Systems and methods for cell programming for the hematopoietic system

Abstract

Provided herein are systems for regulating gene expression, methods of use thereof, and engineered cells thereof for differentiating cells, such as hematopoietic cells.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 446,228, filed February 16, 2023, and U.S. Provisional Patent Application No. 63 / 620,361, filed January 12, 2024, each of which is incorporated by reference in its entirety. [Background technology]

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

[0003] The present disclosure provides a method for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the method comprising culturing the first plurality ex vivo in a medium substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL), wherein within about 14 days after culturing, the conversion rate from the first plurality to the second plurality is at least about 5%, and the second plurality is CD45+.

[0004] The present disclosure provides a method for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the method comprising contacting the first plurality with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to sequentially regulate expression levels of a plurality of distinct target genes, the plurality of gating units comprising a first gating unit that is activatable to regulate expression of the target genes upon activation of the heterologous genetic circuit, and a second gating unit that, upon activation of the first gating unit, activates additional target genes different from the target genes. and a second gating unit, wherein the first gating unit is activatable to regulate expression of an additional target gene, whereby, following regulation of expression of the target gene, regulation of expression of the additional target gene is induced, the target gene comprising one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target gene comprising one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and upon contact, the multiple gating units act in concert to convert the first multiple gating unit to the second multiple gating unit.

[0005] The present disclosure provides a method for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the method comprising contacting the first plurality with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in a coordinated manner to sequentially regulate expression levels of a plurality of distinct target genes, the plurality of gating units comprising a first gating unit that is activatable to regulate expression of the target genes upon activation of the heterologous genetic circuit, and a second gating unit that is activatable to regulate expression of an additional target gene, different from the target genes, upon activation of the first gating unit, whereby modulation of expression of the target genes is induced followed by modulation of expression of the additional target gene, The circuit is programmed such that (x) the target gene comprises one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof, and / or (y) the target gene comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof, and upon contact, the multiple gate units operate in coordination to effect conversion of a first multiple gate unit to a second multiple gate unit.

[0006] The present disclosure provides a method for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the method comprising contacting a first plurality of target genes with heterologous gene regulatory sites to regulate expression levels and / or activity levels of the target genes, the target genes encoding erythroblast transformation specific (ETS) transcription factors, and the contacting results in conversion of the first plurality into the second plurality.

[0007] The present disclosure provides a method for directing a plurality of stem cells to differentiation, the method comprising contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in a coordinated manner to sequentially induce a plurality of distinct modulations of target genes in the plurality of stem cells, each of the plurality of distinct modulations being necessary but individually insufficient to direct the plurality of stem cells to differentiation, the plurality of gating units comprising: (i) a first gating unit that, upon activation of the heterologous genetic circuit, is activated to induce a first distinct modulation of the plurality of distinct modulations; and (ii) a second gating unit, the second gating unit being activatable to induce a second distinct regulation of the plurality of distinct regulations upon activation of the heterologous genetic circuit, wherein the second distinct regulation is induced after the first distinct regulation, whereby both the first distinct regulation and the second distinct regulation enhance or reduce expression and / or activity levels of a target gene in the cell, the target gene encoding an erythroid transformation-specific (ETS) transcription factor, and upon contact, the plurality of gating units operate in coordination to direct the plurality of stem cells toward differentiation.

[0008] The present disclosure provides a system for converting a plurality of stem cells (first plurality) into a plurality of hematopoietic cells (second plurality), the system comprising a medium for ex vivo culture of the first plurality, the medium being substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL), wherein within about 14 days after ex vivo culture of the first plurality in the medium, a conversion rate from the first plurality to the second plurality is at least about 5%, and the second plurality is CD45+.

[0009] The present disclosure provides a system for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the system comprising a heterologous genetic circuit including a plurality of gating units, the plurality of gating units being configured to operate in a coordinated manner to sequentially regulate expression levels of a plurality of distinct target genes upon activation of the heterologous genetic circuit, the plurality of gating units including a first gating unit that is activatable to regulate expression of the target genes upon activation of the heterologous genetic circuit, and a second gating unit that, upon activation of the first gating unit, regulates expression of a different target gene from the target gene. and a second gating unit that is activatable to regulate expression of additional target genes, whereby regulation of expression of the target genes induces regulation of expression of the additional target genes, the target genes comprising one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target genes comprising one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and upon contact of the first plurality by the heterologous genetic circuit, the plurality of gating units operate in coordination to effect conversion of the first plurality to the second plurality.

[0010] The present disclosure provides a system for converting a plurality of stem cells (first plurality) into a plurality of hematopoietic cells (second plurality), the system comprising a heterologous genetic circuit including a plurality of gating units, wherein the plurality of gating units are configured to operate in a coordinated manner to sequentially regulate expression levels of a plurality of distinct target genes upon activation of the heterologous genetic circuit, the plurality of gating units including a first gating unit that is activatable to regulate expression of the target genes upon activation of the heterologous genetic circuit, and a second gating unit that is activatable to regulate expression of an additional target gene that is different from the target genes upon activation of the first gating unit, whereby modulation of expression of the target genes is induced followed by modulation of expression of the additional target gene, and the system The genetic circuit is programmed such that (x) the target gene comprises one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof, and / or (y) the target gene comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof, and upon contact of a first plurality of entities by the heterologous genetic circuit, the plurality of gate units operate in coordination to effect conversion of the first plurality of entities to a second plurality of entities.

[0011] The present disclosure provides a system for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the system comprising heterologous gene regulatory portions that exhibit specific binding to target genes of the first plurality and regulate the expression and / or activity levels of the target genes, the target genes encoding erythroid transformation-specific (ETS) transcription factors, and contacting the target genes by the heterologous gene regulatory portions results in conversion of the first plurality into the second plurality.

[0012] The present disclosure provides a system for directing a plurality of stem cells to differentiate, the system comprising a heterologous genetic circuit including a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in a coordinated manner to sequentially induce a plurality of distinct modulations of target genes in the plurality of stem cells, each of the plurality of distinct modulations being necessary but individually insufficient to direct the plurality of stem cells to differentiate, the plurality of gating units comprising: (i) a first gating unit that is activatable upon activation of the heterologous genetic circuit to induce a first distinct modulation of the plurality of distinct modulations; and (ii) a second gating unit, the second gating unit being activatable upon activation of the heterologous genetic circuit to induce a second distinct regulation of the plurality of distinct regulations, wherein the second distinct regulation is induced subsequent to the first distinct regulation, whereby both the first distinct regulation and the second distinct regulation enhance or reduce the expression and / or activity level of a target gene in the cell, wherein the target gene encodes an erythroid transformation-specific (ETS) transcription factor, and wherein upon contact of the plurality of stem cells by the heterologous genetic circuit, the plurality of gating units operate in coordination to direct the plurality of stem cells toward differentiation.

[0013] The present disclosure provides engineered cells comprising any one of the systems disclosed herein. The present disclosure provides compositions comprising any one of the systems and / or any one of the engineered cells disclosed herein.

[0014] Incorporation by Reference All publications, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material. [Brief explanation of the drawings]

[0015] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figures" and "Figs") [Figure 1] Schematic of a heterologous genetic circuit. An activating moiety can initiate the circuit and activate a gating unit. A gating unit is composed of a gating moiety and / or a gene regulatory moiety. [Figure 2] 1 shows an exemplary heterologous genetic circuit. [Figure 3] Scatter plots (i.e., volcano plots) are shown for days 3, 5, and 9 to identify one or more heterogeneous genetic circuits that drive the differentiation of induced pluripotent stem cells (iPSCs) into hematopoietic progenitor cells. [Figure 4A] Examples of hematopoietic progenitor cell marker analysis data used to generate the scatter plots in Figure 3 are shown. Figure 4A shows the results for the cell marker CD34+. Figure 4B shows the results for the cell marker CD43+. Figure 4C shows the results for the cell marker CD45+. Figure 4D shows the results for the combination of cell markers CD34+ / CD43- / CD45+. The left and right dots represent replicate samples for each condition. The center dot represents the mean value. [Figure 4B] Examples of hematopoietic progenitor cell marker analysis data used to generate the scatter plots in Figure 3 are shown. Figure 4A shows the results for the cell marker CD34+. Figure 4B shows the results for the cell marker CD43+. Figure 4C shows the results for the cell marker CD45+. Figure 4D shows the results for the combination of cell markers CD34+ / CD43- / CD45+. The left and right dots represent replicate samples for each condition. The center dot represents the mean value. [Figure 4C]Examples of hematopoietic progenitor cell marker analysis data used to generate the scatter plots in Figure 3 are shown. Figure 4A shows the results for the cell marker CD34+. Figure 4B shows the results for the cell marker CD43+. Figure 4C shows the results for the cell marker CD45+. Figure 4D shows the results for the combination of cell markers CD34+ / CD43- / CD45+. The left and right dots represent replicate samples for each condition. The center dot represents the mean value. [Figure 4D] Figure 4 shows examples of hematopoietic progenitor cell marker analysis data used to generate the scatter plots in Figure 3. Figure 4A shows the results for cell marker CD34+. Figure 4B shows the results for cell marker CD43+. Figure 4C shows the results for cell marker CD45+. Figure 4D shows the results for the combination of cell markers CD34+ / CD43- / CD45+. The left and right dots represent replicate samples for each condition. The center dot represents the mean value. [Figure 5A] Figure 5A shows cell marker results for CD34+ and CD45+ markers. Figure 5B shows additional cell marker results for CD34+, CD43+, and CD45+ markers. Figure 5C shows a summary of the data in Figure 5B. Figure 5D shows a summary of the results from a repeat experiment. [Figure 5B] Figure 5A shows that heterologous genetic circuits generate substantially more hematopoietic progenitor cells after 5 days of culture compared to directed differentiation. Figure 5A shows cell marker results for CD34+ and CD45+ markers. Figure 5B shows additional cell marker results for CD34+, CD43+, and CD45+ markers. Figure 5C shows a summary of the data in Figure 5B. Figure 5D shows a summary of the results from a repeat experiment. [Figure 5C]Figure 5A shows that heterologous genetic circuits generate substantially more hematopoietic progenitor cells after 5 days of culture compared to directed differentiation. Figure 5A shows cell marker results for CD34+ and CD45+ markers. Figure 5B shows additional cell marker results for CD34+, CD43+, and CD45+ markers. Figure 5C shows a summary of the data in Figure 5B. Figure 5D shows a summary of the results from a repeat experiment. [Figure 5D] Figure 5A shows that heterologous genetic circuits generate substantially more hematopoietic progenitor cells after 5 days of culture compared to directed differentiation. Figure 5A shows cell marker results for CD34+ and CD45+ markers. Figure 5B shows additional cell marker results for CD34+, CD43+, and CD45+ markers. Figure 5C shows a summary of the data in Figure 5B. Figure 5D shows a summary of the results from a repeat experiment. [Figure 6] Developmental cell lineage from pluripotent stem cells (iPSCs) to hematopoietic progenitor cells (HPCs) is shown. Cell surface marker expression (e.g., KDR, CD34, CD43, CD45) indicative of cell state is shown on top of the cells. Genes encoding transcription factors are indicated by arrows, indicating the relative sequential order of activity for lineage progression. Exemplary heterologous genetic circuits utilizing such genes are shown in the appendix table. [Figure 7] We present design concepts for how either three sets of TFs or four sets of TFs are distributed in a 10-step cascade. Spacing paradigms were designed to accommodate the future addition of later steps, temporally disperse gene activation steps, and maximize the efficiency of heterologous gene circuit activation of endogenous genes. [Figure 8A]Figure 8A shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after transduction. Flow cytometry data show the effect on cell surface markers (KDR / CD309) indicative of early stages of lineage differentiation. Figure 8D shows a summary of the percentage of KDR+ cells determined from the flow cytometry data. [Figure 8B] Figure 8A shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after transduction. Flow cytometry data show the effect on cell surface markers (KDR / CD309) indicative of early stages of lineage differentiation. Figure 8D shows a summary of the percentage of KDR+ cells determined from the flow cytometry data. [Figure 8C] Figure 8A shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after transduction. Flow cytometry data show the effect on cell surface markers (KDR / CD309) indicative of early stages of lineage differentiation. Figure 8D shows a summary of the percentage of KDR+ cells determined from the flow cytometry data. [Figure 8D] Figure 8A shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after transduction. Flow cytometry data show the effect on cell surface markers (KDR / CD309) indicative of early stages of lineage differentiation. Figure 8D shows a summary of the percentage of KDR+ cells determined from the flow cytometry data. [Figure 9A] Figure 9D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after introduction. Flow cytometry data show the effect on a cell surface marker (CD34) indicative of an intermediate stage of lineage differentiation. Figure 9D shows a summary of the percentage of KDR-CD34+ (hemogenic endothelial) cells determined from flow cytometry data. [Figure 9B]Figure 9D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after introduction. Flow cytometry data show the effect on a cell surface marker (CD34) indicative of an intermediate stage of lineage differentiation. Figure 9D shows a summary of the percentage of KDR-CD34+ (hemogenic endothelial) cells determined from the flow cytometry data. [Figure 9C] Figure 9D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after introduction. Flow cytometry data show the effect on a cell surface marker (CD34) indicative of an intermediate stage of lineage differentiation. Figure 9D shows a summary of the percentage of KDR-CD34+ (hemogenic endothelial) cells determined from the flow cytometry data. [Figure 9D] Figure 9D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after introduction. Flow cytometry data show the effect on a cell surface marker (CD34) indicative of an intermediate stage of lineage differentiation. Figure 9D shows a summary of the percentage of KDR-CD34+ (hemogenic endothelial) cells determined from the flow cytometry data. [Figure 10A] Figure 10 shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after introduction. Flow cytometry data show the effect on cell surface markers (KDR / CD309, CD34) indicative of intermediate stages of lineage differentiation. Figure 10D shows a summary of the percentage of KDR-CD34+ (hemogenic endothelial) cells determined from flow cytometry data. [Figure 10B] Figure 10 shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after introduction. Flow cytometry data show the effect on cell surface markers (KDR / CD309, CD34) indicative of intermediate stages of lineage differentiation. Figure 10D shows a summary of the percentage of KDR-CD34+ (hemogenic endothelial) cells determined from flow cytometry data. [Figure 10C]Figure 10 shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after introduction. Flow cytometry data show the effect on cell surface markers (KDR / CD309, CD34) indicative of intermediate stages of lineage differentiation. Figure 10D shows a summary of the percentage of KDR-CD34+ (hemogenic endothelial) cells determined from flow cytometry data. [Figure 10D] Figure 10 shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after introduction. Flow cytometry data show the effect on cell surface markers (KDR / CD309, CD34) indicative of intermediate stages of lineage differentiation. Figure 10D shows a summary of the percentage of KDR-CD34+ (hemogenic endothelial) cells determined from flow cytometry data. [Figure 11A] Figure 11D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on iPSC differentiation 7 days after transduction. Flow cytometry data show the effect on cell surface markers (CD43, CD34) indicative of HPC stage. Figure 11D shows a summary of the percentage of double-positive (CD43+CD34+) cells determined from the flow cytometry data. [Figure 11B] Figure 11D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on iPSC differentiation 7 days after transduction. Flow cytometry data show the effect on cell surface markers (CD43, CD34) indicative of HPC stage. Figure 11D shows a summary of the percentage of double-positive (CD43+CD34+) cells determined from the flow cytometry data. [Figure 11C] Figure 11D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on iPSC differentiation 7 days after transduction. Flow cytometry data show the effect on cell surface markers (CD43, CD34) indicative of HPC stage. Figure 11D shows a summary of the percentage of double-positive (CD43+CD34+) cells determined from the flow cytometry data. [Figure 11D]Figure 11D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on iPSC differentiation 7 days after transduction. Flow cytometry data show the effect on cell surface markers (CD43, CD34) indicative of HPC stage. Figure 11D shows a summary of the percentage of double-positive (CD43+CD34+) cells determined from the flow cytometry data. [Figure 12A] Figure 12D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after transduction. Flow cytometry data show the effect on cell surface markers (CD45, CD34) indicative of HPC stage. Figure 12D shows a summary of the percentage of double-positive (CD45+CD34+) cells determined from the flow cytometry data. [Figure 12B] Figure 12D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after transduction. Flow cytometry data show the effect on cell surface markers (CD45, CD34) indicative of HPC stage. Figure 12D shows a summary of the percentage of double-positive (CD45+CD34+) cells determined from the flow cytometry data. [Figure 12C] Figure 12D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after transduction. Flow cytometry data show the effect on cell surface markers (CD45, CD34) indicative of HPC stage. Figure 12D shows a summary of the percentage of double-positive (CD45+CD34+) cells determined from the flow cytometry data. [Figure 12D] Figure 12D shows the effect of heterologous gene circuits (CGO###, listed in Table 4) and control conditions (listed in Table 4) on differentiation of iPSCs 7 days after transduction. Flow cytometry data show the effect on cell surface markers (CD45, CD34) indicative of HPC stage. Figure 12D shows a summary of the percentage of double-positive (CD45+CD34+) cells determined from the flow cytometry data. [Figure 13A]The total cell yields after 6 days of seeding 3,000 cells from the 7-day differentiation procedure (analyzed in Figures 9A-11D and described in Table 4) into DLL4-containing and growth factor-rich expansion media are shown, along with the total hematopoietic (CD45 total, Figure 13A), HPC (CD34+CD45+, Figure 13B), lineage-committed (CD34-CD45+, Figure 13C), and lymphoid (CD7, Figure 13D) cell types. [Figure 13B] The total cell yields after 6 days of seeding 3000 cells from a 7-day differentiation procedure (analyzed in Figures 9A-11D and listed in Table 4) in DLL4-containing, growth factor-rich growth medium are shown, along with total hematopoietic (CD45 total, Figure 13A), HPC (CD34+CD45+, Figure 13B), lineage-committed (CD34-CD45+, Figure 13C), and lymphoid (CD7, Figure 13D) cell types. [Figure 13C] The total cell yields after 6 days of seeding 3000 cells from a 7-day differentiation procedure (analyzed in Figures 9A-11D and listed in Table 4) in DLL4-containing, growth factor-rich growth medium are shown, along with total hematopoietic (CD45 total, Figure 13A), HPC (CD34+CD45+, Figure 13B), lineage-committed (CD34-CD45+, Figure 13C), and lymphoid (CD7, Figure 13D) cell types. [Figure 13D] The total cell yields after 6 days of seeding 3,000 cells from a 7-day differentiation procedure (analyzed in Figures 9A-11D and listed in Table 4) in DLL4-containing, growth factor-rich growth medium are shown, along with the total hematopoietic (CD45 total, Figure 13A), HPC (CD34+CD45+, Figure 13B), lineage-committed (CD34-CD45+, Figure 13C), and lymphoid (CD7, Figure 13D) cell types. [Figure 14A] Figure 14 shows the effect of heterologous gene circuits (CGO###, listed in Table 4) compared to the benchmark protocol on CD34+CD45+CD43+ cells. Figure 14A shows iPSCs at day 7 and CGO178 with transient plasmids. Figure 14B shows the benchmark protocol using a transgenic iPSC line at day 15. [Figure 14B]Figure 14 shows the effect of heterologous gene circuits (CGO###, listed in Table 4) compared to the benchmark protocol on CD34+CD45+CD43+ cells. Figure 14A shows iPSCs at day 7 and CGO178 with transient plasmids. Figure 14B shows the benchmark protocol using a transgenic iPSC line at day 15. Detailed Description of the Invention

[0016] While 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 invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed.

[0017] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "gate unit" includes a plurality of gate units.

[0018] The term "about" or "approximately" generally means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0019] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives.

[0020] definition

[0021] As used interchangeably herein, the terms "genetic circuit," "biological circuit," or "circuit" generally refer to a collection of molecular components (e.g., biological materials such as polypeptides and / or polynucleotides, non-biological materials, etc.) that are operably linked (e.g., operating simultaneously, sequentially, etc.) according to a circuit design. The collection of molecular components may be capable of providing one or more specific outputs (e.g., regulation of one or more genes) in a cell in response to one or more inputs (e.g., a single input or multiple inputs). Such one or more inputs may be sufficient to trigger the molecular components of the genetic circuit to provide one or more specific outputs. For example, a genetic circuit may include one or more molecular switches that are activatable by one or more inputs (FIG. 1).

[0022] A genetic circuit can be a controllable gene expression system comprising an assembly of biological parts that work together (e.g., simultaneously, sequentially, etc.) as a logical function. The genetic circuit can include a plurality of gating units, at least one of which can be activated by an activating moiety (e.g., a heterologous input to a cell) and activates other gating units of the plurality of gating units (e.g., simultaneously, sequentially in a cascading manner, etc.) (FIG. 1). For example, at least one of the gating units can be activated by another of the plurality of gating units (e.g., directly or indirectly) to (i) regulate the expression or activity level of one or more target genes, (ii) activate at least one other of the plurality of gating units, and / or (ii) deactivate at least one other of the plurality of gating units, thereby collectively regulating the expression and / or activity level of one or more target genes in a predetermined and desired manner according to the design of the genetic circuit (FIG. 1). As used herein, the terms "heterologous genetic circuit," "HGC," "cellular algorithm," or "cellgorithm" may be used interchangeably.

[0023] As referred to herein, the term "gate unit" generally refers to a portion of a genetic circuit that can control gene regulation by functioning similarly to a logic gate, where it controls the flow of information and allows the circuit to make multiple decisions at different points. More specifically, the term refers to nucleic acids that encode gene switches and transcription / translation regulatory regions, or a set of regions on which a gene switch acts. The input to a gate unit can be an activating moiety and / or another gate unit. The output to a gate unit can be to activate another gate unit, to deactivate another gate unit, to affect a target gene, and / or any combination of the above. For example, a gate unit can be composed of multiple gate moieties and / or multiple gene regulatory moieties (Figure 1).

[0024] As referred to herein, the term "activating moiety" generally refers to a moiety that can activate multiple gene circuits and / or multiple gating units. The activating moiety can be a heterologous input to a 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 be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) and binds to the polynucleotide sequence of an inactivated gating moiety (e.g., a plasmid encoding another guide nucleic acid molecule), activating such gating moiety (e.g., inducing the expression of a functional form of an additional guide nucleic acid molecule), thereby targeting one or more gene regulatory moieties. As referred to herein, the term "gate moiety" generally refers to a moiety that can affect the function of a gene regulatory moiety within a gating unit. The gating moiety can activate and / or inactivate the gene regulatory moiety. For example, the gating moiety can regulate the expression of the gene regulatory moiety by editing a nucleic acid sequence, thereby activating or inactivating the gene regulatory moiety. For example, a gate moiety can be a guide nucleic acid molecule that can form a complex with an endonuclease (e.g., a Cas protein), bind to a polynucleotide sequence of a gene regulatory moiety (e.g., a plasmid encoding another guide nucleic acid molecule), activate the gene regulatory moiety (e.g., induce expression of a functional form of another guide nucleic acid molecule), and target one or more endogenous genes of a cell. Alternatively, or in addition, a gate moiety can activate and / or deactivate another gate unit of a genetic circuit (Figure 1). For example, a gate moiety can be a guide nucleic acid molecule that can form a complex with an endonuclease (e.g., a Cas protein), bind to a polynucleotide sequence of another gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to be inactivated, and activate the other gate moiety (e.g., induce expression of a functional form of another guide nucleic acid molecule).In another example, a gate moiety can be a guide nucleic acid molecule that can form a complex with an endonuclease (e.g., a Cas protein), bind to a polynucleotide sequence of another gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule) that becomes activated, and inactivates the other gate moiety (e.g., reduces expression of a functional form of the other guide nucleic acid molecule).

[0025] 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 a cell (Figure 1). For example, a gene editing moiety can regulate the expression of a gene by editing a nucleic acid sequence (e.g., CRISPR-Cas, zinc-finger nucleases, TALEN, or siRNA). In some cases, a gene editing moiety can regulate the expression of a gene by editing a genomic DNA sequence. In some cases, a gene editing moiety can regulate the expression of a gene by editing an mRNA template. Editing a nucleic acid sequence can, in some cases, alter the template underlying gene expression (e.g., CRISPR-Cas-inspired RNA targeting systems). Alternatively, a gene editing moiety can suppress the translation of a gene (e.g., Cas13).

[0026] 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 can recruit or contain at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. The gene editing moiety can itself bind to DNA and regulate transcription by physical obstruction, for example, preventing proteins such as RNA polymerase and other related proteins from assembling on the DNA template. The gene editing moiety can regulate gene expression at the translational level, for example, by regulating the production of proteins from an mRNA template. In some cases, the gene editing moiety can regulate gene expression by affecting the stability of mRNA transcripts. In some cases, the gene editing moiety can regulate genes through epigenetic editing (e.g., Cas12).

[0027] In some cases, the plasmid can encode a non-functional form of the gene editing portion. The plasmid can be activated to express a functional form of the gene editing portion, for example, through the activation of a functional gate portion. For example, the gene editing portion can encode a non-functional form of a guide nucleic acid molecule that can otherwise bind to a target gene of a cell. When the functional gate portion (e.g., another guide nucleic acid molecule complexed with a Cas protein) is bound to the plasmid, the plasmid is edited, allowing the expression of a functional form of the gene editing portion (e.g., a functional form of the guide nucleic acid molecule that specifically binds to a target gene of a cell), and allowing the regulation of the target gene in the cell.

[0028] Thus, 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, the gene regulatory portion may include an endonuclease or be operably linked to an endonuclease. An endonuclease may be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. An endonuclease may include a restriction endonuclease that cleaves DNA at specific sites without damaging the bases. Restriction endonucleases may include type I, type II, type III, and type IV endonucleases, and may further include subtypes. In some cases, the endonuclease is Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), or The endonuclease may be a dead endonuclease that exhibits reduced cleavage activity. For example, the endonuclease may be a nuclease-inactivating Cas, such as dCas (e.g., dCas9).

[0029] The gene regulatory portion can be a transcription modulator system (e.g., a gene repressor complex or a gene activator complex). For example, the gene regulatory portion can be a gene repressor complex comprising a dCas protein operably linked (e.g., fused) to a transcription 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, JARID1B / SMCX, JARID1B / SMCY, HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, M.Hal, METI, DRM3, ZMET2, CMT1, CMT2, Lamin A, and Lamin B. Alternatively, the gene regulatory portion 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 can include VP16, VP64, VP48, VP160, p65 subdomain, SET1A, SET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, SYMD2, NSD1, JHDM2a, JHDM2b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRCl, ACTR, P160, CLOCK, TET1CD, TET1, DME, DML1, DML2, and ROS1.

[0030] In some cases, the gene regulatory moiety has an enzymatic activity that modifies the target gene without cleaving the target gene. Modification of the target gene can result in, for example, epigenetic modifications that can modify gene expression and / or activity levels. Examples of enzymatic activities that can be provided by the gene regulatory moiety include nuclease activity, such as that provided by restriction enzymes (e.g., Fokl nuclease), methyltransferases (e.g., Hhal DNA cleavage enzymes), and the like. Methyltransferase activity, such as that provided by m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3, ZMET2, CMT1, and CMT2; demethylase activity, such as that provided by demethylases (e.g., Ten-Eleven Translocation (TET) Dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1); DNA repair activity, DNA damage activity, and deaminases (e.g., cytosine deaminase enzymes such as APOBEC1). These include, but are not limited to, deaminating activity such as that provided by a Gin recombinase, dismutase activity, alkylating activity, depurinating activity, oxidizing activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and / or resolvase (e.g., Gin invertase, e.g., a hyperactive mutant of Gin invertase, GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 degrading enzyme, etc.), transposase activity, recombinase activity such as that provided by a recombinase (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0031] Unless otherwise specified or apparent from context, the terms "polynucleotide," "oligonucleotide," or "nucleic acid," when used interchangeably herein, generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either single-stranded, double-stranded, or multi-stranded. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide can contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci (locuses) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0032] The term "gene" generally refers to nucleic acids (e.g., DNA such as genomic DNA and cDNA) and their corresponding nucleotide sequences involved in encoding an RNA transcript. When used herein with reference to genomic DNA, this term includes intervening non-coding regions and regulatory regions, and may include the 5' and 3' ends. In some uses, this term encompasses the transcribed sequence, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region includes "open reading frames" that encode a polypeptide. In some uses of this term, a "gene" includes only the coding sequence (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, a gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or a native gene in its natural location in the genome of an organism. A gene may refer to an "exogenous gene" or a non-native gene. A non-native gene may refer to a gene that is not normally found in a host organism, but is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene that is not in its natural location in the genome of an organism. A non-native gene may also refer to a naturally occurring nucleic acid or polypeptide sequence that contains mutations, insertions, and / or deletions (e.g., a non-native sequence).

[0033] 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 "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid, is the number of exact matches between the two aligned sequences divided by the length of the longer sequence multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, the advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method discussed in Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), and Altschul, et al., J. Mol. Biol., 215:403-410 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). This program can be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences, for example, using the blastp program.The program also allows for the use of an SEG filter to mask segments of query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired degree of sequence identity ranges from about 50% to 100% and integer values ​​therebetween. 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 to any of the sequences provided herein.

[0034] The term "expression" generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. A transcript and the encoded polypeptide can be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. With respect to expression, "up-regulated" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its expression level in the wild-type state, and "down-regulated" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to its expression in the wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression," the transfected gene is not transferred to daughter cells during cell division. Because its expression is limited to the transfected cells, gene expression is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cells. Such a selection advantage can be resistance to a particular toxin presented to the cells.

[0035] The terms "peptide," "polypeptide," or "protein," when used interchangeably herein, generally refer to a polymer of at least two amino acid residues linked by a peptide bond. The term does not imply a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structure (e.g., domains). The term also encompasses amino acid polymers modified by any other manipulation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and conjugation with a labeling component. The terms "amino acid" and "amino acids," as used herein, generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural and unnatural amino acids, which 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- and L-amino acids.

[0036] The terms "derivative," "variant," or "fragment," when used interchangeably herein with respect to a polypeptide, generally refer to a polypeptide that is related to a wild-type polypeptide, for example, by any of amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Polypeptide derivatives, variants, and fragments can include one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, as compared to the wild-type polypeptide.

[0037] As used herein with respect to polypeptide molecules (e.g., proteins), the terms "engineered," "chimeric," or "recombinant" generally refer to polypeptide molecules having heterologous or altered amino acid sequences as a result of the application of genetic engineering techniques to nucleic acids encoding the polypeptide molecules, as well as cells or organisms that express the polypeptide molecules. As used herein with respect to polynucleotide molecules (e.g., DNA or RNA molecules), the terms "engineered" or "recombinant" generally refer to polynucleotide molecules having heterologous or altered nucleic acid sequences 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, genetically engineered or recombinant polynucleotides (e.g., genomic DNA sequences) can be modified or altered by gene editing moieties.

[0038] Unless otherwise specified or clear from the context, as used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. A nucleotide may include synthetic nucleotides. A nucleotide may include synthetic nucleotide analogs. A nucleotide may be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may 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. As used herein, the term nucleotide may refer to dideoxyribonucleoside triphosphate (ddNTP) and its derivatives. Examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled by well-known techniques. Labeling may be performed using quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.Fluorescent labels for nucleotides may 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, Calif.; Fluorolink deoxynucleotides, Fluorolink Cy3-dCTP, Fluorolink Cy5-dCTP, Fluorolink Fluor X-dCTP, Fluorolink Cy3-dUTP, and Fluorolink Cy5-dUTP, available from Amersham, Arlington Heights, IL; and Fluorolink Cy5-dUTP, available from Boehringer Ingelheim. Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP available from Mannheim, Indianapolis, Ind., and Molecular Chromosomal labeled nucleotides available from Probes, Eugene, Oreg., include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides may also be labeled or unlabeled by chemical modification. The chemically modified single nucleotide may be a biotin-dNTP.Some non-limiting examples of biotinylated dNTPs can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0039] The term "cell" generally refers to a biological cell. A cell may be the basic structural, functional, and / or biological unit of a living organism. A cell may be derived from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, cells from mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidaria, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes the cells are not derived from a natural organism (e.g., the cells can be synthetically produced, sometimes referred to as artificial cells).

[0040] The terms "reprogramming," "dedifferentiation," "increasing cell potency," or "increasing developmental potency," when used interchangeably herein, generally refer to methods of increasing the differentiation potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state.

[0041] The term "differentiation" generally refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as an immune cell. A differentiated or differentiation-induced cell is one that has adopted a more specialized ("committed") position within a cell lineage. The term "committed" generally refers to a cell that, under normal circumstances, will continue to differentiate into a particular cell type or subset of cell types and has progressed down a differentiation pathway to a point where, under normal circumstances, it is unable to differentiate into a different cell type or revert to a less differentiated cell type.

[0042] The term "pluripotent" generally refers to the ability of a cell to form all lineages of the body or soma (i.e., embryo proper). For example, an embryonic stem cell is a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency can be a continuum of developmental potencies ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells) that cannot give rise to a complete organism to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0043] The term "induced pluripotent stem cells" (iPSCs) generally refers to stem cells derived from differentiated cells (e.g., differentiated adult, neonatal, or fetal cells) that have been induced or changed (i.e., reprogrammed) into cells that can differentiate into tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. Produced iPSCs do not refer to cells found in nature. In some cases, iPSCs can be engineered to differentiate directly into committed cells (e.g., natural killer 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 (HPCs)), which can be further induced to differentiate into committed cells (e.g., NK cells).

[0044] The term "embryonic stem cell" (ESC) generally refers to cells derived from naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and, during development, give rise to derivatives of all three primary germ layers: ectoderm, endoderm, and mesoderm. In some cases, ESCs can be engineered to differentiate directly into committed cells (e.g., NK cells). In some cases, ESCs can be engineered to first differentiate into tissue-specific stem cells (e.g., HSCs), which can be further induced to differentiate into committed cells (e.g., NK cells).

[0045] The term "isolated stem cells" generally refers to any type of stem cell disclosed herein (e.g., ESCs, HSCs, mesenchymal endodermal stem cells (MSCs), etc.) that is isolated from a multicellular organism. For example, HSCs can be isolated from a mammalian body, such as a human body. In another example, embryonic stem cells can be isolated from an embryo.

[0046] The term "isolated" generally refers to a cell or population of cells that has been separated from its original environment. For example, the new environment of an isolated cell is substantially free of at least one component that would be found in the environment in which the "un-isolated" reference cell resides. An isolated cell can be a cell that has been removed from some or all components as it would be found in its natural environment, e.g., a cell isolated from a tissue or biopsy sample. The term also includes cells that have been removed from at least one, some, or all components as they would be found in a non-naturally occurring environment, e.g., a cell isolated from a cell culture or cell suspension. Thus, an isolated cell is partially or completely separated from at least one component, including other substances, cells, or cell populations, as it is found in nature or grown, stored, or sustained in a non-naturally occurring environment.

[0047] The term "hematopoietic lineage," as applied to a cell or population, generally refers to cells derived (e.g., differentiated) from mesodermal cells (e.g., derived from pluripotent stem cells), and includes, for example, hemogenic endothelium (HE), pre-hematopoietic stem cells (HSCs), HSCs, and hematopoietic progenitor cells (e.g., multipotent progenitor cells, lymphoid progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T progenitor cells, NK progenitor cells, myeloid progenitor cells, etc.). In some cases, hematopoietic progenitor cells or HSCs can be characterized as being CD34+ (e.g., compared to pluripotent cells). In some cases, hematopoietic progenitor cells or HSCs can be characterized as being CD34+CD45+ or CD34+CD43-CD45+ (e.g., compared to pluripotent cells).

[0048] The terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells," when used interchangeably herein, generally refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., into NK cells) and can be used to differentiate into multipotent hematopoietic stem cells (hematoblasts, myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and erythrocyte progenitors). Hematopoietic stem and progenitor cells (HSCs) refer to cells including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells) lineages. In some cases, HSCs can be CD34+ hematopoietic cells that can give rise to both mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells.

[0049] The term "immune cell" generally refers to a differentiated hematopoietic cell. Non-limiting examples of immune cells include NK cells, T cells, monocytes, innate lymphocytes, tumor-infiltrating lymphocytes, macrophages, granulocytes, and the like.

[0050] In some cases, the term "proGuide" as used generally herein may refer to such a vector (e.g., a plasmid) encoding an activatable gNA. A proGuide may be an example of a gate portion. A proGuide may be an example of a gene regulatory portion.

[0051] In some embodiments, a proGuide provided herein can encode, for example, an activatable guide nucleic acid molecule having an inactivating polynucleotide sequence (e.g., one or more polyX sequences, such as one or more polyT sequences). In some cases, the portion of a proGuide encoding an activatable guide nucleic acid molecule can include various regions linked sequentially (e.g., 5' to 3'), including an upstream stem (e.g., an upstream cleavage site), a polyT unit (or, as used interchangeably herein, a "proUnit" or "proGuide Unit"), and a downstream stem (e.g., a downstream cleavage site). The upstream stem and downstream stem can correspond to "stem region" polynucleotide sequences that are at least partially complementary to each other.

[0052] Overview

[0053] Biological programming, such as cellular programming, allows for the manipulation of cells to produce desired outcomes. The outcomes of cellular programming can include inducing or preventing a wide range of common and / or novel cellular functions, and can also include enhancing or suppressing cellular functions that are already occurring. Cellular programming can be achieved through the use of genetic circuits. Cellular programming can be achieved through the manipulation of biomolecules (e.g., DNA). For example, CRISPR or CRISPR / Cas systems have been adopted for genome editing across many species due to their versatility and easy programmability. Cellular programming can affect endogenous or exogenous genes. Cellular programming can be implemented to function in a time-dependent or time-independent manner.

[0054] Genetic circuits used in cellular programming can be used to control cascades of multiple desired expression and / or activity profiles of multiple genes in a cell. Genetic circuits can be multiplexed to create positive and / or negative feedback systems to allow for better control of specific cellular outcomes.

[0055] While the CRISPR / Cas system is widely used for gene editing, because Cas remains bound to the double-stranded breaks it generates, it is essentially a single-turnover nuclease, leaving many regions of the genome resistant to genome editing. Increased understanding of CRISPR / Cas-based genome editing has facilitated the development of cascade regulatory systems to further exploit this technology for use in the development of genetically engineered cells. By implementing a series of activatable gRNAs, genome editing can be regulated from target site to target site in a more temporal manner, performing sequential genome editing, acting like a domino effect, and barcoding cells. However, this simple barcoding, which often uses exogenous fluorophores, does not allow for the multiplexed regulation of endogenous genes that lead to cell differentiation.

[0056] Thus, there remains an unmet need for an activatable, multiplexed CRISPR / Cas system and its use to edit target polynucleotides (e.g., the genome of a cell, particularly a eukaryotic cell) that uses a cascade of gRNAs to form a genetic circuit containing feedback loops to independently affect gene regulation and, therefore, cell fate decisions. Given their improved multiplexing capabilities through the use of internal positive and / or negative feedback loops, preprogrammed, activatable, self-regulating gRNA cascade CRISPR / Cas systems are useful, for example, in gene therapy, genetic circuits, and / or complex cell fate decisions and / or control.

[0057] The present disclosure provides systems and methods for engineering the CRISPR / Cas9 system, which comprises a Cas endonuclease and an array of activatable cognate single guide RNAs (sgRNAs or gRNAs) with inactivation sequences in non-essential regions, allowing for regulation and modification of the system through the use of positive and negative feedback loops. The present disclosure also provides engineered cells that can include any of the above systems or perform any of the above methods.

[0058] The differentiation of iPSCs into hematopoietic progenitor cells (HPCs) is complicated by several barriers of various etiologies. Attempts to generate HPCs using extracellular control mechanisms (e.g., culture conditions, growth factors, small molecules) have met with limited success on a small scale, but are prone to reproducibility issues and batch-to-batch variability in efficiency and potency. Cells generated from these approaches typically exhibit limited lineage potential and are unable to contribute to important lymphoid lineages. Consistent with other iPSC differentiation procedures based on control of the cellular microenvironment, these approaches are also difficult to scale up to the production and manufacturing levels required for therapeutic products.

[0059] Previous studies have shown that the activity of TF gene products can drive iPSCs to a HPC state with the capacity to generate lymphoid, myeloid, and erythroid cell lineages. Expression of a cohort of seven TFs was achieved by lentiviral insertion of cDNA expression transgenes into the genome of human pluripotent stem cells. The resulting cell lines contained multiple gene insertions, posing significant safety concerns and a barrier to their use in humans. Thus, although uncontrolled genome modifications from lentiviral insertions preclude their use in humans, cells with TF transgene insertions exhibit improved HPC stem cell properties and lymphoid lineage contributions compared to approaches using only extracellular manipulation.

[0060] The approach described herein uses non-integrating plasmid DNAs to deliver genetic instructions to iPSCs. Activation of TF genes provides cells with HPC potential that cannot be readily achieved using external control approaches. Plasmid DNA does not require genomic integration for endogenous gene activation, thus eliminating safety concerns raised by multiple lentiviral transgene insertions. The genetic instructions also have the advantage of being sequentially spaced to improve the transition from one cellular state to the next by timely activation of stage-specific TF gene expression.

[0061] Systems and methods for designing genetic circuits, cells containing genetic circuits, and methods of using same

[0062] Various embodiments of the present disclosure provide systems for inducing a desired expression and / or activity level (or profile) of one or more target genes in a cell. Various embodiments of the present disclosure provide methods for inducing a desired expression and / or activity level (or profile) of one or more target genes in a cell.

[0063] In one aspect, the present disclosure provides a system for inducing a desired expression and / or activity profile of a target gene in a cell. The system can include a heterologous genetic circuit comprising a plurality of gate units. The plurality of 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 plurality of 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, up to about 2, or up to about 1 gate unit. The plurality of gate units can be different (e.g., comprise different polynucleotide sequences).

[0064] The heterogeneous genetic circuits disclosed herein operate multiple gate units in series (e.g., multiple gate units connected sequentially in an end-to-end manner to form a single pathway), in parallel (e.g., multiple gate units connected crosswise to each other to form, e.g., two or more parallel pathways), or a combination thereof.

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

[0066] The cellular outcome may include the regulation of a target gene. The regulation of a target gene may include multiple different regulation of the target gene. Each of the multiple gate units may induce one of multiple distinct regulation of the target gene, whereby the coordinated set of distinct regulation results in the final expression and / or activity profile of the target gene. At least two distinct regulation of the multiple distinct regulation may increase both the expression and / or activity levels of the target gene. At least two distinct regulation of the multiple distinct regulation may decrease both the expression and / or activity levels of the target gene. Alternatively, a first distinct regulation of the multiple distinct regulation may increase the expression and / or activity level of the target gene, and a second distinct regulation of the multiple distinct regulation may decrease the expression and / or activity level of the target gene. In such a case, the first distinct regulation may occur before the second distinct regulation, or vice versa. Alternatively, a separate modulation (e.g., the first and / or second modulation) of the multiple separate modulations can maintain the expression level and / or activity level of the target gene at the expression level and / or activity level before modulation.

[0067] In some cases, each separate regulation of multiple target genes may be necessary to produce the desired expression and / or activity profile of target genes as disclosed herein, but may be individually insufficient.Therefore, the outcome in cells induced by multiple separate regulation of target genes (for example, enhanced cell function, induced cell state, etc.) may not be possible in the absence of any one of the multiple separate regulation of target genes.Alternatively, the degree or scale of the outcome in cells induced by multiple separate regulation of target genes may be greater than the degree or scale of the outcome in control cells, which is not induced by any of the multiple separate regulation of target genes, is induced by one or more but not all of them, and / or is induced by all of the multiple separate regulation of target genes that occur through different sequential orders of events.

[0068] The second gating unit can be activated (e.g., directly or indirectly) by the first gating unit. For example, the second gating unit can be directly activated by the first gating unit. Alternatively, the second gating unit can be activated by one or more additional gating units that are activated (e.g., directly or indirectly) by the first gating unit. The one or more additional gating units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more gating units. The one or more additional gating units may include up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 gating unit. In yet another alternative, the second gating unit may be activated via another moiety (e.g., an activation moiety, a different gating unit, etc.) that is involved in the activation of the first gating unit.

[0069] The second gate unit can be activated to induce the inactivation of the activated first gate unit.The terms " inactivation " and " disruption " can be used interchangeably herein.As disclosed herein, inactivation can be induced by generating modifications (such as single-strand or double-strand breaks, and breaks such as indels) to at least a portion of the first gate unit (for example, the gate portion and / or gene regulatory portion of the first gate unit) that are involved in inducing the first distinct regulation of the target gene.

[0070] As disclosed herein, the inactivation of the first gate unit by the gate portion and / or gene regulatory portion can be achieved through an endonuclease-based system (e.g., a CRISPR / Cas system). Alternatively, or in addition, inactivation can be achieved by using a transcriptional modulator system (e.g., a transcriptional repressor). An endonuclease transcriptional modulator system (e.g., a Cas repressor) can be used to achieve polynucleotide cleavage (e.g., to inactivate the gate portion and / or gene regulatory portion). Polynucleotide cleavage can create nucleic acid modifications such as single-strand breaks, double-strand breaks, insertions, deletions, or indels. Alternatively, or in addition, an endonuclease transcriptional modulator system (e.g., a Cas repressor) can be used to regulate target gene expression.

[0071] Alternatively, the second gate unit can be activated to amplify or enhance the activation of the activated first gate unit. The amplification or enhancement of the first gate unit can be induced by generating modifications (e.g., breaks such as single-strand or double-strand breaks, indels, etc.) in at least a portion of the first gate unit (e.g., the gate portion and / or gene regulatory portion of the first gate unit) that are involved in inducing specific regulation of the target gene.

[0072] The multiple gating units may be pre-configured such that one of the multiple gating units is activatable to regulate the expression and / or activity level of an additional target gene upon activation of the heterologous genetic circuit. The additional target gene may be different from the target gene regulated by the multiple distinct regulation provided herein. For example, the target gene may include an erythroid transformation-specific (ETS) transcription factor, and the additional target gene may include a T-box transcription factor (TBX), a homeobox protein, a GATA, and / or a basic helix-loop-helix (bHLH) transcription factor. In some cases, the first gating unit may be configured to be activated to regulate the expression and / or activity level of the additional target gene. For example, the first gating unit may include multiple activatable gene regulatory moieties to regulate the expression and / or activity level of the target gene and the additional target gene, respectively. In some cases, the second gating unit may be configured to be activated to regulate the expression and / or activity level of the additional target gene. For example, the second gating unit can include multiple activatable gene regulatory moieties to regulate the expression and / or activity levels of the target gene and the additional target gene, respectively. In some cases, the multiple gating units can include at least one additional gating unit configured to be activated to regulate the expression and / or activity level of the additional target gene. For example, the heterologous genetic circuit can be configured such that at least one additional gating unit is activated before activation of the first gating unit, between activation of the first gating unit and the second gating unit, and / or after activation of the second gating unit.

[0073] In some cases, the first gate unit regulates the first target gene. Alternatively, or in addition, the first gate unit can also regulate the second gate unit. The regulation of the second gate unit is determined by rt-qPCR, Western blotting, or other methods to be at least or up to about 1 millisecond, at least or up to about 2 milliseconds, at least or up to about 3 milliseconds, at least or up to about 4 milliseconds, at least or up to about 5 milliseconds, at least or up to about 6 milliseconds, at least or up to about 7 milliseconds, at least or up to about 8 milliseconds, at least or up to about 9 milliseconds, at least or up to about 10 milliseconds, at least or up to about 20 milliseconds, at least or up to about 30 milliseconds, at least or up to about 40 milliseconds, at least or up to about 50 milliseconds, at least or up to about 60 milliseconds, at least or up to about 70 milliseconds, at least or up to about 80 milliseconds, at least or up to about 90 milliseconds, at least or up to about 100 milliseconds, at least or up to about 200 milliseconds, at least or up to about 300 milliseconds, at least or up to about about 400 milliseconds, at least or up to about 500 milliseconds, at least or up to about 600 milliseconds, at least or up to about 700 milliseconds, at least or up to about 800 milliseconds, at least or up to about 900 milliseconds, at least or up to about 1 second, at least or up to about 2 seconds, at least or up to about 3 seconds, at least or up to about 4 seconds, at least or up to about 5 seconds, at least or up to about 6 seconds, at least or up to about 7 seconds, at least or up to about 8 seconds, at least or up to about 9 seconds, at least or up to about 10 seconds, at least or up to about 15 seconds, at least or up to about 20 seconds, at least or up to about 30 seconds, at least or up to about 40 seconds, at least or up to about 50 seconds, at least or up to about 1 minute, at least or up to about 2 minutes, at least or up to about 3 minutes, at least or up to about 4 minutes, at least or up to about 5 minutes, at least or up to about 6 minutes, at least or up to about 7 minutes, at least up to about 8 minutes,This may occur after at least or up to about 9 minutes, at least or up to about 10 minutes, at least or up to about 20 minutes, at least or up to about 30 minutes, at least or up to about 40 minutes, at least or up to about 50 minutes, at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 12 hours, at least or up to about 16 hours, at least or up to about 20 hours, or at least or up to about 24 hours, or more.

[0074] In some cases, the second gating unit can regulate a second target gene, where the regulation of the second target gene is at least or up to about 1 millisecond, at least or up to about 2 milliseconds, at least or up to about 3 milliseconds, at least or up to about 4 milliseconds, at least or up to about 5 milliseconds, at least or up to about 6 milliseconds, at least or up to about 7 milliseconds, at least or up to about 8 milliseconds, at least or up to about 9 milliseconds, at least or up to about 10 milliseconds, at least or up to about 20 milliseconds, at least or up to about 30 milliseconds, at least or up to about 40 milliseconds, at least or up to about 50 milliseconds, at least or up to about 60 milliseconds, at least or up to about 70 milliseconds, at least or up to about 80 milliseconds, at least or up to about 90 milliseconds, at least or up to about 100 milliseconds, at least or up to about 200 milliseconds, at least or up to about 300 milliseconds, at least or up to about 400 milliseconds, at least or up to about 500 milliseconds, of the regulation of the first target gene as confirmed by rt-qPCR, Western blotting, or other methods. seconds, at least or up to about 600 milliseconds, at least or up to about 700 milliseconds, at least or up to about 800 milliseconds, at least or up to about 900 milliseconds, at least or up to about 1 second, at least or up to about 2 seconds, at least or up to about 3 seconds, at least or up to about 4 seconds, at least or up to about 5 seconds, at least or up to about 6 seconds, at least or up to about 7 seconds, at least or up to about 8 seconds, at least or up to about 9 seconds, at least or up to about 10 seconds, at least or up to about 15 seconds, at least or up to about 20 seconds, at least or up to about 30 seconds, at least or up to about 40 seconds, at least or up to about 50 seconds, at least or up to about 1 minute, at least or up to about 2 minutes, at least or up to about 3 minutes, at least or up to about 4 minutes, at least or up to about 5 minutes, at least or up to about 6 minutes, at least or up to about 7 minutes, at least or up to about 8 minutes, at least or up to about 9 minutes, at least or up to about 10 minutes, at least up to about 20 minutes,This may occur after at least or up to about 30 minutes, at least or up to about 40 minutes, at least or up to about 50 minutes, at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 12 hours, at least or up to about 16 hours, at least or up to about 20 hours, or at least or up to about 24 hours, or more.

[0075] In some cases, the modification of the target gene by the gate unit can inactivate the gene. For example, the modification of the gene can stop the expression and / or activity level of the target gene. Alternatively, the modification of the gene can reduce the expression and / or activity level of the target gene. In some cases, the modification of the gene can increase the expression and / or activity level of the target gene. Alternatively, the modification of the gene can maintain the expression and / or activity level of the target gene.

[0076] The at least one additional gating unit can include at least or up to about 1 gating unit, at least or up to about 2 gating units, at least or up to about 3 gating units, at least or up to about 4 gating units, at least or up to about 5 gating units, at least or up to about 6 gating units, at least or up to about 7 gating units, at least or up to about 8 gating units, at least or up to about 9 gating units, at least or up to about 10 gating units, at least or up to about 11 gating units, at least or up to about 12 gating units, at least or up to about 13 gating units, at least or up to about 14 gating units, at least or up to about 15 gating units, at least or up to about 20 gating units, at least or up to about 30 gating units, at least or up to about 40 gating units, or at least or up to about 50 gating units. Each of the at least one additional gate unit can include 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, at least or up to about 15 gene regulatory portions, at least or up to about 20 gene regulatory portions, at least or up to about 30 gene regulatory portions, at least or up to about 40 gene regulatory portions, or at least or up to about 50 gene regulatory portions. Each of the gene regulatory portions of the gate unit can be configured to bind to a different target gene and regulate the expression and / or activity level of the different target gene, respectively. The heterologous genetic circuits provided herein can be configured to regulate the expression and / or activity level of one or more of the target genes provided herein.In some cases, a heterologous genetic circuit may be configured to regulate the expression and / or activity levels of some, but not all, of the target genes provided herein. For example, a heterologous genetic circuit may be pre-configured to regulate the expression and / or activity levels of a first set of one or more target genes (e.g., erythroid transformation-specific (ETS) genes such as ETS1, ETV2, LMO2, etc.), but not pre-configured to regulate the expression and / or activity of a second set of one or more target genes (e.g., TBXT, TBX6, MIXL1, etc.).

[0077] Heterogeneous genetic circuits disclosed herein operate multiple gate units in series (e.g., multiple gate units connected sequentially in an end-to-end manner to form a single pathway), in parallel (e.g., multiple gate units connected crosswise to one another to form, e.g., two or more parallel pathways), or a combination thereof. In some embodiments, multiple gate units in series can operate in a forward cascade. In some embodiments, the forward manner can follow a numerically increasing stepwise order (e.g., stage 1 - stage 2 - stage 3 - stage 4 - stage 5, etc.). In some embodiments, multiple gate units in series can operate in a reverse cascade. In some embodiments, the reverse cascade can follow a numerically decreasing stepwise order (e.g., stage 10 - stage 9 - stage 8 - stage 7 - stage 6, etc.). In some embodiments, the tandem multiple gating units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more gating units. In some embodiments, the tandem multiple gating units can include up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 gating unit. The multiple gating units disclosed herein can operate in concert to induce an outcome in a cell (e.g., as predetermined by the design of the heterologous genetic circuit). Cellular outcomes can include cellular function (e.g., migration, reproduction, response to external stimuli, nutrient production, excretion, respiration, growth) and / or cellular state (e.g., cell fate, differentiation, quiescence, programmed cell death). Such outcomes can be ascertained in vitro, ex vivo, and / or in vivo.For example, the outcomes disclosed herein can be confirmed in vitro by: (i) measuring expression levels of genes of interest by polymerase chain reaction (PCR) or Western blotting; (ii) staining with small molecules or antibodies; (iii) cell sorting based on cell size, morphology, and / or surface protein expression; (iv) using assays to measure phenotypic differentiation and cell function (e.g., cell proliferation assays, metabolic activity assays, cell death assays); (v) microscopy; and / or (iv) screening for molecular and / or genetic differences using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics.

[0078] A heterologous gene circuit may include multiple gate units that are activated sequentially, e.g., serially. The multiple gate units may include preconfigured functional gate units that are activated to regulate (e.g., directly regulate) the expression and / or epigenetic profile of a target gene (e.g., an endogenous target gene). The multiple gate units may further include one or more additional gate units that are preconfigured (i) to be activated before the functional gate unit and (ii) to achieve subsequent activation of the functional gate unit. In some cases, the one or more additional gate units may be preconfigured to be activated to regulate one or more additional target genes. Alternatively, the one or more additional gate units may not be preconfigured to regulate any target gene (e.g., any endogenous target gene) upon activation. Such one or more additional gate units may instead serve to delay (e.g., in terms of time) the activation of the functional gate unit during operation of the heterologous gene circuit, thereby delaying the expression and / or epigenetic profile of the target gene of the functional gate unit; therefore, the one or more additional gate units may be referred to as "blank" gate units.The heterologous genetic circuit may comprise at least or up to about 1 blank gate unit, at least or up to about 2 blank gate units, at least or up to about 3 blank gate units, at least or up to about 4 blank gate units, at least or up to about 5 blank gate units, at least or up to about 6 blank gate units, at least or up to about 7 blank gate units, at least or up to about 8 blank gate units, at least or up to about 9 blank gate units, at least or up to about 10 blank gate units, at least or up to about 11 blank gate units, at least or up to about 12 blank gate units, at least or up to about 13 blank gate units, at least The blank gate units may include at least or up to about 14 blank gate units, at least or up to about 15 blank gate units, at least or up to about 16 blank gate units, at least or up to about 27 blank gate units, at least or up to about 18 blank gate units, at least or up to about 19 blank gate units, at least or up to about 20 blank gate units, at least or up to about 25 blank gate units, at least or up to about 30 blank gate units, at least or up to about 35 blank gate units, at least or up to about 40 blank gate units, at least or up to about 45 blank gate units, or at least or up to about 50 blank gate units.

[0079] In some cases, the use of one or more blank gating units may inhibit activation of a functional gating unit for at least or up to about 1 minute, at least or up to about 5 minutes, at least or up to about 10 minutes, at least or up to about 30 minutes, at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 11 hours, at least or up to about 12 hours, at least or up to about 13 hours, at least or up to about 14 hours, at least or up to about The delay may be by about 15 hours, at least or up to about 16 hours, at least or up to about 17 hours, at least or up to about 18 hours, at least or up to about 19 hours, at least or up to about 20 hours, at least or up to about 21 hours, at least or up to about 22 hours, at least or up to about 23 hours, at least or up to about 24 hours, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, or at least or up to about 7 days (e.g., as determined by measuring the expression / epigenetic profile of the target gene or by measuring the expression of a functional variant or transcript of the functional gating unit).

[0080] In some embodiments, the disclosure provided herein as Appendix A discloses solutions to barriers to therapeutic HPC production. In some embodiments, Appendix A discloses a multi-step cascade for HPC generation. In some embodiments, Appendix A discloses aligning transcription factor gene activation with Cellgorithm library design. In some embodiments, Appendix A discloses a 10-step cascade for HPC generation. In some embodiments, the 10-step cascade can include empty and / or unused steps. In some embodiments, the empty and / or unused steps can space factor activation, leave room for additional genes, maximize the efficiency of Cellgorithm gene target induction, and / or adjust the relative timing of each step. Appendix A discloses Cellgorithms that result in a range of HPC expandability and HPC yields.

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

[0082] In some cases, the modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be caused by a single-strand break where a discontinuity exists in one nucleotide strand.The inactivation of a polynucleotide sequence or a target gene can be caused by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more single-strand breaks.In some cases, the inactivation of a gene can be caused by up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to one single-strand break.

[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 caused by double-strand breaks in which discontinuities exist in both nucleotide strands. In some cases, gene inactivation can be caused by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more double-strand breaks. In some cases, inactivation of a polynucleotide sequence or a target gene can be caused by up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to one double-strand break.

[0084] In some cases, the gNA is at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides or more in length.

[0085] In some cases, the disclosed systems and methods can utilize at least two different endonucleases. A first endonuclease (e.g., a Cas protein not coupled to a transcriptional modulator) can be used in conjunction with a guide nucleic acid to induce cleavage of a target polynucleotide sequence in a gate moiety or gene regulatory moiety plasmid, thereby activating and / or inactivating the gate moiety or gene regulatory moiety, respectively. Furthermore, a second endonuclease (e.g., a Cas protein coupled to a transcriptional modulator) can be used in conjunction with another guide nucleic acid to bind to a target gene (e.g., a target endogenous gene) in a cell and regulate the expression and / or activity of the target gene without causing cleavage in the target gene.

[0086] In some cases, the disclosed systems and methods can utilize a single endonuclease system (e.g., a Cas repressor) to achieve both (i) polynucleotide cleavage (e.g., to activate / inactivate gate and / or gene regulatory moieties) and (ii) target gene expression regulation. When using a single endonuclease-transcription modulator system, unique guide nucleic acid molecules (gNAs) of different spacer sequence lengths can be used to determine whether the single endonuclease-transcription modulator system (i) can hybridize to a polynucleotide sequence and induce Cas-mediated nuclease activity of the polynucleotide sequence, or (ii) can hybridize to a target gene (e.g., genomic DNA) and regulate the expression and / or activity level of the target gene through the action of a transcription activator without mediating Cas nuclease activity, as desired by individual heterologous genetic circuits. For example, the use of gNAs of different spacer sequence lengths that bind to different targets may allow the second gating unit provided herein to induce inactivation of an activated first gating unit and / or induce distinct regulation of a second target gene.

[0087] As described above, the length of the spacer sequence of a gNA can affect the ability of the gNA to mediate Cas nuclease activity. In some cases, gNAs with spacer sequences of different lengths can be used in the same heterologous gene circuit to affect different types of cleavage, activation, inactivation, and / or regulation of one or more target nucleic acids. In some cases, a gNA spacer sequence shorter than a threshold length (e.g., about 16 nucleotides) can eliminate the nuclease activity of a Cas transcriptional regulator while still mediating DNA binding for transcriptional regulation of a target gene. In some cases, a gNA spacer sequence shorter than at least about 25 nucleotides, at least about 20 nucleotides, at least about 19 nucleotides, at least about 18 nucleotides, at least about 17 nucleotides, at least about 16 nucleotides, at least about 15 nucleotides, at least about 15 nucleotides, at least about 14 nucleotides, at least about 13 nucleotides, at least about 12 nucleotides, at least about 11 nucleotides, or at least about 10 nucleotides can eliminate the nuclease activity of a Cas protein while still mediating DNA binding.

[0088] For example, a gNA containing a 20-nucleotide spacer sequence (e.g., a gNA encoded by a gate moiety for targeting a gene regulatory portion plasmid) may be sufficient to promote the nuclease activity of an endonuclease (e.g., a Cas or Cas transcriptional modulator fusion protein) at the target polynucleotide sequence. Alternatively, or in addition, a gNA containing a 14-nucleotide spacer sequence (e.g., a gNA encoded by a gene regulatory portion) may hybridize to DNA but may not be long enough to mediate nuclease activity; it may only promote endonuclease binding to the cognate DNA sequence. Thus, shorter gNAs can selectively allow transcriptional regulation of target genes through the use of an endonuclease transcriptional modulator system (e.g., a Cas activator system, a Cas repressor system) without cleaving the target gene.

[0089] In some cases, the modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can be caused by an indel, also known as an insertion-deletion mutation. Indel mutations can include frameshift or non-frameshift mutations. Indel mutations can also include point mutations, also known as base substitutions, in which only one base or base pair is modified. Indel mutations can comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000 or more bases or base pairs in length. Indel mutations can comprise up to about 2000, up to about 1000, up to about 900, up to about 800, up to about 700, up to about 600, up to about 500, up to about 400, up to about 300, up to about 200, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 base or base pair in length.

[0090] In some cases, the 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 portion (e.g., a nucleic acid molecule and / or an endonuclease, such as a complex comprising a CRISPR / Cas protein and a guide nucleic acid molecule) can specifically bind to a polynucleotide sequence or a target gene so that the expression and / or activity of the polynucleotide sequence or the target gene is modified. The gene regulatory portion can include a transcriptional repressor or a transcriptional activator as provided herein.

[0091] 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 inactivate the gene. For example, modification of a polynucleotide sequence or a target gene can stop the expression and / or activity level of the polynucleotide sequence or the target gene. Alternatively, modification of a polynucleotide sequence or a target gene can reduce the expression and / or activity level of the polynucleotide sequence of the target gene. In some cases, 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. Alternatively, modification of a polynucleotide sequence or a target gene can maintain the expression and / or activity level of the polynucleotide sequence or the target gene.

[0092] In some cases, modification of the polynucleotide sequence (e.g., as a component of a gating unit such as a gating moiety) or target gene may comprise decreasing the expression and / or activity level of the polynucleotide sequence or target gene, respectively, by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. Modification of the polynucleotide sequence or target gene may increase the expression and / or activity level of the polynucleotide sequence or target gene, respectively, 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 3 ... This may include a decrease of 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.

[0093] In some cases, modification of the polynucleotide sequence (e.g., as a component of a gating unit such as a gating moiety) or target gene reduces the expression and / or activity level of the polynucleotide sequence or target gene, respectively, by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 10 ... can also include an increase of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, at least about 1,000,000% or more.Modification of the polynucleotide sequence or target gene may increase the expression and / or activity level of the polynucleotide sequence or target gene, respectively, by up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 400%, up to about 500%, up to about 600%, up to about 600%, up to about 700%, up to about 700%, up to about 800%, up to about 9 ... In some embodiments, the increase may include increasing the chromaticity by about 200%, by about 100%, by about 90%, by about 80%, by about 70%, by about 60%, by about 50%, by about 40%, by about 30%, by about 20%, by about 10%, by about 9%, by about 8%, by about 7%, by about 6%, by about 5%, by about 4%, by about 3%, by about 2%, by about 1%, by about 0.9%, by about 0.8%, by about 0.7%, by about 0.6%, by about 0.5%, by about 0.4%, by about 0.3%, by about 0.2%, by about 0.1%, or less.

[0094] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gating unit such as a gating moiety) or a target gene increases the expression and / or activity level of the polynucleotide sequence or target gene by at least or at most about 0.1 fold, at least or at most about 0.2 fold, at least or at most about 0.3 fold, at least or at most about 0.4 fold, at least or at most about 0.5 fold, at least or at most about 0.6 fold, at least or at most about 0.7 fold, at least or at most about 0.8 fold, at least or at most about 0.9 fold, at least or at most about 1 fold, at least or at most about 2 fold, at least or at most about 3 fold, at least or at most about It may include a 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold decrease.Modification of the polynucleotide sequence or target gene may increase the expression and / or activity level of the polynucleotide sequence or target gene by at most or less than about 10,000 fold, at most or less than about 5,000 fold, at most or less than about 1,000 fold, at most or less than about 500 fold, at most or less than about 100 fold, at most or less than about 90 fold, at most or less than about 80 fold, at most or less than about 70 fold, at most or less than about 60 fold, at most or less than about 50 fold, at most or less than about 40 fold, at most or less than about 30 fold, at most or less than about 40 fold, at most or less than about 5 ... or by less than about 20-fold, by at most or less than about 10-fold, by at most or less than about 9-fold, by at most or less than about 8-fold, by at most or less than about 7-fold, by at most or less than about 6-fold, by at most or less than about 5-fold, by at most or less than about 4-fold, by at most or less than about 3-fold, by at most or less than about 2-fold, by at most or less than about 1-fold, by at most or less than about 0.9-fold, by at most or less than about 0.8-fold, by at most or less than about 0.7-fold, by at most or less than about 0.6-fold, by at most or less than about 0.5-fold, by at most or less than about 0.4-fold, by at most or less than about 0.3-fold, by less than about 0.2-fold, or by less than about 0.1-fold.

[0095] In some cases, modification of the polynucleotide sequence (e.g., as a component of a gating unit such as a gating moiety) or target gene increases the expression and / or activity level of the polynucleotide sequence or target gene, respectively, by at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least or at most about 0.6-fold, at least or at most about 0.7-fold, at least or at most about 0.8-fold, at least or at most about 0.9-fold, at least or at most about 1-fold, at least or at most about 2-fold, at least or at most about 3-fold, at least or at most about This can include an increase of 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.Modification of the polynucleotide sequence or target gene may increase the expression and / or activity level of the target gene by up to or less than about 10,000 fold, up to or less than about 5,000 fold, up to or less than about 1,000 fold, up to or less than about 500 fold, up to or less than about 100 fold, up to or less than about 90 fold, up to or less than about 80 fold, up to or less than about 70 fold, up to or less than about 60 fold, up to or less than about 50 fold, up to or less than about 40 fold, up to or less than about 30 fold, up to or less than about 20 fold, up to or less than about 3 ... or less than about 10-fold, by at most or less than about 9-fold, by at most or less than about 8-fold, by at most or less than about 7-fold, by at most or less than about 6-fold, by at most or less than about 5-fold, by at most or less than about 4-fold, by at most or less than about 3-fold, by at most or less than about 2-fold, by at most or less than about 1-fold, by at most or less than about 0.9-fold, by at most or less than about 0.8-fold, by at most or less than about 0.7-fold, by at most or less than about 0.6-fold, by at most or less than about 0.5-fold, by at most or less than about 0.4-fold, by at most or less than about 0.3-fold, by at most or less than about 0.2-fold, or by at most or less than about 0.1-fold.

[0096] In some cases, (i) a change (e.g., an enhancement or reduction) in the expression and / or activity level of the target gene upon multiple distinct modulations in a sequential manner is (ii) 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%, at least or up to about 10%, at least or up to about 11%, at least or up to about 12%, at least or up to about 13%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 17%, at least or up to about 18%, at least or up to about 19%, at least or up to about 20%, at least or up to about 21%, at least or up to about 22%, at least or up to about 23%, at least or up to about 24%, at least or up to about 25%, at least or up to about 26%, at least or up to about 27%, at least or up to about 28%, at least or up to about 29%, at least or up to about 30%, at least or up to about 31%, at least or up to about 32%, at least or up to about 33%, at least or up to about 34%, at least or up to about 35%, at least or up to about 36%, at least or up to about 37%, at least or up to about 38%, at least or up to about 39%, at least or up to about 40%, at least or up to about 41%, at least or up to about 42%, at least or up to about 43%, at least or up to about 44%, at least or up to about 45%, at least or up to about 46%, at least or up to about 47%, at least or up to It may be about 19%, 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 100%, at least or up to about 125%, at least or up to about 150%, at least or up to about 175%, at least or up to about 200%, at least or up to about 225%, at least or up to about 250%, at least or up to about 275%, or at least or up to about 300% greater.

[0097] In some cases, (i) a change (e.g., an enhancement or reduction) in the expression and / or activity level of the target gene upon multiple distinct modulations in a sequential manner is (ii) 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%, at least or up to about 10%, at least or up to about 11%, at least or up to about 12%, at least or up to about 13%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 17%, at least or up to about 18%, at least or up to about 19% greater than any change in the expression and / or activity level of the target gene upon only or in the absence of one of the multiple distinct modulations. , 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 100%, at least or up to about 125%, at least or up to about 150%, at least or up to about 175%, at least or up to about 200%, at least or up to about 225%, at least or up to about 250%, at least or up to about 275%, or at least or up to about 300% longer duration.

[0098] In some cases, (i) the expression and / or activity level of the target gene upon multiple distinct modulations in a sequential manner (e.g., including sequential interruptions) is (ii) 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%, at least or up to about 10%, at least or up to about 11%, at least or up to about 12%, at least or up to about 13%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 17%, at least or up to about 18%, or at least about at least or up to about 19%, 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 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 up to or about 97%, at least up to or about 98%, at least up to or about 99%, or substantially less than about 100%.

[0099] In some cases, (i) the expression and / or activity level of the target gene upon multiple distinct modulations in a sequential manner (e.g., including sequential interruptions) is (ii) 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%, at least or up to about 10%, at least or up to about 11%, at least or up to about 12%, at least or up to about 13%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 17%, at least or up to about 18%, or at least if or at most about 19%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 40%, at least or at most about 45%, at least or at most about 50%, at least or at most about 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 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 at or at most about 97%, at least at or at most about 98%, at least at or at most about 99%, or substantially about 100% shorter.

[0100] In some cases, the first gate unit and the second gate unit provided herein can be activated to induce a first separate regulation of a common gene and then a second separate regulation (e.g., the expression and / or activity profile of the common gene).The first gate unit and the second gate unit can be activated sequentially (e.g., at different times) to cause the first and second separate regulation of the common gene in a sequential manner.Alternatively, the first gate unit and the second gate unit can be activated simultaneously, and such gate can be pre-configured to still cause the first and second separate regulation of the common gene in such a sequential manner.

[0101] In some cases, activation of the multiple gate units may be the result of a single activation of the heterologous genetic circuit (e.g., by a single activation moiety at a single time point). The multiple gate units may include one of a first gate unit and a second gate unit preconfigured to be sequentially activated upon activation of the heterologous genetic circuit by the single activation. In some cases, one of the first and second gate units may be activated by a single activation moiety (e.g., a guide nucleic acid), while the other of the first and second gate units may be activated by an additional activation moiety (e.g., a different guide nucleic acid) that is different from the activation moiety of the heterologous genetic circuit. The additional activation moiety may be a portion of the heterologous genetic circuit that is generated (e.g., expressed) only upon activation of the heterologous genetic circuit. Alternatively, or in addition, the first and second gate units may each be activated by a different activation moiety that is not the same as the activation moiety of the heterologous genetic circuit. Such a different activation moiety may be a portion of the heterologous genetic circuit that is generated (e.g., expressed) only upon activation of the heterologous genetic circuit.

[0102] In some embodiments of any of the systems disclosed herein, a gating unit may comprise a gating moiety (e.g., at least 1, 2, 3, 4, 5, or more different gating moieties) and / or a gene regulatory moiety (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different gene regulatory moieties). The gating moieties disclosed herein may comprise guide nucleic acid molecules (gNAs) (e.g., at least 1, 2, 3, 4, 5, or more gNAs). The gene regulatory moieties disclosed herein may comprise gNAs (e.g., at least 1, 2, 3, 4, 5, or more gNAs). The guide nucleic acid molecules disclosed herein may include, but are not limited to, DNA, RNA, any analogs of such, or any combination thereof. In some embodiments of any of the systems disclosed herein, the gating moiety and / or gene regulatory moiety may be activatable to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and the complex may be configured or capable of binding to a target polynucleotide, e.g., to regulate the expression and / or activity level of the target polynucleotide or another polynucleotide sequence operably linked to the target polynucleotide. For example, the complex may regulate the expression and / or activity level of a gene comprising the target polynucleotide.

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

[0104] In some embodiments of any of the systems disclosed herein, the gNA of the gate moiety and / or gene regulatory moiety (e.g., the gNA encoded by the gate moiety and / or gene regulatory moiety) can be an activatable gNA. The activatable gNA can be, but is not limited to, any one of the following: a ribonucleotide (e.g., a gRNA), a deoxyribonucleotide, any analog thereof, or any combination thereof. In some embodiments, the activatable gNA molecule can be a self-cleaving gNA (e.g., the gRNA contains a cis ribozyme). For example, when an activatable gNA is expressed in a cell, the activatable gNA can be self-cleavable and non-functional (e.g., not configured to bind to a target gene) unless the gene encoding the activatable gNA is modified prior to expression of the activatable gNA. In some embodiments, the activatable gNA molecule contains a non-canonical transcription termination sequence (e.g., a poly-X sequence, such as a poly-U or poly-T sequence), such that a functional gNA molecule is not expressed until the gene encoding the activatable gNA with the non-canonical transcription termination sequence is modified (e.g., to remove some or all of the transcription termination sequence). Thus, in the absence of transcription termination sequence modification, a non-functional variant (e.g., a non-functional fragment) of the gNA can be expressed. In some embodiments, the gNA can be synthetic. In some embodiments, the gNA can have an attached fluorescent label.

[0105] In some cases, the non-standard termination sequence is at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least The polynucleotide sequence may comprise or consist essentially of a polynucleotide sequence exhibiting 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.

[0106] In some cases, the polynucleotide sequence containing the non-canonical termination sequence (or its complement) has the following structure (II): M-T'-M', wherein (i) T' is a non-canonical termination sequence provided herein (e.g., polyT), and (II) M and M' are as described above for structure (II).

[0107] In some cases, in pairs including M and M' as shown in Structure (II) and / or Structure (III), the pair can form an insulator sequence as provided herein. Alternatively, the pair can be relative to a stem sequence as provided herein.

[0108] In some cases, in the pair comprising M and M' shown in structure (II), the polynucleotide sequence of M and the additional polynucleotide sequence of M' are, respectively, (1) SEQ ID NO:117 and SEQ ID NO:154, (2) SEQ ID NO:118 and SEQ ID NO:155, (3) SEQ ID NO:119 and SEQ ID NO:156, (4) SEQ ID NO:120 and SEQ ID NO:157, (5) SEQ ID NO:121 and SEQ ID NO:158, (6) SEQ ID NO:122 and SEQ ID NO:159, (7) SEQ ID NO:123 and SEQ ID NO:160, (8) SEQ ID NO:124 and SEQ ID NO:161, SEQ ID NO: 124 and SEQ ID NO: 161, (9) SEQ ID NO: 126 and SEQ ID NO: 162, (10) SEQ ID NO: 127 and SEQ ID NO: 163, (11) SEQ ID NO: 128 and SEQ ID NO: 164, (12) SEQ ID NO: 129 and SEQ ID NO: 165, (13) SEQ ID NO: 130 and SEQ ID NO: 166, (14) SEQ ID NO: 131 and SEQ ID NO: 167, (15) SEQ ID NO: 132 and SEQ ID NO: 168, (16) SEQ ID NO: 133 and SEQ ID NO: 169, (17) SEQ ID NO: 134 and SEQ ID NO: 170, and (18) SEQ ID NO: 13 5 and SEQ ID NO: 171, or their complementary sequence pairs, may exhibit at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity.

[0109] In some cases, the size of the polyT sequence is equal to or greater than a threshold length, and the threshold length is sufficient to reduce expression of the guide nucleic acid molecule from the polynucleotide sequence. Thus, a plasmid (e.g., a gate portion or gene regulatory portion) can encode an inactivated gNA containing a polyT sequence equal to or greater than a threshold length, and editing such a plasmid to reduce the length of the polyT to less than the threshold length allows 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 5 to 15 Ts. In some cases, the polyT sequence contains one or more additional nucleotides that are not Ts.

[0110] In some cases, the gene regulatory portion (e.g., guide nucleic acid and / or endonuclease) can be configured to bind to a target polynucleotide sequence operably linked to a target gene in a cell. The target gene can include a coding polynucleotide sequence that encodes a target nucleic acid molecule or a target protein. The target polynucleotide sequence can be a part of the coding polynucleotide sequence. Alternatively, the target polynucleotide sequence may not be a part of the coding polynucleotide sequence. For example, the target polynucleotide sequence can be upstream of the coding polynucleotide sequence (e.g., a part of the promoter of the coding polynucleotide sequence, such as a transcription start site (TSS)).

[0111] In some embodiments, a proGuide may contain a target polynucleotide domain at or adjacent to an inactivating polynucleotide sequence (e.g., at or adjacent to the 5' and / or 3' end of the inactivating polynucleotide sequence), which can be targeted (e.g., via the sequential activation mechanisms of the heterologous genetic circuits provided herein) to modify (e.g., edit, cleave) the inactivating polynucleotide sequence, thereby causing the proGuide to express an activated guide nucleic acid molecule. The target polynucleotide domain of the proGuide does not exhibit sequence identity to any comparable endogenous polynucleotide sequence in the cell, thereby avoiding inadvertent targeting and regulation of endogenous target genes.

[0112] In some embodiments, the inactivation polynucleotide sequence of the proGuide can be placed between two target polynucleotide domains, which may or may not be targetable by a common guide nucleic acid sequence. In some cases, the two target polynucleotide domains can be reverse-complementary to each other, allowing the inactivation polynucleotide sequences to be modified or cleaved by the same mechanism (e.g., the same spacer sequence of the guide nucleic acid molecule).

[0113] In some embodiments, the proGuide is selected from the group consisting of SEQ ID NOs: 190-191 (e.g., targeting BCL11A), SEQ ID NOs: 192-194 (e.g., targeting BCL11B), SEQ ID NOs: 195-198 (e.g., targeting BCL2), SEQ ID NOs: 199-206 (e.g., targeting BCL2L1), SEQ ID NOs: 207-210 (e.g., targeting BMI1), SEQ ID NOs: 211-214 (e.g., targeting CD34), SEQ ID NOs: 215-218 (e.g., targeting DLL4), SEQ ID NOs: 219-224 (e.g., targeting DNMT3B), SEQ ID NOs: 225-227 (e.g., targeting DTX1 targeting), SEQ ID NOS: 228-230 (e.g., EBF1 targeting), SEQ ID NOS: 231-233 (e.g., EOMES targeting), SEQ ID NOS: 234-241 (e.g., ERG targeting), SEQ ID NOS: 242-245 (e.g., ETS1 targeting), SEQ ID NOS: 246-248 (e.g., ETV2 targeting), SEQ ID NOS: 249-252 (e.g., EZH1 targeting), SEQ ID NOS: 253-256 (e.g., FLT1 targeting), SEQ ID NOS: 257-260 (e.g., FOS targeting), SEQ ID NOS: 261-264 (e.g., FOXO1 targeting), SEQ ID NOS: 265-267 (e.g., For example, targeting FOXP3), SEQ ID NOs: 268 to 271 (for example, targeting GATA1), SEQ ID NOs: 272 to 278 (for example, targeting GATA2), SEQ ID NOs: 279 to 282 (for example, targeting GATA3), SEQ ID NOs: 283 to 289 (for example, targeting GFI1), SEQ ID NOs: 290 to 293 (for example, targeting GFI1B), SEQ ID NOs: 294 to 297 (for example, targeting HES1), SEQ ID NOs: 298 to 301 (for example, targeting HHEX), SEQ ID NOs: 302 to 304 (for example, targeting HOXA10), SEQ ID NOs: 305 to 307 (for example, targeting HOXA5), SEQ ID NOs: 308 to 311 (e.g., HOXA7 targeting), SEQ ID NO: 312 (e.g., HOXA9 targeting), SEQ ID NOs: 313 to 315 (e.g., HOXB4 targeting), SEQ ID NOs: 316 to 322 (e.g., ID2 targeting), SEQ ID NOs: 323 to 325 (e.g., ID3 targeting), SEQ ID NOs: 326 to 329 (e.g., IKZF1 targeting), SEQ ID NOs: 330 to 336 (e.g., IKZF2 targeting), SEQ ID NOs: 337 to 340 (e.g., IL7R targeting), SEQ ID NOs: 341 to 347 (e.g., IRF4 targeting), SEQ ID NOs: 348 to 351 (e.g., IRF8 targeting),SEQ ID NOs: 352 to 355 (e.g., KDR targeting), SEQ ID NOs: 356 to 363 (e.g., LCOR targeting), SEQ ID NOs: 364 to 371 (e.g., LEF1 targeting), SEQ ID NOs: 372 to 374 (e.g., LMO2 targeting), SEQ ID NOs: 375 to 378 (e.g., LYL1 targeting), SEQ ID NOs: 379 to 382 (e.g., MEF2C targeting), SEQ ID NOs: 383 to 386 (e.g., MIXL1 targeting), SEQ ID NOs: 387 to 389 (e.g., MYB targeting), SEQ ID NOs: 390 to 393 (e.g., MYC targeting), SEQ ID NOs: 394 to 397 (e.g., NFIL3 targeting), targeting), SEQ ID NOS: 398 to 401 (e.g., targeting NR4A1), SEQ ID NOS: 402 to 405 (e.g., targeting PAX5), SEQ ID NOS: 406 to 409 (e.g., targeting PGF), SEQ ID NOS: 410 to 416 (e.g., targeting RORC), SEQ ID NOS: 417 to 419 (e.g., targeting RUNX1), SEQ ID NOS: 420 to 426 (e.g., targeting RUNX3), SEQ ID NOS: 427 to 430 (e.g., targeting SATB1), SEQ ID NOS: 431 to 434 (e.g., targeting SNAI1), SEQ ID NOS: 435 to 438 (e.g., targeting sox17), SEQ ID NOS: 439 to 446 (e.g., targeting SOX4), targeting), SEQ ID NOs: 447-448 (e.g., SPI1 targeting), SEQ ID NOs: 449-452 (e.g., SUV39H1 targeting), SEQ ID NOs: 453-460 (e.g., TAL1 targeting), SEQ ID NOs: 461-463 (e.g., TBX21 targeting), SEQ ID NOs: 464-470 (e.g., TCF12 targeting), SEQ ID NOs: 471-478 (e.g., TCF3 targeting), SEQ ID NOs: 479-480 (e.g., TCF7 targeting), SEQ ID NOs: 481-484 (e.g., TOX targeting), SEQ ID NOs: 485-488 (e.g., TOX2 targeting), SEQ ID NOs: 489-492 (e.g., , VEGFA targeting), SEQ ID NOs: 493-496 (e.g., VEGFB targeting), SEQ ID NOs: 497-500 (e.g., VEGFC targeting), SEQ ID NOs: 501-504 (e.g., VEGFD targeting), SEQ ID NOs: 505-507 (e.g., ZBTB16 targeting), SEQ ID NOs: 508-511 (e.g., ZBTB17 targeting), SEQ ID NOs: 512-514 (e.g., ZBTB7B targeting), SEQ ID NOs: 515-518 (e.g., ZSCAN4 targeting), or a complementary sequence thereof (shown in Table 5), at least or up to about 50%,The polynucleotide sequences may include polynucleotide sequences exhibiting 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.

[0114] The differential regulation of the target gene may be different (eg, different degrees of change in the expression and / or activity levels of the target gene). For example, the first regulation exerted by the first genetic unit and the second regulation exerted by the second gating unit may differ by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%. The first adjustment and the second adjustment may differ by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, or at most about 0.1%. Alternatively, or in addition, the distinct regulation of the target genes can be substantially the same (eg, the same).

[0115] The multiple separate modulations may be individually sufficient to induce a desired change in the expression and / or activity level of the target gene, or alternatively, the separate modulations may be individually insufficient to induce a desired change in the expression and / or activity level of the target gene.

[0116] The one or more target genes disclosed herein can include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0117] The one or more target genes disclosed herein may include a cell differentiation regulator, a molecular function regulator, a binding factor, a fusion factor, a protein folding chaperone, a protein tag, an RNA folding chaperone, a cell signaling factor, an immune response factor, a sensory receptor, a cell structure factor, a protein binding factor, a cargo receptor, a catalytic factor, or a small molecule sensor.

[0118] The one or more target genes disclosed herein may include cell differentiation regulators, including growth factors, transcription factors, myogenic regulators, immune cell regulators, neuroregulators, stem cell differentiation factors, chondrogenic regulators, osteogenic regulators, senescence factors, stemness factors (e.g., dedifferentiation factors), and the like.

[0119] In some cases, the one or more target genes (e.g., one or more immune cell regulators) are MYCN, PTCRA, BCL11B, HHEX, NOTCH1, TCF3, RAG2, DTX1, RUNX1, HOXA9, HOXA5, HOXB4, RAG1, SPI1, EVT2, SCL, ID2, BCL11A, ID3, TCF7, IKZF1, TCF12, RUNX3, LMO2, LEF1, GFI1, LYL1, AQP3, MEIS1, GATA2, GAT A3, HES1, ST18, NR4A1, C20ORF100, IKAROS, SPIB, RORC, TCF1, LCOR, IRF8, SATB1, BMI1, MYC, AHR, MIXL1, FOXO1, NOTCH1, NOTCH3, IL-2, IL4, IL7, IL15, EBF1, PAX5, TAL1, MYB, ERG, HHEX, E4PB4, GFI1B, PTA, TBXT, TBX6, TBX21, ETS1, ETS2, or MEF2C. For example, one or more target genes for inducing hematopoietic stem cell differentiation may be selected from the group consisting of TBXT, TBX6, MIXL1, ETS1, ETV2, GATA2, SCL, LMO2, HOXA5, HOXA9, ERG, SPI1, MYB, RUNX1, HOXB4, GFI1, LCOR, IRF8, SATB1, and BMI1 (Figure 2). In some cases, the one or more target genes (e.g., one or more hematopoietic stem cell differentiation factors) may not include TBXT, TBX6, and / or MIXL1.

[0120] In some cases, the one or more target genes (e.g., one or more stem cell differentiation factors) may include HOXB4, NOTCH1, SOX11, SOX17, RUNX1, GATA2, FLI1, ERG, WNT, HNF1, HNF3, HNF4, CDX2, and LIN28A.

[0121] In some cases, the one or more target genes (e.g., one or more stemness factors) may include HES1, HES5, CBF1, SOC2, HMGA2, OLIG2, ID2, ID4, HESR1, HESR2, GLI1, GLI2, GLI3, SOXB, and BMI1.

[0122] In some cases, one or more target genes may include a T-box transcription factor (TBX gene). TBX transcription factors are involved in development. T-box proteins have a relatively large DNA binding domain. Non-limiting examples of TBX transcription factors may include TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, TBX22, and TBXT (Brachyury protein). In some cases, one or more target genes may not include one or more TBX transcription factors (e.g., TBXT or TBX6).

[0123] In some cases, the plurality of gate units are pre-configured such that one of the plurality of gate units is activatable to regulate the expression and / or activity level of the TBX gene upon activation of the heterologous genetic circuit. In some cases, the first gate unit is activatable to regulate the expression and / or activity level of the TBX gene. Alternatively or additionally, the second gate unit is activatable to regulate the expression and / or activity level of the TBX gene. Alternatively or additionally, the gate unit is activated before the first gate unit to regulate the expression and / or activity level of the TBX gene. Alternatively or additionally, the gate unit is activated between the first gate unit and the second gate unit to regulate the expression and / or activity level of the TBX gene.

[0124] In some cases, the plurality of gating units are pre-configured such that one of the plurality of gating units is activatable to regulate the expression and / or activity level of TBXT upon activation of the heterologous genetic circuit. In some cases, the first gating unit is activatable to regulate the expression and / or activity level of TBXT. Alternatively or additionally, the second gating unit is activatable to regulate the expression and / or activity level of TBXT. Alternatively or additionally, the gating unit is activated before the first gating unit to regulate the expression and / or activity level of TBXT. Alternatively or additionally, the gating unit is activated between the first gating unit and the second gating unit to regulate the expression and / or activity level of TBXT.

[0125] In some cases, the plurality of gating units are pre-configured such that one of the plurality of gating units is activatable to regulate the expression and / or activity level of TBX6 upon activation of the heterologous genetic circuit. In some cases, the first gating unit is activatable to regulate the expression and / or activity level of TBX6. Alternatively or additionally, the second gating unit is activatable to regulate the expression and / or activity level of TBX6. Alternatively or additionally, the gating unit is activated before the first gating unit to regulate the expression and / or activity level of TBX6. Alternatively or additionally, the gating unit is activated between the first gating unit and the second gating unit to regulate the expression and / or activity level of TBX6.

[0126] In some cases, one or more target genes may include basic helix-loop-helix transcription factors (bHLH genes). bHLH transcription factors are involved in regulating the cell cycle and many other developmental processes. bHLH proteins have a basic helix-loop-helix protein structure. Non-limiting examples of bHLH transcription factors include AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATOH1, ATOH7, ATOH8, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, EPAS1, FERD3L, FIGLA, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2, HIF1A, ID1, ID2, ID3, ID4, KIAA2018, LYL1, MASH1, MATH2, MAX, MESP1, MESP2, MIST1, MITF, MLX, MLXIP, MLXIPL, MNT, MSC, MSGN1, MXD1, MXD3 , MXD4, MXI1, MYC, MYCL1, MYCN, MYF5, MYF6, MYOD1, MYOG, NCOA1, NCOA3, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, NHLH1, NHLH2, NPAS1, NPAS2, NPAS3, NPAS4, OAF1, OLIG1, OLIG2, OLIG3, PTF1A, SCL, SCXB, SIM1, SIM2, SOHLH1, SOHLH2, SREBF1, SREBF2, TAL1, TAL2, TCF12, TCF15, TCF21, TCF3, TCF4, TCFL5, TFAP4, TFE3, TFEB, TFEC, TWIST1, TWIST2, USF1, and USF2.

[0127] In some cases, the plurality of gate units are pre-configured such that one of the plurality of gate units is activatable to regulate the expression and / or activity level of a bHLH gene upon activation of the heterologous genetic circuit. In some cases, the first gate unit is activatable to regulate the expression and / or activity level of a bHLH gene. Alternatively or additionally, the second gate unit is activatable to regulate the expression and / or activity level of a bHLH gene. Alternatively or additionally, the gate unit is activated before the first gate unit to regulate the expression and / or activity level of the bHLH gene. Alternatively or additionally, the gate unit is activated between the first gate unit and the second gate unit to regulate the expression and / or activity level of the bHLH gene.

[0128] In some cases, the plurality of gating units are pre-configured such that one of the plurality of gating units is activatable to regulate the expression and / or activity level of SCL upon activation of the heterologous genetic circuit. In some cases, the first gating unit is activatable to regulate the expression and / or activity level of SCL. Alternatively or additionally, the second gating unit is activatable to regulate the expression and / or activity level of SCL. Alternatively or additionally, a gating unit is activated before the first gating unit to regulate the expression and / or activity level of SCL. Alternatively or additionally, a gating unit is activated between the first gating unit and the second gating unit to regulate the expression and / or activity level of SCL.

[0129] In some cases, one or more target genes may comprise SRY-related box transcription factor (SOX gene).SOX transcription factor is involved in developmental regulation.Non-limiting examples of SOX transcription factor may include SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21, SOX30 and SRY.

[0130] In some cases, one or more target genes may include SOX group A, which includes SRY. In some cases, one or more target genes may include SOX group B1, which includes SOX1, SOX2, and / or SOX3. In some cases, one or more target genes may include SOX group B2, which includes SOX14 and / or SOX21. In some cases, one or more target genes may include SOX group C, which includes SOX4, SOX11, and / or SOX12. In some cases, one or more target genes may include SOX group D, which includes SOX5, SOX6, and / or SOX13. In some cases, one or more target genes may include SOX group E, which includes SOX8, SOX9, and / or SOX10. In some cases, one or more target genes may include SOX group F, which includes SOX7, SOX17, and / or SOX18. In some cases, one or more target genes may include SOX group G, which includes SOX15. In some cases, the one or more target genes may include SOX group H, which includes SOX30.

[0131] In some cases, one or more target genes may comprise Forkhead Box (FOX). FOX is a transcription factor that plays a role in regulating the expression of genes involved in cell growth, proliferation, differentiation and life span. Some FOX genes can bind to chromatin during cell differentiation process. Non-limiting examples of FOX genes may include FOXA, FOXB, FOXC, FOXD, FOXE, FOXF, FOXG, FOXH, FOXI, FOXJ, FOXK, FOXL, FOXM, FOXN, FOXO, FOXP, FOXQ, FOXR and FOXS.

[0132] In some cases, the one or more target genes may include erythroblast transformation specific (ETS) genes, which are transcription factors unique to animals and involved in tissue development. Non-limiting examples of ETS genes can include ELF1, ELF2 (NERF), ELF4 (MEF), GABPα, ERG, FLI1, FEV, ERF (PE2), ETV3 (PE1), ELF3 (ESE1 / ESX), ELF5 (ESE2), ESE3 (EHF), ETS1, ETS2, SPDEF (PDEF / PSE), ETV4 (PEA3 / E1AF), ETV5 (ERM), ETV1 (ER81), ETV2 (ER71), SPI1 (PU.1), SPIB, SPIC, ELK1, ELK4 (SAP1), ELK3 (NET / SAP2), ETV6 (TEL), and ETV7 (TEL2).

[0133] In some cases, one or more target genes may include collagen. Collagen is a fibrous protein and is a major component of skin, bone, tendon, cartilage, blood vessels, and teeth. Collagen forms insoluble fibers with high tensile strength. Non-limiting examples of collagen genes include COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, COL6A6, COL7A1, COL8A1, COL COL1A1, COL1A2, COL1A1, COL1B1, COL1C1, COL1D1, COL1E1, COL1E2, COL1F1, COL1F2, COL1F3, COL1F4, COL1F5, COL1F6, COL1F7, COL1F8, COL1F9, COL1F10, COL1F11, COL1F12, COL1F13, COL1F14, COL1F15, COL1F16, COL1F18, COL1F19, COL20, COL21, COL22, COL23, COL24, COL25, COL26, COL27, and COL28.

[0134] In some cases, one or more target genes may comprise a homeobox gene. Homeobox genes are genes that regulate large-scale anatomical features, for example, in the early stages of embryonic development. Types of homeobox genes include HOX genes, LIM genes, PAX genes, POU genes, CERS genes, HNF genes, SINE genes, CUT genes, ZF genes, paraHOX genes, DLX genes, TALE genes, PRD genes, and NKL genes. Non-limiting examples of homeobox genes include HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXB13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HO XC12, HOXC13, HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13, CDX1, CDX2, CDX4, GSX1, GSX2, PDX 1, EVX1, EVX2, GBX1, GBX2, MEOX1, MEOX2, MNX1, DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, IRX1, IRX2, IRX3, IRX4, IRX5, IRX6 , MEIS1, MEIS2, MEIS3, MKX, PBX1, PBX2, PBX3, PBX4, PKNOX1, PKNOX2, TGIF1, TGIF2, TGIF2LX, TGIF2LY, ISL1, ISL2, LHX 1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1A, LMX1B, HDX, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2, POU 3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1, POU5F1P1, POU5F2, POU6F1, POU6F2, LAS2, LAS3, LAS5, LAS6, HMBOX1, HM BOX1, HNF1A, HNF1B, SIX1, SIX2, SIX3, SIX4, SIX5, SIX6, ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2, SATB1, SATB2, ADNP,ADNP2, TSHZ1, TSHZ2, TSHZ3, ZEB1, ZEB2, ZFHX2, ZFHX3, ZFHX4, ZHX1, HOMEZ, ALX1(CART1), A LX3, ALX4, ARGFX, ARX, DMBX1, DPRX, DRGX, DUXA, DUXBDUX(1, 2, 3, 4, 4c, 5), ESX1, GSC, GSC2, HESX1, HOPX, ISX, LEUTX, MIXL1, NOBOX, OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRRX1, PRRX2, RAX, RAX2, RHOXF1 , RHOXF2 / 2B, SEBOX, SHOX, SHOX2, TPRX1, UNCX, VSX1, VSX2, BARHL1, BARHL2, BARX1, BARX2, BSX, DBX1, DBX2, EMX1, EMX2, EN1, EN2, HHEX, HLX1, LBX1, LBX2, MSX1, MSX2, NANOG, NOTO, TLX1, TLX2, TLX3, TSHZ1, TSHZ2, TSHZ3, VAX1, VAX2, VENTX, NKX2-1NKX2-4, NKX2-2, NKX2-8, NKX3-1, NKX3-2, NKX2-3, NKX2-5, NKX2-6, HMX1, HMX2, HMX3, NKX6-1, NKX6-2, and NKX6-3. In some cases, one or more target genes may not include a homeobox gene (e.g., MIXL1).

[0135] In some cases, the plurality of gating units are pre-configured such that one of the plurality of gating units is activatable to regulate the expression and / or activity level of a homeobox gene upon activation of the heterologous genetic circuit. In some cases, the first gating unit is activatable to regulate the expression and / or activity level of a homeobox gene. Alternatively or additionally, the second gating unit is activatable to regulate the expression and / or activity level of a homeobox gene. Alternatively or additionally, a gating unit is activated before the first gating unit to regulate the expression and / or activity level of a homeobox gene. Alternatively or additionally, a gating unit is activated between the first gating unit and the second gating unit to regulate the expression and / or activity level of a homeobox gene.

[0136] In some cases, the plurality of gate units are pre-configured such that one of the plurality of gate units is activatable to regulate the expression and / or activity level of MIXL1 upon activation of the heterologous genetic circuit. In some cases, the first gate unit is activatable to regulate the expression and / or activity level of MIXL1. Alternatively or additionally, the second gate unit is activatable to regulate the expression and / or activity level of MIXL1. Alternatively or additionally, the gate unit is activated before the first gate unit to regulate the expression and / or activity level of MIXL1. Alternatively or additionally, the gate unit is activated between the first gate unit and the second gate unit to regulate the expression and / or activity level of MIXL1.

[0137] In some cases, one or more target genes may include GATA genes. GATA genes are transcription factors characterized by their ability to bind to the DNA sequence "GATA". Non-limiting examples of GATA genes may include GATA1, GATA2, GATA3, GATA4, GATA5, and GATA6.

[0138] In some cases, the plurality of gating units are pre-configured such that one of the plurality of gating units is activatable to regulate the expression and / or activity level of a GATA gene upon activation of the heterologous genetic circuit. In some cases, the first gating unit is activatable to regulate the expression and / or activity level of a GATA gene. Alternatively or additionally, the second gating unit is activatable to regulate the expression and / or activity level of a GATA gene. Alternatively or additionally, the gating unit is activated before the first gating unit to regulate the expression and / or activity level of the GATA gene. Alternatively or additionally, the gating unit is activated between the first gating unit and the second gating unit to regulate the expression and / or activity level of the GATA gene.

[0139] In some cases, the plurality of gating units are pre-configured such that one of the plurality of gating units is activatable to regulate the expression and / or activity level of GATA2 upon activation of the heterologous genetic circuit. In some cases, the first gating unit is activatable to regulate the expression and / or activity level of GATA2. Alternatively or additionally, the second gating unit is GATA2. Alternatively or additionally, a gating unit is activated before the first gating unit to regulate the expression and / or activity level of GATA2. Alternatively or additionally, a gating unit is activated between the first gating unit and the second gating unit to regulate the expression and / or activity level of GATA2.

[0140] In some cases, the guide nucleic acid (gNA) molecules provided herein can be configured to bind to a target gene (e.g., a coding or non-coding region of a target gene) and regulate the expression and / or activity of the target gene. The gNA molecule can include a polynucleotide sequence (e.g., a spacer sequence) that exhibits specific binding to a target polynucleotide sequence of the target gene. The polynucleotide sequence of the gNA molecule may exhibit at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 1-112 (e.g., any one of SEQ ID NOs: 13-16 for targeting ETS1). The polynucleotide sequence of the gNA molecule that binds to the target gene may contain at least or up to about 10 nucleotides, at least or up to about 11 nucleotides, at least or up to about 12 nucleotides, at least or up to about 13 nucleotides, at least or up to about 14 nucleotides, at least or up to about 15 nucleotides, at least or up to about 16 nucleotides, at least or up to about 17 nucleotides, at least or up to about 18 nucleotides, at least or up to about 19 nucleotides, at least or up to about 20 nucleotides, at least or up to about 21 nucleotides, at least or up to about 22 nucleotides, at least or up to about 23 nucleotides, at least or up to about 24 nucleotides, or at least or up to about 35 nucleotides.

[0141] In some cases, use of the heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), red myeloid progenitor cells (EMPs), mesodermal progenitor cells, mesodermal stem cells) into hematopoietic cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells), such that at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the resulting cells generated by using the heterologous genetic circuits disclosed herein are hematopoietic cells.

[0142] In some cases, use of the heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), red myeloid progenitor cells (EMPs), mesodermal progenitor cells, mesodermal stem cells) into hematopoietic cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells), where at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the resulting cells generated by using the heterologous genetic circuits disclosed herein are of the target cell type. In some embodiments, the target cells can be confirmed by characterizing one or more cell surface markers, which may include KDR, CD34, CD43, CD45, and CD309.

[0143] In some cases, use of the heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), mesenchymal progenitor cells) into hematopoietic cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells) in the absence of, for example, one, two, or all of feeder cells, serum, and one or more exogenous factors (e.g., one or more exogenous growth factors). In some cases, one or more exogenous factors may be required to otherwise effect differentiation of stem cells into hematopoietic cells in the absence of a heterologous genetic circuit as provided herein. Non-limiting examples of the one or more exogenous factors may include bone morphogenetic protein (BMP), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase receptor ligand (FLTL), and / or interleukin (IL).

[0144] Non-limiting examples of BMPs may include BMP1, BMP1b, BMP2, BMP2A, BMP2B, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, and BMP8B. In some embodiments, the BMP comprises BMP4. Non-limiting examples of VEGFs may include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF. Non-limiting examples of FGFs may include FGF1, FGF2 (basic FGF or bFGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. Non-limiting examples of FLTLs may include FLTL-1, FLTL-2, FLTL-3, and FLTL-4. Non-limiting examples of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL- 19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36.

[0145] The use of the heterologous genetic circuits disclosed herein can produce up to about 1×10 6 , up to about 9×10 5 , up to about 8 × 10 6 , up to about 7 × 10 6 , up to about 6 × 10 6 , up to about 5 × 10 6 , up to about 4 × 10 6 , up to about 3 × 10 6 , up to about 2 × 10 6 , up to about 1 × 10 6 , up to about 5 × 10 4 , up to about 2 × 10 4 , up to about 1 × 10 4 , or fewer pluripotent stem cells (e.g., human PSCs), at least about 1 × 10 4, at least about 2 × 10 4 , at least about 5 × 10 4 , at least about 1 x 10 6 , at least about 2 × 10 6 , at least about 5 × 10 6 , at least about 1 x 10 6 , at least about 2 × 10 6 , at least about 5 × 10 6 , at least about 1 x 10 7 , at least about 2 × 10 7 , at least about 5 × 10 7 , at least about 1 x 10 8 , at least about 2 × 10 8 , at least about 5 × 10 8 , at least about 1 × 10, at least about 2 × 10 9 , at least about 5 × 10 9 , at least about 1 x 10 10 , at least about 2 × 10 10 , at least about 5 × 10 10 , at least about 1 x 10 15 , at least about 2 × 10 15 , at least about 5 × 10 15 or more hematopoietic cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells).

[0146] Such generation of hematopoietic cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells) by using the heterologous gene circuits disclosed herein can be accomplished within a span of up to about 60 days, up to about 55 days, up to about 50 days, up to about 45 days, up to about 40 days, up to about 35 days, up to about 30 days, up to about 25 days, up to about 20 days, up to about 15 days, up to about 10 days, up to about 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, or less. When injected into a mouse, the resulting hematopoietic cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells) generated by using the heterologous genetic circuits disclosed herein can extend the mouse lifespan by 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% or more.

[0147] In some cases, the plurality of first type cells can be converted into a plurality of second type cells within at most about 20 days, at most about 19 days, at most about 18 days, at most about 17 days, at most about 16 days, at most about 15 days, at most about 14 days, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, or at most about 1 day after culturing (e.g., ex vivo) the stem cells. In some cases, the rate of conversion of a plurality of first type cells to a plurality of second type cells is at least or at most about 1%, at least or at most about 2%, at least or at most about 3%, at least or at most about 4%, at least or at most about 5%, at least or at most about 6%, at least or at most about 7%, at least or at most about 8%, at least or at most about 9%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 40%, at least or at most about 45%, at least or at most about 50%, at least or at most about 5 ... It 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 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%.

[0148] In some cases, the plurality of stem cells can be converted into a plurality of hematopoietic cells within at most about 20 days, at most about 19 days, at most about 18 days, at most about 17 days, at most about 16 days, at most about 15 days, at most about 14 days, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, or within about 1 day after culturing the stem cells (e.g., ex vivo). In some cases, the conversion rate of the plurality of stem cells to the plurality of hematopoietic cells is at least or at most about 1%, at least or at most about 2%, at least or at most about 3%, at least or at most about 4%, at least or at most about 5%, at least or at most about 6%, at least or at most about 7%, at least or at most about 8%, at least or at most about 9%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 40%, at least or at most about 45%, It 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 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 about 100%.

[0149] In some cases, the plurality of stem cells can be converted into a plurality of hematopoietic cells within at most about 20 days, at most about 19 days, at most about 18 days, at most about 17 days, at most about 16 days, at most about 15 days, at most about 14 days, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, or within about 1 day after culturing the stem cells (e.g., ex vivo), and the plurality of hematopoietic cells are CD45+ (e.g., CD34+CD43−CD45+). In some cases, the conversion rate of a plurality of stem cells to a plurality of hematopoietic cells may be at least or at most about 1%, at least or at most about 2%, at least or at most about 3%, at least or at most about 4%, at least or at most about 5%, at least or at most about 6%, at least or at most about 7%, at least or at most about 8%, at least or at most about 9%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 35%, at least or at most about 40%, at least or at most about 45%, at least or at most about 50%, or at least 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 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 about 100%, wherein the plurality of hematopoietic cells are CD45+ (e.g., CD34+CD43-CD45+).

[0150] In some cases, the plurality of stem cells can be converted into a plurality of hematopoietic cells within at most about 20 days, at most about 19 days, at most about 18 days, at most about 17 days, at most about 16 days, at most about 15 days, at most about 14 days, at most about 13 days, at most about 12 days, at most about 11 days, at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, or within about 1 day after culturing the stem cells (e.g., ex vivo), and the plurality of hematopoietic cells are CD34+. In some cases, the conversion rate of the plurality of stem cells to the plurality of hematopoietic cells that are CD34+ is 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%, 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 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 about 100%.

[0151] In some cases, the use of heterologous genetic circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythroid-myeloid progenitor cells (EMPs), mesenchymal progenitor cells) can induce the stem cells to differentiate into hematopoietic progenitor cells in the absence of feeder cells. In some cases, the use of heterologous genetic circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythroid-myeloid progenitor cells (EMPs), mesenchymal progenitor cells) can induce the stem cells to differentiate into hematopoietic progenitor cells in the absence of serum. In some cases, the use of heterologous genetic circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythroid-myeloid progenitor cells (EMPs), mesenchymal progenitor cells) can induce the stem cells to differentiate into hematopoietic progenitor cells in the absence of one or more exogenous factors (e.g., one or more exogenous growth factors). In some cases, the use of the heterologous genetic circuits disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythromyeloid progenitor cells (EMPs), mesenchymal progenitor cells) into hematopoietic progenitor cells, e.g., in the absence of embryoid bodies and / or serum. The resulting hematopoietic progenitor cells generated by using the heterologous genetic circuits disclosed herein are at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total cell population obtained.

[0152] In some cases, the target gene can be subjected to at least two separate regulation steps, including a first regulation step and a second regulation step.The timing of the first regulation step and the second regulation step can be controlled (for example, as predetermined by the design of the heterologous genetic circuit).For example, the start of the second regulation step (for example, by at least a portion of the second gate unit, such as the second gene regulation part) can be 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 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 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.Initiation of the second regulation (e.g., by at least a portion of a second gating unit, such as a second gene regulatory portion) 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, after initiation of the first regulation (e.g., by at least a portion of a first gating unit, such as a first gene regulatory portion). The time may be 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.

[0153] In some cases, the number of gate units that need to be activated (e.g., sequentially 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 (e.g., substantially) determine the timing between the first regulation and the second regulation. Upon activation of the first regulation of the target gene by the first gate unit, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more additional gate units may need to be activated (e.g., sequentially activated), thereby activating the second gate unit to induce the second regulation. Upon activation of the first regulation of the target gene by the first gating unit, it may be necessary to activate (e.g., sequentially 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 gating units, thereby activating the second gating unit to induce the second regulation.

[0154] The cellular outcome can include the regulation of multiple target genes. For example, the outcome can include the regulation of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more target genes. The outcome can include the regulation of up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 target gene. Each gene disclosed herein may be subject to at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more modulations. Each gene disclosed herein may be subject to up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 modulations. The regulation of one or more target genes (e.g., endogenous genes) as induced by a heterologous gene circuit of the present disclosure can be artificial regulation (or heterologous regulation) that would not otherwise occur in the cell in the absence of (i) the heterologous gene circuit and / or (ii) the activating portion of the heterologous gene circuit.

[0155] The multiple gate units may operate sequentially (e.g., each of the multiple gate units may be activated sequentially). For example, multiple gate units may be activated to activate multiple subsequent gate units. The sequential operation of the gate units may be linear. Alternatively, the sequential operation of the gate units may route back to each other as inputs to form a loop. For example, the multiple gate units may induce a feedback loop, such as a positive feedback loop or a negative feedback loop.

[0156] In some embodiments of any of the systems disclosed herein, the first gating unit may include a first gene regulatory portion that can be activated to exhibit specific binding to a target gene to induce a first distinct regulation. Alternatively, or in addition, the first gating unit may include a first gene regulatory portion that can be activated to exhibit non-specific binding to a target gene to induce a first distinct regulation.

[0157] The first distinct modulation may induce a change (e.g., an increase or decrease) in the expression and / or activity level of the target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. The first distinct modulation of the target gene expression and / or activity level can be up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8% , up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, up to about 0.1%, or less.

[0158] The first distinct modulation (e.g., induced by a first gating unit) disclosed herein can be 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, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or at least about 11-fold, or at least about 12-fold, or at least about 13-fold, or at least about 14-fold, or at least about 15-fold, or at least about 16-fold, or at least about 17-fold, or at least about 18-fold, or at least about 19-fold, or at least about 20-fold, or at least about 21-fold, or at least about 22-fold, or at least about 23-fold, or at least about 24-fold, or at least about 25-fold, or at least about 26-fold, or at least about 27-fold, or at least about 28-fold, or at least about 29-fold, or at least about 30-fold, or at least about 31-fold, or at least about 32-fold, or at least about 33-fold, or at least about 34-fold, or at least about 35-fold, or at least about 36-fold, or at least about 37-fold, The present invention may induce a change (e.g., an increase or decrease) of at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.The first distinct modulation can be up to or less than about 10,000 fold, up to or less than about 5,000 fold, up to or less than about 1,000 fold, up to or less than about 500 fold, up to or less than about 100 fold, up to or less than about 90 fold, up to or less than about 80 fold, up to or less than about 70 fold, up to or less than about 60 fold, up to or less than about 50 fold, up to or less than about 40 fold, up to or less than about 30 fold, up to or less than 20 fold, up to or less than 10 fold, A change (e.g., an increase or decrease) of up to or less than 9-fold, up to or less than 8-fold, up to or less than 7-fold, up to or less than 6-fold, up to or less than about 5-fold, up to or less than 4-fold, up to or less than about 3-fold, up to or less than about 2-fold, up to or less than about 1-fold, up to or less than about 0.9-fold, up to or less than about 0.8-fold, up to or less than about 0.7-fold, up to or less than about 0.6-fold, up to or less than about 0.5-fold, up to or less than about 0.4-fold, up to or less than about 0.3-fold, up to or less than about 0.2-fold, or up to or less than about 0.1-fold may be induced.

[0159] Subsequently, a second separate regulation (e.g., induced by a second gating unit) as disclosed herein may result in 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 75%, at least about 80%, at least about 95%, at least about 10 ... Further alterations (e.g., increases, decreases, or selective attenuation) of 0%, 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% may be induced.The second, distinct modulation of the target gene expression and / or activity level can be 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 A further change (e.g., an increase or decrease) of 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% may be induced.

[0160] Further alterations via the second, separate modulation can include an increase in the expression and / or activity level of the target gene by at least or up to about 0.1 fold, at least or up to about 0.2 fold, at least or up to about 0.3 fold, at least or up to about 0.4 fold, at least or up to about 0.5 fold, at least or up to about 0.6 fold, at least or up to about 0.7 fold, at least or up to about 0.8 fold, at least or up to about 0.9 fold, at least or up to about 1 fold, at least or up to about 2 fold, at least or up to about 3 fold, at least or up to about 4 fold, at least or up to about 5 fold, at least or up to about 6 fold, or at least Further changes (e.g., increases or decreases) of at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold can be induced.The second, distinct modulation can be up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, up to or less than about 10-fold, up to or less than about 10-fold, up to or less than about 10,0 ... Or, a further change (e.g., an increase or decrease) of less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5-fold, at most or less than about 4-fold, at most or less than about 3-fold, at most or less than about 2-fold, at most or less than about 1-fold, at most or less than about 0.9-fold, at most or less than about 0.8-fold, at most or less than about 0.7-fold, at most or less than about 0.6-fold, at most or less than about 0.5-fold, at most or less than about 0.4-fold, at most or less than about 0.3-fold, at most or less than about 0.2-fold, or at most or less than about 0.1-fold may be induced.

[0161] Further changes via a second distinct regulation can occur when the expression and / or activity level of the target gene reaches a target level through the action of a first distinct regulation, for example, by design of a heterologous genetic circuit.

[0162] The further change via the second distinct modulation may be such that the expression and / or activity level of the target gene is increased by at least or at most about 0.1 fold, at least or at most about 0.2 fold, at least or at most about 0.3 fold, at least or at most about 0.4 fold, at least or at most about 0.5 fold, at least or at most about 0.6 fold, at least or at most about 0.7 fold, at least or at most about 0.8 fold, at least or at most about 0.9 fold, at least or at most about 1 fold, at least or at most about 2 fold, at least or at most about 3 fold, at least or at most about 4 fold, at least or at most about 5 fold, at least or at most about 6 fold, at least or at most about 7 fold, at least or at most about 8 fold, at least or at most about 9 fold, at least or at most about 10 fold, at least or at most about 11 fold, at least or at most about 12 fold, at least or at most about 13 fold, at least or at most about 14 fold, at least or at most about 15 fold, at least or at most about 16 fold, at least or at most about 17 fold, at least or at most about 18 fold, at least or at most about 19 fold, at least or at most about 20 fold, at least or at most about 21 fold, at least or at most about 22 fold, at least or at most about 23 fold, at least or at most about 24 fold, at least or at most about 25 fold, at least or at most about 26 fold, at least or at most about 27 fold, at least or at most about 28 fold, at least or at most about 29 fold, at least or at most about 30 fold, at least or at most about 31 fold, at least or at most This may occur when the change (e.g., increase or decrease) is at least about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.The further change via the second distinct modulation may be that the expression and / or activity level of the target gene is increased by at most or less than about 10,000 fold, at most or less than about 5,000 fold, at most or less than about 1,000 fold, at most or less than about 500 fold, at most or less than about 100 fold, at most or less than about 90 fold, at most or less than about 80 fold, at most or less than about 70 fold, at most or less than about 60 fold, at most or less than about 50 fold, at most or less than about 40 fold, at most or less than about 30 fold, at most or less than about 20 fold, at most or less than about or may change (e.g., increase or decrease) by less than about 10-fold, at most or less than about 9-fold, at most or less than about 8-fold, at most or less than about 7-fold, at most or less than about 6-fold, at most or less than about 5-fold, at most or less than about 4-fold, at most or less than about 3-fold, at most or less than about 2-fold, at most or less than about 1-fold, at most or less than about 0.9-fold, at most or less than about 0.8-fold, at most or less than about 0.7-fold, at most or less than about 0.6-fold, at most or less than about 0.5-fold, at most or less than about 0.4-fold, at most or less than about 0.3-fold, at most or less than about 0.2-fold, or at most or less than about 0.1-fold.

[0163] Alternatively, or in addition, a second separate regulation (e.g., induced by a second gating unit) as disclosed herein may decrease the expression and / or activity level of the additional target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 95%, at least about 100%, at least about 105%, at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 135%, at least about 140%, at least about 145%, at least about 150%, at least about 155%, at least about 160%, at least about 165%, at least about 170%, at least about 175%, at least about 180%, at least about 185%, at least about 190%, at least about 205%, at least about 210%, at least about 215%, at least about 220%, at least about 225%, at least about 230%, at least about 235%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 305%, at least about 310 A change (e.g., an increase or decrease) of 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% may be induced.The second, separate modulation of the expression and / or activity level of the additional target gene can be up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, or , up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, or up to about 0.1% change (e.g., increase or decrease). The cell can include a prokaryotic cell, a eukaryotic cell, or an artificial cell.

[0164] In some embodiments, the second plurality comprises hemogenic endothelial cells that are KDR- and CD34+, and optionally, within about 14 days or about 7 days after contact, the conversion rate of the first plurality to the second plurality is at least or at most about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the conversion rate of the first plurality of second plurality via the heterologous gene circuit is at least or at most about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater than the conversion rate in the absence of the heterologous gene circuit.

[0165] In some embodiments, the second plurality comprises hematopoietic cells (HPCs) that are CD34+ and CD43+, and optionally, within about 14 days or about 7 days after contacting, the conversion rate of the first plurality to the second plurality is at least or at most about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the rate of conversion of the first plurality to the second plurality via the heterologous gene circuit is at least or at most about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater than the rate of conversion in the absence of the heterologous gene circuit.

[0166] In some embodiments, the second plurality comprises hematopoietic cells (HPCs) that are CD34+ and CD45+, and optionally, within about 14 days or about 7 days after contacting, the conversion rate of the first plurality to the second plurality is at least or at most about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the rate of conversion of the first plurality to the second plurality via the heterologous gene circuit is at least or at most about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater than the rate of conversion in the absence of the heterologous gene circuit.

[0167] The cells (e.g., initial cells that are modified into the engineered cells disclosed herein, final cell products produced from the engineered cells disclosed herein, etc.) can include muscle cells, immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells, epithelial cells, stem cells, secretory cells, blood cells, germ cells, nurse cells, storage cells, enteroendocrine cells, pituitary cells, neurosecretory cells, duct cells, odontoblasts, cementoblasts, glial cells, or stromal cells.

[0168] Non-limiting examples of such cells include lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, and cytokine-induced killer (CIK) cells (see, e.g., US20080241194), myeloid cells such as granulocytes (basophils, eosinophil granulocytes, neutrophils / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, and dendritic cells, thyroid gland (thyroid epithelial cells, parafollicular cells), parathyroid gland (parathyroid chief cells, oxyphil cells), and the like. Cells from the endocrine system, including cells of the adrenal gland (chromaffin cells), pineal gland (pineal cells), glial cells (astrocytes, microglia), magnocellular neurosecretory cells, stellate cells, Boettcher cells, and pituitary gland (gonadotropes, corticotropes, thyrotropes, somatotropes, lactotropes), cells of the nervous system, including pneumocytes (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells, and Cells of the respiratory system, including myocardiocytes, cells of the circulatory system, including pericytes, cells of the digestive system, including stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, and S cells, enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells),Enteroendocrine cells, including cartilage / bone / muscle cells, osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts), cartilage cells, including chondroblasts and chondrocytes, skin cells, including trichocytes, keratinocytes, and melanocytes (nevus cells), muscle cells, including myocytes, podocytes, juxtaglomerular cells, intraglomerular / extraglomerular mesangial cells, and kidney proximal tubule brush border cells. These cells include urinary system cells, including proximal tubule brush border cells and Macula densa cells, germ line cells, including spermatozoon, Sertoli cells, Leydig cells, and ovum, as well as adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiating epidermal cells), epidermal basal cells (stem cells), keratinocytes of the fingernails and toenails, nail bed basal cells (stem cells), medullary hair shaft cells, cortical hair shaft cells, cuticular hair shaft cells, and cuticular hair root sheath cells. cell), hair root sheath cells in Huxley's layercells of Huxley's layer, hair root sheath cells of Henle's layer, external 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, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina (stem cells), urinary epithelium cells (lining the urinary bladder and urinary ducts), exocrine secretory epithelial cells, salivary gland mucous salivary gland serous cells (polysaccharide-rich secretion), salivary gland serous cells (glycoprotein enzyme-rich secretion), Von Ebner's gland cells in the tongue (cleansing the taste buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), ceruminous gland cells in the ear (wax secretion), eccrine sweat gland dark cells (glycoprotein secretion), eccrine sweat gland clear cells (small molecule secretion)Apocrine sweat gland cells (odor secretion, sex-hormone sensitive), Gland of Moll cells in the eyelid (specialized sweat gland), Sebaceous gland cells (lipid-rich sebum secretion), Bowman's gland cells in the nose (cleansing the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), Seminal vesicle cells (secreting seminal fluid components, including fructose for sperm swimming), Prostate gland cells cells (secreting seminal fluid components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubricant secretion), gland of Littre cells (mucus secretion), endometrium cells (carbohydrate secretion), isolated goblet cells of the respiratory and digestive systems (mucus secretion), stomach lining mucous cells (mucus secretion), gastric gland zymogenic cells (pepsinogen secretion), gastric gland oxyntic cells (hydrochloric acid secretion), pancreatic gland cells Paneth cells in the small intestine (secreting bicarbonate and digestive enzymes), type II pneumocytes in the lungs (secreting surfactant), and Clara cells in the lungs.cells, hormone-secreting cells, anterior pituitary cells, somatotrophs, lactotrophs, thyrotrophs, gonadotrophs, corticotrophs, intermediate pituitary cells, magnocellular neurosecretory cells, intestinal and respiratory system cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxyphil cells, adrenal cells, chromaffin cells, Leydig cells of the testes, theca interna cells of the ovarian follicle, lutein cells of the ruptured follicle, granulosa cells lutein cells, theca lutein cells, juxtaglomerular cells (renin secretion), Macula densa cells, metabolic and storage cells, barrier function cells (lungs, gastrointestinal tract, exocrine glands, and urogenital tract), kidney, immature beta cells, mature beta cells, type I pneumocytes (lining the air spaces of the lungs), pancreatic duct cells (centroacinar cells), non-muscularized duct cells (sweat glands, salivary glands, mammary glands, etc.), bile duct cells, duct cells (seminal vesicles, prostate, etc.), epithelial cells lining closed internal body cavities, ciliated cells with propulsive function, extracellular matrix secreting cells, contractile cellscells), skeletal muscle cells, stem cells, cardiac muscle cells, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglial cells (in the central nervous system), neutrophil granulocytes (N) Eutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural killer T cell, B cell, Natural killer cell, Reticulocyte, Stem cells and committed progenitors for the blood and immune system (various types), Pluripotent stem cells, Totipotent stem cells, Induced pluripotent stem cells, Adult stem cells, Sensory transducer cells, Autonomic neuron cells, Sense organ and peripheral neuron supporting cells, Central nervous system neurons and glial cells Other cells include: lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonium / oocyte, spermatid, spermatid, spermatogonium cell (stem cell of sperm), spermatozoon, nurse cells, ovarian follicular cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, and interstitial kidney cells.

[0169] Stem cells are induced pluripotent stem cells (iPSC), pluripotent stem cells (PSC), embryonic stem cells (ESC), mesenchymal stem cells (MSC), erythro-myeloid progenitor cells (EMP), mesenchymal progenitor cells, hematopoietic stem cells (HSC), hematopoietic progenitor cells, fascial stem cells, neural stem cells, epithelial stem cells, epidermal stem cells, mammary stem cells, intestinal stem cells, neural crest stem cells, and testicular stem cells.

[0170] Stem cells (e.g., pluripotent stem cells) provided herein can be engineered to disrupt expression of a T cell receptor (TCR) (e.g., TCR alpha, TCR beta, CD3 gamma, CD3 delta, CD3 epsilon, and / or CD247) and / or a major histocompatibility complex (MHC) (e.g., MHC class I, MHC class II, and / or MHC class III, where human MHC may be referred to as human leukocyte antigen (HLA)). For example, a gene encoding at least a portion of the TCR of the MHC can be edited (e.g., mutated by non-homologous end joining (NHEJ) or homologous recombination (HR)) to disrupt expression. Upon administration of the differentiated cells provided herein to a subject in need thereof, disruption of TCR and / or MHC expression can reduce or eliminate (i) graft-versus-host disease (GvHD) and / or (ii) host-versus-graft disease (HvGD). For example, disruption of TCR and / or MHC expression can reduce or eliminate the effects of administering the differentiated cells as allogeneic cells (e.g., allogeneic T cells) to an HLA-mismatched (or partially mismatched) subject. Alternatively, the differentiated cells can be administered as allogeneic cells to an HLA-matched subject (e.g., without disruption of TCR and / or MHC expression).

[0171] Disruption of TCR and / or MHC expression can occur before, simultaneously with, or after conversion of a plurality of stem cells (e.g., pluripotent stem cells) into hematopoietic cells (e.g., hematopoietic stem cells). Disruption of TCR and / or MHC expression can occur before, simultaneously with, or after introduction (e.g., expression) of a heterologous genetic circuit into a plurality of stem cells. Disruption of TCR and / or MHC expression can occur before, simultaneously with, or after activation of a heterologous genetic circuit in a plurality of stem cells. In some cases, disruption of TCR and / or MHC expression can occur subsequent to generation of hematopoietic cells.

[0172] Various embodiments of the present disclosure provide engineered cells that are programmed to induce a desired expression and / or activity level (or profile) of one or more target genes in a cell.

[0173] In some embodiments, the engineered cells (e.g., engineered hematopoietic progenitor cells, engineered immune cells) of the present disclosure can be generated from isolated stem cells (e.g., isolated ESCs, iPSCs, EMPs). The heterologous gene circuits and / or components thereof (e.g., gating units, gating moieties, activating moieties, etc.) disclosed herein can be introduced into any stage (or cell state) between (a) the isolated stem cell and (b) its differentiated hematopoietic progenitor cell state.

[0174] In some cases, the engineered cells, hematopoietic progenitor cells, can be derived from PSCs, and heterologous genetic circuits and / or components thereof (e.g., heterologous gating units, heterologous activation moieties, heterologous gating moieties, etc.) can be introduced into cells at (A) a PSC state, (B) a mesodermal stem cell state, (C) a hematopoietic progenitor cell state, or (D) any other intermediate cell state. The heterologous genetic circuits and / or components thereof (e.g., heterologous gating units, heterologous activation moieties, heterologous gating moieties, etc.) can be introduced into cells once during one of (A), (B), (C), or (D). Alternatively, the heterologous genetic circuits and / or components thereof (e.g., heterologous gating units, heterologous activation moieties, heterologous gating moieties, etc.) can be introduced into cells multiple times during two, three, or all of (A), (B), (C), or (D).

[0175] The genetically engineered cells (e.g., engineered hematopoietic progenitor cells) of the present disclosure can be used (e.g., administered) to treat a subject in need thereof. The subject can have or be suspected of having a condition such as a disease (e.g., cancer). Cells (e.g., stem cells or differentiated cells) can be obtained from the subject, and such cells can be cultured ex vivo and genetically modified to generate any of the subject engineered cells (e.g., any immune cell) disclosed herein. The engineered immune cells can then be administered to the subject for adaptive immunotherapy. Thus, the engineered cells can be autologous to the subject in need thereof. Alternatively, the engineered cells can be allogeneic to the subject (e.g., allogeneic stem cell transplantation, allogeneic adoptive immunotherapy, etc.).

[0176] The cells or constructs of the present disclosure may be used (e.g., administered) in a pharmaceutical preparation. The pharmaceutical preparation may further include an additional therapeutic agent. The additional therapeutic agent may include a chemotherapeutic agent, an immunosuppressant, and / or an antibiotic.

[0177] Chemotherapeutic agents, also known as antitumor agents, are a type of cancer treatment that is used to directly or indirectly inhibit the growth and proliferation of cancer cells. Chemotherapeutic agents can be alkylating agents (e.g., oxazaphosphorines, nitrogen mustards, imidazotetrazines, nitrosoureas, alkylsulfonates, hydrazines, or platinum-based agents), antimetabolites (e.g., antifolates, pyrimidine antagonists, purine antagonists, or ribonuclease reductase inhibitors), topoisomerase inhibitors (e.g., topoisomerase I inhibitors or topoisomerase II inhibitors), antibiotics (e.g., bleomycin, actinomycin D, anthracyclines, or mitomycin), mitotic inhibitors (e.g., vinca alkaloids, taxanes, or nontaxane microtubule inhibitors), protein kinase inhibitors (e.g., tyrosine kinase inhibitors, MEK inhibitors, CDK inhibitors), proteasome inhibitors, or PARP inhibitors.

[0178] Immunosuppressants are agents that reduce immune responses. Non-limiting examples of immunosuppressants can include steroids (e.g., prednisone, methylprednisolone, dexamethasone), colchicine, hydroxychloroquine, sulfasalazine, dapsone, methotrexate, mycophenolate mofetil, azathioprine, anti-IL-1 biologics, anti-TNF biologics, anti-IL6 biologics, B-cell growth factor-targeted biologics, T-cell, cytokine, or JAK inhibitors.

[0179] Antibiotics are drugs that destroy or inhibit the growth of microorganisms. Non-limiting examples of antibiotics include tetracycline, oxytetracycline, metacycline, doxycycline, minocycline, erythromycin, lincomycin, penicillin G, and the like.G), clindamycin, kanamycin, chloramphenicol, fradiomycin, streptomycin, norfloxacin, ciprofloxacin, ofloxacin, grepafloxacin, levofloxacin, Sparf Sparfloxacin, ampicillin, carbenicillin, methicillin, cephalosporins, vancomycin, bacitracin, gentamycin, sulfonamides, trimethoprim, dapsone, isoniazid iazid, teicoplanin, avoparcin, synarcid, virginiamycin, piperacillin, ticarcillin, cefepime, cefpirome, rifampicin, pyrazinamide, enrofloxacin, amikacin kacin), netilmycin (netilmycin), imipenem (imipenem), meropenem (meropenem), inezolid furoxime (inezolidcefuroxime), ceftriaxone (ceftriaxone), cefadroxil (cefadroxil), cefazolin (cefazoline), ceftazidime (ceftazidime), cefotaxime (cefotaxime), roxithromycin (roxithromycin), cefaclor (cefaclor),May include cefalexin, cefoxitin, amoxicillin, co-amoxiclav, mupirocin, cloxacillin, and co-trimoxazole.

[0180] The pharmaceutical formulation can further comprise an excipient, which can be a buffer, carrier, stabilizer, solubilizer, filler, preservative, diluent, vehicle, surfactant, salt, peptide, surfactant, oligosaccharide, amino acid, adjuvant, carbohydrate, and / or bulking agent.

[0181] The engineered cells disclosed herein can be administered to a subject prior to, concurrently with, or after activation of the heterologous gene circuit in the engineered stem cells, e.g., the engineered cells can be activated after administration to the subject, e.g., by administering to the subject an activator of the heterologous gene circuit.

[0182] A subject may be treated (e.g., administered) with a population of engineered cells (e.g., engineered hematopoietic progenitor cells) of the present disclosure for at least or up to about 1 dose, at least or up to about 2 doses, at least or up to about 3 doses, at least or up to about 4 doses, at least or up to about 5 doses, at least or up to about 6 doses, at least or up to about 7 doses, at least or up to about 8 doses, at least or up to about 9 doses, or at least or up to about 10 doses. Alternatively, or in addition, a subject may be treated (e.g., administered) with a population of engineered cells (e.g., engineered hematopoietic progenitor cells) of the present disclosure for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, or 100 years.

[0183] Any one of the methods disclosed herein can be utilized to treat a target cell, target tissue, target disease, or target disorder in a subject.

[0184] Non-limiting examples of target tissues can include cells, such as hematopoietic progenitor cells, obtainable from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Examples of samples from a subject from which cells can be derived include, but are not limited to, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, feces, semen, vaginal secretions, interstitial fluid from tumor tissue, ocular fluid, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernail, plasma, nasal swab or nasopharyngeal washing, spinal fluid, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, umbilical cord blood, emphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and / or other excrement or bodily tissue.

[0185] The target disease of interest may be cancer or tumor. Non-limiting examples of cancer include acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, 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, and acute promyelocytic leukemia. leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor tumor), basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinomacarcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, Brown's tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, cancer of unknown primary site, carcinosarcoma, Castleman's disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer Chronic lymphocytic leukemia, Chronic lymphocytic leukemia, Chronic lymphocytic leukemia, Chronic lymphocytic leukemia. leukemia), chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear-cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Dego's disease, dermatofibrosarcoma protuberans. protuberans), dermoid cysts (dermoid cysts) cyst, desmoplastic small round cell tumor, diffuse large B cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinomacarcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial uterine cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumors, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Fallopian tube cancer, Fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri cerebri), glomus tumor (Glomustumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma Melanoma, pancreatic islet cell carcinoma, pancreatic islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, lentigo maligna melanoma, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, maculoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone, malignant fibrous histiocytoma of bone Bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant Triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, spinal germ cell tumor, spinal tumor, medullary thyroidcancer, medulloblastoma, medulloepithelioma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, metastatic urothelial carcinoma, mixed mullerian tumor, monocytic leukemia, mouth cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic disease Disease, Myelodysplastic Syndromes, Myeloid Leukemia, Myeloid Sarcoma, Myeloproliferative Disorders, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Neoplasm, Neuroinoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular Melanoma, Non-Hodgkin's Lymphoma, Non-Melanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular Oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic Nerve Sheath Meningioma, Oral Cancer, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor Malignant Potential Tumor, Paget's disease of the breastbreast, Pancos tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, blood Plasma cell neoplasm, neuropulmonary 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 cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, cancer of the respiratory tract related to the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary tumors neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, Signetling cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, soot wart, spinal cord tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor tumor), surface epithelial-stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyteThe cancer may include, but is not limited to, cells of a cancer, including, but not limited to, leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, terminal lymphatic cancer, testicular cancer, thecoma, throat cancer, thymic cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional cell carcinoma, brachial cancer, urethral cancer, genitourinary neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the target cancer cells represent a subpopulation within a cancer cell population, such as cancer stem cells. In some embodiments, the cancer is a cancer of the hematopoietic lineage, such as lymphoma. The antigen can be a tumor-associated antigen.

[0186] The present disclosure also provides a composition comprising the engineered genetic circuit disclosed herein. The composition may further comprise an actuator of a heterologous genetic circuit. The present disclosure also provides a kit comprising the composition. The kit may further comprise an activator of the heterologous genetic circuit. The activator may be in the same composition as the engineered cells. Alternatively, or in addition, the activator may be in a separate composition different from the engineered cells.

[0187] Example Example 1: Differentiation of hematopoietic progenitor cells

[0188] In this example, induced pluripotent stem cells (iPSCs) are differentiated into hematopoietic progenitor cells using various heterologous genetic circuits (HGCs).

[0189] iPSC maintenance

[0190] Episomal iPSCs (Gibco, Cat. A18945) were maintained in complete Essential 8™ Flex Medium (Gibco, Cat. A2858501) or StemFlex™ Medium (Gibco, Cat. A3349401) on vitronectin (Gibco, Cat. A31804) coated flasks and passaged to maintain 50-90% confluency using Versene (Gibco, Cat. 15040066) to lift cells while maintaining cell clumps.

[0191] Nucleofection

[0192] On experimental day 0, iPSC cells were collected as a single-cell suspension using ACCUTASE™ (Stemcell Technologies, Cat# 07920). Cells were resuspended in P3 Primary Cell Nucleofector™ solution at 1.2e9 cells / ml, and 8e5 cells were nucleofected per well in a 96-well nucleocuvette (Lonza, Cat# V4 SP-3096) on a Lonza 4D Nucleofector (Cat# AAF-1003B / S) with a 96-well shuttle using optimized pulse codes.

[0193] Cells were nucleofected with plasmids encoding Cas9-VPR, the core cascade, and the gene targeting spacer. Cas9-VPR is a Cas transcriptional modulator system that can be used for both cleavage (e.g., cleavage of gate portion plasmids, gene regulatory portion plasmids) and non-cleavage gene regulation (e.g., CRISPR activation of target endogenous genes, CRISPR inhibition of target endogenous genes), depending on the specific gNA used. The only activatable gNA used was the polyT tract, which has a constant core cascade that executes four sequential steps. Nucleotide gNA spacers were used to target the genome according to the sequences in Table 1. Figure 2 lists the gene targets used in each heterologous gene circuit cascade. The steps refer to the order in which genes are expected to be expressed as a result of Cas9-VPR-mediated targeting and activation.

[0194] [Table 1-1]

[0195] [Table 1-2]

[0196] [Table 1-3]

[0197] cell culture

[0198] 1.8e5 nucleofected cells were plated at 500ul / well in 24-well tissue culture plates treated with Anti-adherence rinsing solution (Stemcell Technologies, Cat#07010) rotating at 100 RPM in a humidified 5% CO2 tissue culture incubator. Day 0 medium contains StemPro-34 (Gibco, Cat#10639011), 1X Penicillin-Streptomycin (Gibco Cat#15140122), 2mL Glutamax (Gibco, Cat# 35050061), 50ug / ml Ascorbic Acid (Santa Cruz Biotechnology, Cat#sc-39430), 4mM 1-Thioglycerol (Sigma-Aldrich, Cat#M1753), 1X Insulin-Transferrin-Selenium (ITS-G) (Gibco, Cat# 41400045) and 10mM Y-27632 (Bio-Techne Cat#1254), 50ng / ml rhBMP4 (Peprotec, Cat#120-05ET), 50ng / ml FGF-2 (Peprotech, Cat#3718-FB), and 10 uM CHIR99021 (Bio-Techne, Cat#4423).

[0199] On day 1, cultures were treated with StemPro-34 (Gibco, Cat#10639011), 1X Penicillin-Streptomycin (Gibco Cat#15140122), 2mL Glutamax (Gibco, Cat#35050061), 50ug / ml Ascorbic Acid (Santa Cruz Biotechnology, Cat# sc-39430), 4mM 1 Thioglycerol (Sigma-Aldrich, Cat#M1753), 1X Insulin-Transferrin-Selenium (ITS-G) (Gibco, Cat#41400045), 50ng / ml rhBMP4 (Peprotec, Cat#120-05ET), 50ng / ml FGF-2 (Peprotech, Cat#3718-FB), and 50ng / ml They received a complete media change with VEGF-165 (Peprotech, Cat#100-20). On day 2, 6 uM SB431542 (Selleckchem, Cat#101762-616) was added to the cultures. On day 4, cultures were supplemented with StemPro-34 (Gibco, Cat#10639011), 1X Penicillin-Streptomycin (Gibco Cat#15140122), 2mL Glutamax (Gibco,Cat# 35050061), 50ug / ml Ascorbic Acid (Santa Cruz Biotechnology, Cat#sc-39430), 4mM 1-Thioglycerol (Sigma-Aldrich, Cat# M1753), 1X Insulin-Transferrin-Selenium (ITS-G) (Gibco, Cat#41400045), 50ng / ml FGF-2 (Peprotech, Cat#3718-FB), 50ng / ml VEGF-165 (Peprotech, Cat#100-20), and 50ng / ml The medium was replaced with SCF (Peprotech, Cat#300-07).

[0200] Flow cytometry

[0201] Cultures were analyzed by flow cytometry on a Penteon (Agilent, Cat# 2010284AA) on days 3, 5, and 9. Embryoid bodies were collected into 96 deep-well plates (Nest, Cat# 503162) and disaggregated in 500 ml of TrypLE™ Express Enzyme (Gibco, Cat# 12605028) at 37°C for 20–30 min, followed by mechanical disruption by pipetting. TrypLE™ Express Enzyme was quenched with DMEM-F12 (Stemcell Technologies, Cat# 36254). Cells were stained with Zombie Aqua™ Fixable (Biolegend, Cat# 423102, 1:2000) for 20 minutes at room temperature, washed with 1X DPBS, 2% FBS, 0.02% NaN3, and blocked with Human IgG (Lee Biosolutions, Cat# 340-21) for 10 minutes on ice. Cells were stained with PDGFR-BV421 (BD Bioscienes Cat# 562799, 1:400), CD73-BV785 (Biolegend, Cat# 344028, 1:400), CD144 / VE-Cadherin-PE (Biolegend, Cat# 348505, 1:100), CD34-PE-Dazz (Biolegend, Cat# 343534, 1:400), CD309-PE-Cy7 (Biolegend, Cat# 359912, 1:100), CD43-APC (Biolegend, Cat# 343206, 1:800), and CD45-AF700 (Biolegend, Cat# 304024, 1:200) for 30 minutes on ice. Cells were washed with 1X DPBS, 2% FBS, 0.02% NaN3. For analysis, cells were resuspended in 1X DPBS, 2% FBS, 0.02% NaN3. Data were analyzed using FlowJo™ v10.8 software (BD Life Sciences).Dead cells and doublets were excluded from analysis via Zombie Aqua and doublet discrimination gates.

[0202] Flow cytometry data were compiled to form Figures 4A-4D, which depict the relative frequency of cells among viable singlet cells of appropriate size on day 5 of culture. Scatter plots were generated at various time points to visualize the magnitude of the increase in hematopoietic gene expression induced by HGCs relative to controls (Figure 3).

[0203] Exemplary results of flow cytometry analysis showing the frequency of cells expressing the indicated surface markers in live singlet cells compared to a no DNA control (e.g., nucleofected in the absence of exogenous DNA) for HSCs (Cellgorithms) #7 and #12 at day 5 of culture are shown in Figures 5A-5B. Summary graphs are shown in Figures 5C-5D.

[0204] Example 2: Multi-Step Cascades

[0205] The systems and methods provided herein (e.g., based on a polynucleotide sequence encoding an activatable sgRNA, which polynucleotide sequence includes one or more poly-T sequences) can be utilized to induce sequentially separated multi-step cascade effects, and expression of an endogenous gene product can be activated at any stage of the cascade.

[0206] For example, the multi-step cascade effect can be a 10-step cascade effect, such as a 10-step forward cascade or a 10-step reverse cascade.

[0207] Experiment details

[0208] In summary, experiments involve creating a mixture of plasmid DNAs encoding components of the proGuide cascade, proceeding by introducing those DNAs into cells (e.g., HEK293 cells) via nucleofection, and concluding by assessing the effect on activation of target gene products at various time points using flow cytometric detection of cell surface gene products (e.g., CXCR4).

[0209] The essential components of Cellgorithm (e.g., Cas9-VPR expression plasmid, proGuide-encoding plasmid) are mixed, and a GFP-expressing plasmid is included to identify transfected cells. To construct the plasmid combinations for activating endogenous genes at different stages in the proGuide cascade, the cascade plasmid DNA mixtures used the components listed in Tables 2 and 3. Core cascade plasmids are progressively included in the transfection mixture to add additional stages in the cascade, as follows: For example, the first stage (e.g., stage 1) condition does not include proGuide, and the second stage (e.g., stage 2) includes an sgRNA with a spacer sequence targeting the 5' and 3' cleavage sites in the proGuide plasmid. The second stage (e.g., stage 2) condition includes all the plasmids in the first stage (e.g., stage 1) condition, plus the proGuide plasmid described for the second stage (e.g., stage 2). The third stage (e.g., stage 3) conditions contain all of the plasmids in the second stage (e.g., stage 2) conditions, plus the proGuide described for the third stage (e.g., stage 3). To keep the mass of each proGuide plasmid DNA constant and to keep the mass of total DNA constant for all transfections, a genetically inactive plasmid DNA (e.g., pUC19) is used as a "filler" for conditions with fewer proGuide plasmids.

[0210] To activate expression of an endogenous gene product (e.g., CXCR4), Cas9-VPR is targeted to the promoter region of the gene (e.g., CXCR4) using a 14-nt spacer sequence. For activation in the first stage (e.g., stage 1), gene (e.g., CXCR4) activation is stimulated by an sgRNA with the gene's relevant spacer (e.g., a 14-nt CXCR4 spacer). For subsequent steps, a proGuide plasmid with the gene's relevant spacer (e.g., a 14-nt CXCR4 spacer) is added to the plasmid DNA mixture. By matching the 5' and 3' cleavage sites in the gene (e.g., CXCR4)-activating proGuide for a specific stage in the cascade with the 5' and 3' cleavage sites in the gene (e.g., CXCR4)-activating proGuide, gene (e.g., CXCR4) activation is effectively programmed to occur at one specific stage in the cascade for each condition / mixture of plasmid DNA.

[0211] The plasmid DNA mixture is introduced into cells (e.g., hematopoietic cells) using standard procedures with a nucleofection system (e.g., Lonza 4D). The transfected cells are plated (e.g., in multiwell tissue culture plates) and maintained using standard mammalian tissue culture methods. At specific time points (e.g., 12, 24, 36, 48, and 72 hours) after nucleofection, the cells are processed for flow cytometry and detection of cell surface expression of the gene product (e.g., CXCR4). For each condition, independent replicates (e.g., n=4) (nucleofections) were examined by flow cytometry.

[0212] result

[0213] We generated hematopoietic progenitor cells (HPCs) using the multistep cascade approach described above. The multistep cascade resulted in a range of HPC expandability and yield. HPCs derived from the multistep cascade retained the HPC state and included the ability to acquire lymphoid potential. Some cellgorithms promoted greater loss of CD34 expression, maintained CD45 expression, and lacked CD7 expression. Some cellgorithms allowed HPCs to exit the progenitor state and promoted increased yield of CD7+ cells. The CD34+CD45+, CD34-CD45+, total CD45, and / or CD7 counts after the multistep cascade can be seen in Figures 13A–13D. The triple-positive cell frequencies obtained in vitro were 9.18% for CD34+ cells and 8.77% for CD43+ cells.

[0214] Compared to control conditions, Cellgorithms (listed in Table 4) induced a similar degree of differentiation of iPSCs to early stages of cell lineages, as indicated by the lack of an increase in CD309+ (KDR) cells compared to control conditions (Figures 8A-8D). In contrast, some, but not all, Cellgorithm-treated cells showed a significant increase in hemogenic endothelium stage (KDR-CD34+, Figures 9A-9D, 10A-10D) and HPC stage (CD34+CD43+, Figures 11A-11D, and CD34+CD45+, Figures 12A-12D). CGO178 caused the greatest increase in HPC stage cells.

[0215] Control conditions described herein include "directed differentiation" (e.g., "directed differentiation," "directed differentiation 1," "directed differentiation 2"), in which cells were subjected to handling and addition of DNA but were not treated with an electric field pulse from a nucleofection instrument (e.g., "no zap"). The control condition described herein, "no DNA," is where cells were subjected to handling and electric pulses from a nucleofection instrument, but no plasmid DNA was added to the cells during nucleofection. The control condition described herein, "GFP+core cascade," is where cells were nucleofected with a GFP-expressing plasmid, Cas9-VPR, and proGuide to proceed through all stages, but no gene-targeting proGuide was included. For the control condition described herein, "Cas-VPR control," cells were engineered with a control Cellgorithm gene circuit designed to target one or more genes (e.g., CXCR4 and CD105) different from any of the genes listed in Table 4.

[0216] Both the identity of the TFs activated by cellgorithms and their sequential order of activation were important for their effect on HPC differentiation. Comparison of CGO178 and CGO181 shows that they differ by the inclusion of five TFs activated at stage 6 (Table 4). Addition of these TFs increased the proportion of hemogenic endothelial cells and HPCs in the cultures (Figures 10D, 11D, and 12D). Comparison of CGO176, CGO181, and CGO192 shows that they differ by sequential activation of the same set of TFs (CGO176 and CGO181) versus all-at-once activation of TFs by CGO192 (Table 4). Sequential activation of TFs by CGO176 and CGO181 resulted in a greater proportion of HPC cell types than all-at-once activation by CGO192 (Figures 11D and 12D). Similar improvements in the production of hematopoietic cells, including lymphoid marker cells, were observed from Cellgorithms with sequential activation of TF compared to all-at-once activation and control no-activation of TF (Figures 13A-13D).

[0217] Furthermore, CGO178 was compared to a benchmark protocol as described by Sugimura et al. (Hematopoietic stem and progenitor cells from human pluripotent stem cells, Nature 545, 432-438 (2017)). Compared to the benchmark, the method disclosed herein results in higher and faster conversion rates (Figures 14A-14B).

[0218] [Table 2]

[0219] [Table 3]

[0220] [Table 4-1]

[0221] [Table 4-2]

[0222] [Table 4-3]

[0223] [Table 5-1]

[0224] [Table 5-2]

[0225] [Table 5-3]

[0226] [Table 5-4]

[0227] [Table 5-5]

[0228] Embodiment The following non-limiting embodiments provide illustrative examples of the present invention, but do not limit the scope of the invention.

[0229] Embodiment 1. A method of directing a plurality of stem cells to differentiation, the method comprising: contacting a plurality of stem cells with a heterologous genetic circuit comprising a plurality of gate units, wherein upon activation of the heterologous genetic circuit, the plurality of gate units operate in a coordinated manner to sequentially induce a plurality of distinct modulations of target genes in the plurality of stem cells, each of the plurality of distinct modulations being necessary but individually insufficient to direct the plurality of stem cells toward differentiation, and wherein the plurality of gate units: (i) a first gating unit, the first gating unit being activatable to induce a first distinct regulation of a plurality of distinct regulations upon activation of the heterologous genetic circuit; (ii) a second gating unit, the second gating unit being activatable upon activation of the heterologous genetic circuit to induce a second distinct regulation of the plurality of distinct regulations, wherein the second distinct regulation is induced subsequent to the first distinct regulation, whereby both the first distinct regulation and the second distinct regulation enhance or reduce expression and / or activity levels of a target gene in the cell, wherein the target gene encodes an erythroid transformation-specific (ETS) transcription factor, and upon contact, the plurality of gating units operate in concert to direct the plurality of stem cells to differentiation; and optionally, (1) the plurality of stem cells includes pluripotent stem cells, and / or (2) Upon contact, multiple gate units act in concert to drive the differentiation of multiple stem cells into tissue-specific progenitor cells. Additionally, optionally, i. the tissue-specific progenitor cells include hematopoietic cells; Additionally, optionally, 1. Upon contact, the plurality of gating units act in concert to direct the plurality of stem cells to hematopoietic stem cells; and / or 2. The hematopoietic cells are characterized as being CD45+, and / or 3. The hematopoietic cells are characterized as being CD34+CD43-CD45+, and / or ii. activation of the heterologous genetic circuitry in at least about 10%, at least about 15%, at least about 20%, at least about 30%, or at least about 40% of the stem cells differentiate into tissue-specific progenitor cells; and / or iii. differentiation is observed in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 6 days; and / or (3) ETS transcription factors include ETS1, and / or (4) the ETS transcription factor includes ETV2, and / or (5) ETS transcription factors include LMO2, and / or (6) the plurality of gating units are pre-configured such that one of the plurality of gating units is activatable to regulate the expression and / or activity level of an additional target gene upon activation of the heterologous genetic circuit, the additional target gene comprising a T-box transcription factor (TBX) or a homeobox protein; Additionally, optionally, i. the modulation of the expression and / or activity level of the additional target gene is induced prior to at least one of the multiple distinct modulations of the target gene encoding the ETS transcription factor; and / or ii. The TBX contains at least two different types of TBX; and / or iii. TBX includes TBXT and / or TBX6, and / or iv. the homeobox protein comprises a PRD class homeobox protein; and / or v. the homeobox protein includes MIXL1, and / or (7) the plurality of gating units are not pre-configured to modulate the expression and / or activity levels of one or more members selected from the group consisting of TBXT, TBX6, and MIXL1 prior to the plurality of distinct modulations of target genes encoding ETS transcription factors; and / or (8) the plurality of gate units are pre-configured such that one of the plurality of gate units is activatable to regulate the expression and / or activity levels of different target genes upon activation of the heterologous genetic circuit, the different target genes comprising a GATA transcription factor or a basic helix-loop-helix (bHLH) transcription factor; and / or Additionally, optionally, i. GATA transcription factors include GATA2, and / or ii. the bHLH contains an SCL, and / or (9) the contacting occurs in a medium that is substantially free of exogenous thrombopoietin (TPO) and / or exogenous FLT-3 ligand (FLT3L); and / or (10) the contacting is carried out in a medium that is substantially free of exogenous interleukin (IL); Additionally, optionally, i. the exogenous IL comprises one or more members selected from the group consisting of IL-2, IL-3, IL-7, IL-15, and IL-21; and / or (11) The plurality of gating units includes a third gating unit that is activatable upon activation of the heterologous genetic circuit and that induces a third distinct regulation of the plurality of distinct regulations, wherein the third distinct regulation is induced after the first distinct regulation and the second distinct regulation, whereby the first distinct regulation, the second distinct regulation, and the third distinct regulation all enhance or reduce the expression and / or activity levels of the target gene in the plurality of stem cells; and / or (12) the plurality of gating units are preconfigured such that the expression and / or activity levels of the target genes are sequentially regulated by no more than three distinct gating units of the plurality of gating units; and / or (13) no more than three distinct gating units of the plurality of gating units are preconfigured to sequentially regulate the expression and / or activity levels of target genes; and / or (14) the first and second distinct modulations both enhance the expression and / or activity level of the target gene in the cell; and / or (15) the first and second distinct modulations both reduce the expression and / or activity level of the target gene in the cell, and / or (16)(i) the first gating unit comprises a first gene regulatory portion that is activated upon activation of the first gating unit to induce a first distinct regulation through specific binding of the first gene regulatory portion to a target gene; or (ii) the second gating unit comprises a second gene regulatory portion, the second gene regulatory portion comprising a second gene regulatory portion that is activated upon activation of the second gating unit to induce a second distinct regulation through specific binding of the first gene regulatory portion to a target gene; Further, optionally i. the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to substantially the same polynucleotide sequence of the target gene, and / or ii. the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to different polynucleotide sequences of the target gene; and / or iii. the second gating unit further comprises a second gating portion that is activated upon activation of the second unit to induce activation of the second gene regulatory portion through specific binding of the second gating portion to the second gene regulatory portion; Additionally, optionally, 1. The first gate unit further includes a first gate portion, and the first gate portion is activated when the first gate unit is activated, (a) activation of a first gene regulatory moiety via specific binding of a first gating moiety to the first gene regulatory moiety; and (b) inducing activation of a second gating moiety via specific binding of the first gating moiety to the second gating moiety; and / or 2. The activating portion is: (a) activation of a first gene regulatory moiety via specific binding of an activation moiety to the first gene regulatory moiety; (b) capable of inducing activation of a second gating moiety; and / or (17) The first gate unit, the first gate portion, the first gene regulatory portion, the second gate, the second gate portion, and / or the second gene regulatory portion comprises an activatable gNA, and upon activation of the gNA, the gNA forms a complex with an endonuclease; Additionally, optionally, i. the activatable gNA comprises a non-canonical termination sequence, and / or ii. the endonuclease comprises a CRISPR / Cas protein, and optionally 1. The CRISPR / Cas protein substantially lacks cleavage activity, and / or 2. The CRISPR / Cas protein is coupled to a transcriptional activator, and / or (18) The target gene comprises multiple types of ETS transcription factors, wherein the first distinct regulation via the first gating unit is regulation of a first type of ETS transcription factor, and the second distinct regulation via the second gating unit is regulation of a second type of ETS transcription factor; Further optionally, the method wherein (i) the first type of ETS transcription factors comprises one or more members selected from the group consisting of ETS1, ETV2, LMO2, and combinations thereof, and (ii) the second type of ETS transcription factors comprises ERG.

[0230] Embodiment 2. A method of converting a plurality of stem cells (first plurality) into a plurality of hematopoietic cells (second plurality), the method comprising: culturing the first plurality ex vivo in a medium that is substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL); within about 14 days after culturing, the conversion rate of the first plurality to the second plurality is at least about 3%, and the second plurality is CD45+; Optionally, the culturing comprises contacting a first plurality of target genes with a heterologous gene regulatory moiety to regulate the expression and / or activity levels of the target genes, wherein the target genes encode erythroid transformation-specific (ETS) transcription factors.

[0231] Embodiment 3. A method of converting a plurality of stem cells (first plurality) into a plurality of hematopoietic cells (second plurality), the method comprising: contacting a first plurality of target genes with a heterologous gene regulatory moiety to regulate expression and / or activity levels of the target genes, wherein the target genes encode erythroid transformation-specific (ETS) transcription factors; the contacting results in transformation of the first plurality into a second plurality; Optionally, (1) within about 14 days after contact, the conversion rate from the first plurality to the second plurality is at least about 3%, and the second plurality is CD45+; and / or (2) within about 14 days after contact, the conversion rate from the first plurality to the second plurality is at least about 5%, and the second plurality is CD34+; and / or (3) The method, wherein the contacting occurs ex vivo in a medium that is substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL).

[0232] Embodiment 4. Further optionally, (1) the medium is substantially free of TPO, and / or (2) the medium is substantially free of FLT3L, and / or (3) the medium is substantially free of ILs; Further optionally, the IL comprises one or more members selected from the group consisting of IL-2, IL-3, IL-7, IL-15, and IL-21; and / or (4) the second plurality is CD34+CD43-CD45+, and / or (5) the conversion rate is observed within about 10 days, within about 7 days, or within about 6 days after incubation or contact; and / or (6) the conversion rate is at least about 10% or at least about 15%, and / or (7) additional conversion is observed within about 6 days or within about 5 days; and / or (8) the additional conversion rate is at least about 20%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%, and / or (9) within about 7 days after culturing, the conversion rate from the first plurality to the second plurality is at least about 3%, and the second plurality is CD45+ (or CD34+ and CD45+); and / or (10) within about 7 days after culturing, the conversion rate from the first plurality to the second plurality is at least about 6%, and the second plurality is CD45+ (or CD34+ and CD45+); and / or (11) within about 7 days after culturing, the conversion rate from the first plurality to the second plurality is at least about 5%, and the second plurality is CD34+; and / or (12) within about 7 days after culturing, the conversion rate from the first plurality to the second plurality is at least about 10%, and the second plurality is CD34+; and / or (13) the ETS transcription factor comprises one or more members selected from the group consisting of ETS1, ETV2, and / or LMO2; and / or (14) the heterologous gene regulatory moiety enhances the expression and / or activity level of the target gene; and / or (15) the heterologous gene regulatory moiety reduces the expression and / or activity level of the target gene; and / or (16) the target gene is endogenous to the first plurality; and / or (17) The method further comprises contacting additional target genes in the first plurality with additional heterologous gene regulatory moieties to modulate the expression and / or activity levels of the additional target genes, thereby effecting conversion, wherein the additional target genes comprise one or more members selected from the group consisting of T-box transcription factors (TBXs), homeobox proteins, GATA transcription factors, and basic helix-loop-helix transcription factors (bHLHs); Additionally, optionally, a. the additional heterologous gene regulatory moiety enhances the expression and / or activity level of the additional target gene; and / or b. the additional heterologous gene regulatory moiety reduces the expression and / or activity level of an additional target gene; and / or c. the additional target gene is endogenous to said first plurality; and / or (18) The heterologous gene regulatory portion comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule that exhibits specific binding to a target gene, and / or (19) The additional heterologous gene regulatory portion comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule that exhibits specific binding to an additional target gene; and / or (20) The polypeptide molecule comprises an endonuclease, and the nucleic acid molecule comprises a guide nucleic acid (gNA) that forms a complex with the endonuclease; Further optionally, the endonuclease comprises a CRISPR / Cas protein; Additionally, optionally, i. the CRISPR / Cas protein substantially lacks cleavage activity, and / or ii. the CRISPR / Cas protein is coupled to a transcriptional activator, and / or (21) The method further comprises directing the conversion of a plurality of hematopoietic cells to lymphoid cells; Further optionally, the lymphoid cells are selected from the group consisting of T cells, NK cells, and B cells.

[0233] Embodiment 5. A system for directing a plurality of stem cells to differentiation, the system comprising: a heterologous genetic circuit including a plurality of gate units, wherein upon activation of the heterologous genetic circuit, the plurality of gate units operate in a coordinated manner to sequentially induce a plurality of distinct modulations of target genes in a plurality of stem cells, each of the plurality of distinct modulations being necessary but individually insufficient to direct the plurality of stem cells to differentiation; (i) a first gating unit, the first gating unit being activatable to induce a first distinct regulation of a plurality of distinct regulations upon activation of the heterologous genetic circuit; (ii) a second gating unit that is activatable to induce a second distinct regulation of the plurality of distinct regulations upon activation of the heterologous genetic circuit, wherein the second distinct regulation is induced subsequent to the first distinct regulation, whereby both the first distinct regulation and the second distinct regulation enhance or reduce the expression and / or activity level of the target gene in the cell; The target gene encodes an erythroid transformation-specific (ETS) transcription factor, Upon contact of multiple stem cells through heterogeneous genetic circuits, multiple gate units operate in coordination to direct the multiple stem cells toward differentiation. Optionally, (1) the plurality of stem cells includes pluripotent stem cells, and / or (2) Upon contact, multiple gate units act in a coordinated manner, resulting in the differentiation of multiple stem cells into tissue-specific progenitor cells. Additionally, optionally, i. the tissue-specific progenitor cells include hematopoietic cells; Additionally, optionally, 1. Upon contact, the multiple gating units act in concert to direct the multiple stem cells into hematopoietic stem cells; and / or 2. The hematopoietic cells are characterized as being CD45+, and / or 3. The hematopoietic cells are characterized as CD34+CD43-CD45+, and / or ii. activation of the heterologous genetic circuitry in at least about 10%, at least about 15%, at least about 20%, at least about 30%, or at least about 40% of the stem cells differentiate into tissue-specific progenitor cells; and / or iii. differentiation is observed in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 6 days; and / or (3) ETS transcription factors include ETS1, and / or (4) the ETS transcription factor includes ETV2, and / or (5) ETS transcription factors include LMO2, and / or (6) the plurality of gating units are pre-configured such that one of the plurality of gating units is activatable to regulate the expression and / or activity level of an additional target gene upon activation of the heterologous genetic circuit, the additional target gene comprising a T-box transcription factor (TBX) or a homeobox protein; Additionally, optionally, i. the modulation of the expression and / or activity level of the additional target gene is induced prior to at least one of the multiple distinct modulations of the target gene encoding the ETS transcription factor; and / or ii. The TBX contains at least two different types of TBX; and / or iii. TBX includes TBXT and / or TBX6, and / or iv. the homeobox protein comprises a PRD class homeobox protein, and / or v. the homeobox protein includes MIXL1, and / or (7) the plurality of gating units are not preconfigured to modulate the expression and / or activity levels of one or more members selected from the group consisting of TBXT, TBX6, and MIXL1 prior to the plurality of distinct modulations of target genes encoding ETS transcription factors; and / or (8) the plurality of gate units are pre-configured such that one of the plurality of gate units is activatable to regulate the expression and / or activity levels of different target genes upon activation of the heterologous genetic circuit, the different target genes comprising a GATA transcription factor or a basic helix-loop-helix (bHLH) transcription factor; and / or Additionally, optionally, i. GATA transcription factors include GATA2, and / or ii. the bHLH contains an SCL, and / or (9) the contacting occurs in a medium that is substantially free of exogenous thrombopoietin (TPO) and / or exogenous FLT-3 ligand (FLT3L); and / or (10) the contacting is carried out in a medium that is substantially free of exogenous interleukin (IL); Additionally, optionally, i. the exogenous IL comprises one or more members selected from the group consisting of IL-2, IL-3, IL-7, IL-15, and IL-21; and / or (11) the plurality of gating units includes a third gating unit that is activatable upon activation of the heterologous genetic circuit to induce a third distinct regulation of the plurality of distinct regulations, wherein the third distinct regulation is induced after the first distinct regulation and the second distinct regulation, whereby the first distinct regulation, the second distinct regulation, and the third distinct regulation all enhance or reduce the expression and / or activity level of the target gene in the plurality of stem cells; and / or (12) the plurality of gating units are preconfigured such that the expression and / or activity levels of the target genes are sequentially regulated by no more than three distinct gating units of the plurality of gating units; and / or (13) fewer than three different gate units of the plurality of gate units are preconfigured to sequentially regulate the expression and / or activity levels of the target genes; and / or (14) the first and second distinct modulations both enhance the expression and / or activity level of the target gene in the cell; and / or (15) the first and second distinct modulations both reduce the expression and / or activity level of the target gene in the cell, and / or (16)(i) the first gating unit comprises a first gene regulatory portion that is activated upon activation of the first gating unit to induce a first distinct regulation through specific binding of the first gene regulatory portion to a target gene; (ii) the second gating unit comprises a second gene regulatory portion that is activated upon activation of the second gating unit to induce a second distinct regulation through specific binding of the first gene regulatory portion to a target gene; Additionally, optionally, i. the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to substantially the same polynucleotide sequence of the target gene, and / or ii. the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to different polynucleotide sequences of the target gene; and / or iii. The second gating unit is activated upon activation of the second unit to induce activation of the second gene regulatory moiety through specific binding of the second gating moiety to the second gene regulatory moiety, and further comprises a second gating moiety; Additionally, optionally, 1. The first gate unit, when activated, (a) activation of a first gene regulatory moiety via specific binding of a first gating moiety to the first gene regulatory moiety; (b) activated to induce activation of the second gating moiety by specific binding of the first gating moiety to the second gating moiety; and / or 2. The activation part is (a) activation of a first gene regulatory moiety via specific binding of an activation moiety to the first gene regulatory moiety; (b) capable of inducing activation of a second gating moiety; and / or (17) The first gate unit, the first gate portion, the first gene regulatory portion, the second gate, the second gate portion, and / or the second gene regulatory portion comprises an activatable gNA, and upon activation of the gNA, the gNA forms a complex with an endonuclease; Additionally, optionally, i. the activatable gNA comprises a non-canonical termination sequence, and / or ii. the endonuclease comprises a CRISPR / Cas protein; Additionally, optionally, 1. The CRISPR / Cas protein substantially lacks cleavage activity, and / or 2. The CRISPR / Cas protein is coupled to a transcriptional activator, and / or (18) The target gene comprises multiple types of ETS transcription factors, and the first distinct regulation via the first gate unit is regulation of the first type of ETS transcription factor, and the second distinct regulation via the second gate unit is regulation of the second type of ETS transcription factor; Further optionally, the system includes: (i) the first type of ETS transcription factors includes one or more members selected from the group consisting of ETS1, ETV2, LMO2, and combinations thereof; and (ii) the second type of ETS transcription factors includes ERG.

[0234] Embodiment 6. A system for converting a plurality of stem cells (first plurality) into a plurality of hematopoietic cells (second plurality), the system comprising: a medium for culturing a first plurality of ex vivo cells, the medium being substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL); within about 14 days after ex vivo culture of the first plurality in the medium, the conversion rate of the first plurality to the second plurality is at least about 3%, and the second plurality is CD45+; Optionally, the ex vivo culturing includes contacting a first plurality of target genes with a heterologous gene regulatory moiety to regulate the expression and / or activity levels of the target genes, wherein the target genes encode erythroid transformation-specific (ETS) transcription factors.

[0235] Embodiment 7. A system for converting a plurality of stem cells (first plurality) into a plurality of hematopoietic cells (second plurality), the system comprising: a heterologous gene regulatory portion that exhibits specific binding to a first plurality of target genes and regulates the expression and / or activity levels of the target genes, wherein the target genes encode erythroid transformation-specific (ETS) transcription factors; contacting the target gene with the heterologous gene regulatory moiety results in conversion of the first plurality to a second plurality; Optionally, (1) within about 14 days after contact, the conversion rate from the first plurality to the second plurality is at least about 3%, and the second plurality is CD45+; and / or (2) within about 14 days after contact, the conversion rate from the first plurality to the second plurality is at least about 5%, and the second plurality is CD34+; and / or (3) The system, wherein the contacting occurs ex vivo in a medium substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL).

[0236] Embodiment 8. Further optionally, (1) the medium is substantially free of TPO, and / or (2) the medium is substantially free of FLT3L, and / or (3) the medium is substantially free of ILs, and further optionally: a. the IL comprises one or more members selected from the group consisting of IL-2, IL-3, IL-7, IL-15, and IL-21; and / or (4) the second plurality is CD34+CD43-CD45+, and / or (5) the conversion rate is observed within about 10 days, within about 7 days, or within about 6 days after incubation or contact; and / or (6) the conversion rate is at least about 10% or at least about 15%, and / or (7) additional conversion is observed within about 6 days or within about 5 days; and / or (8) the additional conversion rate is at least about 20%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%, and / or (9) within about 7 days after culturing, the conversion rate from the first plurality to the second plurality is at least about 3%, and the second plurality is CD45+ (or CD34+ and CD45+); and / or (10) within about 7 days after culturing, the conversion rate from the first plurality to the second plurality is at least about 6%, and the second plurality is CD45+ (or CD34+ and CD45+); and / or (11) within about 7 days after culturing, the conversion rate from the first plurality to the second plurality is at least about 5%, and the second plurality is CD34+; and / or (12) within about 7 days after culturing, the conversion rate from the first plurality to the second plurality is at least about 10%, and the second plurality is CD34+; and / or (13) the ETS transcription factor comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, and / or LMO2; and / or (14) The ETS transcription factor comprises one or more members selected from the group consisting of ETS1, ETV2, and / or LMO2; and / or (15) the heterologous gene regulatory moiety enhances the expression and / or activity level of the target gene; and / or (16) the heterologous gene regulatory moiety reduces the expression and / or activity level of the target gene; and / or (17) the target gene is endogenous to the first plurality; and / or (18) The system further includes an additional heterologous gene regulatory moiety configured to bind to the first plurality of additional target genes and regulate the expression and / or activity levels of the additional target genes, thereby effecting conversion; Additional target genes include one or more members selected from the group consisting of T-box transcription factors (TBXs), homeobox proteins, GATA transcription factors, and basic helix-loop-helix transcription factors (bHLHs); Additionally, optionally, a. the additional heterologous gene regulatory moiety enhances the expression and / or activity level of the additional target gene; and / or b. the additional heterologous gene regulatory moiety reduces the expression and / or activity level of an additional target gene, and / or c. the additional target gene is endogenous to the first plurality; and / or (19) The heterologous gene regulatory portion comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule that exhibits specific binding to a target gene, and / or (20) the additional heterologous gene regulatory portion comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule that exhibits specific binding to an additional target gene; and / or (21) The polypeptide molecule comprises an endonuclease, and the nucleic acid molecule comprises a guide nucleic acid (gNA) that forms a complex with the endonuclease; Further optionally, the endonuclease comprises a CRISPR / Cas protein; Additionally, optionally, i. the CRISPR / Cas protein substantially lacks cleavage activity, and / or ii. The system of any one of embodiments 5, 6, and 7, wherein the CRISPR / Cas protein is coupled to a transcriptional activator.

[0237] Embodiment 9. An engineered cell comprising the system of any one of embodiment 5, embodiment 6, embodiment 7, and embodiment 8.

[0238] Embodiment 10. A composition comprising the system described in any one of embodiments 5, 6, 7, and 8, or the artificial cell described in embodiment 9.

[0239] Embodiment 11. A method of converting a plurality of stem cells (first plurality) into a plurality of hematopoietic cells (second plurality), the method comprising: contacting the first plurality with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in a coordinated manner to sequentially regulate the expression levels of a plurality of distinct target genes, and the plurality of gating units a first gating unit, the first gating unit being activatable to regulate expression of a target gene upon activation of the heterologous genetic circuit; a second gating unit that is activatable upon activation of the first gating unit to regulate expression of an additional target gene different from the target gene, whereby regulation of expression of the additional target gene is induced after regulation of expression of the target gene; Heterogeneous genetic circuits are (A) the target gene comprises one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; and / or (B) the target genes include one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target genes include one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (C) the target genes include one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and the additional target genes include one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (D) the plurality of gating units are programmed to include a third gating unit, wherein the third gating unit is activatable upon activation of the second gating unit to regulate expression of another target gene different from the target gene and the additional target gene, whereby the regulation of expression of the another target gene is induced after the regulation of expression of the additional target gene, wherein the target gene comprises one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, wherein the additional target gene comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and wherein the another target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; Upon contact, the plurality of gate units act in concert to effect a transformation of the first plurality of gate units into a second plurality of gate units; Optionally, (1) The heterologous genetic circuit is characterized by (A) and / or (2) the heterologous genetic circuit is characterized by (B), and / or (3) the heterologous genetic circuit is characterized by (C), and / or (4) The heterologous genetic circuit is characterized by (D), and / or (5) the plurality of gating units includes an additional gating unit that is activatable upon activation of the first gating unit, and activation of the additional gating unit is necessary to induce activation of the second gating unit, thereby inducing a time delay between regulation of the target gene by the first gating unit and regulation of the additional target gene by the second gating unit; and / or (6) the second plurality comprises hemogenic endothelial cells that are KDR− and CD34+, and further optionally, a. within about 14 days or within about 7 days after contact, there is at least about 5% conversion of the first plurality to the second plurality; and / or b. within about 14 days or within about 7 days after contacting, the conversion rate of the first plurality to said second plurality is at least about 10%; and / or (7) the second plurality comprises hematopoietic cells (HPCs) that are (i) CD34+ and CD43+, or (ii) CD34+ and CD45+, and optionally: a. within about 14 days or within about 7 days after contact, there is at least about a 3% conversion of the first plurality to the second plurality; and / or b. within about 14 days or within about 7 days after contact, the conversion rate of the first plurality to the second plurality is at least about 6%; and / or (8) the rate of conversion of the first plurality to the second plurality via the heterologous genetic circuit is at least about 1-fold greater than in the absence of the heterologous genetic circuit; and / or (9) the rate of conversion of the first plurality to the second plurality via the heterologous genetic circuit is at least about three times greater than in the absence of the heterologous genetic circuit; and / or (10) the contacting occurs ex vivo in a medium that is substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), interleukin (IL), and combinations thereof; and / or (11) (i) modulation of expression of the target gene, (ii) modulation of expression of an additional target gene, and / or (ii) modulation of expression of another target gene are each necessary, but individually sufficient, to effect conversion of the first plurality to the second plurality; and / or (12) a gating unit of the plurality of gating units comprises a gene regulatory moiety that is activatable upon activation of the gating unit to induce regulation of a corresponding target gene through specific binding of the gene regulatory moiety to the corresponding target gene; and / or (13) the gene regulatory portion comprises a nucleic acid sequence that exhibits complementarity to at least a portion of the corresponding target gene; and / or (14) the gene regulatory portion comprises a guide nucleic acid (gNA) molecule capable of forming a complex with an endonuclease, the complex exhibiting specific binding to the corresponding target gene; and / or (15) The endonuclease comprises a CRISPR / Cas protein, and further optionally, (i) the CRISPR / Cas protein substantially lacks cleavage activity, and / or (ii) the CRISPR / Cas protein is coupled to a transcriptional activator, and / or (16) The method, wherein the plurality of distinct target genes is a first plurality of endogenous target genes.

[0240] Embodiment 12. A system for converting a plurality of stem cells (first plurality) into a plurality of hematopoietic cells (second plurality), the system comprising: The heterologous genetic circuit includes a plurality of gate units, and upon activation of the heterologous genetic circuit, the plurality of gate units are configured to operate in a coordinated manner to sequentially regulate the expression levels of a plurality of distinct target genes, and the plurality of gate units are a first gating unit, the first gating unit being activatable to regulate expression of a target gene upon activation of the heterologous genetic circuit; a second gating unit that is activatable upon activation of the first gating unit to regulate expression of an additional target gene different from the target gene, whereby regulation of expression of the additional target gene is induced after regulation of expression of the target gene; Heterogeneous genetic circuits are (A) the target gene comprises one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; and / or (B) the target genes include one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target genes include one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (C) the target genes include one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and the additional target genes include one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (D) the plurality of gating units include a third gating unit that is activatable and programmed to regulate expression of another target gene, different from the target gene and the additional target gene, upon activation of the second gating unit, whereby regulation of expression of the another target gene is induced after regulation of expression of the additional target gene, wherein the target gene includes one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, wherein the additional target gene includes one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and wherein the another target gene includes one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; Upon contact of the first plurality by the heterologous genetic circuit, the plurality of gating units, operating in concert, effect the first plurality of second plurality of transformations; Optionally, (1) The heterologous genetic circuit is characterized by (A) and / or (2) the heterologous genetic circuit is characterized by (B), and / or (3) the heterologous genetic circuit is characterized by (C), and / or (4) The heterologous genetic circuit is characterized by (D), and / or (5) the plurality of gating units includes an additional gating unit that is activatable upon activation of the first gating unit, and activation of the additional gating unit is necessary to induce activation of the second gating unit, thereby inducing a time delay between regulation of the target gene by the first gating unit and regulation of the additional target gene by the second gating unit; and / or (6) the second plurality comprises hemogenic endothelial cells that are KDR− and CD34+, and optionally, a. the plurality of gate units are configured to operate in concert to provide a conversion rate of the first plurality of gate units to the second plurality of gate units of at least about 5%, within about 14 days, or within about 7 days after contact; and / or b. the plurality of gate units are configured to result in at least about a 10% conversion rate of the first plurality of gate units to the second plurality of gate units within about 14 days or within about 7 days after contact; and / or (7) the second plurality comprises hematopoietic cells (HPCs) that are (i) CD34+ and CD43+, or (ii) CD34+ and CD45+, and optionally: a. the plurality of gate units are configured to operate in concert to result in a conversion rate of at least about 3% from the first plurality of gate units to the second plurality of gate units within about 14 days or within about 7 days after contact; and / or b. the plurality of gate units are configured to operate in concert to result in a conversion rate of at least about 6% from the first plurality of gate units to the second plurality of gate units within about 14 days or within about 7 days after contact; and / or (8) the rate of conversion of the first plurality to the second plurality via the heterologous genetic circuit is at least about 1-fold greater than in the absence of the heterologous genetic circuit; and / or (9) the rate of conversion of the first plurality to the second plurality via the heterologous genetic circuit is at least about three times greater than in the absence of the heterologous genetic circuit; and / or (10) the plurality of gating units are configured to operate in concert to effect conversion ex vivo in a medium substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), interleukin (IL), and combinations thereof; and / or (11) (i) modulation of expression of the target gene, (ii) modulation of expression of an additional target gene, and / or (ii) modulation of expression of another target gene are necessary, but individually sufficient, to effect conversion of the first plurality to the second plurality; and / or (12) a gating unit of the plurality of gating units comprises a gene regulatory moiety that is activatable upon activation of the gating unit to induce regulation of a corresponding target gene through specific binding of the gene regulatory moiety to the corresponding target gene; and / or (13) the gene regulatory portion comprises a nucleic acid sequence that exhibits complementarity to at least a portion of the corresponding target gene; and / or (14) the gene regulatory portion comprises a guide nucleic acid (gNA) molecule capable of forming a complex with an endonuclease, the complex exhibiting specific binding to the corresponding target gene; and / or (15) The endonuclease comprises a CRISPR / Cas protein, and further optionally, (i) the CRISPR / Cas protein substantially lacks cleavage activity, and / or (ii) the CRISPR / Cas protein is coupled to a transcriptional activator, and / or (16) A system in which the plurality of distinct target genes are first endogenous.

[0241] Embodiment 13. An engineered cell comprising the system of embodiment 12.

[0242] Embodiment 14. A composition comprising the system of embodiment 12 or the engineered cells of embodiment 13.

[0243] The systems and methods of the present disclosure may be combined with or modified by other systems and methods, such as those described in International Patent Application No. PCT / US2018 / 052211, International Patent Application No. PCT / US2018 / 052211, International Patent Application No. PCT / US2023 / 028169, International Patent Application No. PCT / US2023 / 028255, and International Patent Application No. PCT / US2023 / 028033, each of which is incorporated herein by reference in its entirety.

[0244] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Accordingly, it is contemplated that the present invention also encompasses 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, and their equivalents, are intended to be encompassed thereby.

Claims

1. 1. A method for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the method comprising: culturing the first plurality ex vivo in a medium that is substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL); The method, wherein the conversion rate from said first plurality to said second plurality is at least about 3% within about 14 days after culturing, and said second plurality is CD45+.

2. The method of claim 1 , wherein the medium is substantially free of the TPO.

3. The method according to any one of claims 1 to 2, wherein the medium is substantially free of FLT3L.

4. The method of any one of claims 1 to 3, wherein the culture medium is substantially free of the IL.

5. 5. The method of claim 4, wherein the IL comprises one or more members selected from the group consisting of IL-2, IL-3, IL-7, IL-15, and IL-21.

6. The method of any one of claims 1 to 5, wherein the second plurality is CD34+CD43-CD45+.

7. 7. The method of any one of claims 1 to 6, wherein the conversion rate of the first plurality to the second plurality is at least about 3% within about 7 days after culturing, and the second plurality is CD45+ (or CD34+ and CD45+).

8. 8. The method of any one of claims 1 to 7, wherein the conversion rate of the first plurality to the second plurality is at least about 6% within about 7 days after culturing, and the second plurality is CD45+ (or CD34+ and CD45+).

9. 9. The method of any one of claims 1 to 8, wherein the additional conversion is at least about 20%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%.

10. 10. The method of any one of claims 1 to 9, wherein culturing comprises contacting the first plurality of target genes with a heterologous gene regulatory moiety to modulate the expression and / or activity levels of the target genes, wherein the target genes encode erythroid transformation-specific (ETS) transcription factors.

11. 11. The method of claim 10, wherein the ETS transcription factor comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, and / or LMO2.

12. 11. The method of claim 10, wherein the ETS transcription factor comprises one or more members selected from the group consisting of ETS1, ETV2, and / or LMO2.

13. The method of claim 10, wherein the heterologous gene regulatory moiety enhances the expression and / or activity level of the target gene.

14. 11. The method of claim 10, wherein the target gene is endogenous to the first plurality.

15. 15. The method of any one of claims 1 to 14, wherein the method further comprises the step of contacting additional target genes in the first plurality with additional heterologous gene regulatory moieties to modulate the expression and / or activity levels of the additional target genes, thereby effecting conversion, wherein the additional target genes comprise one or more members selected from the group consisting of T-box transcription factors (TBX), homeobox proteins, GATA transcription factors, and basic helix-loop-helix transcription factors (bHLH).

16. 16. The method of claim 15, wherein the heterologous gene regulatory moiety enhances the expression and / or activity level of the additional target gene.

17. 16. The method of claim 15, wherein the additional target gene is endogenous to the first plurality.

18. 18. The method of any one of claims 1 to 17, wherein each heterologous gene regulatory moiety comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule that exhibits specific binding to said target gene.

19. 19. The method of claim 18, wherein the polypeptide molecule comprises an endonuclease and the nucleic acid molecule comprises a guide nucleic acid (gNA) that forms a complex with the endonuclease.

20. 20. The method of claim 19, wherein the endonuclease comprises a CRISPR / Cas protein.

21. 21. The method of claim 20, wherein the CRISPR / Cas protein substantially lacks cleavage activity.

22. 21. The method of claim 20, wherein the CRISPR / Cas protein is coupled to a transcriptional activator.

23. The method of any one of claims 1 to 22, further comprising directing the conversion of said plurality of hematopoietic cells to lymphoid cells.

24. 24. The method of claim 23, wherein the lymphoid cell is selected from the group consisting of a T cell, an NK cell, and a B cell.

25. 1. A method of converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the method comprising: contacting the first plurality with a heterologous genetic circuit comprising a plurality of gate units; Upon activation of the heterologous genetic circuit, the plurality of gate units operate in a coordinated manner to sequentially regulate the expression levels of a plurality of distinct target genes, and the plurality of gate units: a first gating unit, the first gating unit being activatable to regulate expression of a target gene upon activation of the heterologous genetic circuit; a second gating unit that is activatable upon activation of the first gating unit to regulate expression of an additional target gene different from the target gene, whereby regulation of the expression of the target gene is followed by regulation of the expression of the additional target gene; the target gene comprises one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; Upon contact, the plurality of gate units act in concert to effect a transformation of the first plurality into the second plurality.

26. 26. The method of any one of claims 1 to 25, wherein the plurality of gating units comprises a third gating unit that is activatable upon activation of the second gating unit to regulate expression of another target gene different from the target gene and the additional target gene, whereby modulation of the expression of the additional target gene is followed by modulation of the expression of the other target gene, wherein the other target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof.

27. 27. The method of any one of claims 1 to 26, wherein the plurality of gating units comprises an additional gating unit that is activatable upon activation of a first gating unit, and activation of the additional gating unit is required to induce activation of a second gating unit, thereby inducing a time delay between the regulation of a target gene by the first gating unit and the regulation of the additional target gene by the second gating unit.

28. 28. The method of any one of claims 1 to 27, wherein the second plurality comprises hemogenic endothelial cells that are KDR- and CD34+.

29. 29. The method of claim 28, wherein the conversion rate of the first plurality to the second plurality is at least about 5% within about 14 days or about 7 days after contacting.

30. 30. The method of claim 28, wherein the conversion rate of the first plurality to the second plurality is at least about 10% within about 14 days or about 7 days after contacting.

31. 31. The method of any one of claims 1-30, wherein the second plurality comprises hematopoietic cells (HPCs) that are (i) CD34+ and CD43+, or (ii) CD34+ and CD45+.

32. 32. The method of claim 31, wherein the conversion rate of the first plurality to the second plurality is at least about 3% within about 14 days or about 7 days after contacting.

33. 32. The method of claim 31, wherein the conversion rate of the first plurality to the second plurality is at least about 6% within about 14 days or about 7 days after contacting.

34. 34. The method of any one of claims 1 to 33, wherein the contacting occurs ex vivo in a medium that is substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), interleukin (IL), and combinations thereof.

35. 35. The method of any one of claims 1 to 34, wherein a gating unit of the plurality of gating units comprises a gene regulatory moiety that is activated upon activation of the gating unit to induce regulation of a corresponding target gene through specific binding of the gene regulatory moiety to the corresponding target gene.

36. 36. The method of any one of claims 1 to 35, wherein the gene regulatory portion comprises a nucleic acid sequence that exhibits complementarity to at least a portion of the corresponding target gene.

37. 37. The method of any one of claims 1 to 36, wherein the gene regulatory moiety comprises a guide nucleic acid (gNA) molecule capable of forming a complex with an endonuclease, and wherein said complex exhibits specific binding to the corresponding target gene.

38. 38. The method of any one of claims 1 to 37, wherein the endonuclease comprises a CRISPR / Cas protein, and further optionally, (i) the CRISPR / Cas protein substantially lacks cleavage activity, and / or (ii) the CRISPR / Cas protein is coupled to a transcriptional activator.

39. 39. The method of any one of claims 1 to 38, wherein the plurality of distinct target genes is endogenous to the first plurality.

40. 1. A method of converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the method comprising: contacting the first plurality with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units act in concert to sequentially regulate the expression levels of a plurality of distinct target genes, and the plurality of gating units: a first gating unit, the first gating unit being activatable to regulate expression of a target gene upon activation of the heterologous genetic circuit; a second gating unit that is activatable upon activation of the first gating unit to regulate expression of an additional target gene different from the target gene, whereby regulation of the expression of the target gene is followed by regulation of the expression of the additional target gene; the heterologous genetic circuit: (x) the target gene comprises one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (y) the target gene comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; Upon contact, the plurality of gate units act in concert to effect a transformation of the first plurality into the second plurality.

41. 1. A method of converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), said method comprising: contacting a target gene in the first plurality with a heterologous gene regulatory moiety to modulate the expression and / or activity level of the target gene, wherein the target gene encodes an erythroid transformation-specific (ETS) transcription factor; wherein said contacting step results in conversion of said first plurality to said second plurality.

42. 1. A method of directing a plurality of stem cells to differentiation, said method comprising: contacting the plurality of stem cells with a heterologous genetic circuit comprising a plurality of gate units, wherein upon activation of the heterologous genetic circuit, the plurality of gate units operate in a coordinated manner to sequentially induce a plurality of distinct modulations of target genes in the plurality of stem cells, each of the plurality of distinct modulations being necessary but individually insufficient to direct the plurality of stem cells to differentiation, and wherein the plurality of gate units: (i) a first gating unit, the first gating unit being activatable to induce a first distinct regulation of the plurality of distinct regulations upon activation of the heterologous genetic circuit; (ii) a second gating unit, the second gating unit being activatable upon activation of the heterologous genetic circuit to induce a second distinct regulation of the plurality of distinct regulations, wherein the second distinct regulation is induced following the first distinct regulation, whereby both the first distinct regulation and the second distinct regulation enhance or reduce the expression and / or activity level of the target gene in a cell; the target gene encodes an erythroid transformation-specific (ETS) transcription factor; Upon contact, the plurality of gating units act in concert to direct the plurality of stem cells to differentiate.

43. 1. A system for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the system comprising: a medium for ex vivo culture of the first plurality, said medium being substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL); A system comprising a culture medium, wherein within about 14 days after ex vivo culture of the first plurality of individuals in the culture medium, a conversion rate from the first plurality to the second plurality of individuals is at least about 3%, and the second plurality of individuals are CD45+.

44. 44. The system of claim 43, wherein the medium is substantially free of the TPO.

45. The system according to any one of claims 43 to 44, wherein the culture medium is substantially free of FLT3L.

46. The system of any one of claims 43 to 45, wherein the culture medium is substantially free of the IL.

47. 47. The system of claim 46, wherein the IL comprises one or more members selected from the group consisting of IL-2, IL-3, IL-7, IL-15, and IL-21.

48. The system of any one of claims 43 to 47, wherein the second plurality is CD34+CD43-CD45+.

49. 49. The system of any one of claims 43 to 48, wherein the conversion rate of the first plurality to the second plurality is at least about 3% within about 7 days after culture, and the second plurality is CD45+ (or CD34+ and CD45+).

50. 50. The system of any one of claims 43 to 49, wherein the conversion rate of the first plurality to the second plurality is at least about 6% within about 7 days after culturing, and the second plurality is CD45+ (or CD34+ and CD45+).

51. 51. The system of any one of claims 43 to 50, wherein the additional conversion rate is at least about 20%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%.

52. 52. The system of any one of claims 43-51, wherein the ex vivo culturing comprises contacting the first plurality of target genes with a heterologous gene regulatory moiety to modulate the expression and / or activity levels of the target genes, wherein the target genes encode erythroid transformation-specific (ETS) transcription factors.

53. 53. The system of claim 52, wherein the ETS transcription factor comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, and / or LMO2.

54. 53. The system of claim 52, wherein the ETS transcription factor comprises one or more members selected from the group consisting of ETS1, ETV2, and / or LMO2.

55. 53. The system of claim 52, wherein the heterologous gene regulatory moiety enhances the expression and / or activity level of the target gene.

56. 53. The system of claim 52, wherein the target gene is endogenous to the first plurality.

57. 57. The system of any one of claims 43-56, wherein the system further comprises an additional heterologous gene regulatory moiety configured to bind to and modulate the expression and / or activity levels of additional target genes in the first plurality, thereby effecting conversion, wherein the additional target genes comprise one or more members selected from the group consisting of T-box transcription factors (TBX), homeobox proteins, GATA transcription factors, and basic helix-loop-helix transcription factors (bHLH).

58. 58. The system of claim 57, wherein the additional heterologous gene regulatory moiety enhances the expression and / or activity level of the additional target gene.

59. 58. The system of claim 57, wherein the additional target gene is endogenous to the first plurality.

60. 60. The system of any one of claims 43 to 59, wherein each heterologous gene regulatory moiety comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule that exhibits specific binding to a target gene.

61. 61. The system of Claim 60, wherein the polypeptide molecule comprises an endonuclease and the nucleic acid molecule comprises a guide nucleic acid (gNA) that forms a complex with the endonuclease.

62. 62. The system of claim 61 , wherein the endonuclease comprises a CRISPR / Cas protein.

63. 63. The system of claim 62, wherein the CRISPR / Cas protein substantially lacks cleavage activity.

64. 63. The system of claim 62, wherein the CRISPR / Cas protein is coupled to a transcriptional activator.

65. 1. A system for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the system comprising: A heterologous genetic circuit including a plurality of gate units, wherein upon activation of the heterologous genetic circuit, the plurality of gate units are configured to operate in concert to sequentially regulate the expression levels of a plurality of distinct target genes, and the plurality of gate units are a first gating unit, the first gating unit being activatable to regulate expression of a target gene upon activation of the heterologous genetic circuit; a second gating unit that is activatable upon activation of the first gating unit to regulate expression of an additional target gene different from the target gene, whereby regulation of the expression of the target gene is followed by regulation of the expression of the additional target gene; the target gene comprises one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; Upon contact of the first plurality by the heterologous genetic circuit, the plurality of gate units operate in coordination to effect transformation of the first plurality into the second plurality.

66. 66. The system of any one of claims 43 to 65, wherein the plurality of gating units comprises a third gating unit that is activatable upon activation of the second gating unit to regulate expression of another target gene different from the target gene and an additional target gene, whereby modulation of the expression of the additional target gene is followed by modulation of the expression of the other target gene, wherein the other target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof.

67. The plurality of gating units comprises an additional gating unit that can be activated upon activation of the first gating unit, and activation of the additional gating unit is necessary to induce activation of the second gating unit, thereby inducing a time delay between the regulation of the target gene by the first gating unit and the regulation of the additional target gene by the second gating unit. The system of any one of claims 43 to 66.

68. 68. The system of any one of claims 43 to 67, wherein the second plurality comprises hemogenic endothelial cells that are KDR- and CD34+.

69. 69. The system of claim 68, wherein the conversion rate of the first plurality to the second plurality is at least about 5% within about 14 days or about 7 days after contact.

70. 69. The system of claim 68, wherein the conversion rate of the first plurality to the second plurality is at least about 10% within about 14 days or about 7 days after contact.

71. 71. The system of any one of claims 43-70, wherein the second plurality comprises hematopoietic cells (HPCs) that are (i) CD34+ and CD43+, or (ii) CD34+ and CD45+.

72. 72. The system of claim 71, wherein the conversion rate of the first plurality to the second plurality is at least about 3% within about 14 days or about 7 days after contact.

73. 72. The system of claim 71, wherein the conversion rate of the first plurality to the second plurality is at least about 6% within about 14 days or about 7 days after contact.

74. 74. The system of any one of claims 43 to 73, wherein the multiple gating units are configured to act in concert to effect ex vivo conversion in a medium that is substantially free of one or more exogenous factors selected from the group consisting of thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), interleukin (IL), and combinations thereof.

75. 75. The system of any one of claims 43 to 74, wherein a gating unit of the plurality of gating units comprises a gene regulatory moiety that is activated upon activation of the gating unit to induce regulation of the corresponding target gene through specific binding of the gene regulatory moiety to the corresponding target gene.

76. 76. The system of any one of claims 43 to 75, wherein the gene regulatory portion comprises a nucleic acid sequence that exhibits complementarity to at least a portion of the corresponding target gene.

77. 77. The system of any one of claims 43 to 76, wherein the gene regulatory moiety comprises a guide nucleic acid (gNA) molecule capable of forming a complex with an endonuclease, and wherein the complex exhibits specific binding to a corresponding target gene.

78. 78. The system of any one of claims 43-77, wherein the endonuclease comprises a CRISPR / Cas protein, and further optionally, (i) the CRISPR / Cas protein substantially lacks cleavage activity, and / or (ii) the CRISPR / Cas protein is coupled to a transcriptional activator.

79. 79. The system of any one of claims 43 to 78, wherein a plurality of distinct target genes are endogenous to said first plurality.

80. 1. A system for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the system comprising: A heterologous genetic circuit including a plurality of gate units, wherein upon activation of the heterologous genetic circuit, the plurality of gate units are configured to operate in concert to sequentially regulate the expression levels of a plurality of distinct target genes, and the plurality of gate units are a first gating unit, the first gating unit being activatable to regulate expression of a target gene upon activation of the heterologous genetic circuit; a second gating unit that is activatable upon activation of the first gating unit to regulate expression of an additional target gene different from the target gene, whereby regulation of the expression of the target gene is followed by regulation of the expression of the additional target gene; the heterologous genetic circuit: (x) the target gene comprises one or more members selected from the group consisting of TBXT, TBX6, MIXL1, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (y) the target gene comprises one or more members selected from the group consisting of ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and the additional target gene comprises one or more members selected from the group consisting of HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; Upon contact of the first plurality by the heterologous genetic circuit, the plurality of gate units operate in coordination to effect transformation of the first plurality into the second plurality.

81. 1. A system for converting a plurality of stem cells (a first plurality) into a plurality of hematopoietic cells (a second plurality), the system comprising: a heterologous gene regulatory moiety that exhibits specific binding to a target gene of said first plurality to regulate the expression and / or activity level of the target gene, wherein said target gene encodes an erythroid transformation specific (ETS) transcription factor; wherein contacting said target gene by said heterologous gene regulatory moiety results in conversion of said first plurality to said second plurality.

82. 1. A system for directing differentiation of a plurality of stem cells, the system comprising: a heterologous genetic circuit including a plurality of gate units, wherein upon activation of the heterologous genetic circuit, the plurality of gate units operate in a coordinated manner to sequentially induce a plurality of distinct modulations of target genes in the plurality of stem cells, each of the plurality of distinct modulations being necessary but individually insufficient to direct the plurality of stem cells toward differentiation, and the plurality of gate units: (i) a first gating unit, the first gating unit being activatable to induce a first distinct regulation of the plurality of distinct regulations upon activation of the heterologous genetic circuit; (ii) a second gating unit, the second gating unit being activatable upon activation of the heterologous genetic circuit to induce a second distinct regulation of the plurality of distinct regulations, wherein the first distinct regulation is followed by the induction of the second distinct regulation, whereby both the first distinct regulation and the second distinct regulation enhance or reduce the expression and / or activity level of the target gene in a cell; the target gene encodes an erythroid transformation-specific (ETS) transcription factor; Upon contact of the plurality of stem cells with the heterologous genetic circuit, the plurality of gate units operate in coordination to direct the plurality of stem cells toward differentiation.

83. An engineered cell comprising the system of any one of claims 43 to 82.

84. A composition comprising the system of any one of claims 43 to 82, or the engineered cell of claim 83.

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