WO2024102860A1 - Cellules ingéniérisées pour une thérapie - Google Patents

Cellules ingéniérisées pour une thérapie Download PDF

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Publication number
WO2024102860A1
WO2024102860A1 PCT/US2023/079157 US2023079157W WO2024102860A1 WO 2024102860 A1 WO2024102860 A1 WO 2024102860A1 US 2023079157 W US2023079157 W US 2023079157W WO 2024102860 A1 WO2024102860 A1 WO 2024102860A1
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cell
coding sequence
cells
essential gene
gene
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PCT/US2023/079157
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Michael NEHIL
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Shoreline Biosciences, Inc.
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    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N2740/16011—Human Immunodeficiency Virus, HIV
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • modified cells include at least one gain-of- function modification within a coding region of an essential gene.
  • engineered cells can exhibit limited tumor cell killing and/or limited persistence. There remains a need for engineered cell therapies for effective treatment of cancer.
  • Some aspects of the present disclosure are based, at least in part, on methods and systems for genetically modifying NK cells and/or pluripotent stem cells (e.g., iPSCs) that are, e.g., differentiated into modified iNK cells, to include one or more gain-of-function modifications (e.g., one or more gain-of-function modifications described herein), and to include one or more loss-of-function modifications (e.g., one or more loss-of-function modifications described herein), as well as modified NK cells and/or modified pluripotent stem cells (e.g., iPSCs) that are, e.g., differentiated into modified iNK cells (and compositions of such cells) that include one or more gain-of-function modifications (e.g., one or more gain-of-function modifications described herein), and that include one or more loss-of-function modifications (e.g., one or more loss-of-function modifications described herein).
  • iPSCs pluripotent stem
  • modified NK cells and/or modified pluripotent stem cells that are, e.g., differentiated into modified iNK cells, include at least one gain-of-function modification within a coding region of an essential gene (e.g., an essential gene described herein).
  • the disclosure features a Natural Killer (NK) cell (or a progeny or daughter cell of such NK cell, or a population of such NK cells) comprising: (a) one or more genomic edits that results in loss of function of one or more of gene products; and/or (b) a genome comprising an exogenous coding sequence, wherein the exogenous coding sequence is in frame with and downstream (3’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence.
  • NK Natural Killer
  • a Natural Killer (NK) cell comprising:
  • a genome comprising a first exogenous coding sequence for Fc ⁇ RIII (CD 16) or variant thereof, a second exogenous coding sequence for a membrane bound interleukin 15 (mb IL-15), a third exogenous coding sequence for HLA-E, a fourth exogenous coding sequence for CXCR2, and a fifth exogenous coding sequence for a CAR (e.g., an EGFR CAR), wherein the exogenous coding sequences are (i) in frame with and downstream (3’) of a coding sequence of an essential gene or (ii) in frame with and upstream (5’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence.
  • the exogenous coding sequences are (i) in frame with and downstream (3’) of a coding sequence of an essential gene or (ii) in frame with and upstream (5’) of a coding sequence of an essential gene, and wherein at least part of the essential gene
  • the NK cell further comprises a genomic edit that results in loss of function of a gene encoding class II major histocompatibility complex transactivator (CIITA).
  • CIITA major histocompatibility complex transactivator
  • one or more of the exogenous coding sequences are (i) in frame with and downstream (3 ’) of a coding sequence of one or more essential genes or (ii) in frame with and upstream (5’) of a coding sequence of one or more essential genes, and wherein at least part of the essential gene comprises an exogenous coding sequence.
  • the first and second exogenous coding sequences are in frame with and downstream (3 ’) of a coding sequence of a first essential gene or are in frame with and upstream (5’) of a coding sequence of a first essential gene;
  • the third exogenous coding sequence is in frame with and downstream (3’) of a coding sequence of a second essential gene or is in frame with and upstream (5’) of a coding sequence of a second essential gene;
  • the fourth and fifth exogenous coding sequences are in frame with and downstream (3’) of a coding sequence of a third essential gene or are in frame with and upstream (5’) of a coding sequence of a third essential gene.
  • the genome comprises: (i) a first regulatory element between the coding sequence of the first essential gene and the first exogenous coding sequence; and/or (ii) a second regulatory element between the first exogenous coding sequence and the second exogenous coding sequence; and/or (iii) a third regulatory element between the second essential gene and the third exogenous coding sequence; and/or (iv) a fourth regulatory element between the third essential gene and the fourth exogenous coding sequence; and/or (v) a fifth regulatory element between the fourth exogenous coding sequence and the fifth exogenous coding sequence.
  • the first, second, third, fourth, and fifth regulatory elements are an IRES or 2A element.
  • the genome comprises a polyadenylation sequence downstream (3’) of the second exogenous coding sequence, downstream (3’) of the third exogenous coding sequence; and/or downstream (3’) of the fifth exogenous coding sequence.
  • the genome comprises a 3’ untranslated region (UTR) sequence downstream (3’) of the second exogenous coding sequence and upstream (5’) of the polyadenylation sequence; a 3’ untranslated region (UTR) sequence downstream (3’) of the third exogenous coding sequence and upstream (5’) of the polyadenylation sequence; and/or a 3’ untranslated region (UTR) sequence downstream (3’) of the fifth exogenous coding sequence and upstream (5’) of the polyadenylation sequence.
  • UTR untranslated region
  • the CD 16 is or comprises the amino acid sequence of SEQ ID NO: 184.
  • the mbIL-15 is or comprises the amino acid sequence of SEQ ID NO: 190.
  • the HLA-E is or comprises the amino acid sequence of SEQ ID N0:124,000; SEQ ID NO:118,200; SEQ ID N0:123,100; SEQ ID N0:124,300; SEQ ID NO: 124,400; SEQ ID NO: 124,500; or SEQ ID NO: 124,600.
  • the CXCR2 is or comprises the amino acid sequence of SEQ ID NO: 125,400.
  • the EGFR CAR is or comprises the amino acid sequence of SEQ ID NO: 194 or SEQ ID NO: 125,300.
  • the NK cell is an induced pluripotent stem cell (iPSC)- derived NK (iNK) cell.
  • iPSC induced pluripotent stem cell
  • iNK derived NK
  • the essential gene encodes a gene product that is required for survival and/or proliferation of the cell.
  • the essential gene is a housekeeping gene, e.g., a gene listed in Table 3.
  • the essential gene encodes glyceraldehyde 3 -phosphate dehydrogenase (GAPDH).
  • the first essential gene, the second essential gene, and/or the third essential gene encodes a gene product that is required for survival and/or proliferation of the cell.
  • the first essential gene, the second essential gene, and/or the third essential gene is a housekeeping gene, e.g., a gene listed in Table 3.
  • one or more of the first essential gene, the second essential gene, and the third essential gene encodes glyceraldehyde 3 -phosphate dehydrogenase (GAPDH).
  • GPDH glyceraldehyde 3 -phosphate dehydrogenase
  • the disclosure features an NK cell, e.g., an NK cell described herein, for use as a medicament.
  • the disclosure features an NK cell, e.g., an NK cell described herein, for use in the treatment of a disease, disorder, or condition, e.g., a tumor and/or a cancer.
  • the disclosure features a progeny or daughter cell of an NK cell described herein.
  • the disclosure features a population of NK cells comprising an NK cell described herein.
  • the population of NK cells is characterized in that, when contacted with tumor cells, a level of killing of tumor cells by the NK cells is increased relative to a reference level of killing of tumor cells by a reference population of NK cells.
  • the population of NK cells is characterized in that, when contacted with tumor cells and an antibody, a level of antibody-dependent cellular cytotoxicity (ADCC) induced by the NK cells is increased relative to a reference level of ADCC induced by a reference population of NK cells.
  • a level of persistence of the population of NK cells is increased relative to a reference level of persistence of a reference population of NK cells. In some embodiments, the level of persistence is measured following contacting with tumor cells.
  • t the reference population of NK cells does not comprise NK cells comprising a genome comprising the first exogenous coding sequence, the second exogenous coding sequence, the third exogenous coding sequence, the fourth exogenous coding sequence, and/or the fifth exogenous coding sequence.
  • the reference population of NK cells does not comprise NK cells comprising a genomic edit that results in loss of function of genes encoding ⁇ -2 microglobulin (B2M), class II major histocompatibility complex transactivator (CIITA), cytokine inducible SH2 containing protein (CISH), and/or an agonist of the TGF beta signaling pathway (e.g., transforming growth factor beta receptor II (TGF ⁇ RII)).
  • B2M microglobulin
  • CIITA class II major histocompatibility complex transactivator
  • CISH cytokine inducible SH2 containing protein
  • TGF beta signaling pathway e.g., transforming growth factor beta receptor II (TGF ⁇ RII)
  • the disclosure features a pharmaceutical composition
  • a pharmaceutical composition comprising an NK cell, a progeny or daughter cell, or a population of NK cells described herein.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
  • the disclosure features a method of treating a condition, disorder, and/or disease, comprising administering to a subject suffering therefrom an NK cell, a progeny or daughter cell, a population of NK cells, or a pharmaceutical composition described herein.
  • the subject is suffering from a tumor, e.g., a solid tumor.
  • the subject is suffering from a cancer.
  • the disclosure features a method comprising administering to a subject an NK cell, a progeny or daughter cell, a population of NK cells, or a pharmaceutical composition described herein.
  • the NK cell, the progeny or daughter cell, or the population of NK cells is allogenic to the subject. In some embodiments, the NK cell, the progeny or daughter cell, or the population of NK cells is autologous to the subject.
  • the disclosure features a method of manufacturing a genetically modified NK cell, the method comprising: (a) knocking-into the genome of an NK cell a first exogenous coding sequence for Fc ⁇ RIII (CD 16) or variant thereof, a second exogenous coding sequence for a membrane bound interleukin 15 (mbIL-15), a third exogenous coding sequence for HLA-E, a fourth exogenous coding sequence for CXCR2, and a fifth exogenous coding sequence for a CAR (e.g., an EGFR CAR), wherein the exogenous coding sequences are (i) in frame with and downstream (3 ’) of a coding sequence of an essential gene or (ii) in frame with and upstream (5’) of a coding sequence of an essential gene, and wherein at least part of the essential gene comprises an exogenous coding sequence; and (b) knocking-out genes of the NK cell encoding ⁇ -2 microglobulin (B2M
  • one or more of the exogenous coding sequences are knocked into the genome of the NK cell (i) in frame with and downstream (3’) of a coding sequence of one or more essential genes or (ii) in frame with and upstream (5’) of a coding sequence of one or more essential genes, and wherein at least part of the essential gene comprises an exogenous coding sequence.
  • knocking-in comprises contacting the NK cell with: (i) a nuclease that causes a break within an endogenous coding sequence of the essential gene, and (ii) a first donor template that comprises a knock-in cassette comprising (a) the first exogenous coding sequence and the second exogenous coding sequence in frame with and downstream (3') of an exogenous coding sequence or partial coding sequence of a first essential gene or (b) the first exogenous coding sequence and the second exogenous coding sequence in frame with and upstream (5') of an exogenous coding sequence or partial coding sequence of a first essential gene, wherein the knock-in cassette is integrated into the genome of the cell by homology- directed repair (HDR) of the break; (iii) a second donor template that comprises a knock-in cassette comprising (a) the third exogenous coding sequence in frame with and downstream (3') of an exogenous coding sequence or partial coding sequence of a second essential gene or
  • HDR homology- directed
  • the nuclease is a CRISPR/Cas nuclease and knocking-in further comprises contacting the NK cell with a guide molecule for the CRISPR/Cas nuclease.
  • knocking-out comprises contacting the NK cell with one or more nucleases that cause a break within an endogenous coding sequence of the genes.
  • the one or more nucleases are CRISPR/Cas nucleases and knocking-out further comprises contacting the NK cell with one or more guide molecules for the CRISPR/Cas nuclease.
  • the nuclease is a Cas (e.g., Cas9, Cas12a, Cas12b, Cas12c, Cas12e, CasX, or Cas ⁇ (Cas12j), or variants thereof).
  • Cas e.g., Cas9, Cas12a, Cas12b, Cas12c, Cas12e, CasX, or Cas ⁇ (Cas12j), or variants thereof).
  • the NK cell is an induced pluripotent stem cell (iPSC)- derived NK (iNK) cell.
  • iPSC induced pluripotent stem cell
  • iNK derived NK
  • the first essential gene, the second essential gene, and the third essential gene encodes a gene product that is required for survival and/or proliferation of the NK cell.
  • the first essential gene, the second essential gene, and the third essential gene is a housekeeping gene, e.g., a gene listed in Table 3.
  • the first essential gene, the second essential gene, and/or the third essential gene encodes glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
  • the disclosure features a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 125,300, or an amino acid sequence having at least 85%, 90%, 92%, 94%, 96%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 125,300.
  • the disclosure features a nucleic acid encoding the amino acid sequence of SEQ ID NO: 125,300, or an amino acid sequence having at least 85%, 90%, 92%, 94%, 96%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 125,300.
  • the disclosure features a vector (e.g., an expression vector) comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO: 125,300, or an amino acid sequence having at least 85%, 90%, 92%, 94%, 96%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 125,300.
  • a Natural Killer (NK) cell comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO: 125,300, or an amino acid sequence having at least 85%, 90%, 92%, 94%, 96%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 125,300.
  • NK Natural Killer
  • the disclosure features an NK cell comprising a vector (e.g., an expression vector) comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO: 125,300, or an amino acid sequence having at least 85%, 90%, 92%, 94%, 96%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 125,300.
  • a vector e.g., an expression vector
  • Fig. 1 shows the locations on the GAPDH gene (SEQ ID NO: 164001) where exemplary AsCpf1 (AsCas12a) guide RNAs bind, and the results of screening the exemplary guide RNAs that target the GAPDH gene three days after transfection. Results are from gDNA from living cells.
  • Fig. 2 shows results of screening the exemplary AsCpf1 (AsCas12a) guide RNAs that target the GAPDH gene, three days after transfection. Results are from gDNA from living cells.
  • Fig. 3 A shows an exemplary integration strategy that targets an essential gene according to certain embodiments of the present disclosure.
  • introducing a double strand break using CRISPR gene editing e.g., by Cas12a, Cas9, Cas12b, Cas12c, Cas12e, CasX, or Cas ⁇ (Cas12j), or a variant thereof, e.g., a variant with a high editing efficiency, e.g., capable of editing about 60% to 100% of cells in a population of cells
  • a terminal exon e.g., within about 500 bp upstream (5') of the stop codon of the essential gene
  • administering a donor plasmid with homology arms designed to mediate homology directed repair (HDR) at the cleavage site results in a population of viable cells carrying a cargo of interest integrated at the essential gene locus.
  • HDR mediate homology directed repair
  • Fig. 3B shows an exemplary integration strategy that targets the GAPDH gene according to certain embodiments of the present disclosure.
  • Fig. 3B shows a strategy wherein the GAPDH gene is modified in an induced pluripotent stem cell (iPSC)
  • iPSC induced pluripotent stem cell
  • this strategy can be applied to a variety of cell types, including primary cells, e.g., T cells, NK cells, stem cells, iPSCs, and cells differentiated from iPSCs, e.g., iPSC-derived T cells or NK cells for treating cancer.
  • primary cells e.g., T cells, NK cells, stem cells, iPSCs, and cells differentiated from iPSCs, e.g., iPSC-derived T cells or NK cells for treating cancer.
  • Fig. 3C shows an exemplary integration strategy that targets the GAPDH gene according to certain embodiments of the present disclosure.
  • the diagram shows that the only cells that should survive over time are those cells that underwent targeted integration of a cassette that restores the GAPDH locus and includes a cargo of interest, as well as unedited cells.
  • the population of unedited cells following CRISPR editing should be small if the nuclease and guide RNA are highly effective at cleaving the essential gene target site and introduce indels that significantly reduce the function of the essential gene product.
  • Fig. 3D shows an exemplary integration strategy that targets an essential gene according to certain embodiments of the present disclosure.
  • introducing a double strand break using CRISPR gene editing e.g., by Cas12a, Cas9, Cas12b, Cas12c, Cas12e, CasX, or Cas ⁇ (Cas12j), or a variant thereof, e.g., a variant with a high editing efficiency, e.g., capable of editing about 60% to 100% of cells in a population of cells
  • a 5' exon e.g., within about 500 bp downstream (3') of a start codon of the essential gene
  • administering a donor plasmid with homology arms designed to mediate homology directed repair (HDR) at the cleavage site results in a population of viable cells carrying a cargo of interest integrated at the essential gene locus.
  • HDR mediate homology directed repair
  • FIG. 4A schematically depicts a knock-in cassette cargo sequence comprising membrane-bound IL15.IL15R ⁇ (mbIL-15) coupled with a GFP sequence, for integration at a target gene as described herein.
  • Fig. 4B schematically depicts a knock-in cassette cargo sequence comprising CD 16, IL 15, and IL15R ⁇ , for integration at a target gene as described herein.
  • Fig. 4C schematically depicts a knock-in cassette cargo sequence comprising CD16 and membrane bound IL15.IL15R ⁇ (mbIL-15), for integration at a target gene as described herein.
  • FIG. 5 A depicts exemplary flow cytometry data from bulk edited iPSC populations seven days after transformation with PLA1829 (see Fig. 4 A) comprising a cargo sequence of membrane-bound IL15.IL15R ⁇ (mbIL-15) coupled with a GFP sequence inserted in the GAPDH gene using RNPs comprising RSQ22337 targeting the GAPDH gene and Cas12a (SEQ ID NO: 62), or control WT cells transformed with RNPs only, measured using ddPCR. Shown on the Y axis is IL-15R ⁇ expression, while GFP expression is shown on the X axis. [0040] Fig.
  • FIG. 5B depicts exemplary flow cytometry data from bulk edited iPSC populations seven days after transformation with PLA1832 or PLA1834 (see Fig. 4B and 4C), comprising a cargo sequence of CD 16, IL-15, and IL15R ⁇ , or comprising a cargo sequence of CD 16 and membrane-bound IL15.IL15R ⁇ (mbIL-15); inserted in the GAPDH gene using RNPs comprising RSQ22337 targeting the GAPDH gene and Cas12a (SEQ ID NO: 62), measured using ddPCR. Shown on the Y axis is IL-15R ⁇ expression, X axis is GFP expression.
  • Fig. 6A is a histogram depicting the genotypes of individual colonies following transformation as described in Fig. 5 A with PLA1829 (5 ⁇ g) and 2 ⁇ M RNPs comprising RSQ22337 targeting the GAPDH gene and Cas12a (SEQ ID NO: 62), measured using ddPCR. Shown are individual homozygous ( ⁇ 100% TI), heterozygous ( ⁇ 50% TI), or wild type ( ⁇ 0% TI) cells.
  • Fig. 6B is a histogram depicting the genotypes of individual colonies following transformation as described in Fig. 5B with PLA1832 (5 ⁇ g) and 2 ⁇ M RNPs comprising RSQ22337 targeting the GAPDH gene and Cas12a (SEQ ID NO: 62), measured using ddPCR. Shown are individual homozygous ( ⁇ 100% TI), heterozygous ( ⁇ 50% TI), or wild type ( ⁇ 0% TI) cells.
  • Fig. 6C is a histogram depicting the genotypes of individual colonies following transformation as described in Fig. 5B with PLA1834 (5 ⁇ g) and 2 ⁇ M RNPs comprising RSQ22337 targeting the GAPDH gene and Cas12a (SEQ ID NO: 62), measured using ddPCR. Shown are individual homozygous ( ⁇ 100% TI), heterozygous ( ⁇ 50% TI), or wild type ( ⁇ 0% TI) cells.
  • FIG. 7 A depicts exemplary flow cytometry data from cells comprising knock-in cargo sequences from PLA1829, PLA1832, or PLA1834 at the GAPDH gene (as described in Fig. 5A-5C) measured at day 32 of differentiation into iNKs; “WT” cells were transformed with RNPs only and were also at day 32 of differentiation into iNKs.
  • the data highlights the efficiency of integration and expression of knock-in cassettes comprising an IL-15R ⁇ protein encoding “cargo” sequence.
  • the Y axis quantifies the percentage of cells from the noted population that are expressing IL-15R ⁇ , while the X axis denotes colony genotype.
  • Fig. 7B depicts exemplary flow cytometry data from cells comprising knock-in cargo sequences from PLA1829, PLA1832, or PLA1834 at the GAPDH gene (as described in Fig. 5A-5C) measured at day 32 of differentiation into iNKs; “WT” cells were transformed with RNPs only and were also at day 32 of differentiation into iNKs.
  • the data highlights the efficiency of integration and expression of knock-in cassettes comprising a CD 16 protein encoding “cargo” sequence.
  • the Y axis quantifies the percentage of cells from the noted population that are expressing CD 16, while the X axis denotes colony genotype.
  • Fig. 7C depicts exemplary flow cytometry data from cells comprising knock-in cargo sequences from PLA1829, PLA1832, or PLA1834 at the GAPDH gene (as described in Fig. 5A-5C) measured at day 32 of differentiation into iNKs; “WT” cells were transformed with RNPs only and were also at day 32 of differentiation into iNKs.
  • the data highlights the efficiency of integration and expression of knock-in cassettes comprising an IL-15R ⁇ protein encoding “cargo” sequence.
  • the Y axis quantifies the median fluorescence intensity (MFI) of a cell population expressing IL-15R ⁇ , while the X axis denotes colony genotype.
  • MFI median fluorescence intensity
  • Fig. 7D depicts exemplary flow cytometry data from cells comprising knock-in cargo sequences from PLA1829, PLA1832, or PLA1834 at the GAPDH gene (as described in Fig. 5A-5C) measured at day 32 of differentiation into iNKs; “WT” cells were transformed with RNPs only and were also at day 32 of differentiation into iNKs.
  • the data highlights the efficiency of integration and expression of knock-in cassettes comprising a CD 16 protein encoding “cargo” sequence.
  • the Y axis quantifies the median fluorescence intensity (MFI) of a cell population expressing CD 16, while the X axis denotes colony genotype.
  • MFI median fluorescence intensity
  • Fig. 7E shows exemplary flow cytometry data from unedited (WT) cells or homozygous cells comprising knock-in cargo sequences from PLA1834 at the GAPDH locus (CD16 +/+ /mbIL-15 +/+ ).
  • the data highlights the efficiency of integration and expression of knock- in cassettes comprising a CD 16 and IL-15Ra protein encoding cargo sequence.
  • the Y axis quantifies the percentage of cells from the noted population that are expressing the selected gene, while the X axis denotes whether the selected gene is CD 16 or IL-15R ⁇ .
  • Fig. 7F depicts exemplary flow cytometry data from iNK cells comprising knock- in cargo sequences from PLA1829 or PLA1834 at the GAPDH gene, or from WT cells, before or after cytotoxicity assay in the absence of trastuzumab (Herceptin).
  • Fig. 7G depicts exemplary flow cytometry data from iNK cells comprising knock- in cargo sequences from PLA1829 or PLA1834 at the GAPDH gene, or from WT cells, before or after cytotoxicity assay in the presence of trastuzumab (Herceptin).
  • Fig. 7H depicts CD 16 surface expression from two independent flow cytometry analyses of homozygous iNK cells comprising knock-in cargo sequences from PLA1834 at the GAPDH gene (CD16 +/+ /mbIL-15 +/+ ), or unedited (WT) cells. CD 16 surface expression was assessed before or after a 2D cell killing (LDH) assay and in absence or presence of trastuzumab.
  • LDH 2D cell killing
  • the Y axis quantifies the percentage of cells from the noted population that are CD56/CD16+, while the X axis denotes whether the sample was before or after the 2D killing assay.
  • Fig. 71 depicts percent cytotoxicity demonstrated by homozygous PL A1834- transformed (CD16 +/+ /mbIL-15 +/+ ) iNK cells or unedited (WT) iNK cells in a 2D cell killing assay (LDH assay). Assays were performed in the presence or absence of 10 ⁇ g/ml trastuzumab at an E:T ratio of 1 (left) or 2.5 (right). The Y axis quantifies the percent cytotoxicity, while the X axis denotes the presence or absence of trastuzumab. *p ⁇ 0.05, **p ⁇ 0.01 (two-way ANOVA). [0053] Fig.
  • FIG. 7J depicts total cell number (left panel) of iNK cells comprising knock-in cargo sequences from PLA1829 or PLA1834 at the GAPDH gene, or of unedited (WT) iNK cells, following an in vitro persistence assay in the absence of the cytokines, IL-2 and IL-15.
  • Fold change of cells comprising a knock-in from PLA1834 relative to cells comprising a homozygous knock-in from PLA1829 is shown in the top right panel.
  • Fold change of cells comprising a homozygous knock-in from PLA1834 (CD16 +/+ /mbIL-15 +/+ ) relative to unedited (WT) cells is shown in the bottom right panel.
  • Fig. 8 A shows the results of a solid tumor killing assay.
  • Clones comprising homozygous CD 16 knock-in at the GAPDH gene were differentiated into iNK cells and functioned to reduce tumor cell spheroid size, particularly following the addition of an antibody, e.g., 10 ⁇ g/mL trastuzumab.
  • the addition of an antibody promotes antibody dependent cellular cytotoxicity (ADCC) and tumor cell killing by iNKs.
  • Control “WT” cells were bulk unedited parental clones that were electroporated without RNPs or plasmids and were at the same stage of iNK cell differentiation as test cells.
  • the Y axis depicts normalized total integrated red object intensity, a proxy for tumor cell abundance, while the X axis depicts the Effector to Target cell (E:T) ratio.
  • the IC50 for “WT” cells was an E:T ratio of 3.0, while the IC50 for SLEEK CD16 KI cells was an E:T ratio of 0.5.
  • Fig. 8B shows the results of a 3D tumor spheroid killing assay.
  • Homozygous PLA1834-transformed (CD16 +/+ /mbIL-15 +/+ ) iNK cells and unedited (WT) iNK cells were introduced to SK-OV-3 tumor cells at an E:T ratio of 10 in the absence (left panels) or presence (right panels) of 10 ⁇ g/ml trastuzumab.
  • the top panels display imaging of the tumor spheroid at 0 hours and 100 hours with visibility of the red object signal used to measure tumor cell abundance.
  • the bottom panels display spheroid size as measured via the integrated red object intensity on the Y axis and time in hours on the X axis.
  • Fig. 8C shows the results of 3D tumor spheroid killing assays.
  • Unedited (WT) iNK cells, peripheral blood NK cells, and two clones of homozygous PLA1834-transformed (CD16 +/+ /mbIL-15 +/+ ) iNK cells were used against SK-OV-3 tumor cells at varying E:T ratios.
  • 5 ng/ml exogenous IL-15 and 10 ⁇ g/ml trastuzumab was present.
  • Two independent experiments were performed for each type of cell or clone with the exception of one experiment for the peripheral blood NK cells.
  • IC50 values based on the top left panel are presented in the table in the bottom left panel and highlight the greater efficacy of the CD16 +/+ /mbIL-15 +/+ iNK cells in killing tumor cells.
  • the right panel displays IC50 values from 3D tumor spheroid killing assays for homozygous PLA1834-transformed (CD16 +/+ /mbIL-15 +/+ ) iNK cells and unedited (WT) iNK cells in the absence and presence of 10 ⁇ g/ml trastuzumab. *p ⁇ 0.05, **p ⁇ 0.01 (unpaired t-test).
  • FIG. 9 A depicts percent cytotoxicity demonstrated by mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells or unedited (WT) iNK cells in a lactate dehydrogenase (LDH) cytotoxicity assay.
  • Three different clones (A2, A4, C4) of mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells were tested.
  • Assays were performed in the presence or absence of 10 ⁇ g/ml trastuzumab and at an E:T ratio of 1.
  • the Y axis quantifies the percent cytotoxicity, while the X axis denotes the iNK cells examined. Error bars denote standard deviation.
  • Fig. 9B depicts flow cytometry data of unedited (WT) and mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells.
  • Two clones (A2, A4) of mbIL-15/CD16 (CD16 +/+ /mbIL- 15 +/+ ) DKI iNK cells were examined. Cells were pre-gated for living hCD45+ cells and further analyzed for CD16/CD56 expression. Approximately 100% of mbIL-15/CD 16 (CD16 +/+ /mbIL- 15 +/+ ) DKI iNK cells displayed high CD 16 expression compared to approximately 50% of WT iNK cells.
  • Fig. 9C is a schematic of an in vivo tumor killing assay. Mice were intraperitoneally inoculated with 0.25 x 10 6 SKOV3-luc cells, and following 2-6 days to allow for tumor establishment, mice were randomized into groups. One day later, mice intraperitoneally received 2 x 10 6 or 5 x 10 6 mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells in combination with 2.5 mpk trastuzumab.
  • mice received an additional dose of 2.5 mpk trastuzumab at 35 days (as indicated by the arrowhead) or at 21, 28, and 35 days (as indicated by the arrows) post-introduction of iNK cells. Mice were followed for up to 90 days post-introduction of iNK cells.
  • the X axis represents time since introduction of NK cells.
  • Fig. 9D shows averaged results with standard error of the mean of the in vivo tumor killing assay described in Fig. 9C.
  • Groups of mice are represented by each horizontal line.
  • the groups included mice that received mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells (DKI iNK) with trastuzumab, trastuzumab alone, or an isotype control.
  • Doses of trastuzumab are indicated by arrows and arrowheads for groups receiving a total of 4 doses or 2 doses, respectively.
  • the X axis represents time since introduction of NK cells, while the Y axis represents tumor burden as measured by bioluminescent imaging (BLI) using an in vivo imaging system (IVIS).
  • Fig. 9E shows the survival of mice subjected to the in vivo tumor killing assay described in Fig. 9C.
  • Groups of mice are represented by each horizontal line.
  • Mice dosed with mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells in combination with trastuzumab (5M DKI iNK + Tras. x 4, 2M DKI iNK + Tras x 2) had prolonged survival compared to mice dosed with trastuzumab alone.
  • the X axis represents time since introduction of NK cells, while the Y axis represents percent survival of the mice.
  • Fig. 9F shows bioluminescent imaging of mice subjected to the in vivo tumor killing assay described in Fig. 9C.
  • the treatment groups of the mice are denoted along the top of the panel, while the time since introduction of NK cells is denoted along the left side of the panel.
  • the right color scale represents the radiance (p/sec/cm 2 /sr) of the bioluminescence (from a minimum of 2.23 x 10 6 to a maximum of 5.57 x 10 7 ) as seen in the images.
  • FIG. 9G shows flow cytometry data of cells obtained by peritoneal lavage of mice subjected to the in vivo tumor killing assay described in Fig. 9C.
  • the top row shows data following sacrifice at day 90, from the mouse that received 5 x 10 6 mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells + trastuzumab according to the in vivo tumor killing assay as described in Fig. 9C.
  • the bottom row shows data following sacrifice at day 118, from the mouse that received 2 x 10 6 mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells + trastuzumab according to the in vivo tumor killing assay as described in Fig. 9C.
  • iNK cells (inset boxes in top left and bottom left) were identified by flow cytometry using the human CD46 (hCD46) marker and further analyzed for expression of CD16/CD56.
  • the data highlights that the mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells persist in vivo for at least 118 days.
  • Fig. 10A is a schematic of an in vivo tumor killing assay. Mice were intraperitoneally inoculated with 0.25 x 10 6 SKOV3-luc cells, and following 2-6 days to allow for tumor establishment, mice were randomized into groups. One day later, mice intraperitoneally received 5 x 10 6 (5M) unedited (WT) or mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells.
  • 5M unedited
  • WT unedited
  • mbIL-15/CD 16 CD16 +/+ /mbIL-15 +/+
  • mice received a single dose of 2.5 mpk trastuzumab at introduction of the iNK cells (day 0) or multiple doses of 2.5 mpk trastuzumab at 0, 7, and 14 days (as indicated by the arrows) post-introduction of iNK cells.
  • Fig. 10B shows tumor burden (median with interquartile range) for the in vivo tumor killing assay described in Fig. 10A.
  • Groups of mice are represented by each horizontal line. Each treatment group had 8 mice.
  • the groups included mice that received unedited (WT) iNK cells, mbit-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells (DKI iNK), or an isotype control.
  • the mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK clone (A2) used corresponds to the A2 clone as identified in Fig. 6C, 9 A, and 9B.
  • the X axis represents time since introduction of NK cells, while the Y axis represents tumor burden as measured by bioluminescent imaging (BLI) using an in vivo imaging system (I VIS).
  • Fig. 10C shows tumor burden (median with interquartile range) for the in vivo tumor killing assay described in Fig. 10A.
  • Groups of mice are represented by each horizontal line. Each treatment group had 8 mice.
  • the groups included mice that received unedited (WT) iNK cells + trastuzumab, mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells (DKI iNK) + trastuzumab, trastuzumab alone, or an isotype control.
  • the mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK clones (A2, A4) used correspond to the A2 and A4 clones as identified in Fig. 6C, 9 A, and 9B.
  • Dosing of trastuzumab on day 0 is indicated by the arrow.
  • the X axis represents time since introduction of NK cells, while the Y axis represents tumor burden as measured by bioluminescent imaging (BLI) using an in vivo imaging system (IVIS).
  • Fig. 10D shows the survival of mice subjected to the in vivo tumor killing assay described in Fig. 10A.
  • Groups of mice are represented by each horizontal line.
  • Mice dosed with mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells in combination with trastuzumab (DKI iNK + Tras. x 1) had significantly prolonged survival compared to mice dosed with trastuzumab alone (Trastuzumab x 1).
  • the X axis represents time since introduction of NK cells, while the Y axis represents percent survival of the mice. ****p ⁇ 0.0001 (Log-rank Mantel-Cox test).
  • Fig. 10E shows tumor burden (median with interquartile range) for the in vivo tumor killing assay described in Fig. 10A.
  • Groups of mice are represented by each horizontal line. Each treatment group had 8 mice.
  • the groups included mice that received unedited (WT) iNK cells in combination with trastuzumab (TRA x 3), mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells in combination with trastuzumab (TRA x 3), trastuzumab (TRA x 3) alone, or an isotype control.
  • the mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK clone used corresponds to the A2 clone as identified in, e.g., Fig. 6C, 9 A, and 9B.
  • Mice dosed with the mbIL-15/CD16 (CD16 +/+ /mbIL-15 +/+ ) DKI iNK cells + trastuzumab had significantly decreased tumor burden as compared to mice dosed with WT iNK cells + trastuzumab.
  • Doses of trastuzumab on day 0, 7, and 14 are indicated by the arrows.
  • the X axis represents time since introduction of NK cells, while the Y axis represents tumor burden as measured by bioluminescent imaging (BLI) using an in vivo imaging system (IVIS). ***p ⁇ 0.001 (unpaired t-test).
  • Fig. 10F shows the survival of mice subjected to the in vivo tumor killing assay described in Fig. 10A. Groups of mice are represented by each horizontal line. Mice dosed with mbIL-15/CD 16 (CD16 +/+ /mIL-15 +/+ ) DKI iNK cells in combination with trastuzumab (x3) had significantly prolonged survival compared to mice dosed with WT iNK cells in combination with trastuzumab (x3).
  • Fig. 10G shows measured tumor burden per mouse on day 33 of the in vivo tumor killing assay described in Fig. 10A.
  • the left panel depicts data for mice receiving a single dose of trastuzumab (on day 0 post-introduction of iNK cells).
  • the right panel depicts data for mice receiving three doses of trastuzumab (on days 0, 7, and 14 post-introduction of iNK cells).
  • the X axis denotes the treatment group, while the Y axis represents tumor burden as measured by bioluminescent imaging (BLI) using an in vivo imaging system (IVIS). **p ⁇ 0.01, ****p ⁇ 0.0001, ns denotes not significant (unpaired t-test).
  • Fig. 10G shows measured tumor burden per mouse on day 33 of the in vivo tumor killing assay described in Fig. 10A.
  • the left panel depicts data for mice receiving a single dose of trastuzumab (on day 0 post-introduction of i
  • FIG. 10H shows measured tumor burden per mouse on day 11 (left panel) and day 54 (right panel) of the in vivo tumor killing assay described in Fig. 10A.
  • the X axis denotes the treatment group, while the Y axis represents tumor burden as measured by bioluminescent imaging (BLI) using an in vivo imaging system (IVIS). ***p ⁇ 0.001, ****p ⁇ 0.0001 (Mann-Whitney test).
  • Fig. 101 shows representative bioluminescent imaging of mice subjected to the in vivo tumor killing assay described in Fig. 10 A.
  • the treatment groups of the mice are denoted along the top of the panel, while the time since introduction of NK cells is denoted along the left side of the panel. Each treatment group had 8 mice.
  • the table below the images displays the number of tumor free mice / total mice in the treatment group (from top of panel) at day 40 postintroduction of NK cells.
  • the bottom color scale represents the radiance (p/sec/cm 2 /sr) of the bioluminescence (from a minimum of 2.30 x 10 5 to a maximum of 3.72 x 10 7 ) as seen in the images.
  • Fig. 10J depicts flow cytometry data of cells obtained by peritoneal lavage of mice subjected to the in vivo tumor killing assay described in Fig. 10A.
  • the top row shows representative data following sacrifice at day 144 from mice that received WT iNK cells + trastuzumab (x3) according to the in vivo tumor killing assay as described in Fig. 10A.
  • the bottom row shows representative data following sacrifice at day 144 from mice that received mbIL-15/CD 16 (CD16 +/+ /mIL-15 +/+ ) DKI iNK cells + trastuzumab (x3) according to the in vivo tumor killing assay as described in Fig. 10A.
  • iNK cells (inset boxes in top left and bottom left) were identified by flow cytometry using the human CD45 (hCD45) marker and further analyzed for expression of human CD16 (hCD16) and human CD56 (hCD56).
  • the data highlights that the mbIL-15/CD 16 (CD16 +/+ /mIL-15 +/+ ) DKI iNK cells persist in vivo for at least 144 days and almost all of these cells continue to express CD 16 on their surface.
  • Fig. 11 A depicts an exemplary flow cytometry chart for a population of NK cells transduced by AAV6 comprising a GFP cargo targeted for knock-in at GAPDH at 5E4 MOI but without the addition of an RNP.
  • Fig. 11B depicts an exemplary flow cytometry chart for a population of NK cells transduced by AAV6 comprising a GFP cargo targeted for knock-in at GAPDH at 5E4 MOI and transformed with 4 ⁇ M of RNPs comprising Cas12a (SEQ NO: 62) and RSQ22337.
  • FIG. 11C depicts an exemplary flow cytometry chart for a population of NK cells transduced by AAV6 comprising a CD 19 CAR cargo targeted for knock-in at GAPDH at 5E4 MOI but without the addition of an RNP.
  • FIG. 11D depicts an exemplary flow cytometry chart for a population of NK cells transduced by AAV6 comprising a CD 19 CAR cargo targeted for knock-in at GAPDH at 5E4 MOI and transformed with 4 ⁇ M of RNPs comprising Cas12a (SEQ NO: 62) and RSQ22337.
  • Fig. 11E depicts a histogram showing genotype data derived from exemplary flow cytometry experiments on populations of NK cells transformed with GAPDH targeting RNPs (comprising Cas12a (SEQ ID NO: 62) and RSQ22337) and transduced with AAV6 comprising either a GFP cargo targeted for knock-in at GAPDH at 5E4 MOI or a CD 19 CAR cargo targeted for knock-in at GAPDH.
  • GAPDH targeting RNPs comprising Cas12a (SEQ ID NO: 62) and RSQ22337
  • AAV6 comprising either a GFP cargo targeted for knock-in at GAPDH at 5E4 MOI or a CD 19 CAR cargo targeted for knock-in at GAPDH.
  • Transgene integration efficiencies greater than 80% at the GAPDH locus were observed in each edited NK cell population.
  • Fig. 11G shows the results of an in vitro tumor killing assay, where NK cells comprising CD 19 CAR knock-in (KI) or GFP knock-in (KI) at the GAPDH gene were challenged with hematological cancer cells (Nalm6 cells). Significantly greater cytotoxicity was observed with NK cells comprising the CD 19 CAR knock-in than the GFP knock-in as assessed by BATDA release following 2 hours of co-culture at an E:T of 1. Average spontaneous BATDA release by Nalm6 cells (dashed horizontal line) and average BATDA released upon treatment with lysis buffer (solid horizontal line) provided for comparison. Each filled circle represents data from eight technical replicates from one biological sample.
  • the X axis denotes NK cell group, while the Y axis quantifies BATDA release as relative fluorescence units (RFUs) as detected by a time-resolved fluorometer.
  • Black horizontal lines represent means. ****p ⁇ 0.0001 (unpaired t-test).
  • Fig. 12A shows the results of an in vitro persistence assay of mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells and unedited (WT) iNK cells.
  • the X axis represents days since removal of exogenous cytokine support, while the Y axis represents the total number of live cells.
  • Fig. 12B shows averaged results of an in vitro persistence assay of mblL- 15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells and CD16/mbIL-15 DKI (DKI) iNK cells.
  • the X axis represents days since removal of exogenous cytokine support, while the Y axis represents the total number of live cells.
  • Fig. 13 A shows averaged results of an in vitro tumor cell killing assay where mbit-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells with or without 10 ⁇ g/ml cetuximab (CTX) were added to Detroit-562 (pharyngeal carcinoma) cells at various E:T ratios (e.g., 1:1, 5:1, 10:1).
  • CTX axis represents time in hours:minutes:seconds from initial seeding of the Detroit-562 cells, while the Y axis represents percent cytolysis as measured by electrical impedance.
  • N 3, error bars represent standard deviation.
  • Fig. 13B shows averaged results of an in vitro tumor cell killing assay where mbit-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells with or without 10 ⁇ g/ml cetuximab (CTX) were added to FaDu (pharyngeal carcinoma) cells at various E:T ratios (e.g., 1:1, 5:1, 10:1).
  • the X axis represents time in hours:minutes:seconds from initial seeding of the FaDu cells, while the Y axis represents percent cytolysis as measured by electrical impedance.
  • N 3, error bars represent standard deviation.
  • Fig. 13C shows averaged results of an in vitro tumor cell killing assay where mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells with or without 10 ⁇ g/ml cetuximab (CTX) were added to HT29 (colorectal adenocarcinoma) cells at various E:T ratios (e.g., 1 :1, 5:1, 10:1).
  • the X axis represents time in hours minutes seconds from initial seeding of the HT29 cells, while the Y axis represents percent cytolysis as measured by electrical impedance.
  • N 3, error bars represent standard deviation.
  • Fig. 13D shows averaged results of an in vitro tumor cell killing assay where mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells with or without 10 ⁇ g/ml cetuximab (CTX) were added to HCT116 (colorectal carcinoma) cells at various E:T ratios (e.g., 1:1, 5:1, 10:1).
  • CTX axis represents time in hours:minutes:seconds from initial seeding of the HCT116 cells, while the Y axis represents percent cytolysis as measured by electrical impedance.
  • N 3, error bars represent standard deviation.
  • Fig. 14A shows averaged results of an in vitro tumor cell killing assay where mbit-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells or unedited (WT) iNK cells added to HT29 (colorectal adenocarcinoma) cells at an E:T ratio of 10: 1.
  • Fig. 14B shows results of an in vitro persistence assay of mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells and unedited (WT) iNK cells.
  • DKI/DKO or WT iNK cells were co-cultured with HT-29 cells for 4 days at a 10: 1 E:T ratio.
  • the X axis denotes evaluation category (e.g., percentage of live NK cells of all cells, percentage of CD 16+ live NK cells), while the Y axis represents the percentage as measured by flow cytometry.
  • Black horizontal lines represent means.
  • FIG. 14C depicts exemplary flow cytometry data from before and after an in vitro persistence assay of mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells and unedited (WT) iNK cells.
  • DKI/DKO or WT iNK cells were cocultured with HT-29 cells for 4 days at a 1 : 1 E:T ratio.
  • FIG. 15 A shows exemplary flow cytometry data from unedited (WT) iNK cells or mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells.
  • the data highlights the efficiency of integration and expression of knock-in cassettes comprising a CD 16 and IL-15R ⁇ protein encoding cargo sequence.
  • the X axis denotes whether the selected gene is CD 16 or IL-15R ⁇ , while the Y axis quantifies the percentage of cells from the noted population that are expressing the selected gene. Horizontal lines represent group means.
  • N 1, ****p ⁇ 0.0001 (two-way ANOV A).
  • Fig. 15B shows the results of 3D tumor spheroid killing assays.
  • Unedited (WT) iNK cells or mbIL-15/CD16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells were used against SK-OV-3 tumor cells at varying E:T ratios.
  • DKI/DKO or WT iNK cells were co-cultured with the tumor spheroids and imaged every 2 hours to measure red object intensity (a proxy for tumor cell abundance) for up to 4 days. Data were normalized to the red object intensity at time of iNK cell addition.
  • IC50 values based on the left panel are presented in the table in the right panel and highlight the greater efficacy of the DKI/DKO iNK cells in killing tumor cells.
  • the X axis represents time in hours since addition of iNK cells to the tumor spheroid, while the Y axis represents normalized spheroid size as measured by red object intensity.
  • N 1, two technical replicates per cell line.
  • Fig. 15C shows the results of a 3D tumor spheroid killing assay.
  • Unedited (WT) iNK cells or mbIL-15/CD16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells were used against SK-OV-3 tumor cells at varying E:T ratios and in the presence of either 10 ⁇ g/ml trastuzumab or IgG (control).
  • DKI/DKO or WT iNK cells were co-cultured with the tumor spheroids and imaged every 2 hours to measure red object intensity (a proxy for tumor cell abundance) for up to 4 days.
  • DKI/DKO iNK cells demonstrate significantly greater antibodydependent cellular cytotoxicity (ADCC) than WT iNK cells.
  • the X axis represents treatment group, while the Y axis represents the calculated IC50 (e.g., the E:T ratio required to reduce the SK-OV-3 spheroids by 50% after 100 hours of killing). Data represents 11 independent experiments. ****p ⁇ 0.0001 (unpaired t-test).
  • Fig. 15D shows the results of an in vitro persistence assay of unedited (WT) iNK cells and mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells in the absence of the cytokines IL-2 and IL-15.
  • the X axis represents days in culture since removal of exogenous cytokine support, while the Y axis represents viability as the percentage of live cells.
  • N 1, two technical replicates per cell line, error bars represent standard deviation.
  • 15E shows the results of an in vitro SMAD2/3 phosphorylation assay of unedited (WT) iNK cells and mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells following treatment with TGF ⁇ (TGFb).
  • DKI/DKO iNK cells or WT iNK cells were plated in a cytokine starved condition and 10 ng/ml of TGF ⁇ was added to the iNK cells the following day. Cells were immediately fixed following the time indicated.
  • the X axis represents time in minutes since addition of the TGF ⁇ , while the Y axis represents normalized level of SMAD2/3 phosphorylation. Data represents one independent experiment. Dashed horizontal line represents level of SMAD2/3 phosphorylation following treatment with vehicle.
  • Fig. 15F shows the results of a 3D tumor spheroid killing.
  • Unedited (WT) iNK cells or mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells were used against SK-OV-3 tumor cells at an E:T ratio of 31.6 and in the presence of either 10 ng/ml TGF ⁇ or IgG (control).
  • DKI/DKO or WT iNK cells were co-cultured with the tumor spheroids and imaged every 2 hours to measure red object intensity (a proxy for tumor cell abundance) for up to 100 days.
  • Results for the DKI/DKO iNK cells are displayed in the left panel, while the results for the WT iNK cells are displayed in the right panel.
  • the X axis represents time in hours since addition of iNK cells to the tumor spheroid, while the Y axis represents normalized spheroid size as measured by red object intensity. N - 1.
  • Fig. 15G shows the results of an in vitro serial killing assay where unedited (WT) iNK cells or mbIL-15/CD16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells were challenged with Nalm6 tumor cells.
  • WT unedited
  • mbIL-15/CD16 CD16 +/+ /mbIL-15 +/+
  • DKI /CKI/TGF ⁇ RII DKO DKI/DKO
  • iNK cells were challenged with Nalm6 tumor cells.
  • 10 x 10 3 Nalm6 tumor cells and 2 x 10 5 iNK cells were plated together in the presence of 10 ng/ml TGF ⁇ .
  • a bolus of 5 x 10 3 Nalm6 tumor cells was added to re-challenge the iNK cell population.
  • the X axis represents the number of challenges that occurred, while the
  • Fig. 16A is a schematic of an in vivo tumor killing assay. Mice were intravenously (IV) inoculated with 0.125 x 10 6 (0.125e6) SKOV3-luc cells, and following 19 days to allow for tumor establishment, on day -2, mice were imaged to establish pre-treatment tumor burden and randomized into two groups.
  • Mice were imaged weekly using an in vivo imaging system (IVIS) to assess tumor burden over time.
  • IVIS in vivo imaging system
  • mice Groups of mice are represented by each horizontal line. Each treatment group had 4 mice.
  • the groups include mice that received mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells in combination with a single dose of trastuzumab (DKI/DKO iNK + Tras.), a single dose of trastuzumab alone (Tras. Only), or an isotype control.
  • Mice dosed with the DKI/DKO iNK cells in combination with trastuzumab had significantly decreased tumor burden as compared to mice dosed with trastuzumab alone.
  • the dose of trastuzumab on day 0 is indicated by the arrow.
  • the dashed vertical line represents the dose of iNK cells.
  • the X axis represents time in days since introduction of NK cells, while the Y axis represents tumor burden as measured by bioluminescent imaging (BLI) using an in vivo imaging system (IVIS).
  • Fig. 16C shows representative bioluminescent imaging of mice subjected to the in vivo tumor killing assay described in Fig. 16 A.
  • the treatment groups of the mice are denoted along the top of the panel, while the time since dosing with iNK cells in combination with trastuzumab or trastuzumab alone is denoted along the left side of the panel.
  • Each treatment group had 4 mice.
  • the color scale at the right represents the radiance (p/sec/cm 2 /sr) of the bioluminescence (from a minimum of 3.94 x 10 4 to a maximum of 7.02 x 10 5 ) as seen in the images.
  • Fig. 17A is a schematic of an in vivo tumor killing assay. Mice were intraperitoneally inoculated with 0.25 x 10 6 SKOV3-luc cells, and following 4 days to allow for tumor establishment, mice were randomized into groups. One day later, some groups of mice intraperitoneally received 5 x 10 6 (5E6) unedited (WT) or mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells.
  • 5E6 unedited
  • mbIL-15/CD 16 CD16 +/+ /mbIL-15 +/+
  • DKI / CISH/TGF ⁇ RII DKO DKI/DKO
  • mice received a dose of 2.5 mpk trastuzumab at 0, 7, and 14 days (as indicated by the arrows) post-introduction of iNK cells, for a total of 3 doses of trastuzumab.
  • Mice were imaged weekly using an in vivo imaging system (IVIS) to assess tumor burden over time.
  • IVIS in vivo imaging system
  • Fig. 17B shows tumor burden (median with interquartile range) for the in vivo tumor killing assay described in Fig. 17A.
  • Groups of mice are represented by each horizontal line. Each treatment group had 5-6 mice.
  • the groups included mice that received unedited iNK cells (WT iNK), mbIL-15/CD16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO iNK cells (DKI/DKO iNK), or an isotype control.
  • the X axis represents time since introduction of NK cells, while the Y axis represents tumor burden as measured by bioluminescent imaging (BLI) using an in vivo imaging system (IVIS).
  • Fig. 17C shows tumor burden (median with interquartile range) for the in vivo tumor killing assay described in Fig. 17A.
  • Groups of mice are represented by each horizontal line. Each treatment group had 5-6 mice.
  • the groups included mice that received unedited (WT) iNK cells in combination with trastuzumab (WT + Tras. x 3), mbIL-15/CD 16 (CD16 +/+ /mbIL-15 +/+ ) DKI / CISH/TGF ⁇ RII DKO (DKI/DKO) iNK cells in combination with trastuzumab (DKI DKO + Tras. x 3), trastuzumab alone, or an isotype control.
  • the X axis represents time since introduction of NK cells, while the Y axis represents tumor burden as measured by bioluminescent imaging (BLI) using an in vivo imaging system (IVIS). ****p ⁇ 0.0001 (one-way ANOVA).
  • Fig. 17D shows the survival of mice subjected to the in vivo tumor killing assay described in Fig. 17 A. Groups of mice are represented by each horizontal line. The X axis represents time since introduction of NK cells, while the Y axis represents percent survival of the mice. *p ⁇ 0.05, **p ⁇ 0.01 (Log-rank Mantel-Cox test).
  • Fig. 17E shows representative bioluminescent imaging of mice subjected to the in vivo tumor killing assay described in Fig. 17 A.
  • the treatment groups of the mice are denoted along the top of the panel, while the time since introduction of NK cells is denoted along the left side of the panel. Each treatment group had 5-6 mice.
  • the table below the images displays the number of mice with complete tumor clearance / total mice in the treatment group (from top of panel) at day 31 post-introduction of NK cells.
  • Fig. 18 depicts the percentage of WT iNK cells or B2M KO iNK cells undergoing specific lysis (y-axis, top panel) or the percentage of live iNK cells (y axis, bottom panel) following in-vitro overnight (16 hour) co-culture exposure to Human Derived Natural Killer (HDNK) cells at various E:T ratios (x axis, both panels); representative data from two HDNK donors and two independent experiments. The data show B2M KO iNKs are more susceptible to HDNK cytotoxicity.
  • Fig. 19 depicts the percentage of HDNKs expressing degranulation marker CD 107a (y-axis) following overnight 1:1 (E:T) co-culture with the noted cell type (x-axis).
  • the myelogenous leukemia cell line, K562 potently activates HDNKs.
  • Fig. 20A depicts K562 cell expression of CD47 isoform 2 (WT or S64A; represented by SEQ ID NO: 18845) driven by an EF1 ⁇ promoter and introduced via lentiviral mediated transduction.
  • K562 cells were transduced with an MOI of 10 using spinfection, stained 48 hours post-transduction, and expression was measured using flow-cytometry (Geometric Mean Fluorescence Intensity (gMFI)).
  • Fig. 20B depicts K562 cell expression of an HLA-E trimer (represented by SEQ ID NO: 18843) driven by an EF1 ⁇ promoter and introduced via lentiviral mediated transduction.
  • K562 cells were transduced with an MOI of 10 using spinfection, stained 48 hours posttransduction, and expression was measured using flow-cytometry (Geometric Mean Fluorescence Intensity (gMFI)).
  • Fig. 20C depicts K562 cell expression of an HLA-G trimer (represented by SEQ ID NO: 18841) driven by an EF1 ⁇ promoter and introduced via lentiviral mediated transduction.
  • K562 cells were transduced with an MOI of 10 using spinfection, stained 48 hours posttransduction, and expression was measured using flow-cytometry (Geometric Mean Fluorescence Intensity (gMFI)).
  • Fig. 21 A depicts the percentage of HDNKs expressing degranulation marker CD 107a (y-axis) following overnight 1:1 (E:T) co-culture with vehicle (NK alone), K562 cells, or K562 cells expressing CD47 (transduced as described in Figure 20A).
  • Fig. 21B depicts the percentage of HDNKs expressing degranulation marker CD 107a (y-axis) following overnight 1:1 (E:T) co-culture with vehicle (NK alone), K562 cells, or K562 cells expressing HLA-G (transduced as described in Figure 20B).
  • Fig. 21C depicts the percentage of HDNKs expressing degranulation marker CD 107a (y-axis) following overnight 1:1 (E:T) co-culture with vehicle (NK alone), K562 cells, or K562 cells expressing HLA-E (transduced as described in Figure 20C); representative data shown, 3 donor HDNK cells, ***p ⁇ 0.001, by ANOVA. These data indicate that expression of HLA-E can effectively shield K562 cells from activating HDNKs, reducing the percentage of HDNKs expressing CD107a.
  • Fig. 21D depicts the percentage of HDNK cells expressing degranulation marker
  • CD 107a in response to overnight 1:1 (E:T) co-culture with vehicle (NK alone), WT K562 cells, or HLA-E expressing K562 cells as a function of HDNK cell NKG2A and/or NKG2C expression status (x-axis).
  • HDNK cell populations labeled NKG2 A+ are NKG2C-
  • HDNK cell populations labeled NKG2C+ are NKG2A-
  • HDNK cell populations labeled NKG2A+ NKG2C+ represent double positive populations for these markers.
  • Fig. 21E depicts the percentage of HDNK cells expressing degranulation marker CD 107a (y-axis) in response to overnight 1:1 (E:T) co-culture with WT K562 cells or HLA-E expressing K562 cells.
  • HDNK cell populations were either NKG2A+ or NKG2A- as indicated. These data indicate that transgenic HLA-E expression (SEQ ID NO: 18843) in K562 cells can effectively inhibit NKG2A+ mediated HDNK degranulation.
  • Fig. 22A depicts the percentage of dead (y-axis) WT K562 cells or CD47 expressing K562 cells following overnight incubation with HDNKs at noted E:T ratios (x-axis); representative data shown, 3 donor HDNK cells.
  • Fig. 22B depicts the percentage of dead (y-axis) WT K562 cells or HLA-G expressing K562 cells following overnight incubation with HDNKs at noted E:T ratios (x-axis); representative data shown, 3 donor HDNK cells.
  • Fig. 22C depicts the percentage of dead (y-axis) WT K562 cells or HLA-E expressing K562 cells following overnight incubation with HDNKs at noted E:T ratios (x-axis); representative data shown, 3 donor HDNK cells. These data indicate that transgenic HLA-E protects K562 cells from HDNK cytotoxicity.
  • Fig. 23A depicts CD56 or MHC class 1 (HLA-1) surface expression in WT iPSCs at day 47 of differentiation to iNK cells; the percentage of cells expressing CD56 was -92%, and the percentage of cells expressing HLA-1 was -85%; representative data from 2 independent experiments, measured using flow cytometry.
  • HLA-1 MHC class 1
  • Fig. 23B depicts CD56 or MHC class 1 (HLA-1) surface expression in B2M KO iPSCs at day 47 of differentiation to iNK cells; the percentage of cells expressing CD56 was -95%, and the percentage of cells expressing HLA-1 was -3%; representative data from 2 independent experiments, measured using flow cytometry.
  • HLA-1 MHC class 1
  • Fig. 24A depicts the percentages of CD4+ T cells that have proliferated (y-axis) following Mixed Lymphocyte Reaction (MLR) experiments comprising PBMC responders Aph10, Aph11, Aph13, or CEL346 (x-axis) that have undergone overnight co-culture at a 2:1 (E:T) ratio (100K PBMC to 50K iNK) with the noted stimulators (vehicle (cytokine only), B2M KO iNKs, WT iNKs, or activation beads).
  • MLR Mixed Lymphocyte Reaction
  • Fig. 24B depicts the percentages of CD8+ T cells that have proliferated (y-axis) following MLR experiments comprising PBMC responders AphlO, Aph11, Aphl3, or CEL346 (x-axis) that have undergone overnight co-culture at a 2:1 (E:T) ratio (100K PBMC to SOK iNK) with the noted stimulators (vehicle (cytokine only), B2M KO iNKs, WT iNKs, or activation beads).
  • Fig. 25A depicts the percentages of CD4+ T cells that have proliferated (y-axis) following MLR experiments comprising PBMC responders AphlO, Aph11, Aphl3, or CEL346 (x-axis) that have undergone overnight co-culture at a 2:1 (E:T) ratio (100K PBMC to 50K iNK) with the noted stimulators (vehicle (cytokine only), B2M KO iNKs Clone 5 (C5), B2M KO iNKs Clone 11 (C11), B2M/CIITA DKO iNKs Clone 10 (C10), WT iNKs, or activation beads).
  • PBMC responders AphlO, Aph11, Aphl3, or CEL346 x-axis
  • the four bars above representing % Proliferated of CD4+ T cells correspond, in order from left to right, to “+ Vehicle (cytokine only)”, “+ B2M KO iPSC iNK, C5”, “+ B2M KO iPSC iNK, C11”, “+ B2M/CIITA DKO iPSC iNK, C10”, “+ WT iPSC iNK”, and “+ Activation Beads”.
  • Fig. 25B depicts the percentages of CD8+ T cells that have proliferated (y-axis) following MLR experiments comprising PBMC responders AphlO, Aph11, Aphl3, or CEL346 (x-axis) that have undergone overnight co-culture at a 2:1 (E:T) ratio (100K PBMC to SOK iNK) with the noted stimulators (vehicle (cytokine only), B2M KO iNKs Clone 5 (C5), B2M KO iNKs Clone 11 (C11), B2M/CIITA DKO iNKs Clone 10 (C10), WT iNKs, or activation beads).
  • PBMC responders AphlO, Aph11, Aphl3, or CEL346 x-axis
  • the four bars above representing % Proliferated of CD4+ T cells correspond, in order from left to right, to “+ Vehicle (cytokine only)”, “+ B2M KO iPSC iNK, C5”, “+ B2M KO iPSC iNK, C11”, “+ B2M/CIITA DKO iPSC iNK, C10”, “+ WT iPSC iNK”, and “+ Activation Beads”.
  • Fig. 25C is a representative flow cytometry plot depicting MHC-1 expression (y- axis) and MHC-II expression (x-axis) in B2M KO iPSC derived iNK cells from Clone 5 (C5). Approximately 96% of cells were negative for both MHC-1 and MHC-II.
  • Fig. 25D is a representative flow cytometry plot depicting MHC-1 expression (y- axis) and MHC-II expression (x-axis) in B2M KO iPSC derived iNK cells from Clone 11 (C11). Approximately 82% of cells were negative for both MHC-1 and MHC-II, while approximately 17% of cells were positive for MHC-II only.
  • Fig. 25E is a representative flow cytometry plot depicting MHC-1 expression (y- axis) and MHC-II expression (x-axis) in B2M/CIITA DKO iPSC derived iNK cells from Clone 10 (C10). Approximately 97% of cells were negative for both MHC-1 and MHC-II.
  • Fig. 26A depicts percentages of cell populations positive (y-axis) for transgenic markers determined by flow cytometry for various B2M KO iPSC clonal cell lines (x-axis) with transgenic CD47 expression (Clones 10 and 12), transgenic HLA-E expression (Clones 2 and 18), or transgenic HLA-G expression (Clones 1 and 16) pre-differentiation (left panel) or at day 31 post-differentiation to iNKs (right panel). A high percentage of Cl 8 derived iNKs expressed HLA-E.
  • Fig. 26B depicts RT-qPCR ddCT values (y-axis) for various B2M KO iPSC derived iNKs expressing transgenic CD47 expression (Clones 10 and 12), transgenic HLA-E expression (Clones 2 and 18), or transgenic HLA-G expression (Clone 1) at day 31 postdifferentiation to iNKs (x-axis). The majority of C18 derived iNKs robustly expressed HLA-E mRNA relative to wild type iNKs.
  • Fig. 27 A depicts the percentage of HDNKs expressing degranulation marker
  • CD 107a (y-axis) following overnight 1:1 (E:T) co-culture with WT iPSC derived iNKs (WT), B2M KO iPSC derived iNKs (B2M KO), or B2M KO iPSC derived iNKs expressing transgenic HLA-E (B2M KO + HLA-E).
  • WT WT
  • B2M KO B2M KO iPSC derived iNKs
  • B2M KO + HLA-E B2M KO + HLA-E
  • Fig. 27B depicts the percentage of HDNK cells expressing degranulation marker CD 107a (y-axis) in response to overnight 1 :1 (E:T) co-culture with WT iPSC derived iNKs (WT), B2M KO iPSC derived iNKs (B2M KO), or B2M KO iPSC derived iNKs expressing transgenic HLA-E (B2M KO + HLA-E).
  • HDNK cell populations labeled NKG2A+ are NKG2C-
  • HDNK cell populations labeled NKG2C+ are NKG2A-
  • HDNK cell populations labeled NKG2A+ NKG2C+ represent double positive populations for these markers.
  • Fig. 28A depicts the mean percentage of PBNKs expressing degranulation marker
  • CD 107a (Y axis) following overnight co-culture at an E:T ratio of 1 :1 with wild-type iNK cells (“+ WT”), B2M KO iNK cells (“+ B2M KO”), or B2M KO iNK cells expressing transgenic HLA-E with a fused HLA-G signal peptide sequence comprising VMAPRTLIL (SEQ ID NO: 18851) (“+ 1737”) or VMAPRTLVL (SEQ ID NO: 18852) (“+ 1738”).
  • PBNKs cultured alone (PBNK alone) were included as a control.
  • Fig. 28C depicts the mean percent lysis of B2M KO iNK cells or B2M KO / HLA-E KI iNK cells (“1737”) (Y axis) following overnight co-culture with PBNKs across various E:T ratios (X axis).
  • Fig. 28D depicts the mean percent lysis of B2M KO iNK cells or B2M KO / HLA-E KI iNK cells (“1738”) (Y axis) following overnight co-culture with PBNKs across various E:T ratios (X axis).
  • Fig. 29 depicts exemplary flow cytometry data of unedited (Control) iNK cells and iNK cells comprising knock-in of a CXCR2 cargo sequence (encoding SEQ ID NO: 18867) at the GAPDHgene, (CXCR2 KI). From left to right, the plots for each iNK cell type display gating for individual cells (singlets), gating for leukocytes, and CXCR2 expression.
  • Fig. 30A depicts exemplary flow cytometry data of unedited (WT) iNK cells (bottom row of plots). Control FMO (fluorescence minus one) data is also shown in the top row of plots. From left to right, the plots in each row display CD45 (Y-axis) and CD56 (X-axis) expression, CD56 (X-axis) expression in samples pre-gated for CD45+, CD 16 (X-axis) and CD56 (Y-axis) expression in samples pre-gated for CD45+, pan-KIR (X-axis) and CD56 (Y- axis) expression in samples pre-gated for CD45+, and CXCR2 (X-axis) and CD56 (Y-axis) expression in samples pre-gated for CD45+.
  • Fig. 30B depicts exemplary flow cytometry data of iNK cells comprising knock- in of a CXCR2 cargo sequence (encoding SEQ ID NO: 18867) at the GAPDH gene (CXCR2 KI).
  • the top row of plots are representative data from CXCR2 KI iNK cells at day 62 (generated as set 1 of CXCR2 KI iNK cells), and the bottom row of plots are representative data from CXCR2 KI iNK cells at day 55 (generated as set 2 of CXCR2 KI iNK cells).
  • the plots in each row display CD45 (Y-axis) and CD56 (X-axis) expression, CD56 (X-axis) expression in samples pre-gated for CD45+, CD 16 (X-axis) and CD 56 (Y-axis) expression in samples pre-gated for CD45+, pan-KIR (X-axis) and CD56 (Y-axis) expression in samples pre- gated for CD45+, and CXCR2 (X-axis) and CD56 (Y-axis) expression in samples pre-gated for CD45+.
  • Fig. 31 shows a schematic of a transwell migration assay.
  • Cells e.g., immune cells expressing a chemokine receptor of interest, e.g., CXCR2, CXCR3
  • the bottom of the upper compartment comprises a microporous membrane with pores of a selected size (e.g., 3.5 uM, 5 uM, 8 uM).
  • the lower compartment contains a media composition comprising a chemokine of interest, e.g., IL8 for CXCR2-expressing cells, CXCL11 for CXCR3 -expressing cells.
  • cells from each compartment are collected and counted (e.g., with a live cell counter, e.g., Nexcelom Celica instrument using AOPI dye).
  • a live cell counter e.g., Nexcelom Celica instrument using AOPI dye.
  • Fig. 32A shows results from a transwell migration assay as depicted in Figure 31.
  • iNK cells at day 55 were seeded into the transwell migration assay apparatus in the presence of IL8 at either 0 ng/ml or 200 ng/ml in the lower compartment. After 4 hours, cells in the lower compartment were collected and counted.
  • the X axis indicates the concentration of IL8 used in the assay, while the Y axis indicates the number of iNK cells that migrated and were counted from the lower compartment. Shown are average results from technical triplicates. Error bars represent standard deviation.
  • Fig. 32B shows results from a transwell migration assay as depicted in Figure 31.
  • the X axis indicates the concentration of IL8 used in the assay, while the Y axis indicates the number of iNK cells that migrated and were counted from the lower compartment. Shown are average results from technical triplicates. Error bars represent standard deviation.
  • Fig. 32C shows results from a transwell migration assay as depicted in Figure 31.
  • Approximately 400,000 CXCR2 KI iNK cells (generated as set 2 of CXCR2 KI iNK cells) at day 55 (D55) were seeded into the transwell migration assay apparatus in the presence of IL8 at either 0 ng/ml or 200 ng/ml in the lower compartment. After 4 hours, cells in the lower compartment were collected and counted.
  • the X axis indicates the concentration of IL8 used in the assay, while the Y axis indicates the number of iNK cells that migrated and were counted from the lower compartment. Shown are average results from technical triplicates. Error bars represent standard deviation.
  • Fig. 33 depicts exemplary flow cytometry data of unedited (WT) iNK cells and iNK cells comprising knock-in of a CXCR2 cargo sequence (encoding SEQ ID NO: 18867) at the GAPDH gene (CXCR2 KI).
  • Control FMO fluorescence minus one
  • CXCR3 expression is shown on the X-axis and CD56 expression is shown on the Y-axis.
  • Approximately 97.2% of CXCR2 KI iNK cells displayed CXCR3 expression at day 55.
  • Fig. 34A shows results from a transwell migration assay as depicted in Figure 31.
  • Approximately 400,000 unedited (WT) iNK cells at day 55 (D55) were seeded into the transwell migration assay apparatus in the presence of CXCL11 at either 0 ng/ml or 200 ng/ml in the lower compartment. After 4 hours, cells in the lower compartment were collected and counted.
  • the X axis indicates the concentration of CXCL11 used in the assay, while the Y axis indicates the number of iNK cells that migrated and were counted from the lower compartment. Shown are average results from technical triplicates. Error bars represent standard deviation.
  • Fig. 34B shows results from a transwell migration assay as depicted in Figure 31.
  • Approximately 400,000 CXCR2 KI iNK cells (generated as set 2 of CXCR2 KI iNK cells) at day 55 (D55) were seeded into the transwell migration assay apparatus in the presence of CXCL11 at either 0 ng/ml or 200 ng/ml in the lower compartment. After 4 hours, cells in the lower compartment were collected and counted.
  • the X axis indicates the concentration of CXCL11 used in the assay, while the Y axis indicates the number of iNK cells that migrated and were counted from the lower compartment. Shown are average results from technical triplicates.
  • Fig. 35A shows results from a transwell migration assay as depicted in Figure 31.
  • Approximately 400,000 unedited (WT) iNK cells or CXCR2 KI iNK cells (generated as set 2 of CXCR2 KI iNK cells) were seeded into the transwell migration assay apparatus in the presence of IL8 at a range of concentrations (0, 50, 100, 200, 400, or 800 ng/ml) in the lower compartment. After 4 hours, cells in the lower compartment were collected and counted.
  • the vertical bars represent, from left to right, results from assaying with 0, 50, 100, 200, 400, and 800 ng/ml IL8.
  • the X axis indicates the iNK cells assayed, while the Y axis indicates the number of iNK cells that migrated and were counted from the lower compartment. Shown are average results from technical triplicates. Error bars represent standard deviation.
  • Fig. 35B shows results from a transwell migration assay as depicted in Figure 31.
  • Approximately 400,000 CXCR2 KI iNK cells (generated as set 2 of CXCR2 KI iNK cells) were seeded into the transwell migration assay apparatus in the presence of IL8 at a range of concentrations (0, 1.5625, 3.125, 6.25, 12.5, 25, or 50 ng/ml) in the lower compartment. After 4 hours, cells in the lower compartment were collected and counted.
  • the X axis indicates the concentration of IL8 used in the assay, while the Y axis indicates the number of iNK cells that migrated and were counted from the lower compartment. Shown are average results from technical triplicates. Error bars represent standard deviation.
  • Fig. 35C shows results from a transwell migration assay as depicted in Figure 31.
  • Unedited (WT) iNK cells or CXCR2 KI iNK cells were seeded into the transwell migration assay apparatus in the presence of IL8 at a range of concentrations (0, 50, 100, or 200 ng/ml) in the lower compartment. After 4 hours, cells in the lower compartment were collected and counted.
  • the X axis indicates the concentration of IL8 used in the assay, while the Y axis indicates the migration ratio, calculated relative to WT.
  • the top horizontal line represents CXCR2 KI iNK cells, while the bottom graphed horizontal line represents WT iNK cells.
  • Fig. 35D shows results from a transwell migration assay as depicted in Figure 31.
  • Unedited (WT) iNK cells or CXCR2 KI iNK cells were seeded into the transwell migration assay apparatus in the presence of IL8 at a range of concentrations (0, 1.56, 3.13, 6.25, 12.5, 25, or 50 ng/ml) in the lower compartment. After 4 hours, cells in the lower compartment were collected and counted.
  • the X axis indicates the concentration of IL8 used in the assay, while the Y axis indicates the migration ratio, calculated relative to WT.
  • the top horizontal line represents CXCR2 KI iNK cells, while the bottom graphed horizontal line represents WT iNK cells.
  • Fig. 36 shows results from a tumor killing assay.
  • SKOV3 tumor cells were seeded into a microplate.
  • Unedited (parental) NK-92 cells or NK-92 cells expressing various fusion proteins (NKG2D with variable combinations of co-stimulatory domains (CD) fused to the C- terminus) were then added to the tumor cells.
  • An xCELLigence real-time cell analysis system (Agilent Technologies, Inc.) was used to track cytolysis of the SK0V3 tumor cells over time.
  • variable combinations of costimulatory domains included 2B4 CD-CD3z CD, 2B4 CD-DAP10 CD, DAP 10 CD-CD3z CD, 2B4 CD- DAP10 CD-CD3z CD, and DAP10 CD-DAP10 CD-CD3z CD.
  • the schematics are positioned to the right of the data lines that correspond to the measured data from NK-92 cells expressing the matching fusion protein.
  • Fig. 37 depicts an exemplary schematic of a chimeric antigen receptor (CAR).
  • the antigen binding domain comprises the single-chain variable fragment (scFv) from cetuximab.
  • TM transmembrane domain;
  • CD costimulatory domain.
  • Fig. 38 depicts exemplary flow cytometry from iNK cells comprising knock-in of an EGFR-CAR cargo sequence (encoding SEQ ID NO: 18866) at the GAPDH gene. As shown, >94% of cells were CD45+ CD56+, >70% of cells were pan-KIR+, and >80% of cells were EGFR-CAR+.
  • the plots display CD56 expression (X-axis) and CD45 expression (Y-axis), CD56 expression (X-axis) and pan-KIR expression (Y-axis), and forward scatter (FSC) (X-axis) and EGFR-CAR expression (as detected by anti-human IgG binding) (Y- axis).
  • Fig. 39 shows results from a tumor-killing assay.
  • EGFR+ or EGFR- SKOV3 tumor cells were seeded into a microplate. After seeding of the tumor cells, unedited (No CAR) iNK cells or iNK cells comprising knock-in of a EGFR-CAR cargo sequence (encoding SEQ ID NO: 18866) at the GAPDH gene (EGFRCAR) were added to the tumor cells.
  • An xCELLigence real-time cell analysis system (Agilent Technologies, Inc.) was used to track cytolysis of the EGFR+ or EGFR- SKOV3 tumor cells over time.
  • cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • Cancerous disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, e.g., malignant tumor growth, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state, e.g., cell proliferation associated with wound repair.
  • pathologic i.e., characterizing or constituting a disease state, e.g., malignant tumor growth
  • non-pathologic i.e., a deviation from normal but not associated with a disease state, e.g., cell proliferation associated with wound repair.
  • CRISPR/Cas nuclease refer to any CRISPR/Cas protein with DNA nuclease activity, e.g., a Cas9 or a Cas12 protein that exhibits specific association (or “targeting”) to a DNA target site, e.g., within a genomic sequence in a cell in the presence of a guide molecule.
  • the strategies, systems, and methods disclosed herein can use any combination of CRISPR/Cas nuclease disclosed herein, or known to those of ordinary skill in the art.
  • Those of ordinary skill in the art will be aware of additional CRISPR/Cas nucleases and variants suitable for use in the context of the present disclosure, and it will be understood that the present disclosure is not limited in this respect.
  • differentiated is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a blood cell.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell.
  • an iPS cell iPSC
  • iPSC can be differentiated into various more differentiated cell types, for example, a hematopoietic stem cell, a lymphocyte, and other cell types, upon treatment with suitable differentiation factors in the cell culture medium.
  • Suitable methods, differentiation factors, and cell culture media for the differentiation of pluri- and multipotent cell types into more differentiated cell types are well known to those of skill in the art.
  • the term “committed”, is applied to the process of differentiation to refer to a cell that has proceeded through a differentiation pathway to a point where, under normal circumstances, it would or will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type (other than a specific cell type or subset of cell types) nor revert to a less differentiated cell type.
  • differentiation marker refers to genes or proteins whose expression are indicative of cell differentiation occurring within a cell, such as a pluripotent cell.
  • differentiation marker genes include, but are not limited to, the following genes: CD34, CD4, CD8, CD3, CD56 (NCAM), CD49, CD45, NK cell receptor (cluster of differentiation 16 (CD16)), natural killer group-2 member D (NKG2D), CD69, NKp30, NKp44, NKp46, CD158b, FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GAT A3, GATA4, ERBB4, GATA6, H0XC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SO
  • differentiation marker gene profile or “differentiation gene profile,” “differentiation gene expression profile,” “differentiation gene expression signature,” “differentiation gene expression panel,” “differentiation gene panel,” or “differentiation gene signature” as used herein refer to expression or levels of expression of a plurality of differentiation marker genes.
  • nuclease refers to any protein that catalyzes the cleavage of phosphodiester bonds.
  • the nuclease is a DNA nuclease.
  • nuclease is a “nickase” which causes a single-strand break when it cleaves double-stranded DNA, e.g., genomic DNA in a cell.
  • nuclease causes a double-strand break when it cleaves double- stranded DNA, e.g., genomic DNA in a cell.
  • the nuclease binds a specific target site within the double-stranded DNA that overlaps with or is adjacent to the location of the resulting break. In some embodiments, the nuclease causes a double-strand break that contains overhangs ranging from 0 (blunt ends) to 22 nucleotides in both 3' and 5* orientations.
  • CRISPR/Cas nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and meganucleases are exemplary nucleases that can be used in accordance with the strategies, systems, and methods of the present disclosure.
  • the term “edited iNK cell” as used herein refers to an iNK cell which has been modified to change at least one expression product of at least one gene at some point in the development of the cell.
  • a modification can be introduced using, e.g., gene editing techniques such as CRISPR-Cas or, e.g., dominant-negative constructs.
  • an iNK cell is edited at a time point before it has differentiated into an iNK cell, e.g., at a precursor stage, at a stem cell stage, etc.
  • an edited iNK cell is compared to a non-edited iNK cell (an NK cell produced by differentiating an iPSC cell, which iPSC cell and/or iNK cell do not have modifications, e.g., genetic modifications).
  • embryonic stem cell refers to pluripotent stem cells derived from the inner cell mass of the embryonic blastocyst.
  • embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm.
  • embryonic stem cells do not contribute to the extra-embryonic membranes or the placenta, i.e., are not totipotent.
  • nucleic acids refers to a native nucleic acid (e.g., a gene, a protein coding sequence) in its natural location, e.g., within the genome of a cell.
  • essential gene refers to a gene that encodes at least one gene product that is required for survival and/or proliferation of the cell.
  • An essential gene can be a housekeeping gene that is essential for survival of all cell types or a gene that is required to be expressed in a specific cell type for survival and/or proliferation under particular culture conditions, e.g., for proper differentiation of iPS or ES cells or expansion of iPS- or ES-derived cells.
  • Loss of function of an essential gene results, in some embodiments, in a significant reduction of cell survival, e.g., of the time a cell characterized by a loss of function of an essential gene survives as compared to a cell of the same cell type but without a loss of function of the same essential gene. In some embodiments, loss of function of an essential gene results in the death of the affected cell. In some embodiments, loss of function of an essential gene results in a significant reduction of cell proliferation, e.g., in the ability of a cell to divide, which can manifest in a significant time period the cell requires to complete a cell cycle, or, in some preferred embodiments, in a loss of a cell’s ability to complete a cell cycle, and thus to proliferate at all.
  • exogenous refers to a nucleic acid (whether native or non-native) that has been artificially introduced into a man-made construct (e.g., a knock-in cassette, or a donor template) or into the genome of a cell using, for example, gene editing or genetic engineering techniques, e.g., HDR based integration techniques.
  • gene editing or genetic engineering techniques e.g., HDR based integration techniques.
  • gene editing system refers to any system having RNA-guided DNA editing activity.
  • guide molecule or “guide RNA” or “gRNA” when used in reference to a CRISPR/Cas system is any nucleic acid that promotes the specific association (or “targeting”) of a CRISPR/Cas nuclease, e.g., a Cas9 or a Cas12 protein to a DNA target site such as within a genomic sequence in a cell.
  • guide molecules are typically RNA molecules it is well known in the art that chemically modified RNA molecules including DNA/RNA hybrid molecules can be used as guide molecules.
  • hematopoietic stem cell refers to CD34-positive (CD34+) stem cells.
  • CD34-positive stem cells are capable of giving rise to mature myeloid and/or lymphoid cell types.
  • the myeloid and/or lymphoid cell types include, for example, T cells, natural killer (NK) cells and/or B cells.
  • iPS cell induced pluripotent stem cell
  • iPS cell differentiated somatic (e.g., adult, neonatal, or fetal) cell by a process referred to as reprogramming (e.g., dedifferentiation).
  • reprogrammed cells are capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. iPSCs are not found in nature.
  • iPS-derived NK cell or “iNK cell” or as used herein refers to a natural killer cell which has been produced by differentiating an iPS cell, which iPS cell may or may not have a genetic modification.
  • iPS-derived T cell or “iT cell” or as used herein refers to a T which has been produced by differentiating an iPS cell, which iPS cell may or may not have a genetic modification.
  • multipotent stem cell refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm and endoderm), but not all three germ layers. Thus, in some embodiments, a multipotent cell may also be termed a “partially differentiated cell.” Multipotent cells are well- known in the art, and examples of multipotent cells include adult stem cells, such as for example, hematopoietic stem cells and neural stem cells. In some embodiments, “multipotent” indicates that a cell may form many types of cells in a given lineage, but not cells of other lineages.
  • multipotent hematopoietic cell can form the many different types of blood cells (red, white, platelets, etc.), but it cannot form neurons. Accordingly, in some embodiments, “multipotency” refers to a state of a cell with a degree of developmental potential that is less than totipotent and pluripotent.
  • pluripotent refers to ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) or a given organism (e.g., human).
  • embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germ layers, the ectoderm, the mesoderm, and the endoderm.
  • pluripotency may be described as a continuum of developmental potencies ranging from an incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell or an induced pluripotent stem cell).
  • an incompletely or partially pluripotent cell e.g., an epiblast stem cell or EpiSC
  • EpiSC epiblast stem cell
  • a complete organism e.g., an embryonic stem cell or an induced pluripotent stem cell
  • pluripotency refers to a cell that has the developmental potential to differentiate into cells of all three germ layers (ectoderm, mesoderm, and endoderm). In some embodiments, pluripotency can be determined, in part, by assessing pluripotency characteristics of the cells.
  • pluripotency characteristics include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers including, but not limited to SSEA1 (mouse only), SSEA3/4, SSEA5, TRA1- 60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, OCT4 (also known as POU5F1), NANOG, SOX2, CD30 and/or CD50; (iv) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm); (v) teratoma formation consisting of the three somatic lineages; and (vi) formation of embryoid bodies consisting of cells from the three somatic lineages.
  • SSEA1 mouse only
  • SSEA3/4 SSEA5- 60/
  • pluripotent stem cell morphology refers to the classical morphological features of an embryonic stem cell.
  • normal embryonic stem cell morphology is characterized as small and round in shape, with a high nucleus-to- cytoplasm ratio, the notable presence of nucleoli, and typical intercell spacing.
  • polycistronic or “multicistronic” when used herein with reference to a knock-in cassette refers to the fact that the knock-in cassette can express two or more proteins from the same mRNA transcript.
  • a “bicistronic” knock-in cassette is a knock-in cassette that can express two proteins from the same mRNA transcript.
  • polynucleotide (including, but not limited to “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide”) as used herein refers to a series of nucleotide bases (also called “nucleotides”) and means any chain of two or more nucleotides.
  • polynucleotides, nucleotide sequences, nucleic acids, etc. can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • a nucleotide sequence typically carries genetic information, including, but not limited to, the information used by cellular machinery to make proteins and enzymes.
  • a nucleotide sequence and/or genetic information comprises double- or single-stranded genomic DNA, RNA, any synthetic and genetically manipulated polynucleotide, and/or sense and/or antisense polynucleotides.
  • nucleic acids contain modified bases.
  • the terms “potency” or “developmental potency” as used herein refer to the sum of all developmental options accessible to the cell (i.e., the developmental potency), particularly, for example in the context of cellular developmental potential.
  • the continuum of cell potency includes, but is not limited to, totipotent cells, pluripotent cells, multipotent cells, oligopotent cells, unipotent cells, and terminally differentiated cells.
  • prevent refers to the prevention of the disease in a mammal, e.g., in a human, including (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; or (c) preventing or delaying the onset of at least one symptom of the disease.
  • protein protein
  • peptide and “polypeptide” as used herein are used interchangeably to refer to a sequential chain of amino acids linked together via peptide bonds.
  • the terms include individual proteins, groups or complexes of proteins that associate together, as well as fragments or portions, variants, derivatives and analogs of such proteins.
  • peptide sequences are presented herein using conventional notation, beginning with the amino or N-terminus on the left, and proceeding to the carboxyl or C- terminus on the right. Standard one-letter or three-letter abbreviations can be used.
  • gene product of interest can refer to any product encoded by a gene including any polynucleotide or polypeptide.
  • the gene product is a protein which is not naturally expressed by a target cell of the present disclosure.
  • the gene product is a protein which confers a new therapeutic activity to the cell such as, but not limited to, a chimeric antigen receptor (CAR) or antigen-binding fragment thereof, a T cell receptor or antigen-binding portion thereof, a non-naturally occurring variant of Fc ⁇ RIII (CD 16), interleukin 15 (IL-15), interleukin 15 receptor (IL-15R) or a variant thereof, interleukin 12 (IL-12), interleukin- 12 receptor (IL-12R) or a variant thereof, human leukocyte antigen G (HLA-G), human leukocyte antigen E (HLA-E), leukocyte surface antigen cluster of differentiation CD47 (CD47), or any combination of two or more thereof.
  • CAR chimeric antigen receptor
  • CD47 non-naturally occurring variant of Fc ⁇ RIII
  • CD 16 non-naturally occurring variant of Fc ⁇ RIII
  • IL-15 interleukin 15
  • IL-15R interleukin 15 receptor
  • IL-12 interleuk
  • reporter gene refers to an exogenous gene that has been introduced into a cell, e.g., integrated into the genome of the cell, that confers a trait suitable for artificial selection.
  • Common reporter genes are fluorescent reporter genes that encode a fluorescent protein, e.g., green fluorescent protein (GFP) and antibiotic resistance genes that confer antibiotic resistance to cells.
  • GFP green fluorescent protein
  • reprogramming or “dedifferentiation” or “increasing cell potency” or “increasing developmental potency” as used herein refer to a method of increasing potency of a cell or dedifferentiating a cell to a less differentiated state.
  • a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state. That is, in some embodiments, a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state.
  • reprogramming refers to dedifferentiating a somatic cell, or a multipotent stem cell, into a pluripotent stem cell, also referred to as an induced pluripotent stem cell, or iPSC.
  • iPSC induced pluripotent stem cell
  • RNA-guided nuclease and “RNA-guided nuclease molecule” are used interchangeably herein.
  • the RNA-guided nuclease is a RNA-guided DNA endonuclease enzyme.
  • the RNA-guided nuclease is a CRISPR nuclease.
  • Non-limiting examples of RNA-guided nucleases are listed in Table 5 below, and the methods and compositions disclosed herein can use any combination of RNA-guided nucleases disclosed herein, or known to those of ordinary skill in the art. Those of ordinary skill in the art will be aware of additional nucleases and nuclease variants suitable for use in the context of the present disclosure, and it will be understood that the present disclosure is not limited in this respect.
  • RNA-guided nucleases e.g., Cas9 and Cas12 nucleases
  • a suitable nuclease is a Cas12a, Cas9, Cas12b, Cas12c, Cas12e, CasX, or Cas ⁇ (Cas12j), or a variant thereof (e.g., a variant with a high editing efficiency, e.g., capable of editing about 60% to 100% of cells in a population of cells) nuclease.
  • nuclease variants e.g., Cas9, Cpf1 (Cas12a, such as the Mad7 Cas12a variant), Cas12b, Cas12e, CasX, or Cas ⁇ (Cas12j) nuclease variants.
  • a nuclease is a nuclease variant, which refers to a nuclease comprising an amino acid sequence characterized by one or more amino acid substitutions, deletions, or additions as compared to the wild type amino acid sequence of the nuclease.
  • a suitable nuclease and/or nuclease variant may also include purification tags (e.g., polyhistidine tags) and/or signaling peptides, e.g., comprising or consisting of a nuclear localization signal sequence.
  • purification tags e.g., polyhistidine tags
  • signaling peptides e.g., comprising or consisting of a nuclear localization signal sequence.
  • suitable nucleases and nuclease variants are described in more detail elsewhere herein and also include those described in PCT application PCT/US2019/22374, filed March 14, 2019, and entitled “Systems and Methods for the Treatment of Hemoglobinopathies," the entire contents of which are incorporated herein by reference.
  • the RNA-guided nuclease is an Acidaminococcus sp.
  • suitable Cpf1 nuclease variants including suitable AsCpf1 variants will be known or apparent to those of ordinary skill in the art based on the present disclosure, and include, but are not limited to, the Cpf1 variants disclosed herein or otherwise known in the art.
  • the RNA-guided nuclease is a Acidaminococcus sp. Cpf1 RR variant (AsCpf1 - RR).
  • the RNA-guided nuclease is a Cpfl RVR variant.
  • suitable Cpf1 variants include those having an M537R substitution, an H800A substitution, and/or an F870L substitution, or any combination thereof (numbering scheme according to AsCpf1 wild-type sequence).
  • a human subject means a human or non-human animal.
  • a human subject can be any age (e.g., a fetus, infant, child, young adult, or adult).
  • a human subject may be at risk of or suffer from a disease, or may be in need of alteration of a gene or a combination of specific genes.
  • a subject may be a non-human animal, which may include, but is not limited to, a mammal.
  • a non-human animal is a non-human primate, a rodent (e.g., a mouse, rat, hamster, guinea pig, etc.), a rabbit, a dog, a cat, and so on.
  • the non-human animal subject is livestock, e.g., a cow, a horse, a sheep, a goat, etc.
  • the non-human animal subject is poultry, e.g., a chicken, a turkey, a duck, etc.
  • treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress, ameliorate, reduce severity of, prevent or delay the recurrence of a disease, disorder, or condition or one or more symptoms thereof, and/or improve one or more symptoms of a disease, disorder, or condition as described herein.
  • a condition includes an injury.
  • an injury may be acute or chronic (e.g., tissue damage from an underlying disease or disorder that causes, e.g., secondary damage such as tissue injury).
  • treatment e.g., in the form of an iPSC-derived NK cell or a population of iPSC-derived NK cells as described herein, may be administered to a subject after one or more symptoms have developed and/or after a disease has been diagnosed.
  • Treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease.
  • treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of genetic or other susceptibility factors).
  • treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • treatment results in improvement and/or resolution of one or more symptoms of a disease, disorder or condition.
  • variant refers to an entity such as a polypeptide or polynucleotide that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity.
  • the terms “functional variant” refer to a variant that confers the same function as the reference entity, e.g., a functional variant of a gene product of an essential gene is a variant that promotes the survival and/or proliferation of a cell. It is to be understood that a functional variant need not be functionally equivalent to the reference entity as long as it confers the same function as the reference entity.
  • Target Cells can be used to edit the genome of any cell.
  • the target cell is a stem cell, e.g., an iPS or ES cell.
  • the target cell can be an iPS- or ES-derived cell, where the genetic modification is made at any stage during the reprogramming process from donor cell to iPSC, during the iPSC stage, and/or at any stage of the process of differentiating the iPSC or ESC to a specialized cell, or even up to or at the final specialized cell state.
  • the target cell can be an iPS-derived NK cell (iNK cell) or iPS-derived T cell (iT cell) where the genetic modification is made at any stage during the reprogramming process from donor cell to iPSC, during the iPSC stage, and/or at any stage of the process of differentiating the iPSC to an iNK or iT state, e.g., at an intermediary state, such as, for example, an iPSC-derived HSC state, or even up to or at the final iNK or iT cell state.
  • iNK cell iPS-derived NK cell
  • iT cell iPS-derived T cell
  • a target cell is one or more of a long-term hematopoietic stem cell, a short term hematopoietic stem cell, a multipotent progenitor cell, a lineage restricted progenitor cell, a lymphoid progenitor cell, a myeloid progenitor cell, a common myeloid progenitor cell, an erythroid progenitor cell, a megakaryocyte erythroid progenitor cell, a retinal cell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmented epithelium cell, a trabecular meshwork cell, a cochlear hair cell, an outer hair cell, an inner hair cell, a pulmonary epithelial cell, a bronchial epithelial cell, an alveolar epithelial cell, a pulmonary epithelial progenitor cell, a striated muscle cell, a cardiac muscle
  • a target cell is a circulating blood cell, e.g., a reticulocyte, megakaryocyte erythroid progenitor (MEP) cell, myeloid progenitor cell (CMP/GMP), lymphoid progenitor (LP) cell, hematopoietic stem/progenitor cell (HSC), or endothelial cell (EC).
  • a reticulocyte e.g., a reticulocyte, megakaryocyte erythroid progenitor (MEP) cell, myeloid progenitor cell (CMP/GMP), lymphoid progenitor (LP) cell, hematopoietic stem/progenitor cell (HSC), or endothelial cell (EC).
  • MEP megakaryocyte erythroid progenitor
  • CMP/GMP myeloid progenitor cell
  • LP lymphoid progenitor
  • HSC hematopoietic stem/progenitor
  • a target cell is one or more of a bone marrow cell (e.g., a reticulocyte, an erythroid cell (e.g., erythroblast), an MEP cell, myeloid progenitor cell (CMP/GMP), LP cell, erythroid progenitor (EP) cell, HSC, multipotent progenitor (MPP) cell, endothelial cell (EC), hemogenic endothelial (HE) cell, or mesenchymal stem cell).
  • a bone marrow cell e.g., a reticulocyte, an erythroid cell (e.g., erythroblast), an MEP cell, myeloid progenitor cell (CMP/GMP), LP cell, erythroid progenitor (EP) cell, HSC, multipotent progenitor (MPP) cell, endothelial cell (EC), hemogenic endothelial (HE) cell, or mesenchymal stem cell).
  • a target cell is one or more of a myeloid progenitor cell (e.g., a common myeloid progenitor (CMP) cell or granulocyte macrophage progenitor (GMP) cell).
  • a target cell is a lymphoid progenitor cell, e.g., a common lymphoid progenitor (CLP) cell.
  • a target cell is one or more of an erythroid progenitor cell (e.g., an MEP cell).
  • a target cell is one or more of a hematopoietic stem/progenitor cell (e.g., a long term HSC (LT-HSC), short term HSC (ST-HSC), MPP cell, or lineage restricted progenitor (LRP) cell).
  • the target cell is a CD34 + cell, CD34 + CD90 + cell, CD34 + CD38" cell, CD34 + CD90 + CD49f + CD38 CD45RA- cell, CD105 + cell, CD31 + , or CD133 + cell, or a CD34 + CD90 + CD133 + cell.
  • a target cell is one or more of an umbilical cord blood CD34 + HSPC, umbilical cord venous endothelial cell, umbilical cord arterial endothelial cell, amniotic fluid CD34 + cell, amniotic fluid endothelial cell, placental endothelial cell, or placental hematopoietic CD34 + cell.
  • a target cell is one or more of a mobilized peripheral blood hematopoietic CD34 + cell (after the subject is treated with a mobilization agent, e.g., G-CSF or Plerixafor).
  • a target cell is a peripheral blood endothelial cell.
  • a target cell is a peripheral blood natural killer cell.
  • a target cell is a primary cell, e.g., a cell isolated from a human subject.
  • a target cell is an immune cell, e.g., a primary immune cell isolated from a human subject.
  • a target cell is part of a population of cells isolated from a subject, e.g., a human subject.
  • the population of cells comprises a population of immune cells isolated from a subject.
  • the population of cells comprises tumor infiltrating lymphocytes (TILs), e.g., TILs isolated from a human subject.
  • TILs tumor infiltrating lymphocytes
  • a target cell is isolated from a healthy subject, e.g., a healthy human donor.
  • a target cell is isolated from a subject having a disease or illness, e.g., a human patient in need of a treatment.
  • a target cell is an immune cell, e.g., a primary immune cell, e.g., a CD8 + T cell, a CD8 + na ⁇ ve T cell, a CD4 + central memory T cell, a CD8 + central memory T cell, a CD4 + effector memory T cell, a CD4 + effector memory T cell, a CD4 + T cell , a CD4 + stem cell memory T cell, a CD8 + stem cell memory T cell, a CD4 + helper T cell, a regulatory T cell, a cytotoxic T cell, a natural killer T cell, a CD4+ na ⁇ ve T cell, a TH 17 CD4 + T cell, a TH1 CD4 + T cell, a TH2 CD4 + T cell, a TH9 CD4 + T cell, a CD4 + Foxp3 + T cell, a CD4 + CD25 + CD127" T cell, or a CD4 + CD25 + CD127
  • a target cell is an alpha-beta T cell, a gamma-delta T cell or a Treg.
  • a target cell is macrophage.
  • a target cell is an innate lymphoid cell.
  • a target cell is a dendritic cell.
  • a target cell is a beta cell, e.g., a pancreatic beta cell.
  • a target cell is isolated from a subject having a cancer.
  • a target cell is isolated from a subject having a cancer, including but not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bile duct cancer; bladder cancer; bone cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma, medulloblastoma); bronchus cancer; carcinosarcoma (e.g
  • Wilms tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); melanoma; midline tract carcinoma; multiple endocrine neoplasia syndrome; muscle cancer; mesothelioma; nasopharynx cancer; neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian
  • a target cell is isolated from a subject having a hematological disorder. In some embodiments, a target cell is isolated form a subject having sickle cell anemia. In some embodiments, a target cell is isolated from a subject having ⁇ - thalassemia.
  • Stem cells are typically cells that have the capacity to produce unaltered daughter cells (self-renewal; cell division produces at least one daughter cell that is identical to the parent cell) and to give rise to specialized cell types (potency).
  • Stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic germ (EG) cells, germline stem (GS) cells, human mesenchymal stem cells (hMSCs), adipose tissue-derived stem cells (ADSCs), multipotent adult progenitor cells (MAPCs), multipotent adult germline stem cells (maGSCs) and unrestricted somatic stem cell (USSCs).
  • ES embryonic stem
  • EG embryonic germ
  • GS germline stem
  • ADSCs adipose tissue-derived stem cells
  • MMCs multipotent adult progenitor cells
  • maGSCs multipotent adult germline stem cells
  • USSCs unrestricted somatic stem cell
  • the stem cell may remain as a stem cell, become a precursor cell, or proceed to terminal differentiation.
  • a precursor cell is a cell that can generate a fully differentiated functional cell of at least one given cell type. Generally, precursor cells can divide. After division, a precursor cell can remain a precursor cell, or may proceed to terminal differentiation.
  • pluripotent stem cells are generally known in the art.
  • the present disclosure provides technologies (e.g., systems, compositions, methods, etc.) related to pluripotent stem cells.
  • pluripotent stem cells are stem cells that: (a) are capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) are capable of differentiating to cell types of all three germ layers (e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); and/or (c) express one or more markers of embryonic stem cells (e.g., human embryonic stem cells express Oct-4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, Sox-2, REXI, etc.).
  • SCID immunodeficient
  • human pluripotent stem cells do not show expression of differentiation markers.
  • ES cells and/or iPSCs edited using methods of the disclosure maintain their pluripotency, e.g., (a) are capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) are capable of differentiating to cell types of all three germ layers, e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); and/or (c) express one or more markers of embryonic stem cells.
  • SCID immunodeficient
  • ES cells e.g., human ES cells
  • ES cells can be derived from the inner cell mass of blastocysts or morulae.
  • ES cells can be isolated from one or more blastomeres of an embryo, e.g., without destroying the remainder of the embryo.
  • ES cells can be produced by somatic cell nuclear transfer.
  • ES cells can be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means to generate ES cells, e.g., with homozygosity in the HLA region.
  • human ES cells can be produced or derived from a zygote, blastomeres, or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce an embryonic cell.
  • Exemplary human ES cells are known in the art and include, but are not limited to, MA01, MA09, ACT-4, No. 3, Hl, H7, H9, H14 and ACT30 ES cells.
  • human ES cells regardless of their source or the particular method used to produce them, can be identified based on, e.g., (i) the ability to differentiate into cells of all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and/or (iii) ability to produce teratomas when transplanted into immunocompromised animals.
  • ES cells have been serially passaged as cell lines.
  • Induced pluripotent stem cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, such as an adult somatic cell (e.g., a fibroblast cell or other suitable somatic cell), by inducing expression of certain genes.
  • iPSCs can be derived from any organism, such as a mammal. In some embodiments, iPSCs are produced from mice, rats, rabbits, guinea pigs, goats, pigs, cows, non-human primates or humans.
  • iPSCs are similar to ES cells in many respects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, potency and/or differentiability.
  • Various suitable methods for producing iPSCs are known in the art.
  • iPSCs can be derived by transfection of certain stem cell-associated genes (such as Oct-3/4 (Pouf51) and Sox-2) into non- pluripotent cells, such as adult fibroblasts. Transfection can be achieved through viral vectors, such as retroviruses, lentiviruses, or adenoviruses.
  • Additional suitable reprogramming methods include the use of vectors that do not integrate into the genome of the host cell, e.g., episomal vectors, or the delivery of reprogramming factors directly via encoding RNA or as proteins has also been described.
  • cells can be transfected with Oct-3/4, Sox-2, Klf4, and/or c- Myc using a retroviral system or with Oct-4, Sox-2, NANOG, and/or LIN28 using a lentiviral system. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and can be isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection.
  • iPSCs from adult human cells are generated by the method described by Yu et al., Science 2007; 318(5854): 1224 or Takahashi et al., Cell 2007; 131:861-72. Numerous suitable methods for reprogramming are known to those of skill in the art, and the present disclosure is not limited in this respect.
  • a target cell for the editing and cargo integration methods described herein is an iPSC, wherein the edited iPSC is then differentiated, e.g., into an iPSC- derived immune cell.
  • the differentiated cell is an iPSC-derived immune cell.
  • the differentiated cell is an iPSC-derived iNK cell, an iPSC-derived T cell (e.g., an iPSC-derived alpha-beta T cell, gamma-delta T cell, Treg, CD4+ T cell, or CD8+ T cell), an iPSC-derived dendritic cell, or an iPSC-derived macrophage.
  • the differentiated cell is an iPSC-derived pancreatic beta cell.
  • the present disclosure provides methods of generating iNK cells (e.g., genetically modified iNK cells), e.g., derived from a genetically modified stem cell (e.g., iPSC).
  • iNK cells e.g., genetically modified iNK cells
  • iPSC genetically modified stem cell
  • genetic modifications present in an iNK cell of the present disclosure can be made at any stage during the reprogramming process from donor cell to iPSC, during the iPSC stage, and/or at any stage of the process of differentiating the iPSC to an iNK state, e.g., at an intermediary state, such as, for example, an iPSC-derived HSC state, or even up to or at the final iNK cell state.
  • one or more genomic modifications present in a genetically modified iNK cell of the present disclosure may be made at one or more different cell stages (e.g., reprogramming from donor to iPSC, differentiation of iPSC to iNK).
  • one or more genomic modifications present in a genetically modified iNK cell provided herein is made before reprogramming a donor cell to an iPSC state.
  • all edits present in a genetically modified iNK cell provided herein are made at the same time, in close temporal proximity, and/or at the same cell stage of the reprogramming/differentiation process, e.g., at the donor cell stage, during the reprogramming process, at the iPSC stage, or during the differentiation process, e.g., from iPSC to iNK.
  • two or more edits present in a genetically modified iNK cell provided herein are made at different times and/or at different cell stages of the reprogramming/differentiation process from donor cell to iPSC to iNK.
  • a first edit is made at the donor cell stage and a second (different) edit is made at the iPSC stage.
  • a first edit is made at the reprogramming stage (e.g., donor to iPSC) and a second (different) edit is made at the iPSC stage.
  • a variety of cell types can be used as a donor cell that can be subjected to reprogramming, differentiation, and/or genetic engineering strategies described herein.
  • the donor cell can be a pluripotent stem cell or a differentiated cell, e.g., a somatic cell, such as, for example, a fibroblast or a T lymphocyte.
  • donor cells are manipulated (e.g., subjected to reprogramming, differentiation, and/or genetic engineering) to generate iNK cells described herein.
  • a donor cell can be from any suitable organism.
  • the donor cell is a mammalian cell, e.g., a human cell or a non-human primate cell.
  • the donor cell is a somatic cell.
  • the donor cell is a stem cell or progenitor cell.
  • the donor cell is not or was not part of a human embryo and its derivation does not involve destruction of a human embryo.
  • a genetically modified iNK cell is derived from an iPSC, which in turn is derived from a somatic donor cell.
  • iPSC iPSC
  • a somatic donor cell Any suitable somatic cell can be used in the generation of iPSCs, and in turn, the generation of iNK cells. Suitable strategies for deriving iPSCs from various somatic donor cell types have been described and are known in the art.
  • a somatic donor cell is a fibroblast cell.
  • a somatic donor cell is a mature T cell.
  • a somatic donor cell from which an iPSC, and subsequently an iNK cell is derived, is a developmentally mature T cell (a T cell that has undergone thymic selection).
  • developmentally mature T cells a T cell that has undergone thymic selection.
  • One hallmark of developmentally mature T cells is a rearranged T cell receptor locus.
  • the TCR locus undergoes V(D)J rearrangements to generate complete V-domain exons. These rearrangements are retained throughout reprogramming of a T cells to an iPSC, and throughout differentiation of the resulting iPSC to a somatic cell.
  • a somatic donor cell is a CD8 + T cell, a CD8 + na ⁇ ve T cell, a CD4 + central memory T cell, a CD8 + central memory T cell, a CD4 + effector memory T cell, a CD4 + effector memory T cell, a CD4 + T cell, a CD4 + stem cell memory T cell, a CD8 + stem cell memory T cell, a CD4 + helper T cell, a regulatory T cell, a cytotoxic T cell, a natural killer T cell, a CD4+ na ⁇ ve T cell, a TH17 CD4 + T cell, a TH1 CD4 + T cell, a TH2 CD4 + T cell, a TH9 CD4 + T cell, a CD4 + Foxp3 + T cell, a CD4 + CD25 + CD127" T cell, or a CD4 + CD25 + CD127" Foxp3 + T cell.
  • T cells can be advantageous for the generation of iPSCs.
  • T cells can be edited with relative ease, e.g., by CRISPR-based methods or other genetic engineering methods.
  • the rearranged TCR locus allows for genetic tracking of individual cells and their daughter cells. For example, if the reprogramming, expansion, culture, and/or differentiation strategies involved in the generation of NK cells a clonal expansion of a single cell, the rearranged TCR locus can be used as a genetic marker unambiguously identifying a cell and its daughter cells. This, in turn, allows for the characterization of a cell population as truly clonal, or for the identification of mixed populations, or contaminating cells in a clonal population.
  • T cells in generating iNK cells carrying multiple edits
  • certain karyotypic aberrations associated with chromosomal translocations are selected against in T cell culture. Such aberrations can pose a concern when editing cells by CRISPR technology, and in particular when generating cells carrying multiple edits.
  • T cell derived iPSCs as a starting point for the derivation of therapeutic lymphocytes can allow for the expression of a pre-screened TCR in the lymphocytes, e.g., via selecting the T cells for binding activity against a specific antigen, e.g., a tumor antigen, reprogramming the selected T cells to iPSCs, and then deriving lymphocytes from these iPSCs that express the TCR (e.g., T cells).
  • This strategy can allow for activating the TCR in other cell types, e.g., by genetic or epigenetic strategies.
  • T cells retain at least part of their “epigenetic memory” throughout the reprogramming process, and thus subsequent differentiation of the same or a closely related cell type, such as iNK cells can be more efficient and/or result in higher quality cell populations as compared to approaches using non-related cells, such as fibroblasts, as a starting point for iNK derivation.
  • a donor cell being manipulated is one or more of a long-term hematopoietic stem cell, a short term hematopoietic stem cell, a multipotent progenitor cell, a lineage restricted progenitor cell, a lymphoid progenitor cell, a myeloid progenitor cell, a common myeloid progenitor cell, an erythroid progenitor cell, a megakaryocyte erythroid progenitor cell, a retinal cell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmented epithelium cell, a trabecular meshwork cell, a cochlear hair cell, an outer hair cell, an inner hair cell, a pulmonary epithelial cell, a bronchial epithelial cell, an alveolar epi
  • a donor cell is one or more of a circulating blood cell, e.g., a reticulocyte, megakaryocyte erythroid progenitor (MEP) cell, myeloid progenitor cell (CMP/GMP), lymphoid progenitor (LP) cell, hematopoietic stem/progenitor cell (HSC), or endothelial cell (EC).
  • a circulating blood cell e.g., a reticulocyte, megakaryocyte erythroid progenitor (MEP) cell, myeloid progenitor cell (CMP/GMP), lymphoid progenitor (LP) cell, hematopoietic stem/progenitor cell (HSC), or endothelial cell (EC).
  • a donor cell is one or more of a bone marrow cell (e.g., a reticulocyte, an erythroid cell (e.g., erythroblast), an MEP cell, myeloid progenitor cell (CMP/GMP), LP cell, erythroid progenitor (EP) cell, HSC, multipotent progenitor (MPP) cell, endothelial cell (EC), hemogenic endothelial (HE) cell, or mesenchymal stem cell).
  • a bone marrow cell e.g., a reticulocyte, an erythroid cell (e.g., erythroblast), an MEP cell, myeloid progenitor cell (CMP/GMP), LP cell, erythroid progenitor (EP) cell, HSC, multipotent progenitor (MPP) cell, endothelial cell (EC), hemogenic endothelial (HE) cell, or mesenchymal stem cell).
  • a donor cell is one or more of a myeloid progenitor cell (e.g., a common myeloid progenitor (CMP) cell or granulocyte macrophage progenitor (GMP) cell).
  • a donor cell is one or more of a lymphoid progenitor cell, e.g., a common lymphoid progenitor (CLP) cell.
  • a donor cell is one or more of an erythroid progenitor cell (e.g., an MEP cell).
  • a donor cell is one or more of a hematopoietic stem/progenitor cell (e.g., a long term HSC (LT-HSC), short term HSC (ST- HSC), MPP cell, or lineage restricted progenitor (LRP) cell).
  • the donor cell is a CD34 + cell, CD34 + CD90 + cell, CD34 + CD38" cell, CD34 + CD90 + CD49f + CD38 CD45RA- cell, CD105 + cell, CD31 + , or CD133 + cell, or a CD34 + CD90 + CD133 + cell.
  • a donor cell is one or more of an umbilical cord blood CD34 + HSPC, umbilical cord venous endothelial cell, umbilical cord arterial endothelial cell, amniotic fluid CD34 + cell, amniotic fluid endothelial cell, placental endothelial cell, or placental hematopoietic CD34 + cell.
  • a donor cell is one or more of a mobilized peripheral blood hematopoietic CD34 + cell (after the subject is treated with a mobilization agent, e.g., G-CSF or Plerixafor).
  • a donor cell is a peripheral blood endothelial cell.
  • a donor cell is a peripheral blood natural killer cell.
  • a donor cell is a dividing cell. In some embodiments, a donor cell is a non-dividing cell.
  • a genetically modified (e.g., edited) iNK cell resulting from one or more methods and/or strategies described herein are administered to a subject in need thereof, e.g., in the context of an immuno-oncology therapeutic approach.
  • donor cells, or any cells of any stage of the reprogramming, differentiating, and/or genetic engineering strategies provided herein can be maintained in culture or stored (e.g., frozen in liquid nitrogen) using any suitable method known in the art, e.g., for subsequent characterization or administration to a subject in need thereof.
  • a target cell described herein e.g., an NK cell or a stem cell (e.g., iPSC) described herein
  • a disruption e.g., a knockout
  • a target cell described herein e.g., an NK cell or a stem cell (e.g., iPSC) described herein
  • a target cell described herein e.g., an NK cell or a stem cell (e.g., iPSC) described herein
  • a gene-editing system e.g., as described herein.
  • a gene-editing system may be or comprise a CRISPR system, a zinc finger nuclease system, a TALEN, and/or a meganuclease.
  • the present disclosure provides methods suitable for high- efficiency knockout (e.g., a high proportion of a cell population comprises a knockout).
  • high-efficiency knockout results in at least 65% of the cells in a population of cells comprising a knockout (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in a population of cells comprise a knockout).
  • a knockout e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in a population of cells comprise a knockout
  • the disclosure provides a genetically engineered target cell described herein (e.g., an NK cell or a stem cell (e.g., iPSC) described herein), and/or progeny cell, comprising a disruption in TGF signaling, e.g., TGF beta signaling.
  • a genetically engineered target cell described herein e.g., an NK cell or a stem cell (e.g., iPSC) described herein
  • progeny cell comprising a disruption in TGF signaling, e.g., TGF beta signaling.
  • TGF signaling e.g., TGF beta signaling
  • TGF beta signaling inhibits or decreases the survival and/or activity of some differentiated cell types that are useful for therapeutic applications, e.g., TGF beta signaling is a negative regulator of natural killer cells, which can be used in immunotherapeutic applications.
  • TGF beta signaling is a negative regulator of natural killer cells, which can be used in immunotherapeutic applications.
  • Modifying a stem cell instead of a differentiated cell has, among others, the advantage of allowing for clonal derivation, characterization, and/or expansion of a specific genotype, e.g., a specific stem cell clone harboring a specific genetic modification (e.g., a targeted disruption of TGF ⁇ RII in the absence of any undesired (e.g., off- target) modifications).
  • a stem cell e.g., a human iPSC, is genetically engineered not to express one or more TGF ⁇ receptor, e.g., TGF ⁇ RII, or to express a dominant negative variant of a TGF ⁇ receptor, e.g., a dominant negative TGF ⁇ RII variant.
  • TGF ⁇ RII Exemplary sequences of TGF ⁇ RII are set forth in KR710923.1, NM_001024847.2, and NM_003242.5.
  • An exemplary dominant negative TGF ⁇ RII is disclosed in Immunity. 2000 Feb;12(2):171-81.
  • the disclosure provides a genetically engineered target cell described herein (e.g., an NK cell or a stem cell (e.g., iPSC) described herein), and/or progeny cell, that additionally or alternatively comprises a disruption in interleukin signaling, e.g., IL-15 signaling.
  • IL-15 is a cytokine with structural similarity to Interleukin-2 (IL-2), which binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (CD 122) and the common gamma chain (gamma-C, CD 132). Exemplary sequences of IL-15 are provided in NG_029605.2.
  • IL-15 signaling may be useful, for example, in circumstances where it is desirable to generate a differentiated cell from a pluripotent stem cell, but with certain signaling pathways (e.g., IL-15) disrupted in the differentiated cell.
  • IL-15 signaling can inhibit or decrease survival and/or activity of some types of differentiated cells, such as cells that may be useful for therapeutic applications.
  • IL-15 signaling is a negative regulator of natural killer (NK) cells.
  • CISH (encoded by the CISH gene) is downstream of the IL-15 receptor and can act as a negative regulator of IL-15 signaling in NK cells.
  • CISH refers to the Cytokine Inducible SH2 Containing Protein (see, e.g., Delconte et al., Nat Immunol. 2016 Jul;17(7):816-24; exemplary sequences for CISH are set forth as NG_023194.1).
  • disruption of CISH regulation may increase activation of Jak/STAT pathways, leading to increased survival, proliferation and/or effector functions of NK cells.
  • genetically engineered NK cells exhibit greater responsiveness to IL-15 -mediated signaling than non-genetically engineered NK cells.
  • genetically engineered NK cells exhibit greater effector function relative to non-genetically engineered NK cells.
  • a genetically engineered NK cell, stem cell and/or progeny cell additionally or alternatively, comprises a disruption and/or loss of function in one or more of B2M, NKG2A, PD1, TIGIT, ADORA2a, CIITA, HLA class II histocompatibility antigen alpha chain genes, HLA class II histocompatibility antigen beta chain genes, CD32B, or TRAC.
  • B2M ⁇ 2 microglobulin refers to a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain on the surface of nearly all nucleated cells. Exemplary sequences for B2M are set forth as NG 012920.2.
  • NKG2A natural killer group 2A refers to a protein belonging to the killer cell lectin-like receptor family, also called NKG2 family, which is a group of transmembrane proteins preferentially expressed in NK cells. This family of proteins is characterized by the type II membrane orientation and the presence of a C-type lectin domain. See, e.g., Kamiya-T et al., J Clin Invest 2019 https://doi.org/10.1172/JCI123955. Exemplary sequences for NKG2A are set forth as AF461812.1.
  • PD 1 Programmed cell death protein 1
  • CD279 cluster of differentiation 279
  • CD279 cluster of differentiation 279
  • PD1 is an immune checkpoint and guards against autoimmunity.
  • Exemplary sequences for PD1 are set forth as NM 005018.3.
  • TIGIT T cell immunoreceptor with Ig and ITIM domains
  • PVR poliovirus receptor
  • ADORA2A refers to the adenosine A2a receptor, a member of the guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) superfamily, which is subdivided into classes and subtypes.
  • G protein guanine nucleotide-binding protein
  • GPCR guanine nucleotide-binding protein-coupled receptor
  • This protein an adenosine receptor of A2A subtype, uses adenosine as the preferred endogenous agonist and preferentially interacts with the G(s) and G(olf) family of G proteins to increase intracellular cAMP levels.
  • Exemplary sequences of ADORA2a are provided in NG 052804.1.
  • CIIT A refers to the protein located in the nucleus that acts as a positive regulator of class II major histocompatibility complex gene transcription, and is referred to as the “master control factor” for the expression of these genes.
  • the protein also binds GTP and uses GTP binding to facilitate its own transport into the nucleus. Mutations in this gene have been associated with bare lymphocyte syndrome type II (also known as hereditary MHC class II deficiency or HLA class Il-deficient combined immunodeficiency), increased susceptibility to rheumatoid arthritis, multiple sclerosis, and possibly myocardial infarction.
  • two or more HLA class II histocompatibility antigen alpha chain genes and/or two or more HLA class II histocompatibility antigen beta chain genes are disrupted, e.g., knocked out, e.g., by genomic editing.
  • two or more HLA class II histocompatibility antigen alpha chain genes selected from HLA-DQA1, HLA-DRA, HLA-DPA1, HL A- DM A, HLA-DQA2, and HLA-DOA are disrupted, e.g., knocked out.
  • two or more HLA class II histocompatibility antigen beta chain genes selected from HLA-DMB, HLA-DOB, HLA-DPB1, HLA-DQB1, HLA-DQB3, HLA-DQB2, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 are disrupted, e.g., knocked out. See, e.g., Crivello et al., J Immunol January 2019, ji 1800257; DOI: https://doi.org/10.4049/jimmunol.1800257, the entire contents of which are incorporated herein by reference.
  • CD32B cluster of differentiation 32B refers to a low affinity immunoglobulin gamma Fc region receptor Il-b protein that, in humans, is encoded by the FCGR2B gene. See, e.g., Rankin-CT et al., Blood 2006 108(7):2384-91, the entire contents of which are incorporated herein by reference.
  • TRAC refers to the T-cell receptor alpha subunit (constant), encoded by the TRAC locus.
  • a target cell described herein e.g., an NK cell or a stem cell (e.g., iPSC) described herein
  • a target cell described herein can additionally be genetically engineered to comprise a genetic modification that leads to expression of one or more gene products of interest described herein using, e.g., a gene-editing system, e.g., as described herein.
  • a gene-editing system may be or comprise a CRISPR system, a zinc finger nuclease system, a TALEN, and/or a meganuclease.
  • a cell is produced by a method of the present disclosure, e.g., a method that comprises contacting the cell with a nuclease that causes a break within an endogenous coding sequence of an essential gene in the cell wherein the essential gene encodes at least one gene product that is required for survival and/or proliferation of the cell.
  • the cell is also contacted with a donor template that comprises a knock-in cassette comprising an exogenous coding sequence for a gene product of interest in frame with and downstream (3') of an exogenous coding sequence or partial coding sequence of the essential gene.
  • the knock-in cassette is integrated into the genome of the cell by homology-directed repair (HDR) of the break, resulting in a genome-edited cell that expresses the gene product of interest and the gene product encoded by the essential gene that is required for survival and/or proliferation of the cell, or a functional variant thereof.
  • HDR homology-directed repair
  • a cell is contacted with a donor template that comprises a knock-in cassette comprising an exogenous coding sequence for a gene product of interest in frame with and upstream (5') of an exogenous coding sequence or partial coding sequence of the essential gene.
  • the cell comprises a genome with an exogenous coding sequence for a gene product of interest in frame with and downstream (3') of a coding sequence of an essential gene, wherein the essential gene encodes a gene product that is required for survival and/or proliferation of the cell.
  • the cell comprises a genome with an exogenous coding sequence for a gene product of interest in frame with and upstream (5') of a coding sequence of an essential gene, wherein the essential gene encodes a gene product that is required for survival and/or proliferation of the cell.
  • the cell comprises a genomic modification, wherein the genomic modification comprises an insertion of an exogenous knock-in cassette within an endogenous coding sequence of an essential gene in the cell’s genome, wherein the essential gene encodes a gene product that is required for survival and/or proliferation of the cell, wherein the knock-in cassette comprises an exogenous coding sequence for a gene product of interest in frame with and downstream ( 3') of an exogenous coding sequence or partial coding sequence encoding the gene product of the essential gene, or a functional variant thereof, and wherein the cell expresses the gene product of interest and the gene product encoded by the essential gene that is required for survival and/or proliferation of the cell, or a functional variant thereof.
  • the gene product of interest and the gene product encoded by the essential gene are expressed from the endogenous promoter of the essential gene.
  • the present disclosure provides a donor template comprising a knock-in cassette with an exogenous coding sequence for a gene product of interest in frame with and downstream (3') of an exogenous coding sequence or partial coding sequence of an essential gene, wherein the essential gene encodes a gene product that is required for survival and/or proliferation of the cell.
  • the present disclosure provides an impetus for designing donor templates comprising a knock-in cassette with an exogenous coding sequence for a gene product of interest in frame with and upstream (5') of an exogenous coding sequence or partial coding sequence of an essential gene, wherein the essential gene encodes a gene product that is required for survival and/or proliferation of the cell; see e.g., Fig. 3D.
  • the donor template is for use in editing the genome of a cell by homology-directed repair (HDR).
  • HDR homology-directed repair
  • Donor template design is described in detail in the literature, for instance in PCT Publication No. W02016/073990A1.
  • Donor templates can be single-stranded or doublestranded and can be used to facilitate HDR-based repair of double-strand breaks (DSBs), and are particularly useful for inserting a new sequence into the target sequence, or replacing the target sequence altogether.
  • the donor template is a donor DNA template.
  • the donor DNA template is double-stranded.
  • donor templates generally include regions that are homologous to regions of DNA within or near (e.g., flanking or adjoining) a target sequence to be cleaved. These homologous regions are referred to herein as “homology arms,” and are illustrated schematically below relative to the knock-in cassette (which may be separated from one or both of the homology arms by additional spacer sequences that are not shown):
  • the homology arms can have any suitable length (including 0 nucleotides if only one homology arm is used), and 5' and 3' homology arms can have the same length, or can differ in length.
  • the selection of appropriate homology arm lengths can be influenced by a variety of factors, such as the desire to avoid homologies or microhomologies with certain sequences such as Alu repeats or other very common elements.
  • a 5' homology arm can be shortened to avoid a sequence repeat element.
  • a 3' homology arm can be shortened to avoid a sequence repeat element.
  • both the 5' and the 3' homology arms can be shortened to avoid including certain sequence repeat elements.
  • a donor template can be a nucleic acid vector, such as a viral genome or circular double-stranded DNA, e.g., a plasmid.
  • Nucleic acid vectors comprising donor templates can include other coding or non-coding elements.
  • a donor template nucleic acid can be delivered as part of a viral genome (e.g., in an AAV, adenoviral, Sendai virus, or lentiviral genome) that includes certain genomic backbone elements (e.g., inverted terminal repeats, in the case of an AAV genome).
  • a donor template is comprised in a plasmid that has not been linearized.
  • a donor template is comprised in a plasmid that has been linearized. In some embodiments, a donor template is comprised within a linear dsDNA fragment.
  • a donor template nucleic acid can be delivered as part of an AAV genome. In some embodiments, a donor template nucleic acid can be delivered as a single stranded oligo donor (ssODN), for example, as a long multi-kb ssODN derived from ml 3 phage synthesis, or alternatively, short ssODNs, e.g., that comprise small genes of interest, tags, and/or probes.
  • ssODN single stranded oligo donor
  • a donor template nucleic acid can be delivered as a DoggyboneTM DNA (dbDNATM) template. In some embodiments, a donor template nucleic acid can be delivered as a DNA minicircle. In some embodiments, a donor template nucleic acid can be delivered as an Integration-deficient Lentiviral Particle (IDLV). In some embodiments, a donor template nucleic acid can be delivered as a MMLV-derived retrovirus. In some embodiments, a donor template nucleic acid can be delivered as a piggyBacTM sequence. In some embodiments, a donor template nucleic acid can be delivered as a replicating EBNA1 episome.
  • IDLV Integration-deficient Lentiviral Particle
  • the 5' homology arm may be about 25 to about 1 ,000 base pairs in length, e.g., at least about 100, 200, 400, 600, or 800 base pairs in length. In certain embodiments, the 5' homology arm comprises about 50 to 800 base pairs, e.g., 100 to 800, 200 to 800, 400 to 800, 400 to 600, or 600 to 800 base pairs. In certain embodiments, the 3' homology arm may be about 25 to about 1,000 base pairs in length, e.g., at least about 100, 200, 400, 600, or 800 base pairs in length.
  • the 3' homology arm comprises about 50 to 800 base pairs, e.g., 100 to 800, 200 to 800, 400 to 800, 400 to 600, or 600 to 800 base pairs.
  • the 5' and 3' homology arms are symmetrical in length. In certain embodiments, the 5' and 3' homology arms are asymmetrical in length.
  • a 5' homology arm is less than about 3,000 base pairs, less than about 2,900 base pairs, less than about 2,800 base pairs, less than about 2,700 base pairs, less than about 2,600 base pairs, less than about 2,500 base pairs, less than about 2,400 base pairs, less than about 2,300 base pairs, less than about 2,200 base pairs, less than about 2,100 base pairs, less than about 2,000 base pairs, less than about 1,900 base pairs, less than about 1,800 base pairs, less than about 1,700 base pairs, less than about 1,600 base pairs, less than about 1,500 base pairs, less than about 1,400 base pairs, less than about 1,300 base pairs, less than about 1,200 base pairs, less than about 1,100 base pairs, less than about 1,000 base pairs, less than about 900 base pairs, less than about 800 base pairs, less than about 700 base pairs, less than about 600 base pairs, less than about 500 base pairs, or less than about 400 base pairs.
  • a 5' homology arm is less than about 1,000 base pairs, less than about 900 base pairs, less than about 800 base pairs, is less than about 700 base pairs, less than about 600 base pairs, less than about 500 base pairs, less than about 400 base pairs, or less than about 300 base pairs.
  • a 5' homology arm is about 400-600 base pairs, e.g., about 500 base pairs.
  • a 3' homology arm is less than about 3,000 base pairs, less than about 2,900 base pairs, less than about 2,800 base pairs, less than about 2,700 base pairs, less than about 2,600 base pairs, less than about 2,500 base pairs, less than about 2,400 base pairs, less than about 2,300 base pairs, less than about 2,200 base pairs, less than about 2,100 base pairs, less than about 2,000 base pairs, less than about 1,900 base pairs, less than about 1,800 base pairs, less than about 1,700 base pairs, less than about 1,600 base pairs, less than about 1,500 base pairs, less than about 1,400 base pairs, less than about 1,300 base pairs, less than about 1,200 base pairs, less than about 1,100 base pairs, less than 1,000 base pairs, less than about 900 base pairs, less than about 800 base pairs, less than about 700 base pairs, less than about 600 base pairs, less than about 500 base pairs, or less than about 400 base pairs.
  • a 3' homology arm is less than about 1,000 base pairs, less than about 900 base pairs, less than about 800 base pairs, less than about 700 base pairs, less than about 600 base pairs, less than about 500 base pairs, less than about 400 base pairs, or less than about 300 base pairs.
  • a 3' homology arm is about 400-600 base pairs, e.g., about 500 base pairs.
  • the 5' and 3' homology arms flank the break and are less than 100, 75, 50, 25, 15, 10 or 5 base pairs away from an edge of the break. In certain embodiments, the 5' and 3' homology arms flank an endogenous stop codon. In certain embodiments, the 5' and 3' homology arms flank a break located within about 500 base pairs (e.g., about 500 base pairs, about 450 base pairs, about 400 base pairs, about 350 base pairs, about 300 base pairs, about 250 base pairs, about 200 base pairs, about 150 base pairs, about 100 base pairs, about 50 base pairs, or about 25 base pairs) upstream (5') of an endogenous stop codon, e.g., the stop codon of an essential gene. In certain embodiments, the 5' homology arm encompasses an edge of the break.
  • the knock-in cassette within the donor template comprises an exogenous coding sequence for the gene product of interest in frame with and downstream (3') of an exogenous coding sequence or partial coding sequence of the essential gene.
  • a knock-in cassette within a donor template comprises an exogenous coding sequence for the gene product of interest in frame with and upstream (5') of an exogenous coding sequence or partial coding sequence of an essential gene.
  • the knock-in cassette is a polycistronic knock-in cassette.
  • the knock-in cassette is a bicistronic knock-in cassette.
  • the knock-in cassette does not comprise a reporter gene, e.g., a fluorescent reporter gene or an antibiotic resistance gene.
  • a single essential gene locus will be targeted by two knock-in cassettes comprising different “cargo” sequences.
  • one allele will incorporate one knock-in cassette, while the other allele will incorporate the other knock-in cassette.
  • a gRNA utilized to generate an appropriate DNA break may be the same for each of the two different knock-in cassettes.
  • gRNAs utilized to generate appropriate DNA breaks for each of the two different knock-in cassettes may be different, such that the “cargo” sequence is incorporated at a different position for each allele. In some embodiments, such a different position for each allele may still be within the ultimate exons coding region.
  • such a different position for each allele may be within the penultimate exon (second to last), and/or ultimate (last) exons coding region. In some embodiments, such a different position for at least one of the alleles may be within the first exon. In some embodiments, such a different position for at least one of the alleles may be within the first or second exon.
  • the knock-in cassette does not need to comprise an exogenous coding sequence that corresponds to the entire coding sequence of the essential gene.
  • a knock-in cassette that comprises a partial coding sequence of the essential gene, e.g., that corresponds to a portion of the endogenous coding sequence of the essential gene that spans the break and the entire region downstream of the break (minus the stop codon), and/or that corresponds to a portion of the endogenous coding sequence of the essential gene that spans the break and the entire region upstream of the break (up to and optionally including the start codon).
  • a base pair’s location in a coding sequence may be defined 3'-to-5' from an endogenous translational stop signal (e.g., a stop codon).
  • an “endogenous coding sequence” can include both exonic and intronic base pairs, and refers to gene sequence occurring 5' to an endogenous functional translational stop signal.
  • a break within an endogenous coding sequence comprises a break within one DNA strand. In some embodiments, a break within an endogenous coding sequence comprises a break within both DNA strands. In some embodiments, a break is located within the last 1000 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 750 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 600 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 500 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 400 base pairs of the endogenous coding sequence.
  • a break is located within the last 300 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 250 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 200 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 150 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 100 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 75 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the last 50 base pairs of the endogenous coding sequence.
  • a break is located within the last 21 base pairs of the endogenous coding sequence.
  • the exogenous partial coding sequence of the essential gene in the knock-in cassette encodes a C-terminal fragment of a protein encoded by the essential gene, e.g., a fragment that is less than 500, 250, 150, 125, 100, 75, 50, 25, 20, 15 or 10 amino acids in length.
  • the exogenous partial coding sequence of the essential gene in the knock-in cassette is codon optimized.
  • the exogenous partial coding sequence of the essential gene in the knock-in cassette is codon optimized to eliminate at least one PAM site.
  • the exogenous partial coding sequence of the essential gene in the knock-in cassette is codon optimized to eliminate more than one PAM site. In some embodiments, the exogenous partial coding sequence of the essential gene in the knock-in cassette is codon optimized to eliminate all relevant nuclease specific PAM sites.
  • a C-terminal fragment of a protein encoded by the essential gene is about 140 amino acids in length. In some embodiments, a C-terminal fragment of a protein encoded by the essential gene is about 130 amino acids in length. In some embodiments, a C-terminal fragment of a protein encoded by the essential gene is about 120 amino acids in length.
  • the C-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence of the essential gene that spans the break. In some embodiments, a C-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 1 exon of the essential gene. In some embodiments, a C-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 2 exons of the essential gene. In some embodiments, a C-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 3 exons of the essential gene.
  • a C-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 4 exons of the essential gene. In some embodiments, a C-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 5 exons of the essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a C-terminal fragment of a protein encoded by an essential gene, e.g., a fragment that is less than 500, 250, 150, 125, 100, 75, 50, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acids in length.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 20 amino acid C-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 19 amino acid C-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes an 18 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 17 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 16 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 1 amino acid C-terminal fragment of a protein encoded by an essential gene.
  • the break within the last exon of the essential gene. In some embodiments, e.g., when the essential gene includes many exons as shown in the exemplary method of Fig. 3 A, it may be advantageous to have the break within the penultimate exon of the essential gene. It is to be understood however that the present disclosure is not limited to any particular location for the break and that the available positions will vary depending on the nature and length of the essential gene and the length of the exogenous coding sequence for the gene product of interest. For example, for essential genes that include a few exons or when the gene product of interest is small it may be possible to locate the break in an upstream exon.
  • an “endogenous coding sequence” can include both exonic and intronic base pairs, and refers to gene sequence occurring 3' to an endogenous functional translational start signal.
  • a break within an endogenous coding sequence comprises a break within one DNA strand. In some embodiments, a break within an endogenous coding sequence comprises a break within both DNA strands. In some embodiments, a break is located within the first 1000 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 750 base pairs of an endogenous coding sequence. In some embodiments, a break is located within the first 600 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 500 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 400 base pairs of the endogenous coding sequence.
  • a break is located within the first 300 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 250 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 200 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 150 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 100 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 75 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 50 base pairs of the endogenous coding sequence. In some embodiments, a break is located within the first 21 base pairs of the endogenous coding sequence.
  • the exogenous partial coding sequence of the essential gene in the knock-in cassette encodes an N-terminal fragment of a protein encoded by the essential gene, e.g., a fragment that is less than 500, 250, 150, 125, 100, 75, 50, 25, 20, 15 or 10 amino acids in length. In some embodiments, an N-terminal fragment of a protein encoded by the essential gene is about 140 amino acids in length. In some embodiments, an N-terminal fragment of a protein encoded by the essential gene is about 130 amino acids in length. In some embodiments, an N-terminal fragment of a protein encoded by the essential gene is about 120 amino acids in length.
  • an N-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence of the essential gene that spans the break. In some embodiments, an N-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 1 exon of the essential gene. In some embodiments, an N-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 2 exons of the essential gene. In some embodiments, an N-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 3 exons of the essential gene.
  • an N-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 4 exons of the essential gene. In some embodiments, an N-terminal fragment includes an amino acid sequence that is encoded by a region of the endogenous coding sequence within 5 exons of the essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes an N-terminal fragment of a protein encoded by an essential gene, e.g., a fragment that is less than 500, 250, 150, 125, 100, 75, 50, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acids in length.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 20 amino acid N-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 19 amino acid N-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes an 18 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 17 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 16 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette encodes a 1 amino acid N-terminal fragment of a protein encoded by an essential gene.
  • the exogenous coding sequence or partial coding sequence of the essential gene in the knock-in cassette is less than 100% identical to the corresponding endogenous coding sequence of the essential gene of the cell, e.g., less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or less than 50% (i.e., when the two sequences are aligned using a standard pairwise sequence alignment tool that maximizes the alignment between the corresponding sequences).
  • the exogenous coding sequence or partial coding sequence of the essential gene in the knock-in cassette is codon optimized relative to the corresponding endogenous coding sequence of the essential gene of the cell, e.g., to prevent further binding of a nuclease to the target site.
  • it may be codon optimized to reduce the likelihood of recombination after integration of the knock-in cassette into the genome of the cell and/or to increase expression of the gene product of the essential gene and/or the gene product of interest after integration of the knock-in cassette into the genome of the cell.
  • a knock-in cassette comprises one or more nucleotides or base pairs that differ (e.g., are mutations) relative to an endogenous knock-in site.
  • such mutations in a knock-in cassette provide resistance to cutting by a nuclease.
  • such mutations in a knock-in cassette prevent a nuclease from cutting the target loci following homologous recombination.
  • such mutations in a knock-in cassette occur within one or more coding and/or non-coding regions of a target gene.
  • such mutations in a knock-in cassette are silent mutations.
  • such mutations in a knock-in cassette are silent and/or missense mutations.
  • such mutations in a knock-in cassette occur within a target protospacer motif and/or a target protospacer adjacent motif (PAM) site.
  • a knock-in cassette includes a target protospacer motif and/or a PAM site that are saturated with silent mutations.
  • a knock-in cassette includes a target protospacer motif and/or a PAM site that are approximately 30%, 40%, 50%, 60%, 70%, 80%, or 90% saturated with silent mutations.
  • a knock-in cassette includes a target protospacer motif and/or a PAM site that are saturated with silent and/or missense mutations.
  • a knock-in cassette includes a target protospacer motif and/or a PAM site that comprise at least one mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, at least 10 mutations, at least 11 mutations, at least 12 mutations, at least 13 mutations, at least 14 mutations, or at least 15 mutations.
  • certain codons encoding certain amino acids in a target site cannot be mutated through codon-optimization without losing some portion of an endogenous proteins natural function. In some embodiments, certain codons encoding certain amino acids in a target site cannot be mutated through codon-optimization.
  • the knock-in cassette is codon optimized in only a portion of the coding sequence.
  • a knock-in cassette encodes a C- terminal fragment of a protein encoded by an essential gene, e.g., a fragment that is less than 500, 250, 150, 125, 100, 75, 50, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acids in length.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 20 amino acid C-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 19 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes an 18 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 17 amino acid C-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 16 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 15 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 14 amino acid C-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 13 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 12 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a l l amino acid C-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 10 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 9 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes an 8 amino acid C -terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 7 amino acid C- terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 6 amino acid C -terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 5 amino acid C-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes an amino acid C-terminal fragment that is less than 5 amino acids of a protein encoded by an essential gene.
  • the knock-in cassette is codon optimized in only a portion of the coding sequence.
  • a knock-in cassette encodes an N- terminal fragment of a protein encoded by an essential gene, e.g., a fragment that is less than 500, 250, 150, 125, 100, 75, 50, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 amino acids in length.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 20 amino acid N -terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 19 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes an 18 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 17 amino acid N-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 16 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 15 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 14 amino acid N-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 13 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 12 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a l l amino acid N-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 10 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 9 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes an 8 amino acid N-terminal fragment of a protein encoded by an essential gene.
  • the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 7 amino acid N- terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 6 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes a 5 amino acid N-terminal fragment of a protein encoded by an essential gene. In some embodiments, the exogenous partial coding sequence of an essential gene in a knock-in cassette that has been codon optimized encodes an amino acid N-terminal fragment that is less than 5 amino acids of a protein encoded by an essential gene.
  • the knock-in cassette comprises one or more sequences encoding a linker peptide, e.g., between an exogenous coding sequence or partial coding sequence of the essential gene and a “cargo” sequence and/or a regulatory element described herein.
  • linker peptides are known in the art, any of which can be included in a knock-in cassette described herein.
  • the linker peptide comprises the amino acid sequence GSG.
  • the knock-in cassette comprises other regulatory elements such as a polyadenylation sequence, and optionally a 3' UTR sequence, downstream of the exogenous coding sequence for the gene product of interest. If a 3'UTR sequence is present, the 3'UTR sequence is positioned 3' of the exogenous coding sequence and 5' of the polyadenylation sequence.
  • the knock-in cassette comprises other regulatory elements such as a 5' UTR and a start codon, upstream of the exogenous coding sequence for the gene product of interest. If a 5'UTR sequence is present, the 5'UTR sequence is positioned 5' of the “cargo” sequence and/or exogenous coding sequence.
  • knock-in cassettes are also described in, e.g., WO2021/226151.
  • HA Homology Arms
  • a donor template comprises a 5' and/or 3' homology arm homologous to region of a GAPDH locus.
  • a donor template comprises a 5' homology arm comprising or consisting of the sequence of SEQ ID NO: 1, 2, or 3.
  • a 5' homology arm comprises or consists of a sequence that is at least 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO: 1, 2, or 3.
  • a donor template comprises a 3' homology arm comprising or consisting of the sequence of SEQ ID NO:4 or 5.
  • a 3' homology arm comprises or consists of a sequence that is at least 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO: 4 or 5.
  • a donor template comprises a 5' homology arm comprising SEQ ID NO: 1, and a 3' homology arm comprising SEQ ID NO: 4. In some embodiments, a donor template comprises a 5' homology arm comprising SEQ ID NO: 2, and a 3' homology arm comprising SEQ ID NO: 4. In some embodiments, a donor template comprises a 5' homology arm comprising SEQ ID NO: 3, and a 3' homology arm comprising SEQ ID NO: 5.
  • a stretch of sequence flanking a nuclease cleavage site may be duplicated in both a 5' and 3' homology arm.
  • such a duplication is designed to optimize HDR efficiency.
  • one of the duplicated sequences may be codon optimized, while the other sequence is not codon optimized.
  • both of the duplicated sequences may be codon optimized.
  • codon optimization may remove a target PAM site.
  • a duplicated sequence may be no more than: 100 bp in length, 90 bp in length, 80 bp in length, 70 bp in length, 60 bp in length, 50 bp in length, 40 bp in length, 30 bp in length, or 20 bp in length.
  • a donor template comprises a 5' and/or 3' homology arm homologous to a region of a TBP locus.
  • a donor template comprises a 5' homology arm comprising or consisting of the sequence of SEQ ID NO:6, 7, or 8.
  • a 5' homology arm comprises or consists of a sequence that is at least 85%, 90%,
  • a donor template comprises a 3' homology arm comprising or consisting of the sequence of SEQ
  • a 3' homology arm comprises or consists of a sequence that is at least 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO:
  • a donor template comprises a 5' homology arm comprising
  • a donor template comprises a 5' homology arm comprising SEQ ID NO: 7, and a 3' homology arm comprising SEQ ID NO: 10. In some embodiments, a donor template comprises a 5' homology arm comprising SEQ ID NO: 8, and a 3' homology arm comprising SEQ ID NO: 11.
  • SEQ ID NO: 6 exemplary 5' HA for knock-in cassette insertion at TBP locus
  • SEQ ID NO: 7 - exemplary 5' HA for knock-in cassette insertion at TBP locus
  • a donor template comprises a 5' and/or 3' homology arm homologous to a region of a G6PD locus. In some embodiments, a donor template comprises a
  • a 5' homology arm comprising or consisting of the sequence of SEQ ID NO: 12.
  • a 5' homology arm comprises or consists of a sequence that is at least 85%, 90%,
  • a donor template comprises a 3' homology arm comprising or consisting of the sequence of SEQ ID NO: 12.
  • a 3' homology arm comprises or consists of a sequence that is at least 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO: 13.
  • a donor template comprises a 5' homology arm comprising
  • SEQ ID NO: 12 and a 3' homology arm comprising SEQ ID NO: 13.
  • a donor template comprises a 5' and/or 3' homology arm homologous to a region of a E2F4 locus.
  • a donor template comprises a 5' homology arm comprising or consisting of the sequence of SEQ ID NO: 14, 15, or 16.
  • a 5' homology arm comprises or consists of a sequence that is at least 85%, 90%,
  • a donor template comprises a 3' homology arm comprising or consisting of the sequence of SEQ ID NO: 17, 18, or 19.
  • a 3' homology arm comprises or consists of a sequence that is at least 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO: 17, 18, or 19.
  • a donor template comprises a 5' homology arm comprising
  • a donor template comprises a 5' homology arm comprising SEQ ID NO: 15, and a 3' homology arm comprising SEQ ID NO: 18. In some embodiments, a donor template comprises a 5' homology arm comprising SEQ ID NO: 16, and a 3' homology arm comprising SEQ ID NO: 19.
  • a donor template comprises a 5' and/or 3' homology arm homologous to a region of a KIF11 locus. In some embodiments, a donor template comprises a
  • a 5' homology arm comprising or consisting of the sequence of SEQ ID NO: 20, 21, or 22.
  • a 5' homology arm comprises or consists of a sequence that is at least 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO: 20, 21, or 22.
  • a donor template comprises a 3' homology arm comprising or consisting of the sequence of SEQ ID NO: 23, 24, or 25.
  • a 3' homology arm comprises or consists of a sequence that is at least 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO: 23, 24, or 25.
  • a donor template comprises a 5' homology arm comprising
  • a donor template comprises a 5' homology arm comprising SEQ ID NO: 21, and a 3' homology arm comprising SEQ ID NO: 24.
  • a donor template comprises a 5' homology arm comprising SEQ ID NO: 22, and a 3' homology arm comprising SEQ ID NO: 25.
  • a donor template comprises an AAV derived sequence.
  • a donor template comprises AAV derived sequences that are typical of an AAV construct, such as cis-acting 5' and 3' inverted terminal repeats (ITRs) (See, e.g., B. J.
  • ITRs are able to form a hairpin.
  • the ability to form a hairpin can contribute to an ITRs ability to self-prime, allowing primase- independent synthesis of a second DNA strand.
  • ITRs also play a role in integration of AAV construct (e.g., a coding sequence) into a genome of a target cell. ITRs can also aid in efficient encapsidation of an AAV construct in an AAV particle.
  • a donor template described herein is included within an rAAV particle (e.g., an AAV6 particle).
  • an ITR is or comprises about 145 nucleic acids.
  • all or substantially all of a sequence encoding an ITR is used.
  • an AAV ITR sequence may be obtained from any known AAV, including presently identified mammalian AAV types.
  • an ITR is an AAV6 ITR.
  • An example of an AAV construct employed in the present disclosure is a “cisacting” construct containing a cargo sequence (e.g., a donor template described herein), in which the donor template is flanked by 5' or “left” and 3' or “right” AAV ITR sequences.
  • 5' and left designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction.
  • a 5' or left ITR is an ITR that is closest to a target loci promoter (as opposed to a polyadenylation sequence) for a given construct, when a construct is depicted in a sense orientation, linearly.
  • 3' and right designations refer to a position of an ITR sequence relative to an entire construct, read left to right, in a sense direction.
  • a 3' or right ITR is an ITR that is closest to a polyadenylation sequence in a target loci (as opposed to a promoter sequence) for a given construct, when a construct is depicted in a sense orientation, linearly.
  • ITRs as provided herein are depicted in 5' to 3' order in accordance with a sense strand. Accordingly, one of skill in the art will appreciate that a 5' or “left” orientation ITR can also be depicted as a 3' or “right” ITR when converting from sense to antisense direction.
  • a given sense ITR sequence e.g., a 5'/left AAV ITR
  • an antisense sequence e.g., 3'/right ITR sequence.
  • One of ordinary skill in the art would understand how to modify a given ITR sequence for use as either a 5'/left or 3'/right ITR, or an antisense version thereof.
  • an ITR e.g., a 5' ITR
  • an ITR e.g., a 3' ITR
  • an ITR includes one or more modifications, e.g., truncations, deletions, substitutions or insertions, as is known in the art.
  • an ITR comprises fewer than 145 nucleotides, e.g., 127, 130, 134 or 141 nucleotides.
  • an ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 ,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 144, or 145 nucleotides.
  • a non-limiting example of 5' AAV ITR sequences includes SEQ ID NO: 158.
  • a non-limiting example of 3' AAV ITR sequences includes SEQ ID NO: 159.
  • the 5' and a 3' AAV ITRs flank a donor template described herein (e.g., a donor template comprising a 5 'HA, a knock-in cassette, and a 3' HA).
  • the ability to modify ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al. “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K.
  • a 5' ITR sequence is at least 85%, 90%, 95%, 98% or 99% identical to a 5' ITR sequence represented by SEQ ID NO: 158.
  • a 3' ITR sequence is at least 85%, 90%, 95%, 98% or 99% identical to a 3' ITR sequence represented by SEQ ID NO: 159.
  • a knock-in cassette described herein includes all or a portion of an untranslated region (UTR), such as a 5' UTR and/or a 3' UTR.
  • UTRs of a gene are transcribed but not translated.
  • a 5' UTR starts at a transcription start site and continues to the start codon but does not include the start codon.
  • a 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • the regulatory and/or control features of a UTR can be incorporated into any of the knock-in cassettes described herein to enhance or otherwise modulate the expression of an essential target gene loci and/or a cargo sequence.
  • Natural 5' UTRs include a sequence that plays a role in translation initiation.
  • a 5' UTR comprises sequences, like Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes.
  • Kozak sequences have the consensus sequence CCR(A/G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), and the start codon is followed by another “G”.
  • the 5' UTRs have also been known to form secondary structures that are involved in elongation factor binding.
  • Non-limiting examples of 5' UTRs include those from the following genes: albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII.
  • a UTR may comprise a non-endogenous regulatory region.
  • a UTR that comprises a non-endogenous regulatory region is a 3’ UTR.
  • a UTR that comprises a non-endogenous regulatory region is a 5’ UTR.
  • a non-endogenous regulatory region may be a target of at least one inhibitory nucleic acid.
  • an inhibitory nucleic acid inhibits expression and/or activity of a target gene.
  • an inhibitory nucleic acid is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), an antisense oligonucleotide, a guide RNA (gRNA), or a ribozyme.
  • an inhibitory nucleic acid is an endogenous molecule.
  • an inhibitory nucleic acid is a non-endogenous molecule.
  • an inhibitory nucleic acid displays a tissue specific expression pattern.
  • an inhibitory nucleic acid displays a cell specific expression pattern.
  • a knock-in cassette may comprise more than one non- endogenous regulatory regions, e.g., two, three, four, five, six, seven, eight, nine, or ten regulatory regions. In some embodiments, a knock-in cassette may comprise four non- endogenous regulatory regions. In some embodiments, a construct may comprise more than one non-endogenous regulatory regions, wherein at least one of the more than one non-endogenous regulatory regions are not the same as at least one of the other non-endogenous regulatory regions.
  • a 3' UTR is found immediately 3' to the stop codon of a gene of interest.
  • a 3' UTR from an mRNA that is transcribed by a target cell can be included in any knock-in cassette described herein.
  • a 3' UTR is derived from an endogenous target loci and may include all or part of the endogenous sequence.
  • a 3' UTR sequence is at least 85%, 90%, 95% or 98% identical to the sequence of SEQ ID NO: 26.
  • a knock-in cassette construct provided herein can include a polyadenylation (poly(A)) signal sequence.
  • poly(A) polyadenylation
  • a poly(A) tail confers mRNA stability and transferability (Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994, which is incorporated herein by reference in its entirety).
  • a poly(A) signal sequence is positioned 3' to a coding sequence.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • a 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
  • a poly(A) tail is added onto transcripts that contain a specific sequence, e.g., a polyadenylation (or poly(A)) signal.
  • a poly(A) tail and associated proteins aid in protecting mRNA from degradation by exonucleases.
  • Polyadenylation also plays a role in transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation typically occurs in the nucleus immediately after transcription of DNA into RNA, but also can occur later in the cytoplasm. After transcription has been terminated, an mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • a cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.
  • a “poly(A) signal sequence” or “polyadenylation signal sequence” is a sequence that triggers the endonuclease cleavage of an mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.
  • poly(A) signal sequences there are several poly(A) signal sequences that can be used, including those derived from bovine growth hormone (bGH) (Woychik et al., Proc. Natl. Acad. Sci. US.A. 81(13):3944-3948, 1984; U.S. Patent No. 5,122,458, each of which is incorporated herein by reference in its entirety), mouse- ⁇ -globin, mouse- ⁇ -globin (Orkin et al., EMBO J 4(2):453-456, 1985; Thein et al., Blood? 1(2):313-319, 1988, each of which is incorporated herein by reference in its entirety), human collagen, polyoma virus (Batt et al., Mol.
  • bGH bovine growth hormone
  • HSV TK Herpes simplex virus thymidine kinase gene
  • IgG heavy-chain gene polyadenylation signal US 2006/0040354, which is incorporated herein by reference in its entirety
  • human growth hormone hGH
  • A SV40 poly
  • the poly(A) signal sequence can be AATAAA.
  • the AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA and that are capable of signaling polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e.g., WO 06/12414, which is incorporated herein by reference in its entirety).
  • a poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., the pCl-neo expression construct of Promega that is based on Levitt et al., Genes Dev. 3(7): 1019- 1025, 1989, which is incorporated herein by reference in its entirety).
  • a poly(A) signal sequence is the polyadenylation signal of soluble neuropilin- 1 (sNRP) (AAATAAAATACGAAATG) (see, e.g., WO 05/073384, which is incorporated herein by reference in its entirety).
  • a poly (A) signal sequence comprises or consists of the SV40 poly(A) site. In some embodiments, a poly(A) signal sequence comprises or consists of SEQ ID NO: 27. In some embodiments, a poly(A) signal sequence comprises or consists of bGHpA. In some embodiments, a poly(A) signal sequence comprises or consists of SEQ ID NO: 28. Additional examples of poly(A) signal sequences are known in the art. In some embodiments, a poly(A) sequence is at least 85%, 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NOs: 27 or 28.
  • the knock-in cassette comprises a regulatory element that enables expression of the gene product encoded by the essential gene and the gene product of interest as separate gene products, e.g., an IRES or 2A element located between the exogenous coding sequence or partial coding sequence of the essential gene and the exogenous coding sequence for the gene product of interest.
  • a regulatory element that enables expression of the gene product encoded by the essential gene and the gene product of interest as separate gene products, e.g., an IRES or 2A element located between the exogenous coding sequence or partial coding sequence of the essential gene and the exogenous coding sequence for the gene product of interest.
  • a knock-in cassette may comprise multiple gene products of interest (e.g., at least two gene products of interest).
  • gene products of interest may be separated by a regulatory element that enables expression of the at least two gene products of interest as more than one gene product, e.g., an IRES or 2A element located between the at least two coding sequences, facilitating creation of at least two peptide products.
  • IRES elements are one type of regulatory element that are commonly used for this purpose. As is well known in the art, IRES elements allow for initiation of translation from an internal region of the mRNA and hence expression of two separate proteins from the same mRNA transcript. IRES was originally discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic cells. Since then, a variety of IRES sequences have been discovered - many from viruses, but also some from cellular mRNAs, e.g., see Mokrejs et al., Nucleic Acids Res. 2006; 34(Database issue):D125-D130.
  • 2A elements are another type of regulatory element that are commonly used for this purpose. These 2A elements encode so-called “self-cleaving” 2 A peptides which are short peptides (about 20 amino acids) that were first discovered in picomaviruses. The term “self- cleaving” is not entirely accurate, as these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at the C -terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream.
  • the “cleavage” occurs between the Glycine (G) and Proline (P) residues found on the C-terminus meaning the upstream cistron, i.e., protein encoded by the essential gene will have a few additional residues from the 2A peptide added to the end, while the downstream cistron, i.e., gene product of interest will start with the Proline (P).
  • Table 2 lists the four commonly used 2A peptides (an optional GSG sequence is sometimes added to the N-terminal end of the peptide to improve cleavage efficiency).
  • 2A peptides that may be suitable for methods and compositions described herein (see e.g., Luke et al., Occurrence, function and evolutionary origins of ‘2A-like’ sequences in virus genomes. J Gen Virol. 2008).
  • Those skilled in the art know that the choice of specific 2 A peptide for a particular knock-in cassette will ultimately depend on a number of factors such as cell type or experimental conditions.
  • nucleotide sequences encoding specific 2A peptides can vary while still encoding a peptide suitable for inducing a desired cleavage event.
  • An essential gene can be any gene that is essential for the survival and/or the proliferation of the cell.
  • an essential gene is a housekeeping gene that is essential for survival of all cell types, e.g., a gene listed in Table 3. See also other housekeeping genes discussed in Eisenberg, Trends in Gen. 2014; 30(3): 119-20 and Moein et al., Adv. Biomed
  • the essential gene is GAPDH and the DNA nuclease causes a break in exon 9, e.g., a double-strand break.
  • the essential gene is GAPDH and the DNA nuclease causes a break in exon 9, e.g., a double-strand break.
  • the essential gene is GAPDH and the DNA nuclease causes a break in exon 9, e.g., a double-strand break.
  • the essential gene is
  • the essential gene is E2F4 and the DNA nuclease causes a break in exon 10, e.g., a double-strand break.
  • the essential gene is G6PD and the DNA nuclease causes a break in exon 13, e.g., a double-strand break.
  • the essential gene is KIF11 and the DNA nuclease causes a break in exon 22, e.g., a double-strand break.
  • a particular essential gene can be selected by analysis of potential off-target sites elsewhere in the genome.
  • only essential genes with one or more gRNA target sites that are unique in the human genome are selected for methods described herein.
  • only essential genes with one or more gRNA target sites that are found in only one other locus in the human genome are selected for methods described herein.
  • only essential genes with one or more gRNA target sites found in only two other loci in the human genome are selected for methods described herein.
  • Gene product of interest [0308]
  • the methods, systems and cells of the present disclosure enable the integration of a gene of interest at an essential gene of a cell.
  • the gene of interest can encode any gene product of interest.
  • a gene product of interest comprises an antibody, an antigen, an enzyme, a growth factor, a receptor (e.g., cell surface, cytoplasmic, or nuclear), a hormone, a lymphokine, a cytokine, a chemokine, a reporter, a functional fragment of any of the above, or a combination of any of the above.
  • sequence for a gene product of interest can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences.
  • a gene of interest may encode an miRNA, an shRNA, a native polypeptide (i.e. a polypeptide found in nature) or fragment thereof; a variant polypeptide (i.e.
  • an exemplary gene product of interest is one that confers therapeutic value, e.g., a new therapeutic activity to the cell.
  • exemplary gene products of interest are polypeptides such as a chimeric antigen receptor (CAR) or antigenbinding fragment thereof, a T cell receptor or antigen binding fragment thereof, a non-naturally occurring variant of Fc ⁇ RIII (CD 16), interleukin 15 (IL-15), interleukin 15 receptor (IL-15R) or a variant thereof, interleukin 12 (IL-12), interleukin- 12 receptor (IL-12R) or a variant thereof, human leukocyte antigen G (HLA-G), human leukocyte antigen E (HLA-E), leukocyte surface antigen cluster of differentiation CD47 (CD47), or any combination of two or more thereof.
  • CAR chimeric antigen receptor
  • IL-15R interleukin 15 receptor
  • IL-15R interleukin 15 receptor
  • IL-12 interleukin 12 receptor
  • IL-12R interleukin-
  • a gene product of interest may be a cytokine.
  • expression of a cytokine from a modified cell generated using a method as described herein allows for localized dosing of the cytokine in vivo (e.g., within a subject in need thereof) and/or avoids a need to systemically administer a high-dose of the cytokine to a subject in need thereof (e.g., a lower dose of the cytokine may be administered).
  • the risk of dose-limiting toxicities associated with administering a cytokine is reduced while cytokine mediated cell functions are maintained.
  • a partial or full peptide of one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, IFN- ⁇ , IFN- ⁇ and/or their respective receptor is introduced to the cell to enable cytokine signaling with or without the expression of the cytokine itself, thereby maintaining or improving cell growth, proliferation, expansion, and/or effector function with reduced risk of cytokine toxicities.
  • the introduced cytokine and/or its respective native or modified receptor for cytokine signaling are expressed on the cell surface.
  • the cytokine signaling is constitutively activated. In some embodiments, the activation of the cytokine signaling is inducible. In some embodiments, the activation of the cytokine signaling is transient and/or temporal.
  • a gene product if interest can be IL2, IL3, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL13, IL15, IL21, GM-CSF, IFN-a, IFN-b, IFN-g, erythropoietin, and/or the respective cytokine receptor.
  • a gene product of interest can be CCL3, TNF ⁇ , CCL23, IL2RB, IL12RB2, or IRF7.
  • a gene product of interest can be a chemokine and/or the respective chemokine receptor.
  • a chemokine receptor can be, but is not limited to, CCR2, CCR5, CCR8, CX3C1, CX3CR1, CXCR1, CXCR2, CXCR3A, CXCR3B, or CXCR2.
  • a chemokine can be, but is not limited to, CCL7, CCL19, or CXL14.
  • chimeric antigen receptor refers to a receptor protein that has been modified to give cells expressing the CAR the new ability to target a specific protein.
  • a cell modified to comprise a CAR or an antigen binding fragment may be used for immunotherapy to target and destroy cells associated with a disease or disorder, e.g., cancer cells.
  • the CAR can bind to any antigen of interest.
  • CARs of interest can include, but are not limited to, a CAR targeting mesothelin, EGFR, HER2 and/or MICA/B.
  • mesothelin-targeted CAR T-cell therapy has shown early evidence of efficacy in a phase I clinical trial of subjects having mesothelioma, non-small cell lung cancer, and breast cancer (NCT02414269).
  • CARs targeting EGFR, HER2 and MICA/B have shown promise in early studies (see, e.g., Li et al. (2016), Cell Death & Disease, 9(177); Han et al. (2016) Am. J. Cancer Res., 8(1): 106-119; and Demoulin 2017) Future Oncology, 13(8); the entire contents of each of which are expressly incorporated herein by reference in their entireties).
  • CARs are well-known to those of ordinary skill in the art and include those described in, for example: WO 13/063419 (mesothelin), WO 15/164594 (EGFR), WO 13/063419 (HER2), WO 16/154585 (MICA and MICE), the entire contents of each of which are expressly incorporated herein by reference in their entireties.
  • a gene product of interest is any suitable CAR, NK cell specific CAR (NK-CAR), T cell specific CAR, or other binder that targets a cell, e.g., an NK cell, to a target cell, e.g., a cell associated with a disease or disorder, may be expressed in the modified cells provided herein.
  • Exemplary CARs, and binders include, but are not limited to, bi-specific antigen binding CARs, switchable CARs, dimerizable CARs, split CARs, multi-chain CARs, inducible CARs, CARs and binders that bind BCMA, androgen receptor, PSMA, PSCA, Mucl, HPV viral peptides (i.e., E7), EBV viral peptides, WT1, CEA, EGFR, EGFRvIII, IL13R ⁇ 2, GD2, CA125, EpCAM, Mucl6, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD10, CD19, CD20, CD22, CD23, CD24, CD26, CD30, CD33, CD34, CD35, CD38 CD41, CD44, CD44V6, CD49f, CD56, CD70, CD92, CD99, CD123, CD133,
  • Additional suitable CARs and binders for use in the modified cells will be apparent to those of skill in the art based on the present disclosure and the general knowledge in the art.
  • Such additional suitable CARs include those described in Figure 3 of Davies and Maher, Adoptive T-cell Immunotherapy of Cancer Using Chimeric Antigen Receptor-Grafted T Cells, Archivum Immunologiae et Therapiae Experimentalis 58(3): 165-78 (2010), the entire contents of which are incorporated herein by reference.
  • CARs suitable for methods described herein include: CD171 -specific CARs (Park et al., Mol Ther (2007) 15(4):825-833), EGFRvIII- specific CARs (Morgan et al, Hum Gene Ther (2012) 23(10): 1043-1053), EGF-R-specific CARs (Kobold et al, J Natl Cancer Inst (2014) 107(1) :364), carbonic anhydrase K-specific CARs (Larners et al., Biochem Soc Trans (2016) 44(3):951-959), FR-a-specific CARs (Kershaw et al., Clin Cancer Res (2006) 12(20):6106- 6015), HER2-specific CARs (Ahmed et al., J Clin Oncol (2015) 33(15)1688-1696; Nakazawa et al., Mol Ther (2011) 19(12):2133-2143; Ahmed et al., Mol Ther (2009) 17(10): 1779-1787
  • a CAR is an anti-EGFR CAR. In some embodiments, a CAR is an antiCD 19 CAR. In some embodiments, a CAR is an anti-BCMA CAR. In some embodiments, a CAR is an anti-CD7 CAR.
  • CD16 refers to a receptor (Fc ⁇ RIII) for the Fc portion of immunoglobulin G, and it is involved in the removal of antigen-antibody complexes from the circulation, as well as other antibody-dependent responses.
  • a CD 16 protein is an hCD16 variant.
  • an hCD16 variant is a high affinity Fl 58V variant.
  • a gene product of interest comprises a high affinity non- cleavable CD 16 (hnCD16) or a variant thereof.
  • a high affinity non- cleavable CD 16 or a variant thereof comprises at least any one of the followings: (a) Fl 76 V and S197P in ectodomain domain of CD16 (see e.g., ling et al., Identification of an ADAM17 Cleavage Region in Human CD 16 (Fc ⁇ RIII) and the Engineering of a Non-Cleavable Version of the Receptor in NK Cells; PLOS One, 2015); (b) a full or partial ectodomain originated from CD64; (c) a non-native (or non-CD16) transmembrane domain; (d) a non-native (or non-CD16) intracellular domain; (e) a non-native (or nonCD 16) signaling domain; (f) a non-native stimulatory
  • the non-native transmembrane domain is derived from CD3D, CD3E, CD3G, CD3s, CD4, CD5, CD5a, CD5b, CD27, CD2S, CD40, CDS4, CD166, 4-1BB, 0X40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) polypeptide.
  • TCR T cell receptor
  • the non-native stimulatory domain is derived from CD27, CD2S, 4-1BB, 0X40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide.
  • the non-native signaling domain is derived from CD3s, 2B4, DAP 10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide.
  • the non-native transmembrane domain is derived from NKG2D
  • the non-native stimulatory domain is derived from 2B4
  • the non-native signaling domain is derived from CD3s.
  • a gene product of interest comprises a high affinity cleavable CD 16 (hnCD16) or a variant thereof.
  • a high affinity cleavable CD 16 or a variant thereof comprises at least Fl 76V.
  • a high affinity cleavable CD 16 or a variant thereof does not comprise an S197P amino acid substitution.
  • IL-15/IL15RA Interleukin-15
  • IL-15 refers to a cytokine with structural similarity to Interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (CD 122) and the common gamma chain (gamma-C, CD 132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following infection by virus(es). This cytokine induces cell proliferation of natural killer cells.
  • IL-15 Receptor alpha specifically binds IL-15 with very high affinity, and is capable of binding IL-15 independently of other subunits (see e.g., Mishra et al., Molecular pathways: Interleukin-15 signaling in health and in cancer, Clinical Cancer Research, 2014). It is suggested that this property allows IL-15 to be produced by one cell, endocytosed by another cell, and then presented to a third party cell.
  • IL15RA is reported to enhance cell proliferation and expression of apoptosis inhibitor BCL2L1/BCL2-XL and BCL2.
  • Exemplary sequences of IL-15 are provided in NG_029605.2, and exemplary sequences of IL-15RA are provided in NM_002189.4.
  • the IL-15R variant is a constitutively active IL-15R variant.
  • the constitutively active IL-15R variant is a fusion between IL- 15R and an IL-15R agonist, e.g., an IL-15 protein or IL-15R-binding fragment thereof.
  • the IL-15R agonist is IL-15, or an IL-15R-binding variant thereof.
  • Exemplary suitable IL-15R variants include, without limitation, those described, e.g., in Mortier E et al, 2006; The Journal of Biological Chemistry 2006281 : 1612-1619; or in Bessard-A et al., Mol Cancer Ther.
  • membrane bound trans-presentation of IL-15 is a more potent activation pathway than soluble IL-15 (see e.g., Imamura et al., Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15, Blood, 2014).
  • IL-15R expression comprises: IL15 and IL15Ra expression using a self-cleaving peptide; a fusion protein of IL 15 andIL15Ra; an IL15/IL15Ra fusion protein with intracellular domain of IL15Ra truncated; a fusion protein of IL 15 and membrane bound Sushi domain of IL15Ra; a fusion protein of IL 15 and IL15R ⁇ ; a fusion protein of IL 15 and common receptor ⁇ C, wherein the common receptor ⁇ C is native or modified; and/or a homodimer of IL15R ⁇ .
  • IL- 12 refers to interleukin- 12, a cytokine that acts on T and natural killer cells.
  • a genetically engineered stem cell and/or progeny cell comprises a genetic modification that leads to expression of one or more of an interleukin 12 (IL12) pathway agonist, e.g., IL-12, interleukin 12 receptor (IL-12R) or a variant thereof (e.g., a constitutively active variant of IL-12R, e.g., an IL-12R fused to an IL-12R agonist (IL-12RA).
  • IL12 interleukin 12
  • IL-12R interleukin 12 receptor
  • IL-12RA IL-12 receptor agonist
  • the gene product of interest comprises a protein or polypeptide whose expression within a cell, e.g., a cell modified as described herein, enables the cell to inhibit or evade immune rejection after transplant or engraftment into a subject.
  • the gene product of interest is HLA-E, HLA-G, CTL4, CD47, or an associated ligand.
  • the gene product of interest is a T cell receptor (TCR) or an antigen-binding fragment thereof, e.g., a recombinant TCR.
  • the recombinant TCR can bind to an antigen of interest, e.g., an antigen selected from, but not limited to, CD279, CD2, CD95, CD152, CD223CD272, TIM3, KIR, A2aR, SIRPa, CD200, CD200R, CD300, LPA5, NY-ESO, PD1, PDL1, or MAGE-A3/A6.
  • the TCR or antigen-binding fragment thereof can bind to a viral antigen, e.g., an antigen from hepatitis A, hepatitis B, hepatitis C (HCV), human papilloma virus (HPV) (e.g., HPV-16 (such as HPV- 16 E6 or HPV- 16 E7), HPV- 18, HPV-31 , HPV-33, or HPV-35), Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8), human T-cell leukemia virusOl (HTLV-1), human T-cell leukemia virus-2 (HTLV-2) or a cytomegalovirus (CMV).
  • a viral antigen e.g., an antigen from hepatitis A, hepatitis B, hepatitis C (HCV), human papilloma virus (HPV) (e.g., HPV-16 (such as HPV- 16 E6 or HPV- 16 E
  • the gene product of interest comprises a single-chain variable fragment that can bind to CD47, PD1, CTLA4, CD28, 0X40, 4- IBB, and ligands thereof.
  • HLA-G refers to the HLA non-classical class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. HLA-G is expressed on fetal derived placental cells. HLA-G is a ligand for NK cell inhibitory receptor KIR2DL4, and therefore expression of this HLA by the trophoblast defends it against NK cell- mediated death.
  • HLA-G Recombinant Proteins: A Comparative Study In Vivo PLOS One 2011, the entire contents of which are incorporated herein by reference.
  • An exemplary sequence of HLA-G is set forth as NG 029039.1.
  • HLA-E refers to the HLA class I histocompatibility antigen, alpha chain E, also sometimes referred to as MHC class I antigen E.
  • the HLA-E protein in humans is encoded by the HLA-E gene.
  • the human HLA-E is a non-classical MHC class I molecule that is characterized by a limited polymorphism and a lower cell surface expression than its classical paralogues.
  • This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane.
  • HLA-E binds a restricted subset of peptides derived from the leader peptides of other class I molecules.
  • HLA-E expressing cells escape allogeneic responses and lysis by NK cells. See e.g., Geomalusse-G et al., Nature Biotechnology 2017 35(8), the entire contents of which are incorporated herein by reference. Exemplary sequences of the HLA-E protein are provided in NM 005516.6.
  • CD47 also sometimes referred to as “integrin associated protein” (IAP) refers to a transmembrane protein that in humans is encoded by the CD47 gene.
  • CD47 belongs to the immunoglobulin superfamily, partners with membrane integrins, and also binds the ligands thrombospondin- 1 (TSP-1) and signal-regulatory protein alpha (SIRPa). CD47 acts as a signal to macrophages that allows CD47-expressing cells to escape macrophage attack. See, e.g., Deuse-T, et al., Nature Biotechnology 2019 37: 252-258, the entire contents of which are incorporated herein by reference.
  • a CD47 gene comprises on or more mutations known to alter CD47 function.
  • a CD47 nucleic acid sequence encoding a transgenic CD47 gene may be fused to one or more non-CD47 gene derived coding sequences.
  • a CD47 coding sequence may be codon-optimized.
  • a gene product of interest comprises a chimeric switch receptor (see e.g., WO2018094244A1 - TGFBeta Signal Converter; Ankri et al., Human T cells Engineered to express a programmed death 1/28 costimulatory retargeting molecule display enhanced antitumor activity, The Journal of Immunology, October 15, 2013, 191; Roth et al., Pooled knockin targeting for genome engineering of cellular immunotherapies, Cell.
  • a chimeric switch receptor see e.g., WO2018094244A1 - TGFBeta Signal Converter; Ankri et al., Human T cells Engineered to express a programmed death 1/28 costimulatory retargeting molecule display enhanced antitumor activity, The Journal of Immunology, October 15, 2013, 191; Roth et al., Pooled knockin targeting for genome engineering of cellular immunotherapies, Cell.
  • chimeric switch receptors are engineered cell-surface receptors comprising an extracellular domain from an endogenous cell-surface receptor and a heterologous intracellular signaling domain, such that ligand recognition by the extracellular domain results in activation of a different signaling cascade than that activated by the wild type form of the cell-surface receptor.
  • a chimeric switch receptor comprises an extracellular domain of an inhibitory cell-surface receptor fused to an intracellular domain that leads to the transmission of an activating signal rather than the inhibitory signal normally transduced by the inhibitory cell-surface receptor.
  • extracellular domains derived from cell-surface receptors known to inhibit immune effector cell activation can be fused to activating intracellular domains. In such an embodiment, engagement of the corresponding ligand may then activate signaling cascades that increase, rather than inhibit, the activation of the immune effector cell.
  • a gene product of interest is a PD1-CD28 switch receptor, wherein the extracellular domain of PD1 is fused to the intracellular signaling domain of CD28 (See e.g., Liu et al., Cancer Res 76:6 (2016), 1578-1590 and Moon et al., Molecular Therapy 22 (2014), S201).
  • encoding gene product of interest is or comprises the extracellular domain of CD200R and the intracellular signaling domain of CD28 (See Oda et al., Blood 130:22 (2017), 2410-2419).
  • a gene product of interest is a reporter gene (e.g., GFP, mCherry, etc.).
  • a reporter gene is utilized to confirm the suitability of a knock-in cassette’s expression capacity.
  • a gene product of interest may be a colored or fluorescent protein such as: blue/UV proteins, e.g. TagBFP, mTagBFP2, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire; cyan proteins, e.g.
  • ECFP Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl; green proteins, e.g. EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, m Wasabi, Clover, mNeonGreen; yellow proteins, e.g. EYFP, Citrine, Venus, SYFP2, TagYFP; orange proteins, e.g. Monomeric Kusabira-Orange, mKOK, mK02, mOrange, m0range2; red proteins, e.g.
  • PA-GFP PAmCherryl, PATagRFP
  • photoconvertible proteins e.g. Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange, PSmOrange, photoswitchable proteins, e.g. Dronpa, and combinations thereof.
  • a gene of interest provided herein can optionally include a sequence encoding a destabilizing domain (“a destabilizing sequence”) for temporal and/or spatial control of protein expression.
  • a destabilizing sequence include sequences encoding a FK506 sequence, a dihydrofolate reductase (DHFR) sequence, or other exemplary destabilizing sequences.
  • protein expression can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays; fluorescent activating cell sorting (FACS) assays; immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).
  • FACS fluorescent activating cell sorting
  • the destabilizing sequence is a FK506- and rapamycin-binding protein (FKBP12) sequence
  • the stabilizing ligand is Shield- 1 (Shldl) (Banaszynski et al. (2012) Cell 126(5): 995-1004, which is incorporated in its entirety herein by reference).
  • a destabilizing sequence is a DHFR sequence
  • a stabilizing ligand is trimethoprim (TMP) (Iwamoto et al. (2010) Chem Biol 17:981-988, which is incorporated in its entirety herein by reference).
  • a destabilizing domain is small molecule-assisted shutoff (SMASh), where a constitutive degron with a protease and its corresponding cleavage site derived from hepatitis C virus are combined.
  • a destabilizing domain comprises a HaloTag system, dTag system, and/or nanobody (see e.g., Luh et al., Prey for the proteasome: targeted protein degradation - a medicinal chemist’s perspective; Angewandte Chemie, 2020).
  • a destabilizing sequence can be used to temporally control a cell modified as described herein.
  • a gene product of interest may be a suicide gene, (see e.g., Zarogoulidis et al., Suicide Gene Therapy for Cancer - Current Strategies; J Genet Syndr Gene Ther. 2013).
  • a suicide gene can use a gene-directed enzyme prodrug therapy (GDEPT) approach, a dimerization inducing approach, and/or therapeutic monoclonal antibody mediated approach.
  • GDEPT gene-directed enzyme prodrug therapy
  • a suicide gene is biologically inert, has an adequate bio-availability profile, an adequate bio-distribution profile, and can be characterized by intrinsic acceptable and/or absence of toxicity.
  • a suicide gene codes for a protein able to convert, at a cellular level, a non-toxic prodrug into a toxic product.
  • a suicide gene may improve the safety profile of a cell described herein (see e.g., Greco et al., Improving the safety of cell therapy with the TK-suicide gene; Front Pharmacology. 2015; Jones et al., Improving the safety of cell therapy products by suicide gene transfer; Frontiers Pharmacology, 2014).
  • a suicide gene is a herpes simplex virus thymidine kinase (HSV-TK).
  • a suicide gene is a cytosine deaminase (CD).
  • a suicide gene is an apoptotic gene (e.g., a caspase).
  • a suicide gene is dimerization inducing, e.g., comprising an inducible FAS
  • a suicide gene is a CD20 antigen, and cells expressing such an antigen can be eliminated by clinical-grade anti-
  • a suicide gene is a truncated human
  • EGFR polypeptide which confers sensitivity to a pharmaceutical-grade anti-EGFR monoclonal antibody, e.g., cetuximab.
  • a suicide gene is a c-myc tag, which confers sensitivity to pharmaceutical-grade anti-cmyc antibodies.
  • a coding sequence for a single gene product of interest may be included in a knock-in cassette.
  • coding sequences for two gene products of interest may be included in a single knock-in cassette; in some embodiments, this may be referred to as a bicistronic or multicistronic construct.
  • coding sequences for more than two gene products of interest may be included in a single knock-in cassette; in some embodiments, this may be referred to as a multicistronic construct.
  • these sequences may have a linker sequence connecting them.
  • Linker sequences are generally known in the art, an exemplary linker sequence is identified in SEQ ID NO: 18871. In some embodiments, where more than one coding sequence for more than one gene product of interest is included in a knock-in cassette, these sequences may be connected by a linker sequence, an IRES, and/or 2A element.
  • a polynucleotide encoding a gene product of interest comprises or consists of the sequence of any one of SEQ ID NOs: 162-163, 165-182, or 18871.
  • a polynucleotide encoding a gene product of interest comprises or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOs: 162- 163, 165- 182, or 18871.
  • a polynucleotide encoding a gene product of interest comprises or consists of a functional variant of any one of SEQ ID NOs: 162-163, 165-182, or 18871.
  • a polynucleotide encoding a gene product of interest comprises or consists of a nucleotide sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (e.g., substitutions, insertions, and/or deletions) relative to any one of SEQ ID NOs: 162-163, 165-182, or 18871.
  • a gene product of interest comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 161, 164, or 183-200. In some embodiments, a gene product of interest comprises or consists of an amino acid sequence that is at least 85%,
  • a gene product of interest comprises or consists of a functional variant of any one of SEQ ID NOs:
  • a gene product of interest comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (e.g., substitutions, insertions, and/or deletions) relative to any one of SEQ ID NOs: 161, 164, or 183-200.
  • a CD47 transgene comprises or is SEQ ID NO: 18845. In some embodiments, a CD47 transgene comprises a coding sequence that is 60%, 65%, 70%,
  • a gene product of interest comprises or consists of an amino acid sequence of SEQ ID NO: 18846.
  • a CD47 transgenic amino acid sequence comprises or is SEQ ID NO: 18846.
  • a CD47 amino acid sequence comprises an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%,
  • a gene product of interest comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations
  • CD 19 gene may be fused to one or more non-CD19 CAR gene derived coding sequences.
  • a CD 19 CAR coding sequence may be codon-optimized.
  • a CD 19 CAR transgene comprises or is SEQ ID NO:
  • a CD 19 CAR transgene comprises a coding sequence that is 60%
  • a gene product of interest comprises or consists of an amino acid sequence of SEQ ID NO: 18850.
  • a CD19 CAR transgenic amino acid sequence comprises or is SEQ ID NO: 18850.
  • a CD19 CAR amino acid sequence comprises an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 18850.
  • a gene product of interest comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (e.g., substitutions, insertions, and/or deletions) relative to SEQ ID NO: 18850.
  • a gene product of interest comprises or consists of an amino acid sequence of SEQ ID NO: 18866.
  • an EGFR CAR transgenic amino acid sequence comprises or is SEQ ID NO: 18866.
  • an EGFR CAR amino acid sequence comprises an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 18866.
  • a gene product of interest comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (e.g., substitutions, insertions, and/or deletions) relative to SEQ ID NO: 18866.
  • a gene product of interest comprises or consists of an amino acid sequence of SEQ ID NO: 18870.
  • a CAR transgenic amino acid sequence comprises or is SEQ ID NO: 18870.
  • a CAR amino acid sequence comprises an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 18870.
  • a gene product of interest comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (e.g., substitutions, insertions, and/or deletions) relative to any one of SEQ ID NO: 18870.
  • a gene product of interest comprises or consists of an amino acid sequence of SEQ ID NO: 18867.
  • a CXCR2 transgenic amino acid sequence comprises or is SEQ ID NO: 18867.
  • a CXCR2 amino acid sequence comprises an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 18867.
  • a gene product of interest comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (e.g., substitutions, insertions, and/or deletions) relative to SEQ ID NO: 18867.
  • HLA-G refers to the HLA non-classical class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. HLA-G is expressed on fetal derived placental cells. HLA-G is a ligand for NK cell inhibitory receptor KIR2DL4, and therefore expression of this HLA by the trophoblast defends it against NK cell- mediated death.
  • HLA-G See e.g., Favier et al., Tolerogenic Function of Dimeric Forms of HLA-G Recombinant Proteins: A Comparative Study In Vivo PLOS One 2011, the entire contents of which are incorporated herein by reference.
  • Exemplary sequences of HLA-G are provided in NG_029039.1 and set forth as SEQ ID NO: 18855.
  • an HLA-G nucleic acid sequence encoding a transgenic HLA-G gene may be fused to one or more non-HLA-G gene derived coding sequences.
  • an HLA-G nucleic acid coding sequence is fused directly or indirectly to a B2M gene derived nucleic acid coding sequence.
  • an HLA-G nucleic acid coding sequence is fused directly or indirectly to a peptide coding sequence.
  • an HLA-G nucleic acid coding sequence is fused directly or indirectly to a linker sequence.
  • an HLA-G nucleic acid coding sequence is comprised within a trimeric construct.
  • a trimeric HLA-G comprising construct comprises (in N to C terminal order) one or more N-terminal peptides, a linker sequence, a B2M gene derived sequence, a linker sequence, and an HLA-G sequence (see e.g., Gomalusse et al., Nature Biotech 2017).
  • a peptide encoding sequence, a B2M gene derived coding sequence, and/or an HLA-G coding sequence may be codon-optimized.
  • a transgenic gene may additionally encode a linker sequence.
  • Linker sequences are generally known in the art. Exemplary linker lengths are, e.g., between 1 and 200 amino acid residues, e.g., 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36- 40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, 96-100, 101- 110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, or 191-200 amino acid residues.
  • a linker comprises about 1 to about 20 amino acid residues (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues). In some embodiments, a linker comprises about 5 to about 30 amino acids in length, e.g., between 10 and 20 amino acids in length, e.g., between 12 and 18 amino acids in length, e.g., 15 amino acids in length. In some embodiments, linkers can include or consist of flexible portions, e.g., regions without significant fixed secondary or tertiary structure.
  • a linker has an increased content of small amino acids, in particular of glycines, alanines, serines, threonines, leucines and/or isoleucines.
  • a linker may comprise at least 50%, 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 more glycine, serine, alanine, and/or threonine residues.
  • Linkers may be glycine-rich linkers, e.g., comprising at least 50%, 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 more glycine residues.
  • Linkers may be serine-rich linkers, e.g., comprising at least 50%, 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 more serine residues.
  • a linker comprises at least 80%, at least 85%, at least 90%, at least 95%, or more glycine, serine, alanine, and/or threonine residues, and the remaining residues, if any, are glutamine, phenylalanine, and/lysine.
  • a linker sequence comprises or consists of the amino acid sequence of SEQ ID NO: 18860 (or an amino acid sequence at least 90%, 95%, 98%, or more identical to SEQ ID NO: 18860). In some embodiments, a linker sequence comprises or consists of the amino acid sequence of SEQ ID NO: 18861 (or an amino acid sequence at least 90%, 95%, 98%, or more identical to SEQ ID NO: 18861).
  • a peptide-B2M-HLA-G transgene comprises or is SEQ ID NO: 18841. In some embodiments, a peptide-B2M-HLA-G transgene comprises a coding sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:
  • a peptide-B2M-HLA-G transgenic amino acid sequence comprises or is SEQ ID NO: 18842.
  • a peptide-B2M-HLA-G amino acid sequence comprises a coding sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
  • a transgenic amino acid sequence comprises or is a functional variant of SEQ ID NO: 18842.
  • a transgenic amino acid sequence comprises or is an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations (e.g., amino acid substitutions, insertions, and/or deletions) as compared to SEQ ID NO: 18842.
  • a peptide-B2M-HLA-G transgenic amino acid comprises or consists of an amino acid sequence of SEQ ID NO: 18842 lacking about 1 to about
  • amino acids at the N-terminus e.g., lacking about 1-24, about 1-23, about 1-22, about 1-21, about 1-20, about 1-19, about 1-18, about 1-17, about 1-16, about 1-15, about 2-24, about 2-23, about 2-22, about 2-21, about 2-20, about 2-19, about 2-18, about 2-17, about 2-16, or about 2- 15 of the amino acids at the N-terminus of SEQ ID NO: 18842).
  • HLA-E refers to the HLA class I histocompatibility antigen, alpha chain E, also sometimes referred to as MHC class I antigen E.
  • the HLA-E protein in humans is encoded by the HLA-E gene.
  • the human HLA-E is a non-classical MHC class I molecule that is characterized by a limited polymorphism and a lower cell surface expression than its classical paralogues.
  • This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane.
  • HLA-E binds a restricted subset of peptides derived from the leader peptides of other class I molecules.
  • HLA-E expressing cells may escape allogeneic responses and lysis by NK cells. See e.g., Geomalusse-G et al., Nature Biotechnology 2017 35(8), the entire contents of which are incorporated herein by reference.
  • Exemplary sequences of the HLA-E protein are provided in NM_005516.6 and set forth as SEQ ID NO: 18853.
  • an HLA-E nucleic acid sequence encoding a transgenic HLA-E gene may be fused to one or more non-HLA-E gene derived coding sequences.
  • an HLA-E nucleic acid coding sequence is fused directly or indirectly to a B2M gene derived nucleic acid coding sequence.
  • an HLA-E nucleic acid coding sequence is fused directly or indirectly to a peptide (e.g., an HLA-G signal peptide) coding sequence.
  • an HLA-E nucleic acid coding sequence is fused directly or indirectly to a linker sequence.
  • an HLA-E nucleic acid coding sequence is comprised within a trimeric construct.
  • a trimeric HLA-E comprising construct comprises (in N to C terminal order) one or more N-terminal peptides (e.g.,
  • HLA-G signal peptides examples include a linker sequence, a B2M gene derived sequence, a linker sequence, and an HLA-E sequence (see e.g., Gomalusse et al., Nature Biotech 2017).
  • a peptide e.g., an HLA-G signal peptide
  • a B2M gene derived coding sequence e.g., a linker sequence
  • an HLA-E sequence e.g., Gomalusse et al., Nature Biotech 2017.
  • a peptide e.g., an HLA-G signal peptide
  • B2M gene derived coding sequence e.g., a B2M gene derived coding sequence
  • an HLA-E coding sequence may be codon-optimized.
  • an HLA-G signal peptide-B2M-HLA-E transgene comprises or is SEQ ID NO: 18843or 18847.
  • an HLA-G signal peptide-B2M-HLA-E transgene comprises or is SEQ ID NO: 18843or 18847.
  • B2M-HLA-E transgene comprises a coding sequence that is 60%, 65%, 70%, 75%, 80%, 85%,
  • an HLA-G signal peptide-B2M-HLA-E transgenic amino acid sequence comprises or is SEQ ID NO: 18844, 18848, 18856, 18857, or 18858.
  • an HLA-G signal peptide-B2M-HLA-E amino acid sequence comprises a coding sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:
  • a transgenic amino acid sequence comprises or is a functional variant of SEQ ID NO: 18844, 18848, 18856, 18857, or
  • a transgenic amino acid sequence comprises or is an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations (e.g., substitutions, insertions, and/or deletions) as compared to SEQ ID NO: 18844, 18848, 18856, 18857, or 18858.
  • an HLA-G signal peptide-B2M-HLA-E transgenic amino acid comprises or consists of an amino acid sequence of SEQ ID NO: 18844 18848, 18856, 18857, or 18858, and lacking about 1 to about 25 amino acids at the N-terminus (e.g., lacking about 1-24, about 1- 23, about 1-22, about 1-21, about 1-20, about 1-19, about 1-18, about 1-17, about 1-16, about 1- 15, about 2-24, about 2-23, about 2-22, about 2-21, about 2-20, about 2-19, about 2-18, about 2- 17, about 2-16, or about 2-15 of the amino acids at the N-terminus of SEQ ID NO: 18844, 18848, 18856, 18857, or 18858).
  • an HLA-E transgenic amino acid sequence comprises or is SEQ ID NO: 18859.
  • an HLA-E transgenic amino acid sequence amino acid sequence comprises a coding sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 18859.
  • a transgenic amino acid sequence comprises or is a functional variant of SEQ ID NO: 18859.
  • a transgenic amino acid sequence comprises or is an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations (e.g., substitutions, insertions, and/or deletions) as compared to SEQ ID NO: 18859.
  • a transgenic amino acid comprises or consists of an amino acid sequence of SEQ ID NO: 18859, and lacking about 1 to about 25 amino acids at the N-terminus (e.g., lacking about 1-24, about 1-23, about 1-22, about 1-21, about 1-20, about
  • residues 21-29 correspond to HLA-G signal peptide
  • residues 1-20 and 45-143 correspond to B2M
  • residues 164-500 correspond to HLA-E
  • residues 21-29 correspond to HLA-G signal peptide
  • residues 1-20 and 45-143 correspond to B2M
  • residues 164-500 correspond to HLA-E
  • residues 21-29 correspond to HLA-G signal peptide
  • residues 1-20 and 45-143 correspond to B2M
  • residues 164-500 correspond to HLA-E
  • an HLA-E transgene encodes an HLA-E polypeptide (e.g., an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18864; or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a portion of SEQ ID NO: 18864 (e.g., lacking 1, 2, 3, 4, or 5 amino acid residues from the N and/or C terminus of SEQ ID NO: 18864)).
  • an HLA-E polypeptide e.g., an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18864; or an amino acid sequence having 80%, 85%, 90%
  • an HLA-E transgene encodes a B2M polypeptide (e.g., an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18863; or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a portion of SEQ ID NO: 18863 (e.g., lacking 1, 2, 3, 4, or 5 amino acid residues from the N and/or C terminus of SEQ ID NO: 18863)).
  • a B2M polypeptide e.g., an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18863; or an amino acid sequence having 80%, 85%, 90%
  • an HLA-E transgene encodes a peptide, e.g., an HLA-G signal peptide.
  • an HLA-E transgene encodes a peptide, e.g., a peptide comprising an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to RIIPRHLQL (SEQ ID NO: 123400), VMAPRTLFL (SEQ ID NO: 123500), VMAPRTLIL (SEQ ID NO: 18851), VMAPRTVLL (SEQ ID NO: 123700), and/or VMAPRTLVL (SEQ ID NO: 18852)).
  • an HLA-E transgene encodes (i) a B2M polypeptide (e.g., an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18863; or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a portion of SEQ ID NO: 18863 (e.g., lacking 1, 2, 3, 4, or 5 amino acid residues from the N and/or C terminus of SEQ ID NO: 18863)); and (ii) an HLA-E polypeptide (e.g., an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18864
  • an HLA-E transgene encodes (i) a peptide, e.g., an HLA-G signal peptide (e.g., an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to VMAPRTLFL (SEQ ID NO: 123500), VMAPRTLIL (SEQ ID NO: 18851), VMAPRTVLL (SEQ ID NO: 123700), and/or VMAPRTLVL (SEQ ID NO: 18852)); (ii) a B2M polypeptide (e.g., an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18863; or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9
  • an HLA-E transgene encodes (i) a peptide comprising an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 123400; (ii) a B2M polypeptide (e.g., an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18863; or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a portion of SEQ ID NO: 18863 (e.g., lacking 1, 2, 3, 4, or 5 amino acid residues from the N and/or C terminus of SEQ ID NO: 18863)); and (iii) an HLA-E polypeptide comprising
  • an HLA-E transgene encodes (i) a signal sequence (e.g., an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18862; or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a portion of SEQ ID NO: 18862 (e.g., lacking 1, 2, 3, 4, or 5 amino acid residues from the N and/or C terminus of SEQ ID NO: 18862)); (ii) an HLA-G signal peptide (e.g., an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 123500, 18851
  • a signal sequence e
  • an HLA-E transgene encodes (i) a signal sequence (e.g., an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18862; or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a portion of SEQ ID NO: 18862 (e.g., lacking 1, 2, 3, 4, or 5 amino acid residues from the N and/or C terminus of SEQ ID NO: 18862)); (ii) a peptide comprising an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 123400; (iii) a B2M polypeptide (e.g.,
  • the present disclosure provides one or more polynucleotide constructs (e.g., knock-in cassettes) packaged into an AAV capsid.
  • an AAV capsid is from or derived from an AAV capsid of an AAV2, 3, 4, 5, 6, 7, 8, 9, or 10 serotype, or one or more hybrids thereof.
  • an AAV capsid is from an AAV ancestral serotype.
  • an AAV capsid is an ancestral (Anc) AAV capsid.
  • An Anc capsid is created from a construct sequence that is constructed using evolutionary probabilities and evolutionary modeling to determine a probable ancestral sequence.
  • an AAV capsid has been modified in a manner known in the art (see e.g., Buning and Srivastava, Capsid modifications for targeting and improving the efficacy of AAV vectors, Mol Ther Methods Clin Dev. 2019)
  • any combination of AAV capsids and AAV constructs may be used in recombinant AAV (rAAV) particles of the present disclosure.
  • an AAV ITR is from or derived from an AAV ITR of AAV2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • an AAV particle is wholly comprised of AAV6 components (e.g., capsid and ITRs are AAV6 serotype).
  • an AAV particle is an AAV6/2, AAV6/8 or AAV 6/9 particle (e.g., an AAV2, AAV8 or AAV9 capsid with an AAV construct having AAV6 ITRs).
  • a donor template is included within an AAV construct.
  • an AAV construct sequence comprises or consists of the sequence of any one of SEQ ID NO: 201-204.
  • an exemplary AAV construct is represented by SEQ ID NO:201.
  • an exemplary AAV construct is represented by SEQ ID NO: 202.
  • an exemplary AAV construct is represented by SEQ ID NO: 203.
  • an exemplary AAV construct is represented by SEQ ID NO: 204.
  • an exemplary AAV construct is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a sequence represented by SEQ ID NO: 201-204.
  • a donor template comprises in 5' to 3' order, a target sequence 5' homology arm (which optionally comprises an optimized sequence that is not a wild type sequence), a second regulatory element that enables expression of a cargo sequence as a separate translational product (e.g., an IRES sequence and/or a 2A element), a cargo sequence
  • a cargo sequence e.g., a gene product of interest
  • a second regulatory element that enables expression of a cargo sequence as a separate translational product e.g., an IRES sequence and/or a 2A element
  • a second cargo sequence e.g., a gene product of interest
  • a poly adenylation signal e.g., a BGHpA signal
  • a target sequence 3' homology arm e.g., a BGHpA signal
  • a donor template comprises or consists of the sequence of any one of SEQ ID NOs: 38-57 and 205-218. In some embodiments, a donor template comprises or consists of a sequence that is at least 85%, 90%, 95%, 98% or 99% identical to any one of SEQ ID NOs: 38-57 and 205-218.
  • SEQ ID NO: 39 exemplary donor template for insertion at GAPDH locus
  • SEQ ID NO: 42 exemplary donor template for insertion at GAPDH locus
  • SEQ ID NO: 43 exemplary donor template for insertion at GAPDH locus
  • SEQ ID NO: 44 - exemplary donor template for insertion at GAPDH locus
  • SEQ ID NO: 45 - exemplary donor template for insertion at GAPDH locus
  • SEQ ID NO: 47 - exemplary donor template for insertion at TBP locus
  • SEQ ID NO: 49 - exemplary donor template for insertion at TBP locus
  • SEQ ID NO: 50 - exemplary donor template for insertion at TBP locus
  • SEQ ID NO: 52 exemplary donor template for insertion at E2F4 locus
  • SEQ ID NO: 53 exemplary donor template for insertion at E2F4 locus
  • SEQ ID NO: 54 - exemplary donor template for insertion at E2F4 locus
  • SEQ ID NO: 55 - exemplary donor template for insertion at KIF11 locus
  • SEQ ID NO: 48 exemplary donor template for insertion at GAPDH locus
  • SEQ ID NO: 215 - exemplary donor template for insertion at GAPDH locus
  • the present disclosure provides methods of editing the genome of a cell.
  • the method comprises contacting the cell with a nuclease that causes a break within an endogenous coding sequence of an essential gene in the cell wherein the essential gene encodes at least one gene product that is required for survival and/or proliferation of the cell.
  • the cell is also contacted with (i) a donor template that comprises a knock-in cassette comprising an exogenous coding sequence for a gene product of interest in frame with and downstream (3') of an exogenous coding sequence or partial coding sequence of the essential gene and/or (ii) a donor template that comprises a knock-in cassette comprising an exogenous coding sequence for a gene product of interest in frame with and upstream ( 5') of an exogenous coding sequence or partial coding sequence of the essential gene (Fig. 3D).
  • the knock-in cassette is integrated into the genome of the cell by homology-directed repair (HDR) of the break, resulting in a genome-edited cell that expresses the gene product of interest and the gene product encoded by the essential gene that is required for survival and/or proliferation of the cell, or a functional variant thereof.
  • HDR homology-directed repair
  • the genetically modified “knock-in” cell survives and proliferates to produce progeny cells with genomes that also include the exogenous coding sequence for the gene product of interest. This is illustrated in Fig. 3 A for an exemplary method.
  • knock-in cassette is not properly integrated into the genome of the cell, undesired editing events that result from the break, e.g., NHEJ-mediated creation of indels, may produce a non-functional, e.g., out of frame, version of the essential gene.
  • this produces a “knock-out” cell when the editing efficiency of the nuclease is high enough to disrupt one allele. Without sufficient functional copies of the essential gene these “knock-out” cells are unable to survive and do not produce any progeny cells.
  • the present disclosure provides methods of editing the genome of a cell.
  • the method comprises contacting the cell with a nuclease that causes a break within an endogenous non-coding sequence of an essential gene in the cell wherein the essential gene encodes at least one gene product that is required for survival and/or proliferation of the cell.
  • a break within an endogenous noncoding sequence alters a functional region of an essential gene that influences post- transcriptional modification patterns, e.g., mRNA splicing, RNA stability, RNA editing, RNA interference, etc.
  • such a break within an endogenous non-coding sequence occurs in a functional region of the essential gene, for example, but not limited to: a splicesome target site (e.g., a 5' splice donor site, an intron branch point sequence, a 3' splice acceptor site, and/or a polypyrimidine tract), an intronic splicing silencer, an intronic splicing enhancer, an exonic splicing silencer, an exonic splicing enhancer, an endogenous RNA interference binding site (e.g., micro RNA, small interfering RNA, etc.), an endogenous RNA editing machinery binding site (e.g., a binding site for adenosine deaminases, cytidine deaminases, etc.), or combinations thereof.
  • the nuclease causes a break at or near where an intron borders an exon in an essential gene, reducing or disrupting the function of the essential gene
  • the method automatically selects for the “knock-in” cells when it is applied to a population of starting cells.
  • the method does not require high knock-in efficiencies because of this automatic selection aspect. It is therefore particularly suitable for methods where the donor template is a dsDNA (e.g., a plasmid) where knock-in efficiencies are often below 5%.
  • the donor template is a dsDNA (e.g., a plasmid) where knock-in efficiencies are often below 5%.
  • some of the cells in the population of starting cells may remain unedited, i.e., unaffected by the nuclease.
  • nuclease editing efficiency is high, e.g., about 60-90%, or higher the percentage of unedited cells will be relatively low as compared to the percentage of genetically modified cells.
  • high nuclease editing efficiencies e.g., greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95%) facilitates efficient population wide transgene integration, as the percentage of unedited cells will be relatively low as compared to the percentage of genetically modified cells.
  • At least about 65% of the cells are edited by a nuclease, e.g., a Cas12a, Cas9, Cas12b, Cas12c, Cas12e, CasX, or Cas ⁇ (Cas12j), or a variant thereof (e.g., a variant with a high editing efficiency).
  • a nuclease e.g., a Cas12a, Cas9, Cas12b, Cas12c, Cas12e, CasX, or Cas ⁇ (Cas12j)
  • a variant thereof e.g., a variant with a high editing efficiency
  • an RNP containing a CRISPR nuclease e.g., Cas12a, Cas9, Cas12b, Cas12c, Cas12e, CasX, or Cas ⁇ (Cas12j), or a variant thereof (e.g., a variant with a high editing efficiency)
  • a guide are capable of cleaving the locus of an essential gene (e.g., a terminal exon in the locus of any essential gene provided in Table 3) in at least 65% of the cells in a population of cells (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in a population of cells).
  • an essential gene e.g., a terminal exon in the locus of any essential gene provided in Table
  • an RNP containing a CRISPR nuclease e.g., Cas12a, Cas9, Cas12b, Cas12c, Cas12e, CasX, or Cas ⁇ (Cas12j), or a variant thereof (e.g., a variant with a high editing efficiency)) and a guide are capable of inducing knock-in cassette integration at a locus of an essential gene (e.g., a terminal exon in the locus of any essential gene provided in Table 3) in at least 65% of the cells in a population of cells (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in a population of cells), e.g., at between 4 days and 10 days (e.g., at between
  • editing efficiency is determined prior to target cell die off, e.g., at day 1 and/or day 2 post transfection or transduction.
  • editing efficiency measured at day 1 and/or day 2 post transfection or transduction may not capture the complete proportion of cells for which editing occurred, as in some embodiments, certain editing events may result in near immediate and/or swift cell death.
  • near immediate and/or swift cell death may be any period of time less than 48 hours post transfection or transduction, for example, less than 48 hours, less than 44 hours, less than 40 hours, less than 36 hours, less than 32 hours, less than 28 hours, less than 24 hours, less than 20 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour after transfection or transduction.
  • the nuclease causes a double-strand break.
  • the nuclease causes a single-strand break, e.g., in some embodiments the nuclease is a nickase.
  • the nuclease is a prime editor which comprises a nickase domain fused to a reverse transcriptase domain.
  • the nuclease is an RNA- guided prime editor and the gRNA comprises the donor template.
  • a dualnickase system is used which causes a double-strand break via two single-strand breaks on opposing strands of a double-stranded DNA, e.g., genomic DNA of the cell.
  • the present disclosure provides methods suitable for high- efficiency knock-in (e.g., a high proportion of a cell population comprises a knock-in allele), overcoming a major manufacturing challenge.
  • high-efficiency knock-in results in at least 65% of the cells in a population of cells comprising a knock-in allele (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in a population of cells comprise a knock-in allele).
  • a knock-in allele e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
  • a gene of interest knocked into a cell may have a role in effector function, specificity, stealth, persistence, homing/chemotaxis, and/or resistance to certain chemicals (see for example, Saetersmoen et al., Seminars in Immunopathology, 2019).
  • the present disclosure provides methods for creation of knock-in cells that maintain high levels of expression regardless of age, differentiation status, and/or exogenous conditions.
  • an integrated cargo is expressed at an optimal level with a desired subcellular localization as a function of an insertion site. In some embodiments, the present disclosure provides such cells.
  • cells of the present disclosure are engineered according to a method described in WO2021/226151.
  • the present disclosure provides systems for editing the genome of a cell.
  • the system comprises the cell, a nuclease that causes a break within an endogenous coding sequence of an essential gene of the cell, wherein the essential gene encodes a gene product that is required for survival and/or proliferation of the cell, and a donor template that comprises a knock-in cassette comprising an exogenous coding sequence for a gene product of interest in frame with and downstream (3') of an exogenous coding sequence or partial coding sequence of the essential gene.
  • the nuclease causes a double-strand break.
  • the nuclease causes a single-strand break, e.g., in some embodiments the nuclease is a nickase.
  • the nuclease is a prime editor which comprises a nickase domain fused to a reverse transcriptase domain.
  • the nuclease is an RNA- guided prime editor and the gRNA comprises the donor template.
  • a dualnickase system is used which causes a double-strand break via two single-strand breaks on opposing strand of a double-stranded DNA, e.g., genomic DNA of the cell.
  • genome editing systems of the present disclosure may be used, for example, to edit stem cells.
  • genome editing systems of the present disclosure include at least two components adapted from naturally occurring CRISPR systems: a guide RNA (gRNA) and an RNA-guided nuclease. These two components form a complex that is capable of associating with a specific nucleic acid sequence and editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and/or a point mutation.
  • gRNA guide RNA
  • RNA-guided nuclease RNA-guided nuclease
  • Naturally occurring CRISPR systems are organized evolutionarily into two classes and five types (Makarova et al. Nat Rev Microbiol. 2011 Jun; 9(6): 467—477 (“Makarova”)), and while genome editing systems of the present disclosure may adapt components of any type or class of naturally occurring CRISPR system, the embodiments presented herein are generally adapted from Class 2, and type II or V CRISPR systems.
  • Class 2 systems which encompass types II and V, are characterized by relatively large, multidomain RNA-guided nuclease proteins (e.g., Cas9 or Cpfl) and one or more guide RNAs (e.g., a crRNA and, optionally, a tracrRNA) that form ribonucleoprotein (RNP) complexes that associate with (i.e., target) and cleave specific loci complementary to a targeting (or spacer) sequence of the crRNA.
  • RNP ribonucleoprotein
  • Genome editing systems similarly target and edit cellular DNA sequences, but differ significantly from CRISPR systems occurring in nature.
  • the unimolecular guide RNAs described herein do not occur in nature, and both guide RNAs and RNA-guided nucleases according to this disclosure may incorporate any number of non-naturally occurring modifications.
  • Genome editing systems can be implemented (e.g., administered or delivered to a cell or a subject) in a variety of ways, and different implementations may be suitable for distinct applications.
  • a genome editing system is implemented, in certain embodiments, as a protein/RNA complex (a ribonucleoprotein, or RNP), which can be included in a pharmaceutical composition that optionally includes a pharmaceutically acceptable carrier and/or an encapsulating agent, such as a lipid or polymer micro- or nano-particle, micelle, liposome, etc.
  • a genome editing system is implemented as one or more nucleic acids encoding the RNA-guided nuclease and guide RNA components described above (optionally with one or more additional components); in certain embodiments, the genome editing system is implemented as one or more vectors comprising such nucleic acids, for instance a viral vector such as an adeno-associated virus; and in certain embodiments, the genome editing system is implemented as a combination of any of the foregoing. Additional or modified implementations that operate according to the principles set forth herein will be apparent to the skilled artisan and are within the scope of this disclosure.
  • the genome editing systems of the present disclosure can be targeted to a single specific nucleotide sequence, or may be targeted to — and capable of editing in parallel — two or more specific nucleotide sequences through the use of two or more guide RNAs.
  • the use of multiple gRNAs is referred to as “multiplexing” throughout this disclosure, and can be employed to target multiple, unrelated target sequences of interest, or to form multiple SSBs or DSBs within a single target domain and, in some cases, to generate specific edits within such target domain.
  • multiplexing can be employed to target multiple, unrelated target sequences of interest, or to form multiple SSBs or DSBs within a single target domain and, in some cases, to generate specific edits within such target domain.
  • Maeder describes a genome editing system for correcting a point mutation (C.2991+1655A to G) in the human CEP290 gene that results in the creation of a cryptic splice site, which in turn reduces or eliminates the function of the gene.
  • the genome editing system of Maeder utilizes two guide RNAs targeted to sequences on either side of (i.e., flanking) the point mutation, and forms DSBs that flank the mutation. This, in turn, promotes deletion of the intervening sequence, including the mutation, thereby eliminating the cryptic splice site and restoring normal gene function.
  • Cotta- Ramusino describes a genome editing system that utilizes two gRNAs in combination with a Cas9 nickase (a Cas9 that makes a single strand nick such as S.
  • the dual-nickase system of Cotta-Ramusino is configured to make two nicks on opposite strands of a sequence of interest that are offset by one or more nucleotides, which nicks combine to create a double strand break having an overhang (5’ in the case of Cotta-Ramusino, though 3’ overhangs are also possible).
  • the overhang in turn, can facilitate homology directed repair events in some circumstances.
  • a gRNA targeted to a nucleotide sequence encoding Cas9 (“referred to as a “governing RNA”), which can be included in a genome editing system comprising one or more additional gRNAs to permit transient expression of a Cas9 that might otherwise be constitutively expressed, for example in some virally transduced cells.
  • governing RNA nucleotide sequence encoding Cas9
  • Genome editing systems can, in some instances, form double strand breaks that are repaired by cellular DNA double-strand break mechanisms such as NHEJ or HDR. These mechanisms are described throughout the literature, for example by Davis & Maizels, PNAS, 111(10):E924-932, March 11, 2014 (“Davis”) (describing Alt-HDR); Frit et al. DNA Repair 17(2014) 81-97 (“Frit”) (describing Alt-NHEJ); and lyama and Wilson III, DNA Repair (Amst.) 2013-Aug; 12(8): 620-636 (“lyama”) (describing canonical HDR and NHEJ pathways generally).
  • genome editing systems operate by forming DSBs
  • such systems optionally include one or more components that promote or facilitate a particular mode of double-strand break repair or a particular repair outcome.
  • Cotta-Ramusino also describes genome editing systems in which a single stranded oligonucleotide “donor template” is added; the donor template is incorporated into a target region of cellular DNA that is cleaved by the genome editing system, and can result in a change in the target sequence.
  • genome editing systems modify a target sequence, or modify expression of a target gene in or near the target sequence, without causing single- or double-strand breaks.
  • a genome editing system may include an RNA-guided nuclease fused to a functional domain that acts on DNA, thereby modifying the target sequence or its expression.
  • an RNA-guided nuclease can be connected to (e.g., fused to) a cytidine deaminase functional domain, and may operate by generating targeted C-to-A substitutions.
  • Exemplary nuclease/deaminase fusions are described in Komor et al. Nature 533, 420-424 (19 May 2016) (“ Komor”).
  • a genome editing system may utilize a cleavage-inactivated (i.e., a “dead”) nuclease, such as a dead Cas9 (dCas9), and may operate by forming stable complexes on one or more targeted regions of cellular DNA, thereby interfering with functions involving the targeted region(s) including, without limitation, mRNA transcription, chromatin remodeling, etc.
  • a cleavage-inactivated nuclease such as a dead Cas9 (dCas9)
  • dCas9 dead Cas9
  • nuclease that causes a break within an endogenous genomic sequence, e.g., a coding sequence of an essential gene of the cell can be used in the methods of the present disclosure.
  • the nuclease is a DNA nuclease.
  • the nuclease causes a single-strand break (SSB) within an endogenous coding sequence of an essential gene of the cell, e.g., in a “prime editing” system.
  • SSB single-strand break
  • DSB double-strand break
  • the double-strand break is caused by a single nuclease.
  • the double-strand break is caused by two nucleases that each cause a singlestrand break on opposing strands, e.g., a dual “nickase” system.
  • the nuclease is a CRISPR/Cas nuclease and the method further comprises contacting the cell with one or more guide molecules for the CRISPR/Cas nuclease. Exemplary CRISPR/Cas nucleases and guide molecules are described in more detail herein.
  • the nuclease (including a nickase) is not limited in any manner and can also be a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, or other nuclease known in the art (or a combination thereof).
  • ZFNs zinc finger nucleases
  • Methods for designing zinc finger nucleases (ZFNs) are well known in the art, e.g., see Umov et al., Nature Reviews Genetics 2010; 11 :636-640 and Paschon et al., Nat. Commun. 2019; 10(1): 1133 and references cited therein.
  • TALENs transcription activator-like effector nucleases
  • a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 50%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 55%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 60%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 65%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 70%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 75%.
  • a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 80%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 85%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 90%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 95%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 96%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 97%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 98%. In some embodiments, a nuclease suitable for methods described herein can have an editing efficiency that is greater than about 99%.
  • the nuclease can be delivered to the cell as a protein or a nucleic acid encoding the protein, e.g., a DNA molecule or mRNA molecule.
  • the protein or nucleic acid can be combined with other delivery agents, e.g., lipids or polymers in a lipid or polymer nanoparticle and targeting agents such as antibodies or other binding agents with specificity for the cell.
  • the DNA molecule can be a nucleic acid vector, such as a viral genome or circular double-stranded DNA, e.g., a plasmid.
  • Nucleic acid vectors encoding a nuclease can include other coding or non-coding elements.
  • a nuclease can be delivered as part of a viral genome (e.g., in an AAV, adenoviral or lentiviral genome) that includes certain genomic backbone elements (e.g., inverted terminal repeats, in the case of an AAV genome).
  • a viral genome e.g., in an AAV, adenoviral or lentiviral genome
  • genomic backbone elements e.g., inverted terminal repeats, in the case of an AAV genome
  • a CRISPR/Cas nuclease can be delivered to the cell as a protein or a nucleic acid encoding the protein, e.g., a DNA molecule or mRNA molecule.
  • the guide molecule can be delivered as an RNA molecule or encoded by a DNA molecule.
  • a CRISPR/Cas nuclease can also be delivered with a guide molecule as a ribonucleoprotein (RNP) and introduced into the cell via nucleofection (electroporation).
  • RNP ribonucleoprotein
  • CRISPR/Cas nucleases include, but are not limited to, naturally-occurring Class 2 CRISPR nucleases such as Cas9, and Cpf1 (Cas12a), as well as other Cas12 nucleases and nucleases derived or obtained therefrom.
  • CRISPR/Cas nucleases are defined as those nucleases that: (a) interact with (e.g., complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that includes (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM,” which is described in greater detail below.
  • PAM protospacer adjacent motif
  • CRISPR/Cas nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations may exist between individual CRISPR/Cas nucleases that share the same PAM specificity or cleavage activity.
  • Skilled artisans will appreciate that some aspects of the present disclosure relate to systems and methods that can be implemented using any suitable CRISPR/Cas nuclease having a certain PAM specificity and/or cleavage activity.
  • the term CRISPR/Cas nuclease should be understood as a generic term, and not limited to any particular type (e.g., Cas9 vs. Cpf1), species (e.g., S.
  • CRISPR/Cas nuclease pyogenes vs. S. aureus or variation (e.g., full-length vs. truncated or split; naturally-occurring PAM specificity vs. engineered PAM specificity, etc.) of CRISPR/Cas nuclease.
  • the PAM sequence takes its name from its sequential relationship to the “protospacer” sequence that is complementary to gRNA targeting domains (or “spacers”). Together with protospacer sequences, PAM sequences define target regions or sequences for specific CRISPR/Cas nuclease and gRNA combinations.
  • CRISPR/Cas nucleases may require different sequential relationships between PAMs and protospacers.
  • Cas9s recognize PAM sequences that are 3' of the protospacer.
  • Cpfl Cas12a
  • Cpfl Cas12a
  • CRISPR/Cas nucleases can also recognize specific PAM sequences.
  • S. aureus Cas9 for instance, recognizes a PAM sequence of NNGRRT or NNGRRV, wherein the N residues are immediately 3' of the region recognized by the gRNA targeting domain.
  • S. pyogenes Cas9 recognizes NGG PAM sequences.
  • F. novicida Cpf1 recognizes a TTN PAM sequence.
  • engineered CRISPR/Cas nucleases can have PAM specificities that differ from the PAM specificities of reference molecules (for instance, in the case of an engineered CRISPR/Cas nuclease, the reference molecule may be the naturally occurring variant from which the CRISPR/Cas nuclease is derived, or the naturally occurring variant having the greatest amino acid sequence homology to the engineered CRISPR/Cas nuclease).
  • CRISPR/Cas nucleases can be characterized by their DNA cleavage activity: naturally-occurring CRISPR/Cas nucleases typically form double-strand breaks (DSBs) in target nucleic acids, but engineered variants called “nickases” have been produced that generate only single-strand breaks (SSBs), e.g., those discussed in Ran et al., Cell 2013; 154(6)r 1380- 1389 (“Ran”), or that that do not cut at all.
  • DSBs double-strand breaks
  • nickases engineered variants called “nickases” have been produced that generate only single-strand breaks (SSBs), e.g., those discussed in Ran et al., Cell 2013; 154(6)r 1380- 1389 (“Ran”), or that that do not cut at all.
  • a naturally occurring Cas9 protein comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which comprise particular structural and/or functional domains.
  • the REC lobe comprises an arginine-rich bridge helix (BH) domain, and at least one REC domain (e.g., a RECI domain and, optionally, a REC2 domain).
  • the REC lobe does not share structural similarity with other known proteins, indicating that it is a unique functional domain.
  • the BH domain appears to play a role in gRNA:DNA recognition, while the REC domain is thought to interact with the repeat:anti-repeat duplex of the gRNA and to mediate the formation of the Cas9/gRNA complex.
  • the NUC lobe comprises a RuvC domain, an HNH domain, and a PAM- interacting (PI) domain.
  • the RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves the non-complementary (i.e., bottom) strand of the target nucleic acid. It may be formed from two or more split RuvC motifs (such as RuvC I, RuvCII, and RuvCIII in S. pyogenes and S. aureus).
  • the HNH domain meanwhile, is structurally similar to HNN endonuclease motifs, and cleaves the complementary (i.e., top) strand of the target nucleic acid.
  • the PI domain as its name suggests, contributes to PAM specificity.
  • Cas9 While certain functions of Cas9 are linked to (but not necessarily fully determined by) the specific domains set forth above, these and other functions may be mediated or influenced by other Cas9 domains, or by multiple domains on either lobe.
  • the repeat: antirepeat duplex of the gRNA falls into a groove between the REC and NUC lobes, and nucleotides in the duplex interact with amino acids in the BH, PI, and REC domains.
  • Some nucleotides in the first stem loop structure also interact with amino acids in multiple domains (PI, BH and RECI), as do some nucleotides in the second and third stem loops (RuvC and PI domains).
  • Cpf1 like Cas9, has two lobes: a REC (recognition) lobe, and a NUC (nuclease) lobe.
  • the REC lobe includes REC 1 and REC2 domains, which lack similarity to any known protein structures.
  • the NUC lobe meanwhile, includes three RuvC domains (RuvC-I, -II and -III) and a BH domain.
  • the Cpf1 REC lobe lacks an HNH domain, and includes other domains that also lack similarity to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED-I, -II and -III), and a nuclease (Nuc) domain.
  • WED Wedge
  • Nuc nuclease
  • Cpf1 While Cas9 and Cpf1 share similarities in structure and function, it should be appreciated that certain Cpf1 activities are mediated by structural domains that are not analogous to any Cas9 domains. For instance, cleavage of the complementary strand of the target DNA appears to be mediated by the Nuc domain, which differs sequentially and spatially from the HNH domain of Cas9. Additionally, the non-targeting portion of Cpf1 gRNA (the handle) adopts a pseudoknot structure, rather than a stem loop structure formed by the repeat:antirepeat duplex in Cas9 gRNAs.
  • CRISPR/Cas nucleases described herein have activities and properties that can be useful in a variety of applications, but the skilled artisan will appreciate that CRISPR/Cas nucleases can also be modified in certain instances, to alter cleavage activity, PAM specificity, or other structural or functional features.
  • nickase variants include Cas9 D10A and Cas9 H840A (numbering scheme according to SpCas9 wild-type sequence). Additional suitable nickase variants, including Cas12a variants, will be apparent to the skilled artisan based on the present disclosure and the knowledge in the art. The present disclosure is not limited in this respect.
  • a nickase may be fused to a reverse transcriptase to produce a prime editor (PE), e.g., as described in Anzalone et al., Nature 2019; 576:149-157, the entire contents of which are incorporated herein by reference.
  • PE prime editor
  • CRISPR/Cas nucleases can be, in certain embodiments, size-optimized or truncated, for instance via one or more deletions that reduce the size of the nuclease while still retaining gRNA association, target and PAM recognition, and cleavage activities.
  • RNA guided nucleases are bound, covalently or non-covalently, to another polypeptide, nucleotide, or other structure, optionally by means of a linker. Exemplary bound nucleases and linkers are described by Guilinger et al., Nature Biotech. 2014; 32:577-582, which is incorporated by reference herein.
  • CRISPR/Cas nucleases also optionally include a tag, such as, but not limited to, a nuclear localization signal, to facilitate movement of CRISPR/Cas nuclease protein into the nucleus.
  • a tag such as, but not limited to, a nuclear localization signal
  • the CRISPR/Cas nuclease can incorporate C- and/or N- terminal nuclear localization signals. Nuclear localization sequences are known in the art.
  • Exemplary suitable nuclease variants include, but are not limited to, AsCpf1
  • a nuclease variant is a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and
  • a Cas12a variant comprises an amino acid sequence having at least about 90%, 95%, or 100% identity to an AsCpf1 sequence described herein.
  • SEQ ID NO: 66 Exemplary AsCpf1 wild-type amino acid sequence
  • Additional suitable nucleases and nuclease variants will be apparent to the skilled artisan based on the present disclosure in view of the knowledge in the art.
  • Exemplary suitable nucleases may include, but are not limited to those provided in Table 5.
  • Guide RNAs of the present disclosure may be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing).
  • gRNAs and their component parts are described throughout the literature, for instance in Briner et al., Molecular Cell 2014; 56(2):333-339 (“Briner”), and in PCT Publication No. W02016/073990A1.
  • type II CRISPR systems generally comprise an CRISPR/Cas nuclease protein such as Cas9, a CRISPR RNA (crRNA) that includes a 5' region that is complementary to a foreign sequence, and a trans-activating crRNA (tracrRNA) that includes a 5' region that is complementary to, and forms a duplex with, a 3' region of the crRNA. While not intending to be bound by any theory, it is thought that this duplex facilitates the formation of — and is necessary for the activity of — the Cas9/gRNA complex.
  • Cas9 CRISPR RNA
  • tracrRNA trans-activating crRNA
  • the crRNA and tracrRNA could be joined into a single unimolecular or chimeric guide RNA, in one non-limiting example, by means of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequence bridging complementary regions of the crRNA (at its 3' end) and the tracrRNA (at its 5' end).
  • GAAA nucleotide
  • linker linker sequence bridging complementary regions of the crRNA (at its 3' end) and the tracrRNA (at its 5' end).
  • Guide RNAs include a “targeting domain” that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired.
  • Targeting domains are referred to by various names in the literature, including without limitation “guide sequences” (Hsu et al., Nat Biotechnol. 2013; 31(9): 827-832, (“Hsu”)), “complementarity regions” (PCT Publication No.
  • targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-24 nucleotides in length (for instance, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the 5' terminus of in the case of a Cas9 gRNA, and at or near the 3' terminus in the case of a Cpf1 gRNA.
  • gRNAs typically (but not necessarily, as discussed below) include a plurality of domains that may influence the formation or activity of gRNA/Cas9 complexes.
  • the duplexed structure formed by first and secondary complementarity domains of a gRNA also referred to as a repeatanti-repeat duplex
  • REC recognition
  • the first and/or second complementarity domains may contain one or more poly- A tracts, which can be recognized by RNA polymerases as a termination signal.
  • first and second complementarity domains are, therefore, optionally modified to eliminate these tracts and promote the complete in vitro transcription of gRNAs, for instance through the use of A-G swaps as described in Briner, or A-U swaps. These and other similar modifications to the first and second complementarity domains are within the scope of the present disclosure.
  • Cas9 gRNAs typically include two or more additional duplexed regions that are involved in nuclease activity in vivo but not necessarily in vitro. See Nishimasu 2015. A first stem-loop one near the 3' portion of the second complementarity domain is referred to variously as the “proximal domain,” (PCT Publication No. W02016/073990A1) “stem loop 1” (Nishimasu 2014 and 2015) and the “nexus” (Briner). One or more additional stem loop structures are generally present near the 3' end of the gRNA, with the number varying by species: S.
  • pyogenes gRNAs typically include two 3' stem loops (for a total of four stem loop structures including the repeat:anti-repeat duplex), while S. aureus and other species have only one (for a total of three stem loop structures).
  • a description of conserved stem loop structures (and gRNA structures more generally) organized by species is provided in Briner.
  • CRISPR/Cas nucleases have been (or may in the future be) discovered or invented which utilize gRNAs that differ in some ways from those described to this point.
  • Cpf1 CRISPR from Prevotella and Franciscella 1
  • Cas12a is a CRISPR/Cas nuclease that does not require a tracrRNA to function (see Zetsche et al., Cell 2015; 163:759-771 (“Zetsche I”)).
  • a gRNA for use in a Cpfl genome editing system generally includes a targeting domain and a complementarity domain (alternately referred to as a “handle”). It should also be noted that, in gRNAs for use with Cpf1, the targeting domain is usually present at or near the 3' end, rather than the 5' end as described above in connection with Cas9 gRNAs (the handle is at or near the 5' end of a Cpf1 gRNA).
  • gRNAs can be defined, in broad terms, by their targeting domain sequences, and skilled artisans will appreciate that a given targeting domain sequence can be incorporated in any suitable gRNA, including a unimolecular or chimeric gRNA, or a gRNA that includes one or more chemical modifications and/or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Thus, for economy of presentation in this disclosure, gRNAs may be described solely in terms of their targeting domain sequences.
  • gRNA should be understood to encompass any suitable gRNA that can be used with any CRISPR/Cas nuclease, and not only those gRNAs that are compatible with a particular species of Cas9 or Cpf1.
  • gRNA can, in certain embodiments, include a gRNA for use with any CRISPR/Cas nuclease occurring in a Class 2 CRISPR system, such as a type II or type V or CRISPR system, or an CRISPR/Cas nuclease derived or adapted therefrom.
  • a method or system of the present disclosure may use more than one gRNA.
  • two or more gRNAs may be used to create two or more double strand breaks in the genome of a cell.
  • a multiplexed editing strategy may be used that targets two or more essential genes at the same time with two or more knock-in cassettes.
  • the two or more knock-in cassettes may comprise different exogenous cargo sequences, e.g., different knock-in cassettes may encode different gene products of interest and thus the edited cells will express a plurality of gene products of interest from different knock-in cassettes targeted to different loci.
  • a double-strand break may be caused by a dual-gRNA paired “nickase” strategy.
  • gRNA pairs should be oriented on the DNA such that PAMs are facing out and cutting with the D10A Cas9 nickase will result in 5' overhangs.
  • a method or system of the present disclosure may use a prime editing gRNA (pegRNA) in conjunction with a prime editor (PE).
  • a pegRNA is substantially larger than standard gRNAs, e.g., in some embodiments longer than 50, 100, 150 or 250 nucleotides, e.g., as described in Anzalone et al., Nature 2019; 576:149- 157, the entire contents of which are incorporated herein by reference.
  • the pegRNA is a gRNA with a primer binding sequence (PBS) and a donor template containing the desired RNA sequence added at one of the termini, e.g., the 3' end.
  • PBS primer binding sequence
  • the PE:pegRNA complex binds to the target DNA, and the nickase domain of the prime editor nicks only one strand, generating a flap.
  • the PBS located on the pegRNA, binds to the DNA flap and the edited RNA sequence is reverse transcribed using the reverse transcriptase domain of the prime editor.
  • the edited strand is incorporated into the DNA at the end of the nicked flap, and the target DNA is repaired with the new reverse transcribed DNA.
  • the original DNA segment is removed by a cellular endonuclease. This leaves one strand edited, and one strand unedited.
  • the unedited strand can be corrected to match the newly edited strand by using an additional standard gRNA.
  • the unedited strand is nicked by a nickase and the newly edited strand is used as a template to repair the nick, thus completing the edit.
  • gRNA design may involve the use of a software tool to optimize the choice of potential target sequences corresponding to a user’s target sequence, e.g., to minimize total off-target activity across the genome.
  • off-target activity is not limited to cleavage
  • the cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme.
  • methods for selection and validation of target sequences as well as off-target analyses can be performed using cas-offinder (Bae et al., Bioinformatics 2014;
  • Cas-offinder is a tool that can quickly identify all sequences in a genome that have up to a specified number of mismatches to a guide sequence.
  • An exemplary score includes a Cutting Frequency Determination (CFD) score, as described by Doench et al., Nat Biotechnol. 2016; 34:184—91.
  • CFD Cutting Frequency Determination
  • gRNAs as used herein may be modified or unmodified gRNAs.
  • a gRNA may include one or more modifications.
  • the one or more modifications may include a phosphorothioate linkage modification, a phosphorodithioate (PS2) linkage modification, a 2’-O-methyl modification, or combinations thereof.
  • the one or more modifications may be at the 5' end of the gRNA, at the 3' end of the gRNA, or combinations thereof.
  • a gRNA modification may comprise one or more phosphorodithioate (PS2) linkage modifications.
  • PS2 phosphorodithioate
  • a gRNA used herein includes one or more or a stretch of deoxyribonucleic acid (DNA) bases, also referred to herein as a “DNA extension.”
  • a gRNA used herein includes a DNA extension at the 5' end of the gRNA, the 3' end of the gRNA, or a combination thereof.
  • the DNA extension may be
  • the DNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 DNA bases long.
  • the DNA extension may include one or more DNA bases selected from adenine (A), guanine (G), cytosine (C), or thymine (T).
  • the DNA extension includes the same DNA bases.
  • the DNA extension may include a stretch of adenine (A) bases.
  • the DNA extension may include a stretch of thymine (T) bases.
  • the DNA extension includes a combination of different DNA bases.
  • a gRNA used herein includes a DNA extension as well as a chemical modification, e.g., one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2’-O-methyl modifications, or one or more additional suitable chemical gRNA modification disclosed herein, or combinations thereof.
  • the one or more modifications may be at the 5' end of the gRNA, at the 3' end of the gRNA, or combinations thereof.
  • any DNA extension may be used with any gRNA disclosed herein, so long as it does not hybridize to the target nucleic acid being targeted by the gRNA and it also exhibits an increase in editing at the target nucleic acid site relative to a gRNA which does not include such a DNA extension.
  • a gRNA used herein includes one or more or a stretch of ribonucleic acid (RNA) bases, also referred to herein as an “RNA extension.”
  • RNA extension includes an RNA extension at the 5' end of the gRNA, the 3' end of the gRNA, or a combination thereof.
  • the RNA extension may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
  • the RNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 RNA bases long.
  • the RNA extension may include one or more RNA bases selected from adenine (rA), guanine (rG), cytosine (rC), or uracil (rU), in which the “r” represents RNA, 2 ’-hydroxy.
  • the RNA extension includes the same RNA bases.
  • the RNA extension may include a stretch of adenine (rA) bases.
  • the RNA extension includes a combination of different RNA bases.
  • a gRNA used herein includes an RNA extension as well as one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2’-O-methyl modifications, one or more additional suitable gRNA modification, e.g., chemical modification, disclosed herein, or combinations thereof.
  • the one or more modifications may be at the 5' end of the gRNA, at the 3' end of the gRNA, or combinations thereof.
  • a gRNA including a RNA extension may comprise a sequence set forth herein.
  • gRNAs used herein may also include an RNA extension and a DNA extension.
  • the RNA extension and DNA extension may both be at the 5' end of the gRNA, the 3' end of the gRNA, or a combination thereof.
  • the RNA extension is at the 5' end of the gRNA and the DNA extension is at the 3' end of the gRNA.
  • the RNA extension is at the 3' end of the gRNA and the DNA extension is at the 5' end of the gRNA.
  • a gRNA which includes a modification is complexed with a CRISPR/Cas nuclease, e.g., an AsCpfl nuclease, to form an RNP, which is then employed to edit a target cell, e.g., a pluripotent stem cell or a progeny thereof.
  • a CRISPR/Cas nuclease e.g., an AsCpfl nuclease
  • Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and/or at or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end).
  • modifications are positioned within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 gRNA, a stem loop structure of a Cas9 or Cpf 1 gRNA, and/or a targeting domain of a gRNA.
  • the 5' end of a gRNA can include a eukaryotic mRNA cap structure or cap analog (e.g., a G(5')ppp(5')G cap analog, a m7G(5')ppp(5')G cap analog, or a 3'- O-Me-m7G(5')ppp(5')G anti reverse cap analog (ARCA)), as shown below:
  • a eukaryotic mRNA cap structure or cap analog e.g., a G(5')ppp(5')G cap analog, a m7G(5')ppp(5')G cap analog, or a 3'- O-Me-m7G(5')ppp(5')G anti reverse cap analog (ARCA)
  • the cap or cap analog can be included during either chemical or enzymatic synthesis of the gRNA.
  • the 5' end of the gRNA can lack a 5' triphosphate group.
  • in vitro transcribed gRNAs can be phosphatase-treated (e.g., using calf intestinal alkaline phosphatase) to remove a 5' triphosphate group.
  • poly A tract can be added to a gRNA during chemical or enzymatic synthesis, using a polyadenosine polymerase (e.g., E, coli Poly(A)Polymerase).
  • a polyadenosine polymerase e.g., E, coli Poly(A)Polymerase
  • Guide RNAs can be modified at a 3' terminal U ribose.
  • the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below: wherein “U” can be an unmodified or modified uridine.
  • the 3' terminal U ribose can be modified with a 2’3' cyclic phosphate as shown below: wherein “U” can be an unmodified or modified uridine.
  • Guide RNAs can contain 3' nucleotides that can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein.
  • uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein;
  • adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein.
  • sugar-modified ribonucleotides can be incorporated into a gRNA, e.g., wherein the 2’ OH-group is replaced by a group selected from H, -OR, -R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN).
  • R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, hetero
  • the phosphate backbone can be modified as described herein, e.g., with a phosphothioate (PhTx) group.
  • one or more of the nucleotides of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2 ’-sugar modified, such as, 2’-O-methyl, 2’-O-methoxyethyl, or 2’-Fluoro modified including, e.g., 2’-F or 2’-O-methyl, adenosine (A), 2’-F or 2’-O-methyl, cytidine (C), 2’-F or 2’-O-methyl, uridine (U), 2’-F or 2’-O-methyl, thymidine (T), 2’-F or 2’-O-methyl, guanosine (G), 2’-O- methoxyethyl-5 -methyluridine (Teo),
  • Guide RNAs can also include “locked” nucleic acids (LN A) in which the 2’ OH- group can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar.
  • LN A locked nucleic acids
  • Any suitable moiety can be used to provide such bridges, including without limitation methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH 2 ) n -amino (wherein amino can be, e.g., NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).
  • amino can be, e.g., NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino
  • a gRNA can include a modified nucleotide which is multicyclic (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R- GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), or threose nucleic acid (TNA, where ribose is replaced with a-L-threofuranosyl-(3' ⁇ 2’)).
  • GNA glycol nucleic acid
  • TAA threose nucleic acid
  • gRNAs include the sugar group ribose, which is a 5-membered ring having an oxygen.
  • Exemplary modified gRNAs can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone).
  • replacement of the oxygen in ribose e.g., with
  • a gRNA comprises a 4’-S, 4’-Se or a 4’-C- aminomethyl-2’-O-Me modification.
  • deaza nucleotides e.g., 7 -deaza-adenosine
  • O- and N-alkylated nucleotides e.g., N6- methyl adenosine
  • one or more or all of the nucleotides in a gRNA are deoxynucleotides.
  • Guide RNAs can also include one or more cross-links between complementary regions of the crRNA (at its 3' end) and the tracrRNA (at its 5' end) (e.g., within a “tetraloop” structure and/or positioned in any stem loop structure occurring within a gRNA).
  • linkers are suitable for use.
  • guide RNAs can include common linking moieties including, without limitation, polyvinylether, polyethylene, polypropylene, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL), and copolymers thereof.
  • a bifunctional cross-linker is used to link a 5' end of a first gRNA fragment and a 3' end of a second gRNA fragment, and the 3' or 5' ends of the gRNA fragments to be linked are modified with functional groups that react with the reactive groups of the cross-linker.
  • these modifications comprise one or more of amine, sulfhydryl, carboxyl, hydroxyl, alkene (e.g., a terminal alkene), azide and/or another suitable functional group.
  • Multifunctional e.g.
  • bifunctional cross-linkers are also generally known in the art, and may be either heterofunctional or homofunctional, and may include any suitable functional group, including without limitation isothiocyanate, isocyanate, acyl azide, an NHS ester, sulfonyl chloride, tosyl ester, tresyl ester, aldehyde, amine, epoxide, carbonate (e.g., Bis(p-nitrophenyl) carbonate), aryl halide, alkyl halide, imido ester, carboxylate, alkyl phosphate, anhydride, fluorophenyl ester, HOBt ester, hydroxymethyl phosphine, O-methylisourea, DSC, NHS carbamate, glutaraldehyde, activated double bond, cyclic hemiacetal, NHS carbonate, imidazole carbamate, acyl imidazole, methylpyridinium ether, azlactone, cyanate este
  • a first gRNA fragment comprises a first reactive group and the second gRNA fragment comprises a second reactive group.
  • the first and second reactive groups can each comprise an amine moiety, which are crosslinked with a carbonate-containing bifunctional crosslinking reagent to form a urea linkage.
  • the first reactive group comprises a bromoacetyl moiety and the second reactive group comprises a sulfhydryl moiety
  • the first reactive group comprises a sulfhydryl moiety and the second reactive group comprises a bromoacetyl moiety, which are crosslinked by reacting the bromoacetyl moiety with the sulfhydryl moiety to form a bromoacetyl-thiol linkage.
  • Suitable gRNA modifications include, for example, those described in PCT Publication No. W02019070762A1 entitled “MODIFIED CPF1 GUIDE RNA;” in PCT Publication No. WO2016089433 Al entitled “GUIDE RNA WITH CHEMICAL MODIFICATIONS;” in PCT Publication No. WO2016164356A1 entitled “CHEMICALLY MODIFIED GUIDE RNAS FOR CRISPR/CAS-MEDIATED GENE
  • Non-limiting examples of guide RNAs suitable for certain embodiments embraced by the present disclosure are provided herein, for example, in the Tables below.
  • suitable guide RNA sequences for a specific nuclease e.g., a Cas9 or Cpf1 nuclease
  • a guide RNA comprising a targeting sequence consisting of RNA nucleotides would include the RNA sequence corresponding to the targeting domain sequence provided as a DNA sequence, and this contain uracil instead of thymidine nucleotides.
  • a guide RNA comprising a targeting domain sequence consisting of RNA nucleotides, and described by the DNA sequence TCTGCAGAAATGTTCCCCGT (SEQ ID NO: 88) would have a targeting domain of the corresponding RNA sequence UCUGCAGAAAUGUUCCCCGU (SEQ ID NO: 89).
  • a targeting sequence would be linked to a suitable guide RNA scaffold, e.g., a crRNA scaffold sequence or a chimeric crRNA/tracrRNA scaffold sequence.
  • Suitable gRNA scaffold sequences are known to those of ordinary skill in the art.
  • a suitable scaffold sequence comprises the sequence UAAUUUCUACUCUUGUAGAU (SEQ ID NO: 90), added to the 5 '-terminus of the targeting domain. In the example above, this would result in a Cpf1 guide RNA of the sequence
  • UAAUUUCUACUCUUGUAGAUUCUGCAGAAAUGUUCCCCGU SEQ ID NO: 91.
  • a DNA extension e.g., in the example above, adding a 25-mer DNA extension as described herein would result, for example, in a guide RNA of the sequence ATGTGTTTTTGTCAAAAGACCTTTTrUrArArUrUrUrCrUrArCrUrUrGrUrArGrArUrUrUrUr
  • the gRNA for use in the disclosure is a gRNA targeting TGF ⁇ RII (TGF ⁇ RII gRNA).
  • TGF ⁇ RII gRNA gRNA targeting TGF ⁇ RII
  • the gRNA targeting TGF ⁇ RII is one or more of the gRNAs described in Table 7.
  • the gRNA for use in the disclosure is a gRNA targeting
  • CISH CISH gRNA
  • the gRNA targeting CISH is one or more of the gRNAs described in Table 8.
  • the gRNA for use in the disclosure is a gRNA targeting
  • B2M B2M gRNA
  • the gRNA targeting B2M is one or more of the gRNAs described in Table 9.
  • the gRNA for use in the disclosure is a gRNA targeting
  • gRNAs targeting B2M and PD1 for use in the disclosure are further described in
  • the gRNA for use in the disclosure is a gRNA targeting
  • NKG2A NKG2A
  • the gRNA targeting NKG2A is one or more of the gRNAs described in Table 10.
  • the gRNA for use in the disclosure is a gRNA targeting
  • TIGIT TIGIT gRNA
  • the gRNA targeting TIGIT is one or more of the gRNAs described in Table 11. Table 11. Exemplary TIGIT gRNAs
  • the gRNA for use in the disclosure is a gRNA targeting ADORA2a (ADORA2a gRNA).
  • the gRNA targeting ADORA2a is one or more of the gRNAs described in Table 12. Table 12. Exemplary ADORA2a gRNAs
  • Methods of characterizing cells including characterizing cellular phenotype are known to those of skill in the art.
  • one or more such methods may include, but not be limited to, for example, morphological analyses and flow cytometry.
  • Cellular lineage and identity markers are known to those of skill in the art.
  • One or more such markers may be combined with one or more characterization methods to determine a composition of a cell population or phenotypic identity of one or more cells.
  • cells of a particular population will be characterized using flow cytometry (for example, see Ye Li et al., Cell Stem Cell. 2018 Aug 2; 23(2): 181-192. e5).
  • a sample of a population of cells will be evaluated for presence and proportion of one or more cell surface markers and/or one or more intracellular markers.
  • cell surface markers may be representative of different lineages.
  • pluripotent cells may be identified by one or more of any number of markers known to be associated with such cells, such as, for example, CD34.
  • markers of differentiated cells may include those associated with differentiated hematopoietic cells such as, e.g., CD43, CD45 (differentiated hematopoietic cells).
  • markers of differentiated cells may be associated with NK cell phenotypes such as, e.g., CD56, NK cell receptor immunoglobulin gamma Fc region receptor III (Fc ⁇ RIII, cluster of differentiation 16 (CD 16)), natural killer group-2 member D (NKG2D), CD69, a natural cytotoxicity receptor, etc.
  • markers may be T cell markers (e.g., CD3, CD4, CD8, etc.).
  • a disease, disorder and/or condition may be treated by introducing genetically modified or engineered cells as described herein (e.g., genetically modified NK or iNK cells) to a subject.
  • genetically modified or engineered cells as described herein (e.g., genetically modified NK or iNK cells)
  • diseases include, but are not limited to, cancer, e.g., solid tumors, e.g., of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testes, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, or esophagus; and hematological malignancies, e.g., acute and chronic leukemias, lymphomas, multiple myeloma and myelodysplastic syndromes.
  • cancer e.g., solid tumors, e.g., of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testes, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, or esophagus
  • hematological malignancies e.g., acute and chronic leukemias, lymphomas, multiple myel
  • the present disclosure provides methods of treating a subject in need thereof by administering to the subject a composition comprising any of the cells described herein.
  • a therapeutic agent or composition may be administered before, during, or after the onset of a disease, disorder, or condition (including, e.g., an injury).
  • the present disclosure provides any of the cells described herein for use in the preparation of a medicament.
  • the present disclosure provides any of the cells described herein for use in the treatment of a disease, disorder, or condition, that can be treated by a cell therapy.
  • the subject has a disease, disorder, or condition, that can be treated by a cell therapy.
  • a subject in need of cell therapy is a subject with a disease, disorder and/or condition, whereby a cell therapy, e.g., a therapy in which a composition comprising a cell described herein, is administered to the subject, whereby the cell therapy treats at least one symptom associated with the disease, disorder, and/or condition.
  • a subject in need of cell therapy includes, but is not limited to, a candidate for bone marrow or stem cell transplant, a subject who has received chemotherapy or irradiation therapy, a subject who has or is at risk of having cancer, e.g., a cancer of hematopoietic system, a subject having or at risk of developing a tumor, e.g., a solid tumor, and/or a subject who has or is at risk of having a viral infection or a disease associated with a viral infection.
  • cancer e.g., a cancer of hematopoietic system
  • a subject having or at risk of developing a tumor e.g., a solid tumor
  • the present disclosure provides pharmaceutical compositions comprising one or more genetically modified or engineered cells described herein, e.g., a genetically modified NK or iNK cell described herein.
  • a pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprises isolated pluripotent stem cell- derived hematopoietic lineage cells comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, CD34+ HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+ HE cells or HSCs.
  • a pharmaceutical composition comprises isolated pluripotent stem cell-derived hematopoietic lineage cells comprising about 95% to about 100% T cells, NK cells, NKT cells, CD34+ HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+ HE cells or HSCs.
  • a pharmaceutical composition of the present disclosure comprises an isolated population of pluripotent stem cell-derived hematopoietic lineage cells, wherein the isolated population has less than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% T cells, NK cells, NKT cells, CD34+ HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+ HE cells or HSCs.
  • an isolated population of pluripotent stem cell-derived hematopoietic lineage cells has more than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% T cells, NK cells, NKT cells, CD34+ HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+ HE cells or HSCs.
  • an isolated population of pluripotent stem cell-derived hematopoietic lineage cells has about 0.1 % to about 1%, about 1% to about 3%, about 3% to about 5%, about 10%-15%, about 15%-20%, about 20%-25%, about 25%-30%, about 30%-35%, about 35%-40%, about 40%-45%, about 45%-50%, about 60%-70%, about 70%-80%, about 80%-90%, about 90%-95%, or about 95% to about 100% T cells, NK cells, NKT cells, CD34+ HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+ HE cells or HSCs.
  • an isolated population of pluripotent stem cell-derived hematopoietic lineage cells comprises about 0.1%, about 1%, about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% T cells, NK cells, NKT cells, CD34+ HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+ HE cells or HSCs.
  • a pharmaceutical composition comprises pluripotent stem cell-derived hematopoietic lineage cells that are allogeneic to a subject.
  • a pharmaceutical composition comprises pluripotent stem cell-derived hematopoietic lineage cells that are autologous to a subject.
  • the isolated population of pluripotent stem cell-derived hematopoietic lineage cells can be either a complete or partial HLA-match with the subject being treated.
  • the pluripotent stem cell-derived hematopoietic lineage cells are not HLA- matched to a subject.
  • pluripotent stem cell-derived hematopoietic lineage cells can be administered to a subject without being expanded ex vivo or in vitro prior to administration.
  • an isolated population of derived hematopoietic lineage cells is modulated and treated ex vivo using one or more agents to obtain immune cells with improved therapeutic potential.
  • the modulated population of derived hematopoietic lineage cells can be washed to remove the treatment agent(s), and the improved population can be administered to a subject without further expansion of the population in vitro.
  • an isolated population of derived hematopoietic lineage cells is expanded prior to modulating the isolated population with one or more agents.
  • an isolated population of derived hematopoietic lineage cells can be genetically modified according to the methods of the present disclosure to express a recombinant TCR, CAR or other gene product of interest.
  • the cells can be activated and expanded using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869;
  • Any cancer can be treated using a cell or pharmaceutical composition described herein.
  • Exemplary therapeutic targets of the present disclosure include cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, eye, gastrointestinal system, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • a cancer may specifically be of the following non-limiting histological type: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma;
  • the cancer is a breast cancer.
  • the cancer is colorectal cancer (e.g., colon cancer).
  • the cancer is gastric cancer.
  • the cancer is RCC.
  • the cancer is non-small cell lung cancer (NSCLC).
  • the cancer is head and neck cancer.
  • solid cancer indications that can be treated with cells described herein (e.g., cells modified using methods of the disclosure, e.g., genetically modified iNK cells), either alone or in combination with one or more additional cancer treatment modality, include: bladder cancer, hepatocellular carcinoma, prostate cancer, ovarian/uterine cancer, pancreatic cancer, mesothelioma, melanoma, glioblastoma, HPV- associated and/or HPV-positive cancers such as cervical and HPV+ head and neck cancer, oral cavity cancer, cancer of the pharynx, thyroid cancer, gallbladder cancer, and soft tissue sarcomas.
  • hematological cancer indications that can be treated with cells described herein (e.g., cells modified using methods of the disclosure, e.g., genetically modified iNK cells), either alone or in combination with one or more additional cancer treatment modalities, include: ALL, CLL, NHL, DLBCL, AML, CML, and multiple myeloma (MM).
  • cells described herein e.g., cells modified using methods of the disclosure, e.g., genetically modified iNK cells
  • additional cancer treatment modalities include: ALL, CLL, NHL, DLBCL, AML, CML, and multiple myeloma (MM).
  • examples of cellular proliferative and/or differentiative disorders of the lung that can be treated with cells described herein (e.g., cells modified using methods of the disclosure) include, but are not limited to, tumors such as bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, metastatic tumors, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.
  • tumors such as bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, metastatic tumors, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.
  • examples of cellular proliferative and/or differentiative disorders of the breast that can be treated with cells described herein (e.g., cells modified using methods of the disclosure) include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget’s disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma,
  • proliferative breast disease
  • examples of cellular proliferative and/or differentiative disorders involving the colon that can be treated with cells described herein (e.g., cells modified using methods of the disclosure) include, but are not limited to, tumors of the colon, such as non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.
  • tumors of the colon such as non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.
  • examples of cancers or neoplastic conditions include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary aden
  • cells described herein are used in combination with one or more cancer treatment modalities.
  • other cancer treatment modalities include, but are not limited to: chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9 -tetrahydrocannabinol (dronabinol, MARINOL®);
  • dynemicin including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin, doxorubicin HC1 liposome injection (DOXIL®) and de
  • anti HGF monoclonal antibodies e.g., AV299 from Aveo, AMG102, from Amgen
  • truncated mTOR variants e.g., CGEN241 from Compugen
  • protein kinase inhibitors that block mTOR induced pathways e.g., ARQ197 from Arqule, XL880 from Exelexis, SGX523 from SGX Pharmaceuticals, MP470 from Supergen, PF2341066 from Pfizer
  • vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine
  • topoisomerase 1 inhibitor e.g., LURTOTECAN®
  • rmRH e.g., ABARELIX®
  • lapatinib ditosylate an ErbB-2 and EGFR dual tyrosine kinase small
  • cells described herein are used in combination with one or more cancer treatment modalities that facilitate the induction of antibody dependent cellular cytotoxicity (ADCC) (see e.g., Janeway’s Immunobiology by K. Murphy and C. weaver).
  • ADCC antibody dependent cellular cytotoxicity
  • such a cancer treatment modality is an antibody.
  • such an antibody is Trastuzumab.
  • such an antibody is Rituximab.

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Abstract

L'invention concerne des stratégies, des systèmes, des compositions et des procédés permettant de modifier génétiquement des cellules en vue d'inclure une ou plusieurs modifications de perte de fonction et/ou d'inclure une ou plusieurs modifications de gain de fonction, ainsi que des cellules modifiées (et des compositions de telles cellules) qui comprennent une ou plusieurs modifications de perte de fonction et/ou qui comprennent une ou plusieurs modifications de gain de fonction. Dans certains aspects, ces cellules modifiées comprennent au moins une modification de gain de fonction dans une région de codage d'un gène essentiel.
PCT/US2023/079157 2022-11-09 2023-11-08 Cellules ingéniérisées pour une thérapie WO2024102860A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020168300A1 (fr) * 2019-02-15 2020-08-20 Editas Medicine, Inc. Cellules tueuses naturelles modifiées (nk) pour l'immunothérapie
US11072781B2 (en) * 2015-11-04 2021-07-27 Fate Therapeutics, Inc. Genomic engineering of pluripotent cells
WO2021226151A2 (fr) * 2020-05-04 2021-11-11 Editas Medicine, Inc. Sélection par knock-in d'un gène essentiel
US20220184131A1 (en) * 2019-05-01 2022-06-16 Juno Therapeutics, Inc. Cells expressing a recombinant receptor from a modified tgfbr2 locus, related polynucleotides and methods
US20220184128A1 (en) * 2019-03-28 2022-06-16 Korea Research Institute Of Bioscience And Biotechnology Method for producing car gene-introduced nk cells and use thereof
US11365394B2 (en) * 2017-12-22 2022-06-21 Fate Therapeutics, Inc. Enhanced immune effector cells and use thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11072781B2 (en) * 2015-11-04 2021-07-27 Fate Therapeutics, Inc. Genomic engineering of pluripotent cells
US11365394B2 (en) * 2017-12-22 2022-06-21 Fate Therapeutics, Inc. Enhanced immune effector cells and use thereof
WO2020168300A1 (fr) * 2019-02-15 2020-08-20 Editas Medicine, Inc. Cellules tueuses naturelles modifiées (nk) pour l'immunothérapie
US20220184128A1 (en) * 2019-03-28 2022-06-16 Korea Research Institute Of Bioscience And Biotechnology Method for producing car gene-introduced nk cells and use thereof
US20220184131A1 (en) * 2019-05-01 2022-06-16 Juno Therapeutics, Inc. Cells expressing a recombinant receptor from a modified tgfbr2 locus, related polynucleotides and methods
WO2021226151A2 (fr) * 2020-05-04 2021-11-11 Editas Medicine, Inc. Sélection par knock-in d'un gène essentiel

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