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Definitions

• Sensitization of a recipient to donor alloantigens is a problem facing clinical transplantation therapies, including cell therapies.
• the propensity for the transplant recipient's immune system to reject allogeneic material greatly reduces the potential efficacy of transplantation therapies and diminishes the possible positive effects surrounding such treatments.
• compositions and methods for producing allogenic cell-based therapies that avoid detection by the recipient’ s immune system.
• an engineered cell comprising modifications that (i) increase expression of one or more tolerogenic factor, (ii) reduce expression of CD 142, and (iii) reduce expression of one or more MHC class I molecules and/or one or more MHC class II molecules, wherein the increased expression of (i) and the reduced expression of (ii) and (iii) is relative to a cell of the same cell type that does not comprise the modifications.
• the modifications in (iii) reduce expression of one or more MHC class I molecules and one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules).
• the one or more 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 one or more tolerogenic factor is selected from the group consisting of CD47, PD-L1, HLA-E or HLA-G, CCL21, FASL, SERPINB9, CD200, MFGE8, and any combination thereof.
• At least one of the one or more tolerogenic factor is CD47. In some embodiments, at least one of the one or more tolerogenic factor is PD-L1. In some embodiments, at least one of the one or more tolerogenic factor is HLA-E. In some embodiments, at least one of the one or more tolerogenic factor is HLA-G.
• the one or more tolerogenic factors is selected from the group consisting of CD47; HLA-E; CD24; PD-L1; CD46; CD55; CD59; CR1; MANF;
• HLA-E and PDL1 HLA-E, PDL1, and A20/TNFAIP, and any combination thereof
• HLA-E, PDL1, and MANF HLA-E, PDL1, A20/TNFAIP, and MANF, and any combination thereof
• the modifications are selected from modifications that reduce expression of MHC I and/or MHC II; reduce expression of CD 142; increase expression of CD47, and optionally CD24 and PD-L1; and increase expression of CD46, CD55, CD59 and CR1.
• the modifications are selected from modifications that reduce expression of MHC class I molecule; reduce expression of CD142; reduce expression of TXNIP; increase expression of PD-L1 and HLA-E; and optionally A20/TNFAIP3 and MANF.
• the modifications are selected from modifications that increase the expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, and MFGE8; and reduce the expression of CD142.
• the modifications are selected from modifications that reduce expression of MHC I and/or MHC II; and increase expression of CD47.
• any of the above modifications are present in a provided engineered cells along with one or more additional edits that increase or decrease expression of a gene in the cell.
• any one or more of the further modifications can be a modification that that reduces expression, such as disrupts, inactivates or knockout expression, of B2M, TAP I, NLRC5, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, RFX5, RFXANK, RFXAP, NFY-A, NFY-B, NFY-C, CTLA- 4, PD-1, IRF1, CD142, MIC-A, MIC-B.
• any one or more of the further modifications can be a modification that reduces expression of a protein that is involved in oxidative or ER stress, TRAC, TRB, CD142, ABO, CD38, PCDH11Y, NLGN4Y and/or RHD.
• proteins that are involved in oxidative or ER stress include thioredoxin- interacting protein (TXNIP), PKR-like ER kinase (PERK), inositol-requiring enzyme la (IRE la), and DJ-1 (PARK7).
• the modification(s) that increase expression comprise increased surface expression, and/or the modifications that reduce expression comprise reduced surface expression.
• the modification that increases expression of the one or more complement inhibitor comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59 and/or an exogenous polynucleotide encoding CD55.
• 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 engineered cell comprises a multicistronic transgene comprising two or more exogenous polynucleotides selected from the group consisting of one or more exogenous polynucleotide encoding the one or more tolerogenic factor, 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.
• each polynucleotide of the multicistronic transgene is operably linked to the same promoter.
• the multicistronic transgene comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD59. In some embodiments, the multicistronic transgene comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59 and an exogenous polynucleotide encoding CD55. In some embodiments, the multicistronic transgene further comprises an exogenous polynucleotide encoding CD47. In some embodiments, the multicistronic transgene is a first transgene and the engineered cell comprises a separate transgene comprising a polynucleotide encoding CD47.
• the promoter is a constitutive promoter.
• the promoter is selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EFla promoter, the PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of moloney virus, Epstein barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter.
• CMV cytomegalovirus
• PGK adenovirus late promoter
• vaccinia virus 7.5K promoter vaccinia virus 7.5K promoter
• SV40 promoter vaccinia virus 7.5K promoter
• SV40 promoter vaccinia virus 7.5K promoter
• SV40 promoter vaccinia virus 7.5K promoter
• SV40 promoter
• the exogenous polynucleotide encoding CD47 is integrated into a first target genomic locus
• the exogenous polynucleotide encoding CD46 is integrated into a second target genomic locus
• the polynucleotide encoding CD59 is integrated into a third target genomic locus.
• the exogenous polynucleotide encoding CD55 is integrated into a fourth target genomic locus.
• at least two of the first, second, and third target genomic locus are the same locus.
• at least two of the first, second, third, and fourth target genomic locus are the same locus.
• the first, second and third target genomic locus are the same locus.
• the first, second, third, and fourth target genomic locus are the same locus. In some embodiments, each of the first, second, and third target genomic locus are different loci. In some embodiments, the first, second, third, and fourth target genomic locus are different loci.
• the modification that reduces expression of CD 142 reduces CD 142 protein expression.
• the modification eliminates CD 142 gene activity.
• the modification comprises inactivation or disruption of both alleles of the CD 142 gene.
• the modification comprises inactivation or disruption of all CD 142 coding sequences in the cell.
• the inactivation or disruption comprises an indel in the CD 142 gene.
• the modification is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the CD 142 gene.
• the CD 142 gene is knocked out.
• the modification is by nuclease-mediated genome editing.
• the nuclease-mediated genome editing is by a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination that targets the CD 142 gene, optionally wherein the Cas is selected from a Cas9 or a Casl2.
• ZFN zinc finger nuclease
• TALEN TAL-effector nuclease
• CRISPR-Cas combination that targets the CD 142 gene, optionally wherein the Cas is selected from a Cas9 or a Casl2.
• the nuclease-mediated genome editing is by a CRISPR-Cas combination and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site within the CD 142 gene, optionally wherein the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.
• gRNA guide RNA
• RNP ribonucleoprotein
• the modification that reduces expression of one or more MHC class I molecules reduces one or more MHC class I molecules protein expression. In some embodiments, the modification that reduces expression of one or more MHC class I molecules comprises reduced expression of B2M. In some embodiments, the modification that reduces expression of one or more MHC class I molecules comprises reduced protein expression of B2M. In some embodiments, the modification eliminates B2M gene activity. In some embodiments, the modification comprises inactivation or disruption of both alleles of the B2M gene. In some embodiments, the modification comprises inactivation or disruption of all B2M coding sequences in the cell. In some embodiments, the inactivation or disruption comprises an indel in the B2M gene.
• the modification is a frameshift mutation or a deletion of a contiguous stretch of genomic DNA of the B2M gene.
• the B2M gene is knocked out.
• the modification is by nuclease-mediated gene editing.
• the nuclease-mediated gene editing is by a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination that targets the B2M gene, optionally wherein the Cas is selected from a Cas9 or a Cas 12.
• the nuclease-mediated gene editing is by a CRISPR-Cas combination and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site within the B2M gene.
• the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.
• the engineered cell is a human cell or an animal cell. In some embodiments, the engineered cell is a human cell. In some embodiments, the engineered cell is a pig (porcine) cell, cow (bovine) cell, or sheep (ovine) cell. In some embodiments, the cell is a cell type that is exposed to the blood or that is able to differentiate into a cell type that is exposed to the blood.
• the engineered cell is a differentiated cell derived from a pluripotent stem cell or a progeny thereof.
• the pluripotent stem cell is an induced pluripotent stem cell.
• the engineered cell is a primary cell isolated from a donor subject.
• the donor subject is healthy or is not suspected of having a disease or condition at the time the donor sample is obtained from the individual donor.
• the engineered cell is selected from a beta islet cell, B cell, T cell, NK cell, retinal pigmented epithelium cell, hepatocyte, thyroid cell, skin cell, glial progenitor cell, neural cell, cardiac cell, and blood cell (e.g., plasma cell or platelet).
• the engineered cell is an endothelial cell.
• the engineered cell is an epithelial cell.
• the engineered cell is a T cell. In some embodiments, the engineered cell is an NK cell. In some embodiments, the engineered cell comprises a chimeric antigen receptor (CAR). In some embodiments, the engineered cell is a beta islet cell. In some embodiments, the engineered cell is a hepatocyte. In some embodiments, the engineered cell is a pluripotent stem cell. In some embodiments, the engineered cell is an induced pluripotent stem cell. In some embodiments, the engineered cell is an embryonic stem cell. In some embodiments, the cell is ABO blood group type O. In some embodiments, the cell is Rhesus factor negative (Rh-). In some embodiments, the cell comprises a functional ABO A allele and/or a functional ABO B allele. In some embodiments, the cell is Rhesus factor positive (Rh+).
• CAR chimeric antigen receptor
• the engineered cell is a beta islet cell.
• the engineered cell is a hepatocyte
• a method of generating an engineered cell comprising: a. reducing or eliminating the expression of one or more MHC class I molecules and/or one or more MHC class II molecules in the cell; b. reducing expression of CD 142 in the cell; and c. increasing the expression of a tolerogenic factor in the cell.
• the one or more 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 one or more tolerogenic factor is selected from the group consisting of CD47, PD-L1, HLA-E or HLA-G, CCL21, FASL, SERPINB9, CD200, MFGE8, and any combination thereof.
• at least one of the one or more tolerogenic factor is CD47.
• at least one of the one or more tolerogenic factor is PD- Ll.
• at least one of the one or more tolerogenic factor is HLA-E.
• at least one of the one or more tolerogenic factor is HLA-G.
• the method comprises reducing the expression of one or more MHC class I molecules and one or more MHC class II molecules.
• a method of generating a hypo-immunogenic 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 CD 142 in 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 reduced expression comprises reduced surface expression and/or the increased expression comprises increased surface expression.
• increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59 and/or an exogenous polynucleotide encoding CD55 to the cell.
• the one or more complement inhibitor is CD46 and CD59, optionally wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD59.
• the one or more complement inhibitor is CD46, CD59 and CD55, optionally wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59 and an exogenous polynucleotide encoding CD55.
• the exogenous polynucleotide encoding CD46 encodes a sequence of amino acid having at least 85% identity to the amino acid sequence of SEQ ID NO: 3 and exhibits complement inhibitory activity In some embodiments, the exogenous polynucleotide encoding CD46 encodes the sequence set forth in SEQ ID NO: 3. In some embodiments, the exogenous polynucleotide encoding CD59 encodes a sequence of amino acids having at least 85% identity to the amino acid sequence of SEQ ID NO: 5 and exhibits complement inhibitory activity In some embodiments, the exogenous polynucleotide encoding CD59 encodes the sequence set forth in SEQ ID NO: 5.
• the exogenous polynucleotide encoding CD55 encodes a sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 8 and exhibits complement inhibitory activity In some embodiments, the exogenous polynucleotide encoding CD55 encodes the sequence set forth in SEQ ID NO: 8. In some embodiments, the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59 and/or the exogenous polynucleotide encoding CD55 is each operably linked to a promoter.
• the modification that increases expression of CD47 comprises an exogenous polynucleotide encoding the CD47 protein.
• the exogenous polynucleotide encoding CD47 encodes a sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 1 and reduces innate immune killing of the engineered cell.
• the exogenous polynucleotide encoding CD47 encodes a sequence set forth in SEQ ID NO: 1.
• the exogenous polynucleotide encoding CD47 is operably linked to a promoter.
• the method comprises introducing to the cell a multicistronic transgene comprising two or more exogenous polypeptides selected from the group consisting of one or more exogenous polynucleotide encoding the one or more tolerogenic factor, 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.
• the two or more exogenous polynucleotides are selected from the group consisting of an exogenous polynucleotide encoding CD47, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD55 polypeptide.
• each polynucleotide of the multicistronic transgene is operably linked to the same promoter.
• the multicistronic transgene comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD59. In some embodiments, the multicistronic transgene comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59 and an exogenous polynucleotide encoding CD55. In some embodiments, the multicistronic transgene further comprises an exogenous polynucleotide encoding CD47. In some embodiments, the engineered cell comprises a separate transgene comprising a polynucleotide encoding CD47.
• the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 is integrated into the genome of the engineered cell.
• the exogenous polynucleotide encoding CD47 is integrated into the genome of the engineered cell.
• the integration is by non-targeted insertion into the genome of the engineered cell, optionally by introduction of the exogenous polynucleotide into the cell using a lentiviral vector.
• the integration is 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 target genomic locus is a safe harbor locus, a B2M gene locus, a CIITA gene locus, a CD 142 gene locus, a TRAC gene locus, or a TRBC gene locus.
• the target genomic locus is selected from the group consisting of: a CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C (also known as AAVST) gene, an albumin gene locus, a SHS231 locus, a CLYBL gene locus, and a ROSA26 gene locus.
• the target genomic locus is a safe harbor locus.
• the nuclease-mediated gene editing is by a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas combination that targets the target genomic locus, optionally wherein the Cas is selected from a Cas9 or a Cas 12.
• ZFN zinc finger nuclease
• TALEN TAL-effector nuclease
• CRISPR-Cas combination that targets the target genomic locus
• the Cas is selected from a Cas9 or a Cas 12.
• the nuclease-mediated gene editing is by a CRISPR-Cas combination and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain that is complementary to a target sequence of the target genomic locus and a homology-directed repair template comprising the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, the exogenous polynucleotide encoding CD55, and/or the exogenous polynucleotide encoding CD47.
• the CRISPR-Cas combination is a ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.
• RNP ribonucleoprotein
• reducing expression of CD 142 reduces CD 142 protein expression.
• reducing expression of CD 142 comprises introducing a modification that reduces CD 142 gene activity.
• the modification that reduces CD142 gene activity comprises inactivation or disruption of both alleles of the CD142 gene.
• the modification that reduces CD 142 gene activity comprises inactivation or disruption of all CD 142 coding sequences in the cell.
• the inactivation or disruption comprises an indel in the CD 142 gene or a deletion of a contiguous stretch of genomic DNA of the CD142 gene.
• the indel is a frameshift mutation.
• the CD142 gene is knocked out.
• the modification that reduces one or more MHC class I molecules expression comprises inactivation or disruption of both alleles of the B2M gene. In some embodiments, the modification that reduces one or more MHC class I molecules protein expression comprises inactivation or disruption of all B2M coding sequences in the cell. In some embodiments, the inactivation or disruption comprises an indel in the B2M gene or a deletion of a contiguous stretch of genomic DNA of the B2M gene. In some embodiments, the indel is a frameshift mutation. In some embodiments, the B2M gene is knocked out. In some embodiments, the modification that reduces one or more MHC class I molecules protein expression is by nuclease- mediated gene editing.
• reducing expression of one or more MHC class II molecules comprises introducing a modification that reduces one or more MHC class II molecules protein expression.
• the modification that reduces one or more MHC class II molecules protein expression comprises reduced expression of CIITA.
• the modification that reduces one or more MHC class II molecules protein expression comprises reduced protein expression of CIITA.
• the modification that reduces one or more MHC class II molecules protein expression reduces CIITA gene activity.
• the modification that reduces one or more MHC class II molecules protein expression comprises inactivation or disruption of both alleles of the CIITA gene.
• the modification comprises inactivation or disruption of all CIITA coding sequences in the cell.
• the inactivation or disruption comprises an indel in the CIITA gene or a deletion of a contiguous stretch of genomic DNA of the CIITA gene.
• the indel is a frameshift mutation.
• the CIITA gene is knocked out.
• the cell is a human cell or an animal cell.
• the animal cell is a pig (porcine) cell, cow (bovine) cell, or sheep (ovine) cell.
• the engineered cell is a human cell.
• the cell is a cell type that is exposed to the blood or that is able to differentiate into a cell type that is exposed to the blood.
• the cell is a primary cell isolated from a donor subject.
• the hypo-immunogenic cell is a differentiated cell derived from the pluripotent stem cell, and the method further comprises differentiating the pluripotent stem cell.
• the pluripotent stem cell is an induced pluripotent stem cell.
• the hypo-immunogenic cell is selected from a beta islet cell, B cell, T cell, NK cell, glial progenitor cell, neural cell, cardiac cell, retinal pigmented epithelium cell, hepatocyte, thyroid cell, skin cell, and blood cell (e.g., plasma cell or platelet).
• the engineered cell is a beta islet cell.
• the engineered cell is a hepatocyte.
• the engineered 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 engineered 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 engineered cell.
• the bicistronic cassette is integrated by non-targeted insertion into the genome of the engineered cell.
• the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the engineered cell.
• the one or more tolerogenic factors is CD47.
• an engineered cell produced according to a method described herein.
• the engineered cell, or progeny or differentiated cells derived from the engineered cell is capable of evading NK cell mediated cytotoxicity upon administration to a recipient patient.
• the engineered cell, or progeny or differentiated cells derived from the engineered cell is protected from cell lysis by mature NK cells upon administration to a recipient patient.
• the engineered cell, or progeny or differentiated cells derived from the engineered cell does not induce an immune response to the cell upon administration to a recipient patient.
• the engineered cell, or progeny or differentiated cells derived from the engineered cell does not induce a systemic inflammatory response to the cell upon administration to a recipient patient. In some embodiments, the engineered cell, or progeny or differentiated cells derived from the engineered cell, does not induce a local inflammatory response to the cell upon administration to a recipient patient.
• the engineered 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).
• CyD cytosine deaminase
• HSV-Tk herpesvirus thymidine kinase
• iCaspase9 inducible caspase 9
• rapamycin-activated caspase 9 rapamycin-activated caspase 9
• 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 engineered cell.
• the bicistronic cassette is integrated by non-targeted insertion into the genome of the engineered 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.
• a population of engineered cells comprising a plurality of the engineered cells described herein.
• the plurality of the engineered primary cells are derived from cells pooled from more than one donor subject.
• each of the more than one donor subjects are healthy subjects or are not suspected of having a disease or condition at the time the donor sample is obtained from the donor subject.
• At least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of cells in the population comprise the modifications.
• At least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of cells in the population comprise an exogenous polynucleotide encoding CD47.
• At least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of cells in the population comprise an exogenous polynucleotide encoding CD46.
• at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of cells in the population comprise an exogenous polynucleotide encoding CD59.
• At least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of cells in the population comprise one or more alterations that inactivate both alleles of a B2M gene.
• At least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of cells in the population comprise one or more alterations that inactivate both alleles of a CIITA gene.
• At least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of cells in the population comprise reduced expression of CD 142 relative to unaltered or unmodified wild type cells.
• At least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of cells in the population comprise one or more alterations that inactivate both alleles of a CD 142 gene.
• composition comprising the population described herein.
• composition comprising a population of engineered beta islet cells, wherein the engineered beta islet cells comprise: (i) a transgene comprising an exogenous polynucleotide encoding CD47, (ii) inactivation or disruption of both alleles of a CD 142 gene, and (iii) inactivation or disruption of both alleles of a B2M gene.
• the engineered beta cells comprise inactivation or disruption of both alleles of a CIITA gene.
• the engineered hepatocyte cells comprise inactivation or disruption of both alleles of a CIITA gene.
• the transgene is a multicistronic transgene, and wherein the transgene further comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD59.
• engineered cells of the population of engineered 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 engineered cells of the population of engineered cells.
• the method further comprises administering to the patient an anti-coagulant agent that reduces coagulation.
• a method of treating a disease, condition, or cellular deficiency in a patient in need thereof comprising: (a) administering to the patient an effective amount of: a population of cells comprising a plurality of engineered cells, wherein the engineered cells comprise modifications that (i) increase expression of one or more complement inhibitor(s) selected from the group consisting of CD46, CD59, and CD55; (ii) increase expression of one or more tolerogenic factor, and (iii) reduce expression of one or more MHC class I molecules and/or one or more MHC class II molecules, wherein the increased expression of (i) and (ii) and the reduced expression of (iii) is relative to a cell of the same cell type that does not comprise the modifications; and (b) administering to the patient an anti-coagulant agent that reduces coagulation.
• the population is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
• the anti-coagulant agent is heparin.
• the heparin is unfractionated heparin.
• the heparin is low molecular weight heparin.
• the heparin is soluble heparin.
• the condition or disease is selected from the group consisting of diabetes, cancer, vascularization disorders, ocular disease, thyroid disease, skin diseases, and liver diseases.
• the population of cells is a population of corneal endothelial progenitor cells or corneal endothelial cells.
• the cellular deficiency is associated with a kidney disease or the cellular therapy is for the treatment of a kidney disease.
• the population of cells is a population of renal precursor cells or renal cells.
• 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 engineered cell.
• the engineered cell is engineered to express the one or more tolerogenic factors.
• the one or more tolerogenic factors is CD47.
• the method further comprises administering one or more additional therapeutic agents to the patient.
• the patient has been administered one or more additional therapeutic agents.
• the method comprises monitoring the therapeutic efficacy of the method.
• the method comprises monitoring the prophylactic efficacy of the method.
• the method is repeated until a desired suppression of one or more disease symptoms occurs.
• the heparin is unfractionated heparin.
• the anti-coagulant is V-acctylcystcinc (NAC).
• the anti-coagulant is an antibody against CD 142.
• a kit comprising the combination described herein.
• FIGS. 1A-1B show expression levels of HLA Class I (HLA-I), HLA Class II (HLA- II), and CD47 as measured by flow cytometry for CIlTA indel/indel ; CD47tg human induced pluripotent stem cells (hiPSCs) (FIG. 1A) and endothelial cells differentiated from B2M indel/indel ; CIlTA indel/indel ; CD47zt hiPSCs (hiECs) (FIG. IB), demonstrating that the cells lack expression of HLA-I and HLA-II and have increased expression of CD47.
• HLA-I HLA Class I
• HLA- II HLA Class II
• CD47 show expression levels of HLA Class I (HLA-I), HLA Class II (HLA- II), and CD47 as measured by flow cytometry for CIlTA indel/indel ; CD47tg human induced pluripotent stem cells (hiPSCs) (FI
• FIGS. 2A-2B show surface expression levels of CD46, CD55, and CD59 in B2M indel/indel ; CIlTA indel/indel ; CD47/g hiPSCs (FIG 2A) and B2M indel/indel ; CUT • CD47tg hiECs (FIG. 2B).
• FIGS. 3A-3B show killing of B2M indel/indel ; CIlTA indel/indel ; CD47/g hiPSCs (FIG.
• FIGS. 4A-4B show killing of a representative B2M indel/indel ; CIlTA indel/indel ; CD47/g CD46+++ hiPSC clone (FIG. 4A) and a representative B2M indel/indel ; CIlTA indel/indel ; CD47/g CD46+++ hiEC clone (FIG. 4B) in an ABO-incompatible CDC assay.
• FIGS. 5A-5B show killing of a representative B2M indel/indel ; cilTA indel/indel ; CD47/g CD55++ hiPSC clone (FIG. 5A) and a representative B2M indel/indel ; CIlTA indel/indel ; CD47/g CD55++ hiEC clone (FIG. 5B) in an ABO-incompatible CDC assay.
• FIGS. 6A-6B show killing of a representative B2M indel/indel ; ciYTA indel/indel ; CD47/g CD59+++ hiPSC clone (FIG. 6A) and a representative B2M indel/indel ; cilTA indel/indel ; CD47/g CD59++ hiEC clone (FIG. 6B) in an ABO-incompatible CDC assay.
• FIGS. 7A-7B show killing of a representative B2M indel/indel ; cilTA indel/indel ; CD47/g CD46++/CD55++ hiPSC clone (FIG. 7A) and a representative B2M indel/indel ; ciYTA indel/indel ; CD47/g CD46++/CD55++ hiEC clone (FIG. 7B) in an ABO-incompatible CDC assay.
• FIGS. 8A-8B show killing of a representative B2M indel/indel ; cilTA indel/indel ; CD47/g CD55++/CD59+++ hiPSC clone and a representative B2M indel/indel ; cilTA indel/indel ; CD47/g CD55++/CD59+++ hiEC clone in an ABO-incompatible CDC assay.
• FIGS. 9A-9B show survival of a representative B2M indel/indel ; CIlTA indel/indel ; CD47tg
• CD46++/CD59+++ hiPSC clone (FIG. 9A) and a representative B2M indel/indel ; CIlTA indel/indel ; CD47tg CD46++/CD59++ hiEC clone(FIG. 9B) in an ABO-incompatible CDC assay.
• an engineered immune-evasive cell e.g., an engineered primary hypo-immunogenic cell
• the engineered cells disclosed herein provide for reduced recognition by the recipient subject's immune system, regardless of the subject's genetic make-up, or any existing response within the subject to one or more previous allogeneic transplants, previous autologous chimeric antigen receptor (CAR) T rejection, and/or other autologous or allogenic therapies wherein a transgene is expressed.
• CAR autologous chimeric antigen receptor
• the engineered cells may include, but are not limited to, beta islet cells, B cells, T cells, NK cells, retinal pigmented epithelium cells, glial progenitor cells, endothelial cells, hepatocytes, thyroid cells, skin cells, and blood cells (e.g., plasma cells or platelets).
• the engineered cells comprise reduced expression of CD 142 and increased expression of one or more tolerogenic factor and/or reduced one or more MHC class I molecules and/or one or more MHC class II molecules expression.
• the cells comprising the modifications described herein survive, engraft, and function following transplant.
• the cells exhibit enhanced survival and/or enhanced engraftment and/or long term function in comparison to cells that do not comprise the modification to CD 142.
• the cells are administered via intravenous infusion, intramuscular injection, or kidney capsule transplant.
• the engineered cells 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, and CD55.
• the engineered 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.
• the engineered cells provided herein are protected from complement-mediated cytotoxicity.
• the engineered cells e.g., overexpressing CD46 and CD59
• the engineered cells are protected from complement reactions that occur independently of IBMIR.
• the anti-coagulant is also administered before or after the transplant therapy.
• the anti-coagulant can be administered between 24 and 12 hours, between 12 hours and 6 hours, between 6 hours and 3 hours, between 3 hours and 1 hour, or within one hour before or after transplant.
• the anti-coagulant is administered concurrently with treatment.
• engineered cells e.g., beta islet cells, hepatocytes, and other cells
• the engineered cells comprise reduced or eliminated expression of CD 142, also known as Coagulation Factor III, Tissue Factor (TF), Thromboplastin, platelet tissue factor, or factor III, a membrane receptor in the blood coagulation pathway that contributes to initiating IBMIR.
• CD 142 also known as Coagulation Factor III, Tissue Factor (TF), Thromboplastin, platelet tissue factor, or factor III, a membrane receptor in the blood coagulation pathway that contributes to initiating IBMIR.
• the engineered cells provided herein utilize expression of tolerogenic factors and can also modulate (e.g., reduce or eliminate) one or more MHC class I molecules and/or one or more MHC class II molecules expression (e.g., surface expression).
• genome editing technologies utilizing rare-cutting endonucleases e.g., the CRISPR/Cas, TALEN, zinc finger nuclease, meganuclease, and homing endonuclease systems
• critical immune genes e.g., by deleting genomic DNA of critical immune genes
• engineered cells provided herein exhibit reduced innate immune cell rejection and/or adaptive immune cell rejection (e.g., hypo-immunogenic cells).
• the engineered cells exhibit reduced susceptibility to NK cell-mediated lysis and/or macrophage engulfment.
• the engineered 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.
• Such hypo-immunogenic cells retain cell-specific characteristics and features upon transplantation.
• endogenous refers to a referenced molecule, such as a polynucleotide (e.g. gene), or polypeptide, that is present in a native or unmodified cell.
• a polynucleotide e.g. gene
• polypeptide that is present in a native or unmodified cell.
• endogenous gene refers to expression of a gene encoded by an endogenous nucleic acid contained within the cell and not exogenously introduced.
• a “gene,” includes a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
• a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
• the sequence of a gene is typically present at a fixed chromosomal position or locus on a chromosome in the cell.
• the term “modification” refers to any change or alteration in a cell that impacts gene expression in the cell.
• the modification is a genetic modification that directly changes the gene or regulatory elements thereof encoding a protein product in a cell, such as by gene editing, mutagenesis or by genetic engineering of an exogenous polynucleotide or transgene.
• indel refers to a mutation resulting from an insertion, deletion, or a combination thereof, of nucleotide bases in the genome.
• an indel typically inserts or deletes nucleotides from a sequence.
• an indel in a coding region of a genomic sequence will result in a frameshift mutation, unless the length of the indel is a multiple of three.
• a CRISPR/Cas system of the present disclosure can be used to induce an indel of any length in a target polynucleotide sequence.
• the alteration results in a knock out of the target polynucleotide sequence or a portion thereof.
• Knocking out a target polynucleotide sequence or a portion thereof using a CRISPR/Cas system of the present disclosure can be useful for a variety of applications. For example, knocking out a target polynucleotide sequence in a cell can be performed in vitro for research purposes.
• a treatment may improve the disease condition, but may not be a complete cure for the disease.
• one or more symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the disease.
• 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.
• Non-limiting examples of such anti-CD20 antibody include obinutuzumab, ublituximab, ocaratuzumab, rituximab, rituximab-Rllb, and biosim ilars thereof.
• Cells that express the safety switch are thus CD20-positive and can be targeted for killing through administration of an anti-CD20 antibody as described.
• the safety switch comprises EGFR, which can be recognized by an anti-EGFR antibody.
• Non- limiting examples of such anti-EGFR antibody include tomuzotuximab, RO5083945 (GA201), cetuximab, and biosimilars thereof.
• 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.
• the modification reduces or eliminates, such as knocks out, the expression of one or more MHC class II genes by targeting Class II transactivator (CIITA) expression.
• the modification occurs using a CRISPR/Cas system.
• CIITA is a member of the LR or nucleotide binding domain (NBD) leucine-rich repeat (LRR) family of proteins and regulates the transcription of one or more MHC class II genes by associating with the MHC enhanceosome.
• NBD nucleotide binding domain
• LRR leucine-rich repeat
• 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 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 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 engineered 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 engineered 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).
• 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 polynucleotide encoding CCL21 is inserted into a B2M gene locus, a CIITA gene locus, or a CD142 gene locus.
• the engineered cell is a T cell and the polynucleotide encoding CCL21 is inserted into a TRAC gene locus, or a TRBC gene locus.
• a suitable gene editing system e.g., CRISPR/Cas system or any of the gene editing systems described herein
• CRISPR/Cas system is used to facilitate the insertion of a polynucleotide encoding CCL21, into a genomic locus of the cell.
• 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 engineered 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 engineered 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. 7036, NCBI Gene ID 4240, Uniprot No.
• the cell is B2M’', CIITA ', CD 142''. CD47tg, CAR+.
• the cell e.g. T cell
• the cell may additional be one in which TRAC is reduced or eliminated (e.g. knocked out).
• the cell is B2' 7 ', CIITA ', CD142'', CD47tg,TRAC' CAR+.
• EphB3, EphB4, and EphB6) CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, Bestrophins, TMEM16A, GABA receptor, glycin receptor, ABC transporters, NAV1.1, NAV1.2, NAVI.3, NAV1.4, NAVI.5, NAV1.6, NAV1.7, NAVI.8, NAV1.9, sphingo sin- 1 -phosphate receptor (S1P1R), NMDA channel, transmembrane protein, multispan transmembrane protein, T-cell receptor motifs; T-cell alpha chains; T-cell P chains; T-cell y chains; T-cell 6 chains, CCR7, CD3, CD4, CD5, CD7, CD8, CDl lb, CDl lc,
• the antigen binding domain targets an antigen characteristic of an infectious disease.
• the ABD binds an antigen associated with an infectious disease.
• the antigen is expressed by a cell affected by an infectious disease.
• the infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 (HHV-8, Kaposi sarcoma- associated herpes virus (KSHV)), Human T-lymphotrophic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian virus 40 (SV40), Epstein-Barr virus, CMV, human papillomavirus.
• expression of a gene is increased by increasing endogenous gene activity (e.g., increasing transcription of the exogenous gene).
• endogenous gene activity is increased by increasing activity of a promoter or enhancer operably linked to the endogenous gene.
• increasing activity of the promoter or enhancer comprises making one or more modifications to an endogenous promoter or enhancer that increase activity of the endogenous promoter or enhancer.
• increasing gene activity of an endogenous gene comprises modifying an endogenous promoter of the gene.
• increasing gene activity of an endogenous gene comprises introducing a heterologous promoter.
• 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. 6,140,081;
• 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 e.g., is engineered to bind to a target site of choice.
• 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.
• 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
• 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.
• the regulatory factor is a zinc finger transcription factor (ZF- TF). In some embodiments, the regulatory factor is VP64-p65-Rta (VPR). [0440] In certain embodiments, the regulatory factor further comprises a transcriptional regulatory domain.
• Common domains include, e.g., transcription factor domains (activators, repressors, co-activators, 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.
• 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
• 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 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. [0457]
• 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.
• 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.
• the homology-mediated gene insertion and replacement can be carried out via specific DNA repair pathways such as homology-directed repair (HDR), synthesis-dependent strand annealing (SDSA), microhomology-mediated end joining (MMEJ), and homology-mediated end joining (HMEJ) pathways.
• HDR homology-directed repair
• SDSA synthesis-dependent strand annealing
• MMEJ microhomology-mediated end joining
• HMEJ homology-mediated end joining
• 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 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. 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 19: 55,115,674.
• the cell is manipulated (e.g., converted or differentiated) into a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, beta islet cell, neuron, cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or brown adipocyte.
• a muscle cell erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, beta islet cell, neuron, cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or brown adipocyte.
• the primary cells are isolated or obtained from an individual or from a pool of primary cells isolated or obtained from more than one individual donor.
• the primary cells may be any type of primary cell described herein, including any described in Section II.C.3.
• the primary cells are selected from T cells, NK cells, beta islet cells, endothelial cells, epithelial cells such as RPE, thyroid, skin, or hepatocytes.
• the primary cells from an individual donor or a pool of individual donors are engineered to contain modifications (e.g. genetic modifications) described herein.
• T cells provided herein are useful for the treatment of suitable cancers including, but not limited to, B cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myeloid lymphoid leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.
• B-ALL B cell acute lymphoblastic leukemia
• diffuse large B-cell lymphoma liver cancer
• pancreatic cancer breast cancer
• breast cancer ovarian cancer
• colorectal cancer lung cancer
• non-small cell lung cancer acute myeloid lymphoid leukemia
• multiple myeloma gastric cancer
• RPE cell types include, but are not limited to, retinal pigmented epithelium (RPE) cell, RPE progenitor cell, immature RPE cell, mature RPE cell, functional RPE cell, and the like.
• RPE retinal pigmented epithelium
• the present technology is directed to engineered RPE cells, such as primary RPE cells isolated from one or more individual donors (e.g. healthy donors) or RPE 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 one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and have reduced CD 142 expression.
• the RPE cells further express one or more complement inhibitors.
• the engineered thyroid cells described herein such as primary thyroid cells isolated from one or more individual donors (e.g. healthy donors) or 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 method of producing a population of hypoimmunogenic cardiac cells from a population of hypoimmunogenic pluripotent (HIP) cells by in vitro differentiation comprises: (a) culturing a population of HIP cells in a culture medium comprising a GSK inhibitor; (b) culturing the population of HIP cells in a culture medium comprising a WNT antagonist to produce a population of pre-cardiac cells; and (c) culturing the population of pre-cardiac cells in a culture medium comprising insulin to produce a population of hypoimmune cardiac cells.
• the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof.
• the GSK inhibitor is at a concentration ranging from about 2 mM to about 10 mM.
• the WNT antagonist is IWR1, a derivative thereof, or a variant thereof. In some instances, the WNT antagonist is at a concentration ranging from about 2 mM to about 10 mM.

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