, a sugar cane, a pumpkin, a hay, a potato, a cotton, a hemp, a tobacco, a flowering plant, a conifer, a gymnosperm, a fern, a clubmoss, a liverwort, a hornwort, a moss), an algal cell (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. Agardh, etc.), a seaweed (e.g., kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate (e.g., a fruit fly, a cnidarian, an echinoderm, a nematode, etc.), a cell from a vertebrate (e.g., a fish, an amphibian, a reptile, a bird, a mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and the like. Sometimes, a cell is not derived from a natural organism (e.g., a cell can be synthetically manufactured, sometimes referred to as an artificial cell).

[0052] The term“differentiation” generally refers to a process by which a non-committed (“uncommitted”) or less committed cell acquires the characteristics of a committed cell such as, for example, an immune cell. A differentiated or differentiation-induced cell is a cell that has attained a more committed (“committed”) position within a cell lineage. The term“committed” generally refers to a cell that has proceeded in a differentiation pathway to a point that, under normal circumstances, it will continue to differentiate into a particular cell type or subset of cell types, and under normal circumstances, cannot differentiate into a different cell type or revert to a less committed cell type.

[0053] The term“dedifferentiation” or“de-differentiation” generally refers to a process by which a committed, committed, or partially committed cell loses the characteristics of a committed cell (e.g., a chondrogenic cell). A dedifferentiated cell or de-differentiation-induced cell is a cell that has attained a less committed position within a cell lineage (e.g., a stem cell or progenitor cell). A dedifferentiated cell (e.g., a stem cell or progenitor cell) can subsequently differentiate into a different cell type or can revert to a less committed cell type.

[0054] The term“pluripotent” generally refers to the ability of a cell to form all lineages of the somatic or body cells (e.g., a blastula). For example, embryonic stem cells are a type of pluripotent stem cell that are capable of forming cells from each of the three germ layers (ectoderm, mesoderm, and endoderm). Pluripotency can be a continuum of developmental potential ranging from cells that are not completely or partially pluripotent (e.g., ectodermal stem cells) that are not capable of generating a complete organism to more primitive, more potent cells (e.g., embryonic stem cells) that are capable of generating a complete organism.

[0055] The term“induced pluripotent stem cell” (iPSC) generally refers to a stem cell derived from a differentiated cell (e.g., a differentiated adult, neonatal, or fetal cell) that has been induced or altered (e.g., reprogrammed) to be capable of differentiating into cells of all three germ layers or layers: mesoderm, endoderm, and ectoderm. The resulting iPSC does not refer to a cell found in nature. In some cases, an iPSC can be engineered to directly differentiate into a committed cell (e.g., a chondrogenic cell). In some cases, an iPSC can be engineered to first differentiate into a tissue-specific stem cell (e.g., a mesenchymal stem cell, a chondroblast), which can be further induced to differentiate into a committed cell (e.g., a chondrogenic cell).

[0056] The term“embryonic stem cell” (ESC) generally refers to a cell that is a naturally occurring pluripotent stem cell derived from the inner cell mass of a blastocyst embryo. Embryonic stem cells are pluripotent and generate all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm during development. In some cases, an ESC can be engineered to directly differentiate into a committed cell (e.g., a chondrogenic cell). In some cases, an ESC can be engineered to first differentiate into a tissue-specific stem cell (e.g., a mesodermal stem cell), which can be further induced to differentiate into a committed cell (e.g., a chondrogenic cell).

[0057] The term“isolated stem cell” generally refers to any type of stem cell disclosed herein (e.g., ESC, HSC, mesenchymal or mesodermal stem cell (MSC), etc.) that is isolated from a multicellular organism. For example, a HSC can be isolated from the body of a mammal, such as a human body. In another example, an embryonic stem cell can be isolated from an embryo.

[0058] The term“isolated” generally refers to a cell or population of cells that has been separated from its original environment. For example, an isolated cell is substantially free of at least one component that is found in the environment of a“non-isolated” reference cell. An isolated cell can be a cell removed from some or all components when found in its natural environment, such as a cell isolated from a tissue or biopsy sample. The term also includes a cell removed from at least one, some, or all components when the cell is found in a non-naturally occurring environment, 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 populations of cells when it is found in nature or when it is grown, stored, or exists in a non-naturally occurring environment.

[0059] The term“chondrogenesis” when applied to a cell or population generally refers to the ability of the cell or population to produce cartilage or stimulate cartilage growth under the right circumstances (e.g., in vivo or ex vivo conditions). The term“chondrogenic cell” generally refers to a cell comprising tissue that produces cartilage or cartilage-related proteins. Non-limiting examples of chondrogenic cells include chondroblasts, chondrocytes, and cells that have differentiated into chondroblasts or chondrocytes, such as chondrogenic precursor cells. Chondrogenic cells develop from mesodermal stem cells (MSCs), which include, but are not limited to, paraxial mesoderm cells, sclerotome cells, and chondroblast progenitor cells.

[0060] The terms“chondroblast progenitor cell,”“chondrogenic precursor cell,” or“chondrogenic progenitor cell,” as used interchangeably herein, generally refer to a chondrogenic specific progenitor cell that is more committed than a stem cell (e.g., ESC, MSC, IPSC, etc.), but less differentiated than a chondroblast or chondrocyte. Chondroblast progenitor cells can be generated ex vivo by engineering isolated stem cells. Chondroblast progenitor cells can be generated in vivo by engineering stem cells in vivo, for example, by administering such stem cells with any one of the heterologous gene circuits disclosed herein.

[0061] Overview

[0062] Bio-programming, such as cell-programming, allows for engineering of cells to produce a desired outcome. The outcome of cell-programming can include inducing or preventing a wide array of common and / or new cell functions; the outcome can also include enhancing or inhibiting a cell function that is already occurring. Cell-programming can be accomplished through the use of gene circuits. Cell-programming can be accomplished through manipulation of biomolecules (e.g., DNA). For example, due to the versatility and ease of programmability of CRISPR or CRISPR / Cas systems, CRISPR or CRISPR / Cas systems have been employed for genome editing across many species. Cell-programming can affect endogenous or exogenous genes. Cell-programming can be implemented to act in a time-dependent manner or a time-independent manner.

[0063] Gene circuits used in cell programming can be used to control the cell fate of a cell or plurality of cells by inducing differentiation or dedifferentiation and transformation from one cell type to another. Cell programming controls by modulating the desired expression and / or activity levels of a plurality of genes in a cell.

[0064] While the CRISPR / Cas system is widely used for gene editing, Cas is essentially a single-turnover nuclease as it remains bound to its generated double-stranded break, and many regions of the genome are resistant to genome editing. Increased understanding of CRISPR / Cas-based genome editing has encouraged the development of cascading regulatory systems to further leverage this technology for engineered cell development. By implementing a series of activatable gRNAs, genome editing can be more temporally regulated from target site to target site, sequential genome editing can be performed to act like a domino effect, and cells can be barcoded. However, this simple barcoding, which typically uses exogenous fluorophores, does not allow for the regulation of endogenous genes to enable cell differentiation.

[0065] Further, differentiation or dedifferentiation of cells is currently achieved by using exogenous serum and growth factors, which bypass the underlying mechanisms of cell programming. Using exogenous serum, growth factors, and other similar methods results in cells being instructed to differentiate, but it lacks the underlying biology (e.g., correct state of chromatin, etc.) that accompanies it. This lack often results in cells experiencing premature terminal differentiation into non-desired cell types or they experience inefficient differentiation, resulting in low yield of target cell types, or the resulting desired differentiated cells are only semi-functional. Semi-functional cells can resemble the cell type of interest, but can lack key biological features necessary for normal function of the desired differentiated cell type.

[0066] Accordingly, there remains an unmet need for an activatable CRISPR / Cas system and its use for editing a target polynucleotide (e.g., a genome of a cell, particularly a eukaryotic cell) that uses a cascade of gRNAs to form a gene circuit in order to influence gene regulation and, in turn, cell fate determination, without the use of serum and exogenous growth factors. The preprogrammed, activatable, and self-regulating gRNA cascade CRISPR / Cas system finds application, for example, in gene therapy, gene circuits, and / or complex cell fate determination and / or control.

[0067] The present disclosure provides systems and methods for engineering a CRISPR / Cas9 system comprising a Cas endonuclease and an array of homologous single guide RNAs (sgRNAs or gRNAs) with inactivation sequences in non-essential regions and activatable to allow for modulation and modification of the system without the need for serum, growth factors, or other additional exogenous signals. The present disclosure also provides engineered cells that can contain any of the above systems or be capable of performing any of the above methods.

[0068] Systems and methods for programming chondrogenic lineage cells

[0069] Various aspects of the present disclosure provide systems for inducing a desired transformation from one type of cell to another type of cell. To this end, various aspects of the present disclosure provide methods for inducing a desired level of expression and / or activity (or profile thereof) of one or more target genes in a cell.

[0070] In an aspect, the present disclosure provides a system for transforming a plurality of cells of a first type into a plurality of cells of a second cell type. The system can comprise a heterologous gene circuit comprising a plurality of gate units. The plurality of gate units can comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gate units. The plurality of gate units can comprise at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 gate unit. The plurality of gate units can be different (e.g., comprise different polynucleotide sequences). Each gate unit in the plurality of gate units can affect modulation of the expression and / or activity level of a different target gene or a plurality of different target genes.

[0071] A heterologous gene circuit as disclosed herein can operate with a plurality of gate units in series (e.g., the plurality of gate units connected sequentially in an end-to-end fashion to form a single pathway), in parallel (e.g., the plurality of gate units cross-connected to each other to form, e.g., two or more parallel sequential pathways), or a combination thereof.

[0072] The plurality of gate units, as disclosed herein, can operate in concert (e.g., as predetermined by the design of the heterologous genetic circuit) to induce an outcome in a cell. The outcome in the cell can include a cell function (e.g., locomotion, reproduction; response to an external stimulus, nutrient output, excretion, respiration, growth) and / or a cell state (e.g., cell fate, differentiation, quiescence, programmed cell death). Such outcomes can be determined in vitro, ex vivo, and / or in vivo. For example, the outcome, as disclosed herein, can be determined in vitro by (i) measuring the expression level of a gene of interest by polymerase chain reaction (PCR) or Western blotting, (ii) via small molecule or antibody staining, (iii) cell sorting based on cell size, morphology, and / or surface protein expression, (iv) using an assay (e.g., a cell proliferation assay or a metabolic activity assay) to measure phenotypic differentiation and cell function, (v) microscopy, and / or (iv) screening for molecular and / or genetic differences using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics.

[0073] The plurality of gate units, as disclosed herein, can be sufficient to effect a transformation from a plurality of cells of a first cell type to a plurality of cells of a second cell type. For example, the plurality of gate units, as disclosed herein, can be sufficient to effect a transformation from a plurality of pluripotent stem cells (PSCs) to a plurality of tissue-specific progenitor cells. Alternatively, the plurality of gate units, as disclosed herein, can be necessary but insufficient to effect a transformation from a plurality of cells of a first cell type to a plurality of cells of a second cell type.

[0074] The outcome in the cell can include modulation of different target genes or different sets of target genes. The plurality of gate units can induce different modulation of the plurality of target genes (e.g., in a sequential manner) such that the collective modulation of the genes cooperatively produces a final expression and / or activity profile of the cell. The final expression and / or activity level profile of the cell can exemplify an outcome, such as a transformation (or a process thereof) of the cell from one cell type to another cell type.

[0075] In some cases, the plurality of gate units, as disclosed herein, can be necessary but individually insufficient to effect a desired expression and / or activity profile of a target cell. Thus, the outcome in the cell induced by the plurality of gate units (e.g., enhanced cell function, induced cell state, etc.) can not be possible in the absence of any of the plurality of gate units. Alternatively, the extent or measure of the outcome in the cell induced by the plurality of gate units can differ (e.g., be greater for a positive marker or be less for a negative marker) from the extent or measure of the outcome in a control cell induced by none, one or more but not all, and / or all of the plurality of gate units, and / or by a different sequential order of events.

[0076] The second gate unit can be activated by the first gate unit (e.g., directly or indirectly). For example, the second gate unit can be activated directly by the first gate unit. Alternatively, the second gate unit can be activated by one or more additional gate units activated (e.g., directly or indirectly) by the first gate unit. The one or more additional gate units can comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gate units. The one or more additional gate units can be at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 gate unit. In yet another alternative, the second gate unit can be activated via another portion responsible for activating the first gate unit (e.g., an activation portion, a different gate unit, etc.). In yet another alternative, the first gate unit and the second gate unit can be activated by different activation portions (e.g., different polynucleotide molecules, such as different guide nucleic acid molecules).

[0077] In some cases, the first gate unit modulates the first target gene. Alternatively or additionally, the first gate unit can also modulate the second gate unit. The modulation of the second gate unit can occur 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, about 600 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, as determined by RT-QPCR, Western blot, or other methods.

[0078] In some cases, the second gate unit can modulate the second target gene. Modulation of the second target gene can occur at least or up to about 1 millisecond, about 2 milliseconds, about 3 milliseconds, about 4 milliseconds, about 5 milliseconds, about 6 milliseconds, about 7 milliseconds, about 8 milliseconds, about 9 milliseconds, about 10 milliseconds, about 20 milliseconds, about 30 milliseconds, about 40 milliseconds, about 50 milliseconds, about 60 milliseconds, about 70 milliseconds, about 80 milliseconds, about 90 milliseconds, about 100 milliseconds, about 200 milliseconds, about 300 milliseconds, about 400 milliseconds, about 500 milliseconds, about 600 milliseconds, 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, after modulation of the first target gene, as determined by RT-QPCR, Western blot, or other methods.

[0079] In some cases, the term“proGuide” as used generally herein can refer to such a vector (e.g., plasmid) that encodes a activatable gNA. The proGuide can be an example of a gate portion. The proGuide can be an example of a gene regulation portion.

[0080] A proGuide as provided herein can encode an activatable guide nucleic acid molecule, e.g., having an inactivating polynucleotide sequence (e.g., one or more polyX sequences, such as one or more polyT sequences). In some embodiments, a portion of a proGuide encoding an activatable guide nucleic acid molecule can comprise various regions connected in order, including a spacer sequence, an additional sequence (e.g., a linker sequence or a backbone sequence), an upstream stem, a polyT unit, and a downstream stem. In some embodiments, the additional 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, a portion of a proGuide encoding an activatable guide nucleic acid molecule can comprise various regions connected in order, including a spacer sequence, an additional sequence (e.g., a linker sequence or a backbone sequence), an upstream stem, a polyT unit, and a downstream stem, as shown in Table 1 (SEQ ID Nos: 1-1932). In some cases, upon modification or removal of the polyT unit, the upstream stem and the downstream stem can form part of a scaffold sequence of a functional guide nucleic acid molecule. In some embodiments, a portion of a proGuide encoding an activatable guide nucleic acid molecule can comprise various regions connected in order, including a spacer sequence, an additional sequence (e.g., a linker sequence or a backbone sequence), an upstream stem, a polyT unit, and a downstream stem, as shown in Table 1 (SEQ ID Nos: 1-1932), with “-” to distinguish between the various regions in a tandem sequence. In some cases, upon modification or removal of the polyT unit, the upstream stem and the downstream stem can form part of a scaffold sequence of a functional guide nucleic acid molecule. In some embodiments, when the spacer sequence does not begin with a G, a G is added before the spacer sequence. In some embodiments, adding a G before the spacer sequence helps to increase expression of the RNA portion from the promoter. In some embodiments, stem 1 and stem 2 are reverse complements of each other. In some embodiments, the upstream stem and the downstream stem are reverse complements of each other. In some embodiments, stem 1 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 a member of the polynucleotide sequences as shown in Table 3 (SEQ ID Nos: 2024-2046).In some embodiments, stem 2 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 a member of the polynucleotide sequences as set forth in Table 3 (SEQ ID Nos: 2047-2069).

[0081] In some embodiments, a proGuide as provided herein can encode an activatable guide nucleic acid molecule, e.g., having an inactivation polynucleotide sequence (e.g., one or more polyX sequences, such as one or more polyT sequences). In some cases, a portion of a proGuide encoding an activatable guide nucleic acid molecule can comprise various regions connected in order (e.g., from 5’ to 3’), including an upstream stem (e.g., an upstream cleavage site), a polyT unit (or as used interchangeably herein, a “proUnit” or “proGuide Unit”), and a downstream stem (e.g., a downstream cleavage site). The upstream stem and the downstream stem can correspond to “stem region” polynucleotide sequences that are at least partially complementary to one another.

[0082] A domain of a polynucleotide sequence encoding (or corresponding to) a molecule of interest can comprise 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, the polyX sequence can be disposed within a domain encoding the molecule of interest (e.g., not at the 5’ end or 3’ end of such a domain), such that expression of the molecule of interest (e.g., transcription of a molecule of interest RNA) will be disrupted (e.g., terminated) in the middle of the expression.

[0083] Accordingly, a polyX sequence (e.g., in a polynucleotide sequence encoding a molecule of interest) can be referred to as a termination sequence (e.g., a non-canonical termination sequence for its sequence and / or its position), a disruption sequence (e.g., for disrupting full expression of a molecule of interest), an inactivation sequence (e.g., for inactivating a function of a polynucleotide sequence or a molecule of interest).

[0084] In some cases, a non-canonical termination sequence can comprise or consist essentially of a polynucleotide sequence that exhibits at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least or up to about 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 the polynucleotide sequence TTTTTTTTcagccaactccaaTTTTTTTT (SEQ ID NO: 1934) or the complement thereof.

[0085] In some cases, a polyX sequence can be located within (e.g., not at a terminus of) a polynucleotide sequence, such as a DNA sequence or an RNA sequence. In some cases, a polyX sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases from a 3’ end of a polynucleotide sequence. In some cases, a polyX sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases from a 5’ end of a polynucleotide sequence. In some cases, a polyX sequence can be located at a terminus of a nucleic acid sequence.

[0086] In some cases, a polyT or polyU sequence can be located within (e.g., not at the terminus of) a polynucleotide sequence, such as a DNA sequence or an RNA sequence. In some cases, a polyT or polyU sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases from the 3’ end of a polynucleotide sequence. In some cases, a polyT or polyU sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases from the 5’ end of a polynucleotide sequence. In some cases, a polyT or polyU sequence can be located at the terminus of a nucleic acid sequence. In some cases, an RNA comprising a polyU sequence can also be represented by a DNA comprising a polyT sequence.

[0087] A polyX sequence (e.g., a polyT sequence or a polyU sequence) can comprise at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 X bases. A polyX sequence can comprise at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, or at most about 2 X bases. A polyX sequence can be represented by a complementary polyX sequence in a corresponding complementary DNA strand (e.g., a polyT as disclosed herein as a DNA sequence can also be referred to as a polyA in a complementary DNA strand). A disclosed polyX sequence can comprise a plurality of X bases. A plurality of X bases can be disclosed sequentially adjacent to one another (e.g., TT, TTT, TTTT, TTTTT, etc.). Alternatively or additionally, a plurality of X bases can be separated by one or more additional nucleotides that are not X. The one or more additional nucleotides can comprise a single type of nucleotide or different types of nucleotides.

[0088] In some embodiments, a proGuide can comprise an inactivated polynucleotide sequence that exhibits at least or more than about 50%, at least or more than about 55%, at least or more than about 60%, at least or more than about 65%, at least or more than about 70%, at least or more than about 75%, at least or more than about 80%, at least or more than about 85%, at least or more than about 86%, at least or more than about 87%, at least or more than about 88%, at least or more than about 89%, at least or more than about 90%, at least or more than about 91%, at least or more than about 92%, at least or more than about 93%, at least or more than about 94%, at least or more than about 95%, at least or more than about 96%, at least or more than about 97%, at least or more than about 98%, at least or more than about 99%, or substantially about 100% sequence identity to TTTTTTTTTT or SEQ ID NO: 1933, or a complement thereof.

[0089] In some embodiments, a proGuide can comprise a target polynucleotide domain at or near (e.g., at or near the 5’ and / or 3’ end of) the inactivation polynucleotide sequence, which target polynucleotide domain can be targeted (e.g., via a sequential activation mechanism of a heterologous genetic circuit as provided herein) to modify (e.g., edit, cleave) the inactivation polynucleotide sequence, thereby activating the guide nucleic acid molecule of the proGuide expression. The target polynucleotide domain of the proGuide can not exhibit sequence identity to any comparable endogenous polynucleotide sequence in the cell, thereby avoiding inadvertent targeting and modulation of the endogenous target gene.

[0090] In some embodiments, the inactivation polynucleotide sequence of a proGuide can be disposed between two target polynucleotide domains, which two target polynucleotide domains can or can not be targetable by a common guide nucleic acid sequence. In some cases, the two target polynucleotide domains can be inverted and complementary to each other, such that the inactivation polynucleotide sequence can be modified or cleaved by the same mechanism (e.g., the same spacer sequence of a guide nucleic acid molecule).

[0091] In some embodiments, a proGuide can comprise one or more members that exhibit 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 a polynucleotide sequence or its complement from one or more of SEQ ID NOs: 1-92 (e.g., ACAN targeting), SEQ ID NOs: 93-184 (e.g., COL2A1 targeting), SEQ ID NOs: 185-276 (e.g., MEF2D targeting), SEQ ID NOs: 277-460 (e.g., pax9 targeting), SEQ ID NOs: 461-644 (e.g., RUNX2 targeting), SEQ ID NOs: 645-920 (e.g., SOX5 targeting), SEQ ID NOs: 921-1288 (e.g., SOX6 targeting), SEQ ID NOs: 1289-1380 (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 a complement thereof.

[0092] The second gate unit can be activatable to induce inactivation of the first gate unit that has been activated. The terms “inactivation” or “disruption” can be used interchangeably herein. Inactivation and as disclosed herein can be induced by producing a modification (e.g., a cleavage, such as a single or double strand break, and an insertion-deletion (indel), etc.) to at least a portion of the first gate unit (e.g., a gate portion and / or a gene regulation portion of the first gate unit) that is responsible for inducing the first different modulation of the target gene.

[0093] Inactivation of the gate portion and / or the gene regulatory portion of the first gate unit as disclosed herein can be achieved by an endonuclease-based system (e.g., CRISPR / Cas system). Alternatively or additionally, inactivation can be achieved by using a transcriptional regulator system (e.g., transcriptional repressor). Alternatively or additionally, inactivation can be achieved by CRISPRi steric hindrance without the need for additional transcriptional regulators. An endonuclease transcriptional regulator system (e.g., Cas-repressor) can be used to achieve polynucleotide cleavage (e.g., for inactivating the gate portion and / or the gene regulatory portion). The polynucleotide cleavage can create a nucleic acid modification such as a single-strand break, a double-strand break, an insertion, a deletion, or an insertion-deletion. Alternatively or additionally, the endonuclease transcriptional regulator system (e.g., Cas-repressor) can be used to modulate target gene expression. Alternatively or additionally, a CAS transcriptional regulator system lacking endonuclease activity (dCAS or naked CAS with a shortened spacer insufficient to support cleavage) targets a region of DNA and physically stops transcriptional elongation, resulting in repression of the target gene (CRISPRi).

[0094] Alternatively, the second gate unit can be activatable to amplify or enhance the activation of the first gate unit that has been activated. The amplification or enhancement of the first gate unit can be induced by producing a modification (e.g., cleavage such as single- or double-strand break, and insertion-deletion, etc.) to at least a portion of the first gate unit (e.g., the gate portion and / or the gene regulatory portion of the first gate unit) that is responsible for inducing the first different regulation of the target gene.

[0095] The heterologous gene circuit can include multiple gate units that are sequentially activated (e.g., activated in series). The multiple gate units can include a functional gate unit that is preconfigured such that it is activated to modulate (e.g., directly modulate) expression and / or epigenetic profile of a target gene (e.g., an endogenous target gene). The multiple gate units can further include one or more additional gate units that are preconfigured to (i) be activated prior to the functional gate unit, and (ii) enable subsequent activation of the functional gate unit. In some cases, the one or more additional gate units can be preconfigured to be activated to modulate one or more additional target genes. Alternatively, the one or more additional gate units can not be preconfigured to modulate any target gene (e.g., any endogenous target gene) when activated. Such one or more additional gate units can instead be used to delay (e.g., in terms of time) activation of the functional gate unit during operation of the heterologous gene circuit, thereby delaying expression and / or epigenetic profile of the target gene of the functional gate unit, and thus the one or more additional gate units can be referred to as “blank” gate units. The heterologous gene circuit can include at least or up to about 1 blank gate unit, at least or up to about 2 blank gate units, at least or up to about 3 blank gate units, at least or up to about 4 blank gate units, at least or up to about 5 blank gate units, at least or up to about 6 blank gate units, at least or up to about 7 blank gate units, at least or up to about 8 blank gate units, at least or up to about 9 blank gate units, at least or up to about 10 blank gate units, at least or up to about 11 blank gate units, at least or up to about 12 blank gate units, at least or up to about 13 blank gate units, at least or up to about 14 blank gate units, at least or up to about 15 blank gate units, at least or up to about 16 blank gate units, at least or up to about 27 blank gate units, at least or up to about 18 blank gate units, at least or up to about 19 blank gate units, at least or up to about 20 blank gate units, at least or up to about 25 blank gate units, at least or up to about 30 blank gate units, at least or up to about 35 blank gate units, at least or up to about 40 blank gate units, at least or up to about 45 blank gate units, at least or up to about 50 blank gate units.

[0096] In some cases, the use of one or more blank gate units can delay activation of a functional gate unit (e.g., as determined by measuring the expression / epigenetic profile of a target gene, or as determined by measuring the expression of a functional variant or transcription product of a functional gate unit) by at least or up to about 1 minute, at least or up to about 5 minutes, at least or up to about 10 minutes, at least or up to about 30 minutes, at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 11 hours, at least or up to about 12 hours, at least or up to about 13 hours, at least or up to about 14 hours, at least or up to about 15 hours, at least or up to about 16 hours, at least or up to about 17 hours, at least or up to about 18 hours, at least or up to about 19 hours, at least or up to about 20 hours, at least or up to about 21 hours, at least or up to about 22 hours, at least or up to about 23 hours, at least or up to about 24 hours, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, or at least or up to about 7 days.

[0097] In some cases, the modification of a target gene by a gate unit can inactivate the gene. For example, the modification of the gene can prevent the expression and / or activity level of the target gene. Alternatively, the modification of the gene can decrease 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.

[0098] In some cases, the modification of the gene can reduce the level of expression and / or activity 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 modification of the gene can reduce the level of expression and / or activity of the target gene by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less.

[0099] In some cases, the modification of the gene can increase the level of expression and / or activity 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%, 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 modification of the gene can increase the level of expression and / or activity of the target gene by at most about 1,000,000%, at most about 100,000%, at most about 9,000%, at most about 8,000%, at most about 7,000%, at most about 6,000%, at most about 5,000%, at most about 4,000%, at most about 3,000%, at most about 2,000%, at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less.

[0100] In some cases, the modification of the gene can reduce the level of expression and / or activity of the target gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to the control level of expression and / or activity. The modification of the gene can reduce the level of expression and / or activity of the target gene by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, up to or less than about 1-fold, up to or less than about 0.9-fold, up to or less than about 0.8-fold, up to or less than about 0.7-fold, up to or less than about 0.6-fold, up to or less than about 0.5-fold, up to or less than about 0.4-fold, up to or less than about 0.3-fold, up to or less than about 0.2-fold, up to or less than about 0.1-fold, as compared to the control level of expression and / or activity.

[0101] In some cases, the modification of the gene can increase the expression and / or activity level of the target gene at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 2-fold, at least or more than about 3-fold, at least or more than about 4-fold, at least or more than about 5-fold, at least or more than about 6-fold, at least or more than about 7-fold, at least or more than about 8-fold, at least or more than about 9-fold, at least or more than about 10-fold, at least or more than about 20-fold, at least or more than about 30-fold, at least or more than about 40-fold, at least or more than about 50-fold, at least or more than about 60-fold, at least or more than about 70-fold, at least or more than about 80-fold, at least or more than about 90-fold, at least or more than about 100-fold, at least or more than about 500-fold, at least or more than about 1,000-fold, at least or more than about 5,000-fold, or at least or more than about 10,000-fold, as compared to the control expression and / or activity level. The modification of the gene can increase the expression and / or activity level of the target gene by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 9-fold, up to or less than about 8-fold, up to or less than about 7-fold, up to or less than about 6-fold, up to or less than about 5-fold, up to or less than about 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, up to or less than about 1-fold, up to or less than about 0.9-fold, up to or less than about 0.8-fold, up to or less than about 0.7-fold, up to or less than about 0.6-fold, up to or less than about 0.5-fold, up to or less than about 0.4-fold, up to or less than about 0.3-fold, up to or less than about 0.2-fold, up to or less than about 0.1-fold, as compared to the control expression and / or activity level.

[0102] 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 level 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, and the like.

[0103] In some cases, activation of the plurality of gate units can be a result of a single activation (e.g., by a single activation moiety) of the heterologous genetic circuit at a single point in time. The plurality of gate units can include one of a first gate unit and a second gate unit that are preconfigured to be sequentially activated upon activation of the heterologous genetic circuit by the single activation. In some cases, one of the first gate unit and the second gate unit can be activated by the single activation moiety (e.g., a guide nucleic acid), while the other of the first gate unit and the second gate unit can be activated by a further activation moiety (e.g., a different guide nucleic acid) that is different from the activation moiety of the heterologous genetic circuit. The further activation moiety can be a portion of the heterologous genetic circuit that is produced (e.g., expressed) only upon activation of the heterologous genetic circuit. Alternatively or additionally, the first gate unit and the second gate unit can each be activated by a different activation moiety that is different from the activation moiety of the heterologous genetic circuit. Such different activation moieties can be portions of the heterologous genetic circuit that are produced (e.g., expressed) only upon activation of the heterologous genetic circuit.

[0104] In some embodiments of any of the systems disclosed herein, a gate unit can comprise a gate portion (e.g., at least or up to about 1 gate portion, at least or up to about 2 gate portions, at least or up to about 3 gate portions, at least or up to about 4 gate portions, at least or up to about 5 gate portions, etc.) and / or a gene regulation portion (e.g., at least or up to about 1 gene regulation portion, at least or up to about 2 gene regulation portions, at least or up to about 3 gene regulation portions, at least or up to about 4 gene regulation portions, at least or up to about 5 gene regulation portions, at least or up to about 6 gene regulation portions, at least or up to about 7 gene regulation portions, at least or up to about 8 gene regulation portions, at least or up to about 9 gene regulation portions, at least or up to about 10 gene regulation portions, etc.). A gate portion as disclosed herein can comprise a guide nucleic acid molecule (gNA) (e.g., at least or up to about 1 gNA molecule, at least or up to about 2 gNA molecules, at least or up to 3 gNA molecules, at least or up to about 4 gNA molecules, at least or up to about 5 gNA molecules, etc.). A gene regulation portion as disclosed herein can comprise a gNA (e.g., at least or up to about 1 gNA molecule, at least or up to about 2 gNA molecules, at least or up to 3 gNA molecules, at least or up to about 4 gNA molecules, at least or up to about 5 gNA molecules, etc.). A guide nucleic acid molecule as disclosed herein can comprise, but is not limited to, DNA, RNA, any analog of such, or any combination thereof. In some embodiments of any of the systems disclosed herein, a gate portion and / or a gene regulation portion can be activated to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and the complex can be configured to or capable of binding a target polynucleotide, e.g., to modulate the expression and / or activity level of a gene comprising the target polynucleotide or another polynucleotide sequence operably coupled to the target polynucleotide. For example, the complex can modulate the expression and / or activity level of a gene comprising the target polynucleotide.

[0105] In some cases, a guide nucleic acid molecule (gNA) (e.g., a functional gNA) expressed by a second gate unit upon activation can produce a modification to at least a portion of a first gate unit. For example, an activated gNA of a second gate unit can produce a modification to a polynucleotide sequence of a first gate unit encoding a gNA (e.g., an activatable gNA) or a promoter sequence of the first gate unit operably coupled to such a gNA of the same first gate unit. Such a modification can render the gNA of the first gate unit inoperable (e.g., reduce or inhibit specific binding to a target gene) when expressed. Alternatively, the modification can reduce (e.g., inhibit) expression of the gNA of the first gate unit.

[0106] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be caused by a single-strand break in which there is a discontinuity in one of the nucleotide strands. Inactivation of a polynucleotide sequence or a target gene can be caused by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or more single-strand breaks. In some cases, inactivation of a gene can be caused by at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 single-strand break.

[0107] In some cases, a gNA can have a size (e.g., including both the spacer sequence and the scaffold sequence) of at least or up to about 60 nucleotides, at least or up to about 70 nucleotides, at least or up to about 80 nucleotides, at least or up to about 85 nucleotides, at least or up to about 90 nucleotides, at least or up to about 95 nucleotides, at least or up to about 100 nucleotides, at least or up to about 105 nucleotides, at least or up to about 110 nucleotides, at least or up to about 120 nucleotides, at least or up to about 130 nucleotides, at least or up to about 140 nucleotides, at least or up to about 150 nucleotides, or at least or up to about 200 nucleotides.

[0108] In some cases, a scaffold sequence of a gNA can have a size of at least or up to about 30 nucleotides, at least or up to about 35 nucleotides, at least or up to about 40 nucleotides, at least or up to about 45 nucleotides, at least or up to about 50 nucleotides, at least or up to about 55 nucleotides, at least or up to about 60 nucleotides, at least or up to about 65 nucleotides, at least or up to about 70 nucleotides, at least or up to about 75 nucleotides, at least or up to about 80 nucleotides, at least or up to about 85 nucleotides, at least or up to about 90 nucleotides, at least or up to about 95 nucleotides, at least or up to about 100 nucleotides, at least or up to about 100 nucleotides, at least or up to about 120 nucleotides, at least or up to about 130 nucleotides, at least or up to about 140 nucleotides, or at least or up to about 150 nucleotides.

[0109] In some cases, the spacer sequence of a gNA can have a size of at least or up to about 10 nucleotides, at least or up to about 11, at least or up to about 12, at least or up to about 13, at least or up to about 14, at least or up to about 15, at least or up to about 16, at least or up to about 17, at least or up to about 18, at least or up to about 19, at least or up to about 20, at least or up to about 21, at least or up to about 22, at least or up to about 23, at least or up to about 24, at least or up to about 25, at least or up to about 26, at least or up to about 27, at least or up to about 28, at least or up to about 29, or at least or up to about 30 nucleotides.

[0110] In some embodiments of any of the systems disclosed herein, an initial (or first) gate unit of a heterologous genetic circuit disclosed herein can be activated partially activated (e.g., directly activated). The activation moiety can directly bind at least a portion of the initial gate unit to activate the initial gate unit, e.g., thereby sequentially activating the heterologous genetic circuit. Alternatively, the activation moiety can activate the initial gate unit without directly binding at least a portion of the initial gate unit (e.g., by using electromagnetic energy). In some cases, the initial gate unit can comprise at least one gate moiety and at least one genetic regulatory moiety. In some cases, the initial gate unit can comprise at least one gate moiety, but can not comprise nor need to comprise a genetic regulatory moiety. In some cases, the initial gate unit can comprise at least one genetic regulatory moiety, but can not comprise nor need to comprise a gate moiety (e.g., the activation moiety can be configured to activate the initial gate unit and at least one additional gate unit).

[0111] In some embodiments of any of the systems disclosed herein, the gNA of the gate portion and / or the gene regulation portion (e.g., the gNA encoded by the gate portion and / or the gene regulation portion) can be an activatable gNA. The activatable gNA can be one of, but is not limited to, any of: a ribonucleotide (e.g., a gRNA), a deoxyribonucleotide, any analog of such, or any combination thereof. In some embodiments, the vector (or expression cassette) encoding the activatable gNA can comprise an inactivation polynucleotide sequence to inactivate the gNA 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 additionally, the inactivation polynucleotide sequence can encode a non-canonical transcriptional termination sequence, as described below. The inactivation polynucleotide sequence can be part of or adjacent to a region of the vector that encodes (i) a spacer sequence of the gNA, (ii) a scaffold sequence of the gNA, and / or (ii) any linker sequence between the spacer sequence and the scaffold sequence. The vector can comprise at least or up to about 1 inactivation polynucleotide sequence, at least or up to about 2 inactivation polynucleotide sequences, at least or up to about 3 inactivation polynucleotide sequences, at least or up to about 4 inactivation polynucleotide sequences, at least or up to about 5 inactivation polynucleotide sequences, at least or up to about 6 inactivation polynucleotide sequences, at least or up to about 7 inactivation polynucleotide sequences, at least or up to about 8 inactivation polynucleotide sequences, at least or up to about 9 inactivation polynucleotide sequences, or at least or up to about 10 inactivation polynucleotide sequences.

[0112] In some cases, the term “proGuide” as used generally herein can refer to such a vector (e.g., plasmid) encoding an activatable gNA. The proGuide can be an example of a gate portion. The proGuide can be an example of a gene regulation portion.

[0113] 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 to become 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 comprises a non-canonical transcription termination sequence (e.g., a polyX sequence, such as a polyU sequence or a 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 can be modified (e.g., to remove some or all of the transcription termination sequence). Thus, in the absence of modification of the transcription termination sequence, a non-functional variant (e.g., a non-functional fragment) of the gNA can be expressed. In some embodiments, the gNA can be synthetic. In some embodiments, the gNA can have a fluorescent label attached.

[0114] In some cases, the polyT sequence is greater than or equal to a threshold length, where the threshold length is sufficient to reduce expression of the guide nucleic acid molecule from the polynucleotide sequence. Thus, a plasmid (e.g., a gate portion or a gene regulation portion) can encode an inactivated gNA comprising a polyT sequence that is greater than or equal to the threshold length, and editing such a plasmid to reduce the length of the polyT to below the threshold length can allow the gNA to be expressed in its entirety without premature termination, thereby activating the gNA. In some cases, the polyT sequence comprises at least 5 T’s. In some cases, the polyT sequence comprises at least 7 T’s. In some cases, the polyT sequence comprises at least 8 T’s. In some cases, the polyT sequence comprises at least 10 T’s. In some cases, the polyT sequence comprises between 5 T’s and 15 T’s. In some cases, the polyT sequence comprises one or more additional nucleotides that are not T’s.

[0115] In some cases, the gene regulation portion (e.g., the guide nucleic acid and / or the endonuclease) can be configured to bind to a target polynucleotide sequence that is operably coupled to a target gene in the cell. The target gene can comprise a coding polynucleotide sequence that encodes a target nucleic acid molecule or a target protein. The target polynucleotide sequence can be part of the coding polynucleotide sequence. Alternatively, the target polynucleotide sequence can not be part of the coding polynucleotide sequence. For example, the target polynucleotide sequence can be upstream of the coding polynucleotide sequence (e.g., part of a promoter of the coding polynucleotide sequence, such as a transcription start site (TSS).

[0116] As provided herein, when the heterogenous gene circuit is activated to induce multiple different modulations of a target gene, the multiple different modulations of the target gene can be different (e.g., different degrees of change in the level of expression and / or activity of the target gene). For example, the different degrees of the first modulation imposed by the first genetic unit and the second modulation imposed by the second gate unit can be at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%. The different degrees of the first modulation and the second modulation can be at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, or at most about 0.1%. Alternatively or additionally, the different modulations of the target gene can be substantially the same (e.g., identical). The multiple different modulations can individually be sufficient to induce a desired change in the level of expression and / or activity of the target gene. Alternatively, the different modulations can individually be insufficient to induce a desired change in the level of expression and / or activity of the target gene.

[0117] One or more target genes as disclosed herein can include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0118] In some cases, a guide nucleic acid molecule (gNA) (e.g., a functional gNA) expressed by the second gate unit upon activation can produce a modification to at least a portion of the first gate unit. For example, the activated gNA of the second gate unit can produce a modification to a polynucleotide sequence of the first gate unit encoding the gNA (e.g., an activatable gNA) or a promoter sequence of the first gate unit operably coupled to such gNA of the same first gate unit. Such modification can render the gNA of the first gate unit inoperable (e.g., reduce or inhibit specific binding to a target gene) when expressed. Alternatively, the modification can reduce (e.g., inhibit) expression of the gNA of the first gate unit.

[0119] In some embodiments of any of the systems disclosed herein, an initial (or first) gate unit of a heterologous genetic circuit as disclosed herein can be activated (e.g., directly activated) by an activation moiety. The activation moiety can directly bind at least a portion of the initial gate unit to activate the initial gate unit, e.g., thereby sequentially activating the heterologous genetic circuit. Alternatively, the activation moiety can activate the initial gate unit without directly binding at least a portion of the initial gate unit (e.g., by using electromagnetic energy). In some cases, the initial gate unit can comprise at least one gate moiety and at least one genetic regulatory moiety. In some cases, the initial gate unit can comprise at least one gate moiety, but can not comprise and need not comprise a genetic regulatory moiety. In some cases, the initial gate unit can comprise at least one genetic regulatory moiety, but can not comprise and need not comprise a gate moiety (e.g., the activation moiety can be configured to activate the initial gate unit and at least one additional gate unit).

[0120] In some embodiments of any of the systems disclosed herein, a gNA of a gate moiety and / or a genetic regulatory moiety (e.g., a gNA encoded by a gate moiety and / or a genetic regulatory moiety) can be an activatable gNA. The activatable gNA can be, but is not limited to, any of the following: a ribonucleotide (e.g., a gRNA), a deoxyribonucleotide, any analog of such, or any combination thereof. In some embodiments, the activatable gNA molecule can be a self-cleaving gNA (e.g., a gRNA contains a cis-cleaving ribozyme). For example, when the activatable gNA is expressed in a cell, the activatable gNA can be self-cleavable to become non-functional (e.g., not configured to bind a target gene) unless the gene encoding the activatable gNA is modified prior to expression of the activatable gNA. In some embodiments, the gNA can be synthetic. In some embodiments, the gNA can have a fluorescent label attached.

[0121] In some cases, a guide nucleic acid molecule (gNA) (e.g., a functional gNA) expressed by a second gate unit can produce a modification to at least a portion of a first gate unit upon activation. For example, an activating gNA of a second gate unit can generate a modification to a polynucleotide sequence of a first gate unit that encodes a gNA (e.g., an activatable gNA) or a promoter sequence of a first gate unit operably coupled to such a gNA of the same first gate unit. Such a modification can render the gNA of the first gate unit inoperable (e.g., reduce or inhibit specific binding to a target gene) when expressed. Alternatively, the modification can reduce (e.g., inhibit) expression of the gNA of the first gate unit.

[0122] In some cases, a modification to a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be caused by a single-strand break in which there is a discontinuity in one strand of nucleotides. Inactivation of a polynucleotide sequence or a target gene can be caused by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or more single-strand breaks. In some cases, gene inactivation can be caused by at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 single-strand break.

[0123] In some cases, a gNA (e.g., including both a spacer sequence and a scaffold sequence) can be at least or up to about 60 nucleotides, at least or up to about 70 nucleotides, at least or up to about 80 nucleotides, at least or up to about 85 nucleotides, at least or up to about 90 nucleotides, at least or up to about 95 nucleotides, at least or up to about 100 nucleotides, at least or up to about 105 nucleotides, at least or up to about 110 nucleotides, at least or up to about 120 nucleotides, at least or up to about 130 nucleotides, at least or up to about 140 nucleotides, at least or up to about 150 nucleotides, or at least or up to about 200 nucleotides in size.

[0124] In some cases, the scaffold sequence of a gNA can be at least or up to about 30 nucleotides, at least or up to about 35 nucleotides, at least or up to about 40 nucleotides, at least or up to about 45 nucleotides, at least or up to about 50 nucleotides, at least or up to about 55 nucleotides, at least or up to about 60 nucleotides, at least or up to about 65 nucleotides, at least or up to about 70 nucleotides, at least or up to about 75 nucleotides, at least or up to about 80 nucleotides, at least or up to about 85 nucleotides, at least or up to about 90 nucleotides, at least or up to about 95 nucleotides, at least or up to about 100 nucleotides, at least or up to about 100 nucleotides, at least or up to about 120 nucleotides, at least or up to about 130 nucleotides, at least or up to about 140 nucleotides, or at least or up to about 150 nucleotides in size.

[0125] In some cases, the spacer sequence of a gNA can be at least or up to about 10 nucleotides, at least or up to about 11 nucleotides, at least or up to about 12 nucleotides, at least or up to about 13 nucleotides, at least or up to about 14 nucleotides, at least or up to about 15 nucleotides, at least or up to about 16 nucleotides, at least or up to about 17 nucleotides, at least or up to about 18 nucleotides, at least or up to about 19 nucleotides, at least or up to about 20 nucleotides, at least or up to about 21 nucleotides, at least or up to about 22 nucleotides, at least or up to about 23 nucleotides, at least or up to about 24 nucleotides, at least or up to about 25 nucleotides, at least or up to about 26 nucleotides, at least or up to about 27 nucleotides, at least or up to about 28 nucleotides, at least or up to about 29 nucleotides, or at least or up to about 30 nucleotides in size.

[0126] In some embodiments of any of the systems disclosed herein, the gNA of the gate portion and / or the gene regulation portion (e.g., the gNA encoded by the gate portion and / or the gene regulation portion) can comprise a spacer sequence. In some cases, the spacer sequence can be specific to a target gene. Alternatively, the spacer sequence can be independent of a target gene.

[0127] 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 having spacer sequences of different lengths can be used in the same heterologous gene circuit to affect different types of cleavage, activation, inactivation, and / or modulation 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 impede nuclease activity of a Cas transcriptional modulator while still mediating DNA binding for transcriptional modulation 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 impede nuclease activity of a Cas protein while still mediating DNA binding.

[0128] For example, a gNA comprising a 20-nucleotide spacer sequence (e.g., a gNA encoded by a gate portion of a targeted gene regulation portion plasmid) can be sufficient to promote nuclease activity of an endonuclease (e.g., a Cas or Cas transcriptional modulator fusion protein). Alternatively or additionally, a gNA comprising a 14-nucleotide spacer sequence (e.g., a gNA encoded by a gene regulation portion) can hybridize to DNA, but can not be long enough to mediate nuclease activity - it can only promote endonuclease binding to a homologous DNA sequence. Thus, shorter gNAs can selectively allow for transcriptional modulation of a target gene, while using a endonuclease transcriptional modulator system (e.g., a Cas activator system, a Cas repressor system), without cleaving the target gene.

[0129] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be caused by a double-strand break in which there is a discontinuity in both nucleotide strands. In some cases, the number of such double-strand breaks (e.g., necessary for such modification) can be at least or up to about 1, at least or up to about 2, at least or up to about 3, at least or up to about 4, at least or up to about 5, at least or up to about 6, at least or up to about 7, at least or up to about 8, at least or up to about 9, or at least or up to about 10.

[0130] In some cases, a modification to a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be caused by an insertion-deletion (also referred to as an indel mutation). An indel mutation can include a frameshift or non-frameshift mutation. An indel mutation can include a point mutation (also referred to as a base substitution), in which only one base or base pair is modified. The length of an indel mutation can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, or more bases or base pairs. The length of an indel mutation can include at most about 2000, at most about 1000, at most about 900, at most about 800, at most about 700, at most about 600, at most about 500, at most about 400, at most about 300, at most about 200, at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 base or base pair.

[0131] In some cases, a modification to a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be achieved without cleaving the polynucleotide sequence or target gene. For example, a gene regulation moiety (e.g., a nucleic acid molecule and / or an endonuclease, such as a complex comprising a CRISPR / Cas protein and a guide nucleic acid molecule) can specifically bind to the polynucleotide sequence or target gene such that expression and / or activity of the polynucleotide sequence or target gene is modified. The gene regulation moiety can comprise a transcriptional repressor or a transcriptional activator as provided herein. Alternatively or additionally, the gene regulation moiety can induce an epigenetic modification (or epigenome modification) as provided herein.

[0132] In some cases, as described herein, modifications to polynucleotide sequences or target genes can inactivate them. For example, modifications can inhibit or reduce the expression and / or activity levels of polynucleotide sequences or target genes. In other cases, as described herein, modifications can activate polynucleotide sequences or target genes. For example, modifications can increase the expression and / or activity levels of polynucleotide sequences or target genes.

[0133] In some cases, as provided herein, modification of a polynucleotide sequence or target gene may include reducing the expression and / or activity level of the polynucleotide sequence or target gene by at least or more than about 0.1%, at least or more than about 0.2%, at least or more than about 0.3%, at least or more than about 0.4%, at least or more than about 0.5%, at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 10%, at least or more than about 15%, at least or more than about 20%, at least or more than about 30%, at least or more than about 40%, at least or more than about 50%, at least or more than about 60%, at least or more than about 70%, at least or more than about 80%, at least or more than about 90%, at least or more than about 95%, at least or more than about 99%, or about 100% (e.g., compared to a control, for example, lacking the modification).

[0134] In some cases, as provided herein, modification of a polynucleotide sequence or target gene may include reducing the expression and / or activity level of the polynucleotide sequence or target gene by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 1.5-fold, at least or more than about 2-fold, at least or more than About 3 times, at least or more than about 4 times, at least or more than about 5 times, at least or more than about 6 times, at least or more than about 7 times, at least or more than about 8 times, at least or more than about 9 times, at least or more than about 10 times, at least or more than about 11 times, at least or more than about 12 times, at least or more than about 13 times, at least or more than about 14 times, at least or more than about 15 times, at least or more than about 20 times, at least or more than about 30 times, at least or more than about 40 times, at least or more than about 50 times, or at least or more than about 100 times (e.g., compared to a control, for example, lacking the modification).

[0135] In some cases, as provided herein, a modification of a polynucleotide sequence or target gene can comprise increasing the expression and / or activity level of the polynucleotide sequence or target gene by at least or more than about 0.1%, at least or more than about 0.2%, at least or more than about 0.3%, at least or more than about 0.4%, at least or more than about 0.5%, at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 10%, at least or more than about 15%, at least or more than about 20%, at least or more than about 30%, at least or more than about 40%, at least or more than about 50%, at least or more than about 60%, at least or more than about 70%, at least or more than about 80%, at least or more than about 90%, at least or more than about 100%, at least or more than about 150%, at least or more than about 200%, at least or more than about 300%, at least or more than about 400%, or at least or more than about 500% (e.g., as compared to a control that, for example, lacks the modification).

[0136] In some cases, as provided herein, a modification of a polynucleotide sequence or target gene can comprise increasing the expression and / or activity level of the polynucleotide sequence or target gene by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 1.5-fold, at least or more than about 2-fold, at least or more than about 3-fold, at least or more than about 4-fold, at least or more than about 5-fold, at least or more than about 6-fold, at least or more than about 7-fold, at least or more than about 8-fold, at least or more than about 9-fold, at least or more than about 10-fold, at least or more than about 11-fold, at least or more than about 12-fold, at least or more than about 13-fold, at least or more than about 14-fold, at least or more than about 15-fold, at least or more than about 20-fold, at least or more than about 30-fold, at least or more than about 40-fold, at least or more than about 50-fold, at least or more than about 100-fold, at least or more than about 200-fold, at least or more than about 300-fold, at least or more than about 400-fold, at least or more than about 500-fold, or at least or more than about 1,000-fold (e.g., as compared to a control that, for example, lacks the modification).

[0137] In some embodiments of any of the systems disclosed herein, the gNA of the gate portion and / or the gene regulation portion (e.g., the gNA encoded by the gate portion and / or the gene regulation portion) can comprise 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 independent of a target gene, but can exhibit specific binding to a target polynucleotide sequence of another gate portion or another gene regulation portion. Example spacer sequences can be found in Table 2 (SEQ ID Nos: 1940-2023).

[0138] Non-limiting examples of 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, a spacer sequence of a guide nucleic acid (e.g., guide RNA) directed to a target gene as provided herein can comprise a polynucleotide sequence (e.g., contiguous 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 one or more members selected from the group consisting of SEQ ID NOs: 1940-1943 (e.g., ACAN targeting), SEQ ID NOs: 1944-1947 (e.g., COL2A1 targeting), SEQ ID NOs: 1948-1951 (e.g., MEF2D targeting), SEQ ID NOs: 1952-1955, 1956-1959 (e.g., pax9 targeting), SEQ ID NOs: 1960-1963, 1964-1967 (e.g., RUNX2 targeting), SEQ ID NOs: 1968-1971, 1972-1975, 1976-1979 (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 NOs: 2020-2023 (e.g., UNCX targeting), or the complement thereof.In some cases, a heterologous gene modulator as provided herein can exhibit specific binding to a target gene comprising a polynucleotide sequence (e.g., a contiguous polynucleotide sequence) exhibiting 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 one or more members selected from the group consisting of SEQ ID NOs: 1940-1943 (e.g., ACAN targeting), SEQ ID NOs: 1944-1947 (e.g., COL2A1 targeting), SEQ ID NOs: 1948-1951 (e.g., MEF2D targeting), SEQ ID NOs: 1952-1955, 1956-1959 (e.g., pax9 targeting), SEQ ID NOs: 1960-1963, 1964-1967 (e.g., RUNX2 targeting), SEQ ID NOs: 1968-1971, 1972-1975, 1976-1979 (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 NOs: 2020-2023 (e.g., UNCX targeting), or a complement thereof (e.g., with uracil to thymine conversion).

[0139] A gene regulation moiety as disclosed herein can comprise an endonuclease, such as a CRISPR-Cas protein exhibiting at least a portion of its nuclease activity. For example, the nuclease activity can be used to activate expression or activity of a guide nucleic acid molecule, thereby activating at least a portion of a heterologous gene circuit as described herein.

[0140] A gene regulatory moiety as disclosed herein can include an endonuclease operably coupled to a transcription effector (including a transcriptional activator or a transcriptional repressor), which transcription effector is heterologous to the cell. The endonuclease can be native or can be engineered to exhibit reduced nuclease activity (or substantially no nuclease activity) such that the endonuclease can be used to specifically bind to a target gene, but not cleave the target gene (e.g., an endogenous gene, such as a TBX, bHLH, SOX, collagen, etc.). In some cases, the nuclease can be a deactivated Cas (dCas). Conversely, once the endonuclease identifies and binds to the target gene, the transcription effector coupled (e.g., covalently or non-covalently coupled) to the endonuclease can interact with the target gene to increase or decrease the expression level of the target gene, thereby increasing or decreasing the activity level of the target gene. For example, the endonuclease and the transcription effector can be part of a fusion protein encoded by the same expression cassette.

[0141] FIG. 10 schematically illustrates the use of a heterologous gene circuit in conjunction with an endonuclease transcription effector fusion (e.g., a CRISPR-Cas-transcriptional activator, such as Cas9-VPR). Each gate moiety can be a modified self-inactivating (e.g., self-destructing) guide RNA that, if not inactivated, would be configured to form a complex with the CRISPR Cas transcription effector fusion. An initial activation moiety (denoted as an activation guide RNA or “aGuide”) can convert the first gate moiety into an activated guide RNA (denoted as a mature Guide). Each mature Guide can target an additional gene regulatory moiety (denoted as a ramGuide) encoding an activatable guide RNA directed to a target gene, to activate such ranGuide. Subsequently, the activated ramGuide can form a complex with the CRISPR-Cas transcription effector fusion protein to bind to the target gene and modulate the expression level of the target gene. The activated mature Guide can also target an additional gate moiety downstream within the heterologous gene circuit signaling cascade to subsequently modulate the expression of one or more additional genes.

[0142] In some embodiments, the transcription effector can be a histone epigenetic modifier (or histone modifier). In some cases, the histone epigenetic modifier can modulate a histone through methylation (e.g., a histone methylation modifier, such as an amino acid methyltransferase, e.g., KRAB). In some cases, the histone epigenetic modifier can modulate a histone through acetylation. In some cases, the histone epigenetic modifier can modulate a histone through phosphorylation. In some cases, the histone epigenetic modifier can modulate a histone through ADP-ribosylation. In some cases, the histone epigenetic modifier can modulate a histone through glycosylation. In some cases, the histone epigenetic modifier can modulate a histone through SUMOylation. In some cases, the histone epigenetic modifier can modulate a histone through ubiquitination. In some cases, the histone epigenetic modifier can modulate a histone through remodeling histone structure, e.g., via ATP hydrolysis-dependent processes.

[0143] In some embodiments, the transcription effector can be a gene epigenetic modifier (or gene modifier). In some cases, the gene modifier can modulate a gene through methylation (e.g., a gene methylation modifier, such as a DNA methyltransferase or DNMT). In some cases, the gene modifier can modulate a gene through acetylation.

[0144] In some embodiments, the transcription effector can be derived from a relevant histone acetyltransferase family. Non-limiting examples of histone acetyltransferases 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.

[0145] In some embodiments, the transcriptional effector can 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, 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.

[0146] Non-limiting examples of transcriptional effectors that enhance expression or activity of a target gene can include, but are not limited to, transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), VP64-p65-rta fusion protein (VPR), and activation domains of EDLL and / or TAL activation domains (e.g., active 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, MORF MYST4, SRC1, ACTR, PI 60, CLOCK; and DNA demethylases such as ten-eleven translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1.

[0147] Non-limiting examples of transcriptional effectors that decrease expression or activity of a target gene can include, but are not limited to, transcriptional repressors such as Kruppel- associated box (KRAB or SKD), KOX1 repressor domain, Mad mSIN3 interaction domain (SID), ERF repressor domain (ERD), SRDX repressor domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID 1A / RBP2, JARID IB / PLU-1, JARID 1C / SMCX, JARID ID / SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases such as M.Hhal, DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant), and the like; and peripheral recruitment elements such as lamin A, lamin B, and the like.

[0148] Various aspects of the present disclosure provide a plurality of heterologous genetic circuits that are individually activatable to modulate expression and / or activity levels of a plurality of different target genes in a sequential manner. In some embodiments, a first heterologous genetic circuit is activated to convert a plurality of cells from a first cell type to a second cell type, and subsequently a second heterologous genetic circuit is activated to convert the plurality of cells from the second cell type to a target cell type.

[0149] In some embodiments, activation of the second genetic circuit can occur immediately after activation of the first heterologous genetic circuit. Alternatively, activation of the second genetic circuit can occur 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.

[0150] One or more target genes as disclosed herein can include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0151] One or more target genes as disclosed herein can include a cell differentiation regulatory factor, a molecular function regulatory factor, a binding factor, a membrane 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.

[0152] One or more target genes as disclosed herein can include a cell differentiation regulatory factor, which includes a growth factor, a transcription factor, a myogenic regulatory factor, an immune cell regulatory factor, a neuronal regulatory factor, a stem cell differentiation factor, a chondrogenic regulatory factor, an osteogenic regulatory factor, a senescence factor, a sternness factor (e.g., a dedifferentiation factor), etc.

[0153] In some cases, one or more target genes (e.g., one or more chondrogenic regulatory factors) can include SOX2, SOX6, SOX9, SHOX2, GLI3, TRPS1, OCT4, NANOG, CHONDROGENIC X3.2, BRACHYURY, MIXL1, TBX6, MSGN1, PARXIS, PAX9, RUNX2, RUNX3, SMAD1, SMAD5, and SMAD8.

[0154] In some cases, the one or more target genes can include homeobox genes. Homeobox genes are genes that regulate, for example, large-scale anatomical features at 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 can include HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXB13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13, HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13, CDX1, CDX2, CDX4, GSX1, GSX2, PDX1, EVX1, EVX2, GBX1, GBX2, MEOX1, MEOX2, MNX1, DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, IRX1, IRX2, IRX3, IRX4, IRX5, IRX6, MEIS1, MEIS2, MEIS3, MKX, PBX1, PBX2, PBX3, PBX4, PKNOX1, PKNOX2, TGIF1, TGIF2, TGIF2LX, TGIF2LY, ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, LMX1B, HDX, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2, POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1, POU5F1P1, POU5F1P4, POU5F2, POU6F1, POU6F2, LASS2, LASS3, LASS4, LASS5, LASS6, HMBOX1, HNF1A, HNF1B, SIX1, SIX2, SIX3, SIX4, SIX5, SIX6, ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2, SATB1, SATB2, ADNP, ADNP2, TSHZ1, TSHZ2, TSHZ3, ZEB1, ZEB2, ZFHX2, ZFHX3, ZFHX4,ZHX1, HOMEZ, ALX1 (CART1), ALX3, ALX4, ARGFX, ARX, DMBX1, DPRX, DRGX, DUXA, DUXB, DUX (1, 2, 3, 4, 4c, 5), ESX1, GSC, GSC2, HESX1, HOPX, ISX, LEUTX, MIXL1, NOBOX, OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, RHOXF1, RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, VSX2, BARHL1, BARHL2, BARX1, BARX2, BSX, DBX1, DBX2, EMX1, EMX2, EN1, EN2, HHEX, HLX1, LBX1, LBX2, MSX1, MSX2, NANOG, NOTO, TLX1, TLX2, 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.

[0155] In some cases, the target gene can include MIXL1. MIXL1 is a transcription factor that preferentially binds to the DNA sequence TAAT on the MIX gene and plays a role in gastrulation mesoderm patterning and tissue specification.

[0156] In some cases, the target gene can include UNCX. UNCX is a transcription factor involved in somitogenesis and neurogenesis. UNCX is also required for specific elements that maintain and differentiate the axial skeleton.

[0157] In some cases, the target gene can include PAX9. PAX9 is a transcription factor that is required for the development of the thymus, parathyroid glands, posterior gill, teeth, skeleton, and limbs.

[0158] In some cases, the one or more target genes can include T-box transcription factors (TBX genes). TBX transcription factors are involved in development. T-box proteins have a relatively large DNA-binding domain. Non-limiting examples of TBX transcription factors can include TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, TBX22, and TBXT (Brachyury protein).

[0159] 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 gene family. TBXT plays a role in determining the midline of bilateral organisms, contributing to the establishment of the anterior-posterior axis. It can also assist in determining mesoderm during gastrulation.

[0160] In some cases, the target gene can include TBX6. TBX6, also known as T-box transcription factor 6, is involved in the segmentation of the paraxial mesoderm into somites.

[0161] In some cases, the one or more target genes can include basic helix-loop-helix transcription factors (bHLH genes). bHLH transcription factors are involved in the regulation of the cell cycle and many other developmental processes. bHLH proteins have a basic helix-loop-helix protein structure. Non-limiting examples of bHLH transcription factors can include AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATOH1, ATOH7, ATOH8, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, EPAS1, FERD3L, FIGLA, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2, HIF1A, ID1, ID2, ID3, ID4, KIAA2018, LYL1, MASH1, MATH2, MAX, MESP1, MESP2, MIST1, MITF, MLX, MLXIP, MLXIPL, MNT, MSC, MSGN1, MXD1, MXD3, MXD4, MXI1, MYC, MYCL1, 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.

[0162] In some cases, the target gene can include MSGN1. MSGN1, also known as mesoderm protein 1, is a WNT-activated BHLH transcription factor involved in mesoderm formation and regulation of transcription by RNA polymerase II. MSGN1 can also be involved in somitogenesis.

[0163] In some cases, the target gene can include TCF15. TCF15 is an early transcription factor that plays a role in somitogenesis, proximal mesoderm development, and stem cell pluripotency regulation.

[0164] In some cases, the one or more target genes can include SRY-related box transcription factors (SOX genes). SOX transcription factors are involved in developmental regulation. Non-limiting examples of SOX transcription factors can include SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21, SOX30, and SRY.

[0165] In some cases, the one or more target genes can include SOX Group A, which includes SRY. In some cases, the one or more target genes can include SOX Group B1, which includes SOX1, SOX2, and / or SOX3. In some cases, the one or more target genes can include SOX Group B2, which includes SOX14 and / or SOX21. In some cases, the one or more target genes can include SOX Group C, which includes SOX4, SOX11, and / or SOX12. In some cases, the one or more target genes can include SOX Group D, which includes SOX5, SOX6, and / or SOX13. In some cases, the one or more target genes can include SOX Group E, which includes SOX8, SOX9, and / or SOX10. In some cases, the one or more target genes can include SOX Group F, which includes SOX7, SOX17, and / or SOX18. In some cases, the one or more target genes can include SOX Group G, which includes SOX15. In some cases, the one or more target genes can include SOX Group H, which includes SOX30.

[0166] In some cases, the target gene can include SOX6. SOX6 is a transcription factor that is required for central nervous system development, cartilage formation, and maintenance of cardiac and skeletal muscle cells.

[0167] In some cases, the target gene can include SOX9. SOX9 is a transcription factor that plays a role in chondrocyte differentiation and regulates transcription of the anti-Mullerian hormone (AMH) gene.

[0168] In some cases, one or more target genes may include collagen. Collagen is a fibrous protein and a major component of skin, bone, tendons, cartilage, blood vessels, and teeth. Collagen forms insoluble fibers with high tensile strength. Non-limiting examples of collagen genes may include COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, COL6A6, COL7A1, COL8A1, and COL8A2. COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, COL12A1, COL13A1, COL14A1, COL15A1, COL16A1, COL17A1, COL18A1, COL19A1, COL20A1, COL21A1, COL22A1, COL23A1, COL24A1, COL25A1, COL26A1, COL27A1, and COL28A1.

[0169] In some cases, target genes may include COL2A1. COL2A1 is a component of the pro-α1 chain of type II collagen. Type II collagen adds structure and strength to the connective tissue that supports the body's muscles, joints, organs, and skin. Type II collagen is primarily found in cartilage.

[0170] In some cases, the use of heterologous gene circuits as disclosed herein can be used to differentiate mesodermal stem cells (MSCs) into chondrocyte progenitor 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 are of the target cell type.

[0171] In some cases, the use of heterologous gene circuits as disclosed herein can be used, for example, to differentiate mesodermal stem cells (MSCs) into chondrocyte progenitor cells in the absence of one, two, or all of feeder cells, serum, and exogenous growth factors. The use of heterologous gene circuits as disclosed herein can differentiate cells from up to approximately 1 x 10⁻⁶ cells. 6 One, at most about 9x10 5 One, at most about 8x10 5 One, at most about 7x10 5 One, at most about 6x105 at most about 5 x 105 5 at most about 4 x 105 5 at most about 3 x 105 5 at most about 2 x 105 5 at most about 1 x 105 5 at most about 5 x 105 4 at most about 2 x 105 4 at most about 1 x 105 4 at least about 1 x 105 4 at least about 2 x 105 4 at least about 5 x 105 4 at least about 1 x 105 5 at least about 2 x 105 5 at least about 5 x 105 5 at least about 1 x 105 6 at least about 2 x 105 6 at least about 5 x 105 6 at least about 1 x 105 7 at least about 2 x 105 7 at least about 5 x 105 7 at least about 1 x 105 8 at least about 2 x 105 8 at least about 5 x 105 8 at least about 1 x 105 9 at least about 2 x 105 9 at least about 5 x 105 9 at least about 1 x 105 10 at least about 2 x 105 10 at least about 5 x 105 10 at least about 1 x 105 15 at least about 2 x 105 15 at least about 5 x 105 15 at least about 1 x 105

[0172] In some cases, use of a heterologous genetic circuit as disclosed herein can be used to differentiate pluripotent stem cells (PSCs, such as induced PSCs or IPSCs) into chondrogenic cells, for example, in the absence of one, two, or all of feeder cells, serum, and exogenous growth factors. Use of a heterologous genetic circuit as disclosed herein can result in the production of at least about 1 x 105 6 at most about 9 x 105 5 at most about 8 x 105 5 at most about 7 x 105 5 at most about 6 x 105 5 at most about 5 x 1055 at least about 1 x 105 5 at least about 2 x 105 5 at least about 5 x 105 5 at least about 1 x 105 5 at least about 2 x 105 4 at least about 5 x 105 4 at least about 1 x 105 4 at least about 2 x 105 4 at least about 5 x 105 4 at least about 1 x 105 4 at least about 2 x 105 5 at least about 5 x 105 5 at least about 1 x 105 5 at least about 2 x 105 6 at least about 5 x 105 6 at least about 1 x 105 6 at least about 2 x 105 7 at least about 5 x 105 7 at least about 1 x 105 7 at least about 2 x 105 8 at least about 5 x 105 8 at least about 1 x 105 8 at least about 2 x 105 9 at least about 5 x 105 9 at least about 1 x 105 9 at least about 2 x 105 10 at least about 5 x 105 10 at least about 1 x 105 10 at least about 2 x 105 15 at least about 5 x 105 15 at least about 1 x 105 15 at least about 2 x 105

[0173] The production of such chondrogenic cells by use of a heterologous genetic circuit as disclosed herein can be achieved over a time span of at most about 60 days, at most about 55 days, at most about 50 days, at most about 45 days, at most about 40 days, at most about 35 days, at most about 30 days, at most about 25 days, at most about 20 days, at most about 15 days, at most about 10 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, at most about 1 day, or less.

[0174] In some cases, chondrogenic cells produced by this method produce more cartilage than chondrogenic cells obtained via directed differentiation. Chondrogenic cells produced 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 additionally, chondrogenic cells produced by this method can have equivalent amounts of produced cartilage as compared to chondrogenic cells obtained via directed differentiation.

[0175] In some cases, chondrogenic cells or chondroprogenitor cells produced by this method exhibit higher expression levels of two or more positive chondroprogenitor cell markers as compared to control chondroprogenitor cells. Non-limiting examples of positive chondroprogenitor cell markers can include CD146, CD73, CD112, and BMPR1.

[0176] In some cases, chondrogenic cells or chondroprogenitor cells produced by this method exhibit at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, 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 level of a positive chondroprogenitor cell marker as compared to control chondroprogenitor cells.

[0177] In some cases, the chondrogenic cells or chondroprogenitor cells produced by this method exhibit a positive chondroprogenitor cell marker expression level that is at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold higher than a control chondroprogenitor cell.

[0178] In some cases, the chondrogenic cells or chondroprogenitor cells produced by this method exhibit a higher expression level of two or more negative chondroprogenitor cell markers compared to a control chondroprogenitor cell. Non-limiting examples of negative chondroprogenitor cell markers can include CD326 and CD309.

[0179] In some cases, the chondrogenic cells or chondroprogenitor cells produced by this method exhibit a negative chondroprogenitor cell marker expression level 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%, 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 than a control chondroprogenitor cell.

[0180] In some cases, the chondrogenic cells or chondroprogenitor cells produced by this method exhibit at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold lower expression levels of a negative chondroprogenitor cell marker than control chondroprogenitor cells.

[0181] In some cases, the expression levels of chondroprogenitor cell markers can be measured using methods such as, but not limited to, RT-PCR, Western blot, Northern blot, protein staining, mRNA staining, and RNA sequencing.

[0182] In some cases, the expression levels can be measured at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 28 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or more days after introduction of the genetic circuit.

[0183] In some cases, the chondrogenic cells or chondroprogenitor cells produced by this method can be chondrocytes. Alternatively, the chondrogenic cells or chondroprogenitor cells produced by this method can be cells other than chondrocytes (e.g., chondroblasts). In some cases, the progenitor cells produced can be substantially mitotically quiescent. Alternatively, the progenitor cells produced can be substantially mitotically active.

[0184] In some cases, the first gate unit can be configured to reduce the expression and / or activity level of one or more target genes. In some cases, the first gate unit can be configured to enhance the expression and / or activity level of one or more target genes. In some cases, the first gate unit can be configured to maintain the expression and / or activity level of one or more target genes.

[0185] In some cases, the modulation of the first target gene can occur before the modulation of the second target gene. In some cases, the modulation of the first target gene can occur after the modulation of the second target gene. In some cases, the modulation of the first target gene can occur about simultaneously with the modulation of the second target gene.

[0186] In some cases, the use of the heterologous genetic circuit can induce differentiation of cells into a cell type of interest in the absence of growth factors, serum (fetal bovine serum, human serum AB, etc.), or other exogenous cell differentiation regulatory factors or media. Serum can include the liquid portion of clotted blood, including nutritional factors and macromolecular factors essential for cell growth.

[0187] Alternatively, the use of the heterologous genetic circuit can induce differentiation of cells into a cell type of interest using a reduced amount of serum and / or growth factors (e.g., reduced 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%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or substantially free of serum). A reduced amount of serum can allow for more consistent cells across experiments or batches, increased growth and / or productivity of differentiated cells, better control of physiological responsiveness, and reduced risk of contamination of cell culture with serum-derived agents.

[0188] In some cases, use of a heterologous genetic circuit in a stem cell (e.g., iSPC, MSC) can induce MSC differentiation into chondrogenic cells in the absence of growth factors, serum (fetal bovine serum, human serum AB, etc.), or other exogenous cell differentiation modulators or media. In some cases, use of a heterologous genetic circuit as disclosed herein can be used to differentiate stem cells into chondrogenic cells in the absence of one or both of growth factors and serum. The resulting chondrogenic cells produced by use of a heterologous genetic circuit as 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.

[0189] In some cases, use of a heterologous genetic circuit to transform from one cell type (e.g., PSC, MSC, or chondroprogenitor cell) to another cell type (e.g., chondrogenic cell) can result in a target cell type. Alternatively, use of a heterologous genetic circuit to transform from one cell type (e.g., PSC, MSC, or chondroprogenitor cell) to another cell type (e.g., chondrogenic cell) can result in an intermediate cell type. The intermediate cell type can undergo a second transformation using a second genetic circuit to result in a target cell type.

[0190] The transformation of a cell from one cell type to another can involve the modulation of multiple target genes. For example, the transformation can involve the modulation of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more target genes. The transformation can involve the modulation of at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 target gene. Each gene disclosed herein can be modulated 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, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more. Each gene disclosed herein can be modulated by at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1. The modulation(s) of the target genes (e.g., endogenous genes) induced by the heterologous gene circuit of the present disclosure can be artificial (or heterologous) modulations that can otherwise not occur in the cell in the absence of (i) the heterologous gene circuit and / or (ii) the activation portion of the heterologous gene circuit.

[0191] As FIG. 3 As demonstrated in the Examples, various heterologous gene circuits can be designed to modulate the expression or activity levels of multiple genes (e.g., multiple endogenous genes) in a cell at multiple different time points.

[0192] For example, the heterologous genetic circuit can be designed to (i) modulate the expression level of a first gene and (ii) subsequently modulate the expression level of a second gene. For example, the heterologous genetic circuit can be designed to (i) activate the expression level of a first gene and (ii) subsequently activate the expression level of a second gene. Alternatively, the heterologous genetic circuit can be designed to (i) activate the expression level of a first gene and (ii) subsequently decrease the expression level of a second gene. Alternatively, the heterologous genetic circuit can be designed to (i) decrease the expression level of a first gene and (ii) subsequently activate the expression level of a second gene. Alternatively, the heterologous genetic circuit can be designed to (i) decrease the expression level of a first gene and (ii) subsequently decrease 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. The heterologous genetic circuit can be designed to modulate the expression level of additional genes. The heterologous genetic circuit can be designed to include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more additional genes. The additional genes can be activated. Alternatively, the additional genes can be decreased. The heterologous genetic circuit can be designed to modulate the expression of additional genes prior to modulating the expression level of both the first gene and the second gene. Alternatively or additionally, the heterologous genetic circuit can be designed to modulate the expression of additional genes after modulating the expression level of the first gene and prior to modulating the expression level of the second gene. Alternatively or additionally, the heterologous genetic circuit can be designed to modulate the expression of additional genes after modulating the expression level of both the first gene and the second gene.

[0193] For example, the heterologous genetic circuit can be designed to (i) modulate the expression level of a first gene and (ii) subsequently modulate the expression level of a second gene. For example, the heterologous genetic circuit can be designed to (i) activate the expression level of a first gene and (ii) subsequently activate the expression level of a second gene. Alternatively, the heterologous genetic circuit can be designed to (i) activate the expression level of a first gene and (ii) subsequently decrease the expression level of a second gene. Alternatively, the heterologous genetic circuit can be designed to (i) decrease the expression level of a first gene and (ii) subsequently activate the expression level of a second gene. Alternatively, the heterologous genetic circuit can be designed to (i) decrease the expression level of a first gene and (ii) subsequently decrease 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. The heterologous genetic circuit can be designed to modulate the expression level of additional genes. The heterologous genetic circuit can be designed to include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more additional genes. The additional genes can be activated. Alternatively, the additional genes can be decreased. The heterologous genetic circuit can be designed to modulate the expression of additional genes prior to modulating the expression level of both the first gene and the second gene. Alternatively or additionally, the heterologous genetic circuit can be designed to modulate the expression of additional genes after modulating the expression level of the first gene and prior to modulating the expression level of the second gene. Alternatively or additionally, the heterologous genetic circuit can be designed to modulate the expression of additional genes after modulating the expression level of both the first gene and the second gene.

[0194] The heterologous gene circuit can be designed to include another gene in addition to the first and second homeodomain genes. The additional gene can be regulated before, concurrently with, or after the first homeodomain protein. Alternatively or additionally, the additional gene can be regulated before, concurrently with, or after the second homeodomain protein. The heterologous gene circuit can be designed to activate the additional gene. Alternatively, the heterologous gene circuit can be designed to repress 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), a SOX, and a collagen.

[0195] The heterologous gene circuit can be designed to include two members selected from the group consisting of a homeodomain protein, a T-box transcription factor (TBX), and a basic helix-loop-helix transcription factor (bHLH). The heterologous gene circuit can be designed to include a homeodomain protein and a TBX. The heterologous gene circuit can be designed to include a homeodomain protein and a bHLH. The heterologous gene circuit can be designed to include a TBX and a bHLH.

[0196] The heterologous gene circuit can be designed to include a first member that is a homeodomain protein, a TBX, or a bHLH, and a second member that is a SOX or a collagen. The heterologous gene circuit can be designed to include a homeodomain protein and a SOX. The heterologous gene circuit can be designed to include a homeodomain protein and a collagen. The heterologous gene circuit can be designed to include a TBX and a SOX. The heterologous gene circuit can be designed to include a TBX and a collagen. The heterologous gene circuit can be designed to include a bHLH and a SOX. The heterologous gene circuit can be designed to include a bHLH and a collagen.

[0197] For example, heterologous gene circuit #10 can be designed to (i) activate expression levels of TBXT and MIXL1 at a time point 1 (denoted as Step 1), (ii) subsequently activate expression levels of MSGN1 and TBX6 at a time point after Step 1 (denoted as Step 2), (iii) subsequently activate expression levels of UNCX, TCF15, and PAX9 at a time point after Step 2 (denoted as Step 3), and (iv) subsequently activate expression levels of SOX6 at a time point after Step 3 (denoted as Step 4). For comparison, a control heterologous gene circuit (denoted as Integration 1) can be designed to activate the same target endogenous genes from heterologous gene circuit #10 at the same time.

[0198] Activating a heterologous gene circuit in a cell as disclosed herein can modulate the expression or activity level of multiple genes at multiple different time points to effect transformation of the cell into different cell types (e.g., stem cells into tissue-specific progenitor cells, etc.). The rate of transformation of such cell types by use of the heterologous gene circuit can be at least or up to about 1 percent (%) higher than the rate of transformation of the cell types by use of a control heterologous gene circuit (e.g., for simultaneously activating multiple target genes) by at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, or at least or up to about 95%.

[0199] Activating a heterologous gene circuit in a cell as disclosed herein can modulate the expression or activity level of multiple genes at multiple different time points to effect transformation of the cell into different cell types (e.g., stem cells into tissue-specific progenitor cells, etc.). The transformation of the cell 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.

[0200] The cell (e.g., an initial cell to be modified into an engineered cell as disclosed herein, a final cell product produced from an engineered cell as disclosed herein, etc.) can comprise a muscle cell, an immune cell, a neuron, an osteoblast, an endothelial cell, a mesenchymal cell, an epithelial cell, a stem cell, a secretory cell, a blood cell, a germ cell, a trophic cell, a storage cell, an enteroendocrine cell, a pituitary cell, a mesodermal cell, a cartilage progenitor cell, a neuroendocrine cell, a duct cell, a dentinoblast, a cementoblast, a glial cell, or an interstitial cell.

[0201] Non-limiting examples of such cells can include lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, cytokine-induced killer (CIK) cells (see, e.g., US20080241194); myeloid cells such as granulocytes (basophils, eosinophils, neutrophils / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, thrombocytes / megakaryocytes, dendritic cells; cells from the endocrine system including thyroid cells (thyroid epithelial cells, parafollicular cells), parathyroid cells (parathyroid chief cells, oxyphil cells), adrenal cells (chromaffin cells), pineal cells (pinealocytes); cells of the nervous system including glial cells (astrocytes, microglia), large cell neuroendocrine cells, stellate cells, Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Boettcher cells (Bocells, including hepatocytes, Kupffer cells, cartilage / bone / muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, tooth cells (osteodentinoblasts, ameloblasts); paraxial mesoderm cells cartilage cells, including blastema cells, chondroblasts, chondrocytes; skin cells, including hair cells, keratinocytes, melanocytes (nevus cells); muscle cells, including muscle cells; urinary system cells, including podocytes, juxtaglomerular cells, intraglomerular mesangial cells / extraglomerular mesangial cells, kidney proximal tubule brush border cells, macula densa cells; reproductive system cells, including sperm, Sertoli cells, Leydig cells, oocyte; and other cells, including adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of the nail and toe nail, nail bed basal cells (stem cells), medulla hair shaft cells, cortex hair shaft cells, keratinized hair shaft cells, keratinized hair root sheath cells, hair root sheath cells of the Huxley layer, hair root sheath cells of the Henle layer, outer hair root sheath cells, hair matrix cells (stem cells), stratified epithelial cells of the wet stratum, 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, uroepithelial cells (lining the urinary bladder and ureter), exocrine epithelial cells, salivary gland mucous cells (polysaccharide-rich secretion), salivary gland serous cells (glycoproteinase-rich secretion), Von Ebner's glands in the tongue, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, taste cells, tasteEbner's) Glandular cells (Washing taste buds), Mammary cells (Lactation), Lacrimal cells (Tear secretion), Ceruminous cells in the ear (Wax secretion), Eccrine sweat dark cells (Glycoprotein secretion), Eccrine sweat clear cells (Small molecule secretion). Apocrine sweat cells (Odor secretion, sex hormone sensitive), Ciliary glands cells in the eyelid (Specialized sweat glands), Sebaceous cells (Lipid-rich sebum secretion), Bowman's glands cells in the nose (Washing olfactory epithelium), Brunner's Glands cells in the duodenum (Enzymes and alkaline mucus), Seminal vesicle cells (Secretion of seminal fluid components, including fructose for motile sperm), Prostate cells (Secretion of seminal fluid components), Bulbourethral gland cells (Mucus secretion), Bartholin's gland cells (Vaginal lubricant secretion), Littre's gland cells (Mucus secretion), Endometrial cells (Carbohydrate secretion), Separated goblet cells of the respiratory and digestive tracts (Mucus secretion), Gastric lining mucus cells (Mucus secretion), Gastric antrum peptic cells (Pepsinogen secretion), Gastric oxyntic cells (Hydrochloric acid secretion), Pancreatic acinar cells (Bicarbonate and digestive enzymes secretion), Small intestinal Paneth cells (Lysozyme secretion), Type II pneumocyte of the lung (Surfactant secretion), Clara cells of the lung, Hormone secreting cells, Anterior pituitary cells, Somatotropic cells, Lactotropes, Thyrotropic, Gonadotropic cells, Adrenocorticotropic cells, Intermediate pituitary cells, Large cell neuroendocrine cells, Intestinal and respiratory cells, Thyroid cells, Thyroid epithelial cells, Follicular paracells, Parathyroid cells, Parathyroid chief cells, Eosinophilic cells, Adrenal cells, Chromaffin cells, Testicular interstitial cells, Endometrial cells of the follicle, Luteal cells of ruptured follicle, Granular luteal cells, Membrane luteal cells, Juxtaglomerular cells (Renin secretion), Dense plaque cells of the kidney, Metabolic and storage cells, Barrier function cells (Lung, intestine, exocrine glands, and urogenital tract), Kidney cells, Type I pneumocyte (Lining air space of the lung), Pancreatic duct cells (Acinar cells), Unstriated duct cells (Sweat glands, salivary glands, mammary glands, etc.), Duct cells (Seminal vesicle, 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, Cardiac muscle cells, Blood and immune system cells, Erythrocyte (Red blood cell), Leukocyte (White blood cell),cell)) megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in 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 and committed progenitor cells (various types) of the blood and immune system, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic neuronal cells, sensory organ and peripheral neuronal support cells, central nervous system neuronal and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, sperm cells, spermatocytes, spermatogonia (stem cells for spermatocytes), spermatozoa, nurse cells, ovarian follicle cells, Sertoli cells (in testes), thymic epithelial cells, interstitial cells, and juxtaglomerular cells.

[0202] In an aspect, the present disclosure provides systems and methods that can transform a plurality of pluripotent stem cells (PSCs) into a plurality of tissue-specific progenitor cells.

[0203] 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, cartilage progenitor cells, neural stem cells, epithelial stem cells, epidermal stem cells, mammary stem cells, intestinal stem cells, neural crest stem cells, or testicular stem cells.

[0204] Various aspects of the present disclosure provide engineered cells programmed to induce a desired level of expression and / or activity (or profile thereof) of one or more target genes in a cell.

[0205] In some embodiments, the engineered cells of the present disclosure (e.g., engineered chondrogenic cells) can be generated from an isolated stem cell (e.g., an isolated MSC or iPSC). As disclosed herein, the heterologous genetic circuit and / or components thereof (e.g., gate units, gate portions, activation portions, etc.) can be introduced during any stage (or cell state) between and including (a) the isolated stem cell, and (b) its differentiated chondrogenic cell state (e.g., a terminally differentiated chondrogenic cell state, such as a chondrocyte).

[0206] 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 can have or can 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 produce any of the subject engineered cells (e.g., chondrogenic cells) as disclosed herein. Subsequently, the engineered immune cells can 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.).

[0207] The engineered cells as disclosed herein can be administered to a subject prior to, concurrently with, or after activation of the heterologous gene circuit in the engineered stem cell. For example, the engineered cells can be activated after administration into the subject, e.g., by administering an activator of the heterologous gene circuit to the subject.

[0208] A subject can 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 additionally, a subject can 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.

[0209] Any of the methods disclosed herein can be utilized to treat a target cell, target tissue, target condition, or target disease of a subject.

[0210] The target disease of the subject can be a disease affecting cartilage. Diseases affecting cartilage can include, but are not limited to, arthritis (e.g., osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis), gout, systemic lupus erythematosus, seronegative spondyloarthropathies, costochondritis, hernias, achondroplasia, or multiple epiphyseal dysplasia. The target condition of the subject can be an injury (e.g., a joint injury, such as a knee injury).

[0211] The target disease of the subject can be a cancer or a tumor. Non-limiting examples of cancers can include cells of a cancer including acanthoma, adenocystic carcinoma, adenoma, adamantinoma, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft-part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large-cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendiceal cancer, astrocytic tumor, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Berringer ductal carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt's lymphoma, cancer of unknown primary, carcinoid tumor, carcinoma, carcinoma in situ, carcinoma of the penis, carcinoma of unknown primary, carcinosarcoma, Castleman's Disease, central nervous system embryonal tumor, cerebellar astrocytic tumor, cerebral astrocytic tumor, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papillary tumor, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, chronic neutrophilic leukemia, clear-cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dematioma, desmoplastic small round cell tumor, desmoplastic melanoma, devascularizing tumor, diffuse large B-cell lymphoma, diffuse large-cell lymphoma, diffuse-type gastric cancer, dysgerminoma, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, enteric cancer, enterocytic tumor, enterogenous polyp, enterogenous tumor, enteric tumor, enteric tumor, enteron cancer, enteron tumor, epithelial carcinoma, epithelial tumor, epithelioid carcinoma, epithelioid sarcoma, ependymal tumor, ependymoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epidermoid carcinoma, epiderDisease), dermatofibrosarcoma protuberans, dermoid cyst, connective tissue proliferative small round cell tumor, diffuse large B-cell lymphoma, dysembryonic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, olfactory neuroblastoma, Ewing's family tumor, Ewing's family sarcoma, Ewing's sarcoma, extracranial germ cell tumor, gonadal extragerminoma, extrahepatic bile duct carcinoma, extramammary Paget's disease, fallopian tube cancer, fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid carcinoma, gallbladder cancer, gallbladder cancer Ganglioglioma, ganglioma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, cerebral glioma, 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, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast cancer and ovarian cancer syndrome, Hodgkin lymphoma. Lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, renal cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, malignant lentigines melanoma, leukemia, lip and oral cancer, liposarcoma, lung cancer, corpus luteum tumor, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, bone malignant fibrous histiocytoma, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdomyosarcoma, malignant salamander tumor Tumor), MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, occult primary metastatic squamous neck cancerPrimary), metastatic urothelial carcinoma, mixed mullerian tumor, monocytic leukemia, oral cancer, myxoid tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoid tumor, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neoplasm, neurofibroma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular oncology, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory tract carcinoma involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sezary Syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, soot wart, squamous cell carcinoma, squamous cell carcinoma, squamous neck cancer, squamous neck cancer, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squwart), spinal cord tumor, spinal column tumor, splenic marginal zone lymphoma, squamous cell carcinoma, stomach cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, advanced lymphoma, testicular cancer, thecoma, throat cancer, thymic carcinoma, thymoma, thyroid cancer, renal pelvis and ureter transitional cell carcinoma, transitional cell carcinoma, Urachal cancer, urethral cancer, Urogenital neoplasm, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, Visual Pathway Glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation within a population of cancer cells, such as cancer stem cells. In some embodiments, the cancer is a cancer of the hematopoietic lineage, such as a lymphoma. The antigen can be a tumor-associated antigen.

[0212] Non-limiting examples of target tissues can include cells that can be obtained from a subject, e.g., chondrogenic cells. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Examples of samples from a subject from which cells can be derived include, but are not limited to, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, feces, semen, vaginal fluid, interstitial fluid derived from tumor tissue, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, nails, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cerebrospinal fluid, tissue, throat swab, biopsy, amniotic fluid, amniotic water, umbilical cord blood, emphatic fluids, lumenal fluids, sputum, pus, microbiota, meconium, breast milk, and / or other excretions or body tissues.

[0213] The present disclosure also provides a composition comprising the engineered genetic circuit as disclosed herein. The composition can further comprise an actuator of a heterologous genetic circuit. The present disclosure also provides a kit comprising the composition. The kit can further comprise an activator of a heterologous genetic circuit. The activator can be in the same composition as the engineered cell. Alternatively or additionally, the activator can be in a different and separate composition from the engineered cell.

[0214] In some cases, the engineered progenitor cells disclosed herein can exhibit (i) comparable or enhanced regenerative capacity; (ii) comparable or enhanced in vitro expression; (iii) comparable or enhanced gene editing capacity; (iv) comparable or enhanced immune tolerance; (v) comparable or shorter manufacturing timeline; (vi) comparable or fewer growth factor or culture requirements; and / or (vii) comparable or enhanced safety compared to control progenitor cells.

[0215] Control progenitor cells can be produced by any method including expansion of progenitor cells (e.g., cartilage progenitor cells) isolated from a tissue, directed iPSC differentiation (e.g., using exogenous growth factors), and / or transgenic iPSC differentiation (e.g., viral transduction of heterologous genes).

[0216] In some cases, tissue-specific progenitor cells can be stored in a container (e.g., a sterile vial). In some cases, 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.

[0217] Pharmaceutical compositions

[0218] In some cases, the methods disclosed herein include 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.

[0219] The pharmaceutical compositions of the present disclosure can be combinations of any of the pharmaceutical compounds described herein with other chemical components such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical compositions facilitate administration of the compounds to an organism. The pharmaceutical compositions can be administered in a therapeutically effective amount as a pharmaceutical composition by various forms and routes, including, for example, intravenous, subcutaneous, intramuscular, inhalation, oral, parenteral, ocular, aural, subcutaneous, transdermal, nasal, intravitreal, intratracheal, intrapulmonary, transmucosal, vaginal, and topical administration.

[0220] The formulation can be modified according to the chosen route of administration. Pharmaceutical compositions containing the compounds described herein can be manufactured, for example, by mixing, dissolving, emulsifying, encapsulating, embedding, or compressing processes.

[0221] Example

[0222] Example 1: Differentiation of chondrocyte progenitor cells

[0223] Tissue-specific cells (e.g., chondrocyte progenitor cells) can be generated from less differentiated cells (e.g., stem cells, such as IPSCs) using the systems and methods disclosed herein.

[0224] A. Generation of chondrocyte progenitor cells.

[0225] In this embodiment, a heterologous gene circuit is used to induce mesodermal stem cells (MSCs) to differentiate into chondrocyte progenitor cells. The differentiation of stem cells (e.g., MSCs) into chondrocyte progenitor cells can be a complex process, requiring the activation of multiple endogenous genes at different time points and the deactivation of multiple endogenous genes at different time points. See also... FIG. 2A Examples of different endogenous genes induced to express at different stages of stem cell differentiation into chondrocyte progenitors and subsequently into chondrocytes. Therefore, one or more heterologous gene circuits as disclosed herein can be used to automatically promote such cascade regulation of different endogenous gene expression. In some cases, each heterologous gene circuit can be configured to regulate the expression levels of multiple genes at multiple different time points when a single such heterologous gene circuit is activated.

[0226] MSC uses encoding such as FIG. 2B or FIG. 3 Transient transfection with plasmid DNA, one of the heterologous gene circuits described herein, for example, targeting combinations of TBXT, MIXL1, TBX6, MSGN1, UNCX, TCF15, PAX9, SOX6, SOX9, PAX3, PAX7, and COL2A1. All targeted genes are activated. Flow cytometry was used to analyze CD146 / CD73 double-positive cells, CD146 / CD112 double-positive cells, and CD326 / CD309 double-negative cells, each of which indicates the formation of chondrogenic progenitor cells. FIG. 6A-6B The study found that top-performing HCGs (such as Cell Algorithm 10) converted approximately 60% of cells into chondrogenic progenitors within four days. FIG. 6C ).

[0227] B. Characterization of chondrocyte progenitor cells generated by heterologous gene circuits.

[0228] Transient plasmid delivery of heterologous gene circuits induced the appearance of double-positive and double-negative chondrogenic progenitor cell markers four days later. FIG. 6C As shown, flow cytometry analysis reveals thatFIG. 3 At least one heterologous gene circuit, heterologous gene circuit 10 (e.g., cell algorithm 10), provided in the study, generates at least approximately 60% conversion of MSCs to chondrocyte progenitor cells within four days. This conversion rate is greater than that of a control population of IPSCs treated simultaneously with the same endogenous gene activated.

[0229] Individually, for positive markers of chondrocyte progenitor cells (e.g., CD146+ / CD73+, CD146+ / CD73+, CD326- / CD309-, etc.), analysis was performed using [data from...]. FIG. 3 Cells treated with various heterologous gene circuits (e.g., via flow cytometry) were then plotted as volcano maps, such as... FIG. 4 As shown. Volcano plots were generated to compare the efficiency of each heterologous gene circuit based on: (i) statistical significance (P-value) compared to the monolithic control circuit that activates the target endogenous gene once, and (ii) the magnitude of change (fold change) of chondrocyte progenitor positive markers compared to the monolithic control circuit.

[0230] FIG. 5A-5D Showing FIG. 4 The representative data used in the volcano map. FIG. 5A-5D In each graph, the y-axis represents the distance from the graph. FIG. 3 When treated with one of the heterologous gene circuits, the proportion of each cell sample expressing the indicated chondrocyte progenitor marker (e.g., CD146+ / CD73+, CD146+ / CD73+, BMPR1+, or CD326 / CD309-) is shown. The x-axis indicates which heterologous gene circuit was utilized for each cell sample. FIG. 5A-5D The figures show that various heterogeneous gene circuits (e.g., Cell Algorithm 9, Cell Algorithm 10, Cell Algorithm 12, etc.) can generate chondrocyte progenitor cells, as indicated by multiple chondrocyte progenitor cell marker groups.

[0231] Example 2: Chondrogenesis assay

[0232] In this predictive embodiment, IPSCs and mesenchymal cells were contacted with top-performing HGCs from Example 1 and allowed to grow in culture medium until chondrocyte formation. A control chondrocyte population was collected from mouse samples and purified. Chondrogenesis in both the HGC and control chondrocyte populations was tested, including the amount of chondrocyte formation and the mechanical properties of the tested cartilage.

[0233] Example 3: Implantation and transplantation of chondrogenic progenitor cells

[0234] Tissue-specific cells (e.g., chondrocyte progenitor cells) prepared using the systems and methods of this disclosure can be applied to subjects in need (e.g., injected into joint tissue) to treat joint-related or cartilage-related conditions.

[0235] A. Generation of Cartilage Progenitor Cells.

[0236] According to the methods described in Example 1, 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 genetic circuit, such as, for example, one of the corresponding heterologous genetic circuits shown in FIG. 1, to facilitate the generation of cartilage progenitor cells. FIG. 3

[0237] B. In vivo administration of cartilage progenitor cells.

[0238] After cartilage progenitor cells are generated by the systems and methods of the present disclosure, the cartilage progenitor cells (e.g., CD146 cells) can be purified using anti-CD146 antibodies. The cells can be concentrated and resuspended in a buffer (e.g., PBS), and then administered to mice via direct injection into a joint or other site of interest. After 8 to 10 weeks, the mice can be sacrificed, and the tissue surrounding the injection area sectioned. The sections can be immunostained for human cartilage to confirm engraftment of the ex vivo generated cartilage progenitor cells.

[0239] Additional protocols for cartilage progenitor cell engraftment are provided herein. After generation as described herein, cartilage progenitor cells can be suspended in a chondrogenic cell culture medium. These cells can be engrafted into a target site of a joint with or without further expansion. For expansion, cartilage progenitor cells can be plated into tissue culture wells containing a hydrogel (flat or patterned) or thin gel coated plastic (flat or patterned) as a sparse culture (e.g., 24-well size plates with 1000-2000 cells per well), and cultured while changing the medium every 3 days. On the day of engraftment, NOD / SCID mice can be anesthetized by intraperitoneal injection of ketamine (2.4 MG / mouse) and xylazine (240 MG / mouse), and the hindlimbs irradiated as previously described (A. SACCO et al. (2008) NATURE 456, 502). The generated cartilage progenitor cells can be counted and resuspended, and subsequently injected into recipient mice.

[0240] Engraftment of the engrafted cartilage progenitor cells (e.g., differentiation and integration into local cartilage tissue) can be visualized by a variety of methods. For example, the cartilage progenitor cells can be engineered to express a heterologous marker (e.g., a fluorescent protein, such as green fluorescent protein) that is not present in the engrafted animal. Alternatively or additionally, the cartilage progenitor cells can be allogeneic to the animal, such that any cartilage differentiated from the cartilage progenitor cells after engraftment can be identified (e.g., immunostained) by an antibody that is not found in the engrafted animal.

[0241] Example 4: In vitro generation of chondrogenic cells​

[0242] To improve cell manufacturing, cells of interest can be engineered to exhibit increased expression of genes, such that their phenotype can be altered to a chondrogenic cell lineage. In some embodiments, improved cell manufacturing can be evidenced by shorter time to differentiate stem cells (e.g., pluripotent stem cells) to target cells (e.g., chondrogenic precursor cells or chondrogenic cells), increased number of target cells compared to other protocols, enhanced levels of cartilage formation (as evidenced by production of collagen, aggrecan, and / or ECM proteins), lower cost to produce similar numbers of target cells, and the like. Upon corec transfection of the cell algorithm into cells of interest, these genes will be expressed sequentially within the cells of interest. The cells of interest can be induced pluripotent stem cells. The engineered cells can exhibit increased presence of chondrogenic cell lineage markers as measured by RNA transcription, can exhibit enhanced DNA accessibility and / or function in chondrogenic cell lineage as indicated by enhanced function in a chondrogenesis assay that measures 1) collagen, aggrecan, and ECM protein production 2) chondron formation, where chondrons express desired levels of collagen, aggrecan, and ECM proteins.

[0243] For example, a library of cell algorithms constructed from sequences described in SEQ ID NOs: 1-1932 with core functional units (including but not limited to the cell algorithms labeled in Table 1) can be introduced into cells of interest. Table 1 shows 1932 unique cartilage constructs (e.g., there are 23 unique stem combinations. There are 21 unique cartilage spacers in the “4-pool”. There are 4 pools because for each gene, all 4 constructs targeting that gene are used together. Thus, there are 21*23 = 483 different combinations. If counting each individual construct, multiply by 4. There are 483*4 = 1932 unique cartilage constructs).

[0244] Example 5: In vivo validation of cell algorithm-derived chondrogenic cells

[0245] Cartilage generating cells produced using the cell algorithmic technology can be used as a regenerative therapy for individuals with cartilage functional defects, cartilage injury, or arthritic cartilage. Delivery of these cells into such individuals can provide pain relief, improved joint function, and improved longevity of function of the corresponding treated joint for the relevant joint. Once produced by the cell algorithmic driven instructions, these cartilage generating cells can be used in the same manner as cartilage generating cells produced by other methods such as isolation from cadaveric donors or differentiation of pluripotent stem cells using protocols that vary growth factors and other small molecules over a period of time (so-called directed differentiation). Regardless of the origin of the cartilage generating cells, they can be evaluated and used in vivo by injecting them into joints of animal models that have undergone acute cartilage injury, or that suffer from arthritis via genetic modeling or induction. The cartilage generating cells can be phenotypically characterized based on gene expression and cell surface marker signatures, and can be functionally characterized for their ability to produce cartilage. Both in vitro and in vivo characterization provide important insights into the quality of the cartilage generating cells.

[0246] To assess the functionality of the produced cartilage generating cells, they can be transplanted into osteochondral defects knees (e.g., full thickness, 1 mm in diameter) of athymic rats (e.g., N=6) or 6 month old Yucatan pigs. Following transplantation of the cartilage generating cells, histological testing can be performed to measure genes of interest (e.g., COL2A1, COL2A2, COL10, AGGN) and extracellular matrix proteins of interest. The cartilage generating cells should be transplanted to produce cartilage that expresses these proteins, without control by the vehicle, to provide healing to the defective cartilage, translating into functional joint outcomes.

[0247] Table 1: proGuide sequences in tandem

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417] Table 2: Sequences of spacer regions and target genes

[0418]

[0419]

[0420]

[0421] Table 3: Sequences of stem 1 and stem 2

[0422]

[0423]

[0424] Embodiments

[0425] The following non-limiting embodiments provide illustrative examples of the present application, but do not limit the scope of the present application.

[0426] 1. A method for converting a plurality of stem cells into a plurality of chondrogenic cells via modulating expression levels of a plurality of different target genes comprising a first homeobox protein and a second homeobox protein, the method comprising:

[0427] a) contacting a first polynucleotide sequence in the plurality of stem cells with a first heterologous gene regulatory moiety to modulate expression levels of the first homeobox protein operably coupled to the first polynucleotide sequence; and

[0428] b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to modulate expression levels of the second homeobox protein operably coupled to the second polynucleotide sequence.

[0429] 2. The method of embodiment 1, wherein (b) is performed after (a), and wherein the steps of (a) and (b) effect modulation of the first homeobox protein and the second homeobox protein in a sequential manner.

[0430] 3. The method of embodiment 1, wherein (i) the first polynucleotide sequence is upstream of or encodes the first homeobox protein, or (ii) the second polynucleotide sequence is upstream of or encodes the second homeobox protein.

[0431] 4. The method of embodiment 1, wherein (iii) the expression level of the first homeobox protein is enhanced upon the contacting by the first heterologous gene regulatory moiety or (iv) the expression level of the second homeobox protein is enhanced upon the contacting by the second heterologous gene regulatory moiety.

[0432] 5. The method of embodiment 1, further comprising modulating expression of an additional target gene in the plurality of target genes, wherein the additional target gene comprises one or more members selected from T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), Sox, and collagens.

[0433] 6. The method of embodiment 5, wherein the expression level of the additional target gene is modulated (i) prior to, (ii) concurrently with, or (iii) subsequent to the second homeobox protein.

[0434] 7. The method of embodiment 5, wherein the expression level of the additional target gene is enhanced.

[0435] 8. The method of embodiment 1, wherein the method comprises contacting the plurality of stem cells with a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to modulate expression levels of the plurality of different target genes in a sequential manner to achieve the transformation, and wherein the plurality of gate units comprises:

[0436] (i) a first gate unit preconfigured to implement the first heterologous gene regulatory moiety to modulate the expression level of the first homeobox protein; and

[0437] (ii) a second gate unit preconfigured to implement the second heterologous gene regulatory moiety to modulate the expression level of the second homeobox protein,

[0438] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to implement the transformation.

[0439] 9. The method of embodiment 8, wherein (i) the first gate unit is activatable to express the first heterologous gene regulatory moiety or (ii) the second gate unit is activatable to express the second heterologous gene regulatory moiety.

[0440] 10. The method of embodiment 9, wherein the plurality of gate units comprises an additional gate unit preconfigured to modulate an expression level of an additional target gene of the plurality of different target genes, wherein the additional target gene encodes one or more members selected from a T-box transcription factor (TBX), a basic helix-loop-helix transcription factor (bHLH), a Sox, and a collagen.

[0441] 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.

[0442] 12. A method for converting a plurality of stem cells into a plurality of chondrogenic cells via modulation of expression levels of a plurality of different target genes comprising a first different target gene and a second different target gene, the method comprising:

[0443] a) contacting a first polynucleotide sequence in the plurality of stem cells with a first heterologous gene regulatory moiety to modulate an expression level of the first different target gene operably coupled to the first polynucleotide sequence; and

[0444] b) contacting a second polynucleotide sequence in the plurality of stem cells with a second heterologous gene regulatory moiety to modulate an expression level of the second different target gene operably coupled to the second polynucleotide sequence,

[0445] wherein the combination of the first and second different target genes is:

[0446] i) a first homeobox protein and a second homeobox protein that are different;

[0447] ii) two different members selected from 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 homeobox protein, the TBX, and the bHLH, and a second member comprising a SOX or a collagen.

[0448] 13. The method of embodiment 12, wherein the combination is (i) the first homeobox protein and the second homeobox protein.

[0449] 14. The method of embodiment 12, wherein the combination is (ii) the two different members selected from the homeobox protein, the TBX, and the bHLH.

[0450] 15. The method of embodiment 12, wherein the combination is (iii) the first member selected from the homeobox protein, the TBX, and the bHLH, and the second member comprising the SOX or the collagen.

[0451] 16. The method of embodiment 12, wherein (b) is performed after (a) to achieve modulation of the first different target gene and the second different target gene in a sequential manner.

[0452] 17. The method of embodiment 12, wherein (i) the first polynucleotide sequence is upstream of or encodes the first different target gene or (ii) the second polynucleotide sequence is upstream of or encodes the second different target gene.

[0453] 18. The method of embodiment 12, wherein (ii) the expression level of the first different target gene is enhanced upon the contacting by the first heterologous gene regulatory moiety or (ii) the expression level of the second different target gene is enhanced upon the contacting by the second heterologous gene regulatory moiety.

[0454] 19. The method of embodiment 12, wherein the method comprises contacting the plurality of stem cells with a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to modulate expression levels of the plurality of different target genes in a sequential manner to achieve the conversion, and wherein the plurality of gate units comprises:

[0455] (i) a first gate unit preconfigured to achieve the first heterologous gene regulatory moiety to modulate the expression level of the first different target gene; and

[0456] (ii) a second gate unit preconfigured to achieve the second heterologous gene regulatory moiety to modulate the expression level of the second different target gene,

[0457] wherein upon activation of the heterologous gene circuit, the plurality of gate units operates to achieve the conversion.

[0458] 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.

[0459] 21. A method for converting a plurality of stem cells to chondrogenic differentiation, the method comprising:

[0460] contacting a polynucleotide sequence in the plurality of stem cells with a heterologous gene regulatory moiety to modulate an expression level of a target gene operably coupled to the polynucleotide sequence,

[0461] wherein a conversion rate from the plurality of stem cells to chondrogenic cells is characterized as at least about 30% within less than 7 days after the contacting.

[0462] 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%.

[0463] 23. The method of embodiment 21, wherein the conversion rate is observed within less than about 6 days after the contacting or within less than about 5 days after the contacting.

[0464] 24. The method of embodiment 21, wherein the expression level of the target gene is enhanced upon the contacting by the heterologous gene regulatory moiety.

[0465] 25. The method of embodiment 21, wherein the target gene comprises one or more members selected from homeobox proteins, T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), SOX, and collagens.

[0466] 26. The method of embodiment 21, wherein the target gene comprises a plurality of different target genes, the plurality of different target genes comprising a first different target gene and a second different target gene, and wherein the contacting comprises:

[0467] (a) contacting a first polynucleotide sequence in the plurality of stem cells by a first heterologous gene regulatory moiety to modulate an expression level of the first different target gene operably coupled to the first polynucleotide sequence; and

[0468] (b) contacting a second polynucleotide sequence in the plurality of stem cells by a second heterologous gene regulatory moiety to modulate an expression level of the second different target gene operably coupled to the second polynucleotide sequence.

[0469] 27. The method of embodiment 26, wherein the combination of the first and second different target genes is:

[0470] (i) a first homeobox protein and a second homeobox protein that are different;

[0471] (ii) two different members selected from homeobox proteins, T-box transcription factors (TBX), and basic helix-loop-helix transcription factors (bHLH); or

[0472] (iii) a first member selected from the homeobox proteins, the TBX, and the bHLH, and a second member comprising SOX or collagens.

[0473] 28. The method of embodiment 26, wherein the steps of (a) and (b) effect modulation of the first different target gene and the second different target gene in a sequential manner.

[0474] 29. The method of embodiment 26, wherein the contacting comprises:

[0475] contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gate units, wherein the heterologous genetic circuit is activatable to induce the plurality of gate units to modulate expression levels of the plurality of different target genes in a sequential manner to effect the conversion, and wherein the plurality of gate units comprises:

[0476] (i) a first gate unit preconfigured to effect the first heterologous genetic regulatory moiety to modulate the expression level of the first different target gene; and

[0477] (ii) a second gate unit preconfigured to effect the second heterologous genetic regulatory moiety to modulate the expression level of the second different target gene,

[0478] wherein upon activation of the heterologous genetic circuit, the plurality of gate units operate to effect the conversion.

[0479] 30. The method of embodiment 21, wherein the target gene is an endogenous target gene.

[0480] 31. A method for treating a subject in need thereof, the method comprising:

[0481] 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,

[0482] wherein a conversion rate from the plurality of stem cells to the plurality of chondrogenic cells is characterized as at least about 30% within less than 7 days after the ex vivo differentiation.

[0483] 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%.

[0484] 33. The method of embodiment 31, wherein the conversion rate is observed within less than about 6 days after the contacting or within less than about 5 days after the contacting.

[0485] 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.

[0486] 35. The method of embodiment 31, wherein the differentiating ex vivo comprises modulating expression levels of a target gene comprising one or more members selected from a homeobox protein, a T-box transcription factor (TBX), a basic helix-loop-helix transcription factor (bHLH), a SOX, and a collagen.

[0487] 36. The method of embodiment 35, wherein the target gene is an endogenous target gene.

[0488] 37. The method of embodiment 35, wherein the target gene comprises a plurality of different target genes comprising:

[0489] (i) different first and second homeobox proteins;

[0490] (ii) two different members selected from a homeobox protein, a T-box transcription factor (TBX), and a basic helix-loop-helix transcription factor (bHLH); or

[0491] (iii) a first member selected from the homeobox protein, the TBX, and the bHLH, and a second member comprising a SOX or a collagen.

[0492] 38. The method of any of the preceding embodiments, wherein the plurality of chondrogenic cells comprises a chondrocyte progenitor cell or a chondrocyte cell.

[0493] 39. The method of any of the preceding embodiments, wherein the plurality of chondrogenic cells are characterized as CD146+ / CD73+, CD146+ / CD112+, or CD326+ / CD309+.

[0494] 40. The method of any of the preceding embodiments, wherein the plurality of stem cells comprises a pluripotent stem cell (PSC) or a mesodermal cell.

[0495] 41. The method of any of the preceding embodiments, wherein the plurality of different target genes are endogenous genes of the plurality of stem cells.

[0496] 42. The method of any of the preceding embodiments, wherein the first or second heterologous gene regulatory moiety comprises (i) an endonuclease or (ii) a guide nucleic acid (gNA) molecule.

[0497] 43. The method of embodiment 42, wherein the endonuclease and the gNA form a complex capable of binding to a corresponding target polynucleotide sequence.

[0498] 44. The method of embodiment 42, wherein the endonuclease is a Cas protein.

[0499] 45. The method of any of the preceding embodiments, wherein the conversion occurs in the substantial absence of (i) serum and / or (ii) an exogenous cellular differentiation regulatory factor.

[0500] 46. The method of embodiment 45, wherein the exogenous cellular differentiation regulatory factor comprises one or more members selected from the group consisting of TGFpi, TGFp2, TGFp3, BMP2, BMP4, BMP6, MP7, and IGF1.

[0501] 47. The method of embodiment 45, wherein the exogenous cellular differentiation regulatory factor is a chondrogenic factor comprising one or more members selected from the group consisting of dexamethasone, ascorbate, insulin, transferrin, and selenite.

[0502] 48. The method of any 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.

[0503] 49. The method of embodiment 48, wherein the PRD-class homeobox protein is MIXLI or UNCX.

[0504] 50. The method of any 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.

[0505] 51. The method of any of the preceding embodiments, wherein the TBX comprises one or more members selected from the group consisting of TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, and TBX22.

[0506] 52. The method of embodiment 51, wherein the TBX is TBXT or TBX6.

[0507] 53. The method of any 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.

[0508] 54. The method of embodiment 53, wherein the bHLH is the Group A bHLH.

[0509] 55. The method of embodiment 53, wherein the bHLH is MSGN1 or TCF15.

[0510] 56. The method of any one of the preceding embodiments, wherein the SOX comprises one or more members selected from SOX A, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH.

[0511] 57. The method of embodiment 56, wherein the SOX is SOXD.

[0512] 58. The method of embodiment 57, wherein the SOXD is SOX6.

[0513] 59. The method of embodiment 56, wherein the SOX is SOXE.

[0514] 60. The method of embodiment 59, wherein the SOXE is SOX9.

[0515] 61. The method of any one of the preceding embodiments, wherein the collagen comprises one or more members selected from collagen type I, collagen type II, collagen type III, collagen type IV, and collagen type V.

[0516] 62. The method of embodiment 61, wherein the collagen is collagen type II.

[0517] 63. The method of embodiment 62, wherein the collagen type II is COL2A1.

[0518] 64. The method 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.

[0519] 65. The method of any one of the preceding embodiments, further comprising storing the plurality of tissue-specific progenitor cells or the plurality of chondrogenic cells in a sterile vial.

[0520] 66. The method of any one of the preceding embodiments, further comprising administering the plurality of tissue-specific progenitor cells or the plurality of chondrogenic cells to a subject in need thereof.

[0521] 67. The method of any one of the preceding embodiments, wherein the administering comprises one or more knee administrations.

[0522] 68. A system for converting a plurality of stem cells into a plurality of chondrogenic cells via modulating expression levels of a plurality of different target genes comprising a first homeobox protein and a second homeobox protein, the system comprising:

[0523] a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in the plurality of stem cells to modulate an expression level of the first homeobox protein operably coupled to the first polynucleotide sequence; and

[0524] b) a second heterologous gene regulatory moiety configured to bind to a second polynucleotide sequence in the plurality of stem cells to modulate an expression level of the second homeobox protein operably coupled to the second polynucleotide sequence.

[0525] 69. The system of embodiment 68, wherein the first and second heterologous gene regulatory moieties are configured to effect modulation of the first homeobox protein and the second homeobox protein in a sequential manner.

[0526] 70. The system of embodiment 68, wherein (i) the first polynucleotide sequence is upstream of or encodes the first homeobox protein or (ii) the second polynucleotide sequence is upstream of or encodes the second homeobox protein.

[0527] 71. The system of embodiment 68, wherein (ii) the expression level of the first homeobox protein is enhanced upon the contacting by the first heterologous gene regulatory moiety or (ii) the expression level of the second homeobox protein is enhanced upon the contacting by the second heterologous gene regulatory moiety.

[0528] 72. The system of embodiment 68, further comprising an additional heterologous gene regulatory moiety configured to modulate expression of an additional target gene in the plurality of target genes, wherein the additional target gene comprises one or more members selected from T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), Sox, and collagens.

[0529] 73. The system of embodiment 72, wherein the expression level of the additional target gene is modulated (i) prior to, (ii) concurrently with, or (iii) subsequent to the second homeobox protein.

[0530] 74. The system of embodiment 72, wherein the expression level of the additional target gene is enhanced.

[0531] 75. The system of embodiment 68, comprising a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to modulate expression levels of the plurality of different target genes in a sequential manner to effect the transformation, and wherein the plurality of gate units comprises:

[0532] (i) a first gate unit preconfigured to implement the first heterologous gene regulatory moiety to modulate the expression level of the first homeobox protein; and

[0533] (ii) a second gate unit preconfigured to implement the second heterologous gene regulatory moiety to modulate the expression level of the second homeobox protein,

[0534] wherein upon activation of the heterologous gene circuit, the plurality of gate units operate to implement the conversion.

[0535] 76. The system of embodiment 75, wherein (i) the first gate unit is activatable to express the first heterologous gene regulatory moiety or (ii) the second gate unit is activatable to express the second heterologous gene regulatory moiety.

[0536] 77. The system of embodiment 75, wherein the plurality of gate units comprises a further gate unit preconfigured to modulate an expression level of a further target gene of the plurality of different target genes, wherein the further target gene encodes one or more members selected from T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), Sox, and collagens.

[0537] 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.

[0538] 79. A system for converting a plurality of stem cells into a plurality of chondrogenic cells via modulation of expression levels of a plurality of different target genes comprising a first different target gene and a second different target gene, the system comprising:

[0539] a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in the plurality of stem cells to modulate an expression level of the first different target gene operably coupled to the first polynucleotide sequence; and

[0540] b) a second heterologous gene regulatory moiety configured for binding to a second polynucleotide sequence in the plurality of stem cells to modulate an expression level of the second different target gene operably coupled to the second polynucleotide sequence,

[0541] wherein the combination of the first and second different target genes is:

[0542] (i) a first and a second different homeobox protein;

[0543] (ii) two different members selected from homeobox proteins, T-box transcription factors (TBX), and basic helix-loop-helix transcription factors (bHLH); or

[0544] (iii) a first member selected from the homeobox protein, the TBX, and the bHLH, and a second member comprising the SOX or the collagen.

[0545] 80. The system of embodiment 79, wherein the combination is (i) the first homeobox protein and the second homeobox protein.

[0546] 81. The system of embodiment 79, wherein the combination is (ii) two different members selected from the homeobox protein, the TBX, and the bHLH.

[0547] 82. The system of embodiment 79, wherein the combination is (iii) a first member selected from the homeobox protein, the TBX, and the bHLH, and a second member comprising the SOX or the collagen.

[0548] 83. The system of embodiment 79, wherein the first and second heterologous genetic regulatory moieties are configured to effectuate the regulation of the first different target gene prior to the regulation of the second different target gene in a sequential manner.

[0549] 84. The system of embodiment 79, wherein (i) the first polynucleotide sequence is upstream of or encodes the first different target gene or (ii) the second polynucleotide sequence is upstream of or encodes the second different target gene.

[0550] 85. The system of embodiment 79, wherein (ii) the expression level of the first different target gene is enhanced upon the contacting by the first heterologous genetic regulatory moiety or (ii) the expression level of the second different target gene is enhanced upon the contacting by the second heterologous genetic regulatory moiety.

[0551] 86. The system of embodiment 79, comprising a heterologous genetic circuit comprising a plurality of gate units, wherein the heterologous genetic circuit is activatable to induce the plurality of gate units to regulate expression levels of the plurality of different target genes in a sequential manner to effectuate the transformation, and wherein the plurality of gate units comprises:

[0552] (i) a first gate unit preconfigured to effectuate the first heterologous genetic regulatory moiety to regulate the expression level of the first different target gene; and

[0553] (ii) a second gate unit preconfigured to effectuate the second heterologous genetic regulatory moiety to regulate the expression level of the second different target gene,

[0554] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion.

[0555] 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.

[0556] 88. A system for converting a plurality of stem cells to chondrogenic differentiation, the system comprising:

[0557] a heterologous gene regulatory moiety configured to bind to a polynucleotide sequence in the plurality of stem cells to modulate an expression level of a target gene operably coupled to the polynucleotide sequence,

[0558] wherein, within less than 7 days after the contacting, a conversion rate from the plurality of stem cells to chondrogenic cells is characterized as at least about 30%.

[0559] 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%.

[0560] 90. The system of embodiment 88, wherein the conversion rate is observed within less than about 6 days after the contacting or within less than about 5 days after the contacting.

[0561] 91. The system of embodiment 88, wherein the expression level of the target gene is enhanced upon the contacting by the heterologous gene regulatory moiety.

[0562] 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 collagens.

[0563] 93. The system of embodiment 88, wherein the target gene comprises a plurality of different target genes, the plurality of different target genes comprising a first different target gene and a second different target gene, and wherein the system comprises:

[0564] a) a first heterologous gene regulatory moiety configured to bind to a first polynucleotide sequence in the plurality of stem cells to modulate an expression level of the first different target gene operably coupled to the first polynucleotide sequence; and

[0565] b) a second heterologous gene regulatory moiety configured to bind to a second polynucleotide sequence in the plurality of stem cells to modulate an expression level of the second different target gene operably coupled to the second polynucleotide sequence.

[0566] 94. The system of embodiment 93, wherein the combination of the first and second different target genes is:

[0567] (i) a different first homeobox protein and a second homeobox protein;

[0568] (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

[0569] (iii) a first member selected from the group consisting of the homeobox protein, the TBX, and the bHLH, and a second member comprising SOX or a collagen.

[0570] 95. The system of embodiment 93, wherein the first and second heterologous gene regulatory moieties are configured to effect regulation of the first different target gene and the second different target gene in a sequential manner.

[0571] 96. The system of embodiment 93, comprising a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to regulate expression levels of the plurality of different target genes in a sequential manner to effect the transformation, and wherein the plurality of gate units comprises:

[0572] (i) a first gate unit preconfigured to effect the first heterologous gene regulatory moiety to regulate the expression level of the first different target gene; and

[0573] (ii) a second gate unit preconfigured to effect the second heterologous gene regulatory moiety to regulate the expression level of the second different target gene,

[0574] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the transformation.

[0575] 97. The system of embodiment 88, wherein the target gene is an endogenous target gene.

[0576] 98. A composition comprising any of the preceding embodiments.

[0577] 99. A composition for use in treating a subject in need thereof, the composition comprising:

[0578] a plurality of chondrogenic cells prepared by differentiating a plurality of stem cells ex vivo,

[0579] wherein a transformation rate from the plurality of stem cells to the plurality of chondrogenic cells is characterized as at least about 30% within less than 7 days after the ex vivo differentiation.

[0580] 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%.

[0581] 101. The composition of embodiment 99, wherein the conversion rate is observed within less than about 6 days after the contacting or within less than about 5 days after the contacting.

[0582] 102. The composition of embodiment 99, wherein the plurality of chondrogenic cells are cultured ex vivo 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.

[0583] 103. The composition of embodiment 99, wherein the ex vivo differentiation is characterized by modulating expression levels of a target gene comprising one or more members selected from a homeobox protein, a T-box transcription factor (TBX), a basic helix-loop-helix transcription factor (bHLH), a SOX, and a collagen.

[0584] 104. The composition of embodiment 99, wherein the target gene is an endogenous target gene.

[0585] 105. The composition of embodiment 99, wherein the target gene comprises a plurality of different target genes comprising:

[0586] (i) a different first homeobox protein and a second homeobox protein;

[0587] (ii) two different members selected from a homeobox protein, a T-box transcription factor (TBX), and a basic helix-loop-helix transcription factor (bHLH); or

[0588] (iii) a first member selected from the homeobox protein, the TBX, and the bHLH, and a second member comprising a SOX or a collagen.

[0589] 106. The system or composition of any of the preceding embodiments, wherein the plurality of chondrogenic cells comprises a chondrocyte progenitor cell or a chondrocyte cell.

[0590] 107. The system or composition of any of the preceding embodiments, wherein the plurality of chondrogenic cells are characterized as CD146+ / CD73+, CD146+ / CD112+, or CD326+ / CD309+.

[0591] 108. The system or composition of any of the preceding embodiments, wherein the plurality of stem cells comprises a pluripotent stem cell (PSC) or a mesodermal cell.

[0592] 109. The system or composition of any of the preceding embodiments, wherein the plurality of different target genes are endogenous genes of the plurality of stem cells.

[0593] 110. The system or composition of any of the preceding embodiments, wherein the first heterologous gene regulatory moiety or the second heterologous gene regulatory moiety comprises (i) an endonuclease or (ii) a guide nucleic acid (gNA) molecule.

[0594] 111. The system or composition of any of the preceding embodiments, wherein the endonuclease and the gNA form a complex capable of binding to a corresponding target polynucleotide sequence.112. The system or composition of any of the preceding embodiments, wherein the endonuclease is a Cas protein.

[0595] 113. The system or composition of any of the preceding embodiments, wherein the transforming occurs in the substantial absence of (i) serum and / or (ii) an exogenous cell differentiation regulatory factor.

[0596] 114. The system or composition of any of the preceding embodiments, wherein the exogenous cell differentiation regulatory factor comprises one or more members selected from TGFpi, TGFp2, TGFp3, BMP2, BMP4, BMP6, MP7, and IGF1.

[0597] 115. The system or composition of any of the preceding embodiments, wherein the exogenous cell differentiation regulatory factor is a chondrogenic factor comprising one or more members selected from dexamethasone, ascorbate, insulin, transferrin, and selenous acid.

[0598] 116. The system or composition of any of the preceding embodiments, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is a paired- like homeobox protein.

[0599] 117. The system or composition of any of the preceding embodiments, wherein the paired- like homeobox protein is MIXLI or UNCX.

[0600] 118. The system or composition of any 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.

[0601] 119. The system or composition of any of the preceding embodiments, wherein the TBX comprises one or more members selected from TBXT, TBR1, TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, and TBX22.

[0602] 120. The system or composition of any of the preceding embodiments, wherein the TBX is TBXT or TBX6.

[0603] 121. The system or composition of any of the preceding embodiments, wherein the bHLH comprises one or more members selected from Group A bHLH, Group B bHLH, Group C bHLH, Group D bHLH, Group E bHLH, and Group F bHLH.

[0604] 122. The system or composition of any of the preceding embodiments, wherein the bHLH is the Group A bHLH.

[0605] 123. The system or composition of any of the preceding embodiments, wherein the bHLH is MSGN1 or TCF15.

[0606] 124. The system or composition of any of the preceding embodiments, wherein the SOX comprises one or more members selected from SOXA, SOXB1, SOXB2, SOXC, SOXD, SOXE, SOXF, SOXG, and SOXH.

[0607] 125. The system or composition of any of the preceding embodiments, wherein the SOX is SOXD.

[0608] 126. The system or composition of any of the preceding embodiments, wherein the SOXD is SOX6.

[0609] 127. The system or composition of any of the preceding embodiments, wherein the SOX is SOXE.

[0610] 128. The system or composition of any of the preceding embodiments, wherein the SOXE is SOX9.

[0611] 129. The system or composition of any of the preceding embodiments, wherein the collagen comprises one or more members selected from Type I collagen, Type II collagen, Type III collagen, Type IV collagen, and Type V collagen.

[0612] 130. The system or composition according to any one of the foregoing embodiments, wherein the collagen is type II collagen.

[0613] 131. The system or composition according to any one of the foregoing embodiments, wherein the type II collagen is COL2A1.

[0614] 132. The system or composition according to any one of the foregoing embodiments, wherein the conversion occurs in less than about 10 days, less than about 7 days, or less than about 5 days.

[0615] The systems and methods disclosed herein may be combined with or modified by other systems and methods for cell programming, such as those described in International Patent Application Nos. PCT / US2018 / 052211, PCT / US2018 / 052211, PCT / US2023 / 028169, PCT / US2023 / 028255 and PCT / US2023 / 028033, each of which is incorporated herein by reference in its entirety.

[0616] Although preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The invention is not intended to be limited to the specific embodiments provided herein. While the invention has been described with reference to the foregoing description, the description and illustration of embodiments herein are not intended to be construed as limiting. Various modifications, alterations, and substitutions will now be conceived by those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, and depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. Therefore, it is contemplated that the invention should also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for converting multiple stem cells into multiple chondrogenic cells by regulating the expression levels of multiple different target genes, including a first homeobox protein and a second homeobox protein, the method comprising: a) The first heterologous gene regulatory portion contacts a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of the first homeobox protein operatively coupled to the first polynucleotide sequence; and b) The second heterologous gene regulatory portion contacts the second polynucleotide sequence in the plurality of stem cells to regulate the expression level of the second homeobox protein operatively coupled to the second polynucleotide sequence.

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

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

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

5. The method of claim 1, further comprising regulating the expression of an additional target gene among the plurality of target genes, wherein the additional target gene comprises one or more members selected from T-box transcription factor (TBX), basic helical-loop-helical transcription factor (bHLH), Sox, and collagen.

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

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

8. The method of claim 1, wherein the method comprises contacting the plurality of stem cells with a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activating to induce the plurality of gate units to sequentially regulate the expression levels of the plurality of different target genes to achieve the transformation, and wherein the plurality of gate units comprises: (i) A first gate unit, which is pre-configured to implement the first heterogene regulatory portion to regulate the expression level of the first homeobox protein; as well as (ii) A second gate unit, pre-configured to implement the second heterogeneous gene regulatory portion to regulate the expression level of the second homeobox protein. When the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

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

10. The method of claim 9, wherein the plurality of gate units includes an additional gate unit pre-configured to regulate the expression level of an additional target gene among the plurality of different target genes, wherein the additional target gene encodes one or more members selected from T-box transcription factor (TBX), basic helical-loop-helical transcription factor (bHLH), Sox, and collagen.

11. The method of claim 1, wherein the conversion occurs within 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 by regulating the expression levels of a plurality of different target genes, including a first different target gene and a second different target gene, the method comprising: a) The first heterologous gene regulatory portion contacts a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of the first different target gene operatively coupled to the first polynucleotide sequence; and b) The second heterologous gene regulatory portion contacts the second polynucleotide sequence in the plurality of stem cells to regulate the expression level of the second different target gene operatively coupled to the second polynucleotide sequence. The combination of the first and second different target genes is: i) Different first and second homeobox proteins; ii) Selected from two different members of homeobox proteins, T-box transcription factor (TBX), and basic helical-loop-helical transcription factor (bHLH); iii) A first member selected from the said homologous box protein, the said TBX and the said bHLH, and a second member containing SOX or collagen.

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

14. The method of claim 12, wherein the combination is (ii) two different members selected from the homologous box protein, the TBX and the bHLH.

15. The method of claim 12, wherein the combination is (iii) a first member selected from the homologous box protein, the TBX and the bHLH, and a second member comprising the SOX or the collagen.

16. The method of claim 12, wherein (b) is performed after (a) to achieve the regulation of the first different target gene and the second different target gene in a sequential manner.

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

18. The method of claim 12, wherein (ii) upon contact with the first heterologous gene-regulated portion, the expression level of the first different target gene is enhanced, or (ii) upon contact with the second heterologous gene-regulated portion, the expression level of the second different target gene is enhanced.

19. The method of claim 12, wherein the method comprises contacting the plurality of stem cells with a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to sequentially regulate the expression levels of the plurality of different target genes to achieve the transformation, and wherein the plurality of gate units comprises: (i) A first gate unit, which is pre-configured to implement the first heterogene regulatory portion to regulate the expression level of the first different target gene; as well as (ii) A second gate unit, pre-configured to implement the second heterologous gene regulatory portion to regulate the expression level of the second different target gene. When the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

20. 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. A method for differentiating multiple stem cells into cartilage, the method comprising: The expression level of a target gene operatively coupled to the plurality of stem cells is regulated by contacting a polynucleotide sequence via a heterologous gene regulatory component to modulate the expression level of the target gene operatively coupled to the polynucleotide sequence, wherein the conversion rate from the plurality of stem cells to chondrogenic cells is characterized as at least about 30% within 7 days after the contact.

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. The method of claim 21, wherein the conversion rate is observed within about 6 days after the contact or within about 5 days after the contact.

24. The method of claim 21, wherein the expression level of the target gene is enhanced after contact with the portion regulated by the heterologous gene.

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

26. The method of claim 21, wherein the target gene comprises a plurality of different target genes, the plurality of different target genes comprising a first different target gene and a second different target gene, and wherein the contact comprises: (a) The first heterogeneous gene regulatory portion contacts the first polynucleotide sequence in the plurality of stem cells to regulate the expression level of the first different target gene operatively coupled to the first polynucleotide sequence. as well as (b) The second heterologous gene regulatory portion contacts the second polynucleotide sequence in the plurality of stem cells to regulate the expression level of the second different target gene operatively coupled to the second polynucleotide sequence.

27. The method of claim 26, wherein the combination of the first and second different target genes is: (i) Different first and second homeobox proteins; (ii) Selected from two different members of the homeobox protein, T-box transcription factor (TBX), and basic helix-loop-helix transcription factor (bHLH); or (iii) A first member selected from the homologous box protein, the TBX and the bHLH, and a second member containing SOX or collagen.

28. The method of claim 26, wherein steps (a) and (b) are performed sequentially to regulate the first different target gene and the second different target gene.

29. The method of claim 26, wherein the contact comprises: The plurality of stem cells are contacted with a heterologous gene circuit comprising multiple gate units, wherein the heterologous gene circuit is activating to induce the multiple gate units to sequentially regulate the expression levels of multiple different target genes to achieve the transformation, and wherein the multiple gate units comprise: (i) a first gate unit pre-configured to implement the first heterologous gene regulatory portion to regulate the expression level of the first different target gene; and (ii) A second gate unit, pre-configured to implement the second heterologous gene regulatory portion to regulate the expression level of the second different target gene. When the heterologous gene circuit is activated, the plurality of gate units operate to achieve the transformation.

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

31. A method for treating a person in need, the method comprising: Multiple chondrogenic cells are applied to the object, wherein the multiple chondrogenic cells are prepared by in vitro differentiation of multiple stem cells. Specifically, within 7 days of the in vitro differentiation, the conversion rate from the plurality of stem cells to the plurality of chondrogenic cells was characterized as at least about 30%.

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. The method of claim 31, wherein the conversion rate is observed within about 6 days after the contact or within about 5 days after the contact.

34. The method of claim 31, wherein the plurality of chondrogenic cells are cultured in vitro 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 claim 31, wherein the in vitro differentiation comprises regulating the expression level of a target gene, said target gene comprising one or more members selected from homeobox proteins, T-box transcription factors (TBX), basic helical-loop-helical transcription factors (bHLH), SOX, and collagen.

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

37. The method of claim 35, wherein the target gene comprises a plurality of different target genes, the plurality of different target genes comprising: (i) Different first and second homeobox proteins; (ii) Selected from two different members of the homeobox protein, T-box transcription factor (TBX), and basic helix-loop-helix transcription factor (bHLH); or (iii) A first member selected from the homologous box protein, the TBX and the bHLH, and a second member containing SOX or collagen.

38. The method according to any one of the preceding claims, wherein the plurality of chondrogenic cells comprise chondrogenic progenitor cells or chondrocytes.

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

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

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

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

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

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

45. The method according to any one of the preceding claims, wherein the transformation occurs under conditions substantially free of (i) serum and / or (ii) exogenous cell differentiation regulators.

46. ​​The method of claim 45, wherein the exogenous cell differentiation regulator comprises one or more members selected from TGFβ1, TGFβ2, TGFβ3, BMP2, BMP4, BMP6, MP7 and IGF1.

47. The method of claim 45, wherein the exogenous cell differentiation regulator is a chondrogenic factor, the chondrogenic factor comprising one or more members selected from dexamethasone, ascorbate, insulin, transferrin, and selenite.

48. The method according to any one of the preceding claims, wherein the first homeobox protein, the second homeobox protein, or the homeobox protein is a pairing box (PRD) type homeobox protein.

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

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

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

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

53. The method according to any one of the preceding claims, wherein the bHLH comprises one or more members selected from group A bHLH, group B bHLH, group C bHLH, group D bHLH, group E bHLH and group F bHLH.

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

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

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

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

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

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

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

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

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

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

64. The method according to any one of the preceding claims, wherein the conversion occurs in less than about 10 days, less than about 7 days, or less than about 5 days.

65. The method according to any one of the preceding claims further comprises storing the plurality of tissue-specific progenitor cells or the plurality of chondrogenic cells in sterile vials.

66. The method according to any one of the preceding claims further comprises administering the plurality of tissue-specific progenitor cells or the plurality of chondrogenic cells to a subject in need.

67. The method according to any one of the preceding claims, wherein the application comprises one or more knee applications.

68. A system for converting multiple stem cells into multiple chondrogenic cells by regulating the expression levels of multiple different target genes, including a first homeobox protein and a second homeobox protein, the system comprising: a) A first heterologous gene regulatory portion, configured to bind a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of the first homeobox protein operatively coupled to the first polynucleotide sequence; as well as b) A second heterologous gene regulatory portion configured to bind a second polynucleotide sequence in the plurality of stem cells to regulate the expression level of a second homeobox protein operatively coupled to the second polynucleotide sequence.

69. A system for converting a plurality of stem cells into a plurality of chondrogenic cells by regulating the expression levels of a plurality of different target genes, including a first different target gene and a second different target gene, said system comprising: a) A first heterologous gene regulatory portion, configured to bind a first polynucleotide sequence in the plurality of stem cells to regulate the expression level of the first different target gene operatively coupled to the first polynucleotide sequence. as well as b) A second heterologous gene regulatory portion, configured to bind a second polynucleotide sequence in the plurality of stem cells to regulate the expression level of a second distinct target gene operatively coupled to the second polynucleotide sequence. The combination of the first and second different target genes is: (i) Different first and second homeobox proteins; (ii) Selected from two different members of the homeobox protein, T-box transcription factor (TBX), and basic helix-loop-helix transcription factor (bHLH); or (iii) A first member selected from the homologous box protein, the TBX and the bHLH, and a second member containing SOX or collagen.

70. A system for differentiating multiple stem cells into cartilage, the system comprising: The heterologous gene regulation component is configured to bind to polynucleotide sequences in the plurality of stem cells to regulate the expression levels of target genes operatively coupled to the polynucleotide sequences. Specifically, within 7 days after the contact, the conversion rate from the plurality of stem cells to chondrogenic cells was characterized as at least about 30%.

71. A composition for treating a subject in need, said composition comprising: Multiple chondrogenic cells were prepared by differentiating multiple stem cells in vitro. in, Within 7 days of the in vitro differentiation, the conversion rate from the plurality of stem cells to the plurality of chondrogenic cells was characterized as at least about 30%.

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