WO2023133568A2 - Hypoimmune beta cells differentiated from pluripotent stem cells and related uses and methods - Google Patents

Hypoimmune beta cells differentiated from pluripotent stem cells and related uses and methods Download PDF

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WO2023133568A2
WO2023133568A2 PCT/US2023/060341 US2023060341W WO2023133568A2 WO 2023133568 A2 WO2023133568 A2 WO 2023133568A2 US 2023060341 W US2023060341 W US 2023060341W WO 2023133568 A2 WO2023133568 A2 WO 2023133568A2
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modified
cell
beta cell
molecules
beta
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WO2023133568A3 (en
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Sonja SCHREPFER
Jeffrey R. Millman
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Sana Biotechnology Inc
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Sana Biotechnology Inc
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Priority to IL314029A priority Critical patent/IL314029A/en
Priority to US18/727,670 priority patent/US20250223564A1/en
Priority to KR1020247026729A priority patent/KR20240142624A/ko
Priority to CA3246902A priority patent/CA3246902A1/en
Priority to CN202380025788.9A priority patent/CN118871590A/zh
Priority to EP23737829.4A priority patent/EP4463171A4/en
Priority to MX2024008571A priority patent/MX2024008571A/es
Priority to AU2023204829A priority patent/AU2023204829A1/en
Priority to JP2024541154A priority patent/JP2025504392A/ja
Publication of WO2023133568A2 publication Critical patent/WO2023133568A2/en
Publication of WO2023133568A3 publication Critical patent/WO2023133568A3/en
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Definitions

  • the present disclosure is directed to modified or engineered stem cell- derived beta (P) cells (SC-beta cells) containing one or more genetic modification, such as genetic modifications, and related methods of their use and generation.
  • the modified cells are hypoimmunogenic cells.
  • the modified SC-beta cells are cells differentiated in vitro from a modified or hypoimmunogenic pluripotent stem cell that contains the one or more modifications.
  • the one or more modifications reduce or eliminate expression of MHC class I and/or MHC class II human leukocyte antigens and also exogenously express one or more tolerogenic factors such as CD47.
  • a method of generating a modified stem cell derived beta cell comprising: (A) providing a modified pluripotent stem cell (PSC) comprising modifications that: (a) inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules; and (b) increase expression of one or more tolerogenic factors in the modified PSC, relative to a control or wild-type PSC; and (B) culturing the modified PSC under conditions sufficient for differentiation of the modified PSC into the modified SC-beta cell.
  • PSC pluripotent stem cell
  • a method of generating a modified stem cell derived beta cell comprising: (A) generating a modified pluripotent stem cell (PSC) comprising: (a) introducing, into a PSC, one or more modifications that inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules; and (b) increasing expression of one or more tolerogenic factors in the PSC, relative to a control or wildtype PSC; and (B) culturing the modified PSC under conditions sufficient for differentiation of the modified PSC into a modified SC-beta cell.
  • MHC major histocompatibility complex
  • a method of generating a modified stem cell derived beta cell comprising (A) providing a modified pluripotent stem cell (PSC) that comprises at least one modification selected from the group consisting of: (a) modifications that inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules; and (b) modifications that increase expression of one or more tolerogenic factors in the modified PSC, relative to a control or wild-type cell of the same cell type that does not comprise the modification; (B) culturing the modified PSC under conditions sufficient for differentiation of the modified PSC into a modified SC-beta cell; and (C) introducing one or more additional modifications into the modified SC-beta cell,
  • PSC pluripotent stem cell
  • SC-beta cell a modified stem cell derived beta cell
  • the method comprising (A) culturing a pluripotent stem cell (PSC) under conditions sufficient for differentiation of the PSC into a SC-beta cell; and (B) generating a modified SC-beta cells comprising: (a) introducing, into the SC-beta cell, one or more modifications that inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules; and (b) increasing expression of one or more tolerogenic factors in the SC-beta cell, relative to a control or wild-type SC-beta cell.
  • MHC major histocompatibility complex
  • a method of generating a generating a modified stem cell derived beta cell comprising (A) providing a modified pluripotent stem cell (PSC) comprising modifications that: (a) reduce expression of one or more major histocompatibility complex (MHC) class I molecule and/or one or more MHC class II molecule in the modified PSC, relative to a control or wildtype PSC; and (b) increase expression of one or more tolerogenic factors in the modified PSC, relative to the control or wild- type PSC; and (B) culturing the modified PSC under conditions sufficient for differentiation of the modified PSC into the modified SC-beta cell.
  • MHC major histocompatibility complex
  • a method of generating a generating a modified stem cell derived beta cell comprising (A) generating a modified pluripotent stem cell (PSC) comprising (a) reducing expression of one or more major histocompatibility complex (MHC) class I molecules and/or one or more MHC class II molecules in a PSC, relative to a control or wild-type PSC; and (b) increasing expression of one or more tolerogenic factors in the PSC, relative to the control or wild-type PSC; and (B) culturing the modified PSC under conditions sufficient for differentiation of the modified PSC into a modified SC-beta cell.
  • PSC pluripotent stem cell
  • reducing expression of the one or more MHC class I molecules and/or the one or more MHC class II molecules comprises introducing modifications that reduce expression of the one or more MHC class I molecule and/or the one or more MHC class II molecules in the modified PSC, relative to the control or wild-type PSC.
  • control or wild- type PSC is an unmodified PSC that does not comprise the modifications.
  • the PSC does not comprise the modifications.
  • expression of one or more MHC class I molecules and one or more MHC class II molecules is reduced in the modified PSC.
  • the modifications in (a) reduce protein expression of the one or more MHC class I molecules. In some of any of the provided embodiments, the modifications in (a) reduce cell surface expression of the one or more MHC class I molecules. In some of any of the provided embodiments, the modifications in (a) reduce a function of the one or more MHC class I molecules. In some embodiments, the function is antigen presentation.
  • the one or more modifications in (a) reduce cell surface trafficking of the one or more MHC class I molecules.
  • the one or more MHC class I molecules is one or more human leukocyte antigen (HLA) class I molecules.
  • HLA human leukocyte antigen
  • the one or more MHC HLA class I molecules is selected from the group consisting of HLA- A, HLA-B, and HLA-C.
  • the one or more molecules that regulate cell surface protein expression of the one or more MHC class I molecules are B2M.
  • the modification that reduce expression of the one or more MHC class I molecules reduce expression of the B-2 microglobulin (B2M) gene and/or the transporter 1, ATP binding cassette subfamily B member (TAPI) gene.
  • the modifications that reduce expression of the one or more MHC class I molecules reduce expression of the B-2 microglobulin (B2M) gene.
  • the modification that reduce expression of the one or more MHC class I molecules reduce expression of the transporter 1, ATP binding cassette subfamily B member (TAPI) gene.
  • the modifications reduce expression of the B-2 microglobulin (B2M) gene and the transporter 1, ATP binding cassette subfamily B member (TAPI) gene.
  • the modifications reduce expression reduce expression reduce expression of the B2M gene.
  • the modification that reduces expression of the one or more MHC class I molecules reduces expression of B2M. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class I molecules reduces mRNA expression of the B2M gene. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class I molecules reduces protein expression of B2M. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class I molecules comprises inactivation or disruption of one allele of the B2M gene.
  • the modification that reduces expression of the one or more MHC class I molecules comprises inactivation or disruption of both alleles of the B2M gene. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class I molecules comprises inactivation or disruption of all B2M coding alleles in the cell. In some of any of the provided embodiments, the inactivation or disruption comprises an indel in the B2M gene. In some of any of the provided embodiments, the inactivation or disruption comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the B2M gene. In some embodiments, the B2M gene is knocked out.
  • the modification that reduces expression of the one or more MHC class I molecules reduces expression of TAPI. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class I molecules reduces mRNA expression of the TAPI gene. In some embodiments, the modification that reduces expression of the one or more MHC class I molecules reduce expression reduces protein expression of a protein encoded by the TAPI gene. In some embodiments, the modification comprises inactivation or disruption of one allele of the TAPI gene. In some embodiments, the modification comprises inactivation or disruption of both alleles of the TAPI gene.
  • the modification comprises inactivation or disruption of all coding sequences of the TAPI gene in the cell.
  • the inactivation or disruption comprises an indel in one allele of the TAPI gene.
  • the inactivation or disruption comprises an indel in both alleles of the TAPI gene.
  • the one or more modifications that reduce expression comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the TAPI gene.
  • the TAPI gene is knocked out.
  • the modifications in (a) reduce protein expression of the one or more MHC class II molecules.
  • the modifications in (a) reduce cell surface expression of the one or more MHC class II molecules. In some of any of the provided embodiments, the modifications in (a) reduce a function of the one or more MHC class II molecules. In some embodiments, the function is antigen presentation.
  • the one or more modifications in (a) reduce cell surface trafficking of the one or more MHC class II molecules.
  • the one or more MHC class II molecules is one or more human leukocyte antigen (HLA) class II molecules.
  • HLA human leukocyte antigen
  • the one or more MHC HLA class II molecules is selected from the group consisting of HLA-DP, HLA-DQ, and/or HLA- DR.
  • the one or more molecules that regulate expression of the one or more MHC class II molecules is/are selected from the group consisting of OITA and CD74.
  • the modification that reduce expression of the one or more MHC class II molecules reduce expression of the OITA gene and/or CD74 gene.
  • the modification is a modification that regulates expression of the one or more MHC class II molecules, and the modification inactivates or disrupts one or more alleles of OITA.
  • the modifications that reduce expression of the one or more MHC class II molecules reduce expression of the CITTA gene.
  • the modification that reduce expression of the one or more MHC class II molecules reduce expression of the CD74 gene.
  • the modifications reduce expression of the OITA gene and the CD74 gene.
  • the modification that reduces expression of the one or more MHC class II molecules reduces expression of CD74. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class II molecules reduces mRNA expression of the CD74 gene. In some embodiments, the modification that reduces expression of the one or more MHC class II molecules reduce expression reduces protein expression of a protein encoded by the CD74 gene. In some embodiments, the modification comprises inactivation or disruption of one allele of the CD74 gene. In some embodiments, the modification comprises inactivation or disruption of both alleles of the CD74 gene. In some embodiments, the modification comprises inactivation or disruption of all coding sequences of the CD74 gene in the cell.
  • the inactivation or disruption comprises an indel in one allele of the CD74 gene. In some embodiments, the inactivation or disruption comprises an indel in both alleles of the CD74 gene. In some embodiments, the one or more modifications that reduce expression comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CD74 gene. In some embodiments, the CD74 gene is knocked out. [0025] In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class II molecules comprises reduced expression of OITA. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class II molecules reduces mRNA expression of the OITA gene.
  • the modification that reduces expression of the one or more MHC class II molecules reduces protein expression of OITA. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class II molecules comprises inactivation or disruption of one allele of the OITA gene. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class II molecules comprises inactivation or disruption of both alleles of the OITA gene. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class II molecules comprises or inactivation or disruption of all OITA coding alleles in the cell. In some of any of the provided embodiments, the inactivation or disruption comprises an indel in the OITA gene. In some of any of the provided embodiments, the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the OITA gene. In some embodiments, the OITA gene is knocked out.
  • expression of all MHC class I molecules and all MHC class II molecules is reduced in the modified PSC.
  • expression of HLA- A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR are reduced in the modified PSC.
  • the one or more tolerogenic factors is selected from the group consisting of CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD- Ll, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, and SERPINB9.
  • at least one of the one or more tolerogenic factors is CD47.
  • the one or more tolerogenic factors is CD47.
  • at least one of the one or more tolerogenic factors is PD-L1.
  • the one or more tolerogenic factors is PD-L1. In some of any of the provided embodiments, at least one of the one or more tolerogenic factors is HLA-E. In some of any of the provided embodiments, the one or more tolerogenic factors is HLA-E. In some of any of the provided embodiments, at least one of the one or more tolerogenic factors is HLA-G. In some of any of the provided embodiments, the one or more tolerogenic factors is HLA-G.
  • increasing expression of the one or more tolerogenic factors comprises introducing a modification that increases expression of the one or more tolerogenic factors in the modified PSC, relative to the control or wild-type PSC.
  • the modification to increase expression of the one or more tolerogenic factors comprises an exogenous polynucleotide encoding the one or more tolerogenic factors.
  • the exogenous polynucleotide encoding the one or more tolerogenic factors is integrated into the genome of the modified PSC.
  • the exogenous polynucleotide is integrated by non-targeted insertion into the genome of the modified PSC.
  • the non-targeted integration is by introduction of the exogenous polynucleotide into the cell using a lentiviral vector.
  • the exogenous polynucleotide is integrated by targeted insertion into a target genomic locus of the cell.
  • the targeted insertion is by nuclease-mediated gene editing with homology-directed repair.
  • increasing expression of the one or more tolerogenic factors comprises introducing a modification that increases expression of the one or more tolerogenic factors in the modified SC-beta cell, relative to the control or wild-type beta cell.
  • the modification to increase expression of the one or more tolerogenic factors comprises an exogenous polynucleotide encoding the one or more tolerogenic factors.
  • the exogenous polynucleotide encoding the one or more tolerogenic factors is integrated into the genome of the modified SC-beta cell.
  • the exogenous polynucleotide is integrated by non-targeted insertion into the genome of the modified SC-beta cell.
  • the non-targeted integration is by introduction of the exogenous polynucleotide into the cell using a lentiviral vector.
  • the exogenous polynucleotide is integrated by targeted insertion into a target genomic locus of the cell.
  • the targeted insertion is by nuclease-mediated gene editing with homology-directed repair.
  • a method of generating a modified stem cell derived beta cell comprising: (A) providing a modified pluripotent stem cell (PSC) comprising knock out of the B2M gene, knock out of the OITA gene, and an exogenous polynucleotide encoding CD47 protein, relative to a control or wild-type PSC; and (B) culturing the modified PSC under conditions sufficient for differentiation of the modified PSC into the modified SC-beta cell.
  • the modified PSC has the phenotype B2M"" 77/ "" 77 ; CIITA' ⁇ ; CD47tg.
  • the modified PSC further comprises a modification to increase expression of an exogenous suicide gene.
  • the modified SC-beta cell further comprises a modification to increase expression of an exogenous suicide gene.
  • SC- beta cell also provided herein is a method of generating a modified stem cell derived beta cell (SC- beta cell), the method comprising: (A) providing a pluripotent stem cell (PSC); (B) culturing the PSC under conditions sufficient for differentiation of the PSC into a SC-beta cell; and (C) generating a modified SC-beta cell from the SC-beta cell by introducing modifications, into the SC-beta cell to knock out the B2M gene and to knock out the OITA gene, and introducing an exogenous polynucleotide encoding CD47 protein.
  • PSC pluripotent stem cell
  • the modified SC-beta cell has the phenotype CIITA''" feZ/m ⁇ feZ ; CD47tg. In some of any embodiments, the modified SC-beta cell further comprises a modification to increase expression of an exogenous suicide gene. In some of any embodiments, the modified SC-beta cell further comprises a modification to increase expression of an exogenous suicide gene.
  • a method of generating a modified stem cell derived beta cell comprising: (A) providing a modified pluripotent stem cell (PSC) comprising knock out of the B2M gene, knock out of the OITA gene, an exogenous polynucleotide encoding CD47 protein, and an exogenous polynucleotide encoding a suicide gene, relative to a control or wild-type PSC; and (B) culturing the modified PSC under conditions sufficient for differentiation of the modified PSC into the modified SC-beta cell.
  • the modified PSC has the phenotype B2M'“ ieZ/ '“ ieZ ; cnTA ⁇ z/ ⁇ /. CD47fg; suic ide genetg.
  • Also provided herein is a method of generating a modified stem cell derived beta cell (SC- beta cell), the method comprising: (A) providing a pluripotent stem cell (PSC); (B) culturing the PSC under conditions sufficient for differentiation of the PSC into a SC-beta cell; and (C) generating a modified SC-beta cell from the SC-beta cell by introducing modifications, into the SC-beta cell to knock out the B2M gene and to knock out the OITA gene, and introducing an exogenous polynucleotide encoding CD47 protein, and an exogenous polynucleotide encoding a safety switch.
  • PSC pluripotent stem cell
  • B pluripotent stem cell
  • C generating a modified SC-beta cell from the SC-beta cell by introducing modifications, into the SC-beta cell to knock out the B2M gene and to knock out the OITA gene, and introducing an exogenous polynucleot
  • the modified SC-beta cell has the phenotype B2M indel/indel - CIITA ⁇ “ CD47tg; safety switch (e.g., suicide gene) transgene.
  • the exogenous polynucleotide encoding CD47 is integrated by non-targeted insertion into the genome of the modified SC-beta cell, optionally by introduction of the exogenous polynucleotide into the cell using a lentiviral vector.
  • the modified SC-beta cell comprises an exogenous polynucleotide encoding a suicide gene or suicide switch.
  • the suicide gene is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase 9 (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).
  • the suicide gene or suicide switch and genes associated with the suicide gene or the safety switch are expressed from a bicistronic cassette integrated into the genome of the modified SC-beta cell.
  • the suicide gene or suicide switch and the one or more tolerogenic factors are expressed from a bicistronic cassette integrated into the genome of the modified SC-beta cell.
  • the bicistronic cassette is integrated by non-targeted insertion into the genome of the modified SC-beta cell.
  • the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the modified SC-beta cell.
  • the one or more tolerogenic factors is CD47.
  • suicide gene is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase 9 (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).
  • CyD cytosine deaminase
  • HSV-Tk herpesvirus thymidine kinase
  • iCaspase9 inducible caspase 9
  • rapamycin-activated caspase 9 rapamycin-activated caspase 9
  • the suicide gene and the one or more tolerogenic factors are expressed from a bicistronic cassette integrated into the genome of the modified PSC.
  • the one or more tolerogenic factor is or comprises CD47 and the suicide gene and the CD47 are expressed from a bicistronic cassette integrated into the genome of the modified PSC.
  • the bicistronic cassette is integrated by non-targeted insertion into the genome of the modified PSC.
  • the non-targeted integration is by introduction of the exogenous polynucleotide into the cell using a lentiviral vector.
  • the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the cell.
  • the targeted insertion is by nuclease-mediated gene editing with homology-directed repair.
  • the safety switch e.g., suicide gene
  • the one or more tolerogenic factors are expressed from a bicistronic cassette integrated into the genome of the modified cell.
  • the safety switch and CD47 are expressed from a bicistronic cassette integrated into the genome of the modified cell.
  • the bicistronic cassette is integrated by non-targeted insertion into the genome of the modified SC-beta cell.
  • the non-targeted integration is by introduction of the exogenous polynucleotide into the cell using a lentiviral vector.
  • the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the cell.
  • the targeted insertion is by nuclease-mediated gene editing with homology-directed repair.
  • the target genomic locus is a safe harbor locus, a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • the safe harbor locus is selected from the group consisting of: a CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C (also known as AAVS1) gene, an albumin gene locus, a SHS231 locus, a CLYBL gene locus, and a ROSA26 gene locus.
  • the modified PSC comprises a modification that reduces expression of CD142, relative to the control or wild-type PSC.
  • the modification reduces mRNA expression of the CD142 gene.
  • the modification reduces protein expression of CD142.
  • the modification comprises inactivation or disruption of one allele of the CD142 gene.
  • the modification comprises inactivation or disruption of both alleles of the CD142 gene.
  • the modification comprises inactivation or disruption of all CD 142 coding alleles in the cell.
  • the inactivation or disruption comprises an indel in the CD142 gene. In some of any of the provided embodiments, the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CD 142 gene.
  • the modified PSC comprises a modification that increases expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, CD55 and CD35, relative to the control or wild-type PSC.
  • the modification to increase expression of the one or more complement inhibitors comprises at least one exogenous polynucleotide selected from the group consisting of an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, an exogenous polynucleotide encoding CD55 and an exogenous polynucleotide encoding CD35.
  • the one or more complement inhibitors is CD46 and CD59. In some of any of the provided embodiments, the one or more complement inhibitor is CD46, CD59 and CD55. In some of any of the provided embodiments, the at least one exogenous polynucleotide is integrated by nontargeted insertion into the genome of the modified PSC. In some embodiments the non-targeted insertion is by introduction of the exogenous polynucleotide into the cell using a lentiviral vector. In some of any of the provided embodiments, the at least one exogenous polynucleotide is integrated by targeted insertion into a target genomic locus of the cell.
  • the targeted insertion is by nuclease-mediated gene editing with homology-directed repair.
  • the target genomic locus is a safe harbor locus, a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • the safe harbor locus is selected from the group consisting of: a CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C (also known as AAVS1) gene, an albumin gene locus, a SHS231 locus, a CLYBL gene locus, and a ROSA26 gene locus.
  • the culturing the PSC under conditions sufficient for differentiation of the PSC into the SC-beta cell comprises one or more of: (i) contacting the PSC with a TGFbeta/Activin agonist and/or, a glycogen synthase kinase 3 (GSK) inhibitor and/or WNT agonist for an amount of time sufficient to form a definitive endoderm cell; (ii) contacting a definitive endoderm cell differentiated from the PSC with a FGFR2b agonist for an amount of time sufficient to form a primitive gut tube cell; (iii) contacting a primitive gut tube cell differentiated from the PSC with a retinoic acid receptor (RAR) agonist, a rho kinase inhibitor, a Smoothened antagonist, a FGFR2b agonist, a protein kinase C activator, and/or a BMP type 1 receptor inhibitor for an amount of time sufficient to form an early pancre
  • RAR retinoic acid receptor
  • the culturing the modified PSC under conditions sufficient for differentiation of the modified PSC into the modified SC-beta cell comprises one or more of (i) contacting the modified PSC with a TGF /Activin agonist or a glycogen synthase kinase 3 (GSK) inhibitor or WNT agonist for an amount of time sufficient to form a definitive endoderm cell; (ii) contacting a definitive endoderm cell differentiated from the modified PSC with a FGFR2b agonist for an amount of time sufficient to form a primitive gut tube cell; (iii) contacting a primitive gut tube cell differentiated from the modified PSC with an RAR agonist, and optionally a rho kinase inhibitor, a Smoothened antagonist, a FGFR2b agonist, a protein kinase C activator, or a BMP type 1 receptor inhibitor for an amount of time sufficient to form an early pancreas progenitor cell; (i) contacting the modified PSC with a TGF
  • the culturing the modified PSC under conditions sufficient for differentiation of the modified PSC into the modified SC-beta cell comprises (i) contacting the modified PSC with a TGF /Activin agonist or a glycogen synthase kinase 3 (GSK) inhibitor or WNT agonist for an amount of time sufficient to form a definitive endoderm cell; (ii) contacting the definitive endoderm cell with a FGFR2b agonist for an amount of time sufficient to form a primitive gut tube cell; (iii) contacting the primitive gut tube cell with an RAR agonist, and optionally a rho kinase inhibitor, a Smoothened antagonist, a FGFR2b agonist, a protein kinase C activator, or a BMP type 1 receptor inhibitor for an amount of time sufficient to form an early pancreas progenitor cell; (iv) incubating the early pancreas progenitor cell for at least about
  • depolymerizing the actin cytoskeleton comprises plating cells on a stiff or soft substrate or introducing a cytoskeletal-modulating agent to cells.
  • the cytoskeletal-modulating agent comprises latrunculin A, latrunculin B, nocodazole, cytochalasin D, jasplakinolide, blebbistatin, y-27632, y-15, gdc-0994, or an integrin modulating agent.
  • the cytoskeletal-modulating agent is latrunculin A.
  • depolymerizing the actin cytoskeleton is initiated at the start of the contacting in (v). In some of any of the provided embodiments, depolymerizing the actin cytoskeleton comprises adding latrunculin A at the start of the contacting for at least at or about the first 24 hours. In some of any of the provided embodiments, resizing the beta cell clusters comprises breaking apart clusters and reaggregating.
  • the TGF /Activin agonist is Activin A.
  • the glycogen synthase kinase 3 (GSK) inhibitor or the WNT agonist is CHIR.
  • the FGFR2b agonist is KGF.
  • the Smoothened antagonist is SANT-1.
  • the RAR agonist is retinoic acid (RA).
  • the protein kinase C activator is TPPB.
  • the BMP type 1 receptor inhibitor is LDN.
  • the rho kinase inhibitor is Y27632.
  • the Alk5 inhibitor is Alk5i.
  • the Erbb4 agonist is betacellulin.
  • the thyroid hormone is T3.
  • the gamma secretase inhibitor is XXI.
  • the PSC is an embryonic stem cell. In some of any of the provided embodiments, the PSC is an induced PSC (iPSC). In some embodiments, the iPSC is a patient-derived iPSC.
  • the modified PSC expresses each of the one or more tolerogenic factors at a first level that is greater than at or about 5-fold over a second level expressed by the control or wild-type PSC.
  • each of the one or more tolerogenic factors is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wild-type PSC.
  • each of the one or more tolerogenic factors is expressed by the modified PSC at greater than at or about 20,000 molecules per cell. In some of any of the provided embodiments, each of the one or more tolerogenic factors is expressed by the modified PSC at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • the one or more tolerogenic factors is or comprises CD47 and the modified PSC expresses CD47 at a first level that is greater than at or about 5- fold over a second level expressed by the control or wild-type PSC.
  • CD47 is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wild-type PSC.
  • the one or more tolerogenic factor is CD47 and CD47 is expressed by the modified PSC at greater than at or about 20,000 molecules per cell.
  • CD47 is expressed by the modified PSC at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • the modified SC-beta cell comprises the modifications of the modified PSC.
  • the modified SC-beta cell comprises modifications that (1) reduce expression of one or more MHC class I molecules and/or one or more MHC class II molecules, relative to a control or wild-type beta cell; and (2) increase expression of one or more tolerogenic factors, relative to the control or wild-type beta cell.
  • the control or wild-type SC-beta cell is an unmodified SC-beta cell differentiated from an unmodified PSC not comprising modifications that reduce expression of the one or more MHC class I molecules and/or the one or more MHC class II molecules or that increase expression of the one or more tolerogenic factors.
  • the control or wild- type beta cell is a wild-type primary beta cell.
  • expression of the one or more MHC class I molecules and the one or more MHC class II molecules is reduced in the modified SC-beta cell.
  • the modifications in (1) reduce protein expression of the one or more MHC class I molecules in the modified SC-beta cell. In some of any of the provided embodiments, the modifications in (1) reduce cell surface expression of the one or more MHC class I molecules in the modified SC-beta cell. In some of any of the provided embodiments, the modifications in (1) reduce a function of the one or more MHC class I molecules in the modified SC-beta cell. In some embodiments, the function is antigen presentation.
  • the one or more MHC class I molecules is one or more human leukocyte antigen (HLA) class I molecules.
  • HLA human leukocyte antigen
  • the one or more MHC HLA class I molecules is selected from the group consisting of HLA-A, HLA-B, and HLA-C.
  • the modification that reduces expression of the one or more MHC class I molecules in the modified SC-beta cell comprises reduced expression of B2M. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class I molecules in the modified SC-beta cell reduces mRNA expression of the B2M gene. In some of any of the provided embodiments, the modification that reduces expression of the one or more MHC class I molecules in the modified SC-beta cell reduces protein expression of B2M.
  • the modification that reduces expression of the one or more MHC class I molecules in the modified SC-beta cell comprises inactivation or disruption of one allele of the B2M gene. In some of any embodiments, the modification that reduces expression of the one or more MHC class I molecules in the modified SC-beta cell comprises inactivation or disruption of both alleles of the B2M gene. In some of any embodiments, the modification that reduces expression of the one or more MHC class I molecules in the modified SC-beta cell comprises inactivation or disruption of all B2M coding alleles in the cell. In some of any embodiments, the inactivation or disruption comprises an indel in the B2M gene. In some of any embodiments, the inactivation or disruption comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the B2M gene.
  • the modifications in (1) reduce protein expression of the one or more MHC class II molecules in the modified SC-beta cell. In some of any embodiments, the modifications in (1) reduce cell surface expression of the one or more MHC class II molecules in the modified SC-beta cell. In some of any embodiments, the modifications in (1) reduce a function of the one or more MHC class II molecules in the modified SC-beta cell. In some embodiments, the function is antigen presentation.
  • the one or more MHC class II molecules is one or more human leukocyte antigen (HLA) class II molecules. In some embodiments, the one or more MHC class II molecules is selected from the group consisting of HLA-DP, HLA-DQ, and/or HLA-DR.
  • the modification that reduces expression of the one or more MHC class II molecules in the modified SC-beta cell comprises reduced expression of OITA. In some of any embodiments, the modification that reduces expression of the one or more MHC class II molecules in the modified SC-beta cell reduces mRNA expression of the OITA gene. In some of any embodiments, the modification that reduces expression of the one or more MHC class II molecules in the modified SC- beta cell reduces protein expression of OITA. In some of any embodiments, the modification that reduces expression of the one or more MHC class II molecules comprises inactivation or disruption of one allele of the OITA gene.
  • the modification that reduces expression of the one or more MHC class II molecules comprises inactivation or disruption of both alleles of the OITA gene. In some of any embodiments, the modification that reduces expression of the one or more MHC class II molecules comprises inactivation or disruption of all OITA coding alleles in the cell. In some of any embodiments, the inactivation or disruption comprises an indel in the OITA gene. In some of any embodiments, the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the OITA gene.
  • expression of all MHC class I molecules and all MHC class II molecules is reduced in the modified SC-beta cell.
  • expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR are reduced in the modified SC-beta cell.
  • the modified SC-beta cell comprises a modification that reduces expression of CD142.
  • the modification reduces mRNA expression of the CD142 gene.
  • the modification reduces protein expression of CD142.
  • the modification comprises inactivation or disruption of one allele of the CD142 gene.
  • the modification comprises inactivation or disruption of both alleles of the CD142 gene.
  • the modification comprises inactivation or disruption of all CD142 coding alleles in the cell.
  • the inactivation or disruption comprises an indel in the CD 142 gene.
  • the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CD 142 gene.
  • the modification to increase expression of the one or more tolerogenic factors in the modified SC-beta cell comprises an exogenous polynucleotide encoding the one or more tolerogenic factors.
  • the exogenous polynucleotide encoding the one or more tolerogenic factors is integrated into the genome of the modified SC-beta cell.
  • the exogenous polynucleotide encoding the one or more tolerogenic factors is integrated into a non-target locus in the genome of the modified SC-beta cell.
  • the exogenous polynucleotide encoding the one or more tolerogenic factors is integrated into a target genomic locus of the modified SC-beta cell.
  • the tolerogenic factor is CD47.
  • the modified SC-beta cell further comprises a modification for expression of an exogenous suicide gene in the modified SC-beta cell.
  • the modified SC-beta cell generated by the provided methods comprises knock out of the B2M gene, knock out of the OITA gene, and an exogenous polynucleotide encoding exogenous CD47 protein, relative to a control or wild-type beta cell.
  • the modified SC-beta cell has the phenotype B2M"" 77/ "" 77 ; ciITA ⁇ e “ CD47tg.
  • the modified SC-beta cell generated by the provided methods comprises knock out of the B2M gene, knock out of the CIITA gene, an exogenous polynucleotide encoding CD47 protein, and an exogenous polynucleotide encoding a suicide gene, relative to a control or wild-type beta cell.
  • the modified SC-beta cell has the phenotype B2M"" 77/ "" 77 ; cnTA ⁇ z/ ⁇ /. CD47fg; suic ide genetg.
  • the exogenous polynucleotide encoding CD47 is integrated into a non-target locus in the genome of the modified SC-beta cell. In some of any embodiments, the exogenous polynucleotide encoding CD47 is integrated into a target genomic locus of the modified SC- beta cell.
  • the exogenous suicide gene is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).
  • the suicide gene and the one or more tolerogenic factors are expressed from a bicistronic cassette integrated into the genome of the modified SC-beta cell.
  • the one or more tolerogenic factors is CD47 and the suicide gene and CD47 are expressed from a bicistronic cassette integrated into the genome of the modified SC-beta cell.
  • the bicistronic cassette is integrated at a non-target locus in the genome of the modified SC-beta cell. In some of any embodiments, the bicistronic cassette is integrated into a target genomic locus of the cell. In some of any embodiments, the target genomic locus is a safe harbor locus, a B2M gene locus, a CIITA gene locus, or a CD142 gene locus.
  • the safe harbor locus is selected from the group consisting of: a CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C (also known as AAVS1) gene, an albumin gene locus, a SHS231 locus, a CLYBL gene locus, and a ROSA26 gene locus.
  • the methods generate a modified SC-beta cell that comprises a modification that increases expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, CD55 and CD35, relative to the control or wild-type beta cell.
  • the modification to increase expression of the one or more complement inhibitors in the modified SC-beta cell comprises at least one exogenous polynucleotide encoding the one or more complement inhibitors selected from the group consisting of an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, an exogenous polynucleotide encoding CD55, and an exogenous polynucleotide encoding CD35.
  • the one or more complement inhibitors is CD46 and CD59.
  • the one or more complement inhibitor is CD46, CD59 and CD55.
  • the reduced expression comprises reduced surface expression.
  • the increased expression comprises increased surface expression.
  • the level of the reduced expression of (1) and the increased expression of (2) by the modified SC-beta cell is retained or is similar compared to the modified PSC.
  • the methods generate a modified SC-beta cell that expresses the one or more tolerogenic factors at a first level that is greater than at or about 5 -fold over a second level expressed by the control or wild-type beta cell.
  • the control or wildtype beta cell is differentiated from an unmodified PSC not comprising modifications that reduce expression of the one or more MHC class I molecules and/or the one or more MHC class II molecules and that increase expression of the one or more tolerogenic factors.
  • the modified SC-beta cell expresses each of the one or more tolerogenic factors at a first level that is greater than at or about 5-fold over a second level expressed by the control or wild-type beta cell.
  • each of the one or more tolerogenic factors is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wild-type beta cell.
  • each of the one or more tolerogenic factors is expressed by the modified SC-beta cell at greater than at or about 20,000 molecules per cell. In some of any embodiments, each of the one or more tolerogenic factors is expressed by the modified SC-beta cell at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • the one or more tolerogenic factors is or comprises CD47 and the modified SC-beta cell expresses CD47 at a first level that is greater than at or about 5-fold over a second level expressed by the control or wild-type beta cell.
  • the control or wild-type beta cell is differentiated from an unmodified PSC not comprising modifications that reduce expression of the one or more MHC class I molecules and/or the one or more MHC class II molecules and that increase expression of the one or more tolerogenic factors.
  • the modified SC-beta cell expresses CD47 at a first level that is greater than at or about 5-fold over a second level expressed by the control or wild-type beta cell.
  • CD47 is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wild-type beta cell.
  • CD47 is expressed by the modified SC-beta cell at greater than at or about 20,000 molecules per cell.
  • CD47 is expressed by the modified SC- beta cell at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • the modified SC-beta cell expresses at least one beta cell marker.
  • the at least one beta cell marker is selected from the group consisting of INS, CHGA, NKX2-2, PDX1, NKX6-1, MAFB, GCK and GLUT1.
  • the modified SC-beta cell exhibits one or more functions of a wild-type or control beta cell.
  • the one or more functions is selected from the group consisting of in vitro glucose-stimulated insulin secretion (GSIS), glucose metabolism, maintaining fasting blood glucose levels, secreting insulin in response to glucose injections in vivo, and clearing glucose after a glucose injection in vivo.
  • GSIS in vitro glucose-stimulated insulin secretion
  • the modified SC-beta cell is capable of glucose-stimulated insulin secretion (GSIS).
  • GSIS glucose-stimulated insulin secretion
  • the insulin secretion is in a perfusion GSIS assay.
  • the GSIS is dynamic GSIS comprising first and second phase dynamic insulin secretion.
  • the GSIS is static GSIS.
  • the static incubation index is greater than at or about 1, greater than at or about 2, greater than at or about 5, greater than at or about 10 or greater than at or about 20.
  • the level of insulin secretion by the modified SC-beta cells is at least 20% of that observed for primary beta islets, such as observed for cadaveric islets.
  • the level of insulin secretion by the modified SC-beta cells is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% of that observed for primary beta islets, such as observed for cadaveric islets.
  • the total insulin content of the modified SC-beta cell is greater than at or about 500 pIU Insulin per 5000 cells, greater than at or about 1000 pIU Insulin per 5000 cells, greater than at or about 2000 pIU Insulin per 5000 cells, greater than at or about 3000 pIU Insulin per 5000 cells or greater than at or about 4000 pIU Insulin per 5000 cells.
  • the proinsulin to insulin ratio of the modified SC-beta cell is between at or about 0.02 and at or about 0.1, optionally at or about 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and any value between any of the foregoing.
  • the modified SC-beta cells exhibit functionality for 1 or more days following transplantation into a subject. In some of any embodiments, the modified SC-beta cells exhibit functionality for more than 1 week following transplantation into a subject. In some of any embodiments, the functionality is selected from the group consisting of maintaining fasting blood glucose levels, secreting insulin in response to glucose injections in vivo, and clearing glucose after a glucose injection in vivo.
  • composition comprising a population of modified SC-beta cells produced by any of the provided methods.
  • SC-beta cell modified stem-cell derived beta cell
  • PSC pluripotent stem cell
  • the modified SC-beta cell has (1) reduced expression of one or more major histocompatibility complex (MHC) class I molecules and/or one or more MHC class II molecules, relative to a control or wild-type beta cell; and (2) increased expression of a tolerogenic factor, relative to the control or wild-type beta cell, and wherein the modified SC-beta cell exhibits glucose-stimulated insulin secretion (GSIS).
  • MHC major histocompatibility complex
  • GSIS glucose-stimulated insulin secretion
  • the tolerogenic factor is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wild-type beta cell.
  • SC-beta cell comprising one or more modifications that: (a) inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules, and/or (b) increase expression of one or more tolerogenic factors, wherein the increased expression is relative to a control or wild-type beta cell that does not comprise the modifications.
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • SC-beta cell modified stem-cell derived beta cell
  • PSC pluripotent stem cell
  • the modified SC-beta cell has modifications that (a) inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules; and (b) increase expression of one or more tolerogenic factors, relative to a control or wild-type beta cell, and wherein the modified SC-beta cell exhibits glucose-stimulated insulin secretion (GSIS).
  • MHC major histocompatibility complex
  • GSIS glucose-stimulated insulin secretion
  • SC-beta cell modified stem cell-derived beta cell
  • PSC pluripotent stem cell
  • the modified SC-beta (1) does not express one or more major histocompatibility complex (MHC) class I molecules and/or one or more MHC class II molecules and (2) overexpresses a tolerogenic factor at a level of greater than at or about 5- fold compared to background, and wherein the modified SC-beta cell exhibits glucose-stimulated insulin secretion (GSIS).
  • MHC major histocompatibility complex
  • GSIS glucose-stimulated insulin secretion
  • the expression of the tolerogenic factor is by flow cytometry with an antibody directed against the tolerogenic factor and the background is determined by flow cytometry staining with an isotype control of the antibody.
  • the tolerogenic factor is expressed at a level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold compared to background expression.
  • SC-beta cell modified stem cell-derived beta cell
  • PSC pluripotent stem cell
  • the modified SC-beta cell (1) does not express one or more major histocompatibility complex (MHC) class I molecules or one or more MHC class II molecules and (2) overexpresses a tolerogenic factor at a first level of greater than at or about 5- fold over a second level expressed by an unmodified cell
  • the unmodified cell is an unmodified PSC that does not comprise modifications to reduce the one or more MHC class I molecules and/or the one or more MHC class II molecules and to overexpress the tolerogenic factor or is an unmodified SC- beta cell differentiated from such unmodified PSC
  • GSIS glucose-stimulated insulin secretion
  • the tolerogenic factor is expressed by the modified SC-beta cell at greater than at or about 20,000 molecules per cell.
  • SC-beta cell modified stem cell-derived beta cell
  • PSC pluripotent stem cell
  • the modified SC-beta cell (1) does not express one or more major histocompatibility complex (MHC) class I molecules or one or more MHC class II molecules and (2) overexpresses a tolerogenic factor, wherein the tolerogenic factor is expressed at greater than at or about 20,000 molecules per cell, and wherein the modified beta cell exhibits glucose- stimulated insulin secretion (GSIS).
  • MHC major histocompatibility complex
  • GSIS glucose- stimulated insulin secretion
  • the tolerogenic factor is expressed by the modified SC-beta at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • the modified SC-beta cell is differentiated from a PSC in which the PSC is a modified PSC comprising modifications that (a) reduce expression of one or more MHC class I molecules and/or one or more MHC class II molecules, relative to a control or wild-type PSC; and (b) increase expression of a tolerogenic factor, relative to the control or wild-type PSC.
  • the control or wild-type PSC is an unmodified PSC that does not comprise the modifications.
  • the modified SC-beta cell expresses the tolerogenic factor at a first level that is greater than at or about 5-fold over a second level expressed by the unmodified PSC or the unmodified SC-beta cell differentiated from the unmodified PSC.
  • the tolerogenic factor is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the unmodified PSC or an unmodified SC-beta differentiated from the unmodified PSC.
  • SC-beta cell a modified stem-cell derived beta cell that has been differentiated in vitro from a modified pluripotent stem cell (PSC), wherein the modified PSC comprises modifications that (a) reduce expression of one or more major histocompatibility complex (MHC) class I molecules or one or more MHC class II molecules, relative to a control or wild-type PSC; and (b) increase expression of a tolerogenic factor, relative to the control or wild-type PSC, and wherein the modified SC-beta cell exhibits glucose-stimulated insulin secretion (GSIS).
  • MHC major histocompatibility complex
  • GSIS glucose-stimulated insulin secretion
  • control or wild- type PSC is an unmodified PSC that does not comprise the modifications.
  • the modified PSC expresses the tolerogenic factor at a first level that is greater than at or about 5 -fold over a second level expressed by the unmodified PSC that does not comprise the modifications.
  • the tolerogenic factor is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over the second level expressed by the unmodified PSC.
  • the modified SC-beta cell comprises modifications that (a) reduce expression of the one or more MHC class I molecules and/or or the one or more MHC class II molecule, relative to the unmodified PSC or an unmodified SC-beta differentiated from the unmodified PSC; and (b) increase expression of a tolerogenic factor, compared to the unmodified PSC or the unmodified SC-beta differentiated from the unmodified PSC.
  • the modified SC-beta expresses the tolerogenic factor at a first level that is greater than at or about 5-fold over a second level expressed by the unmodified PSC or the unmodified SC-beta cell differentiated from an unmodified PSC.
  • the tolerogenic factor is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the unmodified PSC or the unmodified SC-beta cell differentiated from an unmodified PSC.
  • the tolerogenic factor is expressed by the modified PSC at greater than at or about 20,000 molecules per cell.
  • SC-beta cell modified stem cell-derived beta cell
  • PSC pluripotent stem cell
  • the modified PSC comprises modifications such that the modified PSC (a) does not express one or more major histocompatibility complex (MHC) class I molecules and/or or one or more MHC class II molecule; and (b) expresses a tolerogenic factor at greater than at or about 20,000 molecules per cell, and wherein the modified SC- beta cell exhibits glucose-stimulated insulin secretion (GSIS).
  • MHC major histocompatibility complex
  • GSIS glucose-stimulated insulin secretion
  • the tolerogenic factor is expressed by the modified PSC at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • the modified SC-beta cell does not express the one or more MHC class I molecule or the one or more MHC class II molecule and expresses the tolerogenic factor at greater than at or about 20,000 molecules per cell.
  • the tolerogenic factor is expressed by the modified SC-beta cell at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • the tolerogenic factor is selected from the group consisting of CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl- Inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, and SERPINB9, and any combination thereof.
  • the tolerogenic factor comprises CD47.
  • the tolerogenic factor comprises PD-L1.
  • the tolerogenic factor comprises HLA-E.
  • the tolerogenic factor comprises HLA-G.
  • the modified SC-beta cell is differentiated from a modified SC-beta cell
  • the modifications in (a) reduce protein expression of the one or more MHC class I molecules. In some of any embodiments, the modifications in (a) reduce cell surface expression of one or more MHC class I molecules. In some of any embodiments, the one or more modifications in (a) reduce cell surface trafficking of the one or more MHC class I molecules. In some of any embodiments, the modifications in (a) reduce a function of one or more MHC class I molecules. In some embodiments, the function is antigen presentation.
  • the modified SC-beta cell is differentiated from a modified PSC in which the modification that reduces expression of the one or more MHC class I reduces expression of B2M.
  • the modification that reduces expression of the one or more MHC class I molecules reduces mRNA expression of the B2M gene.
  • the modification that reduces expression of the one or more MHC class I molecules reduces protein expression of B2M.
  • the modification that reduces expression of the one or more MHC class I molecules in the modified PSC comprises inactivation or disruption of one allele of the B2M gene.
  • the modification that reduces expression of the one or more MHC class I molecules in the modified PSC comprises inactivation or disruption of both alleles of the B2M gene. In some of any embodiments, the modification that reduces expression of the one or more MHC class I molecules in the modified PSC comprises inactivation or disruption of all B2M coding alleles in the cell. In some of any embodiments, the inactivation or disruption comprises an indel in the B2M gene. In some of any embodiments, the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the B2M gene.
  • the modifications in (a) reduce protein expression of the one or more MHC class II molecules. In some of any embodiments, the modifications in (a) reduce cell surface expression of the one or more MHC class II molecules. In some of any embodiments, the one or more modifications in (a) reduce cell surface trafficking of the one or more MHC class II molecules. In some of any embodiments, the modifications in (a) reduce a function of the one or more MHC class II molecules. In some embodiments, the function is antigen presentation.
  • the modified SC-beta cell is differentiated from a modified PSC in which the modification that reduces expression of the one or more MHC class II comprises reduced expression of OITA.
  • the modification that reduces expression of the one or more MHC class II molecules in the modified PSC reduces mRNA expression of the OITA gene.
  • the modification that reduces expression of the one or more MHC class II molecules in the modified PSC reduces protein expression of OITA.
  • the modification that reduces expression of the one or more MHC class II molecules in the modified PSC comprises inactivation or disruption of one allele of the OITA gene.
  • the modification that reduces expression of the one or more MHC class II molecules in the modified PSC comprises inactivation or disruption of both alleles of the OITA gene. In some of any embodiments, the modification that reduces expression of the one or more MHC class II molecules in the modified PSC comprises inactivation or disruption of all OITA coding alleles in the cell. In some of any embodiments, the inactivation or disruption comprises an indel in the OITA gene. In some of any embodiments, the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the OITA gene.
  • expression of all MHC class I molecules and all MHC class II molecules is reduced in the modified PSC.
  • expression of HLA- A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR are reduced in the modified PSC.
  • the modified SC-beta cell is differentiated from a modified PSC and the modified PSC comprises a modification that reduces expression of CD142.
  • the modification reduces mRNA expression of the CD142 gene.
  • the modification reduces protein expression of CD142.
  • the modification that reduces expression of CD 142 in the modified PSC comprises inactivation or disruption of one allele of the CD142 gene.
  • the modification that reduces expression of CD142 in the modified PSC comprises inactivation or disruption of both alleles of the CD142 gene.
  • the modification that reduces expression of CD142 in the modified PSC comprises inactivation or disruption of all CD142 coding alleles in the cell.
  • the inactivation or disruption comprises an indel in the CD 142 gene.
  • the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CD 142 gene.
  • the modification to increase expression of the tolerogenic factor in the modified PSC comprises an exogenous polynucleotide encoding the tolerogenic factor.
  • the exogenous polynucleotide encoding the tolerogenic factor is integrated into the genome of the modified PSC.
  • the exogenous polynucleotide is integrated by non-targeted insertion into the genome of the modified PSC.
  • the exogenous polynucleotide is integrated by targeted insertion into a target genomic locus of the modified PSC.
  • the modified SC-beta cell is differentiated from a modified PSC in which the modified PSC further comprises an exogenous polynucleotide encoding a suicide gene.
  • the suicide gene is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).
  • the suicide gene and the tolerogenic factor are expressed from a bicistronic cassette integrated into the genome of the modified PSC.
  • the tolerogenic factor is CD47 and the suicide gene and CD47 are expressed from a bicistronic cassette integrated into the genome of the modified PSC.
  • the bicistronic cassette is integrated by non-targeted insertion into the genome of the modified PSC.
  • the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the modified PSC.
  • the target genomic locus is a safe harbor locus, a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • the safe harbor locus is selected from the group consisting of: a CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C (also known as AAVS1) gene, an albumin gene locus, a SHS231 locus, a CLYBL gene locus, and a ROSA26 gene locus.
  • the modified SC-beta cell is differentiated from a modified PSC and the modified PSC comprises a modification that increases expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, CD55 and CD35, relative to the control or wild-type PSC.
  • the modification to increase expression of one or more complement inhibitors comprises at least one exogenous polynucleotide encoding one or more complement inhibitors selected from the group consisting of an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, an exogenous polynucleotide encoding CD55, and an exogenous polynucleotide encoding CD35.
  • the one or more complement inhibitors is CD46 and CD59.
  • the one or more complement inhibitor is CD46, CD59 and CD55.
  • the at least one exogenous polynucleotide is integrated by non-targeted insertion into the genome of the modified PSC. In some of any embodiments, the at least one exogenous polynucleotide is integrated by targeted insertion into a target genomic locus of the cell. In some of any embodiments, the target genomic locus is a safe harbor locus, a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • the safe harbor locus is selected from the group consisting of: a CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C (also known as AAVS1) gene, an albumin gene locus, a SHS231 locus, a CLYBL gene locus, and a ROSA26 gene locus.
  • the expression of one or more MHC class I molecules and one or more MHC class II molecules is reduced in the modified SC-beta cell.
  • the modifications in (1) reduce protein expression of one or MHC class I molecules in the modified SC-beta cell. In some of any embodiments, the modifications in (1) reduce cell surface expression of one or more MHC class I molecules in the modified SC-beta cell. In some of any embodiments, the modifications in (1) reduce a function of MHC class I molecules in the modified SC-beta cell. In some embodiments, the function is antigen presentation. In some of any embodiments, the modification that reduces expression of one or more MHC class I molecules in the modified SC-beta cell comprises reduced expression of B2M.
  • the modification that reduces expression of one or more MHC class I in the modified SC-beta cell reduces mRNA expression of the B2M gene. In some of any embodiments, the modification that reduces expression of one or more MHC class I molecules in the modified SC-beta cell reduces protein expression of B2M. In some of any embodiments, the modification that reduces expression of one or more MHC class I molecules in the modified SC-beta cell comprises inactivation or disruption of one allele of the B2M gene. In some of any embodiments, the modification that reduces expression of one or more MHC class I molecules in the modified SC-beta cell comprises inactivation or disruption of both alleles of the B2M gene.
  • the modification that reduces expression of one or more MHC class I molecules in the modified SC-beta cell comprises inactivation or disruption of all B2M coding alleles in the cell.
  • the inactivation or disruption comprises an indel in the B2M gene.
  • the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the B2M gene.
  • the modifications in (1) reduce protein expression of one or more MHC class II molecules in the modified SC-beta cell. In some of any embodiments, the modifications in (1) reduce cell surface expression of one or more MHC class II molecules in the modified SC-beta cell. In some of any embodiments, the modifications in (1) reduce a function of one or more MHC class II molecules in the SC-beta cell. In some embodiments, the function is antigen presentation. In some of any embodiments, the modification that reduces expression of one or more MHC class II molecules in the modified SC-beta cell comprises reduced expression of OITA.
  • the modification that reduces expression of one or more MHC class II molecules in the modified SC-beta cell reduces mRNA expression of the OITA gene. In some of any embodiments, the modification that reduces expression of one or more MHC class II molecules in the modified SC-beta cell reduces protein expression of OITA. In some of any embodiments, the modification that reduces expression of one or more MHC class II molecules in the modified SC-beta cell comprises inactivation or disruption of one allele of the OITA gene. In some of any embodiments, the modification that reduces expression of one or more MHC class II molecules in the modified SC-beta cell comprises inactivation or disruption of both alleles of the OITA gene.
  • the modification that reduces expression of one or more MHC class II molecules in the modified SC-beta cell comprises inactivation or disruption of all OITA coding alleles in the cell.
  • the inactivation or disruption comprises an indel in the OITA gene.
  • the inactivation or disruption is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the OITA gene.
  • the modified SC-beta cell comprises a modification that reduces expression of CD142, relative to a control or wild-type beta cell. In some of any embodiments, the modification reduces mRNA expression of the CD142 gene. In some of any embodiments, the modification reduces protein expression of CD142.
  • the modifications that reduce expression of CD 142 in the modified SC-beta cell comprises inactivation or disruption of one allele of the CD142 gene. In some of any embodiments, the modifications that reduce expression of CD 142 in the modified SC-beta cell comprises inactivation or disruption of both alleles of the CD 142 gene. In some of any embodiments, the modifications that reduce expression of CD142 in the modified SC-beta cell comprises inactivation or disruption of all CD 142 coding alleles in the cell. In some of any embodiments, the inactivation or disruption comprises an indel in the CD142 gene. In some of any embodiments, the inactivation or disruption comprises a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CD 142 gene.
  • the modification to increase expression of the tolerogenic factor in the modified SC-beta cell comprises an exogenous polynucleotide encoding the tolerogenic factor.
  • the exogenous polynucleotide encoding the tolerogenic factor is integrated into the genome of the modified SC-beta cell.
  • the exogenous polynucleotide is integrated into a non-target locus in the genome of the modified SC-beta cell.
  • the exogenous polynucleotide is integrated into a target genomic locus of the modified SC-beta cell.
  • the tolerogenic factor is CD47 and the modification to increase expression of CD47 in the modified SC-beta cell comprises an exogenous polynucleotide encoding CD47.
  • the exogenous polynucleotide encoding CD47 is integrated into the genome of the modified SC-beta cell.
  • the exogenous polynucleotide is integrated into a non-target locus in the genome of the modified SC-beta cell.
  • the exogenous polynucleotide is integrated into a target genomic locus of the modified SC- beta cell.
  • SC-beta cell modified stem cell derived beta cell
  • PSC pluripotent stem cell
  • the modified SC-beta cell comprises knock out of the B2M gene, knock out of the OITA gene, and an exogenous polynucleotide encoding exogenous CD47 protein, relative to a control or wild-type beta cell.
  • the modified SC-beta cell has the phenotype B2M indel/indel - CIITA ⁇ “ CD47tg.
  • the modified SC-beta cell further comprises an exogenous polynucleotide encoding a suicide gene.
  • SC-beta cell modified stem cell derived beta cell
  • PSC pluripotent stem cell
  • the modified SC-beta cell comprises knock out of the B2M gene, knock out of the OITA gene, an exogenous polynucleotide encoding CD47 protein, and an exogenous polynucleotide encoding a suicide gene, relative to a control or wild-type beta cell.
  • the modified SC-beta cell has the phenotype 2M‘ ndel/mdel cnTAin dei/ in dei. CD47 tg; suic ide gene/g.
  • the suicide gene is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).
  • the suicide gene and the tolerogenic factor are expressed from a bicistronic cassette integrated into the genome of the modified SC-beta cell.
  • the tolerogenic factor is CD47 and the suicide gene and CD47 are expressed from a bicistronic cassette integrated into the genome of the modified SC-beta cell.
  • the bicistronic cassette is integrated at a non-target locus in the genome of the modified SC-beta cell. In some of any embodiments, the bicistronic cassette is integrated into a target genomic locus of the cell. In some of any embodiments, the target genomic locus is a safe harbor locus, a B2M gene locus, a CHTA gene locus, or a CD142 gene locus.
  • the safe harbor locus is selected from the group consisting of: a CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C (also known as AAVS1) gene, an albumin gene locus, a SHS231 locus, a CLYBL gene locus, and a ROSA26 gene locus.
  • the modified SC-beta cell comprises a modification that increases expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, CD55 and CD35 relative to the control or wild-type beta cell.
  • the modification to increase expression of the one or more complement inhibitors in the modified SC-beta cell comprises at least one exogenous polynucleotide encoding one or more complement inhibitors selected from the group consisting of an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, an exogenous polynucleotide encoding CD55, and an exogenous polynucleotide encoding CD35.
  • the one or more complement inhibitors is CD46 and CD59.
  • the one or more complement inhibitor is CD46, CD59 and CD55.
  • the modified SC-beta cell expresses CD47 at a first level that is greater than at or about 5-fold over a second level expressed by the control or wild-type beta cell.
  • the control or wild-type beta cell is differentiated from an unmodified PSC not comprising modifications that reduce expression of one or more MHC class I molecules and/or one or more MHC class II molecules and that increase expression of the one or more tolerogenic factors.
  • the modified SC-beta cell expresses CD47 at a first level that is greater than at or about 5-fold over a second level expressed by the control or wild-type beta cell.
  • CD47 is expressed at a first level that is greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wild-type beta cell.
  • CD47 is expressed by the modified SC-beta cell at greater than at or about 20,000 molecules per cell. In some of any embodiments, CD47 is expressed by the modified SC-beta cell at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • the modified SC-beta cell comprises an exogenous polynucleotide encoding a suicide gene or a suicide switch.
  • the suicide gene or suicide switch is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase 9 (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).
  • the suicide gene or suicide switch and genes associated with the suicide gene or the safety switch are expressed from a bicistronic cassette integrated into the genome of the modified SC-beta cell.
  • the suicide gene or suicide switch and the one or more tolerogenic factors are expressed from a bicistronic cassette integrated into the genome of the modified SC-beta cell.
  • the bicistronic cassette is integrated by non-targeted insertion into the genome of the modified SC-beta cell, optionally by introduction of the exogenous polynucleotide into the cell using a lentiviral vector.
  • the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the cell, optionally wherein the targeted insertion is by nuclease-mediated gene editing with homology-directed repair.
  • the one or more tolerogenic factors is CD47.
  • the modified SC-beta cell expresses at least one beta cell marker.
  • the at least one beta cell marker is selected from the group consisting of INS, CHGA, NKX2-2, PDX1, NKX6-1, MAFB, GCK and GLUT1.
  • the modified SC-beta cell exhibits one or more functions of a wild-type or control beta cell.
  • the one or more functions is selected from the group consisting of in vitro glucose- stimulated insulin secretion (GSIS), glucose metabolism, maintaining fasting blood glucose levels, secreting insulin in response to glucose injections in vivo, and clearing glucose after a glucose injection in vivo.
  • GSIS in vitro glucose- stimulated insulin secretion
  • the GSIS is measured in a perfusion GSIS assay.
  • the GSIS is dynamic GSIS comprising first and second phase dynamic insulin secretion.
  • the GSIS is static GSIS.
  • the static stimulation index is greater than at or about 1, greater than at or about 1.5, greater than at or about 2, greater than at or about 5, greater than at or about 10, greater than at or about 15, or greater than at or about 20.
  • the level of insulin secretion by the modified SC-beta cell is at least 20% of that observed for primary beta islets, such as cadaveric islets.
  • the level of insulin secretion by the modified SC-beta cells is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% of that observed for primary beta islets, such as cadaveric islets.
  • the total insulin content of the modified SC-beta is greater than at or about 500 pIU Insulin per 5000 cells, greater than at or about 1000 pIU Insulin per 5000 cells, greater than at or about 2000 pIU Insulin per 5000 cells, greater than at or about 3000 pIU Insulin per 5000 cells or greater than at or about 4000 pIU Insulin per 5000 cells.
  • the proinsulin to insulin ratio of the modified SC-beta is between at or about 0.02 and at or about 0.1 , optionally at or about 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and any value between any of the foregoing.
  • the modified SC-beta cell exhibits functionality for 1 or more days following transplantation into a subject. In some of any embodiments, the modified SC-beta cell exhibits functionality for more than 1 week following transplantation into a subject. In some of any embodiments, the functionality is selected from the group consisting of maintaining fasting blood glucose levels, secreting insulin in response to glucose injections in vivo, and clearing glucose after a glucose injection in vivo.
  • composition comprising any of the provided SC-beta cells. Also provided herein is a composition comprising a population of any of the provided modified SC-beta cells.
  • the level of the reduced expression of MHC HLA class I and/or MHC HLA class II and/or the level of the increased expression of the tolerogenic factor is retained or is similar compared to the modified PSC in at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population.
  • At least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population are reduced for expression of MHC HLA class I or for B2M. In some of any embodiments, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population are reduced for expression of MHC HLA class II or for OITA.
  • compositions comprising a population of modified SC-beta cells, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population express the tolerogenic factor at a first level that is greater than at or about 5-fold, greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wild-type beta cell.
  • the control or wild-type beta cell is a wild-type primary beta cell.
  • At least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population express the tolerogenic factor at a first level that is greater than at or about 5-fold, greater than at or about 10- fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by an unmodified PSC not comprising the modifications or an unmodified SC- beta cell differentiated from the unmodified PSC.
  • composition comprising a population of modified SC-beta cells, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population expresses the tolerogenic factor at greater than at or about 20,000 molecules per cell, at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • composition comprising a population of modified SC-beta cells, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the cells in the population are reduced for expression of CD142.
  • the provided composition comprises a pharmaceutically acceptable excipient. In some of any embodiments, the provided composition comprises a cryoprotectant.
  • modified SC-beta cells of the population of modified SC-beta cells comprise an exogenous polynucleotide encoding a suicide gene or a suicide switch.
  • the suicide gene or suicide switch is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase 9 (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).
  • the suicide gene and genes associated with the suicide gene or the safety switch are expressed from a bicistronic cassette integrated into the genome of modified SC-beta cells of the population of modified SC-beta cells.
  • the suicide gene or suicide switch and the exogenous CD47 are expressed from a bicistronic cassette integrated into the genome of the modified SC-beta cell.
  • the bicistronic cassette is integrated by non-targeted insertion into the genome, optionally by introduction of the exogenous polynucleotide into modified SC-beta cells of the population of modified SC-beta cells using a lentiviral vector.
  • the bicistronic cassette is integrated by targeted insertion into a target genomic locus of modified SC-beta cells of the population of modified SC-beta cells, optionally wherein the targeted insertion is by nuclease-mediated gene editing with homology-directed repair.
  • a method of treating diabetes in a subject comprising administering any of the modified SC-beta cells or any of the provided compositions to a subject in need of treatment thereof.
  • the diabetes is type I diabetes. In some of any embodiments, the diabetes is type II diabetes. In some of any embodiments, the modified SC-beta cells improve glucose tolerance in the subject.
  • a method for improving glucose tolerance in a subject comprising administering any of the provided modified SC-beta cells or any of the provided compositions to a subject in need of treatment thereof.
  • the subject is a diabetic patient.
  • the diabetic patient has type I diabetes or type II diabetes.
  • glucose tolerance is improved relative to the subject’s glucose tolerance prior to administration of the modified SC-beta cells.
  • administration of the modified SC-beta cells reduces exogenous insulin usage in the subject.
  • glucose tolerance is improved as measured by HbAlc levels.
  • the subject is fasting.
  • administration of the modified SC- beta cells improves insulin secretion in the subject.
  • insulin secretion is improved relative to the subject’s insulin secretion prior to administration of the modified SC-beta cells.
  • the method further comprises administering one or more immunosuppressive agents to the subject.
  • the subject has been administered one or more immunosuppressive agents.
  • the one or more immunosuppressive agents are a small molecule or an antibody.
  • the one or more immunosuppressive agents are selected from the group consisting of cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, a corticosteroids, prednisone, methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentin (thymosin-a), and an immunosuppressive antibody.
  • the one or more immunosuppressive agents comprise cyclosporine. In some embodiments, the one or more immunosuppressive agents comprise mycophenolate mofetil. In some embodiments, the one or more immunosuppressive agents comprise a corticosteroid. In some embodiments, the one or more immunosuppressive agents comprise cyclophosphamide. In some embodiments, the one or more immunosuppressive agents comprise rapamycin. In some embodiments, the one or more immunosuppressive agents comprise tacrolimus (FK- 506). In some embodiments, the one or more immunosuppressive agents comprise anti-thymocyte globulin. In some embodiments, the one or more immunosuppressive agents are one or more immunomodulatory agents.
  • the one or more immunomodulatory agents are a small molecule or an antibody.
  • the antibody binds to one or more of receptors or ligands selected from the group consisting of p75 of the IL-2 receptor, MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-gamma, TNF-alpha, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-
  • the one or more immunosuppressive agents are or have been administered to the subject prior to administration of the modified SC-beta cells. In some embodiments, the one or more immunosuppressive agents are or have been administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to administration of the modified SC-beta cells. In some embodiments, the one or more immunosuppressive agents are or have been administered to the subject at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more prior to administration of the SC-beta cells. In some embodiments, the one or more immunosuppressive agents are or have been administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the one or more immunosuppressive agents are or have been administered to the subject at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more, after administration of the modified SC-beta cells. In some embodiments, the one or more immunosuppressive agents are or have been administered to the subject on the same day as the first administration of the modified SC-beta cells.
  • the one or more immunosuppressive agents are or have been administered to the subject after administration of the modified SC-beta cells. In some embodiments, the one or more immunosuppressive agents are or have been administered to the subject after administration of a first and/or second administration of the modified SC-beta cells. In some embodiments, the one or more immunosuppressive agents are or have been administered to the subject prior to administration of a first and/or second administration of the modified SC-beta cells. In some embodiments, the one or more immunosuppressive agents are or have been administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to administration of a first and/or second administration of the modified SC- beta cells.
  • the one or more immunosuppressive agents are or have been administered to the subject at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more prior to administration of a first and/or second administration of the modified SC-beta cells. In some embodiments, the one or more immunosuppressive agents are or have been administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration of a first and/or second administration of the modified SC-beta cells.
  • the one or more immunosuppressive agents are or have been administered to the subject at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more, after administration of a first and/or second administration of the modified SC-beta cells.
  • the one or more immunosuppressive agents are administered at a lower dosage compared to the dosage of one or more immunosuppressive agents administered to reduce immune rejection of immunogenic cells that do not comprise the modifications of the modified SC-beta cells.
  • the modified SC-beta cell is capable of controlled killing of the modified SC-beta cell.
  • the modified SC-beta cell comprises a suicide gene or a suicide switch.
  • the suicide gene or the suicide switch induces controlled cell death in the presence of a drug or prodrug, or upon activation by a selective exogenous compound.
  • the suicide gene or the suicide switch is an inducible protein capable of inducing apoptosis of the modified SC-beta cell.
  • the inducible protein capable of inducing apoptosis of the modified SC-beta cell is a caspase protein.
  • the caspase protein is caspase 9.
  • the suicide gene or suicide switch is selected from the group consisting of cytosine deaminase (CyD), herpesvirus thymidine kinase (HSV-Tk), an inducible caspase 9 (iCaspase9), and rapamycin-activated caspase 9 (rapaCasp9).
  • the suicide gene or the suicide switch is activated to induce controlled cell death after the administration of the one or more immunosuppressive agents to the subject.
  • the suicide gene or the suicide switch is activated to induce controlled cell death prior to the administration of the one or more immunosuppressive agents to the subject.
  • the suicide gene or the suicide switch is activated to induce controlled cell death after the administration of the modified SC-beta cell to the subject. In some embodiments, the suicide gene or the suicide switch is activated to induce controlled cell death in the event of cytotoxicity or other negative consequences to the subject.
  • the method comprises administering an agent that allows for depletion of a modified SC-beta cell of the population of modified SC-beta cells.
  • the agent that allows for depletion of the modified SC-beta cell is an antibody that recognizes a protein expressed on the surface of the modified SC-beta cell.
  • the antibody is selected from the group consisting of an antibody that recognizes CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8.
  • the antibody is selected from the group consisting of mogamulizumab, AFM13, MOR208, obinutuzumab, ublituximab, ocaratuzumab, rituximab, rituximab-Rllb, tomuzotuximab, RO5083945 (GA201), cetuximab, Hul4.18K322A, Hul4.18- IL2, Hu3F8, dinituximab, c.60C3-Rllc, and biosimilars thereof.
  • the method comprises administering an agent that recognizes the one or more tolerogenic factors on the surface of the modified SC-beta cell.
  • the modified SC-beta cell is engineered to express the one or more tolerogenic factors.
  • the one or more tolerogenic factors is CD47.
  • the method comprises administering one or more additional therapeutic agents to the subject. In some embodiments, the subject has been administered one or more additional therapeutic agents. [0140] In some embodiments, the method comprises monitoring the therapeutic efficacy of the method. In some embodiments, the method comprises monitoring the prophylactic efficacy of the method. In some embodiments, the method is repeated until a desired suppression of one or more disease symptoms occurs.
  • FIG. 1A shows glucose levels measured over time in humanized NSG diabetic mice transplanted with wild-type (WT), B2M mdel/mdel , CIITA mdel/mdel , or B2M mdel/mdel , CIITA mdel/mdel , CD47tg modified SC-beta cells.
  • FIG. IB shows serum levels of human c-peptide in humanized NSG diabetic mice transplanted with wild-type (WT), B2M mdel/mdel , CIITA mdel/mdel , or B2M mdel/mdel , CIITA mdel/mdel , CD47tg modified SC-beta cells and subjected to a glucose challenge on da 29 after transplant, one hour prior to sacrifice.
  • WT wild-type
  • FIG. 2A shows IFN-y spot frequencies enumerated using an Elispot plate reader as an assay for TH1 T cell response in mice administered wild-type, B2M mdel/mdel , CIITA mdel/mdel , or B2M mdel/mdel , CIITA mdel/mdel , CD47tg human SC-beta cells.
  • FIG. 2B shows the mean fluorescence intensity (MFI) of cells labelled with FITC- conjugated goat anti-IgM and analyzed by flow cytometry for cell suspensions of sera from recipient wild-type, B2M indel/indel , ciITA indel/indel , and B2M indel/indel , ciITA indel/indel , CD47tg human SC-beta mice incubated with wild-type, B2M mdel/mdel , CIITA mdel/mdel , and B2M mdel/mdel , CIITA mdel/mdel , CD47tg human cells.
  • MFI mean fluorescence intensity
  • FIG. 2C shows a comparison of NK cell killing using IE-2 stimulated human NK cells as effector and wild-type, B2M mdel/mdel , CIITA mdel/mdel , or B2M mdel/mdel , CIITA mdel/mdel , CD47tg human SC- beta cells as target cells.
  • beta cells differentiated from pluripotent stem cells in which the resulting beta cells contain one or more modifications that make the resulting differentiated beta cells hypoimmune to reduce or evade immune rejection.
  • the modified beta cells that have been differentiated in vitro from PSCs and that contain the one more more modifications are called modified stem cell- derived beta cell (also called “modified SC-beta cell” or “modified SC- cell”).
  • modified SC-beta cell also called “modified SC-beta cell” or “modified SC- cell”.
  • at least one or all of the modifications to the differentiated beta-cell are introduced to the beta-cells after the diffierentiation from the PSCs.
  • the PSCs are first modified with the one or more modifications, or in some embodiments each of the hypoimmune modifications, and then are differentiated to generate the modified SC-beta cells.
  • modified SC-beta cells and hypoimmune PSC derived beta cells can be used interchangeably.
  • a method of generating a hypoimmune beta cell differentiated from a modified pluripotent stem cell (PSC) in vitro that has been modified to evade immune rejection can also encompass mesenchymal stem cells (MSCs) and/or embryonic stem cells (ESCs).
  • the modifications that result in hypoimmune cells are modifications that inactivate or disrupt one or more alleles (e.g. one or both alleles) of one or more major histocompatibility complex (MHC) human leukocyte antigen MHC class I antigens and/or MHC class II antigens, or that inactivate or disrupt one or more alleles (e.g.
  • MHC major histocompatibility complex
  • MHC class I antigens and MHC class II antigens also called human leukocyte antigen (HLA) class I antigens and HLA class II antigens, respectively
  • HLA human leukocyte antigen
  • tolerogenic factors such as CD47.
  • the in vitro differentiated beta cell that has been differentiated from a modified PSC is an example of a modified stem cell-derived beta cell.
  • the provided cells are modified SC-beta cells that are derived from modified PSCs that contain modifications that (a) reduce expression of one or more major histocompatibility complex (MHC) class I molecules and/or one or more of MHC class II molecules; and (b) increase expression of one or more tolerogenic factors in the modified PSC, relative to a control or a wild-type PSC.
  • the modified SC-beta cells are derived from the modified PSC by culture under conditions sufficient for differentiation of the modified PSC into a modified SC-beta cell.
  • the modifications of the modified SC-beta cells make the cells hypoimmune, which in some aspects allow the cells to evade immune rejections compared to control or wild- type beta cells, such as primary human beta cells.
  • the provided embodiments relate to a demonstration of differentiating functional modified SC-beta cells from hypoimmune PSCs in which the modified SC-beta cells retain the hypoimmune modifications of the PSC and exhibit beta cell function such as glucose- stimulated insulin secretion. These results support that the hypoimmune modifications do not impact the differentiation of PSCs into functional beta cells.
  • the provided embodiments provide for a viable source of a transplantable beta cell, and thus provide for an allogeneic cell therapy for improving glucose tolerance in diabetic subjects.
  • rejection of the cells by the recipient subject's immune system is diminished and the cells are able to engraft and function in the host after their administration, regardless of the subject's genetic make-up, or any existing response within the subject to one or more previous allogeneic transplants.
  • the modified SC-beta cells are able to persist without immunosuppression.
  • the modified SC-beta cells are able to persist without immunosuppression course used in allogenic islet transplantation.
  • the cells are genomically stable with respect to the modifications present in the iPSC from which the modified SC-beta cell is differentiated.
  • modified cells provided herein including modified PSCs and modified SC-beta cells (e.g., modified SC-beta cells obtained by in vitro differentiation from the modified PSCs), utilize expression of tolerogenic factors and are also modulated (e.g., reduced or eliminated) for expression (e.g., surface expression) of one or more MHC class I molecules and/or one or more MHC class II molecules.
  • the modification that reduces expression of one or more MHC class I molecules is a modification that reduces expression of b-2 microglobulin (B2M).
  • B2M b-2 microglobulin
  • the modification that reduces expression of one or more MHC class II molecules is a modification that reduces expression of OITA.
  • the modified SC-beta cells comprising the modifications described herein (including reduced or eliminated expression of MHC class I molecules or MHC class II molecules and increased expression of CD47 or other tolerogenic factor) survive, engraft, persist, and function following transplant.
  • the modified SC-beta cells exhibit enhanced survival and/or enhanced engraftment and/or function for a longer term in comparison to control or wild-type beta cells, such as unmodified SC-beta cells that do not comprise the modifications, such as SC-beta cells differentiated from unmodified PSCs that do not contain the modifications rendering the cells hypoimmune.
  • the modified SC-beta cells are administered via intravenous infusion, intramuscular injection, or kidney capsule transplant.
  • a modified SC-beta cell that is hypoimmune, in which the methods include (1) providing a modified PSC with one or more modifications (e.g. genetic modifications) that reduce or eliminate expression of one or more MHC class I molecules (e.g. via reduced or eliminated B2M) and/or one or more MHC class II human leukocyte antigens (e.g. via reduced or eliminated OITA) in the modified PSC and increase expression of a tolerogenic factor (e.g. CD47) in the modified PSC, and (2) differentiating the modified PSC under conditions for differentiation into a beta islet cell.
  • modifications e.g. genetic modifications
  • MHC class I molecules e.g. via reduced or eliminated B2M
  • MHC class II human leukocyte antigens e.g. via reduced or eliminated OITA
  • a tolerogenic factor e.g. CD47
  • modified SC-beta cells obtained by differentiation in vitro from a modified PSC that has one or more modifications (e.g., genetic modifications) to reduce or eliminate expression of one or more MHC class I molecules and/or one or more MHC class II molecules in the modified PSC and increase expression of a tolerogenic factor (e.g., CD47) in the cell.
  • the resulting or obtained modified SC-beta cell also has reduced or eliminated expression of the one or more MHC class I molecules (e.g. via reduced or eliminated B2M) and/or the one or more MHC class II molecules (e.g. via reduced or eliminated OITA) and increased expression of a tolerogenic factor (e.g.
  • CD47 such as compared to a unmodified PSC, including the starting pluripotent stem cell line, or compared to a control or wild-type beta cell such as an SC-beta cell differentiated from an unmodified PSC.
  • the modified SC-beta cells do not express MHC class I molecules and/or MHC class II molecules, and express CD47 at increased levels relative to the starting cell line (e.g., greater than 5-fold over background, greater than 5-fold over a primary beta cell, and/or greater than 5-fold compared to an unmodified PSC or an unmodified SC-beta cell obtained by in vitro differentiation from the unmodified PSC).
  • the modified SC-beta cell expresses CD47 at a level over the expression by the endogenous iPSC and/or beta cell differentiated therefrom.
  • the modified PSC or modified SC-beta cell expresses greater than 20,000 molecules of the tolerogenic factor (e.g., CD47) on its surface.
  • compositions containing the modified SC-beta cells and methods and uses thereof for treating diabetic subjects and/or for improving glucose tolerance in subjects in need thereof are also provided herein.
  • the modified PSCs e.g., modified iPSC
  • the modified SC-beta cells further comprise reduced or eliminated expression of CD 142 (also known as Coagulation Factor III, Tissue Factor (TF), Thromboplastin, platelet tissue factor, or factor III), which is a membrane receptor in the blood coagulation pathway that contributes to initiating IB MIR.
  • CD 142 also known as Coagulation Factor III, Tissue Factor (TF), Thromboplastin, platelet tissue factor, or factor III
  • the modified PSCs comprise reduced or eliminated for expression of CD142 and increased expression of one or more tolerogenic factors (e.g., CD47), and reduced expression of one or more MHC class I molecules and/or MHC class II molecules, such as described above.
  • modified SC-beta cells that are obtained by in vitro differentiation from such modified PSCs.
  • the modified SC-beta cell obtained by in vitro differentiation from a modified PSC is reduced or eliminated for expression of CD142, reduced or eliminated for expression of MHC class I molecules and/or MHC class II molecules and increased for the expression of a tolerogenic factor (e.g. CD47), such as compared to an unmodified PSC or compared to a control or wild-type beta cell such as an SC-beta cell differentiated from an unmodified PSC.
  • a tolerogenic factor e.g. CD47
  • the modified PSCs (e.g., modified iPSC) from which the modified SC-beta cells are differentiated from as described herein further comprise increased expression and/or overexpression of one or more complement inhibitors.
  • the one or more complement inhibitors are selected from CD46, CD59, CD55 and CD35.
  • the modified PSCs comprise increased expression of one or more complement inhibitors and increased expression of one or more tolerogenic factors (e.g., CD47), and reduced expression of one or more MHC class I molecules and/or MHC class II molecules, such as described above.
  • the modified cells comprise increased expression of two or more complement inhibitors in combination, such as increased expression of CD46 and CD59 or increased expression of CD46, CD59, and CD55.
  • modified SC-beta cells that are obtained by in vitro differentiation from such modified PSCs.
  • modified SC-beta cells that are obtained by in vitro differentiation from such modified PSCs.
  • the modified SC-beta cell obtained by in vitro differentiation from a modified PSC is reduced or eliminated for expression of one or more MHC class I molecules and/or one or more MHC class II molecules, increased for the expression of a tolerogenic factor (e.g.
  • CD47 increased for expression of one or more complement inhibitors from CD46, CD59, CD55 and CD35 (e.g. CD46 and CD59 or CD46, CD59 and CD55), such as compared to an unmodified PSC or compared to a control or wild-type beta cell such as a SC-beta cell differentiated from an unmodified PSC.
  • complement inhibitors from CD46, CD59, CD55 and CD35 e.g. CD46 and CD59 or CD46, CD59 and CD55
  • a control or wild-type beta cell such as a SC-beta cell differentiated from an unmodified PSC.
  • the modified SC-beta cells provided herein are protected from complement-mediated cytotoxicity.
  • the modified SC-beta cells e.g., overexpressing one or more complement inhibitors, such as CD46 and CD59
  • CDC complement-dependent cytotoxicity
  • the modified SC-beta cells are protected from CDC that occurs independently of an IB MIR.
  • the altered expression is relative to a similar cell that does not contain the modifications, such as a wild-type cell, the starting cell line to which the modifications are made, or an unmodified cell of the same cell type or a cell that otherwise is the same but that lacks the modifications.
  • a cell that lacks the modifications is any cell as described herein that lacks modifications herein to alter expression of the one or more tolerogenic factors (e.g., CD47), one or more MHC class I molecule and/or one or more MHC class II molecule, CD142 and/or one or more complement inhibitor. Exemplary methods to introduce modifications to a cell to alter expression are described herein.
  • any of a variety of methods for overexpressing or increasing expression of a gene or protein in a pluripotent stem cell may be used, such as by introduction or delivery of an exogenous polynucleotide encoding a protein (i.e., a transgene or “tg”) or introduction of delivery of a fusion protein of a DNA-targeting domain and a transcriptional activator targeting a gene.
  • an exogenous polynucleotide encoding a protein i.e., a transgene or “tg”
  • introduction of delivery of a fusion protein of a DNA-targeting domain and a transcriptional activator targeting a gene i.e., a transgene or “tg”
  • any of a variety of methods for reducing or eliminating expression of a gene or protein in a PSC may be used, including non-gene editing methods such as by introduction or delivery of an inhibitory nucleic acids (e.g., RNAi) or gene editing methods involving introduction or delivery of a targeted nuclease system (e.g., CRISPR/Cas).
  • the method for reducing or eliminating expression is via a nuclease-based gene editing technique.
  • the PSC may then be used to differentiate a modified SC-beta cell that then also is found to contain the similar modifications.
  • description related to editing or modification of a cell relates to editing or modification of the pluripotent stem cell, and that the modified SC-beta cell is derived from such modified pluripotent stem cell by in vitro differentiation therefrom.
  • genome editing technologies utilizing rare-cutting endonucleases are used to reduce or eliminate expression of immune genes (e.g., by deleting genomic DNA of critical immune genes) as described herein, such as genes involved in regulating expression of MHC class I molecules or MHC class II molecules, in PSCs used to derived the modified SC-beta cells.
  • immune genes e.g., by deleting genomic DNA of critical immune genes
  • genome editing technologies or other gene modulation technologies are used to insert tolerance-inducing (tolerogenic) factors (e.g., CD47) into a target genomic locus of PSCs used to derive the modified SC-beta cells, thus producing modified cells that can evade immune recognition upon engrafting into a recipient subject.
  • tolerance-inducing factors e.g., CD47
  • the modified PSCs, and modified SC-beta cells e.g., modified SC-beta cells obtained by in vitro differentiation of the modified PSCs
  • exhibit modulated expression e.g., reduced or eliminated expression
  • modulated expression e.g., reduced or eliminated expression
  • modulated expression e.g., reduced or and modulated expression (e.g., overexpression) of tolerogenic factors, such as CD47, and provide for reduced recognition by the recipient subject’s immune system.
  • the modified cells provided herein also exhibit modulated expression (e.g., reduced expression) of CD142, which, in some aspects, can also be reduced by genome editing technologies (e.g., the CRISPR/Cas, TALEN, zinc finger nuclease, meganuclease, and homing endonuclease systems) to reduce or eliminate expression of CD142 (e.g., by deleting genomic DNA of critical immune genes) in modified PSCs used to derived the modified SC-beta cells.
  • genome editing technologies e.g., the CRISPR/Cas, TALEN, zinc finger nuclease, meganuclease, and homing endonuclease systems
  • the modified SC-beta cells provided herein also exhibit modulated expression (e.g., reduced expression) of CD142, which, in some aspects, can also be reduced by genome editing technologies (e.g., the CRISPR/Cas, TALEN, zinc finger nuclease, meganuclease, and homing endonuclease systems) to reduce or eliminate expression of CD142 (e.g., by deleting genomic DNA of critical immune genes) in modified SC-beta cells.
  • genome editing technologies e.g., the CRISPR/Cas, TALEN, zinc finger nuclease, meganuclease, and homing endonuclease systems
  • the modified cells provided herein exhibit modulated expression (e.g., increased expression) of one or more complement inhibitors selected from CD46, CD59, CD55 and CD35, which, in some aspect, also can be increased by genome editing technologies to insert or integrate an exogenous polynucleotide encoding the one or more complement inhibitors into a genomic locus in modified PSCs used to derive the modified SC-beta cells.
  • modulated expression e.g., increased expression
  • one or more complement inhibitors selected from CD46, CD59, CD55 and CD35 which, in some aspect, also can be increased by genome editing technologies to insert or integrate an exogenous polynucleotide encoding the one or more complement inhibitors into a genomic locus in modified PSCs used to derive the modified SC-beta cells.
  • modulated expression e.g., increased expression
  • CD46, CD59, CD55 and CD35 is increased by genome editing technologies to insert or integrate an exogenous polynucleotide encoding the one or more complement inhibitors into a genomic locus in modified SC-beta cells.
  • the modified SC-beta cells exhibit features that allow them to evade immune recognition.
  • the provided modified SC-beta cells are hypoimmunogenic.
  • modified SC-beta cells provided herein are not subject to an innate immune cell rejection.
  • modified SC-beta cells provided herein exhibit reduced innate immune cell rejection and/or adaptive immune cell rejection (e.g., hypo-immunogenic cells).
  • the modified SC-beta cells exhibit reduced susceptibility to NK cell-mediated lysis and/or macrophage engulfment.
  • the modified SC-beta cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that are transplanted into a recipient subject with little to no immunosuppressant agent needed.
  • universally compatible cells or tissues e.g., universal donor cells or tissues
  • Such hypo-immunogenic cells retain cell-specific characteristics and features upon transplantation.
  • Also provided herein are methods for treating a disorder comprising administering the modified cells that evade immune rejection in an MHC -mismatched allogenic recipient.
  • the modified cells produced from any one of the methods described herein evade immune rejection when repeatedly administered (e.g., transplanted or grafted) to MHC-mismatched allogenic recipient.
  • the modified SC-beta cells are used in methods for treating diabetic subjects (e.g., Type I or Type II diabetes), such as to improve glucose tolerance in the subject.
  • a modified stem-cell derived beta cell (SC-beta cell).
  • the modified SC-beta cell is produced by differentiating a stem or progenitor cell (e.g., a totipotent, pluripotent, or multipotent stem cell) into an SC-beta cell, and then generating a modified SC-beta cell from the SC-beta cell.
  • the modified SC-beta cell is produced from the SC-beta cell by introducing one or more of the modifications disclosed herein.
  • the SC-beta cell is differentiated from a stem cell (e.g., a PSC such as an iPSC) comprising one or more of the modifications, and one or more additional modifications are introduced into the SC-beta cell to generate the modified SC-beta cell.
  • the modified SC-beta cell is differentiated from a stem cell (e.g., a PSC such as an iPSC) comprising the modifications.
  • Pluripotent Stem Cells e.g. iPSCs
  • Methods of Producing e.g. iPSCs
  • the modified stem-cell derived beta cells (SC-beta cells) provided herein can be differentiated from stem or progenitor cells.
  • the stem or progenitor cells are modified.
  • the stem or progenitor cell does not comprise the modifications, and the one or more modifications are introduced into the SC-beta cell to generate the modified SC-beta cell.
  • the cell to be engineered or modified is a stem or progenitor cell that is capable of being differentiated (e.g., the stem cell is totipotent, pluripotent, or multipotent).
  • a stem cell capable of being differentiated is differentiated into an SC-beta cell, which is then modified.
  • the cell is isolated from embryonic or neonatal tissue.
  • the cell is an embryonic stem cell.
  • the cell is an induced pluripotent stem cell derived from somatic cells (e.g., skin or blood cells) and reprogrammed into an embryonic-like pluripotent state.
  • the induced pluripotent stem cell is derived from a fibroblast.
  • the cells that are modified as provided herein are pluripotent stems cells or are cells differentiated from pluripotent stem cells.
  • the cell may be a vertebrate cell, for example, a mammalian cell, such as a human cell or a mouse cell.
  • the cell may also be a vertebrate stem cell, for example, a mammalian stem cell, such as a human stem cell or a mouse stem cell.
  • the cell or stem cell is amenable to modification.
  • the cell or stem cell, or a cell derived from such a stem cell has or is believed to have therapeutic value, such that the cell or stem cell or a cell derived or differentiated from such stem cell may be used to treat a disease, disorder, defect or injury in a subject in need of treatment for same.
  • the modified SC-beta cell is differentiated from a pluripotent stem cell, such as an induced pluripotent stem cell (iPSC), optionally wherein the iPSC is modified as disclosed herein.
  • a pluripotent stem cell such as an induced pluripotent stem cell (iPSC)
  • iPSC induced pluripotent stem cell
  • the iPSC does not comprise the modifications.
  • the cells that are modified as provided herein are modified pluripotent stem cells (e.g., modified iPSC).
  • iPSCs mammalian pluripotent stem cells
  • miPSCs for murine cells or hiPSCs for human cells
  • iPCSs there are a variety of different methods for the generation of iPCSs.
  • the original induction was done from mouse embryonic or adult fibroblasts using the viral introduction of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4; see Takahashi and Yamanaka Cell 126:663-676 (2006), hereby incorporated by reference in its entirety and specifically for the techniques outlined therein.
  • iPSCs are generated by the transient expression of one or more reprogramming factors" in the host cell, usually introduced using episomal vectors. Under these conditions, small amounts of the cells are induced to become iPSCs (in general, the efficiency of this step is low, as no selection markers are used). Without wishing to be bound by theory, it is believed that once the cells are "reprogrammed", and become pluripotent, they lose the episomal vector(s) and produce the factors using the endogenous genes.
  • the number of reprogramming factors that can be used or are used can vary. Commonly, when fewer reprogramming factors are used, the efficiency of the transformation of the cells to a pluripotent state goes down, as well as the "pluripotency", e.g., fewer reprogramming factors may result in cells that are not fully pluripotent but may only be able to differentiate into fewer cell types.
  • a single reprogramming factor, OCT4, is used.
  • two reprogramming factors, OCT4 and KLF4, are used.
  • three reprogramming factors, OCT4, KLF4 and SOX2, are used.
  • four reprogramming factors, OCT4, KLF4, SOX2 and c-Myc are used.
  • 5, 6 or 7 reprogramming factors can be used selected from SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen.
  • these reprogramming factor genes are provided on episomal vectors such as are known in the art and commercially available.
  • the hosts cells used for transfecting the one or more reprogramming factors are non-pluripotent stem cells.
  • iPSCs are made from non- pluripotent cells such as, but not limited to, blood cells, fibroblasts, etc., by transiently expressing the reprogramming factors as described herein.
  • the non-pluripotent cells such as fibroblasts, are obtained or isolated from one or more individual subjects or donors prior to reprogramming the cells.
  • iPSCs are made from a pool of isolated non-pluripotent stems cells, e.g., fibroblasts, obtained from one or more (e.g.
  • the non-pluripotent cells such as fibroblasts
  • the non-pluripotent cells are isolated or obtained from a plurality of different donor subjects (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more), pooled together in a batch, reprogrammed as iPSCs, and are optionally modified in accord with the provided methods.
  • the non-pluripotent cells such as fibroblasts
  • fibroblasts are isolated or obtained from a plurality of different donor subjects (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more), pooled together in a batch, reprogrammed as iPSCs, and differentiated into SC-beta cells, which are then modified in accord with the provided methods.
  • the iPSCs are derived from, such as by transiently transfecting one or more reprogramming factors into cells from a pool of non-pluripotent cells (e.g., fibroblasts) from one or more donor subjects that are different than the recipient subject (e.g., the patient administered the cells).
  • the non-pluripotent cells (e.g., fibroblasts) to be induced to iPSCs can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more donor subjects and pooled together.
  • the non-pluripotent cells e.g., fibroblasts
  • the non-pluripotent cells can be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10, or more 20 or more, 50 or more, or 100 or more donor subjects and pooled together.
  • the non-pluripotent cells are harvested from one or a plurality of individuals, and in some instances, the non-pluripotent cells (e.g., fibroblasts) or the pool of non-pluripotent cells (e.g., fibroblasts) are cultured in vitro and transfected with one or more reprogramming factors to induce generation of iPSCs. In some embodiments, the non-pluripotent cells (e.g., fibroblasts) or the pool of non-pluripotent cells (e.g., fibroblasts) are modified in accord with the methods provided herein.
  • the iPSCs e.g., modified iPSCs
  • a pool of iPSCs e.g., a pool of modified iPSCs
  • pluripotent stem cells e.g., modified pluripotent stem cells
  • Differentiation can be assayed as is known in the art, generally by evaluating the presence of cell-specific markers.
  • the differentiated SC-beta cells generated from PSCs such as modified (e.g., hypoimmunogenic) pluripotent cell derivatives can be transplanted using techniques known in the art that depends on both the cell type and the ultimate use of these cells.
  • the iPSCs may be differentiated to any type of cell described herein.
  • the iPSCs are differentiated into beta islet cells.
  • host cells such as non-pluripotent cells (e.g., fibroblasts) from an individual donor or a pool of individual donors are isolated or obtained, generated into iPSCs in which the iPSCs are then modified to contain modifications (e.g., genetic modifications) described herein and then differentiated into a desired cell type.
  • host cells such as non-pluripotent cells (e.g., fibroblasts) from an individual donor or a pool of individual donors are isolated or obtained, generated into iPSCs in which the iPSCs are then then differentiated into a desired cell type, which is then modified.
  • non-pluripotent cells e.g., fibroblasts
  • the cells as provided herein are beta islet cells derived from iPSCs, such as modified iPSCs that contain modifications (e.g., genetic modifications) described herein and that are differentiated into beta islet cells.
  • modifications e.g., genetic modifications
  • the methods for differentiation depend on the desired cell type using known techniques.
  • the cells differentiated into various beta islet cells may be used for subsequent transplantation or engraftment into subjects (e.g., recipients).
  • pancreatic islet cells are derived from the pluripotent cells (e.g., modified pluripotent cells) described herein.
  • pluripotent cells e.g., modified pluripotent cells
  • Useful methods for differentiating pluripotent stem cells into beta islet cells are described, for example, in U.S. Patent No. 9,683,215; U.S. Patent No. 9,157,062; U.S. Patent No. 8,927,280; U.S. Patent Pub. No. 2021/0207099; Hogrebe et al., “Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells,” Nat.
  • the pluripotent cells e.g., modified pluripotent cells
  • T1DM type I diabetes mellitus
  • Cell systems are a promising way to address T1DM, see, e.g., Ellis et al, Nat Rev Gastroenterol Hepatol. 2017 Oct;14(10):612-628, incorporated herein by reference. Additionally, Pagliuca et al.
  • the method of producing a population of modified pancreatic islet cells from a population of pluripotent cells (e.g., modified pluripotent cells) by in vitro differentiation comprises: (a) culturing the population of iPSCs (e.g., modified iPSCs) in a first culture medium comprising one or more factors selected from the group consisting insulin-like growth factor, transforming growth factor, FGF, EGF, HGF, SHH, VEGF, transforming growth factor-b superfamily, BMP2, BMP7, a GSK inhibitor, an ALK inhibitor, a BMP type 1 receptor inhibitor, and retinoic acid to produce a population of immature pancreatic islet cells; and (b) culturing the population of immature pancreatic islet cells in a second culture medium that is different than the first culture medium to produce a population of pancreatic islet cells (e.g., modified pancreatic islet cells).
  • iPSCs e.g., modified iPSC
  • the method comprise introducing one or more modifications into the pancreatic islet cells.
  • the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 2 mM to about 10 mM.
  • the ALK inhibitor is SB -431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 1 pM to about 10 pM.
  • the first culture medium and/or second culture medium are absent of animal serum.
  • Differentiation is assayed as is known in the art, generally by evaluating the presence of P cell associated or specific markers, including but not limited to, insulin. Differentiation can also be measured functionally, such as measuring glucose metabolism, see generally Muraro et al., Cell Syst. 2016 Oct 26; 3(4): 385-394.e3, hereby incorporated by reference in its entirety, and specifically for the biomarkers outlined there.
  • the beta cells can be transplanted (either as a cell suspension, cell clusters, or within a permeable or semipermeable device or gel matrix as discussed herein) into the portal vein/liver, the omentum, the gastrointestinal mucosa, the bone marrow, a muscle, or subcutaneous pouches.
  • pancreatic islet cells including for use in the present technology are found in W02020/018615, the disclosure is herein incorporated by reference in its entirety.
  • the population of modified beta islet cells such as endothelial cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), are maintained in culture, in some cases expanded, prior to administration. In certain embodiments, the population of modified beta islet cells are cryopreserved prior to administration.
  • pancreatic islet cell types include, but are not limited to, pancreatic islet progenitor cell, immature pancreatic islet cell, mature pancreatic islet cell, and the like.
  • pancreatic cells described herein are administered to a subject to treat diabetes.
  • the pancreatic islet cells modified as disclosed herein such as beta islet cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), secretes insulin.
  • a pancreatic islet cell exhibits at least two characteristics of an endogenous pancreatic islet cell, for example, but not limited to, secretion of insulin in response to glucose, and expression of beta cell markers.
  • beta cell markers or beta cell progenitor markers include, but are not limited to, c- peptide, Pdxl, glucose transporter 2 (Glut2), HNF6, VEGF, glucokinase (GCK), prohormone convertase (PC 1/3), Cdcpl, NeuroD, Ngn3, Nkx2.2, Nkx6.1, Nkx6.2, Pax4, Pax6, Ptfla, Isll, Sox9, Soxl7, and FoxA2.
  • the pancreatic islet cells such as beta islet cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), produce insulin in response to an increase in glucose.
  • the pancreatic islet cells secrete insulin in response to an increase in glucose.
  • the cells have a distinct morphology such as a cobblestone cell morphology and/or a diameter of about 17 pm to about 25 pm.
  • the present technology is directed to modified beta islet cells, such as beta islet cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), that overexpress a tolerogenic factor (e.g., CD47), have reduced expression or lack expression of MHC class I and/or MHC class II human leukocyte antigens, and optionally have reduced CD142 expression.
  • a tolerogenic factor e.g., CD47
  • the beta islet cells further express one or more complement inhibitors.
  • the modified beta islet cells overexpress a tolerogenic factor (e.g., CD47) and harbor a genomic modification in the B2M gene and optionally have reduced CD 142 expression.
  • the beta islet cells further express one or more complement inhibitors.
  • the modified beta islet cells overexpress a tolerogenic factor (e.g., CD47) and harbor a genomic modification in the CIITA gene, and optionally have reduced CD142 expression.
  • the beta islet cells further express one or more complement inhibitors.
  • beta islet cells overexpress a tolerogenic factor (e.g., CD47) and harbor genomic modifications that disrupt one or more of the following genes: the B2M CIITA, and CD142 genes.
  • the provided modified beta islet cells evade immune recognition.
  • the modified beta islet cells described herein such as beta islet cells differentiated from iPSCs derived from one or more individual donors (e.g., healthy donors), do not activate an immune response in the patient (e.g., recipient upon administration).
  • the number of cells administration is at a lower dosage than would be required for immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype but that do not contain the modifications, e.g., genetic modifications, of the modified cells, e.g. with endogenous levels of CD142, MHC class I, and/or MHC class II expression and without increased (e.g., exogenous) expression of CD47).
  • immunogenic cells e.g., a population of cells of the same or similar cell type or phenotype but that do not contain the modifications, e.g., genetic modifications, of the modified cells, e.g. with endogenous levels of CD142, MHC class I, and/or MHC class II expression and without increased (e.g., exogenous) expression of CD47).
  • modified Pluripotent Stem Cells e.g., modified iPSCs
  • the PSCs that are differentiated into beta cells are modified pluripotent stem cells or modified PSCs.
  • pluripotent stem cells that comprise one or more modification (termed “modified pluripotent stem cells”) in which the one or more modification modulates or regulates the expression of one or more target polynucleotide sequences involved in evading or alleviating an immune response.
  • the PSCs such as modified PCSs, are induced pluripotent stem cells (also called “iPSCs,” such as “modified iPSCs”).
  • the one or more modifications modulate or regulate (e.g., reduce or eliminate) the expression of MHC class I molecules, MHC class II molecules, or MHC class I and MHC class II molecules. In some embodiments, the one or more modifications modulate or regulate (e.g., increase) the expression of a tolerogenic factor, such as CD47. In some embodiments, one or more other modifications that modulate or regulate expression of other immune molecules also can be present in the modified pluripotent stem cells, such as a modification that regulates (e.g., reduces or eliminates) the expression of CD142 or a modification that regulates (e.g., increases) the expression of one or more complement inhibitor.
  • the provided modified pluripotent stem cells may also include a modification to increase expression of one or more tolerogenic factors.
  • the tolerogenic factor is one or more of DUX4, B2M-HLA-E, CD16, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc Receptor, IL15-RF, and H2-M3, or any combination thereof.
  • the one or more tolerogenic factors are selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.
  • the modification to increase expression of one or more tolerogenic factors is or includes increased expression of CD47. In some embodiments, the modification to increase expression of one or more tolerogenic factors is or includes increased expression of PD-L1. In some embodiments, the modification to increase expression of one or more tolerogenic factors is or includes increased expression of HLA-E. In some embodiments, the modification to increase expression of one or more tolerogenic factors is or includes increased expression of HLA-G. In some embodiments, the modification to increase expression of one or more tolerogenic factors is or includes increased expression of CCL21, PD-L1, FasL, Serpinb9, H2- M3 (HLA-G), CD47, CD200, and Mfge8.
  • the modified pluripotent stem cells include one or more genomic modifications that reduce expression of MHC class I molecules and a modification that increases expression of CD47.
  • the modified pluripotent stem cells comprise exogenous CD47 proteins and exhibit reduced or silenced surface expression of one or more MHC class I molecules.
  • the cells include one or more genomic modifications that reduce expression of MHC class II molecules and a modification that increases expression of CD47.
  • the modified cells comprise exogenous CD47 nucleic acids and proteins, and exhibit reduced or silenced surface expression of one or more MHC class I molecules.
  • the cells include one or more genomic modifications that reduce or eliminate expression of MHC class II molecules, one or more genomic modifications that reduce or eliminate expression of MHC class II molecules, and a modification that increases expression of CD47.
  • the modified pluripotent stem cells comprise exogenous CD47 proteins, exhibit reduced or silenced surface expression of one or more MHC class I molecules and exhibit reduced or lack surface expression of one or more MHC class II molecules.
  • the cells are B2M indel/indel, CIITAindel/indel, CD47tg cells.
  • the modified pluripotent stem cells may comprise a modification that modulates or regulates the expression of CD142.
  • the modification reduces or eliminates expression of CD142.
  • the modification that reduces expression of CD142 reduces CD142 protein expression.
  • the modification eliminates CD142 gene activity.
  • the modification comprises inactivation or disruption of both alleles of the CD142 gene.
  • the modification comprises inactivation or disruption of all CD142 coding sequences in the cell.
  • the inactivation or disruption comprises an indel in the CD142 gene.
  • the modification is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CD142 gene.
  • the CD142 gene is knocked out.
  • the provided modified pluripotent stem cells may also contain one or more modifications that increase expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, CD55, CD35 and combinations thereof.
  • the modification(s) that increase expression comprise increased surface expression, and/or the modifications that reduce expression comprise reduced surface expression.
  • the modification(s) that increase expression of the one or more complement inhibitor comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, an exogenous polynucleotide encoding CD55 and/or an exogenous polynucleotide encoding CD35.
  • the one or more complement inhibitor is CD46 and CD59, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD59.
  • the one or more complement inhibitor is CD46, CD59 and CD55, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59 and an exogenous polynucleotide encoding CD55.
  • the modified cell comprises a multicistronic vector comprising two or more exogenous polypeptides selected from the group consisting of one or more exogenous polynucleotide encoding the one or more tolerogenic factors, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD55 polypeptide.
  • each of the polynucleotides are separated by an IRES or a self-cleaving peptide.
  • modulation of expression of the one or more target immune molecules e.g. tolerogenic factor (e.g., increased expression)
  • the modulation of expression of the MHC class I molecules and/or MHC class II molecules is relative to the amount of expression of said molecule(s) in a pluripotent stem cell that does not comprise the modification(s) (i.e., unmodified pluripotent stem cell).
  • the cells are engineered or modified to have reduced or increased expression of one or more targets relative to an unaltered or unmodified wild-type cell.
  • the cells are engineered or modified to have constitutive reduced or increased expression of one or more targets relative to an unaltered or unmodified cell. In some embodiments, the cells are engineered or modified to have regulatable reduced or increased expression of one or more targets relative to an unaltered or unmodified cell. In some embodiments, the cells comprise increased expression of a tolerogenic factor (e.g., CD47) and reduced expression of the MHC class I molecules and/or MHC class II molecules relative to a wild-type cell or a control cell of the same cell type. Examples of wild type or control cells include pluripotent cells (e.g., embryonic stem cells or iPSCs).
  • a tolerogenic factor e.g., CD47
  • MHC class I molecules and/or MHC class II molecules relative to a wild-type cell or a control cell of the same cell type. Examples of wild type or control cells include pluripotent cells (e.g., embryonic stem cells or iPSCs).
  • wild-type or control can also mean an engineered cell that may contain nucleic acid changes resulting in reduced expression of MHC I and/or II, but did not undergo the gene editing procedures to result in overexpression of CD47 proteins.
  • wild-type or control also means an iPSC or progeny thereof that may contain nucleic acid changes resulting in pluripotency but did not undergo the gene editing procedures of the present disclosure to achieve reduced expression of MHC I and/or II, and/or overexpression of CD47 proteins.
  • the wild-type cell or the control cell is a starting material.
  • an iPSC cell line starting material is a starting material that is considered a wild-type or control cell as contemplated herein.
  • the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate the engineered cell.
  • reference to an “unmodified cell” can be a control cell that has been engineered in some aspects but does not contain all of the modifications by the gene editing procedures of the present disclosure to achieve reduced expression of MHC I and/or II, and/or overexpression of a tolerogenic protein (e.g., CD47).
  • the unmodified cell or wildtype cell expresses the tolerogenic factor, the MHC class I molecules, and/or the MHC class II molecules. In some embodiments, the unmodified cell or wildtype cell does not express the one or more tolerogenic factors, the MHC class I molecules, and/or the MHC class II molecules. In some embodiments wherein the unmodified cell or wildtype cell does not express the tolerogenic factor is used to generate the engineered primary cell, the provided engineered primary cells include a modification to overexpress the one or more tolerogenic factors or increase the expression of the one or more tolerogenic factors from 0%.
  • the population of modified pluripotent stem cells described elicits a reduced level of immune activation or no immune activation upon administration to a recipient subject.
  • the cells elicit a reduced level of systemic TH1 activation or no systemic TH1 activation in a recipient subject.
  • the cells elicit a reduced level of immune activation of peripheral blood mononuclear cells (PBMCs) or no immune activation of PBMCs in a recipient subject.
  • PBMCs peripheral blood mononuclear cells
  • the cells elicit a reduced level of donor-specific IgG antibodies or no donor specific IgG antibodies against the cells upon administration to a recipient subject.
  • the cells elicit a reduced level of IgM and IgG antibody production or no IgM and IgG antibody production against the cells in a recipient subject. In some embodiments, the cells elicit a reduced level of cytotoxic T cell killing of the cells upon administration to a recipient subject.
  • the modified pluripotent stem cells provided herein comprise a “suicide gene” or “suicide switch.”
  • a suicide gene or suicide switch can be incorporated to function as a “safety switch” that can cause the death of the cell, such as after the modified pluripotent stem cells cell is administered to a subject and if the cells should grow and divide in an undesired manner.
  • the “suicide gene” ablation approach includes a suicide gene in a gene transfer vector encoding a protein that results in cell killing only when activated by a specific compound.
  • a suicide gene may encode an enzyme that selectively converts a nontoxic compound into highly toxic metabolites. The result is specifically eliminating cells expressing the enzyme.
  • the suicide gene is the herpesvirus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir.
  • the suicide gene is a cytosine deaminase (e.g., the Escherichia coli cytosine deaminase (EC-CD)) gene and the trigger is 5- fluorocytosine (5-FC) (Barese et al, Mol. Therap. 20(10): 1932-1943 (2012), Xu et al, Cell Res. 8:73-8 (1998), both incorporated herein by reference in their entirety).
  • modified pluripotent stem cell having (1) reduced expression of MHC I and/or MHC II; and (2) a transgene comprising CD47 and a safety switch inserted at a safe harbor locus, wherein the safe harbor locus is selected from the group consisting of an AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, and SHS231 locus.
  • modified pluripotent stem cells having (1) reduced expression of MHC I and/or MHC II; and (2) a transgene comprising CD47 and HSVtk flanked by CLYBL homology arms, wherein the transgene is inserted at the CLYBL locus.
  • the modified pluripotent stem cell has B2M and/or OITA knockout.
  • the B2M and/or OITA knockout occur in both alleles.
  • the suicide gene is an inducible Caspase protein.
  • An inducible Caspase protein comprises at least a portion of a Caspase protein capable of inducing apoptosis.
  • the inducible Caspase protein is iCasp9. It comprises the sequence of the human FK506-binding protein, FKBP12, with an F36V mutation, connected through a series of amino acids to the gene encoding human caspase 9. FKBP12-F36V binds with high affinity to a small-molecule dimerizing agent, API 903.
  • the suicide function of iCasp9 in the instant invention is triggered by the administration of a chemical inducer of dimerization (CID).
  • CID chemical inducer of dimerization
  • the CID is the small molecule drug API 903. Dimerization causes the rapid induction of apoptosis. (See WO2011146862; Stasi et al, N. Engl. J. Med 365; 18 (2011); Tey et al, Biol. Blood Marrow Transplant. 13:913-924 (2007), each of which are incorporated by reference herein in their entirety.)
  • a safety switch can be incorporated into, such as introduced, into the modified pluripotent stem cells provided herein to provide the ability to induce death or apoptosis of modified cells containing the safety switch, for example if the cells grow and divide in an undesired manner or cause excessive toxicity to the host.
  • the use of safety switches enables one to conditionally eliminate aberrant cells in vivo and can be a critical step for the application of cell therapies in the clinic.
  • Safety switches and their uses thereof are described in, for example, Duzgune ⁇ , Origins of Suicide Gene Therapy (2019); Duzgune ⁇ (eds), Suicide Gene Therapy. Methods in Molecular Biology, vol.
  • the safety switch can cause cell death in a controlled manner, for example, in the presence of a drug or prodrug or upon activation by a selective exogenous compound.
  • the safety switch is selected from the group consisting of herpes simplex virus thymidine kinase (HSV-tk), cytosine deaminase (CyD), nitroreductase (NTR), purine nucleoside phosphorylase (PNP), horseradish peroxidase, inducible caspase 9 (iCasp9), rapamycin-activated caspase 9 (rapaCasp9), CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR8.
  • HSV-tk herpes simplex virus thymidine kinase
  • CyD cytosine deaminase
  • NTR nitroreductase
  • PNP purine
  • the safety switch may be a transgene encoding a product with cell killing capabilities when activated by a drug or prodrug, for example, by turning a non-toxic prodrug to a toxic metabolite inside the cell.
  • cell killing is activated by contacting a modified cell with the drug or prodrug.
  • the safety switch is HSV-tk, which converts ganciclovir (GCV) to GCV-triphosphate, thereby interfering with DNA synthesis and killing dividing cells.
  • the safety switch is CyD or a variant thereof, which converts the antifungal drug 5-fluorocytosine (5-FC) to cytotoxic 5 -fluorouracil (5-FU) by catalyzing the hydrolytic deamination of cytosine into uracil.
  • 5-FU is further converted to potent anti-metabolites (5- FdUMP, 5-FdUTP, 5-FUTP) by cellular enzymes. These compounds inhibit thymidylate synthase and the production of RNA and DNA, resulting in cell death.
  • the safety switch is NTR or a variant thereof, which can act on the prodrug CB 1954 via reduction of the nitro groups to reactive N-hydroxylamine intermediates that are toxic in proliferating and nonproliferating cells.
  • the safety switch is PNP or a variant thereof, which can turn prodrug 6-methylpurine deoxyriboside or fludarabine into toxic metabolites to both proliferating and nonproliferating cells.
  • the safety switch is horseradish peroxidase or a variant thereof, which can catalyze indole-3-acetic acid (IAA) to a potent cytotoxin and thus achieve cell killing.
  • the safety switch may be an iCasp9.
  • Caspase 9 is a component of the intrinsic mitochondrial apoptotic pathway which, under physiological conditions, is activated by the release of cytochrome C from damaged mitochondria. Activated caspase 9 then activates caspase 3, which triggers terminal effector molecules leading to apoptosis.
  • the iCasp9 may be generated by fusing a truncated caspase 9 (without its physiological dimerization domain or caspase activation domain) to a FK506 binding protein (FKBP), FKBP12-F36V, via a peptide linker.
  • FKBP FK506 binding protein
  • the iCasp9 has low dimerindependent basal activity and can be stably expressed in host cells (e.g., human T cells) without impairing their phenotype, function, or antigen specificity.
  • host cells e.g., human T cells
  • CID chemical inducer of dimerization
  • AP1903 rimiducid
  • AP20187 AP20187
  • rapamycin a chemical inducer of dimerization
  • iCasp9 can undergo inducible dimerization and activate the downstream caspase molecules, resulting in apoptosis of cells expressing the iCasp9.
  • CID chemical inducer of dimerization
  • AP1903 rimiducid
  • AP20187 AP20187
  • rapamycin rapamycin
  • rapamycininducible caspase 9 variant is called rapaCasp9. See Stavrou et al., Mai. Ther. 26(5): 1266- 1276 (2016).
  • iCasp9 can be used as a safety switch to achieve controlled killing of the host cells.
  • the safety switch may be a membrane-expressed protein which allows for cell depletion after administration of a specific antibody to that protein.
  • Safety switches of this category may include, for example, one or more transgene encoding CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, or RQR8 for surface expression thereof. These proteins may have surface epitopes that can be targeted by specific antibodies.
  • the safety switch comprises CCR4, which can be recognized by an anti-CCR4 antibody.
  • suitable anti-CCR4 antibodies include mogamulizumab and biosimilars thereof.
  • the safety switch comprises CD 16 or CD30, which can be recognized by an anti-CD16 or anti-CD30 antibody.
  • Non-limiting examples of such antiCD 16 or anti-CD30 antibody include AFM13 and biosimilars thereof.
  • the safety switch comprises CD19, which can be recognized by an antiCD 19 antibody.
  • Non-limiting examples of such anti-CD19 antibody include MOR208 and biosimilars thereof.
  • the safety switch comprises CD20, which can be recognized by an anti- CD20 antibody.
  • Non-limiting examples of such anti-CD20 antibody include obinutuzumab, ublituximab, ocaratuzumab, rituximab, rituximab-Rllb, and biosimilars thereof.
  • the safety switch comprises EGFR, which can be recognized by an anti-EGFR antibody.
  • anti-EGFR antibody include tomuzotuximab, RO5083945 (GA201), cetuximab, and biosimilars thereof.
  • the safety switch comprises GD2, which can be recognized by an anti-GD2 antibody.
  • anti-GD2 antibody include Hul4.18K322A, Hul4.18-IL2, Hu3F8, dinituximab, c.60C3-Rllc, and biosimilars thereof.
  • the safety switch may be an exogenously administered agent that recognizes one or more tolerogenic factors on the surface of the modified cell.
  • the exogenously administered agent is an antibody directed against or specific to a tolerogenic agent, e.g., an anti-CD47 antibody.
  • an exogenously administered antibody may block the immune inhibitory functions of the tolerogenic factor thereby re-sensitizing the immune system to the modified cells.
  • an exogenously administered anti-CD47 antibody may be administered to the subject, resulting in masking of CD47 on the modified cell and triggering of an immune response to the modified pluripotent stem cells.
  • the safety switch can include any of the strategies as described in WO2021146627A1, which is incorporated by reference in its entirety.
  • the method further comprises introducing an expression vector comprising an inducible suicide switch into the cell.
  • the modified pluripotent stem cells are derived from a source cell already comprising one or more of the desired modifications.
  • the modifications of the modified cell may be in any order, and not necessarily the order listed in the descriptive language provided herein.
  • a method of generating a modified pluripotent stem cell comprising: (a) reducing or eliminating the expression of MHC class I and/or MHC class II human leukocyte antigens in the cell; and (b) increasing the expression of a tolerogenic factor in the cell.
  • the one or more tolerogenic factors is selected from DUX4, B2M-HLA-E, CD 16, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc Receptor, IL15- RF, and H2-M3.
  • the one or more tolerogenic factors are selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD- Ll, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2- M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.
  • the one or more tolerogenic factors is CD47.
  • the method comprises reducing or eliminating the expression of MHC class I and MHC class II human leukocyte antigens.
  • the reducing or increasing expression comprise performing one or more modifications to the cell using a guided nuclease (e.g., a CRISPR/Cas system).
  • the method further comprises introducing an expression vector comprising an inducible suicide switch into the cell.
  • the method further comprises increasing the expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55 in said cell.
  • a method of generating a modified pluripotent stem cells cell comprising: (a) increasing the expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, and MFGE8 in the cell, and (b) reducing expression of CD142 in the cell.
  • the reducing or increasing expression comprise performing one or more modifications to the cell using a guided nuclease (e.g., a CRISPR/Cas system).
  • the method further comprises introducing an expression vector comprising an inducible suicide switch into the cell.
  • the method further comprises increasing the expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55 in said cell.
  • the modified iPSCs cells may be assayed for their hypoimmunogenicity and/or retention of pluripotency as is described in W02016183041 and WO2018132783.
  • hypoimmunogenicity is assayed using a number of techniques as exemplified in Figure 13 and Figure 15 of WO2018132783. These techniques include transplantation into allogeneic hosts and monitoring for hypoimmunogenic pluripotent cell growth (e.g., teratomas) that escape the host immune system.
  • hypoimmunogenic pluripotent cell derivatives are transduced to express luciferase and can then followed using bioluminescence imaging.
  • T cell and/or B cell response of the host animal to such cells are tested to confirm that the cells do not cause an immune reaction in the host animal.
  • T cell responses can be assessed by Elispot, ELISA, FACS, PCR, or mass cytometry (CYTOF).
  • B cell responses or antibody responses are assessed using FACS or Luminex.
  • the cells may be assayed for their ability to avoid innate immune responses, e.g., NK cell killing, as is generally shown in Figures 14 and 15 of WO2018132783.
  • the immunogenicity of the cells is evaluated using T cell immunoassays such as T cell proliferation assays, T cell activation assays, and T cell killing assays recognized by those skilled in the art.
  • T cell proliferation assay includes pretreating the cells with interferon-gamma and coculturing the cells with labelled T cells and assaying the presence of the T cell population (or the proliferating T cell population) after a preselected amount of time.
  • the T cell activation assay includes coculturing T cells with the cells outlined herein and determining the expression levels of T cell activation markers in the T cells.
  • In vivo assays can be performed to assess the immunogenicity of the cells outlined herein.
  • the survival and immunogenicity of modified iPSCs or modified SC-beta cells is determined using an allogeneic humanized immunodeficient mouse model.
  • the modified iPSCs are transplanted into an allogeneic humanized NSG-SGM3 mouse and assayed for cell rejection, cell survival, and teratoma formation.
  • grafted modified iPSCs or differentiated cells thereof display long-term survival in the mouse model.
  • pluripotency is assayed by the expression of certain pluripotency-specific factors as generally described herein and shown in Figure 29 of WO2018132783. Additionally or alternatively, the pluripotent cells are differentiated into one or more cell types as an indication of pluripotency.
  • modified pluripotent stem cells (modified iPSCs) have been generated, they can be maintained in an undifferentiated state as is known for maintaining iPSCs.
  • the cells can be cultured on Matrigel using culture media that prevents differentiation and maintains pluripotency.
  • they can be in culture medium under conditions to maintain pluripotency.
  • Target Genes [0216] Once altered, the presence of expression of any of the molecule described herein can be assayed using known techniques, such as Western blots, ELISA assays, FACS assays, and the like. Z Inactivation or Disruption of Target Genes a. Target Genes
  • the provided modified pluripotent stem cells comprise a modification (e.g., genetic modifications) of one or more target polynucleotide or protein sequences (also interchangeably referred to as a target gene) that regulate (e.g., reduce or eliminate) the expression of either MHC class I molecules, MHC class II molecules, or MHC class I and MHC class II molecules.
  • the cell to be modified is an unmodified cell that has not previously been introduced with the one or more modifications.
  • a genetic editing system is used to modify one or more target polynucleotide sequences that regulate (e.g., reduce or eliminate) the expression of either MHC class I molecules, MHC class II molecules, or MHC class I and MHC class II molecules.
  • the genome of the cell has been altered to reduce or delete components required or involved in facilitating HLA expression, such as expression of MHC class I and/or MHC class II molecules on the surface of the cell.
  • expression of a beta-2- microgloublin (B2M), a component of MHC class I molecules, is reduced or eliminated in the cell, thereby reducing or elimination the protein expression (e.g., cell surface expression) of MHC class I by the modified pluripotent stem cells.
  • B2M beta-2- microgloublin
  • any of the described modifications in the modified pluripotent stem cells that regulate (e.g., reduce or eliminate) expression of one or more target polynucleotide or protein in the modified pluripotent stem cells may be combined with one or more modifications to overexpress a polynucleotide (e.g., tolerogenic factor, such as CD47).
  • a polynucleotide e.g., tolerogenic factor, such as CD47
  • reduction of MHC class I and/or MHC class II expression can be accomplished, for example, by one or more of the following: (1) directly targeting the MHC class I genes such as the polymorphic HLA alleles (HLA- A, HLA-B, HLA -C) and/or the MHC class II genes such as HLA-DP, HLA-DQ, and/or HLA-DR; (2) removal of B2M, which will reduce surface trafficking of all MHC class I molecules; and/or (3) deletion of one or more components of the MHC enhanceosomes, such as LRC5, RFX-5, RFXANK, RFXAP, IRF1, NF-Y (including NFY-A, NFY-B, NFY-C), and CIITA that are critical for HLA expression.
  • reduction of MHC class II also may be accomplished by reducing expression, such as by knocking out the gene encoding CD74 in a cell, which is involved in the formation and transport of MHC class
  • HLA expression is interfered with.
  • HLA expression is interfered with by targeting individual HLAs (e.g., knocking out expression of one or more HLA class I molecules such as HLA-A, HLA-B and/or HLA-C and/or knocking out expression of one or more HLA class I molecules such as HLA-DP, HLA-DQ, and/or HLA-DR), targeting transcriptional regulators of HLA expression (e.g., knocking out expression of NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C and/or IRF-1), blocking surface trafficking of MHC class I molecules (e.g., knocking out expression of B2M and/or TAPI), and/or targeting with HLA-Razor (see, e.g., W02016183041).
  • reduction of HLA class II also may be accomplished by reducing expression, such as
  • the modified pluripotent stem cells disclosed herein do not express one or more human leukocyte antigens corresponding to MHC class I (e.g., HLA-A, HLA-B and/or HLA-C) and/or MHC class II (e.g., HLA-DP, HLA-DQ, and/or HLA-DR) and are thus characterized as being hypoimmunogenic.
  • MHC class I e.g., HLA-A, HLA-B and/or HLA-C
  • MHC class II e.g., HLA-DP, HLA-DQ, and/or HLA-DR
  • the modified pluripotent stem cells disclosed herein have been modified such that the cells, including any stem cell or a differentiated stem cell prepared therefrom, do not express or exhibit reduced expression of one or more of the following MHC class I molecules: HLA-A, HLA-B and HLA-C.
  • one or more of HLA-A, HLA-B and HLA-C may be "knocked-out" of a cell.
  • a cell that has a knocked-out HLA-A gene, HLA-B gene, and/or HLA-C gene may exhibit reduced or eliminated expression of each knocked-out gene.
  • the modified pluripotent stem cells disclosed herein have been modified such that the cells, including any stem cell or a differentiated stem cell prepared therefrom, do not express or exhibit reduced expression of one or more of the following MHC class II molecules: HLA-DP, HLA-DQ, and HLA-DR.
  • one or more of HLA-DP, HLA-DQ, and HLA-DR may be "knocked-out" of a cell.
  • a cell that has a knocked-out HLA-DP gene, HLA-DQ gene and/or HLA-DR gene may exhibit reduced or eliminated expression of each knocked-out gene.
  • MHC class I molecules and/or MHC class II molecules is modulated by targeting and deleting a contiguous stretch of genomic DNA, thereby reducing or eliminating expression of a target gene selected from the group consisting of B2M, CIITA, and NLRC5.
  • MHC class I molecules can alternatively or additionally be modulated by reducing or eliminating expression of TAPI.
  • MHC class II molecules can alternatively or additionally be modulated by reducing or eliminating expression of CD74.
  • the provided modified pluripotent stem cells comprise a modification of one or more target polynucleotide sequence that regulate MHC class I. Exemplary methods for reducing expression of MHC class I are described in sections below.
  • the targeted polynucleotide sequence is one or both of B2M and NLRC5.
  • the cell comprises a genetic editing modification (e.g., an indel) to the B2M gene. In some embodiments, the cell comprises a genetic editing modification (e.g., an indel) to the NLRC5 gene. In some embodiments, the cell comprises a genetic editing modification (e.g., an indel) to the TAPI gene.
  • the cell comprises genetic editing modifications (e.g., indels) to the B2M and CIITA genes.
  • a modification that reduces expression of an MHC class I molecule is a modification that reduces expression of B2M.
  • the modification that reduces B2M expression reduces B2M mRNA expression.
  • the reduced mRNA expression of B2M is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the mRNA expression of B2M is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the mRNA expression of B2M is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less. In some embodiments, the mRNA expression of B2M is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the mRNA expression of B2M is eliminated (e.g., 0% expression of B2M mRNA). In some embodiments, the modification that reduces B2M mRNA expression eliminates B2M gene activity.
  • the modification that reduces B2M expression reduces B2M protein expression.
  • the reduced protein expression of B2M is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the protein expression of B2M is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the protein expression of B2M is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less.
  • the protein expression of B2M is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the protein expression of B2M is eliminated (e.g., no detectable expression of B2M protein). In some embodiments, the modification that reduces B2M protein expression eliminates B2M gene activity.
  • the modification that reduces B2M expression comprises inactivation or disruption of the B2M gene. In some embodiments, the modification that reduces B2M expression comprises inactivation or disruption of one allele of the B2M gene. In some embodiments, the modification that reduces B2M expression comprises inactivation or disruption comprises inactivation or disruption of both alleles of the B2M gene.
  • the modification comprises inactivation or disruption of one or more B2M coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption of all B2M coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption comprises an indel in the B2M gene. In some embodiments, the modification is a frameshift mutation of genomic DNA of the B2M gene. In some embodiments, the modification is a deletion of genomic DNA of the B2M gene. In some embodiments, the modification is a deletion of a contiguous stretch of genomic DNA of the B2M gene. In some embodiments, the B2M gene is knocked out.
  • a modification that reduces expression of an MHC class I molecule is a modification that reduces expression of NLRC5.
  • decreased or eliminated expression of NLRC5 reduces or eliminates expression of one or more of the following MHC I molecules - HLA-A, HLA-B, and HLA-C.
  • the modification that reduces NLRC5 expression reduces NLRC5 mRNA expression.
  • the reduced mRNA expression of NLRC5 is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the mRNA expression of NLRC5 is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the mRNA expression of NLRC5 is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less. In some embodiments, the mRNA expression of NLRC5 is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the mRNA expression of NLRC5 is eliminated (e.g., 0% expression of NLRC5 mRNA). In some embodiments, the modification that reduces NLRC5 mRNA expression eliminates NLRC5 gene activity.
  • the modification that reduces NLRC5 expression reduces NLRC5 protein expression.
  • the reduced protein expression of NLRC5 is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the protein expression of NLRC5 is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the protein expression of NLRC5 is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less.
  • the protein expression of NLRC5 is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the protein expression of NLRC5 is eliminated (e.g., no detectable expression of NLRC5 protein). In some embodiments, the modification that reduces NLRC5 protein expression eliminates NLRC5 gene activity.
  • the modification that reduces NLRC5 expression comprises inactivation or disruption of the NLRC5 gene. In some embodiments, the modification that reduces NLCR5 expression comprises inactivation or disruption of one allele of the NLRC5 gene. In some embodiments, the modification that reduces NLRC5 expression comprises inactivation or disruption comprises inactivation or disruption of both alleles of the NLRC5 gene.
  • the modification comprises inactivation or disruption of one or more NLRC5 coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption of all NLRC5 coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption comprises an indel in the NLRC5 gene. In some embodiments, the modification is a frameshift mutation of genomic DNA of the NLRC5 gene. In some embodiments, the modification is a deletion of genomic DNA of the NLRC5 gene. In some embodiments, the modification is a deletion of a contiguous stretch of genomic DNA of the NLRC5 gene. In some embodiments, the NLRC5 gene is knocked out.
  • a modification that reduces expression of an MHC class I molecule is a modification that reduces expression of TAPI.
  • decreased or eliminated expression of TAPI reduces or eliminates expression of one or more of the following MHC I molecules - HLA-A, HLA-B, and HLA-C.
  • the modification that reduces TAPI expression reduces TAPI mRNA expression.
  • the reduced mRNA expression of TAPI is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the mRNA expression of TAPI is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the mRNA expression of TAPI is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less. In some embodiments, the mRNA expression of TAPI is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the mRNA expression of TAPI is eliminated (e.g., 0% expression of TAPI mRNA). In some embodiments, the modification that reduces TAPI mRNA expression eliminates TAPI gene activity.
  • the modification that reduces TAPI expression reduces TAPI protein expression.
  • the reduced protein expression of TAPI is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the protein expression of TAPI is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the protein expression of TAPI is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less.
  • the protein expression of TAPI is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the protein expression of TAPI is eliminated (e.g., no detectable expression of TAPI protein). In some embodiments, the modification that reduces TAPI protein expression eliminates TAPI gene activity.
  • the modification that reduces TAPI expression comprises inactivation or disruption of the TAPI gene. In some embodiments, the modification that reduces TAPI expression comprises inactivation or disruption of one allele of the TAPI gene. In some embodiments, the modification that reduces TAPI expression comprises inactivation or disruption comprises inactivation or disruption of both alleles of the TAPI gene. [0235] In some embodiments, the modification comprises inactivation or disruption of one or more TAPI coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption of all TAPI coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption comprises an indel in the TAPI gene.
  • the modification is a frameshift mutation of genomic DNA of the TAPI gene. In some embodiments, the modification is a deletion of genomic DNA of the TAPI gene. In some embodiments, the modification is a deletion of a contiguous stretch of genomic DNA of the TAPI gene. In some embodiments, the TAPI gene is knocked out.
  • the provided modified pluripotent stem cells comprise a modification of one or more target polynucleotide sequence that regulate MHC class II molecule expression. Exemplary methods for reducing expression of MHC class II are described in sections below.
  • the cell comprises a genetic editing modification to the OITA gene. In some embodiments, the cell comprises a genetic editing modification to the CD74 gene.
  • a modification that reduces expression of an MHC class II molecule is a modification that reduces expression of OITA.
  • the modification that reduces OITA expression reduces OITA mRNA expression.
  • the reduced mRNA expression of OITA is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the mRNA expression of OITA is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the mRNA expression of OITA is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less. In some embodiments, the mRNA expression of OITA is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the mRNA expression of OITA is eliminated (e.g., 0% expression of OITA mRNA). In some embodiments, the modification that reduces OITA mRNA expression eliminates OITA gene activity.
  • the modification that reduces OITA expression reduces OITA protein expression.
  • the reduced protein expression of OITA is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the protein expression of OITA is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the protein expression of OITA is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less.
  • the protein expression of CIITA is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the protein expression of CIITA is eliminated (e.g., 0% expression of OITA protein). In some embodiments, the modification that reduces OITA protein expression eliminates OITA gene activity.
  • the modification that reduces OITA expression comprises inactivation or disruption of the OITA gene. In some embodiments, the modification that reduces OITA expression comprises inactivation or disruption of one allele of the OITA gene. In some embodiments, the modification that reduces OITA expression comprises inactivation or disruption comprises inactivation or disruption of both alleles of the OITA gene.
  • the modification comprises inactivation or disruption of one or more OITA coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption of all OITA coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption comprises an indel in the OITA gene. In some embodiments, the modification is a frameshift mutation of genomic DNA of the OITA gene. In some embodiments, the modification is a deletion of genomic DNA of the OITA gene. In some embodiments, the modification is a deletion of a contiguous stretch of genomic DNA of the OITA gene. In some embodiments, the OITA gene is knocked out.
  • a modification that reduces expression of an MHC class II molecule is a modification that reduces expression of CD74.
  • the modification that reduces CD74 expression reduces CD74 mRNA expression.
  • the reduced mRNA expression of CD74 is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the mRNA expression of CD74 is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the mRNA expression of CD74 is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less. In some embodiments, the mRNA expression of CD74 is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the mRNA expression of CD74 is eliminated (e.g., 0% expression of CD74 mRNA). In some embodiments, the modification that reduces CD74 mRNA expression eliminates CD74 gene activity.
  • the modification that reduces CD74 expression reduces CD74 protein expression.
  • the reduced protein expression of CD74 is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the protein expression of CD74 is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the protein expression of CD74 is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less.
  • the protein expression of CD74 is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the protein expression of CD74 is eliminated (e.g., 0% expression of CD74 protein). In some embodiments, the modification that reduces CD74 protein expression eliminates CD74 gene activity.
  • the modification that reduces CD74 expression comprises inactivation or disruption of the CD74 gene. In some embodiments, the modification that reduces CD74 expression comprises inactivation or disruption of one allele of the CD74 gene. In some embodiments, the modification that reduces CD74 expression comprises inactivation or disruption comprises inactivation or disruption of both alleles of the CD74 gene.
  • the modification comprises inactivation or disruption of one or more CD74 coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption of all CD74 coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption comprises an indel in the CD74 gene. In some embodiments, the modification is a frameshift mutation of genomic DNA of the CD74 gene. In some embodiments, the modification is a deletion of genomic DNA of the CD74 gene. In some embodiments, the modification is a deletion of a contiguous stretch of genomic DNA of the CD74 gene. In some embodiments, the CD74 gene is knocked out.
  • the provided modified cells comprise a modification of one or more target polynucleotide sequence that regulate expression of MHC class I molecules and MHC class II molecules. Exemplary methods for reducing expression of MHC class I molecules and MHC class II molecules including any as described in sections below.
  • the cell comprises genetic editing modifications to the B2M and NLRC5 genes. In some embodiments, the cell comprises genetic editing modifications to the OITA and NLRC5 genes. In some embodiments, the cell comprises genetic editing modifications to the B2M and OITA genes. In particular embodiments, the cell comprises genetic editing modifications to the B2M, OITA and NLRC5 genes.
  • the technology disclosed herein modulate (e.g., reduce or eliminate) the expression of CD142, which is also known as tissue factor, factor III, and F3.
  • the modulation occurs using a CRISPR/Cas system.
  • the target polynucleotide sequence is CD142 or a variant of CD142. In some embodiments, the target polynucleotide sequence is a homolog of CD142. In some embodiments, the target polynucleotide sequence is an ortholog of CD 142.
  • the cells outlined herein comprise a modification targeting the CD142 gene.
  • the modification targeting the CD142 gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the CD142 gene.
  • gRNA guide ribonucleic acid
  • Assays to test whether the CD 142 gene has been inactivated are known and described herein.
  • the resulting modification of the CD 142 gene by PCR and the reduction of CD 142 expression can be assays by FACS analysis.
  • CD142 protein expression is detected using a Western blot of cells lysates probed with antibodies to the CD 142 protein.
  • reverse transcriptase polymerase chain reactions RT-PCR are used to confirm the presence of the inactivating modification.
  • Useful genomic, polynucleotide and polypeptide information about the human CD 142 are provided in, for example, the GeneCard Identifier GC01M094530, HGNC No. 3541, NCBI Gene ID 2152, NCBI RefSeq Nos. NM_001178096.1, NM_001993.4, NP_001171567.1, and NP_001984.1, UniProt No. Pl 3726, and the like.
  • the target polynucleotide sequence is PD-1 or a variant of PD-1. In some embodiments, the target polynucleotide sequence is a homolog of PD-1. In some embodiments, the target polynucleotide sequence is an ortholog of PD-1.
  • the cells outlined herein comprise a genetic modification targeting the gene encoding the programmed cell death protein 1 (PD-1) protein or the PDCD1 gene.
  • primary T cells comprise a genetic modification targeting the PDCD1 gene.
  • the genetic modification can reduce expression of PD-1 polynucleotides and PD-1 polypeptides in T cells includes primary T cells and CAR-T cells.
  • the genetic modification targeting the PDCD1 gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the PDCD1 gene.
  • gRNA guide ribonucleic acid
  • Assays to test whether the PDCD1 gene has been inactivated are known and described herein.
  • the resulting genetic modification of the PDCD1 gene by PCR and the reduction of PD-1 expression can be assays by FACS analysis.
  • PD-1 protein expression is detected using a Western blot of cells lysates probed with antibodies to the PD-1 protein.
  • reverse transcriptase polymerase chain reactions RT-PCR are used to confirm the presence of the inactivating genetic modification.
  • the cells provided herein are modified (e.g., genetically modified) to inactivate or disrupt one or more target polynucleotides or proteins as described. In some embodiments, the cells provided herein are modified (e.g., genetically modified) to reduce expression of the one or more target polynucleotides or proteins as described. In some embodiments, the cell that is modified with the one or more modification to reduce (e.g., eliminate) expression of a polynucleotide or protein is any source cell as described herein. In certain embodiments, the modified pluripotent stem cells (e.g., differentiated cells such as beta islet cells) disclosed herein comprise one or more modifications to reduce expression of one or more target polynucleotides.
  • the modified pluripotent stem cells e.g., differentiated cells such as beta islet cells
  • Non-limiting examples of the one or more target polynucleotides include any as described above, such as CIITA, B2M, CD142, NLRC5, HLA-A, HLA- B, HLA-C, LRC5, RFX-ANK, RFX5, RFX-AP, NFY-A, NFY-B, NFY-C, IRF1, and TAPI.
  • the target polynucleotide may be CD74.
  • the modifications to reduce expression of the one or more target polynucleotides is combined with one or more modifications to increase expression of a desired transgene.
  • the modifications create modified cells that are immune -privileged or hypoimmunogenic cells.
  • such cells By modulating (e.g., reducing or deleting) expression of one or a plurality of the target polynucleotides, such cells exhibit decreased immune activation when engrafted into a recipient subject.
  • the cell is considered hypoimmunogenic, e.g., in a recipient subject or patient upon administration.
  • any method for reducing expression of a target polynucleotide may be used.
  • the modifications result in permanent elimination or reduction in expression of the target polynucleotide.
  • the target polynucleotide or gene is disrupted by introducing a DNA break in the target polynucleotide, such as by using a targeting endonuclease.
  • the modifications result in transient reduction in expression of the target polynucleotide.
  • gene repression is achieved using an inhibitory nucleic acid that is complementary to the target polynucleotide to selectively suppress or repress expression of the gene, for instance using antisense techniques, such as by RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA), and/or ribozymes.
  • RNAi RNA interference
  • siRNA short interfering RNA
  • shRNA short hairpin
  • the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genomic sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence.
  • gene disruption is carried out by induction of one or more doublestranded breaks and/or one or more single-stranded breaks in the gene, typically in a targeted manner.
  • the double-stranded or single-stranded breaks are made by a nuclease, e.g., an endonuclease, such as a gene-targeted nuclease.
  • the targeted nuclease is selected from zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and RNA- guided nucleases such as a CRISPR-associated nuclease (Cas), specifically designed to be targeted to the sequence of a gene or a portion thereof.
  • ZFN zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • Cas RNA- guided nucleases
  • the targeted nuclease generates doublestranded or single-stranded breaks that then undergo repair through error prone non-homologous end joining (NHEJ) or, in some cases, precise homology directed repair (HDR) in which a template is used.
  • NHEJ error prone non-homologous end joining
  • HDR precise homology directed repair
  • the targeted nuclease generates DNA double strand breaks (DSBs).
  • the process of producing and repairing the breaks is typically error prone and results in insertions and deletions (indels) of DNA bases from NHEJ repair.
  • the modification may induce a deletion, insertion, or mutation of the nucleotide sequence of the target gene.
  • the modification may result in a frameshift mutation, which can result in a premature stop codon.
  • the targeted edits occur on both alleles of the gene resulting in a biallelic disruption or edit of the gene.
  • all alleles of the gene are targeted by the gene editing.
  • the nuclease such as a rare-cutting endonuclease
  • the nuclease is introduced into a cell containing the target polynucleotide sequence.
  • the nuclease may be introduced into the cell in the form of a nucleic acid encoding the nuclease.
  • the process of introducing the nucleic acids into cells can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector.
  • the nucleic acid that is introduced into the cell is DNA.
  • the nuclease is introduced into the cell in the form of a protein. For instance, in the case of a CRISPR/Cas system a ribonucleoprotein (RNP) may be introduced into the cell.
  • RNP ribonucleoprotein
  • the modification occurs using a CRISPR/Cas system.
  • Any CRISPR/Cas system that is capable of altering a target polynucleotide sequence in a cell can be used.
  • Such CRISPR-Cas systems can employ a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005; 1 (6)e60).
  • the molecular machinery of such Cas proteins that allows the CRISPR/Cas system to alter target polynucleotide sequences in cells include RNA binding proteins, endo- and exo-nucleases, helicases, and polymerases.
  • the CRISPR/Cas system is a CRISPR type I system.
  • the CRISPR/Cas system is a CRISPR type II system.
  • the CRISPR/Cas system is a CRISPR type V system.
  • the CRISPR/Cas systems include targeted systems that can be used to alter any target polynucleotide sequence in a cell.
  • a CRISPR/Cas system provided herein includes a Cas protein and one or more, such as at least one to two, ribonucleic acids (e.g., guide RNA (gRNA)) that are capable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence.
  • gRNA guide RNA
  • a Cas protein comprises one or more amino acid substitutions or modifications.
  • the one or more amino acid substitutions comprises a conservative amino acid substitution.
  • substitutions and/or modifications can prevent or reduce proteolytic degradation and/or extend the half-life of the polypeptide in a cell.
  • the Cas protein can comprise a peptide bond replacement (e.g., urea, thiourea, carbamate, sulfonyl urea, etc.).
  • the Cas protein can comprise a naturally occurring amino acid.
  • the Cas protein can comprise an alternative amino acid (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.).
  • a Cas protein can comprise a modification to include a moiety (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).
  • a Cas protein comprises a core Cas protein.
  • Exemplary Cas core proteins include, but are not limited to Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 and Cas9.
  • a Cas protein comprises a Cas protein of an E. coli subtype (also known as CASS2).
  • Exemplary Cas proteins of the E. Coli subtype include, but are not limited to Csel, Cse2, Cse3, Cse4, and Cas5e.
  • a Cas protein comprises a Cas protein of the Ypest subtype (also known as CASS3).
  • Exemplary Cas proteins of the Ypest subtype include, but are not limited to Csyl, Csy2, Csy3, and Csy4.
  • a Cas protein comprises a Cas protein of the Nmeni subtype (also known as CASS4).
  • Exemplary Cas proteins of the Nmeni subtype include, but are not limited to Csnl and Csn2.
  • a Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1).
  • Exemplary Cas proteins of the Dvulg subtype include Csdl, Csd2, and Cas5d.
  • a Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7).
  • Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cstl, Cst2, Cas5t.
  • a Cas protein comprises a Cas protein of the Hmari subtype.
  • Exemplary Cas proteins of the Hmari subtype include, but are not limited to Cshl, Csh2, and Cas5h.
  • a Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5).
  • Exemplary Cas proteins of the Apern subtype include, but are not limited to Csal, Csa2, Csa3, Csa4, Csa5, and Cas5a.
  • a Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6).
  • Exemplary Cas proteins of the Mtube subtype include, but are not limited to Csml, Csm2, Csm3, Csm4, and Csm5.
  • a Cas protein comprises a RAMP module Cas protein.
  • Exemplary RAMP module Cas proteins include, but are not limited to, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6. See, e.g., Klompe et al., Nature 571, 219-225 (2019); Strecker et al., Science 365, 48-53 (2019).
  • CRISPR systems of the present disclosure comprise TnpB polypeptides.
  • TnpB polypeptides may comprise a Ruv-C-like domain.
  • the RuvC domain may be a split RuvC domain comprising RuvC-I, RuvC-II, and RuvC-III subdomains.
  • a TnpB may further comprise one or more of a HTH domain, a bridge helix domain, and a zinc finger domain.
  • TnpB polypeptides do not comprise an HNH domain.
  • a TnpB protein comprises, starting at the N-terminus: a HTH domain, a RuvC-I subdomain, a bridge helix domain, a RuvC-II sub-domain, a zinger finger domain, and a RuvC-III sub-domain.
  • a RuvC-III sub-domain forms the C-terminus of a TnpB polypeptide.
  • a TnpB polypeptide is from Epsilonproteobacteria bacterium, Actinoplanes lobatus strain DSM 43150, Actinomadura celluolosilytica strain DSM 45823, Actinomadura namibiensis strain DSM 44197, Alicyclobacillus macrosprangiidus strain DSM 17980, Lipingzhangella halophila strain DSM 102030, or Ktedonobacter recemifer.
  • a TnpB polypeptide is from Ktedonobacter racemifer, or comprises a conserved RNA region with similarity to the 5’ ITR of K. racemifer TnpB loci.
  • a TnpB may comprise a Fanzor protein, a TnpB homolog found in eukaryotic genomes.
  • a CRISPR system comprising a TnpB polypeptide binds a target adjacent motif (TAM) sequence 5’ of a target polynucleotide.
  • TAM is a transposon-associated motif.
  • a TAM sequence comprises TCA.
  • a TAM sequence comprises TTCAN.
  • a TAM sequence comprises TTGAT.
  • a TAM sequence comprises ATAAA.
  • the methods for genetically modifying cells to knock out, knock down, or otherwise modify one or more genes comprise using a site-directed nuclease, including, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TAEENs), meganucleases, transposases, and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems
  • ZFNs are fusion proteins comprising an array of site-specific DNA binding domains adapted from zinc finger-containing transcription factors attached to the endonuclease domain of the bacterial FokI restriction enzyme.
  • a ZFN may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the DNA binding domains or zinc finger domains. See, e.g., Carroll et al., Genetics Society of America (2011) 188:773-782; Kim et al., Proc. Natl. Acad. Sci. USA (1996) 93:1156-1160.
  • Each zinc finger domain is a small protein structural motif stabilized by one or more zinc ions and usually recognizes a 3- to 4-bp DNA sequence. Tandem domains can thus potentially bind to an extended nucleotide sequence that is unique within a cell’s genome.
  • Zinc fingers can be engineered to bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger to bind to a predetermined nucleic acid sequence are known in the art. See, e.g., Sera et al., Biochemistry (2002) 41:7074-7081; Liu et al., Bioinformatics (2008) 24:1850-1857.
  • ZFNs containing FokI nuclease domains or other dimeric nuclease domains function as a dimer.
  • a pair of ZFNs are required to target non-palindromic DNA sites.
  • the two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. See Bitinaite et al., Proc. Natl. Acad. Sci. USA (1998) 95:10570-10575.
  • a pair of ZFNs are designed to recognize two sequences flanking the site, one on the forward strand and the other on the reverse strand.
  • the nuclease domains dimerize and cleave the DNA at the site, generating a DSB with 5' overhangs.
  • HDR can then be utilized to introduce a specific mutation, with the help of a repair template containing the desired mutation flanked by homology arms.
  • the repair template is usually an exogenous double-stranded DNA vector introduced to the cell. See Miller et al., Nat. Biotechnol. (2011) 29:143-148; Hockemeyer et al., Nat. Biotechnol. (2011) 29:731-734.
  • TALENs are another example of an artificial nuclease which can be used to edit a target gene.
  • TALENs are derived from DNA binding domains termed TALE repeats, which usually comprise tandem arrays with 10 to 30 repeats that bind and recognize extended DNA sequences. Each repeat is 33 to 35 amino acids in length, with two adjacent amino acids (termed the repeat-variable diresidue, or RVD) conferring specificity for one of the four DNA base pairs.
  • RVD repeat-variable diresidue
  • TALENs are produced artificially by fusing one or more TALE DNA binding domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a nuclease domain, for example, a FokI endonuclease domain.
  • TALE DNA binding domains e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
  • a nuclease domain for example, a FokI endonuclease domain.
  • the FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI nuclease domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al., Nature Biotech. (2011) 29:143-148.
  • a site-specific nuclease can be produced specific to any desired DNA sequence. Similar to ZFNs, TALENs can be introduced into a cell to generate DSBs at a desired target site in the genome, and so can be used to knock out genes or knock in mutations in similar, HDR-mediated pathways. See Boch, Nature Biotech. (2011) 29:135-136; Boch et al., Science (2009) 326:1509-1512; Moscou et al., Science (2009) 326:3501.
  • Meganucleases are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). Meganucleases are grouped into families based on their structural motifs which affect nuclease activity and/or DNA recognition. The most widespread and best known meganucleases are the proteins in the LAGLID ADG family, which owe their name to a conserved amino acid sequence. See Chevalier et al., Nucleic Acids Res. (2001) 29(18): 3757-3774.
  • the GIY-YIG family members have a GIY-YIG module, which is 70-100 residues long and includes four or five conserved sequence motifs with four invariant residues, two of which are required for activity. See Van Roey et al., Nature Struct. Biol. (2002) 9:806-811.
  • the His-Cys family meganucleases are characterized by a highly conserved series of histidines and cysteines over a region encompassing several hundred amino acid residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774.
  • NHN family are defined by motifs containing two pairs of conserved histidines surrounded by asparagine residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774.
  • Meganucleases can create DSBs in the genomic DNA, which can create a frame-shift mutation if improperly repaired, e.g., via NHEJ, leading to a decrease in the expression of a target gene in a cell.
  • foreign DNA can be introduced into the cell along with the meganuclease. Depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify the target gene. See Silva et al., Current Gene Therapy (2011) 11:11- 27.
  • Transposases are enzymes that bind to the end of a transposon and catalyze its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism.
  • transposases By linking transposases to other systems such as the CRISPR/Cas system, new gene editing tools can be developed to enable site specific insertions or manipulations of the genomic DNA.
  • transposons which use a catalytically inactive Cas effector protein and Tn7-like transposons.
  • the transposase-dependent DNA integration does not provoke DSBs in the genome, which may guarantee safer and more specific DNA integration.
  • the CRISPR system was originally discovered in prokaryotic organisms (e.g., bacteria and archaea) as a system involved in defense against invading phages and plasmids that provides a form of acquired immunity. Now it has been adapted and used as a popular gene editing tool in research and clinical applications.
  • prokaryotic organisms e.g., bacteria and archaea
  • CRISPR/Cas systems generally comprise at least two components: one or more guide RNAs (gRNAs) and a Cas protein.
  • the Cas protein is a nuclease that introduces a DSB into the target site.
  • CRISPR-Cas systems fall into two major classes: class 1 systems use a complex of multiple Cas proteins to degrade nucleic acids; class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV ; class 2 is divided into types II, V, and VI.
  • Cas proteins adapted for gene editing applications include, but are not limited to, Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, and Mad7.
  • Cas9 is a type II Cas protein and is described herein as illustrative. These Cas proteins may be originated from different source species. For example, Cas9 can be derived from S. pyogenes or S. aureus.
  • the type II CRISPR system incorporates sequences from invading DNA between CRISPR repeat sequences encoded as arrays within the host genome. Transcripts from the CRISPR repeat arrays are processed into CRISPR RNAs (crRNAs) each harboring a variable sequence transcribed from the invading DNA, known as the “protospacer” sequence, as well as part of the CRISPR repeat. Each crRNA hybridizes with a second transactivating CRISPR RNA (tracrRNA), and these two RNAs form a complex with the Cas9 nuclease. The protospacer-encoded portion of the crRNA directs the Cas9 complex to cleave complementary target DNA sequences, provided that they are adjacent to short sequences known as “protospacer adjacent motifs” (PAMs).
  • PAMs protospacer adjacent motifs
  • the CRISPR system Since its discovery, the CRISPR system has been adapted for inducing sequence specific DSBs and targeted genome editing in a wide range of cells and organisms spanning from bacteria to eukaryotic cells including human cells.
  • synthetic gRNAs have replaced the original crRNA:tracrRNA complex.
  • the gRNAs can be single guide RNAs (sgRNAs) composed of a crRNA, a tetraloop, and a tracrRNA.
  • the crRNA usually comprises a complementary region (also called a spacer, usually about 20 nucleotides in length) that is user-designed to recognize a target DNA of interest.
  • the tracrRNA sequence comprises a scaffold region for Cas nuclease binding.
  • the crRNA sequence and the tracrRNA sequence are linked by the tetraloop and each have a short repeat sequence for hybridization with each other, thus generating a chimeric sgRNA.
  • the complementary region will direct the Cas nuclease to the target DNA site through standard RNA-DNA complementary base pairing rules.
  • R A or G
  • Y C or T
  • W A or T
  • V A or C or G
  • N any base
  • Cas nucleases may comprise one or more mutations to alter their activity, specificity, recognition, and/or other characteristics.
  • the Cas nuclease may have one or more mutations that alter its fidelity to mitigate off-target effects (e.g., eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9).
  • the Cas nuclease may have one or more mutations that alter its PAM specificity.
  • a Cas protein comprises any one of the Cas proteins described herein or a functional portion thereof.
  • functional portion refers to a portion of a peptide which retains its ability to complex with at least one ribonucleic acid (e.g., guide RNA (gRNA)) and cleave a target polynucleotide sequence.
  • the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.
  • the functional portion comprises a combination of operably linked Casl2a (also known as Cpfl) protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.
  • the functional domains form a complex.
  • a functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain.
  • a functional portion of the Cas9 protein comprises a functional portion of the HNH nuclease domain.
  • a functional portion of the Cas 12a protein comprises a functional portion of a RuvC-like domain.
  • suitable Cas proteins include, but are not limited to, CasO, Casl2a (i.e., Cpfl), Casl2b, Casl2i, CasX, and Mad7.
  • exogenous Cas protein can be introduced into the cell in polypeptide form.
  • Cas proteins can be conjugated to or fused to a cell-penetrating polypeptide or cell-penetrating peptide.
  • cell-penetrating polypeptide and “cellpenetrating peptide” refers to a polypeptide or peptide, respectively, which facilitates the uptake of molecule into a cell.
  • the cell-penetrating polypeptides can contain a detectable label.
  • Cas proteins can be conjugated to or fused to a charged protein (e.g., that carries a positive, negative or overall neutral electric charge). Such linkage may be covalent.
  • the Cas protein can be fused to a superpositively charged GFP to significantly increase the ability of the Cas protein to penetrate a cell (Cronican et al. ACS Chem Biol. 2010; 5(8):747-52).
  • the Cas protein can be fused to a protein transduction domain (PTD) to facilitate its entry into a cell.
  • PTDs protein transduction domain
  • Exemplary PTDs include Tat, oligoarginine, and penetratin.
  • the Cas9 protein comprises a Cas9 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a penetrating domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a superpositively charged GFP.
  • the Casl2a protein comprises a Casl2a polypeptide fused to a cell-penetrating peptide. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to a PTD. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to a tat domain. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to an oligoarginine domain. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to a penetrating domain. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to a superpositively charged GFP.
  • the Cas protein can be introduced into a cell containing the target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein.
  • the process of introducing the nucleic acids into cells can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector.
  • the nucleic acid comprises DNA.
  • the nucleic acid comprises a modified DNA, as described herein.
  • the nucleic acid comprises mRNA.
  • the nucleic acid comprises a modified mRNA, as described herein (e.g., a synthetic, modified mRNA).
  • a CRISPR/Cas system generally includes two components: one or more guide RNA (gRNA) and a Cas protein.
  • the Cas protein is complexed with the one or more, such as one to two, ribonucleic acids (e.g., guide RNA (gRNA)).
  • the Cas protein is complexed with two ribonucleic acids.
  • the Cas protein is complexed with one ribonucleic acid.
  • the Cas protein is encoded by a modified nucleic acid, as described herein (e.g., a synthetic, modified mRNA).
  • gRNAs are short synthetic RNAs composed of a scaffold sequence for Cas binding and a user-designed spacer or complementary portion designated crRNA.
  • the cRNA is composed of a crRNA targeting sequence (herein after also called a gRNA targeting sequence; usually about 20 nucleotides in length) that defines the genomic target to be modified and a region of crRNA repeat (e.g. GUUUUAGAGCUA; SEQ ID NO: 19).
  • a crRNA targeting sequence herein after also called a gRNA targeting sequence; usually about 20 nucleotides in length
  • crRNA repeat e.g. GUUUUAGAGCUA; SEQ ID NO: 19
  • the scaffold sequence for Cas binding is made up of a tracrRNA sequence (e.g.
  • tracrRNA recruits the Cas nuclease (e.g., Cas9) and cleaves upstream of a protospacer- adjacent motif (PAM).
  • Cas nuclease e.g., Cas9
  • PAM protospacer- adjacent motif
  • the specific sequence of PAM varies depending on the species of the Cas gene. For example, the most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG. Other Cas9 variants and other nucleases with alternative PAMs have also been characterized and successfully used for genome editing. Thus, the CRISPR/Cas system can be used to create targeted DSBs at specified genomic loci that are complementary to the gRNA designed for the target loci.
  • the crRNA and tracrRNA can be linked together with a loop sequence (e.g., a tetraloop; GAAA, SEQ ID NO:21) for generation of a gRNA that is a chimeric single guide RNA (sgRNA; Hsu et al. 2013).
  • sgRNA can be generated for DNA-based expression or by chemical synthesis.
  • the complementary portion sequences (e.g., gRNA targeting sequence) of the gRNA will vary depending on the target site of interest.
  • the gRNAs comprise complementary portions specific to a sequence of a gene set forth in Table lb or Table 1c.
  • the genomic locus targeted by the gRNAs is located within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of any of the loci as described.
  • ribonucleic acid that is capable of directing a Cas protein to and hybridizing to a target motif of a target polynucleotide sequence.
  • at least one of the ribonucleic acids comprises
  • the Cas protein is complexed with one to two ribonucleic acids (e.g., guide RNA (gRNA)). In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid, as described herein (e.g., a synthetic, modified mRNA).
  • gRNA guide RNA
  • the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid, as described herein (e.g., a synthetic, modified mRNA).
  • ribonucleic acid that is capable of directing a Cas protein to and hybridizing to a target motif of a target polynucleotide sequence.
  • at least one of the ribonucleic acids comprises tracrRNA.
  • at least one of the ribonucleic acids comprises CRISPR RNA (crRNA).
  • crRNA CRISPR RNA
  • a single ribonucleic acid comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • At least one of the ribonucleic acids comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • both of the one to two ribonucleic acids comprise a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • the ribonucleic acids provided herein can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR/Cas system employed, and the sequence of the target polynucleotide, as will be appreciated by those skilled in the art.
  • the one to two ribonucleic acids can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence.
  • the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell.
  • the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell.
  • the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein.
  • each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs.
  • each of the one to two ribonucleic acids comprises guide RNAs that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.
  • one or two ribonucleic acids are complementary to and/or hybridize to sequences on the same strand of a target polynucleotide sequence.
  • one or two ribonucleic acids are complementary to and/or hybridize to sequences on the opposite strands of a target polynucleotide sequence.
  • the one or two ribonucleic acids are not complementary to and/or do not hybridize to sequences on the opposite strands of a target polynucleotide sequence.
  • the one or two ribonucleic acids are complementary to and/or hybridize to overlapping target motifs of a target polynucleotide sequence. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to offset target motifs of a target polynucleotide sequence.
  • nucleic acids encoding Cas protein and nucleic acids encoding the at least one to two ribonucleic acids are introduced into a cell via viral transduction (e.g., lentiviral transduction).
  • the Cas protein is complexed with 1-2 ribonucleic acids.
  • the Cas protein is complexed with two ribonucleic acids.
  • the Cas protein is complexed with one ribonucleic acid.
  • the Cas protein is encoded by a modified nucleic acid, as described herein (e.g., a synthetic, modified mRNA).
  • gRNA targeting sequences useful for CRISPR/Cas-based targeting of genes described herein are provided in Table lb or Table 1c.
  • gRNA targeting sequences for use in methods of genetic disruption to reduce or eliminate expression of a gene as described.
  • an "inch worming" approach can be used to identify additional loci for targeted insertion of transgenes by scanning the flanking regions on either side of the locus for PAM sequences, which usually occurs about every 100 base pairs (bp) across the genome.
  • the PAM sequence will depend on the particular Cas nuclease used because different nucleases usually have different corresponding PAM sequences.
  • the flanking regions on either side of the locus can be between about 500 to 4000 bp long, for example, about 500 bp, about 1000 bp, about 1500 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 3500 bp, or about 4000 bp long.
  • a new guide can be designed according to the sequence of that locus for use in genetic disruption methods.
  • the CRISPR/Cas system is described as illustrative, any gene-editing approaches as described can be used in this method of identifying new loci, including those using ZFNs, TALENS, meganucleases and transposases.
  • the cells described herein are made using Transcription Activator- Like Effector Nucleases (TALEN) methodologies.
  • TALEN Transcription Activator- Like Effector Nucleases
  • TALEN Transcription Activator- Like Effector Nucleases
  • TALEN Transcription Activator- Like Effector Nucleases
  • TALEN Transcription Activator- Like Effector Nucleases
  • the catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, like for instance I-TevI, ColE7, NucA and Fok-I.
  • the TALE domain can be fused to a meganuclease like for instance I-Crel and I-Onul or functional variant thereof.
  • said nuclease is a monomeric TALE-Nuclease.
  • a monomeric TALE- Nuclease is a TALE-Nuclease that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered TAL repeats with the catalytic domain of I-TevI described in WO2012138927.
  • Transcription Activator like Effector are proteins from the bacterial species Xanthomonas comprise a plurality of repeated sequences, each repeat comprising di-residues in position 12 and 13 (RVD) that are specific to each nucleotide base of the nucleic acid targeted sequence.
  • Binding domains with similar modular base-per-base nucleic acid binding properties can also be derived from new modular proteins recently discovered by the applicant in a different bacterial species.
  • the new modular proteins have the advantage of displaying more sequence variability than TAL repeats.
  • RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A.
  • critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity.
  • TALEN kits are sold commercially.
  • the cells are manipulated using zinc finger nuclease (ZFN).
  • ZFN zinc finger nuclease
  • a "zinc finger binding protein” is a protein or polypeptide that binds DNA, RNA and/or protein, preferably in a sequence-specific manner, as a result of stabilization of protein structure through coordination of a zinc ion.
  • the term zinc finger binding protein is often abbreviated as zinc finger protein or ZFP.
  • the individual DNA binding domains are typically referred to as "fingers.”
  • a ZFP has least one finger, typically two fingers, three fingers, or six fingers. Each finger binds from two to four base pairs of DNA, typically three or four base pairs of DNA.
  • a ZFP binds to a nucleic acid sequence called a target site or target segment.
  • Each finger typically comprises an approximately 30 amino acid, zinc-chelating, DNA- binding subdomain.
  • Studies have demonstrated that a single zinc finger of this class consists of an alpha helix containing the two invariant histidine residues coordinated with zinc along with the two cysteine residues of a single beta turn (see, e.g., Berg & Shi, Science 271:1081-1085 (1996)).
  • the cells described herein are made using a homing endonuclease.
  • a homing endonuclease Such homing endonucleases are well-known to the art (Stoddard 2005). Homing endonucleases recognize a DNA target sequence and generate a single- or double-strand break. Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length.
  • the homing endonuclease may for example correspond to a LAGLID ADG endonuclease, to an HNH endonuclease, or to a GIY-YIG endonuclease.
  • the homing endonuclease can be an I-Crel variant.
  • the cells described herein are made using a meganuclease.
  • Meganucleases are by definition sequence-specific endonucleases recognizing large sequences (Chevalier, B. S. and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). They can cleave unique sites in living cells, thereby enhancing gene targeting by 1000-fold or more in the vicinity of the cleavage site (Puchta et al., Nucleic Acids Res., 1993, 21, 5034-5040; Rouet et al., Mol. Cell. Biol., 1994, 14, 8096-8106; Choulika et al., Mol. Cell.
  • the cells provided herein are made using RNA silencing or RNA interference (RNAi) to knockdown (e.g., decrease, eliminate, or inhibit) the expression of a polypeptide.
  • RNAi RNA silencing or RNA interference
  • Useful RNAi methods include those that utilize synthetic RNAi molecules, short interfering RNAs (siRNAs), PlWI-interacting NRAs (piRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), and other transient knockdown methods recognized by those skilled in the art.
  • RNAi short interfering RNAs
  • piRNAs PlWI-interacting NRAs
  • shRNAs short hairpin RNAs
  • miRNAs microRNAs
  • Reagents for RNAi including sequence specific shRNAs, siRNA, miRNAs and the like are commercially available.
  • a target polynucleotide such as any described above, e.g., OITA, B2M, or NLRC5
  • RNA interference by introducing an inhibitory nucleic acid complementary to a target motif of the target polynucleotide, such as an siRNA, into the cells.
  • a target polynucleotide such as any described above, e.g., CIITA, B2M, or NLRC5
  • RNA interference is employed to reduce or inhibit the expression of at least one selected from the group consisting of CIITA, B2M, and NLRC5.
  • the modification reduces or eliminates, such as knocks out, the expression of MHC class I molecules (e.g., MHC class I genes encoding MHC class I molecules) by targeting the accessory chain B2M.
  • the modification occurs using a CRISPR/Cas system.
  • CRISPR/Cas system By reducing or eliminating, such as knocking out, expression of B2M, surface trafficking of MHC class I molecules is blocked, and such cells exhibit immune tolerance when engrafted into a recipient subject.
  • the cell is considered hypoimmunogenic, e.g., in a recipient subject or patient upon administration.
  • the target polynucleotide sequence provided herein is a variant of B2M. In some embodiments, the target polynucleotide sequence is a homolog of B2M. In some embodiments, the target polynucleotide sequence is an ortholog of B2M.
  • decreased or eliminated expression of B2M reduces or eliminates expression of one or more of the following MHC class I molecules - HLA-A, HLA-B, and HLA-C.
  • the modified pluripotent stem cells cell comprises a modification targeting the B2M gene.
  • the modification targeting the B2M gene is by using a targeted nuclease system that comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene.
  • the at least one guide ribonucleic acid sequence e.g., gRNA targeting sequence
  • the at least one guide ribonucleic acid sequence for specifically targeting the B2M gene is selected from the group consisting of SEQ ID NOS:81240-85644 of Appendix 2 or Table 15 of W02016/183041, the disclosure of which is herein incorporated by reference in its entirety.
  • an exogenous nucleic acid or transgene encoding a polypeptide as disclosed herein is inserted at the B2M gene.
  • a polypeptide as disclosed herein e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein
  • Exemplary transgenes for targeted insertion at the B2M locus include any as described herein.
  • Assays to test whether the B2M gene has been inactivated are known and described herein.
  • the resulting modification of the B2M gene by PCR and the reduction of HLA-I expression can be assays by flow cytometry, such as by FACS analysis.
  • B2M protein expression is detected using a Western blot of cells lysates probed with antibodies to the B2M protein.
  • reverse transcriptase polymerase chain reactions RT-PCR
  • the technologies disclosed herein modulate (e.g., reduce or eliminate) the expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of the NLR family, CARD domain containing 5/NOD27/CLR16.1 (NLRC5).
  • the modulation occurs using a CRISPR/Cas system.
  • NLRC5 is a critical regulator of MHC-I-mediated immune responses and, similar to OITA, NLRC5 is highly inducible by IFN-y and can translocate into the nucleus. NLRC5 activates the promoters of MHC-I genes and induces the transcription of MHC-I as well as related genes involved in MHC-I antigen presentation.
  • the target polynucleotide sequence is a variant of NLRC5. In some embodiments, the target polynucleotide sequence is a homolog of NLRC5. In some embodiments, the target polynucleotide sequence is an ortholog of NLRC5.
  • the cells outlined herein comprise a genetic modification targeting the NLRC5 gene.
  • the genetic modification targeting the NLRC5 gene by the rare- cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene.
  • the at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene is selected from the group consisting of SEQ ID NOS:36353-81239 of Appendix 3 or Table 14 of W02016183041, the disclosure is incorporated by reference in its entirety.
  • RNA expression is detected using a Western blot of cells lysates probed with antibodies to the NLRC5 protein.
  • RT-PCR reverse transcriptase polymerase chain reactions
  • the reduction of the MHC class I expression or function (HLA I when the cells are derived from human cells) in the modified cells can be measured using techniques known in the art; for example, FACS techniques using labeled antibodies that bind the HEA complex; for example, using commercially available HLA-A, B, C antibodies that bind to the alpha chain of the human major histocompatibility HLA Class I antigens.
  • the cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. This may be assayed by FACS analysis using antibodies to one or more HLA cell surface components as discussed above.
  • the modified pluripotent stem cells provided herein have a reduced susceptibility to macrophage phagocytosis and NK cell killing. Methods to assay for hypoimmunogenic phenotypes of the modified cells are described further below.
  • the modification reduces or eliminates, such as knocks out, the expression of MHC class II genes by targeting Class II transactivator (OITA) expression.
  • OITA Class II transactivator
  • the modification occurs using a CRISPR/Cas system.
  • OITA is a member of the LR or nucleotide binding domain (NBD) leucine -rich repeat (LRR) family of proteins and regulates the transcription of MHC class II by associating with the MHC enhanceosome.
  • NBD nucleotide binding domain
  • LRR leucine -rich repeat
  • the target polynucleotide sequence is a variant of OITA. In some embodiments, the target polynucleotide sequence is a homolog of OITA. In some embodiments, the target polynucleotide sequence is an ortholog of OITA.
  • reduced or eliminated expression of OITA reduces or eliminates expression of one or more of the following MHC class II are HLA-DP, HLA-DM, HLA-DOA, HLA- DOB, HLA-DQ, and HLA-DR.
  • the modified cell comprises a modification targeting the OITA gene.
  • the modification targeting the OITA gene is by a targeted nuclease system that comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the OITA gene.
  • the at least one guide ribonucleic acid sequence e.g., gRNA targeting sequence
  • the at least one guide ribonucleic acid sequence for specifically targeting the OITA gene is selected from the group consisting of SEQ ID NOS:5184-36352 of Appendix 1 or Table 12 of W02016183041, the disclosure is incorporated by reference in its entirety.
  • an exogenous nucleic acid or transgene encoding a polypeptide as disclosed herein is inserted at the OITA gene.
  • a polypeptide as disclosed herein e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein
  • exemplary transgenes for targeted insertion at the B2M locus include any as described herein.
  • Assays to test whether the OITA gene has been inactivated are known and described herein.
  • the resulting modification of the OITA gene by PCR and the reduction of HLA-II expression can be assays by flow cytometry, such as by FACS analysis.
  • OITA protein expression is detected using a Western blot of cells lysates probed with antibodies to the OITA protein.
  • reverse transcriptase polymerase chain reactions RT-PCR are used to confirm the presence of the inactivating modification.
  • the reduction of the MHC class II expression or function (HLA II when the cells are derived from human cells) in the modified cells can be measured using techniques known in the art, such as Western blotting using antibodies to the protein, FACS techniques, RT-PCR techniques, etc.
  • the modified cells can be tested to confirm that the HLA II complex is not expressed on the cell surface.
  • Methods to assess surface expression include methods known in the art (See Figure 21 of WO2018132783, for example) and generally is done using either Western Blots or FACS analysis based on commercial antibodies that bind to human HLA Class II HLA- DR, DP and most DQ antigens.
  • the modified pluripotent stem cells provided herein have a reduced susceptibility to macrophage phagocytosis and NK cell killing. Methods to assay for hypoimmunogenic phenotypes of the modified cells are described further below.
  • the modification reduces or eliminates, such as knocks out, the expression of CD142.
  • the modification occurs using a CRISPR/Cas system.
  • CD142 also known as tissue factor (F3) is a membrane-bound protein that initiates blood coagulation by forming a complex with circulating factor VII or factor Vila.
  • the CD142(TF):VIIa complex activates factors IX or X by specific limited proteolysis.
  • CD 142 (TF) plays a role in normal hemostasis by initiating the cellsurface assembly and propagation of the coagulation protease cascade.
  • reducing or eliminating, such as knocking out, expression of CD142 expression of MHC class II molecules is reduced thereby also reducing surface expression.
  • such cells exhibit immune tolerance when engrafted into a recipient subject.
  • the cell is considered hypoimmunogenic, e.g., in a recipient subject or patient upon administration.
  • the target polynucleotide sequence is a variant of CD142. In some embodiments, the target polynucleotide sequence is a homolog of CD 142. In some embodiments, the target polynucleotide sequence is an ortholog of CD 142.
  • the modified pluripotent stem cells comprises a modification targeting the CD142 gene.
  • the modification targeting the CD142 gene is by a targeted nuclease system that comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the CD 142 gene.
  • the target polynucleotide sequence is CD 142 or a variant of CD 142.
  • the target polynucleotide sequence is a homolog of CD 142.
  • the target polynucleotide sequence is an ortholog of CD 142.
  • the cells outlined herein may comprise a modification targeting the CD142 gene.
  • the modification targeting the CD142 gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the CD142 gene.
  • gRNA guide ribonucleic acid
  • Assays to test whether the CD 142 gene has been inactivated are known and described herein.
  • the resulting modification of the CD 142 gene by PCR and the reduction of CD 142 expression can be assays by FACS analysis.
  • CD142 protein expression is detected using a Western blot of cells lysates probed with antibodies to the CD 142 protein.
  • reverse transcriptase polymerase chain reactions RT-PCR
  • Useful genomic, polynucleotide and polypeptide information about the human CD142 are provided in, for example, the GeneCard Identifier GC01M094530, HGNC No.
  • an exogenous nucleic acid or transgene encoding a polypeptide as disclosed herein e.g., a chimeric antigen receptor, CD46, CD59, CD55, or CD47 or another tolerogenic factor disclosed herein
  • a polypeptide as disclosed herein e.g., a chimeric antigen receptor, CD46, CD59, CD55, or CD47 or another tolerogenic factor disclosed herein
  • exemplary transgenes for targeted insertion at the CD 142 locus include any as described herein.
  • the reduction of the CD142 expression or function in the modified cells can be measured using techniques known in the art, such as Western blotting using antibodies to the protein, FACS techniques, RT-PCR techniques, etc.
  • the modified cells can be tested to confirm that CD142 is not expressed on the cell surface. Methods to assess surface expression include methods known in the art (See Figure 21 of WO2018132783, for example) and generally is done using either Western Blots or FACS analysis based on commercial antibodies that bind to human CD142.
  • the modified cells provided herein have a reduced susceptibility to IB MIR. Methods to assay for hypoimmunogenic phenotypes of the modified cells are described further below.
  • the modification that reduces CD142 expression reduces CD142 mRNA expression.
  • the reduced mRNA expression of CD142 is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the mRNA expression of CD142 is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the mRNA expression of CD142 is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less.
  • the mRNA expression of CD142 is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the mRNA expression of CD142 is eliminated (e.g., 0% expression of CD142 mRNA). In some embodiments, the modification that reduces CD142 mRNA expression eliminates CD 142 gene activity.
  • the modification that reduces CD142 expression reduces CD142 protein expression.
  • the reduced protein expression of CD142 is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification.
  • the protein expression of CD142 is reduced by more than about 5%, such as reduced by more than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • the protein expression of CD142 is reduced by up to about 100%, such as reduced by up to about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less.
  • the protein expression of CD142 is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the protein expression of CD142 is eliminated (e.g., 0% expression of CD142 protein). In some embodiments, the modification that reduces CD142 protein expression eliminates CD 142 gene activity.
  • the modification that reduces CD142 expression comprises inactivation or disruption of the CD142 gene. In some embodiments, the modification that reduces CD 142 expression comprises inactivation or disruption of one allele of the CD 142 gene. In some embodiments, the modification that reduces CD142 expression comprises inactivation or disruption comprises inactivation or disruption of both alleles of the CD142 gene.
  • the modification comprises inactivation or disruption of one or more CD142 coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption of all CD142 coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption comprises an indel in the CD142 gene. In some embodiments, the modification is a frameshift mutation of genomic DNA of the CD142 gene. In some embodiments, the modification is a deletion of genomic DNA of the CD142 gene. In some embodiments, the modification is a deletion of a contiguous stretch of genomic DNA of the CD142 gene.
  • Exemplary guide target sequences for CD142 are known, for example: 2. Overexpression ofPoiynucieotides
  • the modified pluripotent stem cells provided herein are genetically modified, such as by introduction of one or more modifications into a cell to overexpress a desired polynucleotide in the cell.
  • the cell to be modified is an unmodified cell that has not previously been introduced with the one or more modifications.
  • the modified pluripotent stem cells provided herein are genetically modified to include one or more exogenous polynucleotides encoding an exogenous protein (also interchangeably used with the term “transgene”).
  • the cells are modified to increase expression of certain genes that are tolerogenic (e.g., immune) factors that affect immune recognition and tolerance in a recipient.
  • the provided modified cells such as T cells or NK cells, also express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the one or more polynucleotides e.g., exogenous polynucleotides, may be expressed (e.g. overexpressed) in the modified pluripotent stem cells together with one or more genetic modifications to reduce expression of a target polynucleotide described above, such as an MHC class I and/or MHC class II molecule or CD142.
  • the provided modified pluripotent stem cells do not trigger or activate an immune response upon administration to a recipient subject.
  • the modified pluripotent stem cell includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different overexpressed polynucleotides.
  • the overexpressed polynucleotide is an exogenous polynucleotide.
  • the modified pluripotent stem cell includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different exogenous polynucleotides.
  • the overexpressed polynucleotide is an exogenous polynucleotide that is expressed episomally in the cells.
  • the overexpressed polynucleotide is an exogenous polynucleotide that is inserted or integrated into one or more genomic loci of the modified cell.
  • expression of a polynucleotide is increased, i.e., the polynucleotide is overexpressed, using a fusion protein containing a DNA-targeting domain and a transcriptional activator.
  • a fusion protein containing a DNA-targeting domain and a transcriptional activator is known to a skilled artisan.
  • the modified pluripotent stem cell contains one or more exogenous polynucleotides in which the one or more exogenous polynucleotides are inserted or integrated into a genomic locus of the cell by non-targeted insertion methods, such as by transduction with a lentiviral vector.
  • the one or more exogenous polynucleotides are inserted or integrated into the genome of the cell by targeted insertion methods, such as by using homology directed repair (HDR). Any suitable method can be used to insert the exogenous polynucleotide into the genomic locus of the modified cell by HDR including the gene editing methods described herein (e.g., a CRISPR/Cas system).
  • the one or more exogenous polynucleotides are inserted into one or more genomic locus, such as any genomic locus described herein (e.g., Table 2). In some embodiments, the exogenous polynucleotides are inserted into the same genomic loci. In some embodiments, the exogenous polynucleotides are inserted into different genomic loci. In some embodiments, the two or more of the exogenous polynucleotides are inserted into the same genomic loci, such as any genomic locus described herein (e.g., Table 2). In some embodiments, two or more exogenous polynucleotides are inserted into a different genomic loci, such as two or more genomic loci as described herein (e.g., Table 2).
  • expression of a tolerogenic factor is overexpressed or increased in the cell.
  • the modified pluripotent stem cell includes increased expression, i.e., overexpression, of at least one tolerogenic factor.
  • the tolerogenic factor is any factor that promotes or contributes to promoting or inducing tolerance to the modified cell by the immune system (e.g., innate or adaptive immune system).
  • the tolerogenic factor is DUX4, B2M-HLA-E, CD 16, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD- Ll, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc Receptor, IL15-RF, and H2-M3.
  • the tolerogenic factor is CD47, PD-L1, HLA-E or HLA-G, CCL21, FasL, Serpinb9, CD200 or Mfge8, or any combination thereof.
  • the one or more tolerogenic factors are selected from the group consisting of CD 16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.
  • the cell includes at least one exogenous polynucleotide that includes a polynucleotide that encodes for a tolerogenic factor.
  • at least one of the exogenous polynucleotides is a polynucleotide that encodes CD47.
  • Provided herein are cells that do not trigger or activate an immune response upon administration to a recipient subject. As described above, in some embodiments, the cells are modified to increase expression of genes and tolerogenic (e.g., immune) factors that affect immune recognition and tolerance in a recipient.
  • the present disclosure provides a cell or population thereof that has been modified to express the tolerogenic factor (e.g., immunomodulatory polypeptide), such as CD47.
  • the present disclosure provides a method for altering a cell genome to express the tolerogenic factor (e.g., immunomodulatory polypeptide), such as CD47.
  • the modified cell expresses an exogenous tolerogenic factor (e.g., immunomodulatory polypeptide), such as an exogenous CD47.
  • overexpression or increasing expression of the exogenous polynucleotide is achieved by introducing into the cell (e.g., transducing the cell) within expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide.
  • the expression vector may be a viral vector, such as a lentiviral vector) or may be a non-viral vector.
  • the cell is modified to contain one or more exogenous polynucleotides in which at least one of the exogenous polynucleotides includes a polynucleotide that encodes for a tolerogenic factor.
  • the tolerogenic factor is DUX4, B2M-HLA-E, CD 16, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl- Inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc Receptor, IL15-RF, and H2-M3.
  • the tolerogenic factor is selected from CD47, PD-L1, HLA-E or HLA-G, CCL21, FasL, Serpinb9, CD200 or Mfge8, or any combination thereof (e.g., all thereof).
  • the one or more tolerogenic factors are selected from the group consisting of CD 16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.
  • at least one of the exogenous polynucleotides is a polynucleotide that encodes CD47.
  • the tolerogenic factor is CD47.
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes CD47, such as human CD47.
  • CD47 is overexpressed in the cell.
  • the expression of CD47 is overexpressed or increased in the modified cell compared to a similar cell of the same cell type that has not been modified with the modification, such as a reference or unmodified cell, e.g. a cell not modified with an exogenous polynucleotide encoding CD47.
  • CD47 is a leukocyte surface antigen and has a role in cell adhesion and modulation of integrins.
  • NP_001768.1, NP_942088.1, NM_001777.3 and NM_198793.2 Useful genomic, polynucleotide and polypeptide information about human CD47 are provided in, for example, the NP_001768.1, NP_942088.1, NM_001777.3 and NM_198793.2.
  • the modified pluripotent stem cell includes increased expression, i.e. overexpression, of at least one tolerogenic factor.
  • the cell includes at least one exogenous polynucleotide that includes a polynucleotide that encodes for a tolerogenic factor.
  • tolerogenic factors include DUX4, B2M- HLA-E, CD 16, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc Receptor, IL15-RF, and H2-M3, or any combination thereof.
  • the one or more tolerogenic factors are selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.
  • at least one of the overexpressed (e.g., exogenous) polynucleotides is a polynucleotide that encodes CD47.
  • the present disclosure provides a cell or population thereof that has been modified to express the tolerogenic factor (e.g., immunomodulatory polypeptide), such as CD47.
  • the present disclosure provides a method for altering a cell genome to express the tolerogenic factor (e.g., immunomodulatory polypeptide), such as CD47.
  • the modified pluripotent stem cell expresses an exogenous tolerogenic factor (e.g., immunomodulatory polypeptide), such as an exogenous CD47.
  • the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide.
  • the modified pluripotent stem cell contains an overexpressed polynucleotide that encodes CD47, such as human CD47.
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes CD47, such as human CD47.
  • CD47 is overexpressed in the cell.
  • the expression of CD47 is increased in the modified pluripotent stem cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding CD47.
  • the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD47 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1. In some embodiments, the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1.
  • the cell comprises an exogenous nucleotide sequence for CD47 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM_198793.2.
  • the cell comprises an exogenous nucleotide sequence for CD47 as set forth in NCBI Ref. Sequence Nos. NM_001777.3 and NM_198793.2.
  • the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD47 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1. In some embodiments, the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1.
  • the cell comprises an exogenous nucleotide sequence for CD47 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM_198793.2.
  • the cell comprises an exogenous nucleotide sequence for CD47 as set forth in NCBI Ref. Sequence Nos. NM_001777.3 and NM_198793.2.
  • the cell comprises an exogenous CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1. In some embodiments, the cell outlined herein comprises an exogenous CD47 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_001768.1 and NP_942088.1.
  • the cell comprises an overexpressed polynucleotide encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 1.
  • the cell comprises an exogenous polynucleotide encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 1.
  • the cell comprises an overexpressed polynucleotide encoding a CD47 polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the cell comprises an exogenous polynucleotide encoding a CD47 polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1.
  • the cell comprises an overexpressed CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 2.
  • the cell comprises an exogenous CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 2.
  • the cell comprises an overexpressed CD47 polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2.
  • the cell comprises an exogenous CD47 polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2.
  • the exogenous nucleotide sequence encoding the CD59 polypeptide is operably linked to a sequence encoding a heterologous signal peptide.
  • an exogenous polynucleotide encoding CD47 is integrated into the genome of the cell by targeted or non-targeted methods of insertion, such as described further below.
  • targeted insertion is by homology-dependent insertion into a target locus, such as by insertion into any one of the gene loci depicted in Table 2, e.g. a B2M gene or a OITA gene.
  • targeted insertion is by homology-independent insertion, such as by insertion into a safe harbor locus.
  • the polynucleotide encoding CD47 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding CD47 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • all or a functional portion of CD47 can be linked to other components such as a signal peptide, a leader sequence, a secretory signal, a label (e.g., a reporter gene), or any combination thereof.
  • the nucleic acid sequence encoding a signal peptide of CD47 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein.
  • the heterologous protein can be, for example, CD8a, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony stimulating factor (GM-CSF), GM-CSF receptor (GM- CSFRa), or an immunoglobulin (e.g., IgE or IgK).
  • tPA tissue plasminogen activator
  • growth hormone granulocyte-macrophage colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • GM-CSFRa GM-CSF receptor
  • immunoglobulin e.g., IgE or IgK
  • the signal peptide is a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g. chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently express a protein by or on a cell.
  • an immunoglobulin such as IgG heavy chain or IgG-kappa light chain
  • a cytokine such as interleukin-2 (IL-2), or CD33
  • a serum albumin protein e.g., HSA or albumin
  • a human azurocidin preprotein signal sequence e.g., a luciferase
  • a trypsinogen e.
  • the exogenous polynucleotide encoding CD47 is operably linked to a promoter.
  • the exogenous polynucleotide encoding CD47 is inserted into any one of the gene loci depicted in Table 2.
  • the exogenous polynucleotide encoding CD47 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the exogenous polynucleotide encoding CD47 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the exogenous polynucleotide encoding CD47 is inserted into a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • CD47 protein expression is detected using a Western blot of cell lysates probed with antibodies against the CD47 protein.
  • reverse transcriptase polymerase chain reactions RT-PCR are used to confirm the presence of the exogenous CD47 mRNA.
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes CD200, such as human CD200.
  • CD200 is overexpressed in the cell.
  • the expression of CD200 is increased in the modified cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding CD200.
  • Useful genomic, polynucleotide and polypeptide information about human CD200 are provided in, for example, the GeneCard Identifier GC03P112332, HGNC No. 7203, NCBI Gene ID 4345, Uniprot No.
  • the polynucleotide encoding CD200 is operably linked to a promoter.
  • the polynucleotide encoding CD200 is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding CD200 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding CD200 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding CD200 is inserted into a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • CD200 protein expression is detected using a Western blot of cell lysates probed with antibodies against the CD200 protein.
  • reverse transcriptase polymerase chain reactions RT-PCR are used to confirm the presence of the exogenous CD200 mRNA.
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes HLA-E, such as human HLA-E.
  • HLA-E is overexpressed in the cell.
  • the expression of HLA-E is increased in the modified pluripotent stem cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding HLA-E.
  • Useful genomic, polynucleotide and polypeptide information about human HLA-E are provided in, for example, the GeneCard Identifier GC06P047281, HGNC No.
  • the polynucleotide encoding HLA-E is operably linked to a promoter.
  • the polynucleotide encoding HLA-E is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding HLA-E is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding HLA-E is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding HLA-E is inserted into a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • HLA-E protein expression is detected using a Western blot of cell lysates probed with antibodies against the HLA-E protein.
  • RT-PCR reverse transcriptase polymerase chain reactions
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes HLA-G, such as human HLA-G.
  • HLA-G is overexpressed in the cell.
  • the expression of HLA-G is increased in the modified pluripotent stem cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding HLA-G.
  • Useful genomic, polynucleotide and polypeptide information about human HLA-G are provided in, for example, the GeneCard Identifier GC06P047256, HGNC No.
  • the polynucleotide encoding HLA-G is operably linked to a promoter.
  • the polynucleotide encoding HLA-G is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding HLA-G is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding HLA-G is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding HLA-G is inserted into a B2M gene locus, a CIITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • HLA-G protein expression is detected using a Western blot of cell lysates probed with antibodies against the HLA-G protein.
  • RT-PCR reverse transcriptase polymerase chain reactions
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes PD-L1, such as human PD-L1.
  • PD-L1 is overexpressed in the cell.
  • the expression of PD-L1 is increased in the modified cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding PD-L1.
  • Useful genomic, polynucleotide and polypeptide information about human PD-L1 or CD274 are provided in, for example, the GeneCard Identifier GC09P005450, HGNC No.
  • the polynucleotide encoding PD-L1 is operably linked to a promoter. [0365] In some embodiments, the polynucleotide encoding PD-L1 is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding PD-L1 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • a safe harbor locus such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding PD-L1 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding PD-L1 is inserted into a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • PD-L1 protein expression is detected using a Western blot of cell lysates probed with antibodies against the PD-L1 protein.
  • RT-PCR reverse transcriptase polymerase chain reactions
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes FasL, such as human FasL.
  • FasL is overexpressed in the cell.
  • the expression of FasL is increased in the modified pluripotent stem cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding FasL.
  • FasL Useful genomic, polynucleotide and polypeptide information about human Fas ligand (which is known as FasL, FASLG, CD178, TNFSF6, and the like) are provided in, for example, the GeneCard Identifier GC01P172628, HGNC No. 11936, NCBI Gene ID 356, Uniprot No. P48023, and NCBI RefSeq Nos. NP_000630.1, NM_000639.2, NP_001289675.1, and NM_001302746.1.
  • the polynucleotide encoding Fas-L is operably linked to a promoter.
  • the polynucleotide encoding Fas-L is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding Fas-L is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding Fas-L is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding Fas-L is inserted into a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • Fas-L protein expression is detected using a Western blot of cell lysates probed with antibodies against the Fas-L protein.
  • reverse transcriptase polymerase chain reactions RT-PCR
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes CCL21, such as human CCL21.
  • CCL21 is overexpressed in the cell.
  • the expression of CCL21 is increased in the modified pluripotent stem cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding CCL21.
  • Useful genomic, polynucleotide and polypeptide information about human CCL21 are provided in, for example, the GeneCard Identifier GC09M034709, HGNC No. 10620, NCBI Gene ID 6366, Uniprot No. 000585, and NCBI RefSeq Nos. NP_002980.1 and NM_002989.3.
  • the polynucleotide encoding CCL21 is operably linked to a promoter.
  • the polynucleotide encoding CCL21 is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding CCL21 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding CCL21 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding CCL21 is inserted into a B2M gene locus, a CIITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • CCL21 protein expression is detected using a Western blot of cell lysates probed with antibodies against the CCL21 protein.
  • RT-PCR reverse transcriptase polymerase chain reactions
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes CCL22, such as human CCL22.
  • CCL22 is overexpressed in the cell.
  • the expression of CCL22 is increased in the modified cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding CCL22.
  • Useful genomic, polynucleotide and polypeptide information about human CCL22 are provided in, for example, the GeneCard Identifier GC16P057359, HGNC No. 10621, NCBI Gene ID 6367, Uniprot No.
  • the polynucleotide encoding CCL22 is operably linked to a promoter.
  • the polynucleotide encoding CCL22 is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding CCL22 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding CCL22 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding CCL22 is inserted into a B2M gene locus, a OITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • CCL22 protein expression is detected using a Western blot of cell lysates probed with antibodies against the CCL22 protein.
  • RT-PCR reverse transcriptase polymerase chain reactions
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes Mfge8, such as human Mfge8.
  • Mfge8 is overexpressed in the cell.
  • the expression of Mfge8 is increased in the modified pluripotent stem cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding Mfge8.
  • Useful genomic, polynucleotide and polypeptide information about human Mfge8 are provided in, for example, the GeneCard Identifier GC15M088898, HGNC No.
  • the polynucleotide encoding Mfge8 is operably linked to a promoter.
  • the polynucleotide encoding Mfge8 is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding Mfge8 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding Mfge8 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding Mfge8 is inserted into a B2M gene locus, a OITA gene locus, a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • Mfge8 protein expression is detected using a Western blot of cell lysates probed with antibodies against the Mfge8 protein.
  • RT-PCR reverse transcriptase polymerase chain reactions
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes SerpinB9, such as human SerpinB9.
  • SerpinB9 is overexpressed in the cell.
  • the expression of SerpinB9 is increased in the modified pluripotent stem cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding SerpinB9.
  • Useful genomic, polynucleotide and polypeptide information about human SerpinB9 are provided in, for example, the GeneCard Identifier GC06M002887, HGNC No.
  • polynucleotide encoding SerpinB9 is operably linked to a promoter.
  • the polynucleotide encoding SerpinB9 is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding SerpinB9 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding SerpinB9 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding SerpinB9 is inserted into a B2M gene locus, a CIITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • SerpinB9 protein expression is detected using a Western blot of cell lysates probed with antibodies against the SerpinB9 protein.
  • reverse transcriptase polymerase chain reactions RT-PCR are used to confirm the presence of the exogenous SerpinB9 mRNA.
  • the tolerogenic factor is CD47 and the cell includes an exogenous polynucleotide encoding a CD47 protein. In some embodiments, the cell expresses an exogenous CD47 polypeptide.
  • a method disclosed herein comprises administering to a subject in need thereof a CD47-SIRPa blockade agent, wherein the subject was previously administered a population of cells engineered to express an exogenous CD47 polypeptide.
  • the CD47-SIRPa blockade agent comprises a CD47-binding domain.
  • the CD47- binding domain comprises signal regulatory protein alpha (SIRPa) or a fragment thereof.
  • the CD47-SIRPa blockade agent comprises an immunoglobulin G (IgG) Fc domain.
  • the IgG Fc domain comprises an IgGl Fc domain.
  • the IgGl Fc domain comprises a fragment of a human antibody.
  • the CD47-SIRPa blockade agent is selected from the group consisting of TTI-621, TTI-622, and ALX148.
  • the CD47-SIRPa blockade agent is TTI-621, TTI-622, and ALX148.
  • the CD47- SIRPa blockade agent is TTI-622.
  • the CD47-SIRPa blockade agent is ALX148.
  • the IgG Fc domain comprises an IgG4 Fc domain.
  • the CD47-SIRPa blockade agent is an antibody.
  • the antibody is selected from the group consisting of MIAP410, B6H12, and Magrolimab. In some embodiments, the antibody is MIAP410. In some embodiments, the antibody is B6H12. In some embodiments, the antibody is Magrolimab. In some embodiments, the antibody is selected from the group consisting of AO- 176, IBI188 (letaplimab), STI-6643, and ZL-1201. In some embodiments, the antibody is AO-176 (Arch). In some embodiments, the antibody is IBI188 (letaplimab) (Innovent). In some embodiments, the antibody is STI-6643 (Sorrento). In some embodiments, the antibody is ZL-1201 (Zai).
  • useful antibodies or fragments thereof that bind CD47 can be selected from a group that includes magrolimab ((Hu5F9-G4)) (Forty Seven, Inc.; Gilead Sciences, Inc.), urabrelimab, CC-90002 (Celgene; Bristol-Myers Squibb), IBI-188 (Innovent Biologies), IBI-322 (Innovent Biologies), TG-1801 (TG Therapeutics; also known as NI-1701, Novimmune SA), ALX148 (ALX Oncology), TJ011133 (also known as TJC4, 1-Mab Biopharma), FA3M3, ZL-1201 (Zai Lab Co., Ltd), AK117 (Akesbio Australia Pty, Ltd.), AO-176 (Arch Oncology), SRF231 (Surface Oncology), GenSci-059 (GeneScience), C47B157 (Janssen Research and Development), C47B161 (Janssen Research
  • the antibody or fragment thereof does not compete for CD47 binding with an antibody selected from a group that includes magrolimab, urabrelimab, CC-90002, IBI-188, IBI-322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO-176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B222, C47B227, Vx-1004, HMBD004, SHR-1603, AMMS4-G4, RTX-CD47, and IMC-002.
  • the antibody or fragment thereof competes for CD47 binding with an antibody selected from magrolimab, urabrelimab, CC-90002, IBI-188, IBI-322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO- 176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B222, C47B227, Vx-1004, HMBD004, SHR-1603, AMMS4-G4, RTX-CD47, and IMC-002.
  • an antibody selected from magrolimab, urabrelimab, CC-90002, IBI-188, IBI-322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO- 176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B
  • the antibody or fragment thereof that binds CD47 is selected from a group that includes a single-chain Fv fragment (scFv) against CD47, a Fab against CD47, a VHH nanobody against CD47, a DARPin against CD47, and variants thereof.
  • scFv single-chain Fv fragment
  • the scFv against CD47, a Fab against CD47, and variants thereof are based on the antigen binding domains of any of the antibodies selected from a group that includes magrolimab, urabrelimab, CC-90002, IBI-188, IBI-322, TG-1801 (NI-1701), ALX148, TJ011133, FA3M3, ZL1201, AK117, AO-176, SRF231, GenSci-059, C47B157, C47B161, C47B167, C47B222, C47B227, Vx-1004, HMBD004, SHR-1603, AMMS4-G4, RTX-CD47, and IMC-002.
  • the CD47 antagonist provides CD47 blockade. Methods and agents for CD47 blockade are described in PCT/US2021/054326, which is incorporated by reference in its entirety.
  • the tolerogenic factor (e.g., CD47) is overexpressed in the modified PSC relative to the control or wild-type PSC.
  • the tolerogenic factor (e.g. CD47) is expressed at a first level that is greater than at or about 3-fold, greater than at or about 5-fold, greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wild-type PSC.
  • the tolerogenic factor (e.g.
  • CD47 is expressed by the modified PSC at greater than at or about 20,000 molecules per cell, at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about
  • the tolerogenic factor (e.g., CD47) is overexpressed in the modified SC-beta cell relative to the control or wild-type beta cell, such as an unmodified SC-beta cell differentiated from an unmodified PSC that does not contain the modifications.
  • the tolerogenic factor e.g., CD47
  • CD47 is expressed at a first level that is greater than at or about 3-fold, greater than at or about 5-fold, greater than at or about 10-fold, greater than at or about 20-fold, greater than at or about 30-fold, greater than at or about 40-fold, greater than at or about 50-fold, greater than at or about 60-fold, or greater than at or about 70-fold over a second level expressed by the control or wildtype beta cell.
  • the tolerogenic factor e.g.
  • CD47 is expressed by the modified SC-beta cell at greater than at or about 20,000 molecules per cell, at greater than at or about 30,000 molecules per cell, greater than at or about 50,000 molecules per cell, greater than at or about 100,000 molecules per cell, greater than at or about 200,000 molecules per cell, greater than at or about 300,000 molecules per cell, greater than at or about 400,000 molecules per cell, greater than at or about 500,000 molecules per cell, or greater than at or about 600,000 molecules per cell.
  • expression of one or more complement inhibitor is increased in the cell.
  • the one or more complement inhibitor is one or more membrane-bound complement inhibitor.
  • at least one of the exogenous polynucleotides includes a polynucleotide that encodes for a complement inhibitor.
  • the one or more complement inhibitor is CD46, CD59, CD55, or CD35 or any combination thereof.
  • the one or more complement inhibitor is CD46, CD59, CD55, or any combination thereof.
  • at least one of the exogenous polynucleotides is a polynucleotide that encodes one or more complement inhibitors, such as CD46.
  • the one or more complement inhibitors are CD46 and CD59, or CD46, CD59, and CD55.
  • expression of CD46 and CD59 or CD46, CD59, and CD55 protects a cell or population thereof from complement-dependent cytotoxicity, including in the presence of antibodies against cell surface antigens expressed by the cell.
  • the present disclosure provides a cell or population thereof that has been modified to express the one or more complement inhibitor, such as CD46, CD59, CD55, or any combination thereof.
  • the one or more complement inhibitor is CD46 and CD59.
  • the one or more complement inhibitor is CD46, CD59, and CD55.
  • the present disclosure provides a method for altering a cell genome to express one or more complement inhibitor.
  • the modified cell expresses one or more exogenous complement inhibitor, such as exogenous CD46 and CD59 or CD46, CD59, and CD55.
  • the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD46 polypeptide.
  • the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD59 polypeptide. In some instances, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD55 polypeptide. In some embodiments, the expression vector comprises nucleotide sequences encoding two or more complement inhibitors in any combination. In some embodiments, the expression vector comprises nucleotide sequences encoding CD46 and CD59. In some embodiments, the expression vector comprises nucleotide sequences encoding CD46, CD59, and CD55.
  • the modified pluripotent stem cells contain an overexpressed polynucleotide that encodes CD46, such as human CD46.
  • the modified pluripotent stem cells contain an exogenous polynucleotide that encodes CD46, such as human CD46.
  • CD46 is overexpressed in the cell.
  • the expression of CD46 is increased in the modified pluripotent stem cells compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding CD46.
  • CD46 is a membrane-bound complement inhibitor.
  • complement factor I a serine protease which protects autologous cells against complement-mediated injury by cleaving C3b and C4b.
  • Useful genomic, polynucleotide and polypeptide information about human CD46 are provided in, for example, the GeneCard Identifier GC01P207752, HGNC No. 6953, NCBI Gene ID 4179, Uniprot No. P15529, and NCBI Ref Seq Nos.
  • the cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD46 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_002380.3, NP_722548.1, NP_758860.1, NP_758861.1, NP_758862.1, NP_758863.1, NP_758869.1, and NP_758871.1.
  • the cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD46 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_002380.3, NPJ722548.1, NP_758860.1, NP_758861.1, NP_758862.1, NP_758863.1, NP_758869.1, and NP_758871.1.
  • the cell comprises an overexpressed nucleotide sequence for CD46 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_002389.4, NM_153826.3, NM_172350.2, NM_172351.2, NM_172352.2 NP_758860.1, NM_172353.2, NM_172359.2, and NM_172361.2.
  • the cell comprises an overexpressed nucleotide sequence for CD46 as set forth in NCBI Ref.
  • the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD46 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_002380.3, NP_722548.1, NP_758860.1, NP_758861.1, NP_758862.1, NP_758863.1, NP_758869.1, and NP_758871.1.
  • the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD46 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_002380.3, NP_722548.1, NP_758860.1, NP_758861.1, NP_758862.1, NP_758863.1, NP_758869.1, and NP_758871.1.
  • the cell comprises an exogenous nucleotide sequence for CD46 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_002389.4, NM_153826.3, NM_172350.2, NM_172351.2, NM_172352.2 NP_758860.1, NM_172353.2, NM_172359.2, and NM_172361.2.
  • the cell comprises an exogenous nucleotide sequence for CD46 as set forth in NCBI Ref.
  • the cell comprises an overexpressed CD46 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_722548.1, NP_758860.1, NP_758861.1, NP_758862.1, NP_758863.1, NP_758869.1, and NP_758871.1.
  • the cell comprises an exogenous CD46 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref.
  • the cell outlined herein comprises an overexpressed CD46 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NPJ722548.1, NP_758860.1, NP_758861.1, NP_758862.1, NP_758863.1, NP_758869.1, and NP_758871.1.
  • the cell outlined herein comprises an exogenous CD46 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NPJ722548.1, NP_758860.1, NP_758861.1, NP_758862.1, NP_758863.1, NP_758869.1, and NP_758871.1.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD46 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 4.
  • a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD46 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 4.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD46 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD46 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 4.
  • a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD46 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 3.
  • a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD46 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 3.
  • the exogenous nucleotide sequence encoding the CD46 polypeptide is operably linked to a sequence encoding a heterologous signal peptide.
  • all or a functional portion of CD46 can be linked to other components such as a signal peptide, a leader sequence, a secretory signal, a label (e.g., a reporter gene), or any combination thereof.
  • the nucleic acid sequence encoding a signal peptide of CD46 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein.
  • the heterologous protein can be, for example, CD8a, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony stimulating factor (GM-CSF), GM-CSF receptor (GM- CSFRa), or an immunoglobulin (e.g., IgE or IgK).
  • the signal peptide is a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g.
  • HSA or albumin a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g., chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently express a protein by or on a cell.
  • trypsinogen e.g., chymotrypsinogen or trypsinogen
  • the exogenous polynucleotide encoding CD46 is operably linked to a promoter.
  • the polynucleotide encoding CD46 is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding CD46 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, SHS231.
  • the polynucleotide encoding CD46 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding CD46 is inserted into a B2M gene locus, a CIITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • CD46 protein expression is detected using a Western blot of cell lysates probed with antibodies against the CD46 protein.
  • reverse transcriptase polymerase chain reactions RT-PCR are used to confirm the presence of the exogenous CD46 mRNA.
  • the modified pluripotent stem cell contains an overexpressed polynucleotide that encodes CD59, such as human CD59.
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes CD59, such as human CD59.
  • CD59 is overexpressed in the cell.
  • the expression of CD59 is increased in the modified pluripotent stem cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding CD59.
  • CD59 is a membrane-bound complement inhibitor. More specifically, CD59 is an inhibitor of complement membrane attack complex (MAC) activity.
  • MAC complement membrane attack complex
  • CD59 acts by binding to the C8 and/or C9 complements of the assembling MAC, thereby preventing incorporation of the multiple copies of C9 required for complete formation of the osmolytic pore.
  • Useful genomic, polynucleotide and polypeptide information about human CD59 are provided in, for example, the GeneCard Identifier GC11M033704, HGNC No. 1689, NCBI Gene ID 966, Uniprot No. P13987, and NCBI RefSeq Nos.
  • the cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD59 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. Nos. NP_000602.1, NP_001120695.1, NP_001120697.1, NP_001120698.1, NP_001120699.1, NP_976074.1, NP_976075.1, and NP_976076.1.
  • the cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD59 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000602.1, NP_001120695.1, NP_001120697.1, NP_001120698.1, NP_001120699.1, NP_976074.1, NP_976075.1, and NP_976076.1.
  • the cell comprises an overexpressed nucleotide sequence for CD59 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_000611.5, NM_001127223.1, NM_001127225.1, NM_001127226.1, NM_001127227.1, NM_203329.2, NM_203330.2, and NM_203331.2.
  • the cell comprises an overexpressed nucleotide sequence for CD59 as set forth in NCBI Ref. Sequence Nos. NM_000611.5, NM_001127223.1, NM_001127225.1, NM_001127226.1, NM_001127227.1, NM_203329.2, NM_203330.2, and NM_203331.2.
  • the cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD59 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. Nos. NP_000602.1, NP_001120695.1, NP_001120697.1, NP_001120698.1, NP_001120699.1, NP_976074.1, NP_976075.1, and NP_976076.1.
  • the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD59 polypeptide has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. Nos. NP_000602.1, NP_001120695.1, NP_001120697.1, NP_001120698.1, NP_001120699.1, NP_976074.1, NP_976075.1, and NP_976076.1.
  • the cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD59 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000602.1, NP_001120695.1, NP_001120697.1, NP_001120698.1, NP_001120699.1, NP_976074.1, NP_976075.1, and NP_976076.1.
  • the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD59 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos.
  • the cell comprises an overexpressed nucleotide sequence for CD59 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos.
  • the cell comprises an exogenous nucleotide sequence for CD59 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos.
  • the cell comprises an overexpressed nucleotide sequence for CD59 as set forth in NCBI Ref. Sequence Nos. NM_000611.5, NM_001127223.1, NM_001127225.1, NM_001127226.1, NM_001127227.1, NM_203329.2, NM_203330.2, and NM_203331.2.
  • the cell comprises an exogenous nucleotide sequence for CD59 as set forth in NCBI Ref. Sequence Nos. NM_000611.5, NM_001127223.1, NM_001127225.1, NM_001127226.1, NM_001127227.1, NM_203329.2, NM_203330.2, and NM_203331.2.
  • the cell comprises an overexpressed CD59 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000602.1, NP_001120695.1, NP_001120697.1, NP_001120698.1, NP_001120699.1, NP_976074.1, NP_976075.1, and NP_976076.1.
  • 95% sequence identity e.g., 95%, 96%, 97%, 98%, 99%, or more
  • the cell comprises an exogenous CD59 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000602.1, NP_001120695.1, NP_001120697.1, NP_001120698.1, NP_001120699.1, NP_976074.1, NP_976075.1, and NP_976076.1.
  • the cell outlined herein comprises an overexpressed CD59 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos.
  • the cell outlined herein comprises an exogenous CD59 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000602.1, NP_001120695.1, NP_001120697.1, NP_001120698.1, NP_001120699.1, NP_976074.1, NP_976075.1, and NP_976076.1.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD59 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 6.
  • a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD59 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 6.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD59 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD59 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 6.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD59 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 5.
  • a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD59 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 5.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD59 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD59 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the exogenous nucleotide sequence encoding the CD59 polypeptide is operably linked to a sequence encoding a heterologous signal peptide.
  • all or a functional portion of CD59 can be linked to other components such as a signal peptide, a leader sequence, a secretory signal, a label (e.g., a reporter gene), or any combination thereof.
  • the nucleic acid sequence encoding a signal peptide of CD59 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein.
  • the heterologous protein can be, for example, CD8a, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony stimulating factor (GM-CSF), GM-CSF receptor (GM- CSFRa), or an immunoglobulin (e.g., IgE or IgK).
  • tPA tissue plasminogen activator
  • growth hormone granulocyte-macrophage colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • GM-CSFRa GM-CSF receptor
  • immunoglobulin e.g., IgE or IgK
  • the signal peptide is a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g. chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently express a protein by or on a cell.
  • an immunoglobulin such as IgG heavy chain or IgG-kappa light chain
  • a cytokine such as interleukin-2 (IL-2), or CD33
  • a serum albumin protein e.g., HSA or albumin
  • a human azurocidin preprotein signal sequence e.g., a luciferase
  • a trypsinogen e.
  • the exogenous polynucleotide encoding CD59 is operably linked to a promoter.
  • the polynucleotide encoding CD59 is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding CD59 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding CD59 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding CD59 is inserted into a B2M gene locus, a CIITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CD59 protein expression is detected using a Western blot of cell lysates probed with antibodies against the CD59 protein.
  • reverse transcriptase polymerase chain reactions are used to confirm the presence of the exogenous CD59 mRNA.
  • the modified pluripotent stem cell contains an overexpressed polynucleotide that encodes CD55, such as human CD55.
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes CD55, such as human CD55.
  • CD55 is overexpressed in the cell.
  • the expression of CD55 is increased in the modified pluripotent stem cell compared to a similar reference or unmodified cell (including with any other modifications) except that the reference or unmodified cell does not include the exogenous polynucleotide encoding CD55.
  • CD55 is a membrane-bound complement inhibitor.
  • interaction of CD55 with cell-associated C4b and C3b polypeptides interferes with their ability to catalyze the conversion of C2 and factor B to enzymatically active C2a and Bb and thereby prevents the formation of C4b2a and C3bBb, the amplification convertases of the complement cascade.
  • CD55 inhibits complement activation by destabilizing and preventing the formation of C3 and C5 convertases.
  • Useful genomic, polynucleotide and polypeptide information about human CD55 are provided in, for example, the GeneCard Identifier GC01P207321, HGNC No. 2665, NCBI Gene ID 1604, Uniprot No.
  • the cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD55 polypeptide that has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000565.1, NP_001108224.1, NP_001287832.1, and NP_001287833.1.
  • the cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD55 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos.
  • the cell comprises an overexpressed nucleotide sequence for CD55 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM_198793.2.
  • the cell comprises an overexpressed nucleotide sequence for CD55 as set forth in NCBI Ref. Sequence Nos. NM_000574.4, NM_001114752.2, NM_001300903.1, and NM_001300904.1.
  • the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD55 polypeptide that has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000565.1, NP_001108224.1, NP_001287832.1, and NP_001287833.1.
  • the cell outlined herein comprises an exogenous nucleotide sequence encoding a CD55 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos.
  • the cell comprises an exogenous nucleotide sequence for CD55 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM_001777.3 and NM_198793.2.
  • the cell comprises an exogenous nucleotide sequence for CD55 as set forth in NCBI Ref. Sequence Nos. NM_000574.4, NM_001114752.2, NM_001300903.1, and NM_001300904.1.
  • the cell comprises an overexpressed CD55 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000565.1, NP_001108224.1, NP_001287832.1, and NP_001287833.1.
  • the cell comprises an exogenous CD55 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence Nos.
  • the cell outlined herein comprises an overexpressed CD55 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000565.1, NP_001108224.1, NP_001287832.1, and NP_001287833.1. In some embodiments, the cell outlined herein comprises an exogenous CD55 polypeptide having an amino acid sequence as set forth in NCBI Ref. Sequence Nos. NP_000565.1, NP_001108224.1, NP_001287832.1, and NP_001287833.1.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD55 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 9.
  • a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD55 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 9.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD55 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD55 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 9.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD55 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 8.
  • a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD55 polypeptide that has at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) to an amino acid sequence as set forth in SEQ ID NO: 8.
  • a cell outlined herein comprises an overexpressed nucleotide sequence encoding a CD55 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, a cell outlined herein comprises an exogenous nucleotide sequence encoding a CD55 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the exogenous nucleotide sequence encoding the CD59 polypeptide is operably linked to a sequence encoding a heterologous signal peptide.
  • all or a functional portion of CD55 can be linked to other components such as a signal peptide, a leader sequence, a secretory signal, a label (e.g., a reporter gene), or any combination thereof.
  • the nucleic acid sequence encoding a signal peptide of CD55 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein.
  • the heterologous protein can be, for example, CD8a, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony stimulating factor (GM-CSF), GM-CSF receptor (GM- CSFRa), or an immunoglobulin (e.g., IgE or IgK).
  • the signal peptide is a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g.
  • HSA or albumin a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g., chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently express a protein by or on a cell.
  • trypsinogen e.g., chymotrypsinogen or trypsinogen
  • the exogenous polynucleotide encoding CD55 is operably linked to a promoter.
  • the polynucleotide encoding CD55 is inserted into any one of the gene loci depicted in Table 2.
  • the polynucleotide encoding CD55 is inserted into a safe harbor locus, such as but not limited to, a gene locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS231.
  • the polynucleotide encoding CD55 is inserted into the CCR5 gene locus, the PPP1R12C (also known as AAVS1) gene locus or the CLYBL gene locus.
  • the polynucleotide encoding CD55 is inserted into a B2M gene locus, a CIITA gene locus, or a CD142 gene locus.
  • a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
  • CRISPR/Cas system or any of the gene editing systems described herein
  • CD55 protein expression is detected using a Western blot of cell lysates probed with antibodies against the CD55 protein.
  • reverse transcriptase polymerase chain reactions RT-PCR
  • RT-PCR reverse transcriptase polymerase chain reactions
  • the cell comprises increased expression of none, one, two, or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55, in any combination.
  • the modified pluripotent stem cell contains an overexpressed polynucleotide that encodes CD46, such as any described above, and an overexpressed polynucleotide that encodes CD59, such as any described above.
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes CD46, such as any described above, and an exogenous polynucleotide that encodes CD59, such as any described above.
  • the modified cell (comprising one or more modifications that increase expression of CD46 and CD59) comprises increased expression of CD46 and CD59 relative to a cell that does not comprise the modifications (e.g., relative to endogenous expression of CD46 and CD59).
  • the modified pluripotent stem cell comprises between 1.5-fold and 2-fold, between 2-fold and 3-fold, between 3-fold and 4-fold, between 4-fold and 5-fold, between 5-fold and 10-fold, between 10-fold and 15-fold, between 15-fold and 20-fold, between 20-fold and 40-fold, between 40-fold and 60-fold, between 60-fold and 80-fold, between 80-fold and 100-fold, or between 100-fold and 200- fold increased expression of CD46 and CD59 compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46 and CD59).
  • the cell without the modification(s) does not have endogenous expression of CD46 and CD59 or does not have detectable expression of CD46 and CD59.
  • the fold increase in expression compared to a cell lacking the modifications is greater than 200-fold.
  • the modified pluripotent stem cells comprises between 2-fold and 200-fold, between 2-fold and 100-fold, between 2-fold and 50-fold, or between 2-fold and 20-fold increased expression of CD46 and CD59 compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46 and CD59).
  • the modified pluripotent stem cell (comprising one or more modifications that increase expression of CD46 and CD59) comprises between 5-fold and 200-fold, between 5-fold and 100-fold, between 5-fold and 50-fold, or between 5-fold and 20- fold increased expression of CD46 and CD59 compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46 and CD59).
  • the modified pluripotent stem cells (comprising one or more modifications that increase expression of CD46 and CD59) comprises increased expression of CD46 and CD59 relative to a cell that does not comprise the modifications (e.g., relative to endogenous expression of CD46 and CD59).
  • the modified pluripotent stem cell comprises at least at or about 2-fold, at least at or about 4-fold, at least at or about 6-fold, at least at or about 10-fold, at least at or about and 15-fold, at least at or about 20-fold, at least at or about 30-fold, at least at or about 50-fold, at least at or about 60-fold, at least at or about 70-fold, at least at or about 80-fold, at least at or about 100-fold, or any value between any of the foregoing values, increased expression of CD46 and CD59 compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46 and CD59).
  • the modified pluripotent stem cell (comprising one or more modifications that increase expression of CD46 and CD59) comprises increased expression of CD46 and CD59 relative to a cell that does not comprise the modifications (e.g., relative to endogenous expression of CD46 and CD59).
  • the modified cell comprises at or about 2-fold, at or about 4- fold, at or about 6-fold, at or about 10-fold, at or about and 15-fold, at or about 20-fold, at or about 30- fold, at or about 50-fold, at or about 60-fold, at or about 70-fold, at or about 80-fold, at or about 100-fold, or any value between any of the foregoing values, increased expression of CD46 and CD59 compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46 and CD59).
  • the cell comprises one or more transgenes encoding the CD46 and CD59.
  • the transgenes are monocistronic or multicistonic vectors, as described below.
  • the CD46 and CD59 are comprised by the same multicistronic vector, optionally in combination with one or more tolerogenic factors such as CD47.
  • the CD46 and CD59 are comprised by different transgenes, optionally in combination with one or more tolerogenic factors such as CD47.
  • the modified pluripotent stem cell contains an overexpressed polynucleotide that encodes CD46, such as any described above, an overexpressed polynucleotide that encodes CD59, such as any described above, and an overexpressed polynucleotide that encodes CD55, such as any described above.
  • the modified pluripotent stem cell contains an exogenous polynucleotide that encodes CD46, such as any described above, an exogenous polynucleotide that encodes CD59, such as any described above, and an exogenous polynucleotide that encodes CD55, such as any described above.
  • the modified pluripotent stem cell (comprising one or more modifications that increase expression of CD46, CD59, and CD55) comprises increased expression of CD46, CD59, and CD55 relative to a cell that does not comprise the modifications (e.g., relative to endogenous expression of CD46, CD59, and CD55).
  • the modified cell comprises between 1.5-fold and 2-fold, between 2-fold and 3-fold, between 3-fold and 4-fold, between 4-fold and 5- fold, between 5 -fold and 10-fold, between 10-fold and 15 -fold, between 15 -fold and 20-fold, between 20- fold and 40-fold, between 40-fold and 60-fold, between 60-fold and 80-fold, between 80-fold and 100- fold, or between 100-fold and 200-fold increased expression of CD46, CD59, and CD55 compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46, CD59, and CD55).
  • the cell without the modification(s) does not have endogenous expression of CD46, CD59, and CD55or does not have detectable expression of CD46, CD59, and CD55.
  • the fold increase in expression compared to a cell lacking the modifications is greater than 200-fold.
  • the modified pluripotent stem cell (comprising one or more modifications that increase expression of CD46, CD59, and CD55) comprises between 2-fold and 200- fold, between 2-fold and 100-fold, between 2-fold and 50-fold, or between 2-fold and 20-fold increased expression of CD46, CD59, and CD55 compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46, CD59, and CD55).
  • the modified pluripotent stem cell (comprising one or more modifications that increase expression of CD46, CD59, and CD55) comprises between 5-fold and 200-fold, between 5-fold and 100-fold, between 5-fold and 50- fold, or between 5-fold and 20-fold increased expression of CD46, CD59, and CD55 compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46, CD59, and CD55).
  • the modified pluripotent stem cell (comprising one or more modifications that increase expression of CD46, CD59, and CD55) comprises increased expression of CD46, CD59, and CD55 relative to a cell that does not comprise the modifications (e.g., relative to endogenous expression of CD46 and CD59).
  • the modified cell comprises at least at or about 2-fold, at least at or about 4-fold, at least at or about 6-fold, at least at or about 10-fold, at least at or about and 15-fold, at least at or about 20-fold, at least at or about 30-fold, at least at or about 50-fold, at least at or about 60-fold, at least at or about 70-fold, at least at or about 80-fold, at least at or about 100-fold, or any value between any of the foregoing values, increased expression of CD46, CD59, and CD55 compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46, CD59, and CD55).
  • modifications e.g., compared to endogenous expression of CD46, CD59, and CD55.
  • the modified pluripotent stem cell (comprising one or more modifications that increase expression of CD46, CD59, and CD55) comprises increased expression of CD46, CD59, and CD55 relative to a cell that does not comprise the modifications (e.g., relative to endogenous expression of CD46, CD59, and CD55).
  • the modified cell comprises at or about 2-fold, at or about 4-fold, at or about 6-fold, at or about 10-fold, at or about and 15-fold, at or about 20-fold, at or about 30-fold, at or about 50-fold, at or about 60-fold, at or about 70-fold, at or about 80-fold, at or about 100-fold, or any value between any of the foregoing values, increased expression of CD46, CD59, and CD55compared to a cell that does not have the modifications (e.g., compared to endogenous expression of CD46, CD59, and CD55).
  • the cell comprises one or more transgenes encoding the CD46, CD59, and CD55.
  • the transgenes are monocistronic or multicistronic vectors, as described below.
  • the CD46, CD59, and CD55 are comprised by the same multicistronic vector, optionally in combination with one or more tolerogenic factors such as CD47.
  • the CD46, CD59, and CD55 are comprised by different transgenes, optionally in combination with one or more tolerogenic factors such as CD47.
  • increased expression of a polynucleotide may be carried out by any of a variety of techniques. For instance, methods for modulating expression of genes and factors (proteins) include genome editing technologies, and RNA or protein expression technologies and the like. For all of these technologies, well known recombinant techniques are used, to generate recombinant nucleic acids as outlined herein.
  • the cell that is modified with the one or more modification for overexpression or increased expression of a polynucleotide is any source cell as described herein.
  • expression of a target gene is increased by expression of fusion protein or a protein complex containing (1) a site-specific binding domain specific for the endogenous CD47, or other gene and (2) a transcriptional activator.
  • the regulatory factor is comprised of a site-specific DNA-binding nucleic acid molecule, such as a guide RNA (gRNA).
  • gRNA guide RNA
  • the method is achieved by site specific DNA-binding targeted proteins, such as zinc finger proteins (ZFP) or fusion proteins containing ZFP, which are also known as zinc finger nucleases (ZFNs).
  • ZFP zinc finger proteins
  • ZFNs zinc finger nucleases
  • the regulatory factor comprises a site-specific binding domain, such as using a DNA binding protein or DNA-binding nucleic acid, which specifically binds to or hybridizes to the gene at a targeted region.
  • the provided polynucleotides or polypeptides are coupled to or complexed with a site-specific nuclease, such as a modified nuclease.
  • the administration is effected using a fusion comprising a DNA-targeting protein of a modified nuclease, such as a meganuclease or an RNA-guided nuclease such as a clustered regularly interspersed short palindromic nucleic acid (CRISPR)-Cas system, such as CRISPR-Cas9 system.
  • a modified nuclease such as a meganuclease or an RNA-guided nuclease such as a clustered regularly interspersed short palindromic nucleic acid (CRISPR)-Cas system, such as CRISPR-Cas9 system.
  • CRISPR clustered regularly interspersed short palindromic nucleic acid
  • the nuclease is modified to lack nuclease activity.
  • the modified nuclease is a catalytically dead dCas9.
  • the site specific binding domain may be derived from a nuclease
  • the recognition sequences of homing endonucleases and meganucleases such as I-Scel, I-Ceul, PI-PspI, Pl-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-Ppol, I-SceIII, I-Crel, I-TevI, I-TevII and I-TevIII. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et al., (1997) Nucleic Acids Res.
  • Zinc finger, TALE, and CRISPR system binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein.
  • Engineered DNA binding proteins are proteins that are non-naturally occurring. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Pat. Nos.
  • the site-specific binding domain comprises one or more zinc-finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner.
  • ZFPs zinc-finger proteins
  • a ZFP or domain thereof is a protein or domain within a larger protein that binds DNA in a sequence-specific manner through one or more zinc fingers, regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • ZFPs are artificial ZFP domains targeting specific DNA sequences, typically 9- 18 nucleotides long, generated by assembly of individual fingers.
  • ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers.
  • sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (-1, 2, 3 and 6) on a zinc finger recognition helix.
  • the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is engineered to bind to a target site of choice.
  • a target site of choice See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos.
  • the site-specific binding domain comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference in its entirety herein.
  • TAL transcription activator-like protein
  • TALE transcription activator-like protein effector
  • the site-specific binding domain is derived from the CRISPR/Cas system.
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.
  • tracrRNA or an active partial tracrRNA a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system, or a “targeting sequence”), and/or other sequences and transcripts from a CRISPR locus.
  • a guide sequence includes a targeting domain comprising a polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • the targeting domain (e.g., targeting sequence) of the gRNA is complementary, e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid.
  • the gRNA may be any as described herein.
  • the gRNA has a targeting sequence that is complementary to a target site of CD47, such as set forth in any one of SEQ ID NOS:200784-231885 (Table 29, Appendix 22 of W02016183041); HLA- E, such as set forth in any one of SEQ ID NOS:189859-193183 (Table 19, Appendix 12 of W02016183041); HLA-F, such as set forth in any one of SEQ ID NOS: 688808-699754 (Table 45, Appendix 38 of W02016183041); HLA-G, such as set forth in any one of SEQ ID NOS:188372-189858 (Table 18, Appendix 11 of W02016183041); or PD-L1, such as set forth in any one of SEQ ID NOS: 193184-200783 (Table 21, Appendix 14 of W02016183041).
  • the target site is upstream of a transcription initiation site of the target gene. In some embodiments, the target site is adjacent to a transcription initiation site of the gene. In some embodiments, the target site is adjacent to an RNA polymerase pause site downstream of a transcription initiation site of the gene.
  • the targeting domain is configured to target the promoter region of the target gene to promote transcription initiation, binding of one or more transcription enhancers or activators, and/or RNA polymerase.
  • One or more gRNA can be used to target the promoter region of the gene.
  • one or more regions of the gene can be targeted.
  • the target sites are within 600 base pairs on either side of a transcription start site (TSS) of the gene.
  • TSS transcription start site
  • gRNA targeting sequence a sequence targeting a gene
  • gRNA targeting sequence a sequence targeting a gene
  • gRNA targeting sequence a sequence targeting a gene
  • a genome-wide gRNA database for CRISPR genome editing is publicly available, which contains exemplary single guide RNA (sgRNA) target sequences in constitutive exons of genes in the human genome or mouse genome (see e.g., genescript.com/gRNA-database.html; see also, Sanjana et al. (2014) Nat. Methods, 11:783-4; www.e- crisp.org/E-CRISP/; crispr.mit.edu/).
  • the gRNA sequence is or comprises a targeting sequence with minimal off-target binding to a non-target gene.
  • the regulatory factor further comprises a functional domain, e.g., a transcriptional activator.
  • the transcriptional activator is or contains one or more regulatory elements, such as one or more transcriptional control elements of a target gene, whereby a site-specific domain as provided above is recognized to drive expression of such gene.
  • the transcriptional activator drives expression of the target gene.
  • the transcriptional activator can be or contain all or a portion of a heterologous transactivation domain.
  • the transcriptional activator is selected from Herpes simplex-derived transactivation domain, Dnmt3a methyltransferase domain, p65, VP16, and VP64.
  • the regulatory factor is a zinc finger transcription factor (ZF-TF). In some embodiments, the regulatory factor is VP64-p65-Rta (VPR).
  • the regulatory factor further comprises a transcriptional regulatory domain.
  • Common domains include, e.g., transcription factor domains (activators, repressors, coactivators, co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and their associated factors and modifiers; chromatin associated proteins and their modifiers (e.g.
  • kinases e.g., kinases, acetylases and deacetylases
  • DNA modifying enzymes e.g., methyltransferases such as members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B, DNMT3L, etc., topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. See, e.g., U.S. Publication No. 2013/0253040, incorporated by reference in its entirety herein.
  • Suitable domains for achieving activation include the HSV VP 16 activation domain (see, e.g., Hagmann et al, J. Virol. 71, 5952-5962 (1 97)) nuclear hormone receptors (see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factor kappa B (Bitko & Bank, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther.
  • HSV VP 16 activation domain see, e.g., Hagmann et al, J. Virol. 71, 5952-5962 (1 97)
  • nuclear hormone receptors see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)
  • chimeric functional domains such as VP64 (Beerli et al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron (Molinari et al., (1999) EMBO J. 18, 6439-6447).
  • Additional exemplary activation domains include, Oct 1, Oct-2A, Spl, AP-2, and CTF1 (Seipel et al, EMBOJ. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, for example, Robyr et al, (2000) Mol. Endocrinol.
  • Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, Cl, API, ARF-5, -6,-1, and -8, CPRF1, CPRF4, MYC-RP/GP, and TRAB1 , See, for example, Ogawa et al, (2000) Gene 245:21-29; Okanami et al, (1996) Genes Cells 1 :87-99; Goff et al, (1991) Genes Dev. 5:298-309; Cho et al, (1999) Plant Mol Biol 40:419-429; Ulmason et al, (1999) Proc. Natl. Acad. Sci.
  • Exemplary repression domains that can be used to make genetic repressors include, but are not limited to, KRAB A/B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B, DNMT3L, etc.), Rb, and MeCP2.
  • TIEG TGF-beta-inducible early gene
  • MBD2 MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B, DNMT3L, etc.), Rb, and MeCP2.
  • DNMT1, DNMT3A, DNMT3B, DNMT3L, etc. members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B, DNMT3L, etc.), Rb, and Me
  • Additional exemplary repression domains include, but are not limited to, R0M2 and AtHD2A. See, for example, Chem et al, (1996) Plant Cell 8:305-321; and Wu et al, (2000) Plant J. 22:19-27.
  • the domain is involved in epigenetic regulation of a chromosome.
  • the domain is a histone acetyltransferase (HAT), e.g., type- A, nuclear localized such as MYST family members MOZ, Ybf2/Sas3, MOF, and Tip60, GNAT family members Gcn5 or pCAF, the p300 family members CBP, p300 or Rttl09 (Bemdsen and Denu (2008) Curr Opin Struct Biol 18(6) :682- 689).
  • HAT histone acetyltransferase
  • the domain is a histone deacetylase (HD AC) such as the class I (HD AC-1, 2, 3, and 8), class II (HDAC IIA (HDAC-4, 5, 7 and 9), HD AC IIB (HDAC 6 and 10)), class IV (HDAC-1 1), class III (also known as sirtuins (SIRTs); SIRT1-7) (see Mottamal et al., (2015) Molecules 20(3):3898- 3941).
  • HD AC histone deacetylase
  • Another domain that is used in some embodiments is a histone phosphorylase or kinase, where examples include MSK1, MSK2, ATR, ATM, DNA-PK, Bubl, VprBP, IKK-a, PKCpi, Dik/Zip, JAK2, PKC5, WSTF and CK2.
  • a methylation domain is used and may be chosen from groups such as Ezh2, PRMT1/6, PRMT5/7, PRMT 2/6, CARMI, set7/9, MLL, ALL-1, Suv 39h, G9a, SETDB1, Ezh2, Set2, Doti, PRMT 1/6, PRMT 5/7, PR-Set7 and Suv4-20h, Domains involved in sumoylation and biotinylation (Lys9, 13, 4, 18 and 12) may also be used in some embodiments (review see Kousarides (2007) Cell 128:693-705).
  • Fusion molecules are constructed by methods of cloning and biochemical conjugation that are well known to those of skill in the art. Fusion molecules comprise a DNA-binding domain and a functional domain (e.g., a transcriptional activation or repression domain). Fusion molecules also optionally comprise nuclear localization signals (such as, for example, that from the SV40 medium T- antigen) and epitope tags (such as, for example, FLAG and hemagglutinin). Fusion proteins (and nucleic acids encoding them) are designed such that the translational reading frame is preserved among the components of the fusion.
  • nuclear localization signals such as, for example, that from the SV40 medium T- antigen
  • epitope tags such as, for example, FLAG and hemagglutinin
  • Fusions between a polypeptide component of a functional domain (or a functional fragment thereof) on the one hand, and a non-protein DNA-binding domain (e.g., antibiotic, intercalator, minor groove binder, nucleic acid) on the other, are constructed by methods of biochemical conjugation known to those of skill in the art. See, for example, the Pierce Chemical Company (Rockford, IL) Catalogue. Methods and compositions for making fusions between a minor groove binder and a polypeptide have been described. Mapp et al, (2000) Proc. Natl. Acad. Sci. USA 97:3930-3935. Likewise, CRISPR/Cas TFs and nucleases comprising a sgRNA nucleic acid component in association with a polypeptide component function domain are also known to those of skill in the art and detailed herein.
  • a non-protein DNA-binding domain e.g., antibiotic, intercalator, minor groove binder, nucleic acid
  • increased expression (i.e., overexpression) of the polynucleotide is mediated by introducing into the cell an exogenous polynucleotide encoding the polynucleotide to be overexpressed.
  • the exogenous polynucleotide is a recombinant nucleic acid.
  • Well-known recombinant techniques can be used to generate recombinant nucleic acids as outlined herein.
  • an exogenous polynucleotide encoding an exogenous polypeptide herein comprises a codon-optimized nucleic acid sequence.
  • the recombinant nucleic acids encoding an exogenous polypeptide may be operably linked to one or more regulatory nucleotide sequences in an expression construct.
  • Regulatory nucleotide sequences will generally be appropriate for the host cell and recipient subject to be treated. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.
  • the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.
  • the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome.
  • the expression vector includes a selectable marker gene to allow the selection of transformed host cells.
  • Certain embodiments include an expression vector comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequence for use herein include promoters, enhancers, and other expression control elements.
  • an expression vector is designed for the choice of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the vector's copy number, the ability to control that copy number, and/or the expression of any other protein encoded by the vector, such as antibiotic markers.
  • the exogenous polynucleotide is operably linked to a promoter for expression of the exogenous polynucleotide in the modified cell.
  • suitable mammalian promoters include, for example, promoters from the following genes: elongation factor 1 alpha (EFla) promoter, ubiquitin/S27a promoter of the hamster (WO 97/15664), Simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, the long terminal repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus Long Terminal repeat region, and the early promoter of human Cytomegalovirus (CMV).
  • EFla elongation factor 1 alpha
  • SV40 Simian vacuolating virus 40
  • SV40 Simian vacuolating virus 40
  • SV40 Simian vacuo
  • heterologous mammalian promoters examples include the actin, immunoglobulin or heat shock promoter(s).
  • promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40).
  • viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40).
  • heterologous mammalian promoters are used. Examples include the actin promoter, an immunoglobulin promoter, and heat-shock promoters.
  • the early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al, Nature 273: 113-120 (1978)).
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll restriction enzyme fragment (Greenaway et al, Gene 18: 355-360 (1982)).
  • the foregoing references are incorporated by reference in their entirety.
  • the expression vector is a bicistronic or multicistronic expression vector.
  • Bicistronic or multicistronic expression vectors may include (1) multiple promoters fused to each of the open reading frames; (2) insertion of splicing signals between genes; (3) fusion of genes whose expressions are driven by a single promoter; and/or (4) insertion of proteolytic cleavage sites between genes (self-cleavage peptide) or insertion of internal ribosomal entry sites (IRESs) between genes.
  • an expression vector or construct herein is a multicistonic construct.
  • multicistronic construct and “multicistronic vector” are used interchangeably herein and refer to a recombinant DNA construct that is to be transcribed into a single mRNA molecule, wherein the single mRNA molecule encodes two or more genes (e.g., two or more transgenes).
  • the multi-cistronic construct is referred to as bicistronic construct if it encodes two genes, and tricistronic construct if it encodes three genes, and quadrocistronic construct if it encodes four genes, and so on.
  • two or more exogenous polynucleotides comprised by a vector or construct are each separated by a multicistronic separation element.
  • the multicistronic separation element is an IRES or a sequence encoding a cleavable peptide or ribosomal skip element.
  • the multicistronic separation element is an IRES, such as an encephalomyocarditis (EMCV) virus IRES.
  • the multicistronic separation element is a cleavable peptide such as a 2A peptide.
  • Exemplary 2A peptides include a P2A peptide, a T2A peptide, an E2A peptide, and an F2Apeptide.
  • the cleavable peptide is a T2A.
  • the two or more exogenous polynucleotides e.g. the first exogenous polynucleotide and second exogenous polynucleotide
  • the first exogenous polynucleotide and the second exogenous polynucleotide are each operably linked to a promoter.
  • the promoter is the same promoter.
  • the promoter is an EFl promoter.
  • an exogenous polynucleotide encoding an exogenous polypeptide encodes a cleavable peptide or ribosomal skip element, such as T2A at the N-terminus or C-terminus of an exogenous polypeptide encoded by a multicistronic vector.
  • a cleavable peptide or ribosomal skip element such as T2A at the N-terminus or C-terminus of an exogenous polypeptide encoded by a multicistronic vector.
  • inclusion of the cleavable peptide or ribosomal skip element allows for expression of two or more polypeptides from a single translation initiation site.
  • the cleavable peptide is a T2A.
  • the T2A is or comprises the amino acid sequence set forth by SEQ ID NO: 11. In some embodiments, the T2A is or comprises the amino acid sequence set forth by SEQ ID NO: 12. In some embodiments, the T2A is or comprises the amino acid sequence set forth by SEQ ID NO: 17. In some embodiments, the T2A is or comprises the amino acid sequence set forth by SEQ ID NO: 18.
  • the vector or construct includes a single promoter that drives the expression of one or more transcription units of an exogenous polynucleotide.
  • such vectors or constructs can be multicistronic (bicistronic or tricistonic, see e.g., U.S. Patent No. 6,060,273).
  • transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products (e.g., one or more tolerogenic factors such as CD47 and/or one or more complement inhibitor such as CD46, CD59, and CD55) from an RNA transcribed from a single promoter.
  • IRES internal ribosome entry site
  • the vectors or constructs provided herein are bicistronic, allowing the vector or construct to express two separate polypeptides.
  • the two separate polypeptides encoded by the vector or construct are tolerogenic factors (e.g., two factors selected from CD47, DUX4, CD24, CD27, CD200, HLA-C, HLA- E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, IL-39, FasL, CCL21, CCL22, Mfge8, and Serpinb9).
  • the one or more tolerogenic factors are selected from the group consisting of CD16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.
  • the two separate polypeptides encoded by the vector or construct are CD46 and CD59. In some embodiments, the two separate polypeptides encoded by the vector or construct are a tolerogenic factor (e.g., CD47) and a complement inhibitor selected from CD46, CD59, and CD55.
  • the vectors or constructs provided herein are tricistronic, allowing the vector or construct to express three separate polypeptides. In some cases, the three nucleic acid sequences of the tricistronic vector or construct are a tolerogenic factor such as CD47, CD46, and CD59. In some cases, the three nucleic acid sequences of the tricistronic vector or construct are CD46, CD59, and CD55.
  • the three nucleic acid sequences of the tricistronic vector or construct are three tolerogenic factors selected from CD47, DUX4, CD24, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, IL-39, FasL, CCL21, CCL22, Mfge8, and Serpinb9.
  • the vectors or constructs provided herein are quadrocistronic, allowing the vector or construct to express four separate polypeptides.
  • the four separate polypeptides of the quadrocistronic vector or construct are CD47, CD46, CD59, and CD55. In some cases, the four separate polypeptides of the quadrocistronic vector or construct are four tolerogenic factors selected from CD47, DUX4, CD24, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, IL-39, FasL, CCL21, CCL22, Mfge8, and Serpinb9.
  • the cell comprises one or more vectors or constructs, wherein each vector or construct is a monocistronic or a multicistronic construct as described above, and the monocistronic or multicistronic constructs encode one or more tolerogenic factors and/or complement inhibitors, in any combination or order.
  • a single promoter directs expression of an RNA that contains, in a single open reading frame (ORF), two, three, or four genes (e.g., encoding a tolerogenic factor (e.g., CD47) and/or one or more complement inhibitors selected from CD46, CD59, and CD55) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2 A sequences) or a protease recognition site (e.g., furin).
  • ORF thus encodes a single polypeptide, which, either during (in the case of 2A) or after translation, is processed into the individual proteins.
  • the peptide such as T2A
  • T2A can cause the ribosome to skip (ribosome skipping) 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 (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)).
  • Many 2 A elements are known in the art.
  • Examples of 2 A sequences that can be used in the methods and nucleic acids disclosed herein include, without limitation, 2A sequences from the foot-and- mouth disease virus (F2A, e.g., SEQ ID NO: 16), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 15), thosea asigna virus (T2A, e.g., SEQ ID NO: 11, 12, 17, or 18), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 13 or 14) as described in U.S. Patent Publication No. 20070116690.
  • F2A foot-and- mouth disease virus
  • E2A equine rhinitis A virus
  • T2A e.g., SEQ ID NO: 11, 12, 17, or 18
  • P2A porcine teschovirus-1
  • the vector or construct (e.g., transgene) contains more than one nucleic acid sequence encoding a protein, e.g., a first exogenous polynucleotide encoding CD46 and a second exogenous polynucleotide encoding CD59, or a first exogenous polynucleotide encoding CD47, a second exogenous polynucleotide encoding CD56, and a third exogenous polynucleotide encoding CD59
  • the vector or construct (e.g., transgene) may further include a nucleic acid sequence encoding a peptide between the first and second exogenous polynucleotide sequences.
  • the nucleic acid sequence positioned between the first and second exogenous polynucleotides encodes a peptide that separates the translation products of the first and second exogenous polynucleotides during or after translation.
  • the peptide contains a self-cleaving peptide or a peptide that causes ribosome skipping (a ribosomal skip element), such as a T2A peptide.
  • inclusion of the cleavable peptide or ribosomal skip element allows for expression of two or more polypeptides from a single translation initiation site.
  • the peptide is a self-cleaving peptide that is a T2A peptide.
  • the T2A is or comprises the amino acid sequence set forth by SEQ ID NO: 11. In some embodiments, the T2A is or comprises the amino acid sequence set forth by SEQ ID NO: 12. In some embodiments, the T2A is or comprises the amino acid sequence set forth by SEQ ID NO: 17. In some embodiments, the T2A is or comprises the amino acid sequence set forth by SEQ ID NO: 18.
  • the process of introducing the polynucleotides described herein into cells can be achieved by any suitable technique.
  • Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, fusogens, and transduction or infection using a viral vector.
  • the polynucleotides are introduced into a cell via viral transduction (e.g., lentiviral transduction) or otherwise delivered on a viral vector (e.g., fusogen-mediated delivery).
  • Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, transposase-mediated delivery, and transduction or infection using a viral vector.
  • the polynucleotides are introduced into a cell via viral transduction (e.g., lentiviral transduction) or otherwise delivered on a viral vector (e.g., fusogen-mediated delivery).
  • viral transduction e.g., lentiviral transduction
  • viral vector e.g., fusogen-mediated delivery
  • vectors that package a polynucleotide encoding an exogenous polynucleotide may be used to deliver the packaged polynucleotides to a cell or population of cells.
  • These vectors may be of any kind, including DNA vectors, RNA vectors, plasmids, viral vectors and particles.
  • lipid nanoparticles can be used to deliver an exogenous polynucleotide to a cell.
  • viral vectors can be used to deliver an exogenous polynucleotide to a cell.
  • Viral vector technology is well known and described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • Viruses, which are useful as vectors include, but are not limited to lentiviral vectors, adenoviral vectors, adeno- associated viral (AAV) vectors, herpes simplex viral vectors, retroviral vectors, oncolytic viruses, and the like.
  • the introduction of the exogenous polynucleotide into the cell can be specific (targeted) or non-specific (e.g., non-targeted).
  • the introduction of the exogenous polynucleotide into the cell can result in integration or insertion into the genome in the cell.
  • the introduced exogenous polynucleotide may be non-integrating or episomal in the cell.
  • a skilled artisan is familiar with methods of introducing nucleic acid transgenes into a cell, including any of the exemplary methods described herein, and can choose a suitable method.
  • an exogenous polynucleotide is introduced into a cell (e.g., source cell) by any of a variety of non-targeted methods.
  • the exogenous polynucleotide is inserted into a random genomic locus of a host cell.
  • viral vectors including, for example, retroviral vectors and lentiviral vectors are commonly used to deliver genetic material into host cells and randomly insert the foreign or exogenous gene into the host cell genome to facilitate stable expression and replication of the gene.
  • the non-targeted introduction of the exogenous polynucleotide into the cell is under conditions for stable expression of the exogenous polynucleotide in the cell.
  • methods for introducing a nucleic acid for stable expression in a cell involves any method that results in stable integration of the nucleic acid into the genome of the cell, such that it may be propagated if the cell it has integrated into divides.
  • the viral vector is a lentiviral vector.
  • Lentiviral vectors are particularly useful means for successful viral transduction as they permit stable expression of the gene contained within the delivered nucleic acid transcript.
  • Lentiviral vectors express reverse transcriptase and integrase, two enzymes required for stable expression of the gene contained within the delivered nucleic acid transcript.
  • Reverse transcriptase converts an RNA transcript into DNA, while integrase inserts and integrates the DNA into the genome of the target cell. Once the DNA has been integrated stably into the genome, it divides along with the host.
  • the gene of interest contained within the integrated DNA may be expressed constitutively or it may be inducible. As part of the host cell genome, it may be subject to cellular regulation, including activation or repression, depending on a host of factors in the target cell.
  • Lentiviruses are subgroup of the Retroviridae family of viruses, named because reverse transcription of viral RNA genomes to DNA is required before integration into the host genome. As such, the most important features of lentiviral vehicles/particles are the integration of their genetic material into the genome of a target/host cell.
  • lentivirus examples include the Human Immunodeficiency Viruses: HIV-1 and HIV -2, the Simian Immunodeficiency Virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia, virus, visna-maedi and caprine arthritis encephalitis virus (CAEV).
  • SIV Simian Immunodeficiency Virus
  • FV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • JDV Jembrana Disease Virus
  • EIAV equine infectious anemia virus
  • CAEV visna-maedi and caprine arthritis encephalitis virus
  • lentiviral particles making up the gene delivery vehicle are replication defective on their own (also referred to as "self-inactivating"). Lentiviruses are able to infect both dividing and non-dividing cells by virtue of the entry mechanism through the intact host nuclear envelope (Naldini L et al., Curr. Opin. Biotechnol, 1998, 9: 457-463). Recombinant lentiviral vehicles/particles have been generated by multiply attenuating the HIV virulence genes, for example, the genes Env, Vif, Vpr, Vpu, Nef and Tat are deleted making the vector biologically safe.
  • lentiviral vehicles for example, derived from HIV- 1 /HIV-2 can mediate the efficient delivery, integration and long-term expression of transgenes into non- dividing cells.
  • Lentiviral particles may be generated by co-expressing the virus packaging elements and the vector genome itself in a producer cell such as human HEK293T cells. These elements are usually provided in three (in second generation lentiviral systems) or four separate plasmids (in third generation lentiviral systems).
  • the producer cells are co-transfected with plasmids that encode lentiviral components including the core (i.e., structural proteins) and enzymatic components of the virus, and the envelope protein(s) (referred to as the packaging systems), and a plasmid that encodes the genome including a foreign transgene, to be transferred to the target cell, the vehicle itself (also referred to as the transfer vector).
  • the plasmids or vectors are included in a producer cell line.
  • the plasmids/vectors are introduced via transfection, transduction or infection into the producer cell line. Methods for transfection, transduction or infection are well known by those of skill in the art.
  • the packaging and transfer constructs can be introduced into producer cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neomyocin (neo), dihydrofolate reductase (DHFR), glutamine synthetase or adenosine deaminase (ADA), followed by selection in the presence of the appropriate drug and isolation of clones.
  • a dominant selectable marker such as neomyocin (neo), dihydrofolate reductase (DHFR), glutamine synthetase or adenosine deaminase (ADA), followed by selection in the presence of the appropriate drug and isolation of
  • the producer cell produces recombinant viral particles that contain the foreign gene, for example, the polynucleotides encoding the exogenous polynucleotide.
  • the recombinant viral particles are recovered from the culture media and titrated by standard methods used by those of skill in the art.
  • the recombinant lentiviral vehicles can be used to infect target cells, such source cells including any described herein.
  • Cells that can be used to produce high-titer lentiviral particles may include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol Ther. 2005, 11: 452- 459), FreeStyleTM 293 Expression System (ThermoFisher, Waltham, MA), and other HEK293T- based producer cell lines (e.g., Stewart et al., Hum Gene Ther. _2011, 2,2.(3):357 ⁇ 369; Lee et al, Biotechnol Bioeng, 2012, 10996): 1551-1560; Throm et al.. Blood. 2009, 113(21): 5104-5110).
  • Additional elements provided in lentiviral particles may comprise retroviral LTR (long- terminal repeat) at either 5' or 3' terminus, a retroviral export element, optionally a lentiviral reverse response element (RRE), a promoter or active portion thereof, and a locus control region (LCR) or active portion thereof.
  • retroviral LTR long- terminal repeat
  • RRE lentiviral reverse response element
  • Other elements include central polypurine tract (cPPT) sequence to improve transduction efficiency in non-dividing cells, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) which enhances the expression of the transgene, and increases titer.
  • WPRE Posttranscriptional Regulatory Element
  • Lentivirus vectors used may be selected from, but are not limited to pLVX, pLenti, pLenti6, pLJMl, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJMl-EGFP, pULTRA, p!nducer2Q, pHIV-EGFP, pCW57.1 , pTRPE, pELPS, pRRL, and pLionll, Any known lentiviral vehicles may also be used (See, U.S. Pat. NOs.
  • the exogenous polynucleotide is introduced into the cell under conditions for transient expression of the cell, such as by methods that result in episomal delivery of an exogenous polynucleotide.
  • polynucleotides encoding the exogenous polynucleotide may be packaged into recombinant adeno-associated viral (rAAV) vectors.
  • rAAV adeno-associated viral
  • Such vectors or viral particles may be designed to utilize any of the known serotype capsids or combinations of serotype capsids.
  • the serotype capsids may include capsids from any identified AAV serotypes and variants thereof, for example, AAV1, AAV2, AAV2G9, AAV3, AAV4, AAV4-4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAVrhlO.
  • the AAV serotype may be or have a sequence as described in United States Publication No. US20030138772; Pulichla et al. Molecular Therapy, 2011, 19(6): 1070-1078; U.S. Pat. Nos. : 6,156,303; 7,198,951; U.S. Patent Publication Nos. : US2015/0159173 and US2014/0359799: and International Patent Publication NOs.: WO1998/011244, W02005/033321 and WO2014/14422.
  • AAV vectors include not only single stranded vectors but self-complementary AAV vectors (scAAVs).
  • scAAV vectors contain DNA which anneals together to form double stranded vector genome. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.
  • the rAAV vectors may be manufactured by standard methods in the art such as by triple transfection, in sf9 insect cells or in suspension cell cultures of human cells such as HEK293 cells.
  • non-viral based methods may be used.
  • vectors comprising the polynucleotides may be transferred to cells by non-viral methods by physical methods such as needles, electroporation, sonoporation, hyrdoporation; chemical carriers such as inorganic particles (e.g., calcium phosphate, silica, gold) and/or chemical methods.
  • synthetic or natural biodegradable agents may be used for delivery such as cationic lipids, lipid nano emulsions, nanoparticles, peptide-based vectors, or polymer-based vectors.
  • the exogenous polynucleotide can be inserted into a specific genomic locus of the host cell.
  • a number of gene editing methods can be used to insert an exogenous polynucleotide (e.g., a transgene) into a specific genomic locus of choice.
  • the exogenous polynucleotide can be inserted into any suitable target genomic loci of the cell.
  • the exogenous polynucleotide is introduced into the cell by targeted integration into a target loci.
  • targeted integration can be achieved by gene editing using one or more nucleases and/or nickases and a donor template in a process involving homology-dependent or homology-independent recombination.
  • Gene editing is a type of genetic engineering in which a nucleotide sequence may be inserted, deleted, modified, or replaced in the genome of a living organism.
  • a number of gene editing methods can be used to insert an exogenous polynucleotide into the specific genomic locus of choice, including for example homology-directed repair (HOR), homology-mediated end-joining (HMEJ), homology-independent targeted integration (HITI), obligate ligation-gated recombination (ObliGaRe), or precise integration into target chromosome (PITCh).
  • HOR homology-directed repair
  • HMEJ homology-mediated end-joining
  • HITI homology-independent targeted integration
  • OFTaRe obligate ligation-gated recombination
  • PITCh precise integration into target chromosome
  • the gene editing technology can include systems involving nucleases, integrases, transposases, and/or recombinases.
  • the gene editing technology mediates single-strand breaks (SSB). In some embodiments, the gene editing technology mediates double-strand breaks (DSB), including in connection with non- homologous end-joining (NHEJ) or homology-directed repair (HDR). In some embodiments, the gene editing technology can include DNA-based editing or prime-editing. In some embodiments, the gene editing technology can include Programmable Addition via Site-specific Targeting Elements (PASTE). In some embodiments, the gene editing technology can include TnpB polypeptides. Many gene editing techniques generally utilize the innate mechanism for cells to repair double-strand breaks (DSBs) in DNA.
  • DSBs single-strand breaks
  • NHEJ non-homologous end-joining
  • HDR homology-directed repair
  • indels insertion/deletion mutations
  • HDR is a preferred pathway for producing targeted knock-ins, knockouts, or insertions of specific mutations in the presence of a repair template with homologous sequences.
  • chemical modulation e.g., treating cells with inhibitors of key enzymes in the NHEJ pathway
  • timed delivery of the gene editing system at S and G2 phases of the cell cycle e.g., cell cycle arrest at S and G2 phases
  • introduction of repair templates with homology sequences e.g., chemical modulation (e.g., treating cells with inhibitors of key enzymes in the NHEJ pathway); timed delivery of the gene editing system at S and G2 phases of the cell cycle; cell cycle arrest at S and G2 phases; and introduction of repair templates with homology sequences.
  • the methods provided herein may utilize HDR-mediated repair, NHEJ-mediated repair, or a combination thereof.
  • the methods provided herein for HDR-mediated insertion utilize a site-directed nuclease, including, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, transposases, and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • meganucleases e.g., transposases
  • CRISPR clustered regularly interspaced short palindromic repeat
  • the nucleases create specific doublestrand breaks (DSBs) at desired locations (e.g., target sites) in the genome, and harness the cell's endogenous mechanisms to repair the induced break.
  • the nickases create specific single-strand breaks at desired locations in the genome.
  • two nickases can be used to create two single-strand breaks on opposite strands of a target DNA, thereby generating a blunt or a sticky end.
  • Any suitable nuclease can be introduced into a cell to induce genome editing of a target DNA sequence including, but not limited to, CRISPR-associated protein (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof.
  • Cas CRISPR-associated protein
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • meganucleases other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof.
  • DNA damage repair pathways can result in integration of the transgene sequence at the target site in the cell. This can occur by a homology-dependent process.
  • the donor template is a circular doublestranded plasmid DNA, single-stranded donor oligonucleotide (ssODN), linear double-stranded polymerase chain reaction (PCR) fragments, or the homologous sequences of the intact sister chromatid.
  • the homology-mediated gene insertion and replacement can be carried out via specific DNA repair pathways such as homology-directed repair (HDR), synthesisdependent strand annealing (SDSA), microhomology-mediated end joining (MMEJ), and homology- mediated end joining (HMEJ) pathways.
  • HDR homology-directed repair
  • SDSA synthesisdependent strand annealing
  • MMEJ microhomology-mediated end joining
  • HMEJ homology- mediated end joining
  • DNA repair mechanisms can be induced by a nuclease after (i) two SSBs, where there is a SSB on each strand, thereby inducing single strand overhangs; or (ii) a DSB occurring at the same cleavage site on both strands, thereby inducing a blunt end break.
  • the target locus with the SSBs or the DSB undergoes one of two major pathways for DNA damage repair: (1) the error-prone non-homologous end joining (NHEJ), or (2) the high-fidelity homology-directed repair (HDR) pathway.
  • NHEJ error-prone non-homologous end joining
  • HDR high-fidelity homology-directed repair
  • a donor template e.g., circular plasmid DNA or a linear DNA fragment, such as a ssODN
  • SSBs or a DSB a donor template introduced into cells in which there are SSBs or a DSB
  • HDR high-density DNA
  • the NHEJ process re-ligates the ends of the cleaved DNA strands, which frequently results in nucleotide deletions and insertions at the cleavage site.
  • HDR is a mechanism for cells to repair double-strand breaks (DSBs) in DNA and can be utilized to modify genomes in many organisms using various gene editing systems, including clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and transposases.
  • CRISPR clustered regularly interspaced short palindromic repeat
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • meganucleases and transposases.
  • the targeted integration is carried by introducing one or more sequence-specific or targeted nucleases, including DNA-binding targeted nucleases and gene editing nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases such as a CRISPR-associated nuclease (Cas) system, specifically designed to be targeted to at least one target site(s) sequence of a target gene.
  • ZFN zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • RNA-guided nucleases such as a CRISPR-associated nuclease (Cas) system, specifically designed to be targeted to at least one target site(s) sequence of a target gene.
  • ZFNs, TALEs, and TALENs are described in, e.g., Lloyd et al., Frontiers in Immunology, 4(221): 1-7 (2013).
  • targeted genetic disruption at or near the target site is carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated proteins
  • ZFNs are fusion proteins comprising an array of site-specific DNA binding domains adapted from zinc finger-containing transcription factors attached to the endonuclease domain of the bacterial FokI restriction enzyme.
  • a ZFN may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the DNA binding domains or zinc finger domains. See, e.g., Carroll et al., Genetics Society of America (2011) 188:773-782; Kim et al., Proc. Natl. Acad. Sci. USA (1996) 93:1156-1160.
  • Each zinc finger domain is a small protein structural motif stabilized by one or more zinc ions and usually recognizes a 3- to 4-bp DNA sequence. Tandem domains can thus potentially bind to an extended nucleotide sequence that is unique within a cell’s genome.
  • Zinc fingers can be engineered to bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger to bind to a predetermined nucleic acid sequence are known in the art. See, e.g., Sera et al., Biochemistry (2002) 41:7074-7081; Liu et al., Bioinformatics (2008) 24:1850-1857.
  • ZFNs containing FokI nuclease domains or other dimeric nuclease domains function as a dimer.
  • a pair of ZFNs are required to target non-palindromic DNA sites.
  • the two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. See Bitinaite et al., Proc. Natl. Acad. Sci. USA (1998) 95:10570-10575.
  • a pair of ZFNs are designed to recognize two sequences flanking the site, one on the forward strand and the other on the reverse strand.
  • the nuclease domains dimerize and cleave the DNA at the site, generating a DSB with 5' overhangs.
  • HDR can then be utilized to introduce a specific mutation, with the help of a repair template containing the desired mutation flanked by homology arms.
  • the repair template is usually an exogenous double-stranded DNA vector introduced to the cell. See Miller et al., Nat. Biotechnol. (2011) 29:143-148; Hockemeyer et al., Nat. Biotechnol. (2011) 29:731-734.
  • TALENs are another example of an artificial nuclease which can be used to edit a target gene.
  • TALENs are derived from DNA binding domains termed TALE repeats, which usually comprise tandem arrays with 10 to 30 repeats that bind and recognize extended DNA sequences. Each repeat is 33 to 35 amino acids in length, with two adjacent amino acids (termed the repeat-variable diresidue, or RVD) conferring specificity for one of the four DNA base pairs.
  • RVD repeat-variable diresidue
  • TALENs are produced artificially by fusing one or more TALE DNA binding domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a nuclease domain, for example, a FokI endonuclease domain.
  • TALE DNA binding domains e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
  • a nuclease domain for example, a FokI endonuclease domain.
  • the FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI nuclease domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al., Nature Biotech. (2011) 29:143-148.
  • a site-specific nuclease can be produced specific to any desired DNA sequence. Similar to ZFNs, TALENs can be introduced into a cell to generate DSBs at a desired target site in the genome, and so can be used to knock out genes or knock in mutations in similar, HDR-mediated pathways. See Boch, Nature Biotech. (2011) 29:135-136; Boch et al., Science (2009) 326:1509-1512; Moscou et al., Science (2009) 326:3501.
  • Meganucleases are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). Meganucleases are grouped into families based on their structural motifs which affect nuclease activity and/or DNA recognition. The most widespread and best known meganucleases are the proteins in the LAGLID ADG family, which owe their name to a conserved amino acid sequence. See Chevalier et al., Nucleic Acids Res. (2001) 29(18): 3757-3774.
  • the GIY-YIG family members have a GIY-YIG module, which is 70-100 residues long and includes four or five conserved sequence motifs with four invariant residues, two of which are required for activity. See Van Roey et al., Nature Struct. Biol. (2002) 9:806-811.
  • the His-Cys family meganucleases are characterized by a highly conserved series of histidines and cysteines over a region encompassing several hundred amino acid residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774.
  • NHN family are defined by motifs containing two pairs of conserved histidines surrounded by asparagine residues. See Chevalier et al., Nucleic Acids Res. (2001) 29(18):3757-3774.
  • Meganucleases can create DSBs in the genomic DNA, which can create a frame-shift mutation if improperly repaired, e.g., via NHEJ, leading to a decrease in the expression of a target gene in a cell.
  • foreign DNA can be introduced into the cell along with the meganuclease. Depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify the target gene. See Silva et al., Current Gene Therapy (2011) 11:11- 27.
  • Transposases are enzymes that bind to the end of a transposon and catalyze its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism.
  • transposases By linking transposases to other systems such as the CRISPR/Cas system, new gene editing tools can be developed to enable site specific insertions or manipulations of the genomic DNA.
  • transposons which use a catalytically inactive Cas effector protein and Tn7-like transposons.
  • the transposase-dependent DNA integration does not provoke DSBs in the genome, which may guarantee safer and more specific DNA integration.
  • the CRISPR system was originally discovered in prokaryotic organisms (e.g., bacteria and archaea) as a system involved in defense against invading phages and plasmids that provides a form of acquired immunity. Now it has been adapted and used as a popular gene editing tool in research and clinical applications.
  • prokaryotic organisms e.g., bacteria and archaea
  • CRISPR/Cas systems generally comprise at least two components: one or more guide RNAs (gRNAs) and a Cas protein.
  • the Cas protein is a nuclease that introduces a DSB into the target site.
  • CRISPR-Cas systems fall into two major classes: class 1 systems use a complex of multiple Cas proteins to degrade nucleic acids; class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV ; class 2 is divided into types II, V, and VI.
  • Cas proteins adapted for gene editing applications include, but are not limited to, Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxl l, Csyl, Csy2, Csy3, and Mad7.
  • Cas9 is a type II Cas protein and is described herein as illustrative. These Cas proteins may be originated from different source species. For example, Cas9 can be derived from .S', pyogenes or S. aureus.
  • the type II CRISPR system incorporates sequences from invading DNA between CRISPR repeat sequences encoded as arrays within the host genome. Transcripts from the CRISPR repeat arrays are processed into CRISPR RNAs (crRNAs) each harboring a variable sequence transcribed from the invading DNA, known as the “protospacer” sequence, as well as part of the CRISPR repeat. Each crRNA hybridizes with a second transactivating CRISPR RNA (tracrRNA), and these two RNAs form a complex with the Cas9 nuclease. The protospacer-encoded portion of the crRNA directs the Cas9 complex to cleave complementary target DNA sequences, provided that they are adjacent to short sequences known as “protospacer adjacent motifs” (PAMs).
  • PAMs protospacer adjacent motifs
  • Cpfl CRISPR from Prevotella and Franciscella 1; also known as Casl2a
  • Casl2a is an RNA-guided nuclease that only requires a crRNA and does not need a tracrRNA to function.
  • the CRISPR system Since its discovery, the CRISPR system has been adapted for inducing sequence specific DSBs and targeted genome editing in a wide range of cells and organisms spanning from bacteria to eukaryotic cells including human cells.
  • synthetic gRNAs have replaced the original crRNA:tracrRNA complexes, including in certain embodiments via a single gRNA.
  • the gRNAs can be single guide RNAs (sgRNAs) composed of a crRNA, a tetraloop, and a tracrRNA.
  • the crRNA usually comprises a complementary region (also called a spacer, usually about 20 nucleotides in length) that is user-designed to recognize a target DNA of interest.
  • the tracrRNA sequence comprises a scaffold region for Cas nuclease binding.
  • the crRNA sequence and the tracrRNA sequence are linked by the tetraloop and each have a short repeat sequence for hybridization with each other, thus generating a chimeric sgRNA.
  • One can change the genomic target of the Cas nuclease by simply changing the spacer or complementary region sequence present in the gRNA.
  • the complementary region will direct the Cas nuclease to the target DNA site through standard RNA-DNA complementary base pairing rules.
  • Cas nucleases may comprise one or more mutations to alter their activity, specificity, recognition, and/or other characteristics.
  • the Cas nuclease may have one or more mutations that alter its fidelity to mitigate off-target effects (e.g., eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9).
  • the Cas nuclease may have one or more mutations that alter its PAM specificity.
  • CRISPR systems of the present disclosure comprise TnpB polypeptides.
  • TnpB polypeptides may comprise a Ruv-C-like domain.
  • the RuvC domain may be a split RuvC domain comprising RuvC-I, RuvC-II, and RuvC-III subdomains.
  • a TnpB may further comprise one or more of a HTH domain, a bridge helix domain and a zinc finger domain.
  • TnpB polypeptides do not comprise an HNH domain.
  • a TnpB protein comprises, starting at the N-terminus: a HTH domain, a RuvC-I subdomain, a bridge helix domain, a RuvC-II sub-domain, a zinger finger domain, and a RuvC-III sub-domain.
  • a RuvC-III sub-domain forms the C-terminus of a TnpB polypeptide.
  • a TnpB polypeptide is from Epsilonproteobacteria bacterium, Actinoplanes lobatus strain DSM 43150, Actinomadura celluolosilytica strain DSM 45823, Actinomadura namibiensis strain DSM 44197, Alicyclobacillus macrosprangiidus strain DSM 17980, Lipingzhangella halophila strain DSM 102030, or Ktedonobacter recemifer.
  • a TnpB polypeptide is from Ktedonobacter racemifer, or comprises a conserved RNA region with similarity to the 5’ ITR of K. racemifer TnpB loci.
  • a TnpB may comprise a Fanzor protein, a TnpB homolog found in eukaryotic genomes.
  • a CRISPR system comprising a TnpB polypeptide binds a target adjacent motif (TAM) sequence 5’ of a target polynucleotide.
  • TAM is a transposon-associated motif.
  • a TAM sequence comprises TCA.
  • a TAM sequence comprises TTCAN.
  • a TAM sequence comprises TTGAT.
  • a TAM sequence comprises ATAAA.
  • the exogenous polynucleotide may function as a DNA repair template to be integrated into the target site through HDR in associated with a gene editing system (e.g., the CRISPR/Cas system) as described.
  • a gene editing system e.g., the CRISPR/Cas system
  • the exogenous polynucleotide to be inserted would comprise at least the expression cassette encoding the protein of interest (e.g., the tolerogenic factor) and would optionally also include one or more regulatory elements (e.g., promoters, insulators, enhancers).
  • the exogenous polynucleotide to be inserted would be flanked by homologous sequence immediately upstream and downstream of the target, i.e., left homology arm (LHA) and right homology arm (RHA), specifically designed for the target genomic locus to serve as template for HDR.
  • LHA left homology arm
  • RHA right homology arm
  • the length of each homology arm is generally dependent on the size of the insert being introduced, with larger insertions requiring longer homology arms.
  • target-primed reverse transcription (TPRT) or prime editing may be used to engineer exogenous genes, such as exogenous transgenes encoding a tolerogenic factor (e.g., CD47) into specific loci.
  • prime editing mediates targeted insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof in human cells without requiring DSBs or donor DNA templates.
  • Prime editing is a genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“PEgRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5' or 3' end, or at an internal portion of a guide RNA).
  • PE prime editing
  • PEgRNA prime editing guide RNA
  • the replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the endogenous strand of the target site to be edited (with the exception that it includes the desired edit).
  • the endogenous strand of the target site is replaced by the newly synthesized replacement strand containing the desired edit.
  • prime editing may be thought of as a “search-and- replace” genome editing technology since the prime editors search and locate the desired target site to be edited, and encode a replacement strand containing a desired edit which is installed in place of the corresponding target site endogenous DNA strand at the same time.
  • prime editing can be adapted for conducting precision CRISPR/Cas-based genome editing in order to bypass double stranded breaks.
  • a homologous protein is or encodes for a Cas protein-reverse transcriptase fusions or related systems to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated with the guide RNA.
  • a prime editor protein is paired with two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences.
  • pegRNAs prime editing guide RNAs
  • a gene editing technology is associated with a prime editor that is a reverse transcriptase, or any DNA polymerase known in the art.
  • a prime editor may comprise Cas9 (or an equivalent napDNAbp) which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., PEgRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA.
  • a specialized guide RNA i.e., PEgRNA
  • methods include any disclosed in Anzalone et al., (doi.org/10.1038/s41586-019-1711-4), or in PCT publication Nos. WO2020191248, WO2021226558, or W02022067130, which are hereby incorporated in their entirety.
  • the base editing technology may be used to introduce singlenucleotide variants (SNVs) into DNA or RNA in living cells.
  • SNVs singlenucleotide variants
  • Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in RNAs or DNAs without generating DSBs.
  • Base editors are typically fusions of a Cas (“CRISPR-associated”) domain and a nucleobase modification domain (e.g., a natural or evolved deaminase, such as a cytidine deaminase that include APOBEC1 (“apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1”), CDA (“cytidine deaminase”), and AID (“activation-induced cytidine deaminase”)) domains.
  • base editors may also include proteins or domains that alter cellular DNA repair processes to increase the efficiency and/or stability of the resulting single -nucleotide change.
  • CBEs cytidine base editors
  • ABEs adenine base editors
  • Base editors are composed by a catalytically dead Cas9 (dCas9) or a nickase Cas9 (nCas9) fused to a deaminase and guided by a sgRNA to the locus of interest.
  • the d/nCas9 recognizes a specific PAM sequence and the DNA unwinds thanks to the complementarity between the sgRNA and the DNA sequence usually located upstream of the PAM (also called protospacer). Then, the opposite DNA strand is accessible to the deaminase that converts the bases located in a specific DNA stretch of the protospacer.
  • base editing is a promising tool to precisely correct genetic mutations as it avoids gene disruption by NHEJ associated with failed HDR-mediated gene correction.
  • Rat deaminase APOBEC1 (rAPOBECl) fused to deactivated Cas9 (dCas9) has been used to successfully convert cytidines to thymidines upstream of the PAM of the sgRNA.
  • this first BE system was optimized by changing the dCas9 to a “nickase” Cas9 D10A, which nicks the strand opposite the deaminated cytidine. Without being bound by theory, this is expected to initiate long-patch base excision repair (BER), where the deaminated strand is preferentially used to template the repair to produce a U:A base pair, which is then converted to T:A during DNA replication.
  • BER base excision repair
  • a base editor is a nucleobase editor containing a first DNA binding protein domain that is catalytically inactive, a domain having base editing activity, and a second DNA binding protein domain having nickase activity, where the DNA binding protein domains are expressed on a single fusion protein or are expressed separately (e.g., on separate expression vectors).
  • a base editor is a fusion protein comprising a domain having base editing activity (e.g., cytidine deaminase or adenosine deaminase), and two nucleic acid programmable DNA binding protein domains (napDNAbp), a first comprising nickase activity and a second napDNAbp that is catalytically inactive, wherein at least the two napDNAbp are joined by a linker.
  • base editing activity e.g., cytidine deaminase or adenosine deaminase
  • napDNAbp nucleic acid programmable DNA binding protein domains
  • a base editor is a fusion protein that comprises a DNA domain of a CRISPR-Cas (e.g., Cas9) having nickase activity (nCas; nCas9), a catalytically inactive domain of a CRISPR-Cas protein (e.g., Cas9) having nucleic acid programmable DNA binding activity (dCas; e.g., dCas9), and a deaminase domain, wherein the dCas is joined to the nCas by a linker, and the dCas is immediately adjacent to the deaminase domain.
  • a CRISPR-Cas e.g., Cas9 having nickase activity
  • dCas e.g., Cas9 having nucleic acid programmable DNA binding activity
  • dCas deaminase domain
  • a base editor is an adenine-to-thymine or “ATBE” (or thymine-to-adenine or “TABE”) transversion base editor.
  • ATBE adenine-to-thymine
  • TABE thymine-to-adenine
  • Exemplary base editor and base editor systems include any as described in patent publication Nos. US20220127622, US20210079366, US20200248169, US20210093667, US20210071163, W02020181202, WO2021158921, WO2019126709, W02020181178, W02020181195, WO2020214842, W02020181193, which are hereby incorporated in their entirety.
  • a gene editing technology is Programmable Addition via Site-specific Targeting Elements (PASTE).
  • PASTE is platform in which genomic insertion is directed via a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase.
  • a serine integrase can be any known in the art.
  • a serine integrase has sufficient orthogonality such that PASTE can be used for multiplexed gene integration, simultaneously integrating at least two different genes at at least two genomic loci.
  • PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events.
  • any of the systems for gene disruption described herein can be used and, when also introduced with an appropriate donor template having with an exogenous polynucleotide, e.g., transgene sequences, can result in targeted integration of the exogenous polynucleotide at or near the target site of the genetic disruption.
  • the genetic disruption is mediated using a CRISPR/Cas system containing one or more guide RNAs (gRNA) and a Cas protein.
  • gRNA guide RNAs
  • Cas protein Exemplary Cas proteins and gRNA are described above, any of which can be used in HDR mediated integration of an exogenous polynucleotide into a target locus to which the Crispr/Cas system is specific for.
  • an appropriate Cas nuclease and gRNA such as depending on the particular target locus and target site for cleavage and integration of the exogenous polynucleotide by HDR. Further, depending on the target locus a skilled artisan can readily prepare an appropriate donor template, such as described further below.
  • the DNA editing system is an RNA-guided CRISPR/Cas system (such as RNA-based CRISPR/Cas system), wherein the CRISPR/Cas system is capable of creating a double-strand break in the target locus (e.g., safe harbor locus) to induce insertion of the transgene into the target locus.
  • the nuclease system is a CRISPR/Cas9 system.
  • the CRISPR/Cas9 system comprises a plasmid-based Cas9.
  • the CRISPR/Cas9 system comprises a RNA-based Cas9.
  • the CRISPR/Cas9 system comprises a Cas9 mRNA and gRNA.
  • the CRISPR/Cas9 system comprises a protein/RNA complex, or a plasmid/RNA complex, or a protein/plasmid complex.
  • methods for generating modified cells which comprises introducing into a source cell (e.g., a pluripotent stem cell, e.g., iPSC) a donor template containing a transgene or exogenous polynucleotide sequence and a DNA nuclease system including a DNA nuclease system (e.g., Cas9) and a locus-specific gRNA.
  • a source cell e.g., a pluripotent stem cell, e.g., iPSC
  • a donor template containing a transgene or exogenous polynucleotide sequence
  • a DNA nuclease system including a DNA nuclease system (e.g.,
  • the donor template to be inserted would comprise at least the transgene cassette containing the exogenous polynucleotide of interest (e.g., the tolerogenic factor or CAR) and would optionally also include the promoter.
  • the transgene cassette containing the exogenous polynucleotide and/or promoter to be inserted would be flanked in the donor template by homology arms with sequences homologous to sequences immediately upstream and downstream of the target cleavage site, i.e., left homology arm (LHA) and right homology arm (RHA).
  • LHA left homology arm
  • RHA right homology arm
  • the homology arms of the donor template are specifically designed for the target genomic locus to serve as template for HDR. The length of each homology arm is generally dependent on the size of the insert being introduced, with larger insertions requiring longer homology arms.
  • a donor template (e.g., a recombinant donor repair template) comprises: (i) a transgene cassette comprising an exogenous polynucleotide sequence (for example, a transgene operably linked to a promoter, for example, a heterologous promoter); and (ii) two homology arms that flank the transgene cassette and are homologous to portions of a target locus (e.g. safe harbor locus) at either side of a DNA nuclease (e.g., Cas nuclease, such as Cas9 or Casl2) cleavage site.
  • the donor template can further comprise a selectable marker, a detectable marker, and/or a purification marker.
  • the homology arms are the same length. In other embodiments, the homology arms are different lengths.
  • the homology arms can be at least about 10 base pairs (bp), e.g., at least about 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 45 bp, 55 bp, 65 bp, 75 bp, 85 bp, 95 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 950 bp, 1000 bp, 1.1 kilobases (kb), 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6
  • the homology arms can be about 10 bp to about 4 kb, e.g., about 10 bp to about 20 bp, about 10 bp to about 50 bp, about 10 bp to about 100 bp, about 10 bp to about 200 bp, about 10 bp to about 500 bp, about 10 bp to about I kb, about 10 bp to about 2 kb, about 10 bp to about 4 kb, about 100 bp to about 200 bp, about 100 bp to about 500 bp, about 100 bp to about 1 kb, about 100 bp to about 2 kb, about 100 bp to about 4 kb, about 500 bp to about I kb, about 500 bp to about 2 kb, about 500 bp to about 4 kb, about 1 kb to about 2 kb, about 1 kb to about 2 kb, about 1 kb to about 4 kb, or about
  • the donor template can be cloned into an expression vector.
  • Conventional viral and non- viral based expression vectors known to those of ordinary skill in the art can be used.
  • the target locus targeted for integration may be any locus in which it would be acceptable or desired to target integration of an exogenous polynucleotide or transgene.
  • a target locus include, but are not limited to, a CXCR4 gene, an albumin gene, a SHS231 locus, an F3 gene (also known as CD142), a MICA gene, a MICB gene, a LRP1 gene (also known as CD91), a HMGB1 gene, an ABO gene, a RHD gene, a FUT1 gene, a KDM5D gene (also known as HY), a B2M gene, a OITA gene, a CCR5 gene, a F3 (i.e., CD142) gene, a MICA gene, a MICB gene, a LRP1 gene, a HMGB1 gene, an ABO gene, a RHD gene, a FUT1 gene, a KDM5D (i.e., CD142) gene, a
  • the exogenous polynucleotide can be inserted in a suitable region of the target locus (e.g., safe harbor locus), including, for example, an intron, an exon, and/or gene coding region (also known as a Coding Sequence, or "CDS").
  • the insertion occurs in one allele of the target genomic locus.
  • the insertion occurs in both alleles of the target genomic locus.
  • the orientation of the transgene inserted into the target genomic locus can be either the same or the reverse of the direction of the gene in that locus.
  • the exogenous polynucleotide is interested into an intron, exon, or coding sequence region of the safe harbor gene locus. In some embodiments, the exogenous polynucleotide is inserted into an endogenous gene wherein the insertion causes silencing or reduced expression of the endogenous gene. Exemplary genomic loci for insertion of an exogenous polynucleotide are depicted in Table 2.
  • Table 2 Exemplary genomic loci for insertion of exogenous polynucleotides
  • the target locus is a safe harbor locus.
  • a safe harbor locus is a genomic location that allows for stable expression of integrated DNA with minimal impact on nearby or adjacent endogenous genes, regulatory element and the like.
  • a safe harbor gene enables sustainable gene expression and can be targeted by engineered nuclease for gene modification in various cell types including pluripotent stem cells, including derivatives thereof, and differentiated cells thereof.
  • Non-limiting examples of a safe harbor locus include, but are not limited to, a CCR5 gene locus, a PPP1R12C (also known as AAVS1) gene locus, a CLYBL gene locus, and/or a Rosa gene locus (e.g., ROSA26 gene locus), n some embodiments, the safe harbor locus is selected from the group consisting of the AAVS1 locus, the CCR5 locus, and the CLYBL locus.
  • SHS231 can be targeted as a safe harbor locus in many cell types.
  • certain loci can function as a safe harbor locus in certain cell types.
  • PDGFRa is a safe harbor for glial progenitor cells (GPCs)
  • OLIG2 is a safe harbor locus for oligodendrocytes
  • GFAP is a safe harbor locus for astrocytes. It is within the level of a skilled artisan to choose an appropriate safe harbor locus depending on the particular modified cell type. In some cases, more than one safe harbor gene can be targeted, thereby introducing more than one transgene into the genetically modified cell.
  • a source cell e.g. a pluripotent stem cell, e.g. iPSC
  • a donor template containing a transgene or exogenous polynucleotide sequence and a DNA nuclease system including a DNA nuclease system (e.g. Cas9) and a locus-specific gRNA that comprise complementary portions (e.g. gRNA targeting sequence) specific to a CCR5 gene locus, a PPP1R12C (also known as AAVS1) gene locus, a CLYBL gene locus, and/or a Rosa gene locus (e.g., ROSA26 gene locus).
  • a source cell e.g. a pluripotent stem cell, e.g. iPSC
  • a donor template containing a transgene or exogenous polynucleotide sequence and a DNA nuclease system including a DNA nuclease system (e.g. Cas9) and a locus
  • the genomic locus targeted by the gRNAs is located within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of any of the loci as described.
  • the gRNAs used herein for HDR-mediated insertion of a transgene comprise a complementary portion (e.g. gRNA targeting sequence) that recognizes a target sequence in AAVS1.
  • the target sequence is located in intron 1 of A A VS 1.
  • AAVS1 is located at Chromosome 19: 55,090,918-55,117,637 reverse strand
  • AAVS1 intron 1 (based on transcript ENSG00000125503) is located at Chromosome 19: 55,117,222-55,112,796 reverse strand.
  • the gRNAs target a genomic locus within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of Chromosome 19: 55, 117,222-55, 112,796.
  • the gRNAs target a genomic locus within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of Chromosome 19: 55,115,674.
  • the gRNA is configured to produce a cut site at Chromosome 19: 55, 115,674, or at a position within 5, 10, 15, 20, 30, 40 or 50 nucleotides of Chromosome 19: 55, 115,674.
  • the gRNA s GET000046 also known as "sgAAVSl-1," described in Li et al., Nat.
  • This gRNA comprises a complementary portion (e.g., gRNA targeting sequence) having the nucleic acid sequence set forth in SEQ ID NO: 36 (e.g., Table 3) and targets intron 1 of AAVS1 (also known as PPP1R12C).
  • gRNA targeting sequence e.g., gRNA targeting sequence having the nucleic acid sequence set forth in SEQ ID NO: 36 (e.g., Table 3) and targets intron 1 of AAVS1 (also known as PPP1R12C).
  • the gRNAs used herein for HDR-mediated insertion of a transgene comprise a complementary portion (e.g., gRNA targeting sequence) that recognizes a target sequence in CLYBL.
  • the target sequence is located in intron 2 of CL YBL.
  • CLYBL is located at Chromosome 13: 99,606,669-99,897, 134 forward strand
  • CLYBL intron 2 (based on transcript ENST00000376355.7) is located at Chromosome 13: 99,773,011-99,858,860 forward strand.
  • the gRNAs target a genomic locus within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of Chromosome 13: 99,773,011-99,858,860. In certain embodiments, the gRNAs target a genomic locus within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of Chromosome 13: 99,822,980.
  • the gRNA is configured to produce a cut site at Chromosome 13: 99,822,980, or at a position within 5, 0, 15, 20, 30, 40 or 50 nucleotides of Chromosome 13: 99,822,980.
  • the gRNA is GET000047, which comprises a complementary portion (e.g., gRNA targeting sequence) having the nucleic acid sequence set forth in SEQ ID NO: 36 (e.g., Table 3) and targets intron 2 of CLYBL.
  • the target site is similar to the target site of the TALENs as described in Cerbini et al., PLoS One, 10(1): eOl 16032 (2015).
  • the gRNAs used herein for HDR-mediated insertion of a transgene comprise a complementary portion (e.g., gRNA targeting sequence) that recognizes a target sequence in CCR5.
  • the target sequence is located in exon 3 of CCR5.
  • CCR5 is located at Chromosome 3: 46,370,854-46,376,206 forward strand
  • CCR5 exon 3 (based on transcript ENST00000292303.4) is located at Chromosome 3: 46,372,892-46,376,206 forward strand.
  • the gRNAs target a genomic locus within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of Chromosome 3: 46,372,892-46,376,206. In certain embodiments, the gRNAs target a genomic locus within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of Chromosome 3: 46,373,180.
  • the gRNA is configured to produce a cut site at Chromosome 3: 46,373,180, or at a position within 5, 10, 15, 20, 30, 40, or 50 nucleotides of Chromosome 3: 46,373,180.
  • the gRNA is GET000048, also known as "crCCR5_D," described in Mandal et al., Cell Stem Cell 15:643-652 (2014).
  • This gRNA comprises a complementary portion having the nucleic acid sequence set forth in SEQ ID NO: 37 (e.g., Table 3) and targets exon 3 of CCR5 (alternatively annotated as exon 2 in the Ensembl genome database). See Gomez-Ospina et al., Nat. Comm. 10( 1 ):4045 (2019).
  • Table 3 sets forth exemplary gRNA targeting sequences.
  • the gRNA targeting sequence may contain one or more thymines in the complementary portion sequences set forth in Table 3 are substituted with uracil.
  • the target locus is a locus that is desired to be knocked out in the cells.
  • a target locus is any target locus whose disruption or elimination is desired in the cell, such as to modulate a phenotype or function of the cell.
  • any of the gene modifications described herein to reduce expression of a target gene may be a desired target locus for targeted integration of an exogenous polynucleotide, in which the genetic disruption or knockout of a target gene and overexpression by targeted insertion of an exogenous polynucleotide may be achieved at the same target site or locus in the cell.
  • the HDR process may be used to result in a genetic disruption to eliminate or reduce expression of (e.g. knock out) any target gene set forth in Table la or Table lb while also integrating (e.g. knocking in) an exogenous polynucleotide into the target gene by using a donor template with flanking homology arms that are homologous to nucleic acid sequences at or near the target site of the genetic disruption.
  • methods for generating modified cells which comprises introducing into a source cell (e.g., a pluripotent stem cell, e.g. iPSC) a donor template containing a transgene or exogenous polynucleotide sequence and a DNA nuclease system including a DNA nuclease system (e.g. Cas9) and a locus-specific gRNA that comprise complementary portions specific to the B2M locus or the CIITA locus.
  • a source cell e.g., a pluripotent stem cell, e.g. iPSC
  • a donor template containing a transgene or exogenous polynucleotide sequence and a DNA nuclease system including a DNA nuclease system (e.g. Cas9) and a locus-specific gRNA that comprise complementary portions specific to the B2M locus or the CIITA locus.
  • the genomic locus targeted by the gRNAs is located within 4000 bp, within 3500 bp, within 3000 bp, within 2500 bp, within 2000 bp, within 1500 bp, within 1000 bp, or within 500 bp of any of the loci as described.
  • the target locus is B2M.
  • the modified cell comprises a genetic modification targeting the B2M gene.
  • the genetic modification targeting the B2M gene is by using a targeted nuclease system that comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene.
  • the at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the B2M gene is selected from the group consisting of SEQ ID NOS: 81240-85644 of Appendix 2 or Table 15 of W02016/183041, the disclosure is incorporated by reference in its entirety.
  • an exogenous polynucleotide is integrated into the disrupted B2M locus by HDR by introducing a donor template containing the exogenous polynucleotide sequence with flanking homology arms homologous to sequences adjacent to the target site targeted by the gRNA.
  • the target locus is OITA.
  • the modified cell comprises a genetic modification targeting the OITA gene.
  • the genetic modification targeting the OITA gene is by a targeted nuclease system that comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the OITA gene.
  • the at least one guide ribonucleic acid sequence for specifically targeting the OITA gene is selected from the group consisting of SEQ ID NOS:5184-36352 of Appendix 1 or Table 12 of W02016183041, the disclosure is incorporated by reference in its entirety.
  • an exogenous polynucleotide is integrated into the disrupted OITA locus by HDR by introducing a donor template containing the exogenous polynucleotide sequence with flanking homology arms homologous to sequences adjacent to the target site targeted by the gRNA.
  • gRNA sequences for use in HDR-mediated integration approaches as described.
  • an "inch worming" approach can be used to identify additional loci for targeted insertion of transgenes by scanning the flanking regions on either side of the locus for PAM sequences, which usually occurs about every 100 base pairs (bp) across the genome.
  • the PAM sequence will depend on the particular Cas nuclease used because different nucleases usually have different corresponding PAM sequences.
  • the flanking regions on either side of the locus can be between about 500 to 4000 bp long, for example, about 500 bp, about 1000 bp, about 1500 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 3500 bp, or about 4000 bp long.
  • a new guide can be designed according to the sequence of that locus for use in genetic disruption methods.
  • the CRISPR/Cas system is described as illustrative, any HDR-mediated approaches as described can be used in this method of identifying new loci, including those using ZFNs, TALENS, meganucleases and transposases.
  • the exogenous polynucleotide encodes an exogenous CD47 polypeptide (e.g., a human CD47 polypeptide) and the exogenous polypeptide is inserted into a safe harbor gene loci or a safe harbor site as disclosed herein or a genomic locus that causes silencing or reduced expression of the endogenous gene.
  • the exogenous polynucleotide encoding CD47 is inserted in a CCR5 gene locus, a PPP1R12C (also known as AAVS1) gene locus, a CLYBL gene locus, and/or a Rosa gene locus (e.g., ROSA26 gene locus).
  • the polynucleotide is inserted in a B2M, OITA, PD1 or CTLA4 gene locus.
  • the modified cell that includes the exogenous polynucleotide is a beta islet cell and includes a first exogenous polynucleotide that encodes a CD47 polypeptide.
  • the modified pluripotent stem cells e.g. modified iPSC
  • the modified pluripotent stem cells comprises one or more additional exogenous polynucleotides that encode one or more complement inhibitors or other tolerogenic polypeptides described herein.
  • the modified pluripotent stem cells (e.g. modified iPSC) comprises reduced expression of CD142 and reduced expression of MHC class I and/or reduced expression of MHC class II.
  • the first exogenous polynucleotide and the one or more additional exogenous polynucleotide are inserted into the same genomic locus. In some embodiments, the first exogenous polynucleotide and the one or more additional exogenous polynucleotide are inserted into different genomic loci.
  • the modified (e.g., hypoimmunogenic) cell is a beta islet cell derived from an modified (e.g., hypoimmunogenic) pluripotent cell (e.g., an iPSC).
  • the cell is a beta islet cell.
  • the cell is an iPSC- derived cell that has been differentiated from a modified iPSC.
  • the cell comprises reduced or eliminated expression of CD142.
  • the cell comprises overexpression or increased expression of one or more complement inhibitor.
  • the cell is an iPSC-derived beta-islet cell that is modified to contain modifications (e.g. genetic modifications) described herein.
  • the cell comprises reduced or eliminated expression of CD142.
  • the cell comprises overexpression or increased expression of one or more complement inhibitor.
  • the modified (e.g. hypoimmunogenic) beta-islet cell can be used to treat a variety of indications with allogenic cell therapy, including any as described herein.
  • the modified (e.g. hypoimmunogenic) betaislet cell can be used to treat diabetes, such as type I diabetes.
  • the cells that are modified as provided herein are cells from a healthy subject, such as a subject that is not known or suspected of having a particular disease or condition to be treated.
  • modified stem cell-derived beta (modified SC-beta) cells obtained by in vitro differentiation of a pluripotent stem cell.
  • modified stem cell-derived beta (modified SC- beta) cells obtained by in vitro differentiation of a modified pluripotent stem cell.
  • the modified pluripotent stem cell can be any as described above, e.g. Section I.
  • the provided modified SC-beta cells are differentiated in vitro from the modified pluripotent stem cell by any method able to generate a functional SC-beta cell.
  • the differentiated modified SC-beta cell is a modified iPSC-derived beta islet cell.
  • the differentiated modified SC-beta cell is an ESC-derived cell.
  • the provided modified SC-beta cells retain the one or more modifications of the modified pluripotent stem cells and/or retain or exhibit similar expression of the target immune molecules (e.g. reduced expression of MHC class I and/or II and increased expression of a tolerogenic factor, such as CD47).
  • the modified SC-beta cells provided herein also are functional and exhibit one or more functions of primary beta cells or beta islet cells, such as the ability to secrete insulin, for example glucose stimulated insulin secretion (GSIS).
  • GSIS glucose stimulated insulin secretion
  • modified stem cell-derived beta (modified SC-beta) cells obtained by in vitro differentiation of a pluripotent stem cell to generate an SC-beta cell, and introduction of the modifications into the SC-beta cell.
  • the modifications introduced in the modified SC-beta cell can be any of the modifications described in Section I (B) for modified PSCs.
  • the provided modified SC-beta cells are differentiated in vitro from the pluripotent stem cell by any method able to generate a functional SC-beta cell, and modified to generate the modified SC-beta cell.
  • the differentiated modified SC-beta cell is an iPSC-derived beta islet cell.
  • the differentiated modified SC-beta cell is an ESC-derived cell.
  • the modified SC-beta cells provided herein also are functional and exhibit one or more functions of primary beta cells or beta islet cells, such as the ability to secrete insulin, for example glucose stimulated insulin secretion (GSIS).
  • GSIS glucose stimulated insulin secretion
  • the modified stem-cell derived beta cell comprises one or more modifications that: (a) inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules, and/or (b) increase expression of one or more tolerogenic factors, wherein the increased expression is relative to a control or wild- type beta cell that does not comprise the modifications.
  • MHC major histocompatibility complex
  • the one or more modifications can be introduced into the SC-beta cell according to any of the methods for inactivating or disrupting genes and/or for overexpression of polynucleotides described in Sections I.B.l and I.B.2 above for modified PSCs.
  • populations of cells containing the modified beta cells are also provided. It is understood that differentiation from a population may not result in 100% having fully differentiated to the same stage in the differentiation pathway. Thus, it should be appreciated that not all cells in a particular population progress through these stages at the same rate, i.e., some cells may have progressed less, or more, down the differentiation pathway than the majority of cells present in the population. Accordingly, a population of beta-cells (e.g. having a b cell marker) may also include cells that are partially differentiated from the modified pluripotent stem cell or is a precursor of the cell stage such as precursor of the differentiated SC-beta cell. In some cases, a percentage or portion of the cells may be at an earlier stage. Exemplary features of provided populations are provided in Section III.
  • the modified SC-beta cells are differentiated in vitro (e.g., from pluripotent stem cells) and are cells that display at least one marker indicative of a pancreatic beta cell (e.g., PDX-1 or NKX6-1), express insulin, and display a GSIS response characteristic of an endogenous mature beta cell both in vitro and in vivo.
  • a pancreatic beta cell e.g., PDX-1 or NKX6-1
  • a marker indicative of a beta cell is a marker selected from INS, CHGA, NKX2-2, PDX1, NKX6-1, MAFB, GCK and GLUTE
  • the GSIS response of the modified SC-beta cell can be observed within two weeks of transplantation of the SC-beta cell into a host (e.g., a human or animal).
  • SC-beta cells need not be derived (e.g., directly) from stem cells, as any method can be used that is capable of deriving SC-beta cells from any endocrine progenitor cell that expresses insulin or precursor thereof using any cell as a starting point in which such starting cell has been modified by the one or more modifications described herein.
  • the starting cell may be a cell according to the present disclosure that is an embryonic stem cells, induced-pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells.
  • embryonic stem cells induced-pluripotent stem cells, progenitor cells
  • partially reprogrammed somatic cells e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived
  • multipotent cells e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived
  • multipotent cells
  • the starting cell does not comprise the one or more modifications that (a) inactivate or disrupt one or more alleles of: (i) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and/or (ii) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules, and/or (b) increase expression of one or more tolerogenic factors.
  • MHC major histocompatibility complex
  • the starting cell may be a modified cell according to the present disclosure that is an embryonic stem cells, induced-pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells.
  • embryonic stem cells induced-pluripotent stem cells, progenitor cells
  • partially reprogrammed somatic cells e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived
  • multipotent cells e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived
  • multipotent cells
  • the modified SC-beta cells have regulated or modulated (e.g. reduced or eliminated) expression of MHC class I molecules, MHC class II molecules, or MHC class I and MHC class II molecules.
  • the regulated or modulated expression of MHC class I and/or Class II is due to gene editing in which the DNA of the gene loci involved in regulation of expression of MHC class I and/or class II have been edited to delete genomic DNA of a gene involved in regulation of expression of the immune molecule.
  • the modified SC-beta cell has an edit to delete genomic DNA of beta-2 microglobulin (B2M) and is thus reduced or eliminated for expression of MHC class I.
  • B2M beta-2 microglobulin
  • the B2M gene is knocked out in the modified SC-beta cell. In some embodiments, both alleles of B2M are knocked out.
  • the modified SC-beta cell has an edit to delete genomic DNA of OITA and is thus reduced or eliminated for expression of MHC class II.
  • the OITA gene is knocked out in the modified SC-beta cell. In some embodiments, both alleles of OITA are knocked out.
  • the modified SC-beta cells have regulated or modulated (e.g. increase) expression of a tolerogenic factor, such as CD47.
  • the tolerogenic factor is one or more of DUX4, B2M-HLA-E, CD16, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc Receptor, IL15-RF, and H2-M3, or any combination thereof.
  • the one or more tolerogenic factors are selected from the group consisting of CD 16, CD24, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL22, CTLA4-Ig, Cl inhibitor, FASL, IDO1, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, IL-10, IL-35, PD-L1, SERPINB9, CCL21, MFGE8, DUX4, B2M-HLA-E, CD27, IL-39, CD16 Fc Receptor, IL15-RF, H2-M3 (HLA-G), A20/TNFAIP3, CR1, HLA-F, and MANF.
  • the increased or overexpressed tolerogenic factor is or includes increased expression of CCL21, PD-L1, FasL, Serpinb9, H2-M3 (HLA-G), CD47, CD200, and Mfge8.
  • the tolerogenic factor is CD47 and the modified SC-beta cell has increased expression of CD47.
  • the tolerogenic factor is PD-L1 and the modified SC-beta cell includes increased expression of PD-L1.
  • the tolerogenic factor is HLA-E and the modified SC-beta cell includes increased expression of HLA-E.
  • the tolerogenic factor is HLA-G and the modified beta-cell includes increased expression of HLA-G.
  • the tolerogenic factor is expressed as an exogenous polynucleotide or transgene in the genome of the modified SC-beta cell.
  • the exogenous polynucleotide or transgene is integrated or inserted into a genome locus of the cells, such as a safe harbor locus.
  • the genomic locus is an ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, or SHS231 locus.
  • modified SC-beta cell e.g. iPSC-derived beta islet cell having (1) reduced expression of MHC I and/or MHC II; and (2) a transgene comprising CD47 and a safety switch inserted at a safe harbor locus, wherein the safe harbor locus is selected from the group consisting of an AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, and SHS231 locus.
  • the safe harbor locus is selected from the group consisting of an AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, and SHS231 locus.
  • modified pluripotent stem cells having (1) reduced expression of MHC I and/or MHC II; and (2) a transgene comprising CD47 and HSVtk flanked by CLYBL homology arms, wherein the transgene is inserted at the CLYBL locus.
  • the modified pluripotent stem cell has B2M and/or OITA knockout.
  • the B2M and/or OITA knockout occur in both alleles.
  • ESC-derived stem cell having (1) reduced expression of MHC I and/or MHC II; and (2) a transgene comprising CD47 and a safety switch inserted at a safe harbor locus, wherein the safe harbor locus is selected from the group consisting of an AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, and SHS231 locus.
  • ESC-derived cells having (1) reduced expression of MHC I and/or MHC II; and (2) a transgene comprising CD47 and HSVtk flanked by CLYBL homology arms, wherein the transgene is inserted at the CLYBL locus.
  • the ESC-derived cell has B2M and/or OITA knockout.
  • the B2M and/or OITA knockout occur in both alleles.
  • a modified SC-beta cell provided herein comprises a safety switch.
  • the introduction of safety switches improves the safety of cell therapies developed using hypoimmunogenic cells (HIP cells, e.g., modified SC-beta cells).
  • HIP cells hypoimmunogenic cells
  • an immunosuppressive factor includes, but is not limited to, CD47, CD24, CD200, HLA-G, HLA- E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35, FASL, Serpmb9, CC121, and Mfge8.
  • the immunosuppressive factor is CD47.
  • the regulatable or inducible expression of an immunosuppressive factor functions to control an immune response by a recipient subject to an engrafted hypoimmunogenic cell.
  • Described herein are methods for the expression of an immunosuppressive factor that requires a mechanism to ‘turn-off expression of the immune regulatory protein in a controlled manner. Also described are modified SC-beta cells possessing controllable expression of one or more immunosuppressive factors. In some cases, the cells overexpress one or more immunosuppressive factors and can be induced to downregulate expression of the one or more immunosuppressive factors. As such, the cells are no longer hypoimmunogenic and are recognized by the recipient's immune cells for cell death.
  • the hypoimmunity of the modified SC-beta cells that are introduced to a recipient subject is achieved through the overexpression of an immunosuppressive molecule including hypoimmunity factors and complement inhibitors accompanied with the repression or genetic disruption of the HLA-I and HLA-II loci.
  • an immunosuppressive molecule including hypoimmunity factors and complement inhibitors accompanied with the repression or genetic disruption of the HLA-I and HLA-II loci.
  • These modifications cloak the cell from the recipient immune system's effector cells that are responsible for the clearance of infected, malignant or non-self cells, such as T cells, B cells, NK cells and macrophages. Cloaking of a cell from the immune system allows for existence and persistence of allogeneic cells within the body.
  • Controlled removal of the engineered cells from the body is crucial for patient safety and can be achieved by uncloaking the cells from the immune system. Uncloaking serves as a safety switch and can be achieved through the downregulation of the immunosuppressive molecules or the upregulation of immune signaling molecules.
  • the level of expression of any of the immunosuppressive molecules described can be controlled on the protein level, mRNA level, or DNA level in the cells.
  • the level of expression of any of the immune signaling molecules described can be controlled on the protein level, mRNA level, or DNA level in the cells.
  • any of the safety switch methods described are used to decrease the level of an immunosuppressive factor in the cells such that the lower level of the immunosuppressive factor is below a threshold level.
  • the level of the immunosuppressive factor in the cells is decreased by about 10-fold, 9- fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1- fold or 0.5-fold below a threshold level of expression.
  • the level of the immunosuppressive factor in the cells is decreased by about 10-fold to 5-fold, 10-fold to 3-fold, 9- fold to 1-fold, 8-fold to 1-fold, 7-fold to 0.5-fold, 6-fold, to 1-fold, 5-fold to 0.5-fold, 4-fold to 0.5-fold, 3-fold to 0.5-fold, 2-fold to 0.5-fold, or 1-fold to 0.5-fold below a threshold level of expression.
  • the threshold level of expression of the immunosuppressive factor is established based on the expression of such factor in an induced pluripotent stem cell.
  • the threshold level of the immunosuppressive factor expression is established based on the expression level of the immunosuppressive factor in a corresponding hypoimmune cell, such as any of the modified SC-beta cells described herein.
  • transcriptional regulation of immunosuppressive factors through employing inducible promoters provides the ability to turn expression of the switch on or off at will through the addition or removal of small molecules, such as, but not limited to, doxycycline. Genetic disruption via targeted nuclease activity can eliminate expression of the immunosuppressive factor to uncloak the cells as well.
  • Exemplary safety switches are described in WO2021146627A1, the content of which is herein incorporated by reference in its entirety.
  • any of the above modified SC-beta cells further have regulated or modulated (e.g. reduced or eliminated) expression of CD142.
  • the regulated or modulated expression of CD 142 is due to gene editing in which the DNA of the CD 142 gene loci has been edited to delete genomic DNA.
  • the modified SC-beta cell has an edit to delete genomic DNA of CD142 and is thus reduced or eliminated for expression of CD142.
  • the CD142 gene is knocked out in the modified SC-beta cell. In some embodiments, both alleles of B2M are knocked out.
  • any of the above modified SC-beta cells further have regulated or modulated (e.g. increased) expression of one or more complement inhibitor.
  • the one or more complement inhibitors is any one of CD46, CD59 and CD55 or is a combination thereof (e.g. CD46 and CD59 or CD46, CD59 and CD55).
  • the one or more complement inhibitor is expressed as an exogenous polynucleotide(s) or transgene(s) in the genome of the modified SC-beta cell.
  • the exogenous polynucleotide(s) or transgene(s) is integrated or inserted into a genome locus of the cells, such as a safe harbor locus.
  • the genomic locus is an ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, or SHS231 locus.
  • the exogenous polynucleotide or transgene is expressed at the same or a different locus from CD47 and/or from a suicide gene.
  • the methods used to differentiate the stem cell-derived P cells are known by one skilled in the art. Such methods are described, for example, in W02019018818, US8507274, US10030229, US10190096, US10253298, US10443042, W02016100925, WO2019217493, US7510876, US8216836, US8633024, US8647873, US10421942, US9404086, US20190359943, US10358628, US8633024, US8647873, US9222069, US10465162, US10370645, US9725699, US10253298, US9499795, US9650610, US9062290, US10494609, US20210060083, US8129182, US8603811, US9328331, US9012218, US9109245, US9982235, US9988604, US10358628, US10138465, US20190211309, US10443042,
  • stage 1 refers to the first step in the differentiation process, the differentiation of pluripotent stem cells into cells expressing markers characteristic of definitive endoderm cells.
  • stage 2 refers to the second step, the differentiation of cells expressing markers characteristic of definitive endoderm cells into cells expressing markers characteristic of gut tube cells.
  • Stage 3 refers to the third step, the differentiation of cells expressing markers characteristic of gut tube cells into cells expressing markers characteristic of early pancreas progenitor cells.
  • Stage 4 refers to the fourth step, the differentiation of cells expressing markers characteristic of early pancreas progenitor cells into cells expressing markers characteristic of pancreatic progenitor cell.
  • Stage 5 refers to the fifth step, the differentiation of cells expressing markers characteristic of pancreatic progenitor cells into cells expressing markers characteristic of pancreatic endoderm cells and/or pancreatic endocrine progenitor cells. It should be appreciated, however, that not all cells in a particular population progress through these stages at the same rate, i.e., some cells may have progressed less, or more, down the differentiation pathway than the majority of cells present in the population.
  • any step reference to a particular stage may include contacting of the particular cell of a given stage with a compound where cells in the contacted population may include a cell that is partially differentiated from the modified pluripotent stem cell or is a precursor of the cell stage.
  • a definitive endoderm cell is a cell that bears the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives.
  • Definitive endoderm cells express at least one of the following markers: FOXA2 (also known as hepatocyte nuclear factor 3P (“HNF3P”)), GATA4, SOX17, CXCR4, Brachyury, Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1. Markers characteristic of the definitive endoderm cells include CXCR4, FOXA2 and SOX17.
  • definitive endoderm cells may be characterized by their expression of CXCR4, FOXA2 and SOX17.
  • an increase in HNF4a may be observed.
  • gut tube cells are cells derived from definitive endoderm that can give rise to all endodermal organs, such as lungs, liver, pancreas, stomach, and intestine.
  • Gut tube cells may be characterized by their substantially increased expression of HNF4a over that expressed by definitive endoderm cells. For example, a ten to forty fold increase in mRNA expression of HNF4a may be observed during Stage 2.
  • early pancreas progenitor cells refer to endoderm cells that give rise to the esophagus, lungs, stomach, liver, pancreas, gall bladder, and a portion of the duodenum.
  • Early pancreatic progenitor cells express at least one of the following markers: PDX1, FOXA2, CDX2, SOX2, and HNF4a.
  • Early pancreatic progenitor cells may be characterized by an increase in expression of PDX1, compared to gut tube cells. For example, greater than fifty percent of the cells in Stage 3 cultures typically express PDX1.
  • pancreatic progenitor cells refer to cells that express at least one of the following markers: PDX1, NKX6.1, HNF6, NGN3, SOX9, PAX4, PAX6, ISL1, gastrin, FOXA2, PTFla, PROXI and HNF4a.
  • Pancreatic progenitor cells may be characterized as positive for the expression of PDX1, NKX6.1, and SOX9.
  • a pancreatic endoderm cell (also sometimes called a pancreatic endocrine progenitor cells) is a cell capable of becoming a pancreatic hormone expressing cell.
  • Pancreatic endoderm cells express at least one of the following markers: NGN3; NKX2.2; NeuroDl; ISL1; PAX4; PAX6; or ARX.
  • Pancreatic endoderm cells may be characterized by their expression of NKX2.2 and NeuroDl.
  • a method of generating insulin-producing beta cells comprising: providing a stem cell (e.g. modified stem cell, such as modified iPSC); providing serum-free media; contacting the stem cell with a TGF /Activin agonist or a glycogen synthase kinase 3 (GSK) inhibitor or WNT agonist for an amount of time sufficient to form a definitive endoderm cell; contacting the definitive endoderm cell with a FGFR2b agonist for an amount of time sufficient to form a primitive gut tube cell; contacting the primitive gut tube cell with an RAR agonist, and optionally a rho kinase inhibitor, a Smoothened antagonist, a FGFR2b agonist, a protein kinase C activator, or a BMP type 1 receptor inhibitor for an amount of time sufficient to form an early pancreas progenitor cell; incubating the early pancreas progenitor cell for at least about 3 days and optionally contacting the early
  • a method of generating insulin-producing beta cells comprising: providing a stem cell (e.g. modified stem cell, such as modified iPSC, or a stem cell that does not comprise one or more modifications); providing serum-free media; contacting the stem cell with a TGF /Activin agonist and/or a glycogen synthase kinase 3 (GSK) inhibitor and/or WNT agonist for an amount of time sufficient to form a definitive endoderm cell; contacting the definitive endoderm cell with a FGFR2b agonist for an amount of time sufficient to form a primitive gut tube cell; contacting the primitive gut tube cell with an RAR agonist, a rho kinase inhibitor, a Smoothened antagonist, a FGFR2b agonist, a protein kinase C activator, and/or a BMP type 1 receptor inhibitor for an amount of time sufficient to form an early pancreas progenitor cell; incubating the early pancreas progenitor cell; incubating
  • the serum-free media comprises one or more selected from the group consisting of: MCDB131, glucose, NaHCOs, BSA, ITS- X, Glutamax, vitamin C, penicillinstreptomycin, CMRL 10666, FBS, Heparin, NEAA, trace elements A, trace elements B, or ZnS04-
  • the TGF /Activin agonist is Activin A; the glycogen synthase kinase 3 (GSK) inhibitor or the WNT agonist is CHIR; the FGFR2b agonist is KGF; the Smoothened antagonist or hedgehog pathway inhibitor is SANT-1 ; the FGF family member/FGFR2b agonist is KGF; the RAR agonist is RA; the protein kinase 3 activator is TPPB; the BMP inhibitor is EDN; the rho kinase inhibitor is Y27632; the Alk5 inhibitor/TGF-b receptor inhibitor is Alk5i; the thyroid hormone is T3; or the gamma secretase inhibitor is XXL
  • the TGF /Activin agonist is Activin A.
  • the concentration of Activin A is between 50 ng/ml-150 ng/ml. In certain embodiments, the concentration of Activin A is 50 ng/ml, 55 ng/ml, 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml, 105 ng/ml, 110 ng/ml, 115 ng/ml, 120 ng/ml, 125 ng/ml, 130 ng/ml, 135 ng/ml, 140 ng/ml, 145 ng/ml, or 150 ng/ml.
  • the concentration of Activin A is between 50 ng/ml-60 ng/ml, 55 ng/ml-65 ng/ml, 60 ng/ml-70 ng/ml, 65 ng/ml-75 ng/ml, 70 ng/ml-80 ng/ml, 75 ng/ml-85 ng/ml, 80 ng/ml-90 ng/ml, 85 ng/ml-95 ng/ml, 90 ng/ml-100 ng/ml, 95 ng/ml-105 ng/ml, 100 ng/ml- 110 ng/ml, 105 ng/ml-115 ng/ml, 110 ng/ml- 120 ng/ml, 115 ng/ml- 125 ng/ml, 120 ng/ml- 130 ng/ml, 125 ng/ml-135 ng/ml, 130 ng/ml-140 ng/ml,
  • the glycogen synthase kinase 3 (GSK) inhibitor or the WNT agonist is CHIR.
  • the concentration of CHIR is between 0.5 pM and 5 pM.
  • the concentration of the CHIR is 0.5 pM, 1.0 pM, 1.5 pM, 2.0pM, 2.5 pM, 3.0 pM, 3.5 pM, 4.0 pM, 4.5 pM, or 5.0 pM.
  • the concentration of CHIR is between 0.5 pM- 1.5 pM, 1.0 pM-2.0 pM, 1.5 pM-2.5 pM, 2.0 pM- 3.0 pM, 2.5 pM-3.5 pM, 3.0 pM-4.0 pM, 3.5 pM-4.5 pM, or 4.0 pM-5.0 pM. In a specific embodiment, the concentration of CHIR is 3.0 pM.
  • the FGFR2b agonist is KGF.
  • the concentration of KGF is between 5 ng/ml-100 ng/ml. In certain embodiments, the concentration of KGF is 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml, 55 ng/ml, 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, or 100 ng/ml.
  • the concentration of KGF is between 5 ng/ml-15 ng/ml, 10 ng/ml-20 ng/ml, 15 ng/ml-25 ng/ml, 20 ng/ml-30 ng/ml, 25 ng/ml-35 ng/ml, 30 ng/ml-40 ng/ml, 35 ng/ml-45 ng/ml, 40 ng/ml-50 ng/ml, 45 ng/ml-55 ng/ml, 50 ng/ml-60 ng/ml, 55 ng/ml-65 ng/ml, 60 ng/ml-70 ng/ml, 65 ng/ml-75 ng/ml, 70 ng/ml-80 ng/ml, 75 ng/ml-85 ng/ml, 80 ng/ml-90 ng/ml, 85 ng/ml-95 ng/ml, 90 ng/ml-100 ng/m
  • the Smoothened antagonist is SANT-1.
  • the concentration of SANT-1 is between 0.05 pM and 0.50 pM. In certain embodiments, the concentration of the SANT-1 is 0.05 pM, 0.10 pM, 0.15 pM, 0.20 pM, 0.25 pM, 0.3 pM, 0.35 pM, 0.4 pM, 0.45 pM, or 0.5 pM.
  • the concentration of SANT-1 is between 0.05 pM-0.15 pM, 0.10 pM-0.20 pM, 0.15 pM-0.25 pM, 0.20 pM-0.30 pM, 0.25 pM-0.35 pM, 0.30 pM-0.40 pM, 0.35 pM- 0.45 pM, or 0.40 pM-0.50 pM. In a specific embodiment, the concentration of SANT-1 is 0.25 pM.
  • the RAR agonist is retinoic acid (RA).
  • the concentration of RA is between 0.05 pM and 2.5 pM. In certain embodiments, the concentration of RA is 0.05 pM, 0.1 pM, 0.15 pM, 0.2 pM, 0.5 pM, 1.0 pM, 1.5 pM, 2.0 pM, or 2.5 pM.
  • the concentration of RA is between 0.005 pM -0.15 pM, 0.10 pM -0.2 pM, 0.15 pM-0.5 pM, 0.2 pM-1.0 pM, 0.5 pM-1.5 pM, 1.0 pM-2.0 pM, or 1.5 pM-2.5 pM.
  • the concentration of RA is 0.10 pM.
  • the concentration of RA is 2.0 pM.
  • the protein kinase C activator is TPPB.
  • the concentration of TPPB is between 0.05 pM and 0.50 pM. In certain embodiments, the concentration of the TPPB is 0.05 pM, 0.10 pM, 0.15 pM, 0.20 pM, 0.25 pM, 0.3 pM, 0.35 pM, 0.4 pM, 0.45 pM, or 0.5 pM.
  • the concentration of TPPB is between 0.05 pM-0.15 pM, 0.10 pM- 0.20 pM, 0.15 pM-0.25 pM, 0.20 pM-0.30 pM, 0.25 pM-0.35 pM, 0.30 pM-0.40 pM, 0.35 pM- 0.45 pM, or 0.40 pM-0.50 pM. In a specific embodiment, the concentration of TPPB is 0.20 pM.
  • the BMP type 1 receptor inhibitor is LDN193189.
  • the concentration of LDN193189 is between 0.05 pM and 0.50 pM.
  • the concentration of the LDN193189 is 0.05 pM, 0.10 pM, 0.15 pM, 0.20 pM, 0.25 pM, 0.3 pM, 0.35 pM, 0.4 pM, 0.45 pM, or 0.5 pM.
  • the concentration of LDN193189 is between 0.05 pM-0.15 pM, 0.10 pM-0.20 pM, 0.15 pM-0.25 pM, 0.20 pM-0.30 pM, 0.25 pM-0.35 pM, 0.30 pM-0.40 pM, 0.35 pM-0.45 pM, or 0.40 pM-0.50 pM. In a specific embodiment, the concentration of LDN193189 is 0.20 pM.
  • the Alk5 inhibitor is Alk5i.
  • the concentration of Alk5i is between 5.0 pM and 15 pM. In certain embodiments, the concentration of Alk5i is 5.0 pM, 6.0 pM, 7.0 pM, 8.0 pM, 9.0 pM, 10.0 pM, 11.0 pM, 12.0 pM, 13.0 pM, 14.0 pM, or 15.0 pM.
  • the concentration of Alk5i is between 5.0 pM-7.0 pM, 6.0 pM -8.0 pM, 7.0 pM -9.0 pM, 8.0 pM -10.0 pM, 9.0 pM -11.0 pM, 10.0 pM -12.0 pM, 11.0 pM -13.0 pM, 12.0 pM -14.0 pM, or 13.0 pM -15.0 pM.
  • the concentration of Alk5i is 10.0 pM.
  • latrunculin A is utilized to chemically depolymerize the actin cytoskeleton.
  • the concentration of latrunculin A is 0.5 pM and 1.5 pM. In certain embodiments, the concentration of latrunculin A is 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 1.1 pM, 1.2 pM, 1.3 pM, 1.4 pM, or 1.5 pM.
  • the concentration of latrunculin A is between 0.5 pM -0.7 pM, 0.6 pM -0.8 pM, 0.7 pM -0.9 pM, 0.8 pM -1.0 pM, 0.9 pM - 1.1 pM, 1.0 pM -1.2 pM, 1.1 pM -1.3 pM, 1.2 pM -1.4 pM, or 1.3 pM -1.5 pM.
  • the concentration of latrunculin A is 1.0 pM.
  • the thyroid hormone is T3.
  • the concentration of T3 is between 0.1 pM and 2 pM.
  • the concentration of T3 is 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 1.1 pM, 1.2 pM, 1.3 pM, 1.4 pM, 1.5 pM, 1.6 pM, 1.7 pM, 1.8 pM, 1.9 pM, or 2.0 pM.
  • the concentration of T3 is between 0.1 pM -0.3 pM, 0.2 pM -0.4 pM, 0.3 pM -0.5 pM, 0.4 pM -0.6 pM, 0.5 pM -0.7 pM, 0.6 pM -0.8 pM, 0.7 pM -0.9 pM, 0.8 pM -1.0 pM, 0.9 pM -1.1 pM, 1.0 pM -1.2 pM, 1.1 pM -1.3 pM, 1.2 pM -1.4 pM, 1.3 pM -1.5 pM, 1.4 pM -1.6 pM, 1.5 pM -1.7 pM, 1.6 pM -1.8 pM, 1.7 pM -1.9 pM, or 1.8 pM -2.0 pM.
  • the concentration of T3 is 1.0 pM.
  • the gamma secretase inhibitor is XXI.
  • the concentration of XXI is between 0.1 pM and 2 pM. In certain embodiments, the concentration of XXI is 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 1.1 pM, 1.2 pM, 1.3 pM, 1.4 pM, 1.5 pM, 1.6 pM, 1.7 pM 1.8 pM, 1.9 pM, or 2.0 pM.
  • the concentration of XXI is between 0.1 pM -0.3 pM, 0.2 pM -0.4 pM, 0.3 pM -0.5 pM, 0.4 pM -0.6 pM, 0.5 pM -0.7 pM, 0.6 pM -0.8 pM, 0.7 pM -0.9 pM, 0.8 pM -1.0 pM, 0.9 pM -1.1 pM, 1.0 pM -1.2 pM, 1.1 pM -1.3 pM, 1.2 pM -1.4 pM, 1.3 pM -1.5 pM, 1.4 pM -1.6 pM, 1.5 pM -1.7 pM, 1.6 pM -1.8 pM, 1.7 pM -1.9 pM, or 1.8 pM -2.0 pM.
  • the concentration of XXI is 1.0 pM.
  • the methods herein detail a differentiation protocol for generating highly functional SC- cells.
  • the methods provided herein comprise six stages that attempt to recreate phases of pancreatic organogenesis by activating and repressing specific developmental pathways with growth factors and small molecules in serum-free medium.
  • hPSCs are seeded onto Matrigel-coated TCP plates at a density of 0.8 x 105 cells/cm2 and cultured in medium.
  • the methods provided herein comprise six stages of stem cell differentiation.
  • Stage 1 comprises incubating a HPSC of Stage 0 in media comprising Activin A and CHIR for about 24 hours followed by about 3 days of incubating the cells in media comprising Activin A in the absence of CHIR.
  • Stage 2 comprises incubating the Stage 1 cells for 2 days in media comprising KGF.
  • the CHIR is CHIR99021.
  • Stage 3 comprises incubating Stage 2 cells for 2 days in media comprising KGF, LDN193189, TPPB, RA (high), and SANT1.
  • Stage 4 comprises incubating Stage 3 cells for about 4 days in media comprising KGF, LDN193189, TPPB, RA (low), and SANT1.
  • Stage 5 comprises incubating the Stage 4 cells in media comprising XXI, Alk5i, T3, SANT1, and RA for 7 days. Additionally, latrunculin A is added to the media for about the first 24 hours of incubation.
  • Stage 6 comprises incubating the cells in an enriched serum-free medium which allows the SC-P cells the time needed to mature before they become glucose responsive.
  • the methods provided herein generate stem cell-derived beta (SC-P) cells that function better (undergoing glucose-stimulated insulin secretion) than cells in the published literature (Pagliuca et al. Cell 2014) and express beta cell markers.
  • the amount of time sufficient to form a definitive endoderm cell, a primitive gut tube cell, an early pancreas progenitor cell, a pancreatic progenitor cell, an endoderm cell, or a beta cell is between about 1 day and about 15 days.
  • stem cell-derived beta (SC-P) cells can be useful as a cellular therapy for diabetes.
  • the presently disclosed method enhances differentiation of human pluripotent stem cells to insulin-producing beta cells. This process is modified from a previously described 6-step differentiation protocol published by Pagliuca et al. Cell 2014. Using the methods disclosed herein, cells that can respond to glucose appropriately to near islet-like levels have been generated, demonstrating both a first phase and second phase response.
  • stage 6 shorten stage 3 to 1 day; (2) allow for TGFbeta signaling in stage 6 by removal of Alk5 inhibitor II (3) remove T3 from stage 6; (4) perform stage 6 in a serum-free basal media; and (5) break apart and reaggregate clusters at the beginning of stage 6.
  • the actin cytoskeleton is a crucial regulator of human pancreatic cell fate.
  • a polymerized cytoskeleton prevents premature induction of NEUROG3 expression in pancreatic progenitors, but also inhibits subsequent differentiation to SC-P cells.
  • Modulation of the actin cytoskeleton and its downstream effector Yes- Associated Protein (YAP) at specific time points during differentiation can enhance differentiation of human pluripotent stem cells to cells of endodermal lineage, pancreatic progenitors, and insulin-producing beta cells.
  • YAP Yes- Associated Protein
  • actin polymerization and YAP activity during Stage 4 enhances generation of pancreatic progenitors (PDX1 +/NKX6-1 +/SOX9+); (2) actin depolymerization and loss of YAP activity during Stage 5, preferentially during the first 24-48 hr of Stage 5, enhances generation of endocrine cells, specifically beta cells that demonstrate enhanced glucose-stimulated insulin secretion (WO2019/222487).
  • the following can be performed: (1) promoting actin polymerization by plating onto stiff surfaces, such as tissue culture plastic with a thin layer of ECM protein to promote attachment; (2) promoting actin depolymerization by plating onto soft surfaces, such as hydrogels, or by treating cells with latrunculin A and/or latrunculin B; (3) promoting YAP transcriptional activity using the same methods to promote actin polymerization; and/or (4) inhibiting YAP transcriptional activity using the same methods to promote actin depolymerization or by treatment with Verteporfin.
  • stem cell-derived beta cells were generated to better perform glucose-stimulated insulin secretion than previous methods and can be generated on attachment culture.
  • stem cell-derived beta cells can be generated but do not function as well as with the presently disclosed approach.
  • the field does not utilize actin cytoskeleton and YAP signaling in their protocols.
  • the field is also unable to generate functional stem cell-derived beta cells with the cells in attachment culture - it must either be done in suspension aggregates (the control for many experiments in the attached data set, first reported in Pagliuca et al. Cell 2014) or in aggregates on an air-liquid- interface (first reported in Rezania et al. Nature Biotechnology 2014).
  • Described herein is the generation of stem cell-derived beta cells that function better (undergoing glucose-stimulated insulin secretion) than cells in the published literature (Pagliuca et al. Cell 2014) and express beta cell markers. Also described herein are methods for the generation of stem cell-derived beta cells in a planar protocol that can undergo glucose-stimulated insulin secretion (GSIS).
  • GSIS glucose-stimulated insulin secretion
  • pancreatic progenitor cells that have reduced endocrine expression (such as expression of NGN3, NEURODI) and increased pancreatic progenitor expression (such as expression of NKX6-1, SOX9).
  • pancreatic progenitors and stem cell-derived beta cells can be useful as a cellular therapy for diabetes.
  • the presently disclosed culture approach can also facilitate enhanced quality and reproducibility of the differentiations and is conducive to automation of the differentiation process for commercialization.
  • differentiation protocols by cytoskeletal modulation can generate cells of several lineages (e.g., SC-b, beta-like cells). It was discovered that the state of the actin cytoskeleton is critical to endodermal cell fate choice. By utilizing a combination of cell-biomaterial interactions as well as small molecule regulators of the actin cytoskeleton (e.g., a cytoskeletal-modulating agent), the timing of endocrine transcription factor expression can be controlled to modulate differentiation fate and develop a two-dimensional protocol for differentiating cells. Importantly, this new planar protocol greatly enhances the function of SC-b cells differentiated from induced pluripotent stem cell (iPSC) lines and forgoes the requirement for three-dimensional cellular arrangements.
  • iPSC induced pluripotent stem cell
  • a cytoskeletal-modulating agent can be any agent that promotes or inhibits actin polymerization or microtubule polymerization.
  • the cytoskeletal-modulating agent can be an actin depolymerization or polymerization agent, a microtubule modulating agent, or an integrin modulating agent (e.g., compounds, such as antibodies and small molecules).
  • the cytoskeletal-modulating agent can be latrunculin A, latrunculin B, nocodazole, cytochalasin D, jasplakinolide, blebbistatin, y-27632, y-15, gdc-0994, or an integrin modulating agent.
  • the cytoskeletal- modulating agent can be any cytoskeletal-modulating agent known in the art (see e.g., Ley et al. Nat Rev Drug Discov. 2016 Mar; 15(3): 173-183).
  • Resizing of cell clusters can be performed by any methods known in the art.
  • cell resizing can comprise breaking apart cell clusters and reaggregating.
  • the cell clusters can be resized by incubating in a cell-dissociating reagent and passed through a cell strainer (e.g., a 100 pm nylon cell strainer).
  • cells can be resized by single cell dispersing with TrypLE and reaggregating.
  • a rotational shaker may be used to induce clustering and reaggregation of these cells.
  • a reaggregation step may facilitate endocrine purification without the need for a more expensive and timeconsuming sorting procedure.

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KR1020247026729A KR20240142624A (ko) 2022-01-10 2023-01-09 만능줄기세포에서 분화된 면역저하 베타세포 및 관련 용도 및 방법
CA3246902A CA3246902A1 (en) 2022-01-10 2023-01-09 DIFFERENTIATED HYPOIMMUNOUS BETA CELLS FROM PURIPOTENTIC STEM CELLS AND RELATED USES AND METHODS
CN202380025788.9A CN118871590A (zh) 2022-01-10 2023-01-09 从多能干细胞分化而来的低免疫β细胞以及相关用途和方法
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WO2024229302A1 (en) * 2023-05-03 2024-11-07 Sana Biotechnology, Inc. Methods of dosing and administration of engineered islet cells
WO2025029671A1 (en) * 2023-07-28 2025-02-06 Vertex Pharmaceuticals Incorporated Cell therapy for diabetes
WO2025117331A1 (en) 2023-12-01 2025-06-05 Eli Lilly And Company Methods of making stem cell-derived islet-like cells, as well as populations and compositions including the same
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HUE057135T2 (hu) * 2015-09-01 2022-04-28 Ncardia B V In vitro módszer egy humán pluripotens õssejtpopuláció kardiomiocita sejtpopulációvá történõ differenciálására
CN110177869A (zh) * 2017-01-13 2019-08-27 加利福尼亚大学董事会 免疫改造的多能细胞
US20210308183A1 (en) * 2018-07-17 2021-10-07 The Regents Of The University Of California Chimeric antigen receptor t cells derived from immunoengineered pluripotent stem cells
BR112021000637A2 (pt) * 2018-07-17 2021-04-13 The Regents Of The University Of California Célula cardíaca hipoimune isolada, método para tratar um paciente que sofre de uma afecção ou doença cardíaca, método para produzir uma população de células cardíacas hipoimunes, célula endotelial hipoimune isolada, método para tratar um paciente que sofre de uma afecção ou doença vascular, método para produzir uma população de células endoteliais hipoimunes, neurônio dopaminérgico hipoimune isolado, método para tratar um paciente que sofre de uma doença ou afecção neurodegenerativa, método para produzir uma população de neurônios dopaminérgicos hipoimunes, célula de ilhota pancreática hipoimune isolada, método para tratar um paciente que sofre de diabetes, método para produzir uma população de células de ilhota pancreática hipoimunes, célula de epitélio pigmentado da retina (rpe) hipoimune isolada, método para tratar um paciente que sofre de uma afecção ocular, e método para produzir uma população de células de epitélio pigmentado da retina (rpe) hipoimunes
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WO2024229302A1 (en) * 2023-05-03 2024-11-07 Sana Biotechnology, Inc. Methods of dosing and administration of engineered islet cells
WO2025029671A1 (en) * 2023-07-28 2025-02-06 Vertex Pharmaceuticals Incorporated Cell therapy for diabetes
CN116732099A (zh) * 2023-08-07 2023-09-12 北赛泓升(北京)生物科技有限公司 一种干细胞多重CRISPR/Cas基因组编辑方法
CN116732099B (zh) * 2023-08-07 2023-11-24 北赛泓升(北京)生物科技有限公司 一种干细胞多重CRISPR/Cas基因组编辑方法
WO2025117331A1 (en) 2023-12-01 2025-06-05 Eli Lilly And Company Methods of making stem cell-derived islet-like cells, as well as populations and compositions including the same
WO2025212859A1 (en) * 2024-04-03 2025-10-09 Solid Biosciences, Inc. Methods and constructs for increasing solid organ transplant survival

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