Systems and methods for stem cell programming

Abstract

Provided herein are systems that modulate gene expression and methods of use thereof for transforming one type of cells to another (e.g., reprogramming differentiated cells into stem cells).

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 390,474, filed Jul. 19, 2022, which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Heterologous proteins and / or nucleic acid molecules can be used to elicit desired responses in cells. Heterologous proteins and / or nucleic acid molecules can regulate genes of interest (e.g., transgenes and / or endogenous genes) to program cells (e.g., stem cells) (e.g., differentiate, dedifferentiate). In some cases, nuclease-based techniques (e.g., clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins or "CRISPR / Cas") have been employed to manipulate polynucleotide sequences, their epigenetic modifications, and / or their expression levels. For example, CRISPR / Cas technology can be characterized by its versatility and ease of programming and can be used to facilitate genome editing across different species. SUMMARY OF THE INVENTION

[0004] The present disclosure provides methods and systems for regulating the expression or activity of a target gene. Some aspects of the present disclosure provide methods and systems for differentiating and dedifferentiating terminally differentiated cells. Some aspects of the present disclosure provide methods and systems for differentiating and dedifferentiating stem cells.

[0005] In one aspect, the present disclosure provides a method for converting a plurality of cells of a first cell type (the first plurality of cells) into a plurality of cells of a second cell type (the second plurality of cells), the method comprising: contacting the first plurality of cells with a heterologous gene regulator that specifically binds to a gene encoding a HERV to regulate the expression level or epigenetic profile of the HERV and effect the conversion from the first plurality of cells to the second plurality of cells.

[0006] In another aspect, the present disclosure provides a system for converting a plurality of cells of a first cell type (the first plurality of cells) into a plurality of cells of a second cell type (the second plurality of cells), the system comprising: a heterologous gene regulator that specifically binds to a gene encoding a HERV to regulate the expression level or epigenetic profile of the HERV and effect the conversion from the first plurality of cells to the second plurality of cells.

[0007] In another aspect, the present disclosure provides a method for converting a plurality of differentiated cells into a plurality of stem cells, the method comprising: contacting the plurality of differentiated 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 or epigenetic profiles of a plurality of different target endogenous genes to effect the conversion, and wherein the plurality of gate units comprise: (i) a first gate unit preconfigured to regulate the expression level or epigenetic profile of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene comprises an Embryonic Genome Activation (EGA)-enriched Alu motif (EEA); and (ii) a second gate unit preconfigured to regulate the expression level or epigenetic profile of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene comprises a cellular dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion.

[0008] In another aspect, the present disclosure provides a system for converting a plurality of differentiated cells into a plurality of stem cells, the system comprising: a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to sequentially regulate the expression levels or epigenetic profiles of a plurality of different target endogenous genes to effect the conversion, wherein the plurality of gate units comprise: (i) a first gate unit preconfigured to regulate the expression level or epigenetic profile of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene comprises an Embryonic Genome Activation (EGA)-enriched Alu motif (EEA); and (ii) a second gate unit preconfigured to regulate the expression level or epigenetic profile of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene comprises a cellular dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion.

[0009] In another aspect, the present disclosure provides a method for converting a plurality of differentiated cells into a plurality of stem cells, the method comprising: contacting the plurality of differentiated 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 or epigenetic profile levels of a plurality of different target endogenous genes to effect the conversion, and wherein the plurality of gate units comprise: (i) a first gate unit preconfigured to regulate the expression level or epigenetic profile level of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene comprises a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC; and (ii) a second gate unit preconfigured to regulate the expression level or epigenetic profile level of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene comprises a different cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion.

[0010] In another aspect, the present disclosure provides a system for converting a plurality of differentiated cells into a plurality of stem cells, the system comprising: a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to sequentially regulate the expression levels or epigenetic profile levels of a plurality of different target endogenous genes to effect the conversion, and wherein the plurality of gate units comprise: (i) a first gate unit preconfigured to regulate the expression level or epigenetic profile level of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene comprises a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC; and (ii) a second gate unit preconfigured to regulate the expression level or epigenetic profile level of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene comprises a different cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion.

[0011] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, which illustrates and describes only illustrative embodiments of the present disclosure. As will be recognized, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0012] Incorporated by reference

[0013] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description of illustrative embodiments that make use of the principles of the invention and the accompanying drawings (also referred to herein as “FIGURES” and “FIG.”), in which:

[0015] Figure 1 Schematically illustrates an example of a heterologous gene circuit.

[0016] Figure 2 Schematically illustrates a network of genes involved in the regulation of cellular pluripotency.

[0017] Figure 3 Examples of heterologous gene circuits are provided, each example showing stepwise regulation of a target gene.

[0018] Figure 4 Shows fluorescence imaging of fibroblasts transfected with various conditions, each condition including a plasmid encoding a fluorescent protein and one or more plasmids required for each of the heterologous gene circuits tested.

[0019] Figure 5 Shows fluorescence imaging (top) and phase contrast imaging, Nanog positive staining imaging, DNA positive staining imaging, and Oct4 positive staining imaging of fibroblasts transfected with a plasmid encoding a fluorescent protein and a plasmid required for a heterologous gene circuit for sequential activation of multiple target genes including an Alu motif enriched for embryonic genome activation (EGA) (EEA) to identify induced pluripotent cell reprogramming (bottom).

[0020] Figure 6 Shows phase contrast imaging of primary fibroblasts engineered with a heterologous gene circuit for targeting human endogenous retrovirus (HERV) and EEA to evaluate iPSC reprogramming and colony formation. DETAILED DESCRIPTION

[0021] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and alternatives will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0022] Unless the context clearly dictates otherwise, as used in this specification and the claims, the singular forms "a", "an", and "the" include plural references. For example, the term "gate unit" includes a plurality of gate units.

[0023] The term "about" or "approximately" generally means within an acceptable error range of a particular value as determined by a person of ordinary skill in the art, and this acceptable error range will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with the practice in the art, "about" can mean within 1 standard deviation or greater than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially for biological systems or processes, the term can mean within an order of magnitude of a certain value, preferably within 5-fold, and more preferably within 2-fold. Where a particular value is described in this application and the claims, unless otherwise stated, it should be assumed that the term "about" means within the acceptable error range of that particular value.

[0024] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. The term "and / or" should be understood to mean either or both of the alternatives.

[0025] Definitions

[0026] 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.) that are operably coupled (e.g., operate simultaneously, sequentially, etc.) according to circuit design. The collection of molecular components may be capable of providing one or more specific outputs (e.g., regulation of one or more genes) in a cell in response to one or more inputs (e.g., a single input or multiple inputs). Such one or more inputs may be sufficient to trigger the molecular components of the gene circuit to provide one or more specific outputs. For example, a gene circuit may include one or more molecular switches ([ Figure 1 ) that are activatable by one or more inputs.

[0027] A gene circuit can be a controllable gene expression system that includes an assembly of biological parts that work together as a logic function (e.g., simultaneously, sequentially, etc.). A gene circuit can include multiple gate units, where at least one of the multiple gate units can be activated by an activating moiety (e.g., a heterologous input to the cell) to activate other ones of the multiple gate units (e.g., all at once simultaneously, sequentially in a cascade, etc.) ( Figure 1 ). For example, at least one of the multiple gate units can be activated by another one of the multiple gate units (e.g., directly or indirectly) to (i) regulate the expression or activity level of one or more target genes, (ii) activate at least one other one of the multiple gate units, and / or (ii) deactivate at least one other one of the multiple gate units, thereby co-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 ( Figure 1 ). As used herein, the terms “heterologous gene circuit,” “HGC,” “cellular algorithm,” or “cellgorithm” may be used interchangeably.

[0028] As mentioned 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 multiplexing decisions at different points. More specifically, the term refers to a nucleic acid encoding a genetic switch and a transcriptional / translational regulatory region or a series of regions on which the genetic switch acts. The input to a gate unit can be an activating moiety and / or another gate unit. The output of a gate unit can be used to 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 multiple gate parts and / or multiple gene regulatory parts ( Figure 1 ).

[0029] As mentioned herein, the term “activating moiety” generally refers to a part that can activate multiple gene circuits and / or multiple gate units. An activating moiety can be a heterologous input to the cell. In some cases, an activating moiety can include, but is not limited to, a guide nucleic acid molecule (e.g., gRNA) or other nucleic acid, polypeptide, polynucleotide, small molecule, light, or a combination thereof. For example, an activating moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to the polynucleotide sequence of an inactivated gate part (e.g., a plasmid encoding another guide nucleic acid molecule) to activate such a gate part that can target one or more gene regulatory parts (e.g., induce the expression of a functional form of another guide nucleic acid molecule).

[0030] As mentioned herein, the term "gate portion" generally refers to a portion that can affect the function of a gene regulatory portion within a gate unit. The gate portion can activate and / or deactivate the gene regulatory portion. For example, the gate portion can regulate the expression of the gene regulatory portion by editing a nucleic acid sequence and thereby activating or deactivating the gene regulatory portion. For example, the gate portion can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of a gene regulatory portion (e.g., a plasmid encoding another guide nucleic acid molecule) to activate a gene regulatory portion that can target one or more endogenous genes of a cell (e.g., induce the expression of a functional form of another guide nucleic acid molecule). Alternatively or additionally, the gate portion can activate and / or deactivate another gate unit of a gene circuit ( Figure 1 ). For example, the gate portion can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of an inactivated another gate portion (e.g., a plasmid encoding another guide nucleic acid molecule) to activate that another gate portion (e.g., induce the expression of a functional form of another guide nucleic acid molecule). In another example, the gate portion can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of an activated another gate portion (e.g., a plasmid encoding another guide nucleic acid molecule) to inactivate that another gate portion (e.g., reduce the expression of a functional form of another guide nucleic acid molecule).

[0031] As used interchangeably herein, the term "gene regulatory portion" or "gene editing portion" generally refers to a portion that can regulate the expression and / or activity profile of a nucleic acid sequence or a protein (whether exogenous or endogenous to a cell) ( Figure 1 ). For example, the gene editing portion can regulate the expression of a gene by editing a nucleic acid sequence (e.g., CRISPR-Cas, zinc finger nuclease, TALEN, or siRNA). In some cases, the gene editing portion can regulate the expression of a gene by editing the genomic DNA sequence. In some cases, the gene editing portion can regulate the expression of a gene by editing the mRNA template. In some cases, editing the nucleic acid sequence can alter the underlying template for gene expression (e.g., an RNA targeting system inspired by CRISPR-Cas). Alternatively, the gene editing portion can inhibit the translation of a gene (e.g., Cas13).

[0032] Alternatively or additionally, the gene editing moiety can be capable of regulating gene expression or activity by specifically binding to a target sequence (or a target sequence within a gene) operably linked to the gene, and regulating the production of mRNA from DNA (such as chromosomal DNA or cDNA). For example, the gene editing moiety can recruit or comprise at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. The gene editing moiety itself can bind to DNA and regulate transcription by physical hindrance, for example, preventing proteins (such as RNA polymerase and other associated proteins) from assembling on the DNA template. The gene editing moiety can regulate gene expression at the translational level, for example, by regulating the production of proteins from the mRNA template. In some cases, the gene editing moiety can regulate gene expression by affecting the stability of the mRNA transcript. In some cases, the gene editing moiety can regulate genes by epigenetic editing (such as Cas12).

[0033] In some cases, a plasmid can encode a non-functional form of the gene editing moiety. The plasmid can be activated (e.g., genetically modified) to express a functional form of the gene editing moiety, for example, via activation of a functional gate moiety. For example, the 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 the cell. When a functional gate moiety (e.g., another guide nucleic acid molecule complexed with a Cas protein) binds to the plasmid, the plasmid can be edited (e.g., cleaved at one or more sites and then repaired via an endogenous mechanism (such as 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 the target gene of the cell), to permit regulation of the target gene in the cell.

[0034] In some cases, the gene regulatory portion 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, the gene regulatory portion can comprise an endonuclease or be operably coupled to an endonuclease. An endonuclease can be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. The endonuclease can comprise a restriction endonuclease that cleaves DNA at specific sites without damaging the bases. Restriction endonucleases can include type I, II, III, and IV endonucleases, which can further include subtypes. In some cases, the endonuclease can be Cas1, Cas2, Cas 3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14 or C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas 13 (C2c2), Cas13b, Cas13c, Cas13d, Cas13x.1, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx10, Csx11, Csf1, Csn2. The endonuclease can be a dead endonuclease that exhibits reduced cleavage activity. For example, the endonuclease can be a nuclease-inactivated Cas, such as dCas (e.g., dCas9).

[0035] The above-mentioned Cas protein can form a complex with a guide nucleic acid (gNA (e.g., guide RNA (gRNA)) and utilize the gNA to specifically bind to a target polynucleotide sequence (e.g., target DNA sequence, target RNA sequence). Thus, in some cases, such Cas proteins can be referred to as "NA-guided nucleases" (e.g., RNA-guided nucleases). As used herein, the term "guide nucleic acid" (gNA) generally can refer to a nucleic acid that can hybridize with another nucleic acid. The guide nucleic acid can be RNA. The guide nucleic acid can be DNA. The guide nucleic acid can be programmed to bind site-specifically to a nucleic acid sequence. The nucleic acid to be targeted or the target nucleic acid can include nucleotides. The guide nucleic acid can include nucleotides. A portion of the target nucleic acid can be complementary to a portion of the guide nucleic acid. The strand of the double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid can be referred to as the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand and thus may not be complementary to the guide nucleic acid can be referred to as the non-complementary strand. The guide nucleic acid can include a polynucleotide chain and can be referred to as a "single guide nucleic acid". The guide nucleic acid can include two polynucleotide chains and can be referred to as a "dual guide nucleic acid". Unless otherwise specified, the term "guide nucleic acid" can be inclusive and refer to both single guide nucleic acids and dual guide nucleic acids. The guide nucleic acid can include a segment, which can be referred to as a "nucleic acid targeting segment" or "nucleic acid targeting sequence" or "spacer sequence". The nucleic acid targeting segment can contain a sub-segment, which can be referred to as a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment" or "scaffold sequence".

[0036] 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 (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 repression domain, Pr-SET7 / 8, SUV4-20H1, RIZ1, JMJD2A, JHDM3A, JMJD2B, JMJD2C, GASC1, JMJD2D, JARID1A, RBP2, JARIDlB / PLU-1, JARIDIC / SMCX, JARIDID / SMCY, HDACl, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDACl1, 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 to) a transcriptional activator. Non-limiting examples of transcriptional activators can include VP16, VP64, VP48, VP160, p65 subdomain, SET1A, SET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, SYMD2, NSD1, JHDM2a, JHDM2b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRCl, ACTR, P160, CLOCK, TET1CD, TET1, DME, DML1, DML2, and ROS1.

[0037] In some cases, the gene regulatory moiety has enzymatic activity that modifies the target gene without cleaving it. Modification of the target gene can result in, for example, epigenetic modifications that can modify gene expression and / or activity levels. Examples of enzymatic activities that can be provided by the gene regulatory moiety can include, but are not limited to: nuclease activity (such as nuclease activity provided by a restriction enzyme (e.g., FokI nuclease)); methyltransferase activity (such as methyltransferase activity provided by a methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), MET1, DRM3, ZMET2, CMT1, CMT2)); demethylase activity (such as demethylase activity provided by a demethylase (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 a deaminase (e.g., a cytosine deaminase such as APOBEC1)), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity (such as integrase activity provided by an integrase and / or a resolvase (e.g., the Gin invertase, such as a hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase, etc.)), transposase activity, recombinase activity (such as recombinase activity provided by a recombinase (e.g., the catalytic domain of the Gin recombinase)), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0038] Unless otherwise specified or obvious from context, as used interchangeably herein, the terms "polynucleotide," "oligonucleotide," or "nucleic acid" generally refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof, whether single-stranded, double-stranded, or multi-stranded. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or 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 known or unknown function. A polynucleotide can include one or more analogs (e.g., altered backbone, sugar, or nucleobase). In the presence of modifications, the nucleotide structure can be modified either before or after polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, brachidial, and wybutosine. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides (including cell-free DNA (cfDNA) and cell-free RNA (cfRNA)), nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0039] The term "gene" generally refers to nucleic acids (e.g., DNA, such as genomic DNA and cDNA) involved in encoding RNA transcripts and their corresponding nucleotide sequences. As used herein, the term with respect to genomic DNA includes intervening non-coding regions as well as regulatory regions and may include 5′ and 3′ termini. In some uses, the term encompasses transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region will contain an "open reading frame" encoding a polypeptide. In some uses of the term, "gene" includes only the coding sequences necessary to encode a polypeptide (e.g., the "open reading frame" or "coding region"). In some cases, genes do not encode polypeptides, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only transcribed sequences but also, in addition, non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or native gene in its natural location in an organism's genome. A gene may refer to an "exogenous gene" or non-native gene. A non-native gene may refer to a gene that is not normally found in a host organism but is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene that is not in its natural location in an organism's genome. A non-native gene may also refer to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-native sequence) containing mutations, insertions, and / or deletions.

[0040] The term "sequence identity" generally refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity 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". The percent identity of two sequences (whether nucleic acid or amino acid sequences) is the number of exact matches between the two aligned sequences divided by the length of the longer sequence, multiplied by 100. The percent identity can also be determined, for example, by comparing sequence information using the BLAST computer program available from the National Institutes of Health (including version 2.2.9). The BLAST program is based on the alignment methods 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). This program can be used to determine the percent identity over the full length of the proteins being compared. Default parameters are provided to optimize the search for short query sequences in, for example, the blastp program. This program also allows the use of the SEG filter to mask-off segments of the query sequence determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The range of the desired degree of sequence identity is approximately 50% to 100% and integer values therebetween. In general, 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 sequence provided herein.

[0041] The term "expression" generally refers to one or more processes of transcription of a polynucleotide from a DNA template (such as transcription into mRNA or other RNA transcripts) and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as "gene product". If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include splicing of the mRNA. In terms of expression, "upregulation" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression level in the wild-type state, while "downregulation" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression in the wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression", the transfected gene is not transferred to daughter cells during cell division. Since its expression is restricted to the transfected cells, the expression of the gene is lost 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 cells. Such a selective advantage can be resistance to a certain toxin presented to the cells.

[0042] As used interchangeably herein, the terms "peptide", "polypeptide", or "protein" generally refer to a polymer of at least two amino acid residues linked by peptide bonds. This term does not imply a specific length of the polymer and is not intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The term applies to both naturally occurring amino acid polymers and amino acid polymers that contain at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structure (e.g., domains). The term also encompasses amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeled component. As used herein, the terms "amino acid" and "amino acids" generally refer to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids may include natural and non-natural amino acids that have been chemically modified to include groups or chemical moieties that do not naturally occur on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0043] As used interchangeably herein, with respect to a polypeptide, the terms "derivative", "variant" or "fragment" generally refer to a polypeptide that is related to a wild-type polypeptide, for example, by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Compared to the wild-type polypeptide, the derivative, variant and fragment of the polypeptide may contain one or more amino acid variations (e.g., mutations, insertions and deletions), truncations, modifications or combinations thereof.

[0044] As used herein, with respect to a polypeptide molecule (e.g., a protein), the terms "engineered", "chimeric" or "recombinant" generally refer to a polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence due to the application of genetic engineering techniques to the nucleic acid encoding the polypeptide molecule and to the cell or organism expressing the polypeptide molecule. As used herein, with respect to a polynucleotide molecule (e.g., a DNA or RNA molecule), the terms "engineered" or "recombinant" generally refer to a polynucleotide molecule having a heterologous nucleic acid sequence or an altered nucleic acid sequence due to the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; transfection, transformation and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, an engineered or recombinant polynucleotide (e.g., a genomic DNA sequence) may be modified or altered by a gene editing moiety.

[0045] Unless otherwise specified or obvious from context, as used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be the monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates such as adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by known techniques. Labeling can also be performed with quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme 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-1-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, which are available from Perkin Elmer, Foster City, Calif.FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, available from Amersham, Arlington Heights, Ill.; fluorescein-15-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-labeled nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, cascade blue-7-UTP, cascade blue-7-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. Chemically modified mononucleotides 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).

[0046] The term "cell" generally refers to biological cells. A cell can be the basic structural, functional, and / or biological unit of a living organism. Cells can be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, stoneworts, liverworts, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. Agardh, etc.), seaweeds (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., Drosophila, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes, cells are not derived from natural organisms (e.g., cells can be synthetically manufactured, sometimes referred to as artificial cells).

[0047] As used interchangeably herein, the terms "reprogramming", "dedifferentiation", "increasing cell potential", or "increasing developmental potential" generally refer to methods of increasing the potential of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is in a less differentiated state than the same cell in a non-reprogrammed state.

[0048] The term "differentiation" generally refers to the process by which non-specialized ("undetermined") or less specialized cells acquire the characteristics of specialized cells (such as, for example, immune cells). Differentiated or differentiation-induced cells are cells that have obtained a more specialized ("determined") position within a cell lineage. The term "determination" generally refers to a cell at a point in the differentiation pathway where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types and, under normal circumstances, cannot differentiate into different cell types or revert to a less differentiated cell type.

[0049] The term "pluripotent" generally refers to the ability of a cell to form all lineages of the body or somatic cells (i.e., the embryo proper). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers (ectoderm, mesoderm, and endoderm). Pluripotency can be a continuum of developmental potential ranging from cells that are incompletely or partially pluripotent (e.g., ectodermal stem cells) that cannot give rise to a complete organism to more primitive, pluripotent cells that can give rise to a complete organism (e.g., embryonic stem cells).

[0050] 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 (i.e., reprogrammed) to a cell capable of differentiating into tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. The resulting iPSCs do not refer to cells found in nature. In some cases, iPSCs can be engineered to directly differentiate into committed cells (e.g., natural killer (NK) cells). In some cases, iPSCs can be engineered to first differentiate into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) or hematopoietic progenitor cells), which can then be further induced to differentiate into committed cells (e.g., NK cells).

[0051] Review

[0052] Biological programming such as cell programming (e.g., generating iPSCs) allows cells to be engineered to produce desired outcomes. The outcomes of cell programming can include inducing or preventing a wide range of common and / or novel cellular functions; the outcomes can also include enhancing or suppressing existing cellular functions. Cell programming can be accomplished by using genetic circuits. Cell programming can be accomplished by manipulating biomolecules (e.g., endogenous DNA). For example, due to the versatility and easy programmability of the CRISPR or CRISPR / Cas system, the CRISPR or CRISPR / Cas system has been adopted 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 or time-independent manner.

[0053] The genetic circuits used in cell programming can be used to control cascades of multiple desired expression and / or activity profiles of multiple genes in a cell. To allow for better control of specific cellular outcomes, genetic circuits can be multiplexed to create positive and / or negative feedback systems.

[0054] Although CRISPR / Cas systems can be used for gene editing, Cas is essentially a single-turnover nuclease because it remains bound to the double-strand breaks it creates, 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 exploit 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 multiplexed regulation of endogenous genes to achieve cell differentiation.

[0055] Yamanaka factors are a group of protein transcription factors that play an important role in the generation of iPSCs, and their overexpression can be used to induce pluripotency. However, the methods for overexpressing Yamanaka factors are inefficient, slow, and random. In addition to these problems, epigenetic markers can remain in the original differentiated cells, which can exacerbate problems in iPSCs prepared by this method.

[0056] For example, there remains an unmet need for activatable multiplexed CRISPR / Cas systems and their use for editing target polynucleotides (e.g., the genomes of cells, particularly eukaryotic cells), which use cascades of gRNAs to form gene circuits in order to independently affect gene regulation and thereby reprogram cells to efficiently generate iPSCs and improve the viability and use of iPSCs in a manner that removes epigenetic markers.

[0057] Accordingly, aspects of the present disclosure provide systems, compositions, and methods for modulating a target gene (e.g., an endogenous target gene) that (i) is derived from a virus and (ii) is integrated into the genome of a non-viral cell, e.g., to modulate the fate of the non-viral cell (e.g., to convert the non-viral cell from a first cell type to a second cell type, such as dedifferentiation). Other aspects of the present disclosure provide systems, compositions, and methods for modulating other target genes (e.g., additional endogenous target genes) to modulate non-viral cell fate. In some embodiments, the systems, compositions, and methods provided herein may not utilize and do not require the use of a heterologous gene encoding such a target gene, but rather rely on modulating endogenous genes. In some embodiments, the present disclosure provides systems and methods for engineering a CRISPR / Cas9 system that includes a Cas endonuclease and a homologous single guide RNA (sgRNA or gRNA) array, the homologous single guide RNA having an inactivating sequence in a non-essential region and being activatable to permit derivation of iPSCs from differentiated cells. The present disclosure also provides engineered cells that may contain any of the foregoing systems or be capable of performing any of the foregoing methods.

[0058] Systems and methods for converting one type of cell to another type of cell

[0059] Various aspects of the present disclosure provide systems for inducing a desired level of expression and / or activity (or profile thereof) of one or more target genes in a cell. 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.

[0060] In one aspect, the present disclosure provides a system for inducing a desired expression and / or activity profile of a target gene (e.g., a target gene derived from a virus) in a cell. The system may include a heterologous gene regulator that exhibits specific binding to the target gene. In some cases, the heterologous gene regulator may be part of a heterologous gene circuit introduced into the cell to induce the desired expression. For example, the heterologous gene regulator may include an endonuclease and / or a polynucleotide sequence (e.g., a Cas protein, a guide nucleic acid molecule, and / or a combination thereof).

[0061] In some cases, a heterologous gene circuit can include at least one gate unit (e.g., a single gate unit or multiple gate units). The multiple gate units can include at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gate units. The multiple gate units can include at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, or at most about 2 gate units. The multiple gate units can be different (e.g., contain different polynucleotide sequences).

[0062] Alternatively, the expression and / or activity profile of a target gene of a cell as disclosed herein can be regulated in the absence of a heterologous gene circuit. For example, a gene regulatory moiety configured to bind to the target gene (e.g., a CRISPR / Cas protein and a guide nucleic acid molecule directed against the target gene) can be introduced (e.g., transfected or transduced) into the cell such that the introduction can be sufficient to regulate the expression and / or activity profile of the target gene without any further regulation of the activity of the gene regulatory moiety.

[0063] A heterologous gene circuit as disclosed herein can be operated with a single gate unit such that activation of the single gate unit can regulate the expression or activity level of a target gene (e.g., a target endogenous gene) such as a virus-derived gene (e.g., HERV) in a mammalian genome. Alternatively or additionally, a heterologous gene circuit as disclosed herein can be operated with a single gate unit such that activation of the single gate unit can regulate the epigenetic profile of a target gene (e.g., a target endogenous gene) such as a virus-derived gene (e.g., HERV) in a mammalian genome.

[0064] A heterologous gene circuit as disclosed herein can be operated with multiple gate units in series (e.g., multiple gate units are sequentially connected end-to-end to form a single path), multiple gate units in parallel (e.g., multiple gate units are cross-connected to each other to form, for example, two or more parallel paths), or a combination thereof, e.g., to sequentially regulate the expression or activity of multiple target genes (e.g., endogenous target genes) such as virus-derived genes and additional target genes.

[0065] Without wishing to be bound by theory, modulating (e.g., modifying) the gene expression level and / or epigenetic profile of virus-derived genes (e.g., HERVs) in the mammalian genome can prime cells for enhanced regulation of cell fate. In some cases, modulation of the expression level and / or epigenetic profile of virus-derived genes can effect opening of at least a portion of the cell genome and / or resetting of at least a portion of the cell, such that epigenetic markers (e.g., DNA histone markers) can be generally removed. Alternatively, modulation of the expression level and / or epigenetic profile of virus-derived genes can effect opening of at least a portion of the cell genome and / or resetting of at least a portion of the cell, such that epigenetic markers (e.g., DNA histone markers) can be selectively added to specific locations. Alternatively, modulation of the expression level and / or epigenetic profile of virus-derived genes can effect opening of at least a portion of the cell genome and / or resetting of at least a portion of the cell, such that epigenetic markers (e.g., DNA histone markers) can be generally added. Alternatively, modulation of the expression level and / or epigenetic profile of virus-derived genes can effect opening of at least a portion of the cell genome and / or resetting of at least a portion of the cell, such that epigenetic markers (e.g., DNA histone markers) can be selectively removed from specific locations.

[0066] In some cases, modulation of the expression level and / or epigenetic profile of virus-derived genes can effect opening of at least a portion of the cell genome and / or resetting of at least a portion of the cell, such that the process of differentiating cells can be targeted specifically according to a targeting element. In some cases, modulation of the expression level and / or epigenetic profile of virus-derived genes can effect opening of at least a portion of the cell genome and / or resetting of at least a portion of the cell, such that the process of differentiating cells can be targeted specifically according to the target cell lineage.

[0067] In some cases, modulation of the expression level and / or epigenetic profile of virus-derived genes can effect opening of at least a portion of the cell genome and / or resetting of at least a portion of the cell, such that the process of dedifferentiating cells can be targeted specifically according to a targeting element. In some cases, modulation of the expression level and / or epigenetic profile of virus-derived genes can effect opening of at least a portion of the cell genome and / or resetting of at least a portion of the cell, such that the process of dedifferentiating cells can be targeted specifically according to the initial cell lineage.

[0068] Multiple gate units as disclosed herein can operate in concert (e.g., as predetermined by the design of a heterologous genetic circuit) to induce an outcome in a cell. The outcome in the cell can include cell functions (e.g., motility, reproduction; response to external stimuli, nutrient output, excretion, respiration, growth) and / or cell states (e.g., cell fate, differentiation, quiescence, programmed cell death). Such outcomes can be determined in vitro, ex vivo, and / or in vivo. For example, an 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) staining via small molecules or antibodies, (iii) cell sorting based on cell size, morphology, and / or surface protein expression, (iv) using assays (e.g., cell proliferation assays, metabolic activity assays, cell killing assays) to measure phenotypic differentiation and cell functions, (v) microscopy, and / or (iv) screening for molecular and / or genetic differences using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics.

[0069] The outcome in the cell can include regulation of a target gene. Regulation of the target gene can include multiple different modulations of the target gene. Multiple gate units can each induce one of the multiple different modulations of the target gene such that the set of different modulations synergistically produces the final expression and / or activity profile of the target gene. At least two different modulations of the multiple different modulations can both increase the expression and / or activity level of the target gene. At least two different modulations of the multiple different modulations can both decrease the expression and / or activity level of the target gene. Alternatively, a first different modulation of the multiple different modulations can increase the expression and / or activity level of the target gene while a second different modulation of the multiple different modulations can decrease the expression and / or activity level of the target gene. In such a case, the first different modulation can occur before the second different modulation or vice versa. Alternatively, different modulations of the multiple different modulations (e.g., the first modulation and / or the second modulation) can maintain the expression and / or activity level of the target gene at the expression and / or activity level prior to the modulation.

[0070] The results in the cell can include regulation of the epigenetic profile of a target gene. The regulation of the epigenetic profile of the target gene can include multiple different regulations of the target gene. Multiple gate units can each induce one of the multiple different regulations of the epigenetic profile of the target gene, such that the set of different regulations synergistically produces the final epigenetic profile of the target gene. At least two different regulations of the multiple different regulations can both modify the epigenome of the target gene. The epigenome of the target gene can be modified by regulating methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ribosylation, or citrullination. In this case, the first different regulation can occur before the second different regulation or vice versa. Alternatively, different regulations of the multiple different regulations (e.g., the first regulation and / or the second regulation) can maintain the epigenetic state of the target gene.

[0071] In some cases, as disclosed herein, each different regulation of the multiple different regulations of the target gene can be necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene. Thus, in the absence of any one of the multiple different regulations of the target gene, the results in the cell induced by the multiple different regulations of the target gene (e.g., enhanced cell function, induced cell state, etc.) can be impossible. Alternatively, the degree or measurement of the results in the cell induced by the multiple different regulations of the target gene can be greater than the degree or measurement of the results in control cells induced by none, one or more but not all, and / or all of the multiple different regulations of the target gene occurring in a different order of events.

[0072] In some cases, as disclosed herein, each different regulation of the multiple different regulations of the target gene can be necessary but individually insufficient to achieve the desired epigenetic profile of the target gene. Thus, in the absence of any one of the multiple different regulations of the target gene, the results in the cell induced by the multiple different regulations of the target gene (e.g., enhanced cell function, induced cell state, etc.) can be impossible. Alternatively, the degree or measurement of the results in the cell induced by the multiple different regulations of the epigenome of the target gene can be greater than the degree or measurement of the results in control cells induced by none, one or more but not all, and / or all of the multiple different regulations of the epigenome of the target gene occurring in a different order of events.

[0073] The second gate unit can be activated by the first gate unit (e.g., directly or indirectly). For example, the second gate unit can be directly activated by the first gate unit. Alternatively, the second gate unit can be activated by one or more additional gate units activated by the first gate unit (e.g., directly or indirectly). The one or more additional gate units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gate units. The one or more additional gate units are at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 gate unit. In yet another alternative, the second gate unit can be activated via another part (e.g., activation part, different gate unit, etc.) responsible for activating the first gate unit.

[0074] The second gate unit can be activatable to induce inactivation of the already-activated first gate unit. The terms "inactivation" or "disruption" can be used interchangeably herein. Inactivation and as disclosed herein can be induced by creating a modification (e.g., cleavage, such as single-strand or double-strand breaks, and insertion-deletion (indel), etc.) to at least a part of the first gate unit (e.g., the gate part and / or gene regulatory part of the first gate unit), which part is responsible for inducing a first different regulation of the target gene.

[0075] Inactivation of the gate part and / or gene regulatory part of the first gate unit as disclosed herein can be achieved by an endonuclease-based system (e.g., the CRISPR / Cas system). Alternatively or additionally, inactivation can be achieved by using a transcriptional regulator system (e.g., a transcriptional repressor). The endonuclease transcriptional regulator system (e.g., a Cas inhibitor) can be used to effect polynucleotide cleavage (e.g., for inactivating the gate part and / or gene regulatory part). Polynucleotide cleavage can create nucleic acid modifications such as single-strand breaks, double-strand breaks, insertions, deletions, or insertion-deletions (indel). Alternatively or additionally, the endonuclease transcriptional regulator system (e.g., a Cas inhibitor) can be used to regulate target gene expression.

[0076] Alternatively, the second gate unit can be activatable to amplify or enhance the activation of the already-activated first gate unit. The amplification or enhancement of the first gate unit can be induced by generating a modification (e.g., cleavage, such as single-strand or double-strand breaks, and indels, 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), which portion is responsible for inducing the first distinct regulation of the target gene.

[0077] In some cases, the first gate unit regulates a first target gene. Alternatively or additionally, the first gate unit can also regulate the second gate unit. The regulation of the second gate unit can occur at least or up to about 1 millisecond, at least or up to about 2 milliseconds, at least or up to about 3 milliseconds, at least or up to about 4 milliseconds, at least or up to about 5 milliseconds, at least or up to about 6 milliseconds, at least or up to about 7 milliseconds, at least or up to about 8 milliseconds, at least or up to about 9 milliseconds, at least or up to about 10 milliseconds, at least or up to about 20 milliseconds, at least or up to about 30 milliseconds, at least or up to about 40 milliseconds, at least or up to about 50 milliseconds, at least or up to about 60 milliseconds, at least or up to about 70 milliseconds, at least or up to about 80 milliseconds, at least or up to about 90 milliseconds, at least or up to about 100 milliseconds, at least or up to about 200 milliseconds, at least or up to about 300 milliseconds, at least or up to about 400 milliseconds, at least or up to about 500 milliseconds, at least or up to about 600 milliseconds, at least or up to about 700 milliseconds, at least or up to about 800 milliseconds, at least or up to about 900 milliseconds, at least or up to about 1 second, at least or up to about 2 seconds, at least or up to about 3 seconds, at least or up to about 4 seconds, at least or up to about 5 seconds, at least or up to about 6 seconds, at least or up to about 7 seconds, at least or up to about 8 seconds, at least or up to about 9 seconds, at least or up to about 10 seconds, at least or up to about 15 seconds, at least or up to about 20 seconds, at least or up to about 30 seconds, at least or up to about 40 seconds, at least or up to about 50 seconds, at least or up to about 1 minute, at least or up to about 2 minutes, at least or up to about 3 minutes, at least or up to about 4 minutes, at least or up to about 5 minutes, at least or up to about 6 minutes, at least or up to about 7 minutes, at least or up to about 8 minutes, at least or up to about 9 minutes, at least or up to about 10 minutes, at least or up to about 20 minutes, at least or up to about 30 minutes, at least or up to about 40 minutes, at least or up to about 50 minutes, at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 12 hours, at least or up to about 16 hours, at least or up to about 20 hours, or at least or up to about 24 hours, or occurs after the regulation of the first gate unit, as determined by rt-qPCR, Western blotting, or other methods.

[0078] In some cases, the second gate unit can modulate a second target gene. The modulation of the second target gene can occur at least or up to about 1 millisecond, at least or up to about 2 milliseconds, at least or up to about 3 milliseconds, at least or up to about 4 milliseconds, at least or up to about 5 milliseconds, at least or up to about 6 milliseconds, at least or up to about 7 milliseconds, at least or up to about 8 milliseconds, at least or up to about 9 milliseconds, at least or up to about 10 milliseconds, at least or up to about 20 milliseconds, at least or up to about 30 milliseconds, at least or up to about 40 milliseconds, at least or up to about 50 milliseconds, at least or up to about 60 milliseconds, at least or up to about 70 milliseconds, at least or up to about 80 milliseconds, at least or up to about 90 milliseconds, at least or up to about 100 milliseconds, at least or up to about 200 milliseconds, at least or up to about 300 milliseconds, at least or up to about 400 milliseconds, at least or up to about 500 milliseconds, at least or up to about 600 milliseconds, at least or up to about 700 milliseconds, at least or up to about 800 milliseconds, at least or up to about 900 milliseconds, at least or up to about 1 second, at least or up to about 2 seconds, at least or up to about 3 seconds, at least or up to about 4 seconds, at least or up to about 5 seconds, at least or up to about 6 seconds, at least or up to about 7 seconds, at least or up to about 8 seconds, at least or up to about 9 seconds, at least or up to about 10 seconds, at least or up to about 15 seconds, at least or up to about 20 seconds, at least or up to about 30 seconds, at least or up to about 40 seconds, at least or up to about 50 seconds, at least or up to about 1 minute, at least or up to about 2 minutes, at least or up to about 3 minutes, at least or up to about 4 minutes, at least or up to about 5 minutes, at least or up to about 6 minutes, at least or up to about 7 minutes, at least or up to about 8 minutes, at least or up to about 9 minutes, at least or up to about 10 minutes, at least or up to about 20 minutes, at least or up to about 30 minutes, at least or up to about 40 minutes, at least or up to about 50 minutes, at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 12 hours, at least or up to about 16 hours, at least or up to about 20 hours, or at least or up to about 24 hours, or longer after the modulation of the first target gene, as determined by rt-qPCR, Western blotting, or other methods.

[0079] In some cases, the modification of the target gene by the gate unit can inactivate the gene. For example, the modification of the gene can terminate the expression and / or activity level of the target gene. Alternatively, the modification of the gene can reduce the expression and / or activity level of the target gene. In some cases, the modification of the gene can increase the expression and / or activity level of the target gene. Alternatively, the modification of the gene can maintain the expression and / or activity level of the target gene.

[0080] The expression and / or activity profile of a gene of interest (e.g., a differentiation marker) can be compared to a control gene (e.g., a housekeeping gene such as GAPDH), the relative expression levels of two or more genes of interest (e.g., the ratio of the expression or activity levels between a stem cell marker and a differentiation marker), the relative average expression level of a gene of interest compared to the average expression level of the same gene of interest in a cell type of interest, etc.

[0081] In some cases, a guide nucleic acid molecule (gNA) (e.g., a functional gNA) expressed by a second gate unit upon activation can create a modification to at least a portion of the first gate unit. For example, the activated gNA of the second gate unit can create a modification to the polynucleotide sequence of the first gate unit encoding the gNA (e.g., an activatable gNA) or the promoter sequence of the first gate unit operably coupled to such gNA. Such a modification can render the gNA of the first gate unit inoperable when expressed (e.g., reduce or inhibit specific binding to a target gene). Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0082] In some cases, a 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 nucleotide strand. Inactivation of a polynucleotide sequence or a target gene can be caused by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or more single-strand breaks. In some cases, inactivation of a gene can be caused by 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.

[0083] In some cases, the size of the gNA (e.g., including both the spacer sequence and the 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.

[0084] In some cases, the size of the scaffold sequence of the 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 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.

[0085] In some cases, the size of the spacer sequence of the gNA can be 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.

[0086] In some cases, the systems and methods of the present disclosure can utilize a single endonuclease system (e.g., a Cas inhibitor) to achieve both (i) polynucleotide cleavage (e.g., for activating / inactivating a gate portion and / or a gene regulatory portion) and (ii) regulation of target gene expression. When using a single endonuclease transcriptional regulator system, unique guide nucleic acid molecules (gNAs) with different spacer sequence lengths can be used to determine whether the single endonuclease transcriptional regulator system can (i) hybridize to a polynucleotide sequence to induce Cas-mediated nuclease activity of the polynucleotide sequence, or (ii) can hybridize to a target gene (e.g., genomic DNA) to regulate the expression and / or activity level of the target gene via the action of a transcriptional activator without mediating Cas nuclease activity, as desired by a single heterologous gene circuit. For example, using gNAs with different spacer sequence lengths that bind to different targets can allow a second gate unit as provided herein to induce inactivation of an already activated first gate unit and / or induce different regulation of a second target gene.

[0087] As described above, the length of the spacer sequence of the gNA can affect the ability of the gNA to mediate Cas nuclease activity. In some cases, gNAs with spacer sequences of different lengths can be used in the same heterologous gene circuit to effect different types of cleavage, activation, inactivation, and / or regulation of one or more target nucleic acids. In some cases, a gNA spacer sequence shorter than a threshold length (e.g., about 16 nucleotides) can impede the nuclease activity of the Cas transcriptional regulator while still mediating DNA binding for transcriptional regulation of the 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 the nuclease activity of the Cas protein while still mediating DNA binding.

[0088] For example, a gNA comprising a 20-nucleotide spacer sequence (e.g., a gNA encoded by the gate portion for targeting the gene regulatory portion plasmid) can be sufficient to promote the nuclease activity of an endonuclease (e.g., a Cas or Cas transcriptional regulator fusion protein). Alternatively or additionally, a gNA comprising a 14-nucleotide spacer sequence (e.g., a gNA encoded by the gene regulatory portion) can hybridize to DNA but may not be long enough to mediate nuclease activity—it may only promote the binding of the endonuclease to the homologous DNA sequence. Thus, shorter gNAs can selectively permit transcriptional regulation of the target gene while using an endonuclease transcriptional regulator system (e.g., a Cas activator system, a Cas repressor system) without cleaving the target gene.

[0089] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as the gate portion) or a target gene can be caused by a double-strand break in which discontinuities exist in both nucleotide strands. In some cases, the number of such double-strand breaks (e.g., required for such modification) can be at least or 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.

[0090] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate moiety) or a target gene can be caused by an insertion-deletion (also referred to as an indel mutation). Indel mutations can include frameshift or non-frameshift mutations. Indel mutations can include point mutations (also referred to as base substitutions), where 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.

[0091] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate moiety) or a target gene can be achieved without cleaving the polynucleotide sequence or the target gene. For example, a gene regulatory 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 a polynucleotide sequence or a target gene such that the expression and / or activity of the polynucleotide sequence or the target gene is modified. The gene regulatory moiety can comprise a transcriptional repressor or a transcriptional activator as provided herein. Alternatively or additionally, the gene regulatory moiety can induce an epigenetic modification (or epigenomic modification) as provided herein.

[0092] In some cases, such as provided herein, modification of a polynucleotide sequence or a target gene can inactivate the polynucleotide sequence or the target gene. For example, modification of a polynucleotide sequence or a target gene can inhibit or reduce the expression and / or activity level of the polynucleotide sequence or the target gene. In some cases, such as provided herein, modification of a polynucleotide sequence or a target gene can activate the polynucleotide sequence or the target gene. For example, modification of a polynucleotide sequence or a target gene can increase the expression and / or activity level of the polynucleotide sequence or the target gene.

[0093] In some cases, such as provided herein, modification of a polynucleotide sequence or a target gene can include reducing the expression and / or activity level of the polynucleotide sequence or the target gene by at least or up to about 0.1%, at least or up to about 0.2%, at least or up to about 0.3%, at least or up to about 0.4%, at least or up to about 0.5%, at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 95%, at least or up to about 99%, or about 100% (e.g., as compared to a control lacking the modification, for example).

[0094] In some cases, such as provided herein, modification of a polynucleotide sequence or a target gene can include reducing the expression and / or activity level of the polynucleotide sequence or the target gene to 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 1.5-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 11-fold, at least or up to about 12-fold, at least or up to about 13-fold, at least or up to about 14-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold or at least or up to about 100-fold (e.g., as compared to a control lacking the modification, for example).

[0095] In some cases, such as those provided herein, modification of a polynucleotide sequence or target gene can include increasing the expression and / or activity level of the polynucleotide sequence or target gene by at least or up to about 0.1%, at least or up to about 0.2%, at least or up to about 0.3%, at least or up to about 0.4%, at least or up to about 0.5%, at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500% (e.g., as compared to a control lacking the modification, for example).

[0096] In some cases, such as those provided herein, modification of a polynucleotide sequence or target gene can include increasing the expression and / or activity level of the polynucleotide sequence or 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 1.5-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 11-fold, at least or up to about 12-fold, at least or up to about 13-fold, at least or up to about 14-fold, at least or up to about 15-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 100-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, at least or up to about 500-fold, or at least or up to about 1,000-fold (e.g., as compared to a control lacking the modification, for example).

[0097] In some cases, the activation of multiple gate units can be the result of a single activation of a heterologous genetic circuit (e.g., by a single activation moiety at a single time point). The multiple gate units can include one of a first gate unit and a second gate, which are preconfigured to be sequentially activated upon activation of the heterologous genetic circuit by a single activation. In some cases, one of the first gate unit and the second gate unit can be activated by a 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 different activation moiety (e.g., a different guide nucleic acid) that is different from the activation moiety of the heterologous genetic circuit. The different activation moiety can be part of the heterologous genetic circuit that is only produced (e.g., expressed) 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 parts of the heterologous genetic circuit that are only produced (e.g., expressed) upon activation of the heterologous genetic circuit.

[0098] In some embodiments of any of the systems disclosed herein, the gate unit can include 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.). The gate portion as disclosed herein can include 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 about 3 gNA molecules, at least or up to about 4 gNA molecules, at least or up to about 5 gNA molecules, etc.). The gene regulation portion as disclosed herein can include gNA (e.g., at least or up to about 1 gNA molecule, at least or up to about 2 gNA molecules, at least or up to about 3 gNA molecules, at least or up to about 4 gNA molecules, at least or up to about 5 gNA molecules, etc.). The guide nucleic acid molecule as disclosed herein can include, but is not limited to, DNA, RNA, any analog thereof, or any combination thereof. In some embodiments of any of the systems disclosed herein, the gate portion and / or the gene regulation portion can be activatable to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and the complex can be configured to or capable of binding to a target polynucleotide, e.g., to regulate the expression and / or activity level of the target polynucleotide or another polynucleotide sequence operably coupled to the target polynucleotide. For example, the complex can regulate the expression and / or activity level of a gene containing the target polynucleotide.

[0099] In some cases, a single gate unit can contain a single gene regulation portion for each target gene (e.g., a target endogenous gene). For example, activation of a single gate unit can enable the gene regulation portion to express a functional guide nucleic acid molecule for binding to and modulating the expression / epigenetic profile of the target gene. Alternatively, a single gate unit can contain multiple gene regulation portions for each target gene for multiplexed targeting. For example, the multiple gene regulation portions can encode multiple different guide nucleic acid molecules that have multiple different spacer sequences targeting a common target gene, such that activation of a single gate unit can enable the multiple gene regulation portions to express multiple different guide nucleic acid molecules (e.g., substantially simultaneously based on separate activation of the single gate unit) for multiplexed targeting and modulating the expression / epigenetic profile of the common target gene.

[0100] In some cases, when a heterologous gene circuit is designed to sequentially regulate the expression / epigenetic profiles of multiple target genes, each of the multiple target genes can be regulated via a single gene regulatory moiety at a given step of the heterologous gene circuit. In some cases, each of the multiple target genes can be regulated via multiplexed targeting at a given step of the heterologous gene circuit. In some cases, one target gene can be regulated via a single gene regulatory moiety at a given step of the heterologous gene circuit, while a different target gene can be regulated via multiplexed targeting at a given step of the heterologous gene circuit.

[0101] In some embodiments of any of the systems disclosed herein, an initial (or first) gate unit of a heterologous gene circuit as disclosed herein can be activated (e.g., directly activated) by an activation moiety. The activation moiety can directly bind to at least a portion of the initial gate unit to activate the initial gate unit, e.g., thereby sequentially activating the heterologous gene circuit. Alternatively, the activation moiety (e.g., electromagnetic energy) can activate the initial gate unit without directly binding to at least a portion of the initial gate unit. In some cases, the initial gate unit can comprise at least one gate portion and at least one gene regulatory moiety. In some cases, the initial gate unit can comprise at least one gate portion, but can not comprise and need not comprise a gene regulatory moiety. In some cases, the initial gate unit can comprise at least one gene regulatory moiety, but can not comprise and need not comprise a gate portion (e.g., the activation moiety can be configured to activate the initial gate unit and at least one additional gate unit).

[0102] 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, but is not limited to, any one of the following: ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogs thereof, or any combination thereof. In some embodiments, the vector (or expression cassette) encoding the activatable gNA can include an inactivating polynucleotide sequence to inactivate the gNA until it is activated (e.g., until the inactivating polynucleotide sequence is modified or removed from the vector). For example, the inactivating polynucleotide sequence can encode a self-cleaving polynucleotide molecule (e.g., a ribozyme). Alternatively or additionally, the inactivating polynucleotide sequence can encode a non-canonical transcription termination sequence, as described below. The inactivating polynucleotide sequence can be part of or adjacent to a region of the vector that encodes (i) the spacer sequence of the gNA, (ii) the scaffold sequence of the gNA, and / or (iii) any linker sequence between the spacer sequence and the scaffold sequence. The vector can contain at least or up to about 1 inactivating polynucleotide sequence, at least or up to about 2 inactivating polynucleotide sequences, at least or up to about 3 inactivating polynucleotide sequences, at least or up to about 4 inactivating polynucleotide sequences, at least or up to about 5 inactivating polynucleotide sequences, at least or up to about 6 inactivating polynucleotide sequences, at least or up to about 7 inactivating polynucleotide sequences, at least or up to about 8 inactivating polynucleotide sequences, at least or up to about 9 inactivating polynucleotide sequences, or at least or up to about 10 inactivating polynucleotide sequences.

[0103] In some cases, as used generally herein, the term "proGuide" can refer to a vector (e.g., a 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.

[0104] The proGuide can contain a linker sequence between (i) a domain encoding the spacer sequence of the guide nucleic acid and (ii) a domain containing the scaffold sequence of the guide nucleic acid (the domain containing one or more inactivating polynucleotide sequences (e.g., one or more polyT sequences)). Alternatively, the proGuide can not contain a linker sequence between the two domains (i) and (ii).

[0105] The proGuide can comprise a target polynucleotide domain at or adjacent to the inactivated polynucleotide sequence (e.g., at or adjacent to the 5' and / or 3' ends of the inactivated polynucleotide sequence), and the target polynucleotide domain can be targeted (e.g., via the sequential activation mechanism of the heterologous gene circuit provided herein) to modify (e.g., edit, cleave) the inactivated polynucleotide sequence, thereby enabling the guide nucleic acid molecule whose expression is activated by the proGuide. The target polynucleotide domain of the proGuide may not exhibit sequence identity with any comparable endogenous polynucleotide sequence in the cell, thereby avoiding inadvertently targeting and regulating endogenous target genes.

[0106] In some embodiments, the inactivated polynucleotide sequence of the proGuide can be arranged between two target polynucleotide domains, and the two domains may or may not be targetable by a common guide nucleic acid sequence. In some cases, the two target polynucleotide domains can be reverse and complementary to each other such that the inactivated polynucleotide sequence can be modified or cleaved by the same mechanism (e.g., the same spacer sequence of the guide nucleic acid molecule).

[0107] 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 the target gene) unless the gene encoding the activatable gNA is modified before the 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.

[0108] In some cases, the size of the polyT sequence is greater than or equal to a threshold length, where the threshold length is sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence. Thus, a plasmid (e.g., a gate portion or a gene regulatory portion) can encode an inactivated gNA comprising a polyT sequence 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 Ts. In some cases, the polyT sequence comprises at least 7 Ts. In some cases, the polyT sequence comprises at least 8 Ts. In some cases, the polyT sequence comprises at least 10 Ts. In some cases, the polyT sequence comprises from 5 Ts to 15 Ts. In some cases, the polyT sequence comprises one or more additional nucleotides that are not Ts.

[0109] In some cases, a gene regulatory portion (e.g., a guide nucleic acid and / or an endonuclease) can be configured to bind to a target polynucleotide sequence operably coupled to a target gene in a cell. The target gene can comprise a coding polynucleotide sequence encoding a target nucleic acid molecule or a target protein. The target polynucleotide sequence can be a part of the coding polynucleotide sequence. Alternatively, the target polynucleotide sequence can not be a part of the coding polynucleotide sequence. For example, the target polynucleotide sequence can be upstream of the coding polynucleotide sequence (e.g., a part of the promoter of the coding polynucleotide sequence, such as a transcription start site (TSS).

[0110] As provided herein, when a heterologous gene circuit is activated to induce multiple different modulations of a target gene as provided herein, the multiple different modulations of the target gene can be different (e.g., different degrees of alteration of the expression and / or activity level of the target gene). For example, the different degrees of a first modulation imposed by a first gene unit and a second modulation imposed by a 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., the same).

[0111] The multiple different modulations can each be sufficient to induce a desired alteration of the expression and / or activity level of the target gene. Alternatively, the different modulations can each be insufficient to induce a desired alteration of the expression and / or activity level of the target gene.

[0112] 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.

[0113] As provided herein, the conversion of a plurality of cells of a first cell type to a plurality of cells of a second cell type via the use of a heterologous gene regulator (e.g., as part of a heterologous gene circuit) can occur in the absence of one or more heterologous factors that are typically required to induce a comparable conversion in the absence of the heterologous gene regulator. In a comparable conversion, one or more heterologous factors can be added exogenously to the cell culture medium or can be expressed in the cells. In some cases, to convert differentiated cells to stem cells (e.g., iPSCs), one or more heterologous factors can include one or more reprogramming factors such as Oct4, Sox2, Nanog, Lin28, L-Myc, Klf4, and / or SV40LT.

[0114] In some cases, the use of a heterologous gene circuit as disclosed herein can be used to convert a plurality of cells of a first cell type to a plurality of cells of a second cell type. In some cases, the first plurality of cells can be differentiated cells and the second plurality of cells can be stem cells (e.g., iPSCs). Alternatively, the first plurality of cells can be stem cells and the second plurality of cells can be differentiated cells. In some cases, differentiated cells are converted to stem cells (e.g., iPSCs). In some cases, stem cells are converted to different types of stem cells. In some cases, stem cells are converted to differentiated cells. In some cases, differentiated cells are converted to different types of differentiated cells. In some cases, the differentiated cells are terminally differentiated cells.

[0115] Terminally differentiated cells (e.g., the initial cells to be modified into engineered cells as disclosed herein, the final cell products produced from the engineered cells as disclosed herein, etc.) can include muscle cells, immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells, epithelial cells, stem cells, secretory cells, blood cells, germ cells, nurse cells, storage cells, enteroendocrine cells, pituitary cells, neurosecretory cells, duct cells, odontoblasts, cementoblasts, glial cells, or stromal cells.

[0116] 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; 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 (chief cells of the parathyroid gland, oxyphil cells), adrenal cells (chromaffin cells), pinealocytes (Pinealocytes); cells of the nervous system, including glial cells (astrocytes, microglia), magnocellular neurosecretory cells, astrocytes, Boettcher cells, and pituitary cells (gonadotrophs, corticotrophs, thyrotrophs, somatotrophs, lactotrophs); cells of the respiratory system, including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells; cells of the circulatory system, including cardiomyocytes, pericytes; cells of the digestive system, including gastric cells (chief cells of the stomach, marginal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffin cells, APUD cells, liver cells (hepatocytes, Kupffer cells), cartilage / bone / muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, dental cells (cementoblasts, ameloblasts); chondrocytes, including chondroblasts, chondrocytes; skin cells, including hair cells, keratinocytes, melanocytes (nevus cells); muscle cells, including myocytes; urinary system cells, including podocytes, juxtaglomerular cells, mesangial cells (intraglomerular / mesangial cells), brush border cells of the proximal renal tubule, macula densa cells; reproductive system cells, including spermatozoa, Sertoli cells, Leydig cells, oocytes; and other cells,including adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of nails and toenails, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, cuticular hair stem cells, cuticular 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), wet stratified barrier epithelial cells, surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urothelial cells (lining the urinary bladder and ureters), exocrine epithelial cells, salivary gland mucous cells (polysaccharide-rich secretion), salivary gland serous cells (glycoprotease-rich secretion), von Ebner's gland cells in the tongue (washing taste buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), ceruminous gland cells in the ear (wax secretion), dark cells of eccrine sweat glands (glycoprotein secretion), light cells of eccrine sweat glands (small molecule secretion). Apocrine sweat gland cells (odor secretion, sex hormone-sensitive), ciliary gland cells in the eyelids (specialized sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Bowman's gland cells in the nose (washing the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting semen components,including spermatozoa, fructose), prostatic cells (secreting semen components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubricant secretion), Littre's gland cells (mucus secretion), endometrial cells (carbohydrate secretion), isolated goblet cells of the respiratory and digestive tracts (mucus secretion), gastric mucosa mucus cells (mucus secretion), gastric gland zymogenic cells (pepsinogen secretion), gastric gland oxyntic cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), small intestinal Paneth cells (lysozyme secretion), type II pneumocytes of the lung (surfactant secretion), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, somatotrophs, lactotropes, thyrotrophs, gonadotrophs, corticotrophs, intermediate pituitary cells, magnocellular neurosecretory cells, intestinal and respiratory tract cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief cells of the parathyroid gland, oxyphil cells, adrenal cells, chromaffin cells, Leydig cells of the testis, theca cells of the follicle, luteal cells of the ruptured follicle, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands and urogenital tract), kidney cells, type I pneumocytes (lining air space of the lung), pancreatic duct cells (centroacinar cells), nonstriated duct cells (sweat glands, salivary glands, mammary glands, etc.), duct cells (seminal vesicles, prostate, etc.), epithelial cells lining closed internal body cavities, ciliated cells with propulsion function, extracellular matrix-secreting cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells (e.g.,CD34+ cells, peripheral blood mononuclear cells), erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (of various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in the central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, sensory transducer cells, autonomic neuron cells, sensory organ and peripheral neuron supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, nurse cells, follicular cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells and interstitial kidney cells.,

[0117] Stem cells can include induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs) (e.g., from umbilical cord blood), muscle stem cells, neural stem cells, epithelial stem cells, epidermal stem cells, mammary stem cells, intestinal stem cells, neural crest stem cells or testicular stem cells.

[0118] Stem cells such as induced pluripotent stem cells can be identified by cell surface markers. Non-limiting examples of iPSC cell surface markers include 5T4, ABCG2, activin RIB / ALK4, activin RIIB, alkaline phosphatase / ALPL, B18R, E-cadherin, Cbx2, CD9, CD30 / TNFRSF8, CD117, CDX2, CDH3, CHD1, Cripto, DNMT3B, DPPA2, DPPA4, DPPA5, ESG1, EpCAM, TROP1, ERRβ, NR3B2, ERVMER34-1, ESGP, FBXO15, FGF4, FGF5, FOXD3, GCTM2, GBX2, GCNF, GDF3, CD49, CD29, KLF4, KLF5, L1TD1, Lefty1, LIN28, LIN41, c-Maf, c-Myc, Nanog, OCT3, OCT4, Polocalyxin, SMAD2, SMAD3, SOX2, SSEA1, SSEA3, SSEA4, STAT3, SUZ12, TBX2, TBX3, TBX5, TERT, TEX19, THAP11, TRA-1-60, TRA-1-81TROP, UTF1, VISTA, and / or ZIC3.

[0119] Aspects of the present disclosure provide for contacting a first plurality of cells with a heterologous gene regulator that exhibits specific binding to a target gene derived from a non-mammalian genome (e.g., a non-human genome) to effect conversion of the first plurality of cells of a first cell type into a plurality of cells of a second cell type (e.g., from a differentiated cell into a stem cell). In some embodiments, the target gene can (i) be derived from a virus (e.g., a DNA virus, an RNA virus, or a retrovirus (e.g., a retrovirus or a pararetrovirus)) and (ii) be integrated in a non-viral genome (e.g., a mammalian genome, such as the human genome). In some cases, the target gene can be derived from a retrovirus, such as, for example, a human endogenous retrovirus (HERV) gene. The target gene can comprise at least a portion of the HERV gene. At least a portion of the HERV gene can include one or more members selected from a long terminal repeat (LTR) (e.g., U3, R, U5), a group-specific antigen (GAG) gene (e.g., a matrix (MA) domain, a capsid (CA) domain, a nucleocapsid (NC) domain), a protease (PR) gene, a polymerase (POL) gene (e.g., a reverse transcriptase (RT), a ribonuclease H (RH), an integrase (IN)), and an envelope (ENV) gene (e.g., a surface component (SU), a transmembrane component (TM)). The LTR as disclosed herein can be an LTR arranged upstream of the target gene, such as, for example, a 5’ LTR. Non-limiting examples of LTRs of HERVs can include LTR5HS, LTR5A, and LTR5B. Non-limiting examples of LTR5HS can include LTR5HS-1, LTR5HS-2, LTR5HS-3, LTR5HS-4, LTR5HS-5, LTR5HS-6, LTR5HS-7, LTR5HS-8, LTR5HS-9, LTR5HS-10, LTR5HS-11, and LTR5HS-12.

[0120] LTR5HS is a subgroup of regulatory elements that regulate all human HERV-K elements. LTR5HS elements can be regulated by DNA hypermethylation. Alternatively, LTR5HS elements can be regulated by DNA hypomethylation or DNA demethylation.

[0121] In some cases, the heterologous gene regulator exhibits specific binding to a gene (or target gene) encoding a mobile genetic element (MGE). An MGE is a segment of genetic material that can move within a genome or that can be transferred from one species or replicon to another species or replicon. Non-limiting examples of MGEs are plasmids, transposons, integrons, viral agents, and introns.

[0122] Transposons, also known as jumping genes, are a group of mobile genetic elements that are DNA sequences. Transposons can move to different positions within the genome. Alternatively, some transposons always remain at specific insertion sites within the genome. Transposons are divided into two major categories: retrotransposons and DNA transposons. Retrotransposons are typically found in eukaryotes. Non-limiting examples of retrotransposons include long terminal repeats or LTRs, and non-long terminal repeats or non-LTRs (e.g., long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs)). DNA transposons can be found in both eukaryotes and prokaryotes. Non-limiting examples of DNA transposons include cut-and-paste DNA transposons, rolling circle DNA transposons (also known as helitrons), and self-synthesizing DNA transposons (also known as polintons).

[0123] Long interspersed nuclear elements (LINEs) are transposable elements that are typically about 7,000 base pairs in length. LINEs generally function by being transcribed into mRNA and translated into a protein that acts as a reverse transcriptase to integrate a DNA copy into the genome at a new site. Non-limiting examples of LINEs include LINE-1, L1H, RTE, Tad, and CRE.

[0124] Short interspersed nuclear elements (SINEs) are transposable elements that are typically about 100 - 700 base pairs in length. SINEs are generally lineage-specific. An example of a SINE is the Alu element, which is a repetitive sequence specific to primates. The Alu element is approximately 11% of the human genome. The Alu element is typically about 300 base pairs in length. The Alu element is closely associated with several human diseases such as cancer.

[0125] In some cases, heterologous gene regulators exhibit specific binding to a gene (or target gene) encoding (or containing or operably coupled to, as used interchangeably herein) an Alu motif enriched in embryonic genome activation (EGA) (EEA). The EEA or EEA motif is an Alu element enriched near genes involved in embryonic genome activation. The EEA motif-associated mechanism is closely related to cell reprogramming by activating genes such as NANOG and REX1.

[0126] Long terminal repeats are pairs of DNA sequences hundreds of bases in length that appear at either end of retrotransposons or endogenous retroviruses in eukaryotic genomes. LTRs typically encode reverse transcriptase and integrase, allowing the retrotransposon to be copied and inserted into different positions. Non-limiting examples of LTRs include HERV, MLT2A1, and MLT2A2.

[0127] In some cases, heterologous gene regulators exhibit specific binding to genes (or target genes) encoding human endogenous retroviruses or HERVs. HERVs are stable elements in DNA and are remnants of ancient infections that affected the primate germ line over the past 100 million years. HERVs and their genetic products, including RNA, cytoplasmic DNA, and proteins, can regulate the immune system and are affected by it. HERVs are closely related to assisting the immune system in defense against exogenous infections. Non-limiting examples of HERVs include HERV-H (e.g., RTVL-L, RGH), HERV-F, HERV-R (e.g., ERV9, ERV3), HERV-P (e.g., HuERS-P, HuRRS-p), HERV-L, HERV-I (e.g., RTVL-I), HERV-IP-T47D (e.g., ERV-FTD), HERV-K (e.g., HML-1, HML-1.1, HML-2, HERV-K10, HERV-K-HTDV, HML-3, HML-3.1, HML-4, HERV-K-T47D, HML-5, HERV-K-NMWV2, HML-6, HML-6p, HML-7, KERV-K-NMWV7, HML-8, HERV-K-NMWV3, HML-9, HERV-K-NMWV9, HML-10), HERV-W, HERV-E (e.g., 4-1, ERVA, NP-2), 51-1, RRHERV-I, HERV-T (e.g., S71, CRTK1, CRTK6), and ERV-FRD.

[0128] In some cases, heterologous gene regulators exhibit specific binding to genes (or target genes) encoding HERV-K. HERV-K is a family of 30 - 50 sequences that are highly conserved in primates. HERV-K proteins have protease enzymatic activity but do not have reverse transcriptase enzymatic activity. HERV-K is typically expressed at low steady-state levels in many human tissues and tumors.

[0129] In some cases, the heterologous gene regulator includes an endonuclease (e.g., Cas9) capable of forming a complex with a nucleic acid molecule. In some cases, the heterologous gene regulator further includes a gene regulatory factor. The gene regulatory factor can be a gene activator, a DNA-binding protein that has positive control over gene expression. Non-limiting examples of gene activators can include VP16, VP64, p65, p53, E2F1, TAT, E2A, NFAT, GAL4, CGN4, HAP1, MLL, TRG3, GLN3, OAF1, PIP2, PDR1, PDR3, PHO4, LEU3, RTA, and the VP64-p65-RTA fusion (VPR). The gene activator can be coupled to the endonuclease. The gene regulatory factor can be a gene repressor, a DNA-binding protein that has negative control over gene expression. Non-limiting examples of gene repressors can be the tetracycline repressor, the AMP early repressor (ICER), the Kruppel-associated box (KRAB), the glycine-rich repressor of YY1, the Sp1-like repressor, the E(spI) repressor, the IκB repressor, or MeCP2.

[0130] Aspects of the present disclosure provide engineered cells that are programmed to induce a desired level of expression and / or activity (or profile thereof) of one or more target genes in a cell.

[0131] One or more target genes targeted by the heterologous gene regulator as disclosed herein can include cell dedifferentiation factors (e.g., Yamanaka factors). In some cases, the one or more target genes can include Oct3, Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, HERV-K, EEA, ZSCAN4, DUX4, OTX2, ABCE1, COL5A1, GAL4NT13, DUXA, DUXB, ARGFX, CPHX1, CPHX2, TPRX1, DPPA3, NASP, ATP2B1, NFAT, H1FOO, and / or CAMKII.

[0132] In some cases, one or more target genes of a heterologous gene circuit can include OCT4, SOX2, KLF4, and / or MYC. In some cases, the heterologous gene circuit can be preconfigured such that (i) regulation of the expression level or epigenetic profile level of a first target endogenous gene from OCT4, SOX2, KLF4, and / or MYC occurs before (ii) regulation of the expression level or epigenetic profile level of a second target endogenous gene from OCT4, SOX2, KLF4, and / or MYC. For example, the heterologous gene circuit can be programmed such that activation of a first gate unit preconfigured to target and regulate the expression / epigenetic level of a first target endogenous gene can occur before activation of a second gate unit preconfigured to target and regulate the expression / epigenetic level of a second target endogenous gene (e.g., without human intervention or any secondary activation of the heterologous gene circuit after activation of the first gate unit or after activation of the heterologous gene circuit). The first target endogenous gene can be OCT4, and the second target endogenous gene can be SOX2, KLF4, and / or MYC. The first target endogenous gene can be SOX2, and the second target endogenous gene can be OCT4, KLF4, and / or MYC. The first target endogenous gene can be KLF4, and the second target endogenous gene can be OCT4, SOX2, and / or MYC. The first target endogenous gene can be MYC, and the second target endogenous gene can be OCT4, SOX2, and / or KLF4. Without wishing to be bound by theory, guiding and controlling the sequential regulation of target genes while minimizing intervention (e.g., human intervention) or disruption to the cell can (i) maintain cellular characteristics of the cell, such as viability, proliferative capacity, and / or therapeutic potency and / or (ii) enhance the desired outcome of cell engineering (e.g., cell reprogramming as provided herein).

[0133] One or more target genes can include HERV (e.g., HERV-K), POU family transcription factors (e.g., Oct4), KLF4, MYC, SOX2, EEA, and miR-302. In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) provided herein for a target gene can include a polynucleotide sequence (e.g., a 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 with one or more members selected from SEQ ID NO: 1-63 or its complementary sequences. In some cases, a heterologous gene regulator provided herein can exhibit specific binding to a target gene that includes a polynucleotide sequence (e.g., a 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 with one or more members selected from SEQ ID NO: 1-63 or its complementary sequences (e.g., with conversion of uracil to thymine).

[0134] One or more target genes can include HERV (e.g., HERV-K). In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) for a target gene as provided herein can include a polynucleotide sequence (e.g., a 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 SEQ ID NO: 1-12 or its complementary sequences. In some cases, a heterologous gene regulator as provided herein can exhibit specific binding to a target gene that includes a polynucleotide sequence (e.g., a 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 SEQ ID NO: 1-12 or its complementary sequences (e.g., with conversion of uracil to thymine).

[0135] One or more target genes can include POU family transcription factors (e.g., Oct4). OCT4 is a transcription factor in the POU family. OCT4 is involved in the self-renewal of undifferentiated embryonic stem cells and is a commonly used marker for undifferentiated cells. In some cases, a heterologous gene regulator as provided herein can exhibit specific binding to a target gene that includes a polynucleotide sequence (e.g., a 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 SEQ ID NO: 13-17 or its complementary sequence. In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) as provided herein can include a polynucleotide sequence (e.g., a 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 SEQ ID NO: 13-17 or its complementary sequence.

[0136] One or more target genes can include SOX2. SOX2 is involved in the self-renewal of undifferentiated embryonic stem cells and is a common marker for undifferentiated cells. In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) targeting a target gene as provided herein can include a polynucleotide sequence (e.g., a 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 with one or more members selected from SEQ ID NO: 42-58 or its complementary sequences. In some cases, a heterologous gene regulator as provided herein can exhibit specific binding to a target gene that includes a polynucleotide sequence (e.g., a 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 with one or more members selected from SEQ ID NO: 42-58 or its complementary sequences (e.g., with the conversion of uracil to thymine).

[0137] One or more target genes can include MYC (or C-Myc). C-Myc is a regulatory gene and transcription factor involved in cell proliferation. In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) provided herein for a target gene can include a polynucleotide sequence (e.g., a 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 SEQ ID NO: 30-41 or its complementary sequence. In some cases, a heterologous gene regulator provided herein can exhibit specific binding to a target gene that includes a polynucleotide sequence (e.g., a 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 SEQ ID NO: 30-41 or its complementary sequence (e.g., with conversion of uracil to thymine).

[0138] One or more target genes can include KLF4. KLF4, also known as Kruppel-like factor 4, is a zinc finger transcription factor involved in regulating proliferation, differentiation, apoptosis, and somatic cell reprogramming. In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) provided herein for a target gene can include a polynucleotide sequence (e.g., a 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 SEQ ID NO: 25-29 or its complementary sequences. In some cases, a heterologous gene regulator provided herein can exhibit specific binding to a target gene that includes a polynucleotide sequence (e.g., a 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 SEQ ID NO: 25-29 or its complementary sequences (e.g., with conversion of uracil to thymine).

[0139] One or more target genes can include miR-302. miR-302 is a polycistronic miRNA cluster that can induce and maintain pluripotency. In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) provided herein for a target gene can include a polynucleotide sequence (e.g., a 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 SEQ ID NO: 18-24 or its complementary sequences. In some cases, a heterologous gene regulator provided herein can exhibit specific binding to a target gene that includes a polynucleotide sequence (e.g., a 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 SEQ ID NO: 18-24 or its complementary sequences (e.g., with conversion of uracil to thymine).

[0140] One or more target genes can include EEA. In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) for a target gene as provided herein can include a polynucleotide sequence (e.g., a 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 SEQ ID NO: 59 - 63 or its complementary sequences. In some cases, a heterologous gene regulator as provided herein can exhibit specific binding to a target gene that includes a polynucleotide sequence (e.g., a 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 SEQ ID NO: 59 - 63 or its complementary sequences (e.g., with conversion of uracil to thymine).

[0141] In some cases, contacting multiple cells with a heterologous gene regulator can dedifferentiate the cells into iPSCs. Using a heterologous gene regulator as disclosed herein can result in dedifferentiation of up to about 1x10 6 、9x10 5 、8x10 5 、7x10 5 、6x10 5 、5x10 5 、4x10 5 、3x10 5 、2x10 5 、1x10 5 、5x10 4 、2x10 4, 1 x 10 4 or fewer terminally differentiated cells generate at least about 1 x 10 4 , 2 x 10 4 , 5 x 10 4 , 1 x 10 5 , 2 x 10 5 , 5 x 10 5 , 1 x 10 6 , 2 x 10 6 , 5 x 10 6 , 1 x 10 7 , 2 x 10 7 , 5 x 10 7 , 1 x 10 8 , 2 x 10 8 , 5 x 10 8 , 1 x 10 9 , 2 x 10 9 , 5 x 10 9 , 1 x 10 10 , 2 x 10 10 , 5 x 10 10 , 1 x 10 15 , 2 x 10 15 , 5 x 10 15 or more iPSCs.

[0142] Such generation of iPSCs by using a heterologous gene regulator as disclosed herein can be achieved within a span of up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 20 days, up to about 25 days, up to about 30 days, up to about 35 days, up to about 40 days, up to about 45 days, up to about 50 days, up to about 55 days, up to about 60 days or up to about 65 days.

[0143] Converting a plurality of cells of a first cell type into a plurality of cells of a second cell type (e.g., reprogramming differentiated cells into stem cells, such as pluripotent stem cells) can be characterized as having an efficiency of conversion (or conversion rate) of 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 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95%, at least or up to about 99%, or about 100%.

[0144] In some cases, a heterologous gene regulator and an additional heterologous gene regulator are introduced into the cell substantially simultaneously. In some cases, the heterologous gene regulator is introduced before the additional heterologous gene regulator. In some cases, the heterologous gene regulator is introduced after the additional heterologous gene regulator.

[0145] In some cases, the heterologous gene regulator and the additional heterologous gene regulator may or may not be part of the same heterologous gene circuit. The heterologous gene regulator and the additional heterologous gene regulator may be part of different heterologous gene circuits. Alternatively, neither the heterologous gene regulator nor the additional heterologous gene regulator may be part of any heterologous gene circuit.

[0146] In some cases, one or more cells may additionally be contacted with an inhibitor. In some cases, one or more cells may be contacted with the inhibitor before the introduction of the heterologous gene regulator. Alternatively, one or more cells may be contacted with the inhibitor simultaneously with the introduction of the heterologous gene regulator. Alternatively, one or more cells may be contacted with the inhibitor after the introduction of the heterologous gene regulator. An inhibitor is a gene whose presence prevents the expression of another gene at a different locus. The inhibitor may be a p53 inhibitor, for example in the form of a small molecule drug or p53 short hairpin RNA (shRNA).

[0147] In some cases, a target gene can be subjected to one (e.g., a single) gene regulation (e.g., a single targeted activation step). In some cases, the target gene can be subjected to at least two heterologous gene regulators including a first regulator that effects a first regulation and a second (or additional) regulator that effects a second (or additional) regulation. The timing of the first regulation and the second regulation can be controlled (e.g., as predetermined by the design of the heterologous gene circuit). For example, the initiation of the second regulation (e.g., by at least a portion of a second gate unit, such as a second gene regulatory portion) can occur at least about 1 second, at least about 2 seconds, at least about 3 seconds, at least about 4 seconds, at least about 5 seconds, at least about 6 seconds, at least about 7 seconds, at least about 8 seconds, at least about 9 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days or at least about 10 days after the initiation of the first regulation (e.g., by at least a portion of a first gate unit, such as a first gene regulatory portion). The initiation of the second regulation (e.g., by at least a portion of a second gate unit, such as a second gene regulatory portion) can occur at most about 10 days, at most about 9 days, at most about 8 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, at most about 20 hours, at most about 10 hours, at most about 9 hours, at most about 8 hours, at most about 7 hours, at most about 6 hours, at most about 5 hours, at most about 4 hours, at most about 3 hours, at most about 2 hours, at most about 1 hour, at most about 50 minutes, at most about 40 minutes, at most about 30 minutes, at most about 20 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6 minutes, at most about 5 minutes, at most about 4 minutes, at most about 3 minutes, at most about 2 minutes, at most about 1 minute, at most about 50 seconds, at most about 40 seconds, at most about 30 seconds, at most about 20 seconds, at most about 10 seconds, at most about 9 seconds, at most about 8 seconds, at most about 7 seconds, at most about 6 seconds, at most about 5 seconds, at most about 4 seconds, at most about 3 seconds, at most about 2 seconds or at most about 1 second after the initiation of the first regulation (e.g., by at least a portion of a first gate unit, such as a first gene regulatory portion).

[0148] In some cases, the number of gate units that need to be activated (e.g., sequentially activated) between the activation of a first regulation by a first gate unit and the subsequent activation of a second regulation by a second gate unit can at least partially determine (e.g., substantially determine) the timing between the first regulation and the second regulation. When activating a first regulation of a target gene by a first gate unit, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more additional gate units may need to be activated (e.g., sequentially activated) to activate the second gate unit for inducing the second regulation. When activating a first regulation of a target gene by a first gate unit, 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 additional gate unit may need to be activated (e.g., sequentially activated) to activate the second gate unit for inducing the second regulation.

[0149] The results of the cells can include the regulation of multiple target genes. For example, the results can include the regulation of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more target genes. The results can include the regulation 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. 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 regulations can be performed on each gene disclosed herein. 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 regulation can be performed on each gene disclosed herein. One or more regulations of a target gene (e.g., an endogenous gene) induced by a heterologous gene circuit disclosed herein can be an artificial regulation (or heterologous regulation), which otherwise may not occur in a cell in the absence of (i) a heterologous gene regulator and / or (ii) an activating portion of a heterologous gene regulator.

[0150] A first different regulator can induce a change (e.g., an increase or decrease) in the expression and / or activity level of a target gene (e.g., HERV-K) by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500% or more. The first different regulation can induce a change (e.g., an increase or decrease) in the expression and / or activity level of a target gene (e.g., HERV-K) 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.

[0151] Compared to a control expression and / or activity level, a first different modulation (e.g., induced by a first gate unit) as disclosed herein can induce a change (e.g., an increase or decrease) in the expression and / or activity level of a target gene to 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. Compared to a control expression and / or activity level, the first different modulation can induce a change (e.g., an increase or decrease) in the expression and / or activity level of a target gene to at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5-fold, at most or less than about 4-fold, at most or less than about 3-fold, at most or less than about 2-fold, at most or less than about 1-fold, at most or less than about 0.9-fold, at most or less than about 0.8-fold, at most or less than about 0.7-fold, at most or less than about 0.6-fold, at most or less than about 0.5-fold, at most or less than about 0.4-fold, at most or less than about 0.3-fold, at most or less than about 0.2-fold, or at most or less than about 0.1-fold.

[0152] Subsequently, a second different modulation as disclosed herein (e.g., induced by a second gate unit) can induce an additional change (e.g., increase, decrease, or alternative attenuation) in the expression and / or activity level of a target gene of at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, 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%. The second different modulation can induce an additional change (e.g., increase or decrease) in the expression and / or activity level of the target gene of 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%, or at most about 0.1%.

[0153] Compared to the control expression and / or activity level, additional alterations via a second different regulation can induce an additional change (e.g., increase or decrease) in the expression and / or activity level of the target gene to 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. Compared to the control expression and / or activity level, the second different regulation can induce an additional change (e.g., increase or decrease) in the expression and / or activity level of the target gene to at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5-fold, at most or less than about 4-fold, at most or less than about 3-fold, at most or less than about 2-fold, at most or less than about 1-fold, at most or less than about 0.9-fold, at most or less than about 0.8-fold, at most or less than about 0.7-fold, at most or less than about 0.6-fold, at most or less than about 0.5-fold, at most or less than about 0.4-fold, at most or less than about 0.3-fold, at most or less than about 0.2-fold, or at most or less than about 0.1-fold.

[0154] When the expression and / or activity level of the target gene reaches the target level via the action of a first different regulation (e.g., through the design of a heterologous gene circuit), additional alterations via a second different regulation can occur.

[0155] When the expression and / or activity level of a target gene is altered (e.g., increased or decreased) by the action of a first distinct modulation to 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 compared to a control expression and / or activity level, an additional alteration via a second distinct modulation can occur. When the expression and / or activity level of a target gene is altered (e.g., increased or decreased) by the action of a first distinct modulation to at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5-fold, at most or less than about 4-fold, at most or less than about 3-fold, at most or less than about 2-fold, at most or less than about 1-fold, at most or less than about 0.9-fold, at most or less than about 0.8-fold, at most or less than about 0.7-fold, at most or less than about 0.6-fold, at most or less than about 0.5-fold, at most or less than about 0.4-fold, at most or less than about 0.3-fold, at most or less than about 0.2-fold, or at most or less than about 0.1-fold compared to a control expression and / or activity level, an additional alteration via a second distinct modulation can occur.

[0156] Alternatively or additionally, a second different modulation as disclosed herein (e.g., induced by a second gate unit) can induce an alteration (e.g., increase or decrease) in the expression and / or activity level of an additional 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% or at least about 1,000,000%. The second different modulation can induce an alteration (e.g., increase or decrease) in the expression and / or activity level of an additional 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% or at most about 0.1%. The cells can include prokaryotic cells, eukaryotic cells or artificial cells.

[0157] Stem cells (e.g., iPSCs) generated by the systems and methods of the present disclosure may be capable of differentiating into cell types found in one or more tissues. Non-limiting examples of such tissues may include skin, heart, lung, kidney, bone, cartilage, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, blood, serum, reproductive organs, and the like.

[0158] The engineered cells of the present disclosure (e.g., engineered iPSCs) or any further engineered variants thereof (e.g., differentiation by-products of such iPSCs) can be used (e.g., administered) to a subject in need. The subject may have or may be suspected of having a condition, such as a disease (e.g., cancer). Cells (e.g., differentiated cells) can be obtained from the subject, and such cells can be cultured ex vivo and genetically modified to generate iPSCs as disclosed herein. Subsequently, the engineered iPSCs can be administered to the subject for adoptive immunotherapy. Thus, the engineered cells or any further engineered variants thereof can be autologous to the subject in need. Alternatively, the engineered cells or any further engineered variants thereof can be allogeneic to the subject (e.g., allogeneic stem cell transplantation, allogeneic adoptive immunotherapy, etc.).

[0159] The engineered cells of the present disclosure or any further engineered variants thereof can be administered to a subject before, simultaneously with, or after activation of the heterologous gene circuit and / or heterologous gene regulator in the engineered stem cells. For example, the engineered cells or any further engineered variants thereof can be activated after being administered into the subject, e.g., by administering an activator of the heterologous gene circuit to the subject.

[0160] An object can be treated (e.g., administered) with the engineered cells (e.g., engineered iPSCs) of the present disclosure or any group of their further engineered variants with 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, an object can be treated (e.g., administered) with the engineered cells (e.g., engineered iPSCs) of the present disclosure or any group of their further engineered variants 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.

[0161] Any of the methods disclosed herein can be utilized to treat target cells, target tissues, target conditions, or target diseases of an object.

[0162] Non-limiting examples of objects include humans, dogs, cats, mice, rats, and their transgenic species. Examples of samples from an object from which cells can be derived include, but are not limited to, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric juice and digestive fluid, tears, feces, semen, vaginal fluid, interstitial fluid derived from tumor tissue, eye 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, fetal water, amniotic fluid, cord blood, emphatic fluid, cavity fluid, sputum, pus, microbiota, meconium, breast milk, and / or other excretions or body tissues.

[0163] The target disease of the subject can be cancer or a tumor. Non-limiting examples of cancers can include cancer cells of cancers including acanthoma, acinar cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloid leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendiceal cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary, carcinoid tumor, carcinoma, carcinoma in situ, carcinoma of the penis, carcinoma of unknown primary, carcinosarcoma, Castleman's Disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease (Degosdisease), dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial carcinoma, endometroid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, olfactory neuroblastoma, Ewing family tumors, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget disease, fallopian tube cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma, hemangiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, lentigo maligna melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel Cell Carcinoma, mesothelioma, mesothelioma, metastatic squamous neck cancer with occultPrimary), metastatic urothelial carcinoma, mixed Mullerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplasia, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular oncology, oligodendroastrocytoma, oligodendroglioma, oxyphilic cell tumor, 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, superior sulcus tumor of the lung, 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-lymphocyte 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 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 (Sootwart), spinal cord tumors, spinal tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficially spreading melanoma, supratentorial primitive neuroectodermal tumors, surface epithelial stromal tumors, 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, theca cell tumor, laryngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, urogenital neoplasm, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subset 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 lymphoma. The antigen can be a tumor-associated antigen.

[0164] The present disclosure also provides a composition comprising an engineered gene regulator and / or an engineered gene circuit as disclosed herein. The composition can further comprise an actuator of a heterologous gene circuit. The present disclosure also provides a kit comprising the composition. The kit can further comprise an activator of the heterologous gene circuit. The activator can be in the same composition as the engineered gene regulator and / or the engineered gene circuit. Alternatively or additionally, the activator can be in a composition that is different and separate from the engineered gene regulator and / or the engineered gene circuit.

[0165] Examples

[0166] Example 1: Heterologous Gene Circuit for Transforming Cell Types via Modulating Endogenous Genes

[0167] Modulation of one or more endogenous genes (e.g., regulating their expression or activity) can be used to transform cells of a first cell type (the first plurality of cells) into a plurality of cells of a second cell type (the second plurality of cells). In some cases, such modulation can transform differentiated cells (e.g., terminally differentiated cells, such as primary fibroblasts) into more stem cell-like cells (e.g., induced pluripotent stem cells or "iPSCs"). In some cases, the one or more genes can include one or more members from the pluripotency regulatory gene network (PGRN) genes, such as Figure 2As shown. Alternatively or additionally, one or more genes can be non-PGRN genes (e.g., human endogenous retrovirus (HERV), embryo genome activation (EGA) enriched Alu motif (EEA), etc.). In some cases, HERV can include HERV-K. In some cases, targeting HERV-K can include targeting regulatory elements of HERV-K, such as LTR5HS.

[0168] In some cases, one or more genes can each be regulated one or more times (e.g., sequentially). In some cases, one or more genes can include multiple different genes, and the multiple different genes can be regulated substantially simultaneously or sequentially.

[0169] In some cases, one or more heterologous gene regulators can be used to induce the regulation of one or more endogenous genes to achieve transformation. For example, the heterologous gene regulator can include a guide nucleic acid that exhibits specific binding to an endogenous gene (e.g., coding region or non-coding region), and the guide nucleic acid can form a complex with a Cas protein or its variant to induce such regulation. In some cases, the heterologous gene regulator can be part of a heterologous gene circuit, as provided herein.

[0170] Heterologous gene circuit

[0171] The heterologous gene circuit (HGC) can be designed such that each heterologous gene circuit (e.g., except for the control heterologous gene circuit) can sequentially activate multiple heterologous gene regulators to target multiple endogenous genes in primary fibroblasts to achieve the transformation (e.g., reprogramming) of primary fibroblasts into iPSCs. Table 1 shows a library of different HGCs for targeting various endogenous genes. The endogenous genes include EEA, Oct4 (O), Klf4 (K), c-Myc (M), Sox2 (S), and miR-302 (e.g., an inhibitor of p53; denoted as "miRNA" herein).

[0172] The HGC can be designed such that a guide nucleic acid containing a spacer sequence that exhibits specific binding to a designated target endogenous gene can be activated (e.g., expressed) at the corresponding step. Examples of spacer sequences for endogenous genes are provided in Table 2. The guide nucleic acid is also designed with a scaffold sequence to allow formation of a complex with a Cas-based gene activator (e.g., dCas9-VPR) to specifically bind and activate the target gene.

[0173] In some cases, one guide nucleic acid sequence having one spacer sequence for a target gene can be used to regulate the target gene. In some cases, multiple (or a pool) of nucleic acid sequences having different spacer sequences for a common target gene can be used to regulate the target gene (e.g., multiplexing).

[0174] Cells (e.g., primary fibroblasts) can be transfected with an HGC plasmid and a gene encoding a Cas-based gene activator. Subsequently, the cells can be transfected with the activator for each HGC, thereby activating the HGC and triggering sequential gene regulation.

[0175] In some cases, the cells can also be transfected with additional gene regulatory factors that are not part of the HGC. For example, cells (e.g., primary fibroblasts) can be transfected with a gene encoding a short hairpin RNA (shRNA) against p53 (denoted herein as "p53"), e.g., to promote or enhance p53 regulation.

[0176] Table 1: Heterologous gene circuits.

[0177]

[0178] Table 2: Heterologous gene regulators (e.g., guide nucleic acids)

[0179]

[0180]

[0181] Example 2: Reprogramming primary fibroblasts to iPSCs by targeting endogenous genes (e.g., EEA and / or miR-302)

[0182] According to Example 1, the HGC was designed to reprogram primary fibroblasts to iPSCs. Figure 3 A library of different HGCs tested was shown, each HGC being designed to be able to sequentially target one or more endogenous genes through four steps (Step 1, Step 2, Step 3, and Step 4). HGC#1 and 2 were designed as controls, and HGC#3-9 were designed to test the sequential targeting of endogenous genes in various combinations and orders.

[0183] Reprogram primary fibroblasts into iPSCs by targeting, for example, EEA and / or miR-302.

[0184] Primary fibroblasts were transfected with the corresponding HGC plasmid or treated as a control (e.g., cells only without any treatment, nucleofector alone, etc.), and then observed (e.g., for 5 days). Each condition had sufficient replicates (e.g., n = 4) at one or more time points for evaluation (e.g., via RNA isolation or staining to evaluate the expression levels of target endogenous genes and / or embryonic development markers such as Nanog). Visual assessment of colony formation / expansion was also performed on the cells, which indicated the reprogramming of fibroblasts to iPSCs.

[0185] For transfection, each condition also included a gene encoding GFP to track transfection efficiency. AsFigure 4 Neutralization Figure 5 As shown at the top, the transfection efficiency for this condition is sufficient (e.g., greater than 80%). At Figure 4 The three controls shown from left to right at the top correspond to conditions #1-3 in Table 1, respectively.

[0186] As Figure 5 at the bottom and Figure 3 as shown in the comparison results in the rightmost column, compared with other HGC conditions, HGC#6 produced the highest iPSC reprogramming rate, as determined by the colony formation rate (e.g., after 5 days).

[0187] As Figure 3 shown, sequential activation of endogenous genes (e.g., condition 6) is more effective (e.g., up to 4-fold) in iPSC reprogramming than activating endogenous genes substantially together (e.g., condition 2).

[0188] As Figure 3 shown, sequential (e.g., prior) activation of EEA is more effective in iPSC reprogramming than activating EEA and at least one of the other genes (e.g., O, K, M, and / or S) substantially together (e.g., conditions 7, 8, and 9). Additionally, even when EEA is sequentially (e.g., prior) activated with any one of the other genes, sequential activation of the other genes (e.g., condition 6) is more effective in iPSC reprogramming than activating all of the other genes substantially together (e.g., conditions 3-5).

[0189] As Figure 3 shown, sequential activation of M and / or S twice (e.g., conditions 6, 7, and 8) is more effective in iPSC reprogramming than activating S only once (e.g., conditions 2, 3, 4, and 5).

[0190] Example 3: Reprogramming of primary fibroblasts into iPSCs by targeting endogenous genes (e.g., HERV)

[0191] Target HERV and EEA

[0192] According to Example 1 and similar to the procedures described in Example 2, HGC condition #4 was used to target and regulate HERV (e.g., HERV-K) and EEA to reprogram primary fibroblasts into iPSCs. At the time of transfection and subsequent culture (e.g., 5 days), targeting HERV and EEA in the presence of anti-p53 shRNA was sufficient to induce iPSC colony formation ( Figure 6 ).

[0193] Target HERV

[0194] According to the procedures described in Example 1 and similar to those in Example 2, HGC can be designed to reprogram primary fibroblasts into iPSCs. HGC can be designed to sequentially activate multiple genes. In some cases, genes among the multiple genes can be targeted via multiplexed guide nucleic acid targeting (e.g., a single gate unit containing multiple different gene regulatory parts proGuide for targeting different regions of the gene), while at least one additional gene among the multiple genes can be targeted via single nucleic acid targeting (e.g., a single gate unit containing a single gene regulatory part proGuide for targeting a single region of the additional gene).

[0195] Briefly, primary fibroblasts were transfected with the corresponding HGC plasmids to regulate the following endogenous genes: Oct4 (O), Klf4 (K), c-Myc (M), Sox2 (S), and HERV-K elements (e.g., LTR5HS; denoted as "H" herein).

[0196] Table 3 shows a library of different HGCs tested, each HGC being designed to be able to sequentially target one or more endogenous genes through four steps (Step 1, Step 2, Step 3, and Step 4). HGC#1-1 to 1-3 were designed as controls lacking HERV regulation. HGC#2-1 to 2-4 were designed to test the sequential targeting of endogenous genes (including HERV) in various combinations and orders. Here, at each corresponding step of the HGC, KLF4 or MYC (e.g., KLF4 and MYC) was activated with a single guide nucleic acid target, while the other genes were activated via multiplexed guide nucleic acid targeting.

[0197] Primary fibroblasts (e.g., approximately 500k fibroblasts per well) were transfected with the corresponding HGC plasmids. In addition, fibroblasts under each condition were also transfected with multiple plasmids, each plasmid encoding (i) Cas9 VPR, anti-p53 shRNA, miR-302, and GFP. After subsequent culture (e.g., 7 days), the cells were visually evaluated to identify the number of colonies showing iPSC-like morphology, which indicates iPSC reprogramming.

[0198] As shown in the last column of Table 3, activating HERV-K activation (e.g., condition 2-4) without activating other OSKM factors was able to generate iPSC morphology colonies.

[0199] As shown in the last column of Table 3, activating HERV-K followed by delayed activation of other OSKM factors (e.g., condition 2-2) was more effective in iPSC reprogramming than delayed activation of other OSKM factors without prior HERV-K activation (e.g., condition 1-2).

[0200] As shown in the last column of Table 3, substantially simultaneous activation of HERV-K and O (e.g., Condition 2-3) is less effective for iPSC reprogramming than comparable sequential activation of the OSKM factors in the absence of HERV-K activation (e.g., Condition 1-3). Similarly, substantially simultaneous activation of HERV-K and other OSKM factors (e.g., Condition 2-1) is ineffective for iPSC reprogramming, similar to comparable activation of the OSKM factors in the absence of HERV-K activation (e.g., Condition 1-1).

[0201] Individually, in the absence of HERV-K activation, sequential activation of the OSKM factors (e.g., Condition 1-3) is more effective for iPSC reprogramming than substantially simultaneous activation of all the OSKM factors (e.g., Conditions 1-1 and 1-2).

[0202] Table 3: Heterologous gene circuits.

[0203] Condition # Step 1 Step 2 Step 3 Step 4 Number of iPSC morphological colonies 1-1 OSKM - - - 0 1-2 - OSKM - - 2 1-3 O KM S S 8 2-1 H + OSKM - - - 0 2-2 H OSKM - - 5 2-3 H + O KM S S 0 2-4 H - - - 2

[0204] Embodiments

[0205] The following non-limiting embodiments provide illustrative examples of the invention but do not limit the scope of the invention.

[0206] Embodiment 1. A method for converting a plurality of cells of a first cell type (first plurality of cells) into a plurality of cells of a second cell type (second plurality of cells), the method comprising:

[0207] Contacting the first plurality of cells with a heterologous gene regulator that specifically binds to a gene encoding HERV to regulate the expression level or epigenetic profile of HERV and effect the conversion from the first plurality of cells to the second plurality of cells,

[0208] Optionally wherein:

[0209] (1) (i) the first plurality of cells comprise terminally differentiated cells and (ii) the second plurality of cells comprise pluripotent stem cells; and / or

[0210] (2) the contacting enhances the expression level of HERV; and / or

[0211] (3) the heterologous gene regulator specifically binds to a gene encoding HERV-K; and / or

[0212] (4) the heterologous gene regulator specifically binds to a gene encoding LTR5HS,

[0213] Further optionally wherein:

[0214] (a) The heterologous gene regulator exhibits specific binding to a polynucleotide sequence that exhibits at least about 80% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences, optionally with conversion of uracil to thymine; and / or

[0215] (b) The heterologous gene regulator exhibits specific binding to a polynucleotide sequence that exhibits at least about 90% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences, optionally with conversion of uracil to thymine; and / or

[0216] (c) The heterologous gene regulator comprises a polynucleotide sequence that exhibits at least about 80% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences; and / or

[0217] (d) The heterologous gene regulator comprises a polynucleotide sequence that exhibits at least about 90% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences; and / or

[0218] (5) The contacting effects or is sufficient to effect the conversion without using an additional heterologous gene regulator that exhibits specific binding to an additional target gene, the additional target gene comprising one or more cell dedifferentiation factors selected from OCT4, SOX2, KLF4, and MYC,

[0219] Optionally further wherein the additional target gene comprises OCT4, SOX2, KLF4, and MYC; and / or

[0220] (6) The method further comprises contacting the first plurality of cells with an additional heterologous gene regulator that exhibits specific binding to an additional gene, the additional gene comprising one or more cell dedifferentiation factors selected from OCT4, SOX2, KLF4, and MYC, to effect sequential regulation of the gene and the additional gene,

[0221] Optionally further wherein:

[0222] (a) Regulation of the gene occurs before, simultaneously with, or after regulation of the additional gene; and / or

[0223] (b) Regulation of the gene occurs before regulation of the additional gene; and / or

[0224] (c) The additional gene includes OCT4; and / or

[0225] (7) The method further includes contacting the first plurality of cells with an additional heterologous gene regulator that exhibits specific binding to an Alu motif enriched in embryonic genome activation (EGA) (EEA) to effect the transformation; and / or

[0226] (8) The contacting includes contacting the first plurality of 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 regulate the expression levels or epigenetic profiles of the gene and different genes in a sequential manner to effect the transformation, and wherein the plurality of gate units includes:

[0227] (i) A first gate unit that includes the heterologous gene regulator, wherein the first gate unit is preconfigured to regulate the expression level or epigenetic profile of the gene; and

[0228] (ii) A second gate unit that is preconfigured to regulate the expression level or epigenetic profile of the different gene,

[0229] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the transformation,

[0230] Further optionally wherein:

[0231] (a) The heterologous gene circuit is preconfigured to regulate the expression level of the epigenetic profile of the gene before regulating the expression level of the epigenetic profile of the different gene; and / or

[0232] (b) The different gene includes one or more members selected from the following: Oct3, Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, EEA, ZSCAN4, DUX4, OTX2, ABCE1, COL5A1, GAL4NT13, DUXA, DUXB, ARGFX, CPHX1, CPHX2, TPRX1, DPPA3, NASP, ATP2B1, NFAT, H1FOO, and CAMKII; and / or

[0233] (c) The different gene includes one or more members selected from the following: Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, and EEA; and / or

[0234] (9) The heterologous gene regulator includes a heterologous endonuclease for the specific binding to the gene encoding HERV

[0235] Further optionally wherein:

[0236] (a) The heterologous endonuclease is a Cas protein; and / or

[0237] (b) The heterologous gene regulator further comprises a gene activator; and / or

[0238] (c) The gene activator is selected from VP16, VP64, p65, RTA, and VP64-p65-RTA fusion (VPR); and / or

[0239] (d) The gene activator is coupled to the heterologous endonuclease.

[0240] Embodiment 2. A system for converting a plurality of cells of a first cell type (first plurality of cells) into a plurality of cells of a second cell type (second plurality of cells), the system comprising:

[0241] A heterologous gene regulator that exhibits specific binding to a gene encoding a HERV to regulate the expression level or epigenetic profile of the HERV and effect conversion from the first plurality of cells to the second plurality of cells,

[0242] Optionally wherein:

[0243] (1)(i) The first plurality of cells comprise terminally differentiated cells and (ii) the second plurality of cells comprise pluripotent stem cells; and / or

[0244] (2) The specific binding of the heterologous gene regulator to the gene effects an enhanced expression level of the HERV; and / or

[0245] (3) The heterologous gene regulator exhibits specific binding to a gene encoding HERV-K; and / or

[0246] (4) The heterologous gene regulator exhibits specific binding to a gene encoding LTR5HS,

[0247] Further optionally wherein:

[0248] (a) The heterologous gene regulator exhibits specific binding to a polynucleotide sequence that exhibits at least about 80% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NOs: 1-12 and their complementary sequences, optionally with uracil to thymine conversion; and / or

[0249] (b) The heterologous gene regulator exhibits specific binding to a polynucleotide sequence that exhibits at least about 90% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences, optionally with conversion of uracil to thymine; and / or

[0250] (c) The heterologous gene regulator comprises a polynucleotide sequence that exhibits at least about 80% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences; and / or

[0251] (d) The heterologous gene regulator comprises a polynucleotide sequence that exhibits at least about 90% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences; and / or

[0252] (5) The specific binding of the heterologous gene regulator to the gene is configured or sufficient to effect the conversion without using an additional heterologous gene regulator that exhibits specific binding to an additional target gene, the additional target gene comprising one or more cell dedifferentiation factors selected from OCT4, SOX2, KLF4, and MYC,

[0253] further optionally wherein the additional target gene comprises OCT4, SOX2, KLF4, and MYC; and / or

[0254] (6) The system further comprises an additional heterologous gene regulator that exhibits specific binding to an additional gene, the additional gene comprising one or more cell dedifferentiation factors selected from OCT4, SOX2, KLF4, and MYC, to effect sequential regulation of the gene and the additional gene,

[0255] further optionally wherein:

[0256] (a) Regulation of the gene occurs before, simultaneously with, or after regulation of the additional gene; and / or

[0257] (b) Regulation of the gene occurs before regulation of the additional gene; and / or

[0258] (c) The additional gene comprises OCT4; and / or

[0259] (7) The system further comprises an additional heterologous gene regulator that exhibits specific binding to an Alu motif enriched in embryonic genome activation (EGA) (EEA) to effect the conversion; and / or

[0260] (8) The system includes 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 the expression levels or epigenetic profiles of the gene and different genes in a sequential manner to effect the transformation, and wherein the plurality of gate units includes:

[0261] (i) A first gate unit that includes the heterologous gene regulator, wherein the first gate unit is preconfigured to regulate the expression level or epigenetic profile of the gene; and

[0262] (ii) A second gate unit that is preconfigured to regulate the expression level or epigenetic profile of the different gene,

[0263] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the transformation,

[0264] Further optionally wherein:

[0265] (a) The heterologous gene circuit is preconfigured to regulate the expression level of the epigenetic profile of the gene before regulating the expression level of the epigenetic profile of the different gene; and / or

[0266] (b) The different gene includes one or more members selected from the following: Oct3, Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, EEA, ZSCAN4, DUX4, OTX2, ABCE1, COL5A1, GAL4NT13, DUXA, DUXB, ARGFX, CPHX1, CPHX2, TPRX1, DPPA3, NASP, ATP2B1, NFAT, H1FOO, and CAMKII; and / or

[0267] (c) The different gene includes one or more members selected from the following: Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, and EEA; and / or

[0268] (9) The heterologous gene regulator includes a heterologous endonuclease for specific binding to the gene encoding HERV,

[0269] Further optionally wherein:

[0270] (a) The heterologous endonuclease is a Cas protein; and / or

[0271] (b) The heterologous gene regulator further includes a gene activator; and / or

[0272] (c) The gene activator is selected from VP16, VP64, p65, RTA, and the VP64-p65-RTA fusion (VPR); and / or

[0273] (d) The gene activator is conjugated to the heterologous endonuclease.

[0274] Embodiment 3. A method for converting a plurality of differentiated cells into a plurality of stem cells, the method comprising:

[0275] Contacting the plurality of differentiated 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 regulate the expression levels or epigenetic profiles of a plurality of different target endogenous genes in a sequential manner to effect the conversion, and wherein the plurality of gate units comprise:

[0276] (i) A first gate unit preconfigured to regulate the expression level or epigenetic profile of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene comprises an embryo genome activation (EGA)-enriched Alu motif (EEA); and

[0277] (ii) A second gate unit preconfigured to regulate the expression level or epigenetic profile of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene comprises a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC,

[0278] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion,

[0279] Optionally wherein:

[0280] (1) The plurality of stem cells comprise a plurality of pluripotent stem cells; and / or

[0281] (2) The heterologous gene circuit is preconfigured such that upon activation of the heterologous gene circuit, (i) the expression level or epigenetic profile of the first target endogenous gene is regulated before (ii) the expression level or epigenetic profile of the second target endogenous gene; and / or

[0282] (3) The plurality of gate units comprise a third gate unit preconfigured to regulate the expression level or epigenetic profile of a third target endogenous gene among the plurality of different target endogenous genes, wherein the third target endogenous gene comprises a different cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC,

[0283] Wherein, when activating the heterologous gene circuit, the expression levels or epigenetic profiles of the cell dedifferentiation factor and the different cell dedifferentiation factors are sequentially regulated,

[0284] Further optionally, wherein:

[0285] (a) The second target endogenous gene includes OCT4; and / or

[0286] (b) The third target endogenous gene includes SOX2; and / or

[0287] (4) The plurality of gate units includes a third gate unit that is preconfigured to regulate the expression level or epigenetic profile of the second target endogenous gene,

[0288] Wherein activating the heterologous gene circuit achieves two or more sequential regulations of the expression level or epigenetic profile of the cell dedifferentiation factor,

[0289] Further optionally, wherein:

[0290] (a) The cell dedifferentiation factor is MYC; and / or

[0291] (b) The cell dedifferentiation factor is SOX2; and / or

[0292] (5) The cell dedifferentiation factor includes OCT4; and / or

[0293] (6) The cell dedifferentiation factor includes SOX2; and / or

[0294] (7) The cell dedifferentiation factor includes KLF4; and / or

[0295] (8) The cell dedifferentiation factor includes MYC.

[0296] Embodiment 4. A system for converting a plurality of differentiated cells into a plurality of stem cells, the system comprising:

[0297] 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 or epigenetic profiles of a plurality of different target endogenous genes to effect the conversion, wherein the plurality of gate units includes:

[0298] (i) A first gate unit that is preconfigured to regulate the expression level or epigenetic profile of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene includes an Alu motif enriched in embryonic genome activation (EGA) (EEA); and

[0299] (ii) A second gate unit, which is preconfigured to regulate the expression level or epigenetic profile of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene includes a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC,

[0300] Wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the transformation,

[0301] Optionally wherein:

[0302] (1) The plurality of stem cells includes a plurality of pluripotent stem cells; and / or

[0303] (2) The heterologous gene circuit is preconfigured such that upon activation of the heterologous gene circuit, (i) the expression level or epigenetic profile of the first target endogenous gene is regulated before (ii) the expression level or epigenetic profile of the second target endogenous gene; and / or

[0304] (3) The plurality of gate units includes a third gate unit, which is preconfigured to regulate the expression level or epigenetic profile of a third target endogenous gene among the plurality of different target endogenous genes, wherein the third target endogenous gene includes a different cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC,

[0305] Wherein, upon activation of the heterologous gene circuit, the expression levels or epigenetic profiles of the cell dedifferentiation factor and the different cell dedifferentiation factor are sequentially regulated,

[0306] Further optionally wherein:

[0307] (a) The second target endogenous gene includes OCT4; and / or

[0308] (b) The third target endogenous gene includes SOX2; and / or

[0309] (4) The plurality of gate units includes a third gate unit, which is preconfigured to regulate the expression level or epigenetic profile of the second target endogenous gene,

[0310] Wherein activation of the heterologous gene circuit effects two or more sequential regulations of the expression level or epigenetic profile of the cell dedifferentiation factor,

[0311] Further optionally wherein:

[0312] (a) The cell dedifferentiation factor is MYC; and / or

[0313] (b) The cell dedifferentiation factor is SOX2; and / or

[0314] (5) The cell dedifferentiation factors include OCT4; and / or

[0315] (6) The cell dedifferentiation factors include SOX2; and / or

[0316] (7) The cell dedifferentiation factors include KLF4; and / or

[0317] (8) The cell dedifferentiation factors include MYC.

[0318] Embodiment 5. A method for converting a plurality of differentiated cells into a plurality of stem cells, the method comprising:

[0319] Contacting the plurality of differentiated 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 regulate the expression levels or epigenetic profile levels of a plurality of different target endogenous genes in a sequential manner to effect the conversion, and wherein the plurality of gate units comprise:

[0320] (i) A first gate unit preconfigured to regulate the expression level or epigenetic profile level of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene comprises a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC; and

[0321] (ii) A second gate unit preconfigured to regulate the expression level or epigenetic profile level of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene comprises a different cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC,

[0322] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion,

[0323] Optionally wherein:

[0324] (1) The heterologous gene circuit is preconfigured such that activation of the first gate unit effects activation of the second gate unit, such that (i) the regulation of the expression level or epigenetic profile level of the first target endogenous gene occurs before (ii) the regulation of the expression level or epigenetic profile level of the second target endogenous gene,

[0325] Further optionally wherein the first target endogenous gene is OCT4, such that the regulation of the expression level or epigenetic profile level of OCT4 occurs before the regulation of the expression level or epigenetic profile level of KLF4; and / or

[0326] (2) The plurality of stem cells includes a plurality of pluripotent stem cells; and / or

[0327] (3) The first target endogenous gene includes OCT4, and the second target endogenous gene includes one or more members selected from SOX2, KLF4, and MYC; and / or

[0328] (4) The first target endogenous gene includes one or more members selected from OCT4, KLF4, and MYC, and the second target endogenous gene includes SOX2; and / or

[0329] (5) The plurality of gate units further includes a third gate unit that is preconfigured to regulate the expression level or epigenetic profile level of the first target endogenous gene or the second target endogenous gene to respectively achieve two or more sequential regulations of the first target endogenous gene or the second target endogenous gene,

[0330] further optionally wherein SOX2 is subject to the two or more sequential regulations; and / or

[0331] (6) Regulating the expression level or epigenetic profile level of the plurality of different target endogenous genes includes enhancing the expression level or epigenetic profile level of the plurality of different target endogenous genes.

[0332] Embodiment 6. A system for converting a plurality of differentiated cells into a plurality of stem cells, the system comprising:

[0333] 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 level or epigenetic profile level of a plurality of different target endogenous genes to effect the conversion, and wherein the plurality of gate units includes:

[0334] (i) A first gate unit that is preconfigured to regulate the expression level or epigenetic profile level of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene includes a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC; and

[0335] (ii) A second gate unit that is preconfigured to regulate the expression level or epigenetic profile level of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene includes a different cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC,

[0336] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion,

[0337] Optionally wherein:

[0338] (1) The heterologous gene circuit is preconfigured such that activation of the first gate unit enables activation of the second gate unit, such that (i) regulation of the expression level or epigenetic profile level of the first target endogenous gene occurs before (ii) regulation of the expression level or epigenetic profile level of the second target endogenous gene.

[0339] Further optionally, wherein the first target endogenous gene is OCT4, such that regulation of the expression level or epigenetic profile level of OCT4 occurs before regulation of the expression level or epigenetic profile level of KLF4; and / or

[0340] (2) The plurality of stem cells includes a plurality of pluripotent stem cells; and / or

[0341] (3) The first target endogenous gene includes OCT4, and the second target endogenous gene includes one or more members selected from SOX2, KLF4, and MYC; and / or

[0342] (4) The first target endogenous gene includes one or more members selected from OCT4, KLF4, and MYC, and the second target endogenous gene includes SOX2; and / or

[0343] (5) The plurality of gate units further includes a third gate unit, which is preconfigured to regulate the expression level or epigenetic profile level of the first target endogenous gene or the second target endogenous gene to achieve two or more sequential regulations of the first target endogenous gene or the second target endogenous gene, respectively.

[0344] Further optionally, wherein:

[0345] (a) SOX2 undergoes the two or more sequential regulations; and / or

[0346] (b) Regulating the expression level or epigenetic profile level of the plurality of different target endogenous genes includes enhancing the expression level or epigenetic profile level of the plurality of different target endogenous genes.

[0347] Additional details of the heterologous gene circuit (HGC) and its uses are provided in International Application No. PCT / US2018 / 052211, titled "CRISPR / CAS SYSTEM AND METHOD FOR GENOME EDITING AND MODULATING TRANSCRIPTION", International Application No. PCT / US2023 / 013240, titled "SYSTEMS FOR CELL PROGRAMMING AND METHODS THEREOF", and Clarke et al., Molecular Cell, 81, 226-238, 2021, titled "Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities", each of which is incorporated herein by reference in its entirety.

[0348] It should be understood that the various aspects of the present invention can be understood individually, jointly, or in combination with each other. The various aspects of the present invention described herein can be applied to any specific application disclosed herein. A composition of matter comprising a compound of any of the formulas disclosed herein in the composition portion of the present disclosure can be used in the method portion comprising the methods of use and production disclosed herein, or vice versa.

[0349] Although the preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided within this specification. While the present invention has been described with reference to the foregoing specific description, the description and illustration of the embodiments herein are not to be construed in a limiting sense. Various variations, changes, and alternatives will now occur to those skilled in the art without departing from the present invention. Additionally, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein can be used to practice the present invention. Accordingly, it is contemplated that the present invention should also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the present invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for converting a plurality of cells of a first cell type (the first plurality of cells) into a plurality of cells of a second cell type (the second plurality of cells), the method comprising: contacting the first plurality of cells with a heterologous gene regulator that specifically binds to a gene encoding a HERV to regulate the expression level or epigenetic profile of the HERV and effect the conversion from the first plurality of cells to the second plurality of cells.

2. The method of claim 1, wherein (i) the first plurality of cells comprise terminally differentiated cells and (ii) the second plurality of cells comprise pluripotent stem cells.

3. The method of claim 1, wherein the contacting enhances the expression level of the HERV.

4. The method of claim 1, wherein the heterologous gene regulator specifically binds to a gene encoding HERV-K.

5. The method of claim 1, wherein the heterologous gene regulator specifically binds to a gene encoding LTR5HS.

6. The method of claim 5, wherein the heterologous gene regulator specifically binds to a polynucleotide sequence that exhibits at least about 80% sequence identity with a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences, optionally with conversion of uracil to thymine.

7. The method of claim 5, wherein the heterologous gene regulator specifically binds to a polynucleotide sequence that exhibits at least about 90% sequence identity with a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences, optionally with conversion of uracil to thymine.

8. The method of claim 5, wherein the heterologous gene regulator comprises a polynucleotide sequence that exhibits at least about 80% sequence identity with a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences.

9. The method of claim 5, wherein the heterologous gene regulator comprises a polynucleotide sequence that exhibits at least about 90% sequence identity with a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences.

10. The method of claim 1, wherein the contacting effects or is sufficient to effect the conversion without using an additional heterologous gene regulator that specifically binds to an additional target gene, the additional target gene comprising one or more cell dedifferentiation factors selected from OCT4, SOX2, KLF4, and MYC.

11. The method of claim 10, wherein the additional target gene comprises OCT4, SOX2, KLF4, and MYC.

12. The method according to claim 1, further comprising contacting the first plurality of cells with an additional heterologous gene regulator that exhibits specific binding to an additional gene, the additional gene including one or more cell dedifferentiation factors selected from OCT4, SOX2, KLF4, and MYC, to effect sequential regulation of the gene and the additional gene.

13. The method according to claim 12, wherein the regulation of the gene occurs before, simultaneously with, or after the regulation of the additional gene.

14. The method according to claim 13, wherein the regulation of the gene occurs before the regulation of the additional gene.

15. The method according to claim 13, wherein the additional gene includes OCT4.

16. The method according to claim 1, further comprising contacting the first plurality of cells with an additional heterologous gene regulator that exhibits specific binding to an Alu motif enriched in embryonic genome activation (EGA) (EEA) to effect the transformation.

17. The method according to claim 1, wherein the contacting comprises contacting the first plurality of 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 or epigenetic profiles of the gene and different genes to effect the transformation, and wherein the plurality of gate units comprises: (i) a first gate unit that includes the heterologous gene regulator, wherein the first gate unit is preconfigured to regulate the expression level or epigenetic profile of the gene; and (ii) a second gate unit that is preconfigured to regulate the expression level or epigenetic profile of the different gene, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the transformation.

18. The method according to claim 17, wherein the heterologous gene circuit is preconfigured to regulate the expression level of the epigenetic profile of the gene before regulating the expression level of the epigenetic profile of the different gene.

19. The method according to claim 17, wherein the different gene includes one or more members selected from the following: Oct3, Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, EEA, ZSCAN4, DUX4, OTX2, ABCE1, COL5A1, GAL4NT13, DUXA, DUXB, ARGFX, CPHX1, CPHX2, TPRX1, DPPA3, NASP, ATP2B1, NFAT, H1FOO, and CAMKII.

20. The method according to claim 19, wherein the different gene includes one or more members selected from the following: Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, and EEA.

21. The method according to claim 1, wherein the heterologous gene regulator comprises a heterologous endonuclease for specifically binding to the gene encoding the HERV.

22. The method according to claim 21, wherein the heterologous endonuclease is a Cas protein.

23. The method according to claim 21, wherein the heterologous gene regulator further comprises a gene activator.

24. The method according to claim 23, wherein the gene activator is selected from VP16, VP64, p65, RTA, and the VP64-p65-RTA fusion (VPR).

25. The method according to claim 23, wherein the gene activator is coupled to the heterologous endonuclease.

26. A system for converting a plurality of cells of a first cell type (the first plurality of cells) into a plurality of cells of a second cell type (the second plurality of cells), the system comprising: a heterologous gene regulator that exhibits specific binding to a gene encoding an HERV to regulate the expression level or epigenetic profile of the HERV and effect the conversion from the first plurality of cells to the second plurality of cells.

27. The system according to claim 26, wherein (i) the first plurality of cells comprises terminally differentiated cells and (ii) the second plurality of cells comprises pluripotent stem cells.

28. The system according to claim 26, wherein the specific binding of the heterologous gene regulator to the gene results in an enhanced expression level of the HERV.

29. The system according to claim 26, wherein the heterologous gene regulator exhibits specific binding to a gene encoding HERV-K.

30. The system according to claim 26, wherein the heterologous gene regulator exhibits specific binding to a gene encoding LTR5HS.

31. The system according to claim 30, wherein the heterologous gene regulator exhibits specific binding to a polynucleotide sequence that exhibits at least about 80% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences, optionally with the conversion of uracil to thymine.

32. The system according to claim 30, wherein the heterologous gene regulator exhibits specific binding to a polynucleotide sequence that exhibits at least about 90% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences, optionally with the conversion of uracil to thymine.

33. The system according to claim 30, wherein the heterologous gene regulator comprises a polynucleotide sequence that exhibits at least about 80% sequence identity to a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences.

34. The system according to claim 30, wherein the heterologous gene regulator comprises a polynucleotide sequence that exhibits at least about 90% sequence identity with a polypeptide sequence of one or more members selected from SEQ ID NO: 1-12 and their complementary sequences.

35. The system according to claim 26, wherein the specific binding of the heterologous gene regulator to the gene is configured to or sufficient to effect the transformation without using an additional heterologous gene regulator that exhibits specific binding to an additional target gene, the additional target gene comprising one or more cell dedifferentiation factors selected from OCT4, SOX2, KLF4, and MYC.

36. The system according to claim 35, wherein the additional target gene comprises OCT4, SOX2, KLF4, and MYC.

37. The system according to claim 26, further comprising an additional heterologous gene regulator that exhibits specific binding to an additional gene, the additional gene comprising one or more cell dedifferentiation factors selected from OCT4, SOX2, KLF4, and MYC, to effect sequential regulation of the gene and the additional gene.

38. The system according to claim 37, wherein the regulation of the gene occurs before, simultaneously with, or after the regulation of the additional gene.

39. The system according to claim 38, wherein the regulation of the gene occurs before the regulation of the additional gene.

40. The system according to claim 38, wherein the additional gene comprises OCT4.

41. The system according to claim 26, further comprising an additional heterologous gene regulator that exhibits specific binding to an Alu motif enriched in embryonic genome activation (EGA) (EEA) to effect the transformation.

42. The system according to claim 26, wherein the system comprises 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 or epigenetic profiles of the gene and different genes to effect the transformation, and wherein the plurality of gate units comprises: (i) a first gate unit that comprises the heterologous gene regulator, wherein the first gate unit is preconfigured to regulate the expression level or epigenetic profile of the gene; and (ii) a second gate unit that is preconfigured to regulate the expression level or epigenetic profile of the different gene, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the transformation.

43. The system according to claim 42, wherein the heterologous gene circuit is preconfigured to regulate the expression level of the epigenetic profile of the gene before regulating the expression level of the epigenetic profile of the different gene.

44. The system according to claim 42, wherein the different genes include one or more members selected from the following: Oct3, Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, EEA, ZSCAN4, DUX4, OTX2, ABCE1, COL5A1, GAL4NT13, DUXA, DUXB, ARGFX, CPHX1, CPHX2, TPRX1, DPPA3, NASP, ATP2B1, NFAT, H1FOO, and CAMKII.

45. The system according to claim 44, wherein the different genes include one or more members selected from the following: Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, and EEA.

46. The system according to claim 26, wherein the heterologous gene regulator includes a heterologous endonuclease for specifically binding to the gene encoding HERV.

47. The system according to claim 46, wherein the heterologous endonuclease is a Cas protein.

48. The system according to claim 46, wherein the heterologous gene regulator further includes a gene activator.

49. The system according to claim 48, wherein the gene activator is selected from VP16, VP64, p65, RTA, and the VP64-p65-RTA fusion (VPR).

50. The system according to claim 48, wherein the gene activator is coupled to the heterologous endonuclease.

51. A method for converting a plurality of differentiated cells into a plurality of stem cells, the method comprising: contacting the plurality of differentiated 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 or epigenetic profiles of a plurality of different target endogenous genes to effect the conversion, and wherein the plurality of gate units include: (i) a first gate unit preconfigured to regulate the expression level or epigenetic profile of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene includes an Alu motif enriched in embryonic genome activation (EGA) (EEA); and (ii) a second gate unit preconfigured to regulate the expression level or epigenetic profile of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene includes a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion.

52. The method according to claim 51, wherein the plurality of stem cells include a plurality of pluripotent stem cells.

53. The method according to claim 51, wherein the heterologous gene circuit is preconfigured such that upon activation of the heterologous gene circuit, (i) the expression level or epigenetic profile of the first target endogenous gene is regulated before (ii) the expression level or epigenetic profile of the second target endogenous gene.

54. The method according to claim 51, wherein the plurality of gate units includes a third gate unit that is preconfigured to regulate the expression level or epigenetic profile of a third target endogenous gene among the plurality of different target endogenous genes, wherein the third target endogenous gene includes a different cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC, wherein, upon activation of the heterologous gene circuit, the expression level or epigenetic profile of the cell dedifferentiation factor and the different cell dedifferentiation factors are sequentially regulated.

55. The method according to claim 54, wherein the second target endogenous gene includes OCT4.

56. The method according to claim 54, wherein the third target endogenous gene includes SOX2.

57. The method according to claim 51, wherein the plurality of gate units includes a third gate unit that is preconfigured to regulate the expression level or epigenetic profile of the second target endogenous gene, wherein activation of the heterologous gene circuit effects two or more sequential regulations of the expression level or epigenetic profile of the cell dedifferentiation factor.

58. The method according to claim 57, wherein the cell dedifferentiation factor is MYC.

59. The method according to claim 57, wherein the cell dedifferentiation factor is SOX2.

60. A system for converting a plurality of differentiated cells into a plurality of stem cells, the system comprising: a heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to sequentially regulate the expression level or epigenetic profile of a plurality of different target endogenous genes to effect the conversion, wherein the plurality of gate units includes: (i) a first gate unit that is preconfigured to regulate the expression level or epigenetic profile of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene includes an embryo genome activation (EGA)-enriched Alu motif (EEA); and (ii) a second gate unit that is preconfigured to regulate the expression level or epigenetic profile of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene includes a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the conversion.

61. A method for converting a plurality of differentiated cells into a plurality of stem cells, the method comprising: Contacting the plurality of differentiated 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 regulate the expression levels or epigenetic profile levels of a plurality of different target endogenous genes in a sequential manner to effect the transformation, and wherein the plurality of gate units comprise: (i) A first gate unit preconfigured to regulate the expression level or epigenetic profile level of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene comprises a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC; and (ii) A second gate unit preconfigured to regulate the expression level or epigenetic profile level of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene comprises a different cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the transformation.

62. The method of claim 61, wherein the heterologous gene circuit is preconfigured such that activation of the first gate unit effects activation of the second gate unit, such that (i) regulation of the expression level or epigenetic profile level of the first target endogenous gene occurs prior to (ii) regulation of the expression level or epigenetic profile level of the second target endogenous gene.

63. The method of claim 62, wherein the first target endogenous gene is OCT4, such that regulation of the expression level or epigenetic profile level of OCT4 occurs prior to regulation of the expression level or epigenetic profile level of KLF4.

64. The method of claim 61, wherein the plurality of stem cells comprise a plurality of pluripotent stem cells.

65. The method of claim 61, wherein the first target endogenous gene comprises OCT4, and the second target endogenous gene comprises one or more members selected from SOX2, KLF4, and MYC.

66. The method of claim 61, wherein the first target endogenous gene comprises one or more members selected from OCT4, KLF4, and MYC, and the second target endogenous gene comprises SOX2.

67. The method of claim 61, wherein the plurality of gate units further comprise a third gate unit preconfigured to regulate the expression level or epigenetic profile level of the first target endogenous gene or the second target endogenous gene to effect two or more sequential regulations of the first target endogenous gene or the second target endogenous gene, respectively.

68. The method of claim 67, wherein SOX2 undergoes the two or more sequential regulations.

69. The method of claim 61, wherein regulating the expression levels or epigenetic profile levels of the plurality of different target endogenous genes comprises enhancing the expression levels or epigenetic profile levels of the plurality of different target endogenous genes.

70. A system for transforming a plurality of differentiated cells into a plurality of stem cells, the system comprising: A heterologous gene circuit comprising a plurality of gate units, wherein the heterologous gene circuit is activatable to induce the plurality of gate units to regulate the expression levels or epigenetic profile levels of a plurality of different target endogenous genes in a sequential manner to effect the transformation, and wherein the plurality of gate units comprises: (i) A first gate unit preconfigured to regulate the expression level or epigenetic profile level of a first target endogenous gene among the plurality of different target endogenous genes, wherein the first target endogenous gene comprises a cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC; and (ii) A second gate unit preconfigured to regulate the expression level or epigenetic profile level of a second target endogenous gene among the plurality of different target endogenous genes, wherein the second target endogenous gene comprises a different cell dedifferentiation factor selected from OCT4, SOX2, KLF4, and MYC, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate to effect the transformation.

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