System and method for stem cell programming

Abstract

Systems for regulating gene expression to convert cells of one cell type to another (e.g., reprogramming differentiated cells to stem cells), and methods of using the same, are provided herein.

Description

Technical Field

[0001] Cross-reference 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 Art

[0002] Heterologous proteins and / or nucleic acid molecules can be utilized to induce desirable responses in cells. Heterologous proteins and / or nucleic acid molecules can regulate a target gene (e.g., a transgene and / or an endogenous gene) to program (e.g., differentiate, dedifferentiate) a cell (e.g., a stem cell). In some cases, endonuclease-based techniques (e.g., clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins or "CRISPR / Cas") have been employed for the manipulation of polynucleotide sequences, their epigenetic modification, and / or their expression levels. For example, CRISPR / Cas technology can be characterized by its versatility and ease of programmability and can be used to facilitate genome editing across different species.

Summary of the Invention

[0003] 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.

[0004] 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 modulator that exhibits specific binding to a gene encoding a HERV to regulate the expression level or epigenetic profile of the HERV and achieve conversion of the first plurality of cells into the second plurality of cells.

[0005] 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 modulator that exhibits specific binding to a gene encoding a HERV to regulate the expression level or epigenetic profile of the HERV and achieve conversion of the first plurality of cells into the second plurality of cells.

[0006] 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 genetic circuit comprising a plurality of gate units, the heterologous genetic circuit being activatable to achieve conversion by inducing the plurality of gate units to sequentially regulate the expression level or epigenetic profile of a plurality of distinct target endogenous genes, the plurality of gate units comprising (i) a first gate unit preconfigured to regulate the expression level or epigenetic profile of a first target endogenous gene of the plurality of distinct target endogenous genes, the first target endogenous gene comprising 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 of the plurality of distinct target endogenous genes, the second target endogenous gene comprising a cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, and wherein upon activation of the heterologous genetic circuit, the plurality of gate units operate to achieve conversion.

[0007] 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 genetic circuit comprising a plurality of gate units, wherein the heterologous genetic circuit is activatable to achieve the conversion by inducing the plurality of gate units to sequentially regulate the expression levels or epigenetic profiles of a plurality of distinct target endogenous genes, 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 distinct target endogenous genes, the first target endogenous gene comprising 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 distinct target endogenous genes, the second target endogenous gene comprising a cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC. Upon activation of the heterologous genetic circuit, the plurality of gate units operate to achieve the conversion.

[0008] 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 genetic circuit comprising a plurality of gate units, wherein the heterologous genetic circuit is activatable to achieve the conversion by inducing the plurality of gate units to sequentially regulate the expression levels or epigenetic profile levels of a plurality of distinct target endogenous genes, 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 distinct target endogenous genes, the first target endogenous gene comprising a cell dedifferentiation factor selected from the group consisting of 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 distinct target endogenous genes, the second target endogenous gene comprising a different cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, and wherein, upon activation of the heterologous genetic circuit, the plurality of gate units operate to achieve the conversion.

[0009] 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 genetic circuit including a plurality of gate units, the heterologous genetic circuit being activatable to achieve the conversion by inducing the plurality of gate units to sequentially regulate the expression levels or epigenetic profile levels of a plurality of distinct target endogenous genes, the plurality of gate units including: (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 distinct target endogenous genes, the first target endogenous gene including a cell dedifferentiation factor selected from the group consisting of 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 distinct target endogenous genes, the second target endogenous gene including a different cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, and upon activation of the heterologous genetic circuit, the plurality of gate units operate to achieve the conversion.

[0010] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which illustrates only exemplary embodiments of the present disclosure. As will be understood, the present disclosure is capable of other embodiments and different implementations, and some of the details thereof are capable of modification in various obvious respects without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0011] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material.

Brief Description of the Drawings

[0012] The novel features of the invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the invention will be obtained from the following detailed description which sets forth illustrative embodiments in which the principles of the invention are utilized, and from the appended drawings (also referred to herein as "Figure" and "FIG.").

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[0013] 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. Numerous variations, changes, and substitutions may 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.

[0014] As used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "gate unit" includes a plurality of gate units.

[0015] The term "about" or "approximately" generally means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to the convention in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude of the value, preferably within five-fold, more preferably within two-fold. When a particular value is recited in this application and the claims, the term "about" should be assumed to mean within the acceptable error range of the particular value, unless otherwise stated.

[0016] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination of them. The term "and / or" should be understood to mean either one or both of the alternatives.

[0017] Definitions

[0018] When the terms "genetic circuit", "biological circuit", or "circuit" are used interchangeably herein, they generally refer to an assembly of molecular components (e.g., biological materials such as polypeptides and / or polynucleotides, non-biological materials, etc.) operably coupled (e.g., operating simultaneously, sequentially, etc.) according to circuit design. The assembly of molecular components can be capable of providing one or more specific outputs (e.g., regulation of one or more genes) into a cell in response to one or more inputs (e.g., a single input or multiple inputs). Such one or more inputs can be sufficient to induce the molecular components of the genetic circuit to provide one or more specific outputs. For example, a genetic circuit can include one or more molecular switches that can be activated by one or more inputs (Figure 1).

[0019] A genetic circuit can be a controllable gene expression system that includes an assembly of biological parts that function together as a logic function (e.g., simultaneously, sequentially, etc.). A genetic circuit can include a plurality of gate units, and at least one of the plurality of gate units can be activated by an activation portion (e.g., a heterologous input to a cell) that activates another of the plurality of gate units (e.g., simultaneously at once, sequentially in a cascade manner, etc.) (Figure 1). For example, at least one of the plurality of gate units can, as predetermined by the design of the genetic circuit, (i) regulate the expression or activity level of one or more target genes, (ii) activate at least one other gate unit of the plurality of gate units, and / or (ii) inactivate at least one other gate unit of the plurality of gate units, thereby overall regulating the expression and / or activity level of one or more target genes in a desired manner and can be activated by another of the plurality of gate units (e.g., directly or indirectly) (Figure 1). The terms “heterologous genetic circuit,” “HGC,” “cellular algorithm,” or “cellgorithm” as used herein can be used interchangeably.

[0020] The term “gate unit” as referred to herein generally refers to a part of a genetic circuit that can control gene regulation by functioning in a manner similar to a logic gate, which can control the flow of information and enable the circuit to multiplex decisions at different points. More specifically, this term refers to a nucleic acid encoding a gene switch and the transcription / translation regulatory region or series of regions on which the gene switch acts. The input of a gate unit can be an activation portion and / or another gate unit. The output of a gate unit can be used to activate another gate unit, to inactivate another gate unit, to affect a target gene, and / or for any combination of these. For example, a gate unit can be composed of a plurality of gate portions and / or a plurality of gene regulatory portions (Figure 1).

[0021] As used herein, the term "activating moiety" generally refers to a moiety that can activate multiple genetic circuits and / or multiple gate units. The activating moiety can be a heterologous input to the cell. In some cases, the activating moiety can include, but is not limited to, a guide nucleic acid molecule (e.g., gRNA) or other nucleic acid, polypeptide, polynucleotide, small molecule, light, or a combination thereof. For example, the activating moiety can form a complex with an endonuclease (e.g., a Cas protein) and bind to the polynucleotide sequence of an inactivated gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to activate such a gate moiety that can target one or more gene regulatory moieties (e.g., induce the expression of a functional form of an additional guide nucleic acid molecule), and can be a guide nucleic acid molecule.

[0022] As used herein, the term "gate moiety" generally refers to a portion that can affect the function of a gene regulatory portion within a gate unit. The gate moiety can activate and / or inactivate the gene regulatory portion. For example, the gate moiety can regulate the expression of a gene regulatory portion by editing a nucleic acid sequence, thereby activating or inactivating the gene regulatory portion. For example, the gate moiety can form a complex with an endonuclease (e.g., a Cas protein) and bind to the 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 in addition, the gate moiety can activate and / or inactivate another gate unit of a genetic circuit (Figure 1). For example, the gate moiety can form a complex with an endonuclease (e.g., a Cas protein) and bind to the polynucleotide sequence of another gate moiety to be inactivated (e.g., a plasmid encoding another guide nucleic acid molecule) to activate another gate moiety (e.g., induce the expression of a functional form of another guide nucleic acid molecule). In another example, the gate moiety can form a complex with an endonuclease (e.g., a Cas protein) and bind to the polynucleotide sequence of another gate moiety to be activated (e.g., a plasmid encoding another guide nucleic acid molecule) to inactivate another gate moiety (e.g., reduce the expression of a functional form of another guide nucleic acid molecule).

[0023] As used interchangeably herein, the terms "gene regulating moiety" or "gene editing moiety" generally refer to a moiety that can regulate the expression and / or activity profile of a nucleic acid sequence or protein, whether exogenous or endogenous to the cell (Figure 1). For example, a gene editing moiety can regulate gene expression by editing a nucleic acid sequence (e.g., CRISPR-Cas, zinc finger nuclease, TALEN, or siRNA). In some cases, a gene editing moiety can regulate gene expression by editing genomic DNA sequences. In some cases, a gene editing moiety can regulate gene expression by editing an mRNA template. Editing of the nucleic acid sequence can, in some cases, modify the template underlying gene expression (e.g., CRISPR-Cas-inspired RNA targeting systems). Alternatively, a gene editing moiety can suppress gene translation (e.g., Cas13).

[0024] Alternatively, or in addition, the gene editing moiety may be capable of regulating gene expression or activity by specifically binding to a target sequence (or a target sequence within a gene) operably linked to the gene and regulating the production of mRNA from DNA such as chromosomal DNA or cDNA. For example, the gene editing moiety may 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 obstruction, 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 translation level, for example, by regulating the production of protein 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 via epigenetic editing (e.g., Casl2).

[0025] In some cases, the plasmid can encode a non-functional form of the gene editing moiety. The plasmid can be activated (e.g., gene 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 able to bind to a target gene of the cell. Upon binding of a functional gate moiety (e.g., another guide nucleic acid molecule complexed with a Cas protein) to the plasmid, the plasmid is edited (e.g., cleaved at one or more sites and then repaired via an endogenous mechanism such as homologous recombination, non-homologous end joining) to enable expression of a functional form of the gene editing moiety (e.g., a functional form of a guide nucleic acid molecule having specific binding to the target gene of the cell), allowing for regulation of the target gene in the cell.

[0026] In some cases, the gene regulatory portion may include a nucleic acid molecule (e.g., a guide nucleic acid molecule that forms a complex with an endonuclease such as a Cas protein). Alternatively, or in addition thereto, the gene regulatory portion may include an endonuclease or be operably linked to an endonuclease. An endonuclease can be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. The endonuclease can include a restriction endonuclease that cleaves DNA at specific sites without damaging the bases. The restriction endonucleases can include type I, type II, type III, and type IV endonucleases, and can further include subtypes. In some cases, the endonuclease can be Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14 or C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas13 (C2c2), Cas13b, Cas13c, Cas13d, Cas13x.1, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx10, Csx11, Csf1, Csn2. The endonuclease can be an inactive endonuclease (dead endonuclease) that exhibits a decrease in cleavage activity. For example, the endonuclease can be a nuclease-inactivated Cas such as dCas (e.g., dCas9).

[0027] The above-mentioned Cas protein can form a complex with a guide nucleic acid (gNA (e.g., guide RNA (gRNA))) and specifically bind to a target polynucleotide sequence (e.g., target DNA sequence, target RNA sequence) using the gNA. Thus, in some cases, such a Cas protein can be referred to as a "nucleic acid-guided nuclease" (e.g., RNA-guided nuclease). As used herein, the term "guide nucleic acid" (gNA) generally can refer to a nucleic acid that can hybridize to another nucleic acid. The guide nucleic acid can be RNA. The guide nucleic acid can also be DNA. The guide nucleic acid can be programmed to specifically bind to a site in the nucleic acid sequence. The nucleic acid to be targeted or the target nucleic acid can contain nucleotides. The guide nucleic acid can contain 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 contain a polynucleotide strand and can be referred to as a "single guide nucleic acid". The guide nucleic acid can contain two polynucleotide strands and can be referred to as a "double guide nucleic acid". Unless otherwise specified, the term "guide nucleic acid" can be inclusive and can refer to both single guide nucleic acids and double guide nucleic acids. The guide nucleic acid can contain a segment that can be referred to as a "nucleic acid-targeting segment" or a "nucleic acid-targeting sequence" or a "spacer sequence".The nucleic acid targeting segment may include a sub-segment that may be referred to as a "protein binding segment", or a "protein binding sequence", or a "Cas protein binding segment", or a "scaffold sequence".

[0028] The gene regulatory moiety can be a transcriptional modulator 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 linked (e.g., bound or fused) to a transcriptional repressor. Non-limiting examples of transcriptional repressors include KRAB, SID, MBD2, MBD3, DNMT1, DNMT2A, DNMT3A, DNMT3B, DNMT3L, Mecp2, FOG1, ROM2, LSD1, ERD, SRDX repression domain, Pr-SET7 / 8, SUV4-20H1, RIZ1, JMJD2A, JHDM3A, JMJD2B, JMJD2C, GASC1, JMJD2D, JARID1A, RBP2, JARID1B / PLU-1, 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 linked (e.g., fused) to a transcriptional activator. Non-limiting examples of transcriptional activators include VP16, VP64, VP48, VP160, p65 subdomain, SET1A, SET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, SYMD2, NSD1, JHDM2a, JHDM2b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, p1 60, CLOCK, TET1CD, TET1, DME, DML1, DML2, and ROS1.

[0029] In some cases, the gene regulatory moiety has an enzymatic activity that modifies the target gene without cleaving it. Modification of the target gene can result in epigenetic modifications, for example, that can modify gene expression and / or activity levels. Examples of enzymatic activities that can be provided by the gene regulatory moiety include nuclease activities such as those provided by restriction enzymes (e.g., FokI nuclease), methyltransferase activities such as those provided by methyltransferases (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3, ZMET2, CMT1, CMT2), demethylase activities such as those provided by demethylases (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1), DNA repair activities, DNA damage activities, deamination activities such as those provided by deaminases (e.g., cytosine deaminase enzymes such as APOBEC1), dismutase activities, alkylation activities, depurination activities, oxidation activities, pyrimidine dimer formation activities, integrase and / or resolvase activities such as those provided by integrases and resolvases (e.g., GinH106Y, which is a hyperactive mutant of the Gin invertase, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase, etc., such as the Gin invertase), transposase activities, recombinase activities such as those provided by recombinases (e.g., the catalytic domain of the Gin recombinase), polymerase activities, ligase activities, helicase activities, photolyase activities, and glycosylase activities, but are not limited thereto.

[0030] Unless otherwise specified or not apparent from the context, the terms "polynucleotide", "oligonucleotide", or "nucleic acid" used interchangeably herein generally refer to a polymeric form of nucleotides of any length, in any form of single-stranded, double-stranded, or multi-stranded, which are either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can be exogenous or endogenous to the cell. Polynucleotides can exist in a cell-free environment. Polynucleotides can be genes or fragments thereof. Polynucleotides can be DNA. Polynucleotides can be RNA. Polynucleotides can assume any three-dimensional structure and can perform any known or unknown function. Polynucleotides can contain one or more analogs (e.g., modified backbone, sugar, or nucleobase). When present, modifications to the nucleotide structure can be imparted before or after polymerization of the polynucleotide. 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, queuosine, and wyosine.Non-limiting examples of polynucleotides include the coding or non-coding regions of genes or gene fragments, loci determined from 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 nucleotide sequence can be interfered with by non-nucleotide components.

[0031] The term "gene" generally refers to nucleic acids (e.g., DNA such as genomic DNA and cDNA) involved in the coding of RNA transcripts and their corresponding nucleotide sequences. This term as used herein with respect to genomic DNA includes regulatory regions in addition to intervening non-coding regions and may include the 5' and 3' termini. In some applications, this term encompasses transcribed sequences that include 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region contains an "open reading frame" that encodes a polypeptide. In some applications of this term, "gene" includes only the coding sequences necessary to encode a polypeptide (e.g., an "open reading frame" or "coding region"). In some cases, a gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions that include upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene," i.e., a native gene in its natural location in an organism's genome. A gene can refer to an "exogenous gene" or a non-native gene. A non-native gene can refer to a gene that is not normally found in the host organism but is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene that is not in its natural location in an organism's genome. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-native sequence) that includes mutations, insertions, and / or deletions.

[0032] The term "sequence identity" generally refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. The percent identity can also be determined, for example, by comparing sequence information using an advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), and is 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). Using this program, the percent identity over the full length of the proteins being compared can be determined. Default parameters are provided, for example, for optimizing searches with short query sequences using the blastp program. The program also allows the use of a SEG filter to mask-off segments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993).The desirable range of sequence identity is approximately 50% to 100% and integer values therebetween. Generally, the present disclosure encompasses sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any sequence provided herein.

[0033] The term “expression” generally refers to one or more processes by which a polynucleotide is transcribed (such as into mRNA or other RNA transcripts) from a DNA template, and / or the process by which the transcribed mRNA is later translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can be collectively referred to as “gene products”. When the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in eukaryotic cells. “Upregulated” with respect to expression generally refers to an increase in the expression level of a polynucleotide (such as an RNA, e.g., mRNA) and / or polypeptide sequence compared to the expression level in the wild-type state, while “downregulated” generally refers to a decrease in the expression level of a polynucleotide (such as an RNA, e.g., mRNA) and / or polypeptide sequence compared to the 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 limited to the transfected cells, the expression of the gene decreases over time. In contrast, stable expression of a transfected gene can occur when a 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 particular toxin presented to the cells.

[0034] As used interchangeably herein, the terms "peptide", "polypeptide", or "protein" generally refer to a polymer of at least two amino acid residues joined by peptide bonds. This term is not intended to imply a polymer of a specific length, nor is it intended to suggest or distinguish whether the peptide is produced using recombinant techniques, chemical synthesis, or enzymatic synthesis, or whether it is naturally occurring. This term applies to amino acid polymers that contain at least one modified amino acid in addition to naturally occurring amino acid polymers. In some cases, the polymer may be interrupted by non-amino acids. This term includes amino acid chains of any length, including full-length proteins, as well as proteins that do or do not have secondary and / or tertiary structures (e.g., domains). This term also encompasses amino acid polymers modified by any other operations, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and conjugation with a labeling component. As used herein, the terms "amino acid" and "amino acids" generally refer to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural and non-natural amino acids that have been chemically modified to include groups or chemical moieties that do not naturally occur on the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0035] As used interchangeably herein with respect to polypeptides, the terms "derivative", "variant", or "fragment" generally refer to a polypeptide that is related to a wild-type polypeptide by, for example, any of amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of a polypeptide can include one or more amino acid mutations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof as compared to the wild-type polypeptide.

[0036] As used herein, the terms "engineered," "chimeric," or "recombinant" with respect to a polypeptide molecule (e.g., a protein) generally refer to a polypeptide molecule having a non-homologous amino acid sequence or a modified amino acid sequence, as well as a cell or organism that expresses such polypeptide molecule, as a result of the application of genetic engineering techniques to the nucleic acid encoding the polypeptide molecule. The terms "engineered" or "recombinant" as used herein with respect to a polynucleotide molecule (e.g., a DNA molecule or an RNA molecule) generally refer to a polynucleotide molecule having a non-homologous nucleic acid sequence or a modified nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques, transfection, transformation, and other gene transfer techniques, homologous recombination, site-directed mutagenesis, and gene fusion. In some cases, an engineered polynucleotide or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or altered by a gene editing moiety.

[0037] Unless otherwise specified or not apparent from the context, the term "nucleotide" as used herein 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 adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Examples of such derivatives 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. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may not be labeled or may be detectably labeled by well-known techniques. Labeling can also be performed using quantum dots. Detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.Examples of fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2’7’-dimethoxy-4’5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N’,N’-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4’-dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2’-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from Perkin Elmer, Foster City, California; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, available from Amersham, Arlington Heights, Illinois; fluorescein-15-d ATP, 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, Indiana; 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, Oregon. Nucleotides can also be labeled or marked by chemical modification.A chemically modified single nucleotide can be a biotin-dNTP. Some non-limiting examples of biotinylated dNTPs 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).

[0038] The term "cell" generally refers to a biological cell. A cell can be the basic structural, functional, and / or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells derived from plants (e.g., plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hemp, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, horsetails, mosses, liverworts), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardlii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. Agardh, etc.), seaweeds (e.g., kelp), fungal cells (e.g., yeast cells, cells derived from mushrooms), animal cells, cells derived from invertebrates (e.g., flies, cnidarians, echinoderms, nematodes, etc.), cells derived from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells derived from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes, a cell does not originate from a natural organism (e.g., a cell can be made synthetically and is sometimes called an artificial cell).

[0039] The terms "reprogramming," "dedifferentiation," "increased cellular potency," or "increased developmental potency," which are used interchangeably herein, generally refer to methods of increasing the potency of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cellular potency has more developmental flexibility (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 a cell in a less differentiated state than the same cell in a non-reprogrammed state.

[0040] The term "differentiation" generally refers to the process by which unspecialized (or "uncommitted") or less specialized cells acquire the characteristics of specialized cells, such as immune cells. Differentiated cells or cells induced to differentiate are cells obtained at a more specialized (or "committed") position within a cell lineage. The term "committed" generally refers to a cell that, in a differentiation pathway, has progressed to the point where it will continue to differentiate into a specific cell type or subset of cell types under normal circumstances, but cannot differentiate into a different cell type or return to a less differentiated cell type under normal circumstances.

[0041] The term "pluripotency" 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 capable of forming cells from each of the three germ layers: the ectoderm, mesoderm, and endoderm. Pluripotency can range from less primitive and less pluripotent cells (e.g., epiblast stem cells) that cannot give rise to a complete organism to more primitive and more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism, and can represent a continuum of developmental potential.

[0042] The term "induced pluripotent stem cell" (iPSC) generally refers to stem cells derived from differentiated cells (e.g., differentiated adult cells, neonatal cells, or fetal cells) that have been induced or changed (i.e., reprogrammed) into cells that can differentiate into all three germ layers: mesoderm, endoderm, and ectoderm, or tissues of the dermal layer. The iPSCs produced do not refer to cells found naturally. In some cases, iPSCs can be engineered to differentiate directly into specified 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 induced to differentiate into specified cells (e.g., NK cells).

[0043] Overview

[0044] Biological programming such as cell programming (e.g., generation of iPSCs) enables the manipulation of cells to achieve desired results. The results of cell programming can include inducing or preventing a wide range of common and / or new cell functions. The results can also include enhancing or suppressing cell functions that have already occurred. Cell programming can be achieved by the use of genetic circuits. Cell programming can be achieved by the manipulation of biomolecules (e.g., endogenous DNA). For example, the CRISPR or CRISPR / Cas system has been adopted for genome editing across many species due to its versatility and easy programmability. Cell programming can affect endogenous or exogenous genes. Cell programming can be implemented to function either time-dependently or non-time-dependently.

[0045] Genetic circuits used in cell programming can be used to control cascades of multiple desired expression and / or activity profiles of multiple genes in cells. To enable better control of specific cell outcomes, genetic circuits can be multiplexed to create positive feedback systems and / or negative feedback systems.

[0046] The CRISPR / Cas system can be used for gene editing, but Cas is essentially a single-turnover nuclease as it remains bound to the double-strand breaks it generates, and many regions of the genome are resistant to genome editing. With the increasing understanding of CRISPR / Cas-based genome editing, the development of cascade regulatory systems for further leveraging this technology for use in the generation of engineered cells has been encouraged. By implementing a series of activatable gRNAs, genome editing can be regulated more temporally from target site to target site, sequential genome editing can be carried out to function like a domino effect, and cells can be barcoded. However, this simple barcoding, which often uses exogenous fluorophores, does not enable multiplex regulation of endogenous genes to achieve cell differentiation.

[0047] Pluripotency can be induced using the overexpression of the Yamanaka factors, a group of protein transcription factors that play an important role in the generation of iPSCs. However, the methods for overexpressing the Yamanaka factors are inefficient, slow, and stochastic. In addition to these problems, epigenetic markers can remain derived from the original differentiated cells, which can exacerbate issues when using iPSCs generated by this method.

[0048] For example, there is still a need for an activatable multiplex CRISPR / Cas system for editing a target polynucleotide (e.g., the genome of a cell, particularly a eukaryotic cell) and its use, the use of a cascade of gRNAs to form a genetic circuit to affect gene regulation alone, then the reprogramming of cells to efficiently generate iPSCs, and methods for removing epigenetic markers to improve the survival rate and use of iPSCs.

[0049] Accordingly, various aspects of the present disclosure provide, for example, a system, composition, and method therefor for regulating the fate of non-viral cells (e.g., the conversion of non-viral cells from a first cell type to a second cell type such as dedifferentiation) by (i) regulating a target gene (e.g., an endogenous target gene) that is derived from a virus and (ii) integrated into the genome of a non-viral cell. Further aspects of the present disclosure provide a system, composition, and method therefor for regulating the fate of non-viral cells by regulating other target genes (e.g., additional endogenous target genes). In some embodiments, the systems, compositions, and methods provided herein may not utilize, and need not utilize, a heterologous gene encoding such a target gene, and instead may rely on the regulation of endogenous genes. In some embodiments, the present disclosure provides a system and method for manipulating a CRISPR / Cas9 system comprising an array of Cas endonucleases and cognate single-guide RNAs (sgRNAs or gRNAs) that have an inactivating sequence in a non-essential region, are activatable, and enable the induction of iPSCs from differentiated cells. The present disclosure also provides engineered cells that can comprise any of the above systems or can perform any of the above methods.

[0050] Systems and methods for converting one type of cell to another type of cell

[0051] Various aspects of the present disclosure provide systems for inducing desired expression and / or activity levels (or profiles thereof) of one or more target genes in a cell. Various aspects of the present disclosure provide methods for inducing desired expression and / or activity levels (or profiles thereof) of one or more target genes in a cell.

[0052] In certain aspects, 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 can include a heterologous gene modulator that exhibits specific binding to the target gene. Optionally, the heterologous gene modulator can be part of a heterologous genetic circuit that is introduced into the cell to induce the desired expression. For example, the heterologous gene modulator can include an endonuclease and / or a polynucleotide sequence (e.g., a Cas protein, a guide nucleic acid molecule, and / or a combination thereof).

[0053] Optionally, the heterologous genetic 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 up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, or up to about 2 gate units. The multiple gate units can be different (e.g., include different polynucleotide sequences).

[0054] Alternatively, the expression and / or activity profile of the target gene of the cells disclosed in this specification can be regulated in the absence of a heterologous genetic circuit. For example, a gene regulatory moiety (e.g., a CRISPR / Cas protein and a guide nucleic acid molecule for the target gene) configured to bind to the target gene can be introduced (e.g., transfected or transduced) into the cell, and as a result, 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.

[0055] The heterologous genetic circuits disclosed in this specification can operate 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 the mammalian genome. Alternatively, or in addition thereto, the heterologous genetic circuits disclosed in this specification can operate 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 the mammalian genome.

[0056] The heterologous genetic circuits disclosed in this specification operate using multiple gate units in series (e.g., a plurality of gate units are sequentially connected end-to-end to form a single pathway), in parallel (e.g., a plurality of gate units are cross-connected to each other to form, for example, two or more parallel pathways), or a combination thereof, and can sequentially regulate the expression or activity of multiple target genes (e.g., endogenous target genes) such as virus-derived genes and additional target genes, respectively.

[0057] Although not bound by theory, by modulating (e.g., modifying) the gene expression levels and / or epigenetic profiles of virus-derived genes (e.g., HERVs) in the mammalian genome, cells can be primed for improved regulation of cell fate. In some cases, modulation of the expression levels and / or epigenetic profiles of virus-derived genes can achieve opening of at least a portion of the cell's genome and / or resetting of at least a portion of the cell such that epigenetic markers (e.g., DNA histone markers) can be ubiquitously removed. Alternatively, modulation of the expression levels and / or epigenetic profiles of virus-derived genes can achieve opening of at least a portion of the cell's 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 at specific positions. Alternatively, modulation of the expression levels and / or epigenetic profiles of virus-derived genes can achieve opening of at least a portion of the cell's genome and / or resetting of at least a portion of the cell such that epigenetic markers (e.g., DNA histone markers) can be ubiquitously added. Alternatively, modulation of the expression levels and / or epigenetic profiles of virus-derived genes can achieve opening of at least a portion of the cell's 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 positions.

[0058] In some cases, the regulation of the expression level of virus-derived genes and / or the epigenetic profile can achieve the opening of at least a part of the cell genome and / or the reset of at least a part of the cell so as to specifically target the process of differentiating cells according to the target elements. In some cases, the regulation of the expression level of virus-derived genes and / or the epigenetic profile can achieve the opening of at least a part of the cell genome and / or the reset of at least a part of the cell so as to specifically target the process of differentiating cells according to the target cell lineage.

[0059] In some cases, the regulation of the expression level of virus-derived genes and / or the epigenetic profile can achieve the opening of at least a part of the cell genome and / or the reset of at least a part of the cell so as to specifically target the process of dedifferentiating cells according to the targeting elements. In some cases, the regulation of the expression level of virus-derived genes and / or the epigenetic profile can achieve the opening of at least a part of the cell genome and / or the reset of at least a part of the cell so as to specifically target the process of dedifferentiating cells according to the initial cell lineage.

[0060] The multiple gate units disclosed herein can operate in concert (e.g., as predetermined by the design of a non-homologous genetic circuit) to induce a fate in a cell. Fates in a cell can include cell functions (e.g., responses to movement, regeneration, external stimuli, nutrient production, excretion, respiration, growth) and / or cell states (e.g., cell fate, differentiation, dormancy, programmed cell death). Such fates can be confirmed in vitro, ex vivo, and / or in vivo. For example, the fates disclosed herein can be confirmed in vitro by (i) measuring the expression level of a gene of interest by polymerase chain reaction (PCR) or Western blotting, (ii) staining with a small molecule or an antibody, (iii) cell sorting based on cell size, morphology, and / or surface protein expression, (iv) use of an assay to measure phenotypic differentiation and cell function (e.g., a cell proliferation assay, a metabolic activity assay, a cell death assay), (v) microscopy, and / or (iv) screening for molecular and / or genetic differences using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics.

[0061] Intracellular fate can include regulation of a target gene. Regulation of the target gene can include a plurality of distinct regulations of the target gene. The plurality of gate units can each induce one of the plurality of distinct regulations of the target gene such that the collection of distinct regulations cooperate to achieve the final expression and / or activity profile of the target gene. At least two of the plurality of distinct regulations can increase both the expression and / or activity level of the target gene. At least two of the plurality of distinct regulations can decrease both the expression and / or activity level of the target gene. Alternatively, a first of the plurality of distinct regulations can increase the expression and / or activity level of the target gene, and a second of the plurality of distinct regulations can decrease the expression and / or activity level of the target gene. In such a case, the first distinct regulation can occur before the second distinct regulation, and vice versa. Alternatively, one of the plurality of distinct regulations (e.g., the first and / or second regulation) can maintain the expression and / or activity level of the target gene at the pre-regulation expression and / or activity level.

[0062] Fates in cells can include regulation of the epigenetic profile of a target gene. Regulation of the epigenetic profile of a target gene can include a plurality of distinct regulations of the target gene. Each of the plurality of gate units can induce one of the plurality of distinct regulations on the epigenetic profile of the target gene, whereby a collection of distinct regulations cooperatively achieves the final epigenetic profile of the target gene. At least two distinct regulations of the plurality of distinct regulations can both modify the epigenome of the target gene. The epigenome of the target gene can be modified by regulation of methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ribosylation, or citrullination. In such cases, a first distinct regulation can occur before a second distinct regulation, or vice versa. Alternatively, a distinct regulation (e.g., a first and / or a second regulation) of the plurality of distinct regulations can maintain the epigenetic state of the target gene.

[0063] In some cases, each distinct regulation of the plurality of distinct regulations of the target gene disclosed herein may be necessary to achieve the desired expression and / or activity profile of the target gene, but may be insufficient individually. For this reason, the fate in the cell induced by a plurality of distinct regulations of the target gene (e.g., improvement of cell function, induction of cell state, etc.) may not be possible in the absence of any one of the plurality of distinct regulations of the target gene. Alternatively, the degree or scale of the fate in the cell induced by a plurality of distinct regulations of the target gene may be higher than the degree or scale of the fate in the control cell induced by any one or more, and / or all of the plurality of distinct regulations of the target gene occurring through different sequential events.

[0064] In some cases, each separate regulation of a plurality of separate regulations of a target gene may be necessary to achieve the desired epigenetic profile of the target gene as disclosed herein, but may be insufficient individually. For this reason, the outcome in a cell induced by a plurality of distinct regulations of a target gene (e.g., improvement of cell function, induction of cell state, etc.) may not be possible in the absence of any one of the plurality of distinct regulations of the target gene. Alternatively, the degree or scale of the outcome in a cell induced by a plurality of separate regulations of the epigenome of a target gene may be higher than the degree or scale of the outcome in a control cell induced by not all, but any one or more, and / or all of the plurality of separate regulations of the epigenome of the target gene via events in a different sequential order.

[0065] 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 that are 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 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, 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 involved in the activation of the first gate unit (e.g., an activation part, a different gate unit, etc.).

[0066] The second gate unit can be made activatable to induce inactivation of the activated first gate unit. The terms "inactivation" or "disruption" may be used interchangeably herein. The inactivation disclosed herein can be induced by generating a modification (e.g., a cleavage such as a single-stranded or double-stranded break, and an indel, etc.) in 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) involved in inducing a first distinct regulation of the target gene.

[0067] Inactivation of the gate portion and / or the gene regulatory portion of the first gate unit disclosed herein can be achieved by an endonuclease-based system (e.g., the CRISPR / Cas system). Alternatively, or in addition thereto, inactivation can be achieved by the use of a transcriptional regulator system (e.g., a transcriptional repressor). The endonuclease transcriptional regulator system (e.g., a Cas repressor) can be used to achieve polynucleotide cleavage (e.g., to inactivate the gate portion and / or the gene regulatory portion). Polynucleotide cleavage can create nucleic acid modifications such as single-stranded breaks, double-stranded breaks, insertions, deletions, or insertions-deletions (indels). Alternatively, or in addition thereto, the endonuclease transcriptional regulator system (e.g., a Cas repressor) can be used to regulate target gene expression.

[0068] Alternatively, the second gate unit can be made activatable to amplify or enhance the activation of the activated first gate unit. The amplification or enhancement of the first gate unit can be induced by generating a modification (e.g., a cleavage such as a single-stranded or double-stranded break, and an indel, etc.) in 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) involved in inducing a first distinct regulation of the target gene.

[0069] In some cases, the first gate unit regulates the first target gene. Alternatively, or in addition thereto, the first gate unit can also modulate the second gate unit. The modulation of the second gate unit is for a minimum or maximum of about 1 millisecond, minimum or maximum of about 2 milliseconds, minimum or maximum of about 3 milliseconds, minimum or maximum of about 4 milliseconds, minimum or maximum of about 5 milliseconds, minimum or maximum of about 6 milliseconds, minimum or maximum of about 7 milliseconds, minimum or maximum of about 8 milliseconds, minimum or maximum of about 9 milliseconds, minimum or maximum of about 10 milliseconds, minimum or maximum of about 20 milliseconds, minimum or maximum of about 30 milliseconds, minimum or maximum of about 40 milliseconds, minimum or maximum of about 50 milliseconds, minimum or maximum of about 60 milliseconds, minimum or maximum of about 70 milliseconds, minimum or maximum of about 80 milliseconds, minimum or maximum of about 90 milliseconds, minimum or maximum of about 100 milliseconds, minimum or maximum of about 200 milliseconds, minimum or maximum of about 300 milliseconds, minimum or maximum of about 400 milliseconds, minimum or maximum of about 500 milliseconds, minimum or maximum of about 600 milliseconds, minimum or maximum of about 700 milliseconds, minimum or maximum of about 800 milliseconds, minimum or maximum of about 900 milliseconds, minimum or maximum of about 1 second, minimum or maximum of about 2 seconds, minimum or maximum of about 3 seconds, minimum or maximum of about 4 seconds, minimum or maximum of about 5 seconds, minimum or maximum of about 6 seconds, minimum or maximum of about 7 seconds, minimum or maximum of about 8 seconds, minimum or maximum of about 9 seconds, minimum or maximum of about 10 seconds, minimum or maximum of about 15 seconds, minimum or maximum of about 20 seconds, minimum or maximum of about 30 seconds, minimum or maximum of about 40 seconds, minimum or maximum of about 50 seconds, minimum or maximum of about 1 minute, minimum or maximum of about 2 minutes, minimum or maximum of about 3 minutes, minimum or maximum of about 4 minutes, minimum or maximum of about 5 minutes, minimum or maximum of about 6 minutes, minimum or maximum of about 7 minutes, minimum or maximum of about 8 minutes, minimum or maximum of about 9 minutes, minimum or maximum of about 10 minutes, minimum or maximum of about 20 minutes, minimum or maximum of about 30 minutes, minimum or maximum of about 40 minutes, minimum or maximum of about 50 minutes, minimum or maximum of about 1 hour, minimum or maximum of about 2 hours, minimum or maximum of about 3 hours, minimum or maximum of about 4 hours, minimum or maximum of about 5 hours, minimum or maximum of about 6 hours, minimum or maximum of about 7 hours, as confirmed by rt-qPCR, Western blotting, or other methods.It may occur after a minimum or maximum of approximately 8 hours, a minimum or maximum of approximately 9 hours, a minimum or maximum of approximately 10 hours, a minimum or maximum of approximately 12 hours, a minimum or maximum of approximately 16 hours, a minimum or maximum of approximately 20 hours, or a minimum or maximum of approximately 24 hours, or after modulation of the first gate unit.,

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

[0071] In some cases, modification of the target gene by the gate unit may inactivate the gene. For example, the modification of the gene may stop the expression and / or activity level of the target gene. Alternatively, the modification of the gene may reduce the expression and / or activity level of the target gene. In some cases, the modification of the gene may increase the expression and / or activity level of the target gene. Alternatively, the modification of the gene may maintain the expression and / or activity level of the target gene.

[0072] The expression and / or activity profile of a target gene (e.g., a differentiation marker) can be compared to that of a control gene (e.g., a housekeeping gene such as GAPDH), the relative expression levels of two or more target genes (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 the target gene compared to the average expression level of the same target gene in the target cell type, etc.

[0073] In some cases, the guide nucleic acid molecule (gNA) (e.g., a functional gNA) expressed by the second gate unit can create a modification in at least a portion of the first gate unit upon activation. For example, the activated gNA of the second gate unit can create a modification in the polynucleotide sequence of the first gate unit encoding the gNA (e.g., an activatable gNA), or in the promoter sequence of the first gate unit operably linked to such gNA of the same first gate unit. Such a modification can render the gNA of the first gate unit inoperable when expressed (e.g., reducing or inhibiting specific binding to the target gene). Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0074] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can occur by a single-strand break that results in discontinuity in one nucleotide strand. Inactivation of a polynucleotide sequence or a target gene can occur by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or more single-strand breaks. In some cases, gene inactivation can occur 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.

[0075] In some cases, the gNA can have a size of at least or at most about 60 nucleotides, at least or at most about 70 nucleotides, at least or at most about 80 nucleotides, at least or at most about 85 nucleotides, at least or at most about 90 nucleotides, at least or at most about 95 nucleotides, at least or at most about 100 nucleotides, at least or at most about 105 nucleotides, at least or at most about 110 nucleotides, at least or at most about 120 nucleotides, at least or at most about 130 nucleotides, at least or at most about 140 nucleotides, at least or at most about 150 nucleotides, or at least or at most about 200 nucleotides (e.g., including both a spacer sequence and a scaffold sequence).

[0076] In some cases, the scaffold sequence of the gNA can have a size of at least or at most about 30 nucleotides, at least or at most about 35 nucleotides, at least or at most about 40 nucleotides, at least or at most about 45 nucleotides, at least or at most about 50 nucleotides, at least or at most about 55 nucleotides, at least or at most about 60 nucleotides, at least or at most about 65 nucleotides, at least or at most about 70 nucleotides, at least or at most about 75 nucleotides, at least or at most about 80 nucleotides, at least or at most about 85 nucleotides, at least or at most about 90 nucleotides, at least or at most about 95 nucleotides, at least or at most about 100 nucleotides, at least or at most about 100 nucleotides, at least or at most about 120 nucleotides, at least or at most about 130 nucleotides, at least or at most about 140 nucleotides, or at least or at most about 150 nucleotides.

[0077] In some cases, the spacer sequence of the gNA can have a size of at least or at most about 10 nucleotides, at least or at most about 11, at least or at most about 12, at least or at most about 13, at least or at most about 14, at least or at most about 15, at least or at most about 16, at least or at most about 17, at least or at most about 18, at least or at most about 19, at least or at most about 20, at least or at most about 21, at least or at most about 22, at least or at most about 23, at least or at most about 24, at least or at most about 25, at least or at most about 26, at least or at most about 27, at least or at most about 28, at least or at most about 29, or at least or at most about 30 nucleotides.

[0078] In some cases, the systems and methods of the present disclosure 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 are 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) hybridize to a target gene (e.g., genomic DNA) and regulate the expression and / or activity level of the target gene by the action of a transcriptional activator without mediating Cas nuclease activity as desired by individual non-homologous genetic circuits. For example, the use of gNAs with different spacer sequence lengths that bind to different targets can enable the second gate unit provided herein to induce inactivation of the activated first gate unit and / or induce separate regulation of a second target gene.

[0079] As described above, the length of the spacer array of the gNA can affect the ability of the gNA to mediate Cas nuclease activity. In some cases, gNAs with different lengths of spacer arrays can be used in the same non-homologous genetic circuit to affect the cleavage, activation, inactivation, and / or regulation of different types of one or more target nucleic acids. In some cases, a gNA spacer array shorter than a threshold length (e.g., about 16 nucleotides) can eliminate the nuclease activity of the Cas transcriptional regulator while always mediating DNA binding for transcriptional regulation of the target gene. In some cases, a gNA spacer array shorter than at least about 25 nucleotides, at least about 20 nucleotides, at least about 19 nucleotides, at least about 18 nucleotides, at least about 17 nucleotides, at least about 16 nucleotides, at least about 15 nucleotides, at least about 15 nucleotides, at least about 14 nucleotides, at least about 13 nucleotides, at least about 12 nucleotides, at least about 11 nucleotides, or at least about 10 nucleotides can eliminate the nuclease activity of the Cas protein while always mediating DNA binding.

[0080] For example, a gNA containing a 20-nucleotide spacer array (e.g., a gNA encoded by a gate portion to target a gene regulatory portion plasmid) may be sufficient to promote the nuclease activity of an endonuclease (e.g., Cas or a Cas transcriptional regulator fusion protein) in a target polynucleotide sequence. Alternatively, or in addition, a gNA containing a 14-nucleotide spacer array (e.g., a gNA encoded by a gene regulatory portion) may be able to hybridize to DNA but may not be long enough to mediate nuclease activity and may only be able to promote endonuclease binding to cognate DNA sequences. Thus, shorter gNAs can selectively enable transcriptional regulation of a target gene by use of an endonuclease transcriptional regulator system (e.g., a Cas activator system, a Cas repressor system) without cleaving the target gene.

[0081] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can occur 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., those 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.

[0082] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can occur by an indel, also known as an insertion-deletion mutation. Indel mutations can include frameshift mutations or non-frameshift mutations. Indel mutations can include point mutations, also referred to as base substitutions, in which only one base or a base pair is modified. Indel mutations can include bases or base pairs of a length 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, 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. Indel mutations can include bases or base pairs of a length of up to about 2000, up to about 1000, up to about 900, up to about 800, up to about 700, up to about 600, up to about 500, up to about 400, up to about 300, up to about 200, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1.

[0083] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can be achieved without cleaving the polynucleotide sequence or the target gene. For example, a gene regulatory moiety (e.g., a nucleic acid molecule such as a complex comprising a CRISPR / Cas protein and a guide nucleic acid molecule and / or an endonuclease) can specifically bind to the polynucleotide sequence or the 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 activator provided herein. Alternatively, or in addition thereto, the gene regulatory moiety can induce an epigenetic modification (or epigenomic modification) provided herein.

[0084] In some cases, modification of a polynucleotide sequence or a target gene provided herein can inactivate the polynucleotide sequence or the target gene. For example, modification of a polynucleotide sequence or a target gene can suppress or reduce the expression and / or activity level of the polynucleotide sequence or the target gene. In some cases, modification of a polynucleotide sequence or a target gene provided herein 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.

[0085] In some cases, the modification of the polynucleotide sequence or target gene provided herein may reduce 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 95%, at least or up to about 99%, or about 100% (e.g., compared to a control lacking the modification).

[0086] In some cases, the modification of the polynucleotide sequence or target gene provided herein may reduce 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, or at least or up to about 100-fold (e.g., compared to a control lacking the modification).

[0087] In some cases, the modification of the polynucleotide sequence or target gene provided herein may increase the expression and / or activity level of the polynucleotide sequence or target gene by at least or up to about 0.1%, at least or up to about 0.2%, at least or up to about 0.3%, at least or up to about 0.4%, at least or up to about 0.5%, at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500% (e.g., compared to a control lacking the modification).

[0088] In some cases, the modification of the polynucleotide sequence or target gene provided herein can increase 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., compared to a control lacking the modification).

[0089] In some cases, the activation of multiple gate units can be the result of a single activation of a non-homologous genetic circuit (e.g., by a single activation moiety at a single point in time). The multiple gate units can include one of a first gate unit and a second gate unit that are preconfigured to be sequentially activated upon activation of the non-homologous genetic circuit by a single activation. In some cases, one of the first and second gate units can be activated by a single activation moiety (e.g., a guide nucleic acid), while the other of the first and second gate units can be activated by an additional activation moiety different from the activation moiety of the non-homologous genetic circuit (e.g., a different guide nucleic acid). The additional activation moiety can be a part of the non-homologous genetic circuit that is generated (e.g., expressed) only upon activation of the non-homologous genetic circuit. Alternatively, or in addition, the first and second gate units can each be activated by different activation moieties that are not the same as the activation moiety of the non-homologous genetic circuit. Such different activation moieties can be parts of the non-homologous genetic circuit that are generated (e.g., expressed) only upon activation of the non-homologous genetic circuit.

[0090] In some embodiments of any one 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 regulatory portion (e.g., at least or up to about 1 gene regulatory portion, at least or up to about 2 gene regulatory portions, at least or up to about 3 gene regulatory portions, at least or up to about 4 gene regulatory portions, at least or up to about 5 gene regulatory portions, at least or up to about 6 gene regulatory portions, at least or up to about 7 gene regulatory portions, at least or up to about 8 gene regulatory portions, at least or up to about 9 gene regulatory portions, at least or up to about 10 gene regulatory portions, etc.). The gate portion 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 regulatory portion disclosed herein can include a gNA (e.g., at least or up to about 1 gNA molecule, at least or up to about 2 gNA molecules, at least or up to about 3 gNA molecules, at least or up to about 4 gNA molecules, at least or up to about 5 gNA molecules, etc.). The guide nucleic acid molecule disclosed herein can include, but is not limited to, DNA, RNA, any analogs thereof, or any combination thereof. In some embodiments of any one of the systems disclosed herein, the gate portion and / or the gene regulatory portion can be activatable to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and the complex is configured or capable of binding to a target polynucleotide, for example, to regulate the expression and / or activity level of the target polynucleotide or another polynucleotide sequence operably linked to the target polynucleotide.For example, the complex can regulate the expression and / or activity level of a gene containing a target polynucleotide.

[0091] In some cases, a single gate unit may contain a single gene regulatory moiety for each target gene (e.g., a target endogenous gene). For example, activation of a single gate unit can cause the gene regulatory moiety to express a functional guide nucleic acid molecule that binds to the target gene and regulates the expression / epigenetic profile of the target gene. Alternatively, a single gate unit may contain multiple gene regulatory moieties per target gene for multiplex targeting. For example, the multiple gene regulatory moieties can be such that activation of a single gate unit expresses multiple different guide nucleic acid molecules (e.g., based substantially simultaneously on activation of the single gate unit alone) that have multiple different spacer sequences for a common target gene, so as to achieve multiplex targeting and regulation of the expression / epigenetic profile of the common target gene.

[0092] In some cases, when a heterologous genetic 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 in a given step of the heterologous genetic circuit. In some cases, each of the multiple target genes can be regulated via multiplex targeting in a given step of the heterologous genetic circuit. In some cases, one target gene can be regulated via a single gene regulatory moiety in a given step of the heterologous genetic circuit, while a different target gene can be regulated via multiplex targeting in a given step of the heterologous genetic circuit.

[0093] In some embodiments of any one of the systems disclosed herein, the first (or first) gate unit of the non-homologous genetic circuit disclosed herein may be activated (e.g., directly activated) by an activation moiety. The activation moiety may be directly coupled to at least a portion of the first gate unit to activate the first gate unit, thereby enabling, for example, continuous activation of the non-homologous genetic circuit. Alternatively, the activation moiety (e.g., electromagnetic energy) may activate the first gate unit without directly coupling to at least a portion of the first gate unit. In some cases, the first gate unit may include at least one gate portion and at least one gene regulatory portion. In some cases, the first gate unit may include at least one gate portion, but does not include, or need not include, a gene regulatory portion. In some cases, the first gate unit may include at least one gene regulatory portion, but does not include, or need not include, a gate portion (e.g., the activation moiety may be configured to activate the first gate unit and at least one additional gate unit).

[0094] In some embodiments of any one of the systems disclosed herein, the gNA of the gate portion and / or the gene regulatory portion (e.g., the gNA encoded by the gate portion and / or the gene regulatory portion) can be an activatable gNA. The activatable gNA can be any one of the following, but is not limited thereto: ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogs thereof, or any combination thereof. In some embodiments, a 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). Alternatively, or in addition thereto, the inactivating polynucleotide sequence can encode a non-standard 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 (ii) any linker sequence between the spacer sequence and the scaffold sequence. The vector can include 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.

[0095] Optionally, the term "proGuide" as generally used herein can refer to such 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 regulatory portion.

[0096] The pro-guide may include 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, and this domain contains one or more inactivating polynucleotide sequences (e.g., one or more poly-T sequences). Alternatively, the pro-guide may not include a linker sequence between the two domains (i) and (ii).

[0097] The pro-guide can include a target polynucleotide domain in or adjacent to (e.g., at and / or adjacent to the 5' and / or 3' ends of the inactivating polynucleotide sequence) the inactivating polynucleotide sequence, and by targeting this target polynucleotide domain (e.g., via the sequential activation mechanism of the heterologous genetic circuit provided herein), the inactivating polynucleotide sequence can be modified (e.g., edited, cleaved), thereby enabling the pro-guide to express an activating guide nucleic acid molecule. The target polynucleotide domain of the pro-guide does not exhibit sequence identity to any comparable endogenous polynucleotide sequence in the cell, thereby avoiding inadvertent targeting and regulation of the endogenous target gene.

[0098] In some embodiments, the inactivating polynucleotide sequence of the pro-guide can be located between two target polynucleotide domains, which may or may not be targetable by a common guide nucleic acid sequence. In some cases, the two target polynucleotide domains can be reverse complementary to each other such that the inactivating polynucleotide sequence can be modified or cleaved by the same mechanism (e.g., the same spacer sequence of the guide nucleic acid molecule).

[0099] In some embodiments of any one of the systems disclosed herein, the gNA of the gate portion and / or the gene regulatory portion (e.g., the gNA encoded by the gate portion and / or the gene regulatory portion) can be an activatable gNA. The activatable gNA can be any one of, but not limited to, ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogs thereof, or any combination thereof. In some embodiments, the activatable gNA molecule can be a self-cleaving gNA (e.g., the gRNA contains a ribozyme). For example, when the activatable gNA is expressed intracellularly, the activatable gNA may be self-cleavable such that it is non-functional (e.g., not configured to bind to a target gene) unless the gene encoding the activatable gNA is modified prior to the expression of the activatable gNA. In some embodiments, the activatable gNA molecule includes a non-standard transcription termination sequence such that a functional gNA molecule is not expressed until the gene encoding the activatable gNA having the non-standard transcription termination sequence (e.g., a polyX sequence such as a polyU sequence or a polyT 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 bound fluorescent label.

[0100] In some cases, the size of the polyT sequence is equal to or greater than a threshold length, which 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 containing a polyT sequence of at least the threshold length, and editing such a plasmid to reduce the length of the polyT to less than the threshold length enables the expression of the entire gNA without premature termination, thereby activating the gNA. In some cases, the polyT sequence contains at least 5 Ts. In some cases, the polyT sequence contains at least 7 Ts. In some cases, the polyT sequence contains at least 8 Ts. In some cases, the polyT sequence contains at least 10 Ts. In some cases, the polyT sequence contains from 5 to 15 Ts. In some cases, the polyT sequence contains one or more additional nucleotides other than T.

[0101] 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 linked to a target gene in a cell. The target gene can include a coding polynucleotide sequence encoding a target nucleic acid molecule or a target protein. The target polynucleotide sequence can be part of the coding polynucleotide sequence. Alternatively, the target polynucleotide sequence may not be part of the coding polynucleotide sequence. For example, the target polynucleotide sequence can be upstream of the coding polynucleotide sequence (e.g., part of a promoter of the coding polynucleotide sequence such as a transcription start site (TSS)).

[0102] As provided herein, when a heterologous genetic circuit is activated to induce multiple different modulations of a target gene, as provided herein, the multiple distinct modulations of the target gene can be different (e.g., different degrees of change in the expression and / or activity level of the target gene). For example, the first modulation exerted by the first gate unit and the second modulation exerted by the second gate unit can be different by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%. The first modulation and the second modulation can be different by up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, or up to about 0.1%. Alternatively, or in addition, the distinct modulations of the target gene can be substantially the same (e.g., the same).

[0103] The multiple distinct modulations can each be sufficient to induce a desired change in the expression and / or activity level of the target gene. Alternatively, the distinct modulations can each be insufficient to induce a desired change in the expression and / or activity level of the target gene.

[0104] One or more target genes disclosed herein can include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0105] 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 modulator (e.g., as part of a heterologous genetic circuit) can be performed in the absence of one or more heterologous factors that are normally required to induce a comparable conversion in the absence of the heterologous gene modulator. In a comparable conversion, one or more heterologous factors can be added exogenously to the cell culture medium or 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.

[0106] In some cases, the use of a heterologous genetic circuit disclosed herein can be used to convert a plurality of cells of a first cell type to a plurality of second cell types. 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, differentiated cells are terminally differentiated cells.

[0107] Final differentiated cells (e.g., the first cells to be modified into the engineered cells disclosed herein, the final cell products generated from the engineered cells disclosed herein, etc.) can include muscle cells, immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells, epithelial cells, stem cells, secretory cells, blood cells, germ cells, nurse cells, storage cells, enteroendocrine cells, pituitary cells, neurosecretory cells, duct cells, odontoblasts, cementoblasts, glial cells, or stromal cells.

[0108] Non-limiting examples of such cells include cells derived from the lymphatic system, such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, cytokine-induced killer (CIK) cells; bone marrow cells, such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes / hyper-segmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, dendritic cells; cells derived from the endocrine system, including cells of the thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, oxyphil cells), adrenal (chromaffin cells), pineal (pinealocytes); cells of the nervous system, including glial cells (astrocytes, microglia), megalocytic neurosecretory cells, stellate cells, Betz cells, and cells of the pituitary (gonadotrophs, corticotrophs, thyrotrophs, somatotrophs, lactotrophs); cells of the respiratory system, such as lung cells (Pneumocytes) (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, such as cells of the stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteric chromaffin cells, APUD cells, cells of the intestine endocrine, including the liver (hepatocytes, Kupffer cells), cartilage / bone / muscle; cells of the bone, including osteoblasts, osteocytes, osteoclasts, teeth (osteoblasts, ameloblasts); cells of the cartilage, including chondroblasts, chondrocytes; skin cells (nevus cells), including trichocytes, keratinocytes, melanocytes; muscle cells, including myocytes; cells of the urinary system, including podocytes, juxtaglomerular cells, mesangial cells within the glomerulus / mesangial cells outside the glomerulus, renal proximal tubule brush border cells, macula densa cells; sperm (spermatozoon), Sertoli cells, Leydiggerm cells, including sperm cells, ova (eggs); and adipocytes, fibroblasts, tendon cells, epithelial keratinocytes (differentiated epithelial cells), epithelial basal cells (stem cells), keratinocytes of the nails and toes, nail bed basal cells (stem cells), medullary hair shaft cells, cortical hair shaft cells, cuticular hair shaft cells, reticular 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 of the epithelium of the cornea (stem cells), tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urothelial cells (lining the bladder and ureters), exocrine epithelial cells, salivary gland mucosal cells (secreting polysaccharide-rich secretions), salivary gland serous cells (secreting glycoprotein enzyme-rich secretions), von Ebner gland cells in the tongue (flushing taste buds), mammary gland cells (secreting milk), lacrimal gland cells (secreting tears), skin gland cells in the ear (secreting wax), dark cells of eccrine sweat glands (secreting glycoproteins), clear cells of eccrine sweat glands (secreting small molecules), apocrine sweat gland cells (secreting odors, sensitive to sex hormones), glands of Moll cells in the eyelids (specialized sweat glands), sebaceous gland cells (secreting lipid-rich sebum), Bowman's gland cells of the nose (flushing the olfactory epithelium), Brunner gland cells of the duodenum (secreting enzymes and alkaline mucus), seminal vesicle cells (secreting semen components containing fructose for sperm motility), prostate cells (secreting semen components), globulonet gland cells (secreting mucus), Bartholin gland cells (secreting vaginal lubricant), Littre gland cells (secreting mucus), endometrial cells (secreting carbohydrates), isolated goblet cells of the airways and digestive tract (secreting mucus), stomach lining mucus cells (secreting mucus), gastric gland zymogen cells (secreting pepsinogen), gastric gland oxyntic cells (secreting hydrochloric acid), pancreatic acinar cells (secreting bicarbonate and digestive enzymes), Paneth cells of the small intestine (secreting lysozyme), type II pneumocytes of the lung (secreting surfactant), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, somatotrophs, lactotrophs, thyrotrophs, gonadotrophs, corticotrophs, intermediate pituitary cells, giant neurosecretory cells, intestinal and airway cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxyphil cells, adrenal cells, chromaffin cells, Leydig cells of the testis, inner theca cells of follicles (Theca internacells), lutein cells of ruptured follicles, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (renin secretion), macular densa cells of the kidney, metabolic and storage cells, barrier function cells (lungs, intestines, exocrine glands, and urogenital tract), the kidney, type I pneumocytes (lining the air spaces of the lung), pancreatic duct cells (centroacinar cells), ductless gland duct cells (sweat glands, salivary glands, mammary glands, etc.), duct cells (seminal vesicles, prostate, etc.), epithelial cells lining closed body cavities, ciliated cells with a 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 (various types), epidermal Langerhans cells, osteoclasts (bone), dendritic cells (lymphoid tissue), microglial cells (central nervous system), neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, sensory transducer cells, autonomic nerve cells, sensory organ and peripheral nerve support cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonium / oocyte, spermatid, spermatocyte, spermatogonium cell (stem cell for spermatocytes), sperm, nurse cells, ovarian follicle cells, Sertoli cells (in the testis), thymic epithelial cells, stromal cells, and interstitial kidney cells, and can include other cells.

[0109] Stem cells can include induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), mesenchymal stem cells (MSC), hematopoietic stem cells (HSC) (e.g., derived from umbilical cord blood), muscle stem cells, neural stem cells, epithelial stem cells, mammary stem cells, intestinal stem cells, neural crest stem cells, or testicular stem cells.

[0110] 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, ERR beta, 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, Porocarcinoma calicin, 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.

[0111] Various aspects of the present disclosure provide for contacting a first plurality of cells with a heterologous gene modulator that exhibits specific binding to a target gene derived from a non-mammalian genome (e.g., a non-human genome) to achieve conversion of the first plurality of cells of a first cell type to a plurality of cells of a second cell type (e.g., from differentiated cells to stem cells). In some embodiments, the target gene is (i) derived from a virus (e.g., a DNA virus, an RNA virus, or a retrovirus (e.g., a retrovirus or a pararetrovirus)), and (ii) can be integrated into 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 a human endogenous retrovirus (HERV) gene. The target gene can include at least a portion of the HERV gene. At least a portion of the HERV gene can include one or more members selected from the group consisting of one or more long terminal repeat sequences (LTRs) (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., reverse transcriptase (RT), ribonuclease H (RH), integrase (IN)), and an envelope (ENV) gene (e.g., a surface component (SU), a transmembrane component (TM)). The LTRs disclosed herein can be LTRs located upstream of the target gene, such as 5’ LTRs. Non-limiting examples of HERV LTRs include LTR5HS, LTR5A, and LTR5B. Non-limiting examples of LTR5HS 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.

[0112] LTR5HS is a subgroup of regulatory elements that regulate all human HERV-K elements. LTR5HS elements can be regulated via DNA hypermethylation. Alternatively, LTR5HS elements can be regulated via DNA hypomethylation or DNA demethylation.

[0113] In some cases, the heterologous gene modulator exhibits specific binding to a gene encoding a mobile genetic element (MGE) (or target gene). An MGE is a segment of genetic material that can move around with the genome or be transferred from one species or replicon to another. Non-limiting examples of MGEs are plasmids, transposons, integrons, viral agents, and introns.

[0114] Transposons, also known as jumping genes, are a group of mobile genetic elements that are DNA sequences. Transposons can move to different locations within the genome. Alternatively, some transposons are always retained at specific insertion sites within the genome. Transposons are divided into two major groups: retrotransposons and DNA transposons. Retrotransposons are often found in eukaryotes. Non-limiting examples of retrotransposons include long terminal repeats, i.e., LTRs, and non-long terminal repeats, i.e., 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 called helitrons), and self-synthesizing DNA transposons (also called polintons).

[0115] Long interspersed nuclear elements (LINEs) are transposable elements typically about 7000 base pairs in length. LINEs often function by being transcribed into mRNA and translated into proteins that act as reverse transcriptases to incorporate DNA copies into the genome at new sites. Non-limiting examples of LINEs include LINE-1, L1H, RTE, Tad, and CRE.

[0116] Short interspersed nuclear elements (SINEs) are transposable elements typically about 100 - 700 base pairs in length. SINEs are often sequence-specific. One example of a SINE is the Alu element, which is primate-specific repetitive. The Alu element makes up about 11% of the human genome. The Alu element is typically about 300 base pairs in length. The Alu element is involved in several human diseases such as cancer.

[0117] In some cases, the heterologous gene modulator exhibits specific binding to a gene (or target gene) that encodes (or, as used interchangeably herein, includes or is operably linked to) an embryo genome activation (EGA) enriched Alu motif (EEA). The EEA or EEA motif is an Alu element enriched near genes involved in embryo genome activation. The EEA motif-related mechanism is involved in cell reprogramming by activation of genes such as Nanog and Rex1.

[0118] Long terminal repeats (LTRs) are pairs of DNA sequences hundreds of bases in length that are present at the ends of either retrotransposons or endogenous retroviruses in the eukaryotic genome. LTRs typically encode reverse transcriptase and integrase and enable the retrotransposon to be copied and inserted at different positions. Non-limiting examples of LTRs include HERV, MLT2A1, and MLT2A2.

[0119] In some cases, the heterologous gene modulator exhibits specific binding to a gene (or target gene) encoding a human endogenous retrovirus or HERV. HERVs are stable elements in DNA left from ancient infections that have affected the primate germline over the past 100 million years. HERVs and their gene products, including RNA, cytosolic DNA, and proteins, can regulate and be affected by the immune system. HERVs are involved in 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.

[0120] In some cases, the heterologous gene modulator exhibits specific binding to a gene (or target gene) encoding HERV-K. HERV-K is a family of 30 - 50 sequences that are highly conserved in primates. The HERV-K protein has protease activity but no reverse transcriptase activity. HERV-K is often expressed at low steady-state levels in many human tissues and tumors.

[0121] In some cases, the heterologous gene modulator includes an endonuclease (e.g., Cas9) that can form a complex with a nucleic acid molecule. In some cases, the heterologous gene modulator further includes a gene regulator. The gene regulator can be a gene activator, a DNA-binding protein that has positive control over gene expression. Non-limiting examples of gene activators include VP16, VP64, p65, p53, E2F1, TAT, E2A, NFAT, GAL4, CGN4, HAP1, MLL, TRG3, GLN3, 0AF1, PIP2, PDR1, PDR3, PHO4, LEU3, RTA, and the VP64-p65-RTA fusion (VPR). The gene activator can bind to the endonuclease, and the gene regulator can be a gene repressor, a DNA-binding protein that has negative control over gene expression. Non-limiting examples of gene repressors include the tetracycline repressor, the AMP early repressor (ICER), the Kruppel-associated box (KRAB), the YY1 glycine-rich repressor, the Sp1-like repressor, the E(spI) repressor, the IκB repressor, or MeCP2.

[0122] Various aspects of the present disclosure provide engineered cells programmed to induce desired expression and / or activity levels (or profiles thereof) of one or more target genes in a cell.

[0123] One or more target genes targeted by the heterologous gene modulators 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.

[0124] In some cases, one or more target genes of the heterologous genetic circuit may include OCT4, SOX2, KLF4, and / or MYC. In some cases, the heterologous genetic circuit may be preconfigured such that (i) the regulation of the expression level or epigenetic profile level of a first target endogenous gene derived from OCT4, SOX2, KLF4, and / or MYC occurs before (ii) the regulation of the expression level or epigenetic profile level of a second target endogenous gene derived from OCT4, SOX2, KLF4, and / or MYC. For example, the heterologous genetic circuit may be programmed such that the activation of a first gate unit preconfigured to target and regulate the expression / epigenetic level of the first target endogenous gene occurs before the activation of a second gate unit preconfigured to target and regulate the expression / epigenetic level of a second target endogenous gene (e.g., after activation of the first gate unit or after activation of the heterologous genetic circuit, in the absence of human intervention or any secondary activation of the heterologous genetic circuit). The first target endogenous gene may be OCT4, and the second target endogenous gene may be SOX2, KLF4, and / or MYC. The first target endogenous gene may be SOX2, and the second target endogenous gene may be OCT4, KLF4, and / or MYC. The first target endogenous gene may be KLF4, and the second target endogenous gene may be OCT4, SOX2, and / or MYC. The first target endogenous gene may be MYC, and the second target endogenous gene may be OCT4, S0X2, and / or KLF4. Without being bound by theory, by targeting and controlling the sequential regulation of target genes while minimizing cell intervention (e.g., human intervention) or disruption, (i) cell characteristics such as viability, proliferative ability, and / or therapeutic efficacy can be maintained, and / or (ii) desirable results of cell engineering (e.g., reprogramming of the cells provided herein) can be improved.

[0125] One or more target genes may include HERV (e.g., HERV-K), POU family transcription factors (e.g., Oct4), KLF4, MYC, SOX2, EEA, and miR-302. Optionally, the spacer sequence of the guide nucleic acid (e.g., guide RNA) for the target genes provided herein is at least or at most about 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 86%, at least or at most about 87%, at least or at most about 88%, at least or at most about 89%, at least or at most about 90%, at least or at most about 91%, at least or at most about 92%, at least or at most about 93%, at least or at most about 94%, at least or at most about 95%, at least or at most about 96%, at least or at most about 97%, at least or at most about 98%, at least or at most about 99%, or substantially about 100% sequence identity to one or more members selected from SEQ ID NOs: 1-63 or their complementary sequences (e.g., continuous polynucleotide sequences). Optionally, the heterologous gene modulator provided herein shows specific binding to a target gene comprising a polynucleotide sequence (e.g., continuous polynucleotide sequence) that shows at least or at most about 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 86%, at least or at most about 87%, at least or at most about 88%, at least or at most about 89%, at least or at most about 90%, at least or at most about 91%, at least or at most about 92%, at least or at most about 93%, at least or at most about 94%, at least or at most about 95%, at least or at most about 96%, at least or at most about 97%, at least or at most about 98%, at least or at most about 99%, or substantially about 100% sequence identity to one or more members selected from SEQ ID NOs: 1-63 or their complementary sequences (e.g., with conversion from uracil to thymine).

[0126] One or more target genes may include HERV (e.g., HERV-K). In some cases, the spacer sequence of the guide nucleic acid (e.g., guide RNA) for the target genes provided herein is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least or up to about 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 NOs: 1-12 or their complementary sequences, and may include a polynucleotide sequence (e.g., a continuous polynucleotide sequence).In some cases, the heterologous gene modulators provided herein may exhibit specific binding to a target gene comprising a polynucleotide sequence (e.g., a continuous 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 NOs: 1-12 or their complementary sequences (e.g., with the conversion from uracil to thymine).

[0127] One or more target genes may include POU family transcription factors (e.g., Oct4). OCT4 is a transcription factor of the POU family. OCT4 is involved in the self-renewal of undifferentiated embryonic stem cells and is a marker commonly used for undifferentiated cells. In some cases, the heterologous gene modulators provided herein are 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 (e.g., contiguous polynucleotide sequence) to one or more members selected from SEQ ID NOs: 13-17 or their complementary sequences, and may exhibit specific binding to target genes containing such polynucleotide sequences.In some cases, the spacer sequence of the guide nucleic acid (e.g., guide RNA) provided herein may include a polynucleotide sequence (e.g., a continuous 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 NOs: 13 - 17 or their complementary sequences.

[0128] One or more target genes may include SOX2. SOX2 is involved in the self-renewal of undifferentiated embryonic stem cells and is a marker commonly used for undifferentiated cells. In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) for a target gene provided herein is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least or up to about 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 NOs: 42-58 or their complementary sequences, and may include a polynucleotide sequence (e.g., a continuous polynucleotide sequence).In some cases, the heterologous gene modulators provided herein may exhibit specific binding to a target gene comprising 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 NOs: 42 - 58 or their complementary sequences (e.g., with the conversion from uracil to thymine).

[0129] One or more target genes may include MYC (or C-Myc). C-Myc is a regulatory gene and a transcription factor involved in cell proliferation. In some cases, the spacer sequence of the guide nucleic acid (e.g., guide RNA) for the target gene provided herein is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least or up to about 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 NOs: 30-41 or their complementary sequences, and may include a polynucleotide sequence (e.g., a continuous polynucleotide sequence).In some cases, the heterologous gene modulators provided herein may exhibit specific binding to a target gene comprising 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 NOs: 30-41 or its complementary sequence (e.g., with conversion from uracil to thymine).

[0130] One or more target genes may include KLF4. KLF4, also known as Kruppel-like factor 4, is a zinc finger transcription factor involved in the regulation of proliferation, differentiation, apoptosis, and somatic cell reprogramming. In some cases, the spacer sequence of a guide nucleic acid (e.g., guide RNA) for a target gene provided herein is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least or up to about 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 NOs: 25-29 or their complementary sequences, and may include a polynucleotide sequence (e.g., a continuous polynucleotide sequence).In some cases, the heterologous gene modulators provided herein may exhibit specific binding to a target gene comprising 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 NOs: 25-29 or their complementary sequences (e.g., with the conversion from uracil to thymine).

[0131] One or more target genes may include miR-302. miR-302 is a polycistronic miRNA cluster that can induce and maintain pluripotency. Optionally, the spacer sequence of the guide nucleic acid (e.g., guide RNA) for the target genes provided herein is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least or up to about 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 NOs: 18-24 or their complementary sequences, and may include a polynucleotide sequence (e.g., a continuous polynucleotide sequence).In some cases, the heterologous gene modulators provided herein may exhibit specific binding to a target gene comprising 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 NOs: 18-24 or their complementary sequences (e.g., with the conversion from uracil to thymine).

[0132] One or more target genes may include EEA. In some cases, the spacer sequence of the guide nucleic acid (e.g., guide RNA) for the target genes provided herein may be 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 NOs: 59-63 or their complementary sequences, and may include a polynucleotide sequence (e.g., a continuous polynucleotide sequence).In some cases, the heterologous gene modulators provided herein may exhibit specific binding to a target gene comprising 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 NOs: 59 - 63 or its complementary sequence (e.g., with the conversion from uracil to thymine).

[0133] In some cases, cells can be dedifferentiated into iPSCs by contacting a plurality of cells with a heterologous gene modulator. By using the heterologous gene modulators disclosed herein, at least about 1x10 4 、2x10 4 、5x10 4 、1x10 5 、2x10 5 、5x10 5 、1x10 6 、2x10 6 、5x10 6 、1x10 7 、2x10 7 、5x10 7 、1x10 8 、2x10 8 、5x10 8 、1x10 9 、2x10 9 、5x10 9 、1x10 10 、2x1010 , 5 x 10 10 , 1 x 10 15 , 2 x 10 15 , 5 x 10 15 , or iPSCs of more than that, at most about 1 x 10 6 , 9 x 10 5 , 8 x 10 5 , 7 x 10 5 , 6 x 10 5 , 5 x 10 5 , 4 x 10 5 , 3 x 10 5 , 2 x 10 5 , 1 x 10 5 , 5 x 10 4 , 2 x 10 4 , 1 x 10 4 , or can be generated from terminally differentiated cells of less than that.

[0134] Such generation of iPSCs by using the heterologous gene modulators disclosed herein can be achieved within a span of at most about 1 day, at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 8 days, at most about 9 days, at most about 10 days, at most about 11 days, at most about 12 days, at most about 13 days, at most about 14 days, at most about 15 days, at most about 20 days, at most about 25 days, at most about 30 days, at most about 35 days, at most about 40 days, at most about 45 days, at most about 50 days, at most about 55 days, at most about 60 days, or at most about 65 days.

[0135] Conversion of a plurality of cells of a first cell type to a plurality of cells of a second cell type (e.g., reprogramming of differentiated cells to stem cells, e.g., pluripotent stem cells) can be characterized by having a conversion efficiency (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%.

[0136] In some cases, a heterologous gene modulator and an additional heterologous gene modulator are introduced into the cell substantially simultaneously. In some cases, the heterologous gene modulator is introduced before the additional heterologous gene modulator. In some cases, the heterologous gene modulator is introduced after the additional heterologous gene modulator.

[0137] In some cases, the heterologous gene modulator and the additional heterologous gene modulator may or may not be part of the same heterologous genetic circuit. The heterologous gene modulator and the additional heterologous gene modulator may be part of different heterologous genetic circuits. Alternatively, both the heterologous gene modulator and the additional heterologous gene modulator may not be part of any heterologous genetic circuit.

[0138] In some cases, one or more cells can be further contacted with an inhibitor. In some cases, one or more cells can be contacted with an inhibitor prior to the introduction of a heterologous gene modulator. Alternatively, one or more cells can be contacted with an inhibitor substantially simultaneously with the introduction of a heterologous gene modulator. Alternatively, one or more cells can be contacted with an inhibitor after the introduction of a heterologous gene modulator. An inhibitor is a gene whose presence inhibits the expression of another gene at a different locus. The inhibitor can be, for example, a small molecule drug or a p53 inhibitor in the form of p53 short hairpin RNA (shRNA).

[0139] In some cases, the target gene can be subjected to one (e.g., a single) gene regulation (e.g., a single targeting activation step). In some cases, the target gene can be subjected to at least two different heterologous gene modulators including a first modulator that performs a first regulation and a second (or additional) modulator that performs a second (or additional) regulation. The timing of the first regulation and the second regulation is controllable (e.g., as predetermined by the design of a heterologous genetic circuit). For example, the initiation of the second regulation (e.g., by at least a part of a second gate unit such as a second gene regulation part) 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 part of a first gate unit such as a first gene regulation part).The initiation of this second regulation (e.g., by at least a part of a second gate unit such as a second gene regulation part) can occur up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, up to about 1 day, up to about 20 hours, up to about 10 hours, up to about 9 hours, up to about 8 hours, up to about 7 hours, up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2 hours, up to about 1 hour, up to about 50 minutes, up to about 40 minutes, up to about 30 minutes, up to about 20 minutes, up to about 10 minutes, up to about 9 minutes, up to about 8 minutes, up to about 7 minutes, up to about 6 minutes, up to about 5 minutes, up to about 4 minutes, up to about 3 minutes, up to about 2 minutes, up to about 1 minute, up to about 50 seconds, up to about 40 seconds, up to about 30 seconds, up to about 20 seconds, up to about 10 seconds, up to about 9 seconds, up to about 8 seconds, up to about 7 seconds, up to about 6 seconds, up to about 5 seconds, up to about 4 seconds, up to about 3 seconds, up to about 2 seconds, or up to about 1 second after the initiation of the first regulation (e.g., by at least a part of a first gate unit such as a first gene regulation part).

[0140] In some cases, the number of gate units that need to be activated (e.g., continuously activated) between the activation of the first regulation by the first gate unit and the subsequent activation of the second regulation by the second gate unit can at least partially determine (e.g., substantially determine) the timing between the first regulation and the second regulation. When activating the first regulation of the target gene by the first gate unit, to activate the second gate unit to induce the second regulation, it may be necessary to activate (e.g., continuously activate) 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. When activating the first regulation of the target gene by the first gate unit, to activate the second gate unit to induce the second regulation, it may be necessary to activate (e.g., continuously activate) up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 additional gate unit.

[0141] Cell fate can include the regulation of multiple target genes. For example, fate 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. Fate 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. Each of the genes disclosed herein can be subject to at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more regulations. Each of the genes disclosed herein can be subject to 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. One or more regulations of a target gene (e.g., an endogenous gene) induced by the heterologous genetic circuit of the present disclosure can be an artificial regulation (or heterologous regulation) and otherwise cannot occur in a cell in the absence of (i) a heterologous gene modulator and / or (ii) an activating portion of a heterologous gene modulator.

[0142] The first distinct modulator can induce a change (e.g., an increase or a decrease) 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%, or more with respect to the expression and / or activity level of a target gene (e.g., HERV-K). The first distinct modulation can induce a change (e.g., an increase or a decrease) of 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 with respect to the expression and / or activity level of a target gene (e.g., HERV-K).

[0143] (For example, induced by the first gate unit) The first distinct regulation disclosed herein can induce a change (e.g., increase or decrease) in the expression and / or activity level of a target gene that is at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least or at most about 0.6-fold, at least or at most about 0.7-fold, at least or at most about 0.8-fold, at least or at most about 0.9-fold, at least or at most about 1-fold, at least or at most about 2-fold, at least or at most about 3-fold, at least or at most about 4-fold, at least or at most about 5-fold, at least or at most about 6-fold, at least or at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 60-fold, at least or at most about 70-fold, at least or at most about 80-fold, at least or at most about 90-fold, at least or at most about 100-fold, at least or at most about 500-fold, at least or at most about 1,000-fold, at least or at most about 5,000-fold, or at least or at most about 10,000-fold lower or higher compared to the expression and / or activity level of a control. The first distinct regulation can induce a change (e.g., increase or decrease) in the expression and / or activity level of a target gene that is at most about 10,000-fold, at most about 5,000-fold, at most about 1,000-fold, at most about 500-fold, at most about 100-fold, at most about 90-fold, at most about 80-fold, at most about 70-fold, at most about 60-fold, at most about 50-fold, at most about 40-fold, at most about 30-fold, at most about 20-fold, at most about 10-fold, at most about 9-fold, at most about 8-fold, at most about 7-fold, at most about 6-fold, at most about 5-fold, at most about 4-fold, at most about 3-fold, at most about 2-fold, at most about 1-fold, at most about 0.9-fold, at most about 0.8-fold, at most about 0.7-fold, at most about 0.6-fold, at most about 0.5-fold, at most about 0.4-fold, at most about 0.3-fold, at most about 0.2-fold, or at most about 0.1-fold lower or higher compared to the expression and / or activity level of a control.

[0144] Subsequently, a second distinct modulation disclosed herein (e.g., induced by a second gate unit) can induce a further change (e.g., an increase, decrease, or selective attenuation) 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% in the expression and / or activity level of the target gene. The second distinct modulation can induce a further change (e.g., an increase or decrease) of up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, or up to about 0.1% in the expression and / or activity level of the target gene.

[0145] A further change by the second separate regulation can induce a further change (e.g., increase or decrease) of at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least or at most about 0.6-fold, at least or at most about 0.7-fold, at least or at most about 0.8-fold, at least or at most about 0.9-fold, at least or at most about 1-fold, at least or at most about 2-fold, at least or at most about 3-fold, at least or at most about 4-fold, at least or at most about 5-fold, at least or at most about 6-fold, at least or at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 60-fold, at least or at most about 70-fold, at least or at most about 80-fold, at least or at most about 90-fold, at least or at most about 100-fold, at least or at most about 500-fold, at least or at most about 1,000-fold, at least or at most about 5,000-fold, or at least or at most about 10,000-fold in the expression and / or activity level of the target gene compared to the expression and / or activity level of the control. The second separate regulation can induce a further change (e.g., increase or decrease) of at most about 10,000-fold or less, at most about 5,000-fold or less, at most about 1,000-fold or less, at most about 500-fold or less, at most about 100-fold or less, at most about 90-fold or less, at most about 80-fold or less, at most about 70-fold or less, at most about 60-fold or less, at most about 50-fold or less, at most about 40-fold or less, at most about 30-fold or less, at most about 20-fold or less, at most about 10-fold or less, at most about 9-fold or less, at most about 8-fold or less, at most about 7-fold or less, at most about 6-fold or less, at most about 5-fold or less, at most about 4-fold or less, at most about 3-fold or less, at most about 2-fold or less, at most about 1-fold or less, at most about 0.9-fold or less, at most about 0.8-fold or less, at most about 0.7-fold or less, at most about 0.6-fold or less, at most about 0.5-fold or less, at most about 0.4-fold or less, at most about 0.3-fold or less, at most about 0.2-fold or less, at most about 0.1-fold or less in the expression and / or activity level of the target gene compared to the expression and / or activity level of the control.

[0146] A further change by the second distinct regulation can occur when the expression and / or activity level of the target gene reaches the target level, for example, by the action of the first distinct regulation by the design of a non-homologous genetic circuit.

[0147] A further change due to the second distinct regulation can occur when the expression and / or activity level of the target gene is changed (e.g., increased or decreased) by a factor of at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least or at most about 0.6-fold, at least or at most about 0.7-fold, at least or at most about 0.8-fold, at least or at most about 0.9-fold, at least or at most about 1-fold, at least or at most about 2-fold, at least or at most about 3-fold, at least or at most about 4-fold, at least or at most about 5-fold, at least or at most about 6-fold, at least or at most about 7-fold, at least or at most about 8-fold, at least or at most about 9-fold, at least or at most about 10-fold, at least or at most about 20-fold, at least or at most about 30-fold, at least or at most about 40-fold, at least or at most about 50-fold, at least or at most about 60-fold, at least or at most about 70-fold, at least or at most about 80-fold, at least or at most about 90-fold, at least or at most about 100-fold, at least or at most about 500-fold, at least or at most about 1,000-fold, at least or at most about 5,000-fold, or at least or at most about 10,000-fold as compared to the expression and / or activity level of the control by the action of the first distinct regulation. A further change due to the second distinct regulation can occur when the expression and / or activity level of the target gene is changed (e.g., increased or decreased) by a factor of at most about 10,000-fold or less, at most about 5,000-fold or less, at most about 1,000-fold or less, at most about 500-fold or less, at most about 100-fold or less, at most about 90-fold or less, at most about 80-fold or less, at most about 70-fold or less, at most about 60-fold or less, at most about 50-fold or less, at most about 40-fold or less, at most about 30-fold or less, at most about 20-fold or less, at most about 10-fold or less, at most about 9-fold or less, at most about 8-fold or less, at most about 7-fold or less, at most about 6-fold or less, at most about 5-fold or less, at most about 4-fold or less, at most about 3-fold or less, at most about 2-fold or less, at most about 1-fold or less, at most about 0.9-fold or less, at most about 0.8-fold or less, at most about 0.7-fold or less, at most about 0.6-fold or less, at most about 0.5-fold or less, at most about 0.4-fold or less, at most about 0.3-fold or less, at most about 0.2-fold or less, or at most about 0.1-fold or less as compared to the expression and / or activity level of the control by the action of the first distinct regulation.

[0148] Alternatively, or in addition thereto, a second distinct modulation as disclosed herein (e.g., induced by a second gate unit) can induce a change (e.g., an increase or decrease) 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% in the expression and / or activity level of a further target gene. The second distinct modulation can induce a change (e.g., an increase or decrease) 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% in the expression and / or activity level of a further target gene. The cell can include a prokaryotic cell, a eukaryotic cell, or an artificial cell.

[0149] Stem cells (e.g., iPSCs) generated by the systems and methods of the present disclosure can differentiate into cell types found in one or more tissues. Non-limiting examples of such tissues 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, genitalia, and the like.

[0150] An engineered cell of the present disclosure (e.g., an engineered iPSC) or any further engineered variant thereof (e.g., a differentiation byproduct of such an iPSC) can be used (e.g., administered) to treat a subject in need of treatment. The subject can have or be suspected of having a disease such as a disorder (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 the iPSCs disclosed herein. Subsequently, the engineered iPSCs can be administered to the subject for adoptive immunotherapy. Thus, the engineered cell or any further engineered variant thereof can be autologous to the subject in need thereof. Alternatively, the engineered cell or any further engineered variant thereof can be allogeneic to the subject (e.g., allogeneic stem cell transplantation, allogeneic adoptive immunotherapy, etc.).

[0151] The engineered cells or any further engineered variants thereof disclosed herein can be administered to a subject before, simultaneously with, or after activation of a heterologous genetic circuit and / or a heterologous gene modulator in the engineered stem cell. For example, the engineered cell or any further engineered variant thereof can be activated after being administered to the subject, for example, by administering an activator of the heterologous genetic circuit to the subject.

[0152] The subject can be treated (e.g., administered) with a population of the engineered cells (e.g., engineered iPSCs) of the present disclosure or any further engineered variants thereof over a minimum or maximum of about 1 dose, minimum or maximum of about 2 doses, minimum or maximum of about 3 doses, minimum or maximum of about 4 doses, minimum or maximum of about 5 doses, minimum or maximum of about 6 doses, minimum or maximum of about 7 doses, minimum or maximum of about 8 doses, minimum or maximum of about 9 doses, or minimum or maximum of about 10 doses. Alternatively, or in addition thereto, the subject can be treated (e.g., administered) with a population of the engineered cells (e.g., engineered iPSCs) of the present disclosure or any further engineered variants thereof over a minimum of about 1 week, minimum of about 2 weeks, minimum of about 3 weeks, minimum of about 4 weeks, minimum of about 5 weeks, minimum of about 6 weeks, minimum of about 2 months, minimum of about 3 months, minimum of about 4 months, minimum of about 5 months, minimum of about 6 months, minimum of about 7 months, minimum of about 8 months, minimum of about 9 months, minimum of about 10 months, minimum of about 11 months, minimum of about 1 year, minimum of about 2 years, minimum of about 3 years, minimum of about 4 years, minimum of about 5 years, minimum of about 6 years, minimum of about 7 years, minimum of about 8 years, minimum of about 9 years, minimum of about 10 years, minimum of about 15 years, minimum of about 20 years, minimum of about 30 years, minimum of about 40 years, minimum of about 50 years, minimum of about 60 years, minimum of about 70 years, minimum of about 80 years, minimum of about 90 years, or minimum of about 100 years.

[0153] Any one of the methods disclosed herein can be utilized to treat a target cell, target tissue, target disease, or target disorder of a subject.

[0154] Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. Examples of samples from subjects 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 from tumor tissue, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, cerebrospinal fluid, hair, fingernails, plasma, nasal swab or nasopharyngeal wash, cerebrospinal fluid, spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, cord blood, lung fluid, cavity fluid, sputum, pus, microbiota, meconium, breast milk, and / or other excretions or body tissues.

[0155] The target disease can be cancer or a tumor. Non-limiting examples of cancer can include cancer cells, such as acanthoma, acinar cell carcinoma, acoustic neuroma, acral lentiginous melanoma, apocrine hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, 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 carcinoma, angioimmunoblastic T-cell lymphoma, angioleiomyolipoma, angiosarcoma, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, bile duct 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 site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, carcinoma of unknown primary site, carcinosarcoma, Castleman 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, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, embryonal dysplastic neuroepithelial tumor, fetal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial carcinoma of the uterus, endometroid tumor, enteropathy-associated T-cell lymphoma, epithelioblastoma, epithelioma, epithelioid sarcoma, erythroleukemia, esophageal cancer, ganglioneuroma, Ewing family tumor, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget disease, fallopian tube cancer, hydatidiform mole, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid carcinoma, gallbladder cancer, gallbladder carcinoma, 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, pericytic tumor, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, insulinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi sarcoma, Kaposi sarcoma, kidney cancer, cratoskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, malignant acral lentiginous melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, corpus luteum cyst, 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, recurrent metastatic squamous cell carcinoma of the neck of unknown primary origin, metastatic urothelial carcinoma, mixed Müllerian duct tumor, monocytic leukemia, oral cavity cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, fungating polypoid tumor, fungating polypoid tumor, myelodysplastic disorder, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorder, mucocele, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, tumor, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, nerve tumor, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, Ocular oncology, anaplastic astrocytoma, anaplastic glioma, tumor cell tumor, optic nerve sheath meningioma, oral cavity cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, low-grade ovarian tumor, mammary Paget disease, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epitheloid cell tumor, pharyngeal cancer, pheochromocytoma, intermediate-differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasmacytoma, pleuropulmonary blastoma, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primary neuroectodermal tumor, prostate cancerPseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory cancer associated with the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous adenocarcinoma, secondary tumor, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord stromal tumor, Sézary syndrome, signet ring cell carcinoma, skin cancer, small round blue cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, soot disease, spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, stomach cancer, superficial spreading malignant melanoma, primitive neuroectodermal tumor above the tentorium, surface epithelial stromal tumor, synovial sarcoma, T cell acute lymphoblastic leukemia, T cell large granular lymphocyte leukemia, T cell leukemia, T cell lymphoma, T cell prelymphocytic leukemia, teratoma, terminal lymphoma, testicular cancer, myoma, pharyngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, ureteral cancer, urethral cancer, urogenital tumor, uterine sarcoma, choroidal melanoma, vaginal cancer, Werner-Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenström macroglobulinemia, Warthin tumor, Wilms tumor, and combinations thereof are included. 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 hematopoietic cancer such as lymphoma. The antigen can be a tumor-associated antigen.,

[0156] The present disclosure also provides a composition comprising an engineered gene modulator and / or an engineered genetic circuit disclosed herein. The composition can further comprise an actuator of a heterologous genetic circuit. The present disclosure also provides a kit comprising the composition. The kit can further comprise an activator of the heterologous genetic circuit. The activator can be of the same composition as the engineered gene modulator and / or the engineered genetic circuit. Alternatively, or in addition thereto, the activator can be in a separate composition different from the engineered gene modulator and / or the engineered genetic circuit.,

Examples

[0157] Example 1: Heterologous Genetic Circuit for Converting Cell Types via Regulation of Endogenous Genes

[0158] By utilizing the regulation of one or more endogenous genes (e.g., regulation of expression or activity), cells of a first cell type (a plurality of first cells) can be converted into a plurality of cells of a second cell type (a plurality of second cells). In some cases, such regulation can convert differentiated cells (e.g., terminally differentiated cells such as primary fibroblasts) into more stem-like cells (e.g., induced pluripotent stem cells or "iPSCs"). In some cases, one or more genes can include one or more members from the pluripotency regulatory gene network (PGRN) genes, as shown in FIG. 2. Alternatively, or in addition, one or more genes may not be PGRN genes (e.g., human endogenous retrovirus (HERV), embryonic genome activation (EGA) enriched Alu motif (EEA), etc.). In some cases, HERV can include HERV-K. In some cases, targeting of HERV-K can include targeting regulatory elements of HERV-K such as LTR5HS.

[0159] In some cases, each of the one or more genes can be regulated one or more times (e.g., sequentially). In some cases, one or more genes can include a plurality of different genes, and the plurality of different genes can be regulated substantially simultaneously or sequentially.

[0160] In some cases, one or more heterologous gene modulators can be used to induce the regulation of one or more endogenous genes to achieve conversion. For example, a heterologous gene modulator can include a guide nucleic acid that exhibits specific binding to an endogenous gene (e.g., a coding region or a non-coding region), and this guide nucleic acid can form a complex with a Cas protein or a variant thereof to induce such regulation. In some cases, a heterologous gene modulator can be part of a heterologous gene circuit provided herein.

[0161] Heterologous gene circuit

[0162] A heterologous genetic circuit (HGC) can be designed such that each heterologous genetic circuit (e.g., excluding a control heterologous genetic circuit) can sequentially activate a plurality of heterologous gene modulators to target a plurality of endogenous genes in primary fibroblasts and achieve the conversion (e.g., reprogramming) of primary fibroblasts into iPSCs. Table 1 shows a library of different HGCs for targeting various endogenous genes. Endogenous genes include EEA, Oct4 (O), Klf4 (K), c-Myc (M), Sox2 (S), and miR-302 (e.g., a suppressor of p53, denoted herein as "miRNA").

[0163] An 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) in the corresponding step. Examples of spacer sequences for endogenous genes are provided in Table 2. The guide nucleic acid is also designed using a scaffold sequence that enables the formation of a complex with a Cas-based gene activator (e.g., dCas9-VPR) to specifically bind to the target gene and activate the target gene.

[0164] In some cases, a target gene can be regulated using one guide nucleic acid sequence having one spacer sequence for the target gene. In some cases, a target gene can be regulated using a plurality of nucleic acid sequences (or a pool of nucleic acid sequences) having different spacer sequences for a common target gene (e.g., multiplexing).

[0165] 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 of each HGC, thereby activating the HGC and inducing sequential gene regulation.

[0166] 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 (referred to herein as "p53") to, for example, promote or enhance the regulation of p53.

[0167]

Table 1

[0168]

Table 2-1

[0169]

Table 2-2

[0170] Example 2: Reprogramming of primary fibroblasts into iPSCs by targeting endogenous genes (e.g., EEA and / or miR-302)

[0171] According to Example 1, HGCs were designed to reprogram primary fibroblasts into iPSCs. Figure 3 shows a library of different HGCs tested, each of which is designed to be able to sequentially target one or more endogenous genes over 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 sequential targeting of endogenous genes in various combinations and orders.

[0172] For example, reprogramming of primary fibroblasts into iPSCs by targeting EEA and / or miR-302.

[0173] Primary fibroblasts were transfected with each HGC plasmid or treated as a control (e.g., cells without any treatment, nucleofection reagent 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., evaluating the expression levels of target endogenous genes and / or embryonic markers such as Nanog via RNA isolation or staining). Cells were also visually evaluated for colony formation / growth indicating reprogramming of fibroblasts into iPSCs.

[0174] For transfection, each condition also included a gene encoding GFP to track transfection efficiency. As shown at the top of Figures 4 and 5, the transfection efficiency for the conditions was sufficient (e.g., > 80%). The three controls shown from left to right at the top of Figure 4 correspond to conditions #1 - 3 in Table 1, respectively.

[0175] As shown in the lower part of Figure 5 and the compared results in the rightmost column of Figure 3, HGC#6 resulted in the highest rate of iPSC reprogramming as confirmed by colony formation yield (e.g., after 5 days) compared to other HGC conditions.

[0176] As shown in Figure 3, sequential activation of endogenous genes (e.g., condition 6) was more effective (e.g., > 4 - fold more effective) in iPSC reprogramming than simultaneous activation of endogenous genes (e.g., condition 2).

[0177] As shown in FIG. 3, activating EEA sequentially (e.g., prior to) with respect to activating one or more of other genes (e.g., O, K, M, and / or S) (e.g., condition 6) was more effective in iPSC reprogramming than activating EEA and at least one of the other genes substantially simultaneously (e.g., conditions 7, 8, and 9). Further, even when EEA was activated sequentially (e.g., previously) with respect to any of the other genes, activating the other genes sequentially (e.g., condition 6) was more effective in iPSC reprogramming than activating all of the other genes substantially simultaneously (e.g., conditions 3-5).

[0178] As shown in FIG. 3, activating M and / or S sequentially twice (e.g., conditions 6, 7, and 8) was more effective in iPSC reprogramming than activating S only once (e.g., conditions 2, 3, 4, and 5).

[0179] Example 3: Reprogramming of Primary Fibroblasts into iPSCs by Targeting Endogenous Genes (e.g., HERV)

[0180] Targeting of HERV and EEA

[0181] According to Example 1, in the same procedure as described in Example 2, using HGC condition #4, HERV (e.g., HERV-K) and EEA were targeted and regulated, and primary fibroblasts were reprogrammed into iPSCs. Targeting of HERV and EEA in the presence of anti-p53 shRNA was sufficient to induce iPSC colony formation during transfection and subsequent culture (e.g., for 5 days) (FIG. 6).

[0182] Targeting of HERV

[0183] According to Example 1, following the procedure described in Example 2, HGCs can be designed to reprogram primary fibroblasts into iPSCs. HGCs can be designed for sequential activation of multiple genes. In some cases, one of the multiple genes can be targeted via multiplexed guide nucleic acid targeting (e.g., a single gate unit comprising multiple different gene regulatory sub-guide RNAs for targeting different regions of the gene), while at least one additional gene of the multiple genes can be targeted via single nucleic acid targeting (e.g., a single gate unit comprising a single gene regulatory sub-guide RNA for targeting a single region of the additional gene).

[0184] Briefly, primary fibroblasts were transfected with each HGC plasmid to regulate the following endogenous genes: Oct4 (O), Klf4 (K), c-Myc (M), Sox2 (S), and the HERV-K element (e.g., LTR5HS, designated herein as "H").

[0185] Table 3 shows a library of different HGCs tested, each designed to sequentially target one or more endogenous genes over 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 sequential targeting of endogenous genes including HERV in various combinations and orders. Here, in each step of the HGCs, KLF4 or MYC (e.g., KLF4 and MYC) was activated by single guide nucleic acid targeting, and the other genes were activated by multiplexed guide nucleic acid targeting.

[0186] Primary fibroblasts (e.g., about 500,000 fibroblasts per well) were transfected with each HGC plasmid. Furthermore, the fibroblasts under each condition were also transfected with a plurality of plasmids that each encode (i) Cas9-VPR, anti-p53 shRNA, miR-302, and GFP. After subsequent culture (e.g., for 7 days), the cells were visually evaluated to identify the number of colonies showing iPSC-like morphology, which is an indicator of iPSC reprogramming.

[0187] As shown in the last column of Table 3, activation of HERV-K activation without activation of other OSKM factors (e.g., Conditions 2-4) was able to generate iPSC morphology colonies.

[0188] As shown in the last column of Table 3, activation of HERV-K with continued delayed activation of other OSKM factors (e.g., Conditions 2-2) was more effective in iPSC reprogramming than delayed activation of other OSKM factors without prior activation of HERV-K (e.g., Conditions 1-2).

[0189] As shown in the last column of Table 3, substantially simultaneous activation of HERV-K and O (e.g., Conditions 2-3) was not as effective in iPSC reprogramming as comparable sequential activation of OSKM factors in the absence of HERV-K activation (e.g., Conditions 1-3). Similarly, substantially simultaneous activation of HERV-K and other OSKM factors (e.g., Conditions 2-1) was not effective in iPSC reprogramming, similar to comparable activation of OSKM factors in the absence of HERV-K activation (e.g., Conditions 1-1).

[0190] Separately, in the absence of HERV-K activation, sequential activation of OSKM factors (e.g., Conditions 1-3) was more effective for iPSC reprogramming compared to activating all of the OSKM factors substantially simultaneously (e.g., Conditions 1-1 and 1-2).

[0191] [Table 3]

[0192] Embodiment The following non-limiting embodiments provide exemplary examples of the present invention but do not limit the scope of the present invention.

[0193] Embodiment 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 modulator that exhibits specific binding to a gene encoding HERV to regulate the expression level or epigenetic profile of HERV and achieve conversion of the first plurality of cells into the second plurality of cells, optionally, (1)(i) the first plurality of cells includes terminally differentiated cells, (ii) the second plurality of cells includes pluripotent stem cells, and / or (2) contacting increases the expression level of HERV, and / or (3) the heterologous gene modulator exhibits specific binding to a gene encoding HERV-K, and / or (4) the heterologous gene modulator exhibits specific binding to a gene encoding LTR5HS, and further optionally, (a) the heterologous gene modulator 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 the group consisting of SEQ ID NOs: 1-12 and their complementary sequences, optionally with a conversion from uracil to thymine; (b) the heterologous gene modulator 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 the group consisting of SEQ ID NOs: 1-12 and their complementary sequences, optionally with a conversion from uracil to thymine; (c) The heterologous gene modulator comprises a polynucleotide sequence that exhibits at least about 80% sequence identity to the polypeptide sequence of one or more members selected from the group consisting of SEQ ID NOs: 1 to 12 and their complementary sequences, (d) The heterologous gene modulator comprises a polynucleotide sequence that exhibits at least about 90% sequence identity to the polypeptide sequence of one or more members selected from the group consisting of SEQ ID NOs: 1 to 12 and their complementary sequences, and / or (5) Contacting is achieved or is sufficient to achieve conversion without using a further heterologous gene modulator that exhibits specific binding to a further target gene comprising one or more cell dedifferentiation factors selected from the group consisting of OCT4, SOX2, KLF4, and MYC, Optionally further, the further target gene comprises OCT4, SOX2, KLF4, and MYC, and / or (6) The method further comprises the step of contacting a first plurality of cells with a further heterologous gene modulator that exhibits specific binding to a further gene comprising one or more cell dedifferentiation factors selected from the group consisting of OCT4, SOX2, KLF4, and MYC to achieve sequential regulation of the gene and the further gene, Optionally further, (a) Regulation of the gene occurs before, simultaneously with, or after regulation of the further gene, (b) Regulation of the gene occurs before regulation of the further gene, and / or (c) The further gene comprises OCT4, and / or (7) The method further comprises the step of contacting a first plurality of cells with a further heterologous gene modulator that exhibits specific binding to an embryo genome activation (EGA) enriched Alu motif (EEA) to achieve conversion, and / or (8) Contacting comprises contacting the first plurality of cells with a heterologous genetic circuit comprising a plurality of gate units, the heterologous genetic circuit being activatable to achieve conversion by inducing the plurality of gate units and sequentially regulating the expression levels or epigenetic profiles of genes and different genes, and the plurality of gate units are (i) a first gate unit comprising a heterologous gene modulator, the first gate unit being preconfigured to regulate the expression level or epigenetic profile of a gene, and (ii) a second gate unit preconfigured to regulate the expression level or epigenetic profile of a different gene, and upon activation of the heterologous genetic circuit, the plurality of gate units operate to achieve conversion, Optionally further, (a) the heterologous gene circuit is preconfigured to regulate the expression level of the epigenetic profile of a gene before regulating the expression level of the epigenetic profile of a different gene, (b) the different genes comprise one or more members selected from the group consisting of Oct3, Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, EEA, ZSCAN4, DUX4, 0TX2, ABCE1, C0L5A1, GAL4NT13, DUXA, DUXB, ARGFX, CPHX1, CPHX2, TPRX1, DPPA3, NASP, ATP2B1, NFAT, H1F00, and CAMKII, (c) the different genes comprise one or more members selected from the group consisting of Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, and EEA, and / or (9) the heterologous gene modulator comprises a heterologous endonuclease for specific binding to a gene encoding HERV, Optionally further, (a) the heterologous endonuclease is a Cas protein, (b) the heterologous gene modulator further comprises a gene activator, (c) The gene activator is selected from the group consisting of VP16, VP64, p65, RTA, and the VP64-p65-RTA fusion (VPR), (d) The gene activator is bound to a heterologous endonuclease.

[0194] Embodiment 2. 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 modulator that modulates the expression level or epigenetic profile of HERV and exhibits specific binding to a gene encoding HERV to achieve conversion of the first plurality of cells to the second plurality of cells, (1) (i) The first plurality of cells includes terminally differentiated cells, (ii) The second plurality of cells includes pluripotent stem cells, and / or (2) The specific binding of the heterologous gene modulator to the gene achieves an increase in the expression level of HERV, and / or (3) The heterologous gene modulator exhibits specific binding to a gene encoding HERV-K, and / or (4) The heterologous gene modulator exhibits specific binding to a gene encoding LTR5HS, Optionally further, (a) The heterologous gene modulator 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 the group consisting of SEQ ID NOs: 1-12 and their complementary sequences, optionally with conversion from uracil to thymine, (b) The heterologous gene modulator 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 the group consisting of SEQ ID NOs: 1-12 and their complementary sequences, optionally with conversion from uracil to thymine, (c) The heterologous gene modulator comprises a polynucleotide sequence that exhibits at least about 80% sequence identity to the polypeptide sequence of one or more members selected from the group consisting of SEQ ID NOs: 1 to 12 and their complementary sequences, (d) The heterologous gene modulator comprises a polynucleotide sequence that exhibits at least about 90% sequence identity to the polypeptide sequence of one or more members selected from the group consisting of SEQ ID NOs: 1 to 12 and their complementary sequences, and / or (5) Specific binding to the gene of the heterologous gene modulator is configured to achieve conversion or is sufficient to achieve conversion without using a further heterologous gene modulator that exhibits specific binding to a further target gene comprising one or more cell dedifferentiation factors selected from the group consisting of OCT4, SOX2, KLF4, and MYC, Optionally further, the further target gene comprises OCT4, SOX2, KLF4, and MYC, and / or (6) The system further comprises a further heterologous gene modulator that specifically binds to a further gene comprising one or more cell dedifferentiation factors selected from the group consisting of OCT4, SOX2, KLF4, and MYC to achieve sequential regulation of the gene and the further gene, Optionally further, (a) Regulation of the gene occurs before, simultaneously with, or after regulation of the further gene, (b) Regulation of the gene occurs before regulation of the further gene, and / or (c) The further gene comprises OCT4, and / or (7) The system further comprises a further heterologous gene modulator that exhibits specific binding to the embryo genome activation (EGA) enriched Alu motif (EEA) to achieve conversion, and / or (8) The system is a heterologous genetic circuit comprising a plurality of gate units, the heterologous genetic circuit being activatable to induce the plurality of gate units to sequentially regulate genes and the expression levels or epigenetic profiles of different genes to achieve a conversion, the plurality of gate units being (i) a first gate unit comprising a heterologous gene modulator, the first gate unit being preconfigured to regulate the expression level or epigenetic profile of a gene, and (ii) a second gate unit preconfigured to regulate the expression level or epigenetic profile of a different gene, the heterologous genetic circuit comprising Upon activation of the heterologous genetic circuit, the plurality of gate units operate to achieve a conversion, Optionally further, (a) the heterologous gene circuit is preconfigured to regulate the expression level of the epigenetic profile of a gene before regulating the expression level of the epigenetic profile of a different gene, (b) the different genes include one or more members selected from the group consisting of Oct3, Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, EEA, ZSCAN4, DUX4, 0TX2, ABCE1, C0L5A1, GAL4NT13, DUXA, DUXB, ARGFX, CPHX1, CPHX2, TPRX1, DPPA3, NASP, ATP2B1, NFAT, H1F00, and CAMKII, (c) the different genes include one or more members selected from the group consisting of Oct4, Sox2, Klf4, c-Myc, miR-302, miR-307, and EEA, and / or (9) The heterologous gene modulator includes a heterologous endonuclease for specific binding to a gene encoding HERV, Optionally further, (a) the heterologous endonuclease is a Cas protein, (b) the heterologous gene modulator further includes a gene activator, (c) The gene activator is selected from the group consisting of VP16, VP64, p65, RTA, and the VP64-p65-RTA fusion (VPR). (d) The gene activator is bound to a heterologous endonuclease.

[0195] Embodiment 3. A method for converting a plurality of differentiated cells into a plurality of stem cells, the method comprising: contacting a plurality of differentiated cells with a heterologous genetic circuit comprising a plurality of gate units, the heterologous genetic circuit being activatable to achieve conversion by inducing the plurality of gate units to sequentially regulate the expression levels or epigenetic profiles of a plurality of distinct target endogenous genes, the plurality of gate units comprising: (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, the first target endogenous gene comprising an embryo genome activation (EGA) enriched Alu motif (EEA), the first gate unit; 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, the second target endogenous gene comprising a cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, the method comprising the steps of: upon activation of the heterologous genetic circuit, the plurality of gate units operate to achieve conversion; Optionally, (1) the plurality of stem cells comprises a plurality of pluripotent stem cells and / or (2) upon activation of the heterologous genetic circuit, the heterologous genetic circuit is preconfigured such that (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 (3) 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 distinct target endogenous genes, the third target endogenous gene including a different cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, Upon activation of the heterologous genetic circuit, the cell dedifferentiation factor and the expression level or epigenetic profile of the different cell dedifferentiation factor are sequentially regulated, Optionally, (a) the second target endogenous gene includes OCT4, and / or (b) the third target endogenous gene includes SOX2, and / or (4) The plurality of gate units includes a third gate unit that is preconfigured to regulate the expression level or epigenetic profile of a second target endogenous gene, and activation of the heterologous genetic circuit achieves two or more sequential regulations of the expression level or epigenetic profile of the cell dedifferentiation factor, Optionally, (a) the cell dedifferentiation factor is MYC, and / or (b) the cell dedifferentiation factor is S0X2, and / or (5) The cell dedifferentiation factor includes OCT4, and / or (6) The cell dedifferentiation factor includes SOX2, and / or (7) The cell dedifferentiation factor includes KLF4, and / or (8) The cell dedifferentiation factor includes MYC.

[0196] Embodiment 4. A system for converting a plurality of differentiated cells into a plurality of stem cells, the system comprising a heterologous genetic circuit including a plurality of gate units, the heterologous genetic circuit being activatable to achieve the conversion by inducing the plurality of gate units to sequentially regulate the expression level or epigenetic profile of a plurality of distinct target endogenous genes, the plurality of gate units being (i) A first gate unit preconfigured to regulate the expression level or epigenetic profile of a first target endogenous gene among a plurality of different target endogenous genes, wherein the first target endogenous gene comprises a first gate unit containing an embryogenic genome activation (EGA) enriched Alu motif (EEA), (ii) A second gate unit preconfigured to regulate the expression level or epigenetic profile of a second target endogenous gene among a plurality of different target endogenous genes, wherein the second target endogenous gene comprises a second gate unit containing a cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, and comprising a heterologous genetic circuit, Upon activation of the heterologous genetic circuit, the plurality of gate units operate to achieve a conversion. Optionally, (1) The plurality of stem cells comprises a plurality of pluripotent stem cells, and / or (2) Upon activation of the heterologous genetic circuit, the heterologous genetic circuit is preconfigured such that (i) the expression level or epigenetic profile of the first target endogenous gene is regulated prior to (ii) the expression level or epigenetic profile of the second target endogenous gene, and / or (3) The plurality of gate units comprises a third gate unit preconfigured to regulate the expression level or epigenetic profile of a third target endogenous gene among a plurality of distinct target endogenous genes, wherein the third target endogenous gene comprises a different cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, Upon activation of the heterologous genetic circuit, the expression levels or epigenetic profiles of the cell dedifferentiation factor and the different cell dedifferentiation factor are sequentially regulated. Further optionally, (a) The second target endogenous gene comprises OCT4, and / or (b) The third target endogenous gene comprises SOX2, and / or (4) The plurality of gate units includes a third gate unit that is preconfigured to regulate the expression level or epigenetic profile of a second target endogenous gene, and activation of the heterologous genetic circuit achieves two or more sequential regulations of the expression level or epigenetic profile of the cell dedifferentiation factor, Optionally, (a) the cell dedifferentiation factor is MYC, and / or (b) the cell dedifferentiation factor is SOX2, and / or (5) the cell dedifferentiation factor includes OCT4, and / or (6) the cell dedifferentiation factor includes SOX2, and / or (7) the cell dedifferentiation factor includes KLF4, and / or (8) the cell dedifferentiation factor includes MYC.

[0197] Embodiment 5. 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 genetic circuit comprising a plurality of gate units, wherein the heterologous genetic circuit is activatable to achieve conversion by inducing the plurality of gate units to sequentially regulate the expression level or epigenetic profile level of a plurality of distinct target endogenous genes, and 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, the first target endogenous gene comprising a cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, (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, the second target endogenous gene comprising a different cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, Upon activation of the heterologous genetic circuit, a plurality of gate units operate to perform the conversion, Optionally, (1) The heterologous genetic circuit is preconfigured such that activation of the first gate unit achieves activation of the second gate unit such that 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. Optionally further, the first target endogenous gene is OCT4 and / or such that regulation of the expression level or epigenetic profile level of OCT4 occurs before regulation of KLF4. (2) The plurality of stem cells includes a plurality of pluripotent stem cells and / or (3) The first target endogenous gene includes OCT4 and the second target endogenous gene includes one or more members selected from the group consisting of SOX2, KLF4, and MYC, and / or (4) The first target endogenous gene includes one or more members selected from the group consisting of OCT4, KLF4, and MYC and the second target endogenous gene includes SOX2, and / or (5) The plurality of gate units further includes 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 and achieve two or more sequential regulations of each of the first target endogenous gene or the second target endogenous gene. Optionally further, SOX2 undergoes two or more sequential regulations and / or (6) Regulation of the expression level or epigenetic profile level of the plurality of distinct target endogenous genes includes improving the expression level or epigenetic profile level of the plurality of distinct target endogenous genes.

[0198] Embodiment 6. A system for converting a plurality of differentiated cells into a plurality of stem cells, the system comprising A heterologous genetic circuit comprising a plurality of gate units, wherein the heterologous genetic circuit is activatable to achieve conversion by inducing a plurality of gate units and sequentially regulating the expression levels or epigenetic profile levels of a plurality of distinct target endogenous genes, and the plurality of gate units are (i) A first gate unit preconfigured to regulate the expression level or epigenetic profile level of a first target endogenous gene among a plurality of different target endogenous genes, wherein the first target endogenous gene comprises a cell dedifferentiation factor selected from the group consisting of 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 a plurality of different target endogenous genes, wherein the second target endogenous gene comprises a different cell dedifferentiation factor selected from the group consisting of OCT4, SOX2, KLF4, and MYC, and the heterologous genetic circuit comprises Upon activation of the heterologous genetic circuit, the plurality of gate units operate to effect conversion. Optionally, (1) The heterologous genetic circuit is preconfigured such that activation of the first gate unit achieves activation of the second gate unit such that 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. Optionally further, the first target endogenous gene is OCT4 and / or such that regulation of the expression level or epigenetic profile level of OCT4 occurs before regulation of KLF4. (2) The plurality of stem cells comprise a plurality of pluripotent stem cells and / or (3) The first target endogenous gene comprises OCT4 and the second target endogenous gene comprises one or more members selected from the group consisting of SOX2, KLF4, and MYC and / or (4) The first target endogenous gene includes one or more members selected from the group consisting of OCT4, KLF4, and MYC, the second target endogenous gene includes SOX2, and / or (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 and achieve two or more sequential regulations of each of the first target endogenous gene or the second target endogenous gene, Optionally, further, (a) SOX2 is subject to two or more sequential regulations, and / or (b) The regulation of the expression level or epigenetic profile level of a plurality of distinct target endogenous genes includes improving the expression level or epigenetic profile level of the plurality of distinct target endogenous genes.

[0199] Further details of the heterologous genetic circuit (HGC) and its use are provided in International Application PCT / US2018 / 052211 (entitled "CRISPR / CAS SYSTEM AND METHOD FOR GENOME EDITING AND MODULATING TRANSCRIPTION"), International Application PCT / US2023 / 0132,40 (entitled "SYSTEMS FOR CELL PROGRAMMING and METHODS THEREOF"), and Clarke et al., Molecular CELL, 81, 226 - 238, 2021 (entitled "Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities"), each of which is hereby incorporated by reference in its entirety.

[0200] It should be understood that the different aspects of the present invention can be understood individually, collectively, or in combination with each other. The various aspects of the invention described herein may be applied to any of the specific uses disclosed herein. Compositions comprising a compound of any of the formulas disclosed herein in the Compositions section of the present disclosure may be utilized in the Methods section, including the methods of use and production disclosed herein, or vice versa.

[0201] Preferred embodiments of the invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The invention is not intended to be limited by the specific examples provided herein. The invention is described with reference to the foregoing specification, but the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative ratios set forth herein, depending on various conditions and variables. It should be understood that various alternatives of the embodiments of the invention described herein may be employed in practicing the invention. Therefore, the invention is also intended to encompass any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and methods and structures within the scope of these claims, as well as their equivalents, are intended to be embraced by these claims.

Claims

[Claim 1] A method for converting multiple cells of a first cell type (first multiple cells) into multiple cells of a second cell type (second multiple cells), A method comprising the step of contacting the first plurality of cells with a heterogene modulator that exhibits specific binding to a gene encoding HERV, thereby regulating the expression level or epigenetic profile of HERV and achieving conversion from the first plurality of cells to the second plurality of cells.

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